TW201219115A - Microfluidic test module with flexible membrane for internal microenvironment pressure-relief - Google Patents

Abstract

A microfluidic test module having an outer casing for hand held portability, and, a microfluidic device mounted within the outer casing for processing a biological sample, the outer casing providing a microenvironment adjacent the microfluidic device, wherein, the outer casing has a membrane seal of flexible material between the microenvironment and atmosphere for reducing pressure changes in the microenvironment resulting from atmospheric pressure fluctuations.

Description

201219115 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatments and assays for molecular diagnostics. [Prior Art]

Molecular diagnostics have been used to provide an early indication of disease detection before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the incidence of ineffective health care, Improve patient outcomes 'Improve disease management and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobase allows the short sequence (oligonucleotide) of the synthesized DN A to bind (hybridize) to a particular nucleic acid sequence for use in nucleic acid assays. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will get in the future, to determine the type and pathogenicity of the infectious pathogen, or to determine the individual's response to the drug. 核酸 Nucleic acid-based molecular diagnostic test-5-201219115 Nucleic acid-based test There are four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine, sputum and tissue samples, for gene analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are typically required at the beginning of the nucleic acid assay. Blood is one of the most commonly sought sample types. It has three main components: white blood cells, red blood cells, and platelets. Platelets accelerate coagulation and remain active outside the body. To inhibit coagulation, the sample was mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. The red blood cells of the sample are typically removed to concentrate the target cells. In the human body, red blood cells account for about 99% of cellular material, but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by dissolving the red blood cells by differential lysis solution, leaving the remaining remaining cellular material'. The remaining cellular material can then be separated from the sample by centrifugation. This provides a concentration of cells from which the nucleic acid can be extracted. The exact procedure used to extract nucleic acids depends on the sample and the diagnostic analysis to be performed. For example, the protocol used to extract viral RN A is quite different from the protocol used to extract the genome 201219115 DN A. However, self-labeled cell-extracted nucleic acids typically comprise a cell lysis step and subsequent nucleic acid purification. The cell lysis step ruptures the cell and nuclear membrane and releases the genetic material. This is often accomplished using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells.

The nucleic acid is then purified by an alcohol precipitation step, which is usually carried out using ice ethanol or isopropanol, or via a solid phase purification step, which is usually carried out in a column of cerium oxide substrate, resin or high. The concentration is carried out on paramagnetic beads in the presence of a chaotropic salt, followed by washing of the nucleic acid, followed by elution with a buffer of low ionic strength. A selective step prior to precipitation of the nucleic acid is the addition of a protease which digests the protein to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to disrupt thermal lysis of the cell membrane. The target DNA or RNA can be present in the extracted material in very small amounts, φ especially if the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target at a low concentration to a detectable amount. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR) » PCR is well known in the art, and a complete description of this type of reaction is provided in E, van Pelt-Verkuil et al. Principles and Technical Aspects of PCR Amplification, Springer, 2008 ° PCR is a useful technique for amplifying target DNA sequences in the context of complex DNA. If RNA is to be amplified (by PCR), the RNA must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, at 201219115, the obtained cdNA was amplified by PCR. PCR is an exponential method and can be continued as long as the conditions for maintaining the reaction are acceptable. The components of the PCR reaction are:

1. A primer that is complementary to a region of the flanking target sequence and has a short single strand of DNA of about 1 to 30 nucleotides. 2. DN A polymerase-synthetic DNA thermostable enzyme

3. Deoxyribonucleoside triphosphate (dNTP) - provides nucleotides that are incorporated into newly synthesized DNA strands - 4-buffer - provides the ideal chemical environment for DNA synthesis.

PCR typically involves placing these reactants in a vial (about 10 to 50 microliters) containing the extracted nucleic acid. The tube is placed in a thermal cycler; the reactor is a device that allows the reaction to take unequal time at a range of different temperatures. The standard procedures for each thermal cycle involve the denaturing phase, the annealing phase, and the extended phase. The extension phase is sometimes referred to as the primer extension phase. In addition to these three-step procedures, a two-step thermal procedure can also be used in which the annealing phase and the extension phase are combined. The denatured phase typically involves raising the reaction temperature to 90 to 95 ° C to denature the DNA strand; in the annealed phase, the temperature is lowered to 50 to 60 ° C to bond the primer; then in the extended phase, the temperature is raised to The optimal DN A polymerase activity temperature is 60 to 72 °C for primer extension. This process is repeated approximately 20 to 40 times, with the end result of generating millions of sets of sequence copies between the primers.

Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multiple primer set PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR -8 - 201219115 And reverse transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single trial by a single target (targeting 'multiple genes) (in other ways, several trials are required). The optimization of multi-initiator PCR is difficult because it requires the selection of primers with approximate binding temperature and amplicon of similar length and base composition to ensure the amplification efficiency of each amplicon.

Linker-primed PCR, also known as ligation adaptor PCR, is a nucleic acid that amplifies virtually all DNA sequences in a complex DNA mixture without the need for a labeled-specific primer. method. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). A double-stranded oligonucleotide linker (also referred to as a conjugate) having a suitable overhang is then ligated to the end of the target DN A fragment using a ligase. Next, nucleic acid amplification is carried out using an oligonucleotide primer specific for the linker sequence. Thereby, all DNA-derived fragments adjacent to the linker oligonucleotide can be amplified. Direct PCR describes a system that performs PCR directly on a sample without any (or minimal) nucleic acid extraction. Many components present in unpurified biosamples, such as protohemoglobin components in the blood, have long been thought to inhibit PCR reactions. Therefore, it is customary to enhance the purification of the target nucleic acid prior to preparation of the PCR reaction mixture. However, with appropriate changes in chemical properties and sample concentrations, it is possible to perform PCR with minimal DNA purification or direct PCR. Modifications in PCR chemistry for direct PCR include increasing buffer -9 - 201219115 fluid strength, using highly active and processivity polymerases and additives chelated with potential polymerase inhibitors.

Repeated sequence PCR utilizes two separate nucleic acid amplification cycles to increase the probability of amplifying the correct amplicon. One type of repetitive PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in a different nucleic acid amplification cycle. The first pair of primers hybridize to a nucleic acid sequence located in a region other than the target nucleic acid sequence. A second pair of primers (nested primers) is used for the second amplification, the pair of primers being incorporated into the first PCR product to produce a second PCR product containing the target nucleic acid, and the second product is shorter than the first product . The rationale used in this strategy is that if the wrong locus is amplified due to a mistake during the first nucleic acid amplification, the probability that the wrong locus is re-amplified by the second pair of primers is very low, thus ensuring specificity.

Real-time PCR or quantitative PCR was used to measure the amount of PCR product in real time. The initial amount of nucleic acid in the sample can be determined by using a probe containing a fluorophore or a fluorescent dye and a set of standards in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the amount of pathogen in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. The reverse transcription system is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. The isothermal amplification is another form of nucleic acid amplification that does not rely on the thermal denaturation of the target DNA during the -10-201219115 amplification reaction, thus eliminating the need for sophisticated instruments. Therefore, the thermostatic nucleic acid amplification method can be carried out in a field place or simply in an environment outside the laboratory. Some constant temperature nucleic acid amplification methods have been described

Said, including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Normal Amplification (Loop-

Mediated Isothermal Amplification), where constant temperature nucleic acid amplification does not rely on continuous heating to denature template DNA to produce a single strand of the molecule as a template for continued amplification, but to generate a single strand of molecules using other methods at a constant temperature, such as by specific limitations Nucleases perform enzymatic cleavage of DN A molecules or separate DN A double strands using enzymes. The ability of strand-substituted amplification (SDA) to cleave unmodified strands of herni-modified DNA by specific restriction enzymes, as well as the ability of polymerase extension and substitution of downstream stocks lacking 5'-3' exonuclease activity . An exponential nucleic acid amplification is then achieved by coupling sense and antisense, wherein the strand from the sense reaction replaces the template for the antisense reaction. The nicking enzyme used in this reaction does not cleave DNA in a conventional manner, but produces nicks on the strands of DNA, such as N. Alwl, N. BstNBl, and Mlyl. SDA is improved by using a combination of a thermostable restriction enzyme (Aval) and a thermostable outer polymerase (Bst 201219115 polymerase). This combination has been shown to increase the amplification efficiency of the reaction from 1 to 8 fold amplification to 101 (fold) amplification, so it is possible to utilize this technique to amplify unique single copy molecules.

Transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA) RNA polymerase is used to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase, and selective RNase Η (if reverse transcriptase does not have RNase activity). One of the primers contains a promoter sequence of RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to the target ribosomal RNA (rRNA) at a defined site. The reverse transcriptase then extends from the 3' end of the promoter to produce a DNA copy of the target rRNA. The RNA in the formed RNA:DNA duplex is decomposed by the RNase activity (if any) of the reverse transcriptase or the additional RNase. In the next step, the second primer binds to the DNA copy. The new DN Α strand is synthesized from the end of this primer by reverse transcriptase to produce a double-stranded DN A molecule. RN A polymerase recognizes the promoter sequence in the DN A template and begins transcription. Each newly synthesized RN A amplicon is re-entered as a template for a new replication cycle. In recombinase polymerase amplification (RPA), isothermal amplification of a specific DNA fragment is achieved by binding an oppositely directed oligonucleotide primer to the template DNA and then extending the primers by a DNA polymerase. . Denaturation of the double-stranded DNA (dsDNA) template does not require heating. Instead, RP A uses a recombinase-introduction complex to scan dsDN A to facilitate stock exchange at homologous sites. The resulting structure is stabilized by the interaction of a single DN A binding protein with the substituted template strand, thereby preventing the primer from exiting via branch migration -12-201219115. Recombinase unwinding allows the strand-substituted DNA polymerase (such as a large fragment of Bacillus subtilis Pol I (Bsu) to be accessible near the 3' end of the oligonucleotide followed by primer extension. Exponential nucleic acid amplification is accomplished through repeated cycles of this process.

The helicase-dependent amplification (HDA) mimics the in vivo system in which DN A helicase is used to generate a single-strand template for primer hybridization followed by extension of the primer by DNA polymerase. In the first step of the HDA reaction, the helicase passes through the target DNA to interrupt the hydrogen bond between the two strands, which in turn binds to the single-stranded binding protein. The primer is bonded by exposing the single-stranded region by helicase. The DN A polymerase then uses the free deoxyribonucleoside triphosphate (dNTP) to extend the 3 ends of each primer to make two copies of the DNA strand. The two dsDN A replicated strands each enter the next HD A cycle, resulting in amplification of the exponential nucleic acid of the target sequence. Other DNA-based thermostating techniques include rolling circle amplification (RCA), in which a DNA polymerase extends the primer around a circular DNA template to produce a long DNA product consisting of many repeating copies of the loop. Prior to the end of the reaction, the polymerase produced tens of thousands of copies of the circular template, and the copies of the copies were tethered to the original target DNA. This approach allows for spatial dissociation of the target and rapid nucleic acid amplification of the signal. A copy of the template can be generated in one hour at most. A variant of the branched-type amplification system, RC A, which uses a closed circular probe (C-probe) or a padlock probe and a DNA polymerase with high processivity for exponential amplification under constant temperature conditions. The C-probe. Circular Thermostat Amplification (LAMP) provides high selectivity and uses DNA aggregation 201219115

The enzyme and a set of four specially designed primers that recognize a total of six different sequences on the target DNA. The primers within the sense and antisense strands containing the underlying DNA initiate the LAMP. The strands initiated by the exogenous primers then replace the DNA synthesis to release a single strand of DNA. The single-stranded DNA can serve as a template for DNA synthesis initiated by a second primer and an external primer, and the second primer and the foreign primer are hybridized to the other end of the target, and the DNA synthesis produces a stem-loop DNA structure. . In the subsequent LAMP cycle, an internal primer hybridizes to the loop on the product and initiates the synthesis of the substituted DN A, resulting in the original stem loop DN A and the new stem loop DNA with twice as long stems. The cycle reaction continued to accumulate 9 copies of the target within one hour. The final product is a stem-loop DNA having a plurality of inverted repeats of the target and a cauliflower-like structure in which a plurality of loop systems are formed by adhering the overlapping repeating repeats of the same strand to each other.

After completion of the nucleic acid amplification, the amplification product must be analyzed to determine whether or not the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product can be carried out by simply measuring the size of the amplicon by gel electrophoresis to hybridization with DN A to identify the nucleotide composition of the amplicon. Gel electrophoresis is the easiest way to check whether a nucleic acid amplification method produces the desired amplicon. Gel electrophoresis utilizes an electric field applied to a gel matrix to separate DNA fragments. Negatively charged DNA fragments will move in the matrix at different rates, depending on the fragment size. After electrophoresis is completed, the fragments in the gel are stained for visualization. Ethidium bromide is a commonly used dye which exhibits fluorescence under ultraviolet light. The size of the fragments is determined by comparison with DNA ladders -14 - 201219115, which contain DNA fragments of known size and are run side by side with the amplicons on the gel. Since the oligonucleotide primer binds to a specific anchoring point adjacent to the DN A, the size of the amplified product can be predicted and detected as a band of known size on the gel. To determine the correctness of the amplicon, or if several amplicons are produced, a DN A probe that hybridizes to the amplicon is typically employed.

DN A hybridization refers to the formation of double stranded DNA by complementary base pairing. The DN A hybrid used to clearly identify a particular amplification product requires the use of a DN A probe of about 20 nucleotides in length. If the probe has a sequence that is complementary to the amplicon (target) DN A sequence, hybridization will occur at the appropriate temperature, pH and ion concentration. If hybridization occurs, the gene of interest or the DN A sequence is present in the original sample. Optical detection systems are most commonly used to detect hybridization methods. One of the amplicon and probe is labeled with a fluorescent agent or an electrochemiluminescent agent to emit light. These methods differ in that they cause the photo-generating group to produce an excited state, but both can be used to covalently label nucleotide strands. In the case of electrochemiluminescence (ECL), the light system is generated by current stimulating luminescent group molecules or complexes. In the case of fluorescence, it is irradiated with excitation light to cause light to be emitted. The fluorescent system is detected by a light source and a detection unit that provides excitation light of a wavelength absorbed by the fluorescent molecule. The detection unit includes a light sensor (such as a photomultiplier tube or a charge coupled device (CCD) array) that detects a transmitted signal and a device that prevents excitation light from being included in the output of the light sensor (such as a wavelength selective filter). The fluorescent molecules emit Stokes shifted light in response to the excitation light, which is collected by the detection unit. The Stokes shift is the -15-201219115 frequency difference or wavelength difference between the emitted light and the absorbed excitation light. The ECL emission system is detected using a light sensor that is sensitive to the emission wavelength of the E C L species used. For example, transition metal-ligand complexes emit light of a visible wavelength, so conventional photodiodes and CCDs can be used as light sensors. One of the advantages of ECL is that if the emitted light of the ambient light ' E C L · is the only light in the detection system, the sensitivity is increased.

Microarrays allow hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are powerful molecular diagnostic tools that screen thousands of genetic diseases or detect the presence of numerous infectious pathogens in a single experiment. Microarrays are composed of a number of different DNA probes that are fixed at the point of conformation. The target DN A (amplicon) is first labeled with a fluorescent or luminescent molecule (either during nucleic acid amplification or after nucleic acid amplification), followed by administration of the target DN A to the probe microarray. The microarray is cultured in a temperature controlled, humid environment for hours or days to allow hybridization between the probe and the amplicon. After incubation, the microarray must be washed through a series of buffers to remove unbound strands. The surface of the microarray is dried with a gas stream (usually nitrogen) after cleaning. The rigor of miscellaneous and cleaning is critical. Insufficient stringency may result in highly non-specific binding. Excessive rigor may result in inability to properly combine, resulting in reduced sensitivity. Hybridization is identified by detecting the light emitted by the amplicon labeled with the complementary probe forming hybrid. Fluorescent systems from microarrays are detected using a microarray scanner, which is typically a computer-controlled inverted scanning fluorescent conjugated focus microscope that typically uses laser-excited fluorescent dyes and light sensors (such as optoelectronics). The emission signal is detected by the 倍-16-201219115 pipette or CCD). The fluorescent molecules emit Stokes shifted light (as described above) which is collected by the detection unit.

The emitted fluorescent light must be collected, separated from the unabsorbed excitation wavelength and transmitted to the detector. In a microarray scanner, a conjugate focal configuration of a conjugated focal hole aperture mounted on the image side is typically used to eliminate non-focusing information. This device allows only the light of the focused portion to be detected. Light from above and below the focal plane of the target cannot enter the detector, thus increasing the signal to noise ratio. The detected fluorescent photons are converted into electrical energy by a detector and then converted into a digital signal. This digital signal is translated into a number that represents the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the target sequence to which it is hybridized can be simultaneously identified and analyzed. More information on fluorescent probes can be found at: http://www.premierbiosoft.com/tech_notes/FRET_prob e.html

Http ://www.invitrogen.com/site/us/en/home/References/Mo lecular-Probes-The-Handbook/Technical-Notes-and-

Product-Highlights/Fluorescence-Resonance-Energy-

Transfer-FRET.html POINT-OF-CARE Molecular Diagnostics Although molecular diagnostic tests offer many benefits, the growth of such tests in clinical laboratories is still slower than expected, not laboratory medical tests -17-201219115 The mainstream. This is primarily due to the higher complexity and cost of nucleic acid detection compared to assays that do not involve nucleic acid methods. The extensive use of molecular diagnostic testing in clinical settings is closely related to the development of instrumentation, which must significantly reduce costs, provide rapid (automatic) analysis from initial (sample processing) to final (resulting), and does not require The operation of substantial human intervention.

Point-of-care technology can provide care in the physician's office, on the hospital bed side, or even in a consumer-oriented home environment. This technology offers many advantages including: - Quick results, immediate treatment and improved care quality - from very small Sample testing gains laboratory counts - reduces clinical workload - reduces laboratory workload and reduces office productivity by reducing administrative effort

- Improve cost per patient by reducing hospital stays, outpatients can be diagnosed at the time of initial diagnosis and reducing sample handling, storage and delivery - contributing to clinical management decisions such as infection control and antibiotic use in on-wafer laboratories ( Iab-on-a-chip (LOC)-based molecular diagnostics provides a device for automated and accelerated molecular diagnostic analysis of molecular diagnostic systems based on microfluidics. The shorter detection time is primarily due to the fact that the required sample volume is less active, automated, and low-cost built-in cascaded diagnostic method steps within the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are common -18- 201219115

Microfluidic device form. The LOC device has an MST structure within the MST layer for integrating fluid processing onto a single supporting substrate (usually helium). The unit cost of each LOC device is very low by manufacturing the VLSI (Ultra Large Integrated Circuit) lithography technology of the semiconductor industry. However, large external piping and electronics are required to control the flow of fluid through the apparatus, to add reagents, to control reaction conditions, and the like. Connecting the LOC devices to these external devices substantially limits the molecular diagnostic use of the LOC devices in a laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as an option in a fixed-point care environment. In view of this, there is a need for a molecular diagnostic system based on a LOC device for use in point-of-care. SUMMARY OF THE INVENTION Various aspects of the invention are now described by the following numbered paragraphs. GBS001.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a microsystem technology (MST) layer on the support substrate; covering the upper cover of the MST layer, the upper cover having a plurality of upper covers A fluid connection between the MST layer and the MST layer flows from the MST layer to the upper cap and fluid from the cap to the MS T layer. GBS00 1.2 Preferably, the upper cover has passages connecting at least some of the fluid connections, the passages being configured to attract fluid flow between the fluid connections by capillary action. 19·201219115 GBS001.3 Preferably, the reservoir is configured to retain reagents. GBS001.4 Preferably, at least two of the body connectors are formed with the layers of the GBS001.5. GBS001.6 is preferably such that the outer surface of the MST layer GBS001.7 preferably has an outer surface with an entrance to the channel. Preferably, wherein the fluid comprises components of different sizes smaller than the size of the valve and large GBS001.9, preferably at least one of the dialysis portions is an array of holes. GBS00 1.10 Preferably, the waste receptacle for collection from the blood GBS00 1.il is preferably configured to allow the blood to enter. GBS001.12 Preferably, the upper cover has fluid communication with the passageway to maintain fluid communication between the reservoir via the MST layer and the flow passage by surface tension of the meniscus of the reagent. The channel and the reservoir are attached to a single material. The channel is formed on one surface of the layer to form the channel. The upper cover has a dialysis portion on the opposite side of the surface for receiving fluid and feeding the fluid to the microfluidic device, and the dialysis portion is configured to be a component of the size valve. The dialysis section includes the fluid connection member for filtering out blood of the flow system larger than the valve, and the upper cover has red blood cells removed by the dialysis portion. The reagent is an anticoagulant, and the microcapsule device is mixed with the anticoagulant before the dialysis portion is capped.

-20- 201219115 Nucleic acid amplification unit of the nucleic acid sequence in the fluid. GBS001.13 Preferably, the upper cap has a lysis reagent reservoir for containing a lysis reagent to dissolve cells in the blood and to release the nucleic acid sequence within the cell. GBS001.14 Preferably, the nucleic acid amplification part is a polymerase chain reaction (PCR) part, and the upper cover has a PCR reagent reservoir containing dNTPs and primers for amplifying the nucleic acid sequence Before with the blood

GBS001.15 Preferably, the cap has a polymerase reservoir containing a polymerase to mix the polymerase with the fluid prior to amplification of the nucleic acid sequence. GBS001.16 Preferably, the microfluidic device also has a hybridization unit comprising a hybridization chamber containing a probe nucleic acid sequence for binding to a target nucleic acid sequence in the fluid. GBS001.17 Preferably, the probe nucleic acid sequence is comprised of a fluorescence resonance energy transfer (FRET) probe. GBS001.18 Preferably, each of the hybridization chambers has a light sensor for detecting fluorescence from the FRET probe after attachment to the target nucleic acid sequence after fluorescence excitation. GBS001.19 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the fluid. GBS00 1.20 Preferably, the microfluidic device also has a CMOS circuit between the support substrate and the MST layer for operational control of the heater. -21 - 201219115 The surface micromachined layer provides a small feature of high density. The upper cover provides the larger features required. The surface micromachined layer and the upper cover together provide the various structures necessary. GSR001.1 This aspect of the invention provides a test module comprising: a container for receiving an untreated biological sample through an opening;

a cover movable between an open and closed position, exposing an opening when the cover is in an open position, closing the opening when the cover is in an off position; and a microfluidic device for processing the biological sample, the microfluidic device There is a sample inlet; wherein the container is configured to be in fluid communication with the sample inlet to cause the biological sample to flow to the sample inlet by capillary action. GSR001.2 Preferably, the cover is a sealing tape having a low viscosity adhesive for sealing the opening in the closed position. GSR001.3 Preferably, the test module also has a hand movement

The outer casing. GSR001.4 Preferably, the microfluidic device has a lid-on-lab (LOC) device having a support substrate and a microsystem technology (MST) layer on the support substrate, the MST layer having The MST channel and the plurality of fluid connections are in fluid communication with the upper cover, and the upper cover has the sample inlet and the upper cover passage for fluid communication with the fluid connections. GSR001.5 Preferably, the MST channels each have a cross-sectional area of from 1 square micron to 400 square micrometers for biochemical treatment of components within the biological sample, and the upper cover channels each have a width greater than 400 squares - 22-201219115 The cross-sectional area of the micrometer is used to accept the biological sample and the cells transported within the biological sample to a predetermined location in the MST channel. GSR001.6 Preferably, the housing has a lance for puncturing a patient to obtain a blood sample for introduction into the sample inlet. GSR00 1.7 Preferably, the lancet is moveable between a retracted and extended position, and the housing has an offset mechanism to bias the lance toward the extended position and a user activated catch to cause the lancet Retained in the retracted position

Until it is started by the user. GSR001.8 Preferably, the upper cover is configured to draw fluid through the upper cover passage by capillary action. GSR001.9 Preferably, the upper lid has a reservoir in fluid communication with the upper lid passage, the reservoir being configured to retain reagent by surface tension of the meniscus of the reagent. GSR001.10 Preferably, the LOC device has a dialysis section for accepting sample fluids having components of different sizes and dividing the sample fluid into two fluid streams depending on the size of the components. GSR001.il Preferably, the components in the sample comprise cells, and one of the two fluid streams has only cells smaller than a predetermined valve size. GSR001.12 Preferably, the LOC device has a nucleic acid probe. A hybridization portion of a needle sequence configured to detect hybridization of a target nucleic acid sequence within a cell of the sample fluid with the nucleic acid probe sequence. GSR001.13 Preferably, the upper cover has a layered structure, wherein the upper cover passage is formed in an outer layer, and the receptacle is formed on the opposite outer layer. -23- 201219115 GSR00114 Preferably, the upper cover channel is formed on an outer surface of the upper cover. The outer surface is in contact with the MST layer of the LOC device to close the upper cover channel. GSR001M5 Preferably, the upper cover has a waste receptacle for collecting one of the two fluid streams. GSR001 16 Preferably, the reagent in the reservoir is an anticoagulant' and the cap is configured to mix the blood with the anticoagulant prior to entering the dialysis section. GSR001.17 Preferably, the LOC device has a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence in the sample fluid. GSR00 1.18 Preferably, the reservoir is a lysis reagent reservoir for containing a lysis reagent to dissolve cells in the fluid and to release any nucleic acid sequences within the cell. GSR001.19 Preferably, the cap has a PCR reagent reservoir containing dNTPs, buffers and primers to mix the PCR reagents with the fluid prior to amplification of the nucleic acid sequence. GSR00 1.20 Preferably, the cap has a polymerase reservoir containing polymerase' to polymerize the polymerase with the fluid prior to amplification of the nucleic acid sequence. The sample container can introduce the sample into the microfluidic test module and direct the sample into the microscopic sample inlet of the microfluidic device that is inserted into the test module. GSR002.1 This aspect of the invention provides a test module comprising: -24 - 201219115 a housing for hand movement; and a microfluidic device mounted to the housing for processing a biological sample, the microfluidic device having a top-on-lab (LOC) device having a support substrate and a microsystem technology (MST) layer on the support substrate, the MST layer having an MST channel and a plurality of fluid connections to fluid with the cap The cover has a sample inlet and an upper cover passage for fluid communication with the fluid connections.

GSR002.2 Preferably, the outer casing has a container for receiving an untreated biological sample through the opening and a cover movable between the open and closed positions, the opening being exposed when the cover is in the open position, when the cover is closed The opening is closed in position, the container being configured to be in fluid communication with the sample inlet to cause the biological sample to flow to the sample inlet by capillary action. GSR002.3 Preferably, the cover is a sealing tape having a low viscosity adhesive for sealing the opening in the closed position. GSR002.4 Preferably, the MST layer has a heater for heating the fluid within the MST channel. GSR002.5 Preferably, the MST channels each have a cross-sectional area of from 1 square micron to 400 square micrometers for biochemical treatment of components within the biological sample, and the upper cap channels respectively have greater than 400 square microns The cross-sectional area is for accepting the biological sample and the cells transported within the biological sample to a predetermined location in the MST channel. GSR002.6 Preferably, the housing has a lance for puncturing a patient to obtain a blood sample for introduction into the sample inlet. GSR002.7 Preferably, the needle is movable between -25 and 201219115 in the retracted and extended position, and the housing has an offset mechanism to bias the needle to the extended position, and a user-actuated lock (catch) ) to keep the needle in the retracted position until activated by the user. GSR002.8 Preferably, the upper cover is configured to draw fluid through the upper cover passage by capillary action. GSR002.9 Preferably, the upper cover has a fluid with the upper cover channel

In the case of a reservoir, the reservoir is configured to retain the reagent by surface tension of the meniscus of the reagent. GSR002.1 0 Preferably, the LOC device has a dialysis section for accepting sample fluids having components of different sizes and dividing the sample fluid into two streams of fluid depending on the size of the components. GSR002.il Preferably, the components in the sample comprise cells, and one of the two fluid streams has only cells smaller than a predetermined valve size

GSR002.1 2 Preferably, the LOC device has a hybridization portion comprising a probe nucleic acid sequence configured to detect hybridization of a target nucleic acid sequence within a cell of the sample fluid with the probe nucleic acid sequence . GSR002.1 3 Preferably, the upper cover has a layered structure, wherein the upper cover passage is formed in an outer layer, and the receptacle is formed on the opposite outer layer. GSR002. 1 4 Preferably, the upper cover channel is formed on the outer surface of the upper cover. The outer surface is in contact with the MST layer of the LOC device to close the upper cover channel. GSR002.1 5 Preferably, the upper cover has a waste receptacle for collecting one of the two fluid streams. -26-201219115 GSR002.1 6 Preferably, the reagent in the reservoir is an anticoagulant and the cap is configured to mix the blood with the anticoagulant prior to entering the dialysis section. GSR002.1 7 Preferably, the LOC device has a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence in the sample fluid. GSR002.1 8 Preferably, the reservoir is a lysis reagent reservoir for containing a lysis reagent to dissolve cells in the fluid and release the intracellular

Any nucleic acid sequence. GSR002.1 9 Preferably, the upper cap has a PCR reagent reservoir containing dNTPs and primers to mix the PCR reagents with the fluid prior to amplification of the nucleic acid sequence. GSR002.20 Preferably, the cap has a polymerase reservoir containing a polymerase to mix the polymerase with the fluid prior to amplification of the nucleic acid sequence.

The surface micromachined layer provides a lower density of features. The upper cover provides the larger features required. The surface micromachined layer and the upper cover together provide the various features necessary. The sample container can introduce the sample into the microfluidic test module, and guide the sample into a microscopic sample inlet of the microfluidic device that is inserted into the test module by funnel. GAS001.1 This aspect of the invention provides a microfluidic A device comprising: a support substrate; a microsystem technology (MST) layer for processing a fluid sample; and a light sensor adjacent to the MST layer for detecting photons emitted by the MST layer -27-201219115. GAS001.2 Preferably, the fluid sample has a target component. The MST layer has a probe array for reacting with the target molecule, and a probe-target complex that emits photons when excited. GAS001.3 Preferably, the target molecule is a labeled nucleic acid and the probe is configured to form a probe-target hybrid to enable the probe to detect the probe-target hybrid within the probe array. GAS001.4 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate, and the probe resonant energy transfer (FRET) probe, wherein the light sensor is a photodiode in a CMOS circuit Polar body array. Preferably, the microfluidic device also has a polylock reaction (PCR) portion and an array of hybridization chambers containing the FRET probe for amplifying the nucleic acid sequence in the fluid prior to hybridization with the FRET probe. Each of the hybridization chambers has a light window to expose the FRET light. GAS001.6 Preferably, the dipole system is registered with each of the hybrid chambers in the photodiode array. GAS001.7 Preferably, the CMOS circuit has a pad for electrical connection of the device and a memory for storing identification data of each FRET probe. The CM0s circuit is configured to convert the photodiode Outputting a signal indicating that the probe-target hybrid β probe has been formed, and providing the signal to the pad for transmission to the external child, and forming a sequence, the light sensation is between the fluorophores The enzyme is linked to the target of the PCR

Photoelectric and external types of FRET device from the spoon -28 - 201219115 GAS001.8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. GAS.001.9 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS001.10 Preferably, the fluorophore is a transition metal-ligand complex. GAS001.il Preferably, the luminescent group is a lanthanide metal-ligand error

Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS001.13 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS001.14 Preferably, the CMOS circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence. GAS001.15 Preferably, the microfluidic device also has a hybrid heating φ device controlled by the CMOS circuit to provide thermal energy for hybridization. GAS001.16 Preferably, the hybridization portion has the PCR portion. The fluid flow path to the end liquid sensor, the hybrid chamber being disposed along two sides of the fluid flow path. GAS001.17 Preferably, the fluid flow path is configured to draw fluid from the PCR portion to the liquid endpoint sensor by capillary action, and each of the hybrid chambers is configured to be filled with the fluid from the fluid by capillary action The flow path fluid is such that during use, the CMOS circuit reacts from the liquid end -29-201219115 point sensor to indicate that the fluid has reached the output of the liquid end point sensor to activate the hybrid; G AS00 1 .18 Preferably, the volume of each of the hybridization chambers is less than 9,000 cubic microns. GAS001.19 Preferably, the distance between the photodiode and the FRET probe is less than 24.9 microns. GAS00 1.20 Preferably, the microfluidic device also has a

a sample inlet of the fluid sample and a plurality of reagent reservoirs for processing different reagents required for the fluid sample, wherein the fluid sample is attracted to the endpoint sensor from the inlet by capillary action, and no need to add from the micro a liquid from a source other than the fluid device.

The integrated image sensor eliminates the need for expensive external imaging systems, offers a comprehensive solution that is both mass-produced and inexpensive, and its low system components represent a lightweight, highly mobile system. The integrated image sensor has the benefit of increased read sensitivity due to large angle light collection and eliminates the need to use optical components in the string of light collecting elements. GAS002.1 Aspects of the invention provide a microfluidic device for detecting hybridization of a probe to a target nucleic acid sequence, the microfluidic device comprising: a sample inlet for receiving a fluid sample having a labeled nucleic acid sequence; An array for hybridization with the target nucleic acid sequence; and a flow path from the sample inlet to the array; wherein the flow path is configured to attract the fluid sample from the sample inlet to the probe by capillary action -30- 201219115 GAS 002.2 Preferably, the microfluidic device also has: a support substrate; a microsystem technology (MST) layer for processing a fluid sample, the MST layer having the flow path; and light adjacent to the MST layer An inductor for detecting photons emitted by the MST layer;

The probe is configured to hybridize to the target nucleic acid sequence to form a probe-target hybrid that emits photons. GAS002.3 Preferably, the microfluidic device also has a polymerase ligation reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS002.4 Preferably, the microfluidic device also has a CMOS circuit between the MS T layer and the support substrate, and the probe is a fluorescence resonance energy transfer (FRET) probe, wherein the light sensor The system is incorporated into the photodiode array in the CMOS circuit [J. GAS 0021.5 Preferably, the microfluidic device also has an array of hybridization chambers containing the FRET probes, each having a light window to expose the FRET probe to excitation light. GAS002.6 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the hybrid portion by capillary action. GAS002.7 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and a memory for storing identification data identifying each FRET probe type, the CMOS circuit being configured to convert from the 201219115 The output of the photodiode is a signal indicative of the FRET probe that has formed the probe-target hybrid and is provided to the pad for transmission to the external device GAS002.8. Preferably, the CMOS circuit is configured The photodiode is activated after a predetermined delay after the excitation light is extinguished. GAS002.9 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.

GAS002.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS002.il Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS002.1 2 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS002.1 3 Preferably, the quencher does not have natural emission light that is responsive to the excitation light.

GAS002.1 4 Preferably, the C Μ Ο S circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence. GAS002.1 5 Preferably, the microfluidic device also has a hybrid heater controlled by the CMOS circuit to provide thermal energy for hybridization.

GAS002.1 6 Preferably, the microfluidic device also has a fluid flow path from the PCR portion to the end liquid sensor, the hybrid chamber being disposed along both sides of the fluid flow path. GAS002.1 7 Preferably, the fluid flow path is configured to draw fluid from the PCR portion to the liquid endpoint sensor by capillary-32-201219115, and each of the hybrid chambers is configured to The capillary action fills the fluid from the fluid flow path such that during use, the CMOS circuit activates the hybridization heater from the output of the liquid endpoint sensor indicating that the fluid has reached the output of the liquid endpoint sensor. GAS002.1 8 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns.

GAS002.1 9 Preferably, the photodiode is at a distance of less than 249 microns from the FRET probe. GAS002.20 Preferably, the microfluidic device also has a reagent reservoir for receiving a sample inlet of the fluid sample and a plurality of different reagents required to process the fluid sample, wherein the fluid sample is acted upon by capillary action The inlet is attracted to the endpoint sensor and there is no need to add liquid from a source other than the microfluidic device. The sample inlet can introduce the sample into the microfluidic test module, which delivers a small amount of sample to a designated portion of the microfluidic device with a high volumetric efficiency of φ. The probe hybrid provides analysis of the target via hybridization. GAS003.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a microsystem technology (MST) layer provided with a hybridization portion having a probe array for hybridization with the target nucleic acid sequence Forming a probe-target hybrid; and a light sensor' is used to detect the probe-target hybridization-33-201219115. GAS003.2 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate, wherein the photo sensor is shunted into the photodiode array in the CMOS circuit. GAS003.3 Preferably, the microfluidic device also has an array of hybridization chambers containing the probe, wherein the probe is a fluorescence resonance energy transfer (FRET) probe, and the hybridization chamber has a light window to expose the FRET Probe to excitation light.

GAS003.4 Preferably, the microfluidic device also has a polymerase linkage reaction (PCR) portion for amplifying the target nucleic acid sequence prior to hybridization to the FRET probe. GAS003.5 Preferably, the MST layer has a plurality of MST channels configured to attract fluid containing the target nucleic acid sequence through the PC R portion and into the hybridization chamber by capillary action. GAS 003.6 Preferably, the CMOS circuit has an external

a pad electrically connected to the device, wherein the CMOS circuit is configured to convert an output from the photodiode to display a signal that the FRET probe hybridizes to the target nucleic acid sequence and provide the signal to the pad for transmission to The external device. GAS 003.7 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS003.8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. GAS003.9 Preferably, the FRET probe has a fluorophore -34 - 201219115 and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS003.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS 003.1 1 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS003.12 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate.

GAS003.1 3 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS003.14 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS 003.1 5 Preferably, the microfluidic device also has a hybrid heater controlled by the CMOS circuit to provide thermal energy for hybridization. GAS 003.1 6 Preferably, the microfluidic device also has a fluid flow path from the PCR φ portion to the end liquid sensor, the hybrid chamber being disposed along two sides of the fluid flow path. GAS003.1 7 preferably 'the fluid flow path configured to draw fluid from the PCR portion to the liquid endpoint sensor by capillary action, and each of the hybrid chambers is configured to be filled by capillary action The fluid flow path 'cures' the CMOS circuit reacts during use to activate the hybrid heater from the liquid end point sensor indicating that the fluid has reached the output of the liquid end point sensor. GAS 003.1 8 Preferably, the volume of each of the hybridization chambers is less than 9,000 • 35 - 201219115 cubic microns. GAS 003.1 9 Preferably, the photodiode is less than 249 microns from the FRET probe. GAS 003.20 preferably the microfluidic device also has a plurality of reagent reservoirs for treating the different reagents required for the fluid, wherein the flow system is attracted to the endpoint sensor from the inlet by capillary action and does not need to be added Liquid from a source other than the microfluidic device

The probe hybridization provides analysis of the target via hybridization. The integrated image sensor eliminates the need for costly external imaging systems, provides a comprehensive solution that is both mass-produced and inexpensive, and its low system components represent a lightweight, highly mobile system. The integrated image sensor has the benefit of increased read sensitivity due to large angle light collection and eliminates the need to use optical components in the string of light collecting elements. GAS004.1 This aspect of the invention provides a microfluidic device comprising:

a support substrate; a microsystem technology (MST) layer covering the support substrate for processing a fluid containing a target nucleic acid sequence, the MS T layer having an array of hybridization chambers, respectively, for containing the nucleic acid sequence a probe for hybridization; a temperature sensor for sensing the temperature of the array of hybridization chambers; and a hybridization heater for heating the array of hybridization chambers; wherein, when used, from the temperature sensor The output is used to feedback control the hybrid heater. Preferably, the microfluidic device also has a CMOS circuit between the support substrate and the MST layer, the CMOS circuit having a light sensor. GAS004.3 Preferably, the light sensor is an array of photodiodes respectively registered with each of the hybrid chambers. GAS004.4 Preferably, the CMOS circuit has a storage off

A digital memory of data processed by the fluid, the data including the probe details and the location of each probe in the array. GAS004.5 Preferably, the microfluidic device also has a reagent reservoir that integrally contains all of the reagents used to treat the fluid.

GAS004.6 Preferably, the hybridization chamber has a heater controlled by the CMOS circuit, respectively, for maintaining the hybridization temperature of the probe and the target nucleic acid sequence. GAS004.7 is less than 249 microns. GAS004.8 cubic micron. GAS004.9 cubic micron. GAS004.10 square micron. GAS004.il micron. Preferably, the distance between the photodiode and the hybridization chamber is preferably, the volume of the hybridization chamber is less than 900,000. Preferably, the volume of the hybridization chamber is less than 200,000. Preferably, the volume of the hybridization chamber is smaller than Preferably, the volume of the hybridization chamber is less than 9000 cubic centimeters. Preferably, the CMOS derives a single result from a photodiode corresponding to a hybridization chamber comprising the same probe -37-201219115. GAS004.1 3 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS004.1 4 Preferably, the hybridization chamber has a light window positioned to expose the FRET probe. To the excitation light.

GAS004.1 5 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured such that when the FRET probe system forms a hybrid with one of the target nucleic acid probes, The fluorophore emits a fluorescent signal to the light sensor in response to the excitation light, the CMOS circuit being configured to activate the light sensor after a predetermined delay after the excitation light is extinguished. GAS004.1 6 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid. GAS004.1 7 Preferably, the CMOS circuit has an external

The device is electrically connected to the pad, and the CMOS circuit is configured to convert the output from the photodiode to display a signal that the FRET probe hybridizes to the target nucleic acid sequence and provide the signal to the pad for transmission to The external device. GAS004.1 8 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the array of the hybrid chamber by capillary action. GAS004.1 9 Preferably, the LOC device has an upper cover that defines the reagent reservoir. GAS004.20 Preferably, each of the reagent reservoirs has a surface tension valve -38 - 201219115, each surface tension valve having a meniscus anchor for forming a meniscus to retain reagent therein. The probe hybridization provides analysis of the target via hybridization. The temperature feedback control ensures control of the temperature within the hybridization chamber to achieve optimal hybridization temperature and subsequent optimal detection temperature. GAS006.1 This aspect of the invention provides a microfluidic device' which comprises:

a support substrate; a microsystem technology (MS T) layer covering the support substrate for processing a fluid containing a target nucleic acid sequence, the MST layer having an array of hybridization chambers respectively containing nucleic acid sequences for use with the target A hybrid probe that is configured to respond to excitation light to emit a fluorescent signal upon formation of hybridization; wherein the hybridization chamber has a wall portion that is optically transmissive by the fluorescent signal. GAS006.2 Preferably, the microfluidic device also has a light sensor, wherein the wall portion is interposed between the probe and the light sensor. GAS 006.3 Preferably, the microfluidic device also has a CMOS circuit between the support substrate and the MST layer, the CMOS circuit having the light sensor. GAS006.4 Preferably, the microfluidic device also has an array of hybridization chambers wherein the light sensors are respectively located in an array of photodiodes registered at each of the hybridization chambers. GAS006.5 Preferably, the CMOS circuit has a digital body for storing data relating to the processing of the fluid, the data including the details of the probe -39 - 201219115 and the position of each probe in the array. GAS006.6 Preferably, the CMOS circuit has a temperature sensor for sensing the temperature of the hybridization chamber array, wherein the output from the temperature sensor is used to feedback control the hybridization heater. GAS006.7 Preferably, the microfluidic device also has a heater controlled by the CMOS circuit for maintaining the hybridization temperature of the probe and the target nucleic acid sequence.

GAS006.8 Preferably, the distance between the light sensor and the hybridization chamber is less than 249 microns. GAS006.9 Preferably, the wall is not penetrated by the excitation light. GAS006.1 0 Preferably, the probe is a fluorescence resonance energy transfer (F R E T) probe. GAS006.il Preferably, the hybridization chamber has a light window positioned to expose the FRET probe to the excitation light. GAS006.1 2 Preferably, the FRET probes each have a fluorophore and

a quencher, the fluorophore being configured such that when the FRET probe system hybridizes to one of the target nucleic acid sequences, the fluorophore emits a fluorescent signal in response to the excitation light, and the CMOS circuit is configured The light sensor is activated after a predetermined delay after the excitation light is extinguished. GAS006.1 3 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid. GAS006.1 4 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert the output of the photodiode from -40 to 201219115 to display the FRET probe a signal that hybridizes to the target nucleic acid sequence and provides the signal to the pad for transmission to the external device

GAS 006.1 5 Preferably, the microfluidic device also has a reagent reservoir that integrally contains all of the reagents used to treat the fluid. GAS 0 0 6.1 6 Preferably, the volume of the reagent reservoir is less than 20,000,000 cubic microns each.

GAS006.1 7 Preferably, the microfluidic device also has an upper cover defining the reagent reservoir. GAS006.1 8 Preferably, each of the reagent reservoirs has a surface tension valve, each of the surface tension valves having a meniscus anchor for forming a meniscus to retain reagent therein. GAS006.1 9 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the array of hybridization chambers by capillary action. The probe hybridization provides analysis of the target via hybridization. The light-transmissive hybridization chamber provides for the transmission of a fluorescent signal for detecting hybridization of the target with the probe. GAS007.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a microsystem technology (MS T) layer covering the support substrate for processing a fluid containing a target nucleic acid sequence, the MST layer Having multiple probe spottings' each of the probe spots separately contains a probe for hybridization with the target nucleic acid sequence - 41 - 201219115 needle: wherein the probe mass in each of the probe spots is less than 270 picograms . GAS007.2 preferably has a probe mass of less than 60 picograms in each of the probe spots. GAS007.3 Preferably, the probe mass in each of the probe spots is less than 12 picograms. GAS007.4 preferably has a probe mass of less than 2.7 picograms in each of the probe spots.

GAS 007.5 preferably 'the microfluidic device also has a photodiode array, wherein the MST layer has an array of hybridization chambers, each of the hybridization chambers respectively containing one of the probe spots, and the position of the photodiode array At least one of the photodiodes is associated with each of the hybridization chambers. GAS007.6 Preferably, the microfluidic device also has a CMOS circuit between the support substrate and the MST layer, the CMOS circuit having an array of the photodiodes.

GAS007.7 Preferably, each of the hybridization chambers has a volume of less than 200,000 cubic microns. GAS007.8 Preferably, each of the hybridization chambers has a volume of less than 40,000 cubic microns. GAS007.9 Preferably, each of the hybridization chambers has a volume of less than 9000 cubic microns. GAS007.1 0 Preferably, the CMOS circuit has digital memory for storing data relating to the processing of the fluid, the data including the probe details and the position of each of the probes in the array. -42- 201219115 GAS007.1 1 Preferably, the microfluidic device also has a heater controlled by the CMOS circuit for maintaining the hybridization temperature of the probe and the target nucleic acid sequence. GAS007.1 2 Preferably, the CMOS circuit has a temperature sensor for sensing the temperature of the hybridization chamber array, wherein the output from the temperature sensor is used to feedback control the hybridization heater. GAS 007.1 3 preferably 'the distance of the light sensor from the hybridization chamber

GAS007.1 4 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 007.1 5 Preferably, the hybridization chamber has a light window positioned to expose the FRET probe to the excitation light. GAS007.1 6 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured such that when the FRET probe line forms a hybrid with one of the target nucleic acid sequences, the fluorescein The photocell emits a fluorescent light signal in response to the excitation light, the CMOS circuit being configured to activate the light sensor after a predetermined delay after the excitation light is extinguished, the digital memory including the predetermined delay. GAS007.1 7 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid. GAS 007.1 8 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence -43- 201219115 Column hybridization signal and provides this signal to the pad for transmission to the external device. GAS007.1 9 Preferably, the microfluidic device also has a reagent reservoir that integrally contains all of the reagents used to treat the fluid. GAS007.20 Preferably, the volume of the reagent reservoir is less than 20,000,000 cubic microns each.

This low probe volume represents a low probe cost, which in turn allows for inexpensive detection systems. GAS008.1 This aspect of the invention provides a microfluidic device comprising: a support substrate: a microsystem technology (MST) layer provided with a hybridization portion having an array of hybridization chambers, each hybridization chamber containing Different probe types for hybridization of the target nucleic acid sequence: wherein each of the hybridization chambers has a volume of less than 900,000 cubic microns.

GAS008.2 Preferably, each of the hybridization chambers has a volume of less than 200,000 cubic microns. GAS008.3 Preferably, each of the hybridization chambers has a volume of less than 40,000 cubic microns. GAS008.4 Preferably, each of the hybridization chambers has a volume of less than 9000 cubic microns. GAS008.5 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate; wherein the CMOS circuit has a probe for detecting the hybrid in the hybrid cell array - 44 - 201219115 Photodiode array. GAS 008.6 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 008.7 Preferably, each of the hybridization chambers has a light window for exposing the FRET probe to excitation light. GAS 008.8 Preferably, the microfluidic device also has a polymerase linkage

A lock reaction (PCR) portion for amplifying a target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS008.9 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the hybrid portion by capillary action. GAS 008.1 0 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence Hybridizing the signal and providing the signal to the pad for transmission to the external device

GAS008.il Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS008.1 2 Preferably, the CMOS circuit is configured to activate the inductor after a predetermined delay after the excitation light is extinguished. GAS 008.1 3 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 〇〇 nanosecond. GAS 00 8.14 Preferably, the fluorophore is a transition metal-ligand complex. -45- 201219115 GAS008.1 5 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS008.1 6 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS 008.1 7 Preferably, the CMOS circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence.

GAS 008.1 8 Preferably, each of the hybridization chambers has hybridization heaters for providing hybridization heat energy, each of the hybridization heaters being controlled by the CMOS circuit. GAS008.1 9 Preferably, the hybridization portion has a fluid flow path from the PCR portion to the end point liquid sensor, the hybridization chamber being disposed along both sides of the fluid flow path. GAS 008.20 Preferably, the fluid flow path is configured to be by hair

Finely acting from the PCR portion to draw fluid flow to the liquid endpoint sensor, and each of the hybrid chambers is configured to be filled with fluid from the fluid flow path by capillary action such that during use, the CMOS circuit reacts from the liquid The endpoint sensor indicates that the fluid has reached the output of the liquid endpoint sensor to activate the hybrid heater. This low volume hybridization chamber represents, to some extent, a low probe volume, thereby providing low probe cost and an inexpensive detection system. GAS009.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a sample of biological material containing a target nucleic acid sequence; and -46-201219115 Microsystem Technology (MST) layer, the MST The layer is provided with a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence and a probe array for hybridizing with the target nucleic acid sequence. GAS009.2 Preferably, the microfluidic device also has a support substrate and a CMOS. Circuit;

The probe is configured to form a probe-target hybrid upon hybridization with the target nucleic acid sequence, the probe-target hybridization system configured to emit photons in response to an excitation source, and the CMOS circuit is interposed between the MST layers A photodiode array is provided between the support substrate and a hybrid for detecting the probe-target. GAS 009.3 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS009.4 Preferably, each of the hybridization chambers has a means for exposing the

I FRET probe to the light window of the excitation light. GAS009.5 Preferably, the MST layer has a plurality of MST channels, and the MST channels are configured to attract fluid containing the sample through the PCR portion and into the hybridization chamber by capillary action. GAS009.6 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence The signal is hybridized and the signal is provided to the pad for transmission to the external device. GAS009.7 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. -47-201219115 GAS009.8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. GAS009.9 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 nanosecond. GAS009.1 0 Preferably, the PCR portion has at least one elongated PCR chamber;

At least one elongated heater element for heating a nucleic acid sequence within the elongate PCR chamber; wherein the elongate heater element extends GAS009.il parallel to a longitudinal length of the PCR chamber. Preferably, the PCR portion has The PCR inlet and the microchannel of the PCR outlet, and the elongate PCR chamber is part of the microchannel. GAS009.1 2 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action.

GAS009.1 3 Preferably, the PCR portion has a plurality of elongate PCR chambers, and the microchannels have a curved configuration formed by a series of wide curves, each of the wide curves forming the elongate PCR chamber One of the channel departments. GAS 009.14 Preferably, each of the channel portions along each of the wide curves has a plurality of elongated heaters. GAS009.1 5 Preferably, the plurality of elongated heaters are arranged end to end along the channel portion. GAS009.1 6 Preferably, the plurality of elongated heaters are independently operated -48-201219115 GAS009.1 7 Preferably, the microfluidic device also has at least one temperature sensor for feedback control Long heater. GAS009.1 8 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the polymerase, the dNTP and the buffer in the elongated heater to amplify the The liquid in the PCR portion is retained in the nucleic acid sequence.

GAS009.1 9 Preferably, the active valve has a meniscus anchor and a heater for anchoring the meniscus such that the liquid is retained in the PCR portion, the heating The device is used to boil the liquid at the meniscus anchor to release the meniscus, and the capillary drive flow is restored to flow out of the PCR portion. GAS009.20 Preferably, the PCR portion has a thermal cycle time of less than 30 seconds. The PCR section is amplified by the target to provide the sensitivity required for the target detection. The probe hybridization provides analysis of the target via hybridization. The integrated PCR and probe hybridization section substantially reduces the possibility of introducing contamination during the assay, simplifies the analysis phase, and provides a lightweight and inexpensive single-device analysis solution. GAS0 1 0.1 This aspect of the invention provides a microfluidic device comprising: a sample flow path for a sample flow path of a biological sample containing a target nucleic acid sequence; an array of hybridization chambers, each hybridization chamber containing for use with the target a probe for hybridization of a nucleic acid sequence, each of the hybridization chambers having a chamber inlet for fluid communication between the sample stream and the -49-201219115 probe: wherein the chamber inlet is configured as a diffusion barrier to prevent hybridization The needle spreads between the hybrid chambers, causing erroneous hybridization assay results. GAS010.2 Preferably, the chamber inlet defines a tortuous flow path. GAS01 0.3 Preferably, the tortuous flow path has a curved configuration. GAS01 0.4 Preferably, the microfluidic device also has a support substrate

a microsystem technology (MST) layer having the sample flow path and the hybrid chamber array, and a CMOS circuit interposed between the MST layer and the support substrate; wherein the CMOS circuit has a hybridization for detecting the probe Photodiode array. GAS010.5 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS01 0.6 Preferably, each of the hybridization chambers has a light window for exposing the FRET probe to excitation light. GAS010.7 Preferably, the microfluidic device also has a polymerase linkage

A lock reaction (PCR) portion for amplifying a target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS 010.8 Preferably, the sample flow path is configured to attract the sample fluid through the PCR portion and into the hybridization chamber by capillary action. GAS010.9 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence The signal is hybridized and the signal is provided to the pad for transmission to the external device. -50- 201219115 GAS01 0.1 0 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 010.il Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS01 0.1 3 Preferably, the fluorophore is a transition metal-ligand error

Compound. GAS 010.14 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS01 0.1 5 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 010.16 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS 010.17 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS 010.18 Preferably, each of the hybridization chambers has hybridization heaters for providing hybridization heat energy, each of the hybridization heaters being controlled by the CMOS circuit. GAS 010.19 Preferably, the sample fluid flow path has an endpoint liquid sensor such that during use, the CMOS circuit is activated in response to output from the endpoint liquid sensor indicating that the sample fluid has reached the output of the liquid endpoint sensor The hybrid heater. GAS010.20 Preferably, the -51 - 201219115 spacing between the photodiode and the probe is less than 249 microns. The probe hybridization provides analysis of the target via hybridization. The diffusion barrier substantially eliminates the reflux of the probe prior to and after hybridization to prevent signal loss and provide a high degree of detection sensitivity. GAS012.1 This aspect of the invention provides a microfluidic device comprising: a support substrate;

a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to excitation light; and on the substrate a CMOS circuit having a light sensor for returning a fluorescent signal to produce an output signal; wherein the CMOS circuit is configured to trigger a time delay when the excitation light is deactivated until the light sensor is activated . GAS012.2 Preferably, the light sensor is a photodiode and the

The spacing between the probe and the photodiode is less than 24 9 microns. GAS 012.3 Preferably, the microfluidic device also has a probe array and a corresponding photodiode array. GAS012.4 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorescence lifetime of the fluorophore is greater than 〇〇 nanosecond. GAS 012.6 Preferably, the fluorophore is a transition metal-ligand complex. -52- 201219115 GAS012.7 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS012.8 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 012.9 Preferably, the quencher does not have natural emission light that is responsive to the excitation light.

GAS012.10 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying a target nucleic acid sequence from a biological sample. GAS012.11 Preferably, the microfluidic device also has an array of hybridization chambers for containing the FRET probes, each having a light window to expose the FRET probe to the excitation light. GAS012.12 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS012.13 Preferably, the probe array has more than 1 000 probes

GAS012.14 Preferably, the time delay is between 100 picoseconds and 10 milliseconds. GAS 01 2.15 Preferably, the microfluidic device also has a trigger photodiode that is responsive to the excitation light and configured to provide an indication that the excitation light is deactivated. GAS012.16 Preferably, the CMOS circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence. GAS 0 1 2. 17 Preferably, each of the hybridization chambers has hybridization heaters for providing hybrid energy of -53 - 201219115, each of which is controlled by operation of the CMOS circuit. GAS 0 1 2.1 8 Preferably, the sample fluid flow path has an end point liquid sensor such that during use, the C Ο 电路 S circuit responds to the liquid sensor from the end point indicating that the sample fluid has reached the end point liquid sensing The hybrid heater is activated by the output of the device.

GAS012.19 Preferably, the PCR portion has an active valve for retaining the PCR portion when the elongated heater heats the circulation of the nucleic acid sequence and a mixture of primers, dNTPs, polymerase and buffer to amplify the nucleic acid sequence Liquid in the middle. GAS012.20 Preferably, the active valve has a meniscus anchor and a heater for anchoring a meniscus such that the liquid is retained in the PCR portion, the heater It is used to boil the liquid at the meniscus anchor to release the meniscus, and the capillary drive flow is restored to flow out of the PCR portion.

This time delayed fluorescence detection eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. GAS013.1 This aspect of the invention provides a microfluidic device comprising: a probe for hybridizing with a target nucleic acid sequence to form a probe-target hybrid having a reporter fluorophore a fluorescent light emitting signal for responding to the excitation light; a detecting photodiode for exposing to the excitation light and the fluorescent signal: and -54-201219115 for triggering the photodiode to be exposed to the excitation light, The trigger photodiode system is configured to activate the detection photodiode after the excitation light is extinguished. GAS013.2 Preferably, the microfluidic device also has a hybrid comprising a probe for hybridization to the target nucleic acid sequence. The probe is fixed to an inner surface of the hybridization chamber, and the detection photodiode and the trigger photodiode system are adjacent to the inner surface.

GAS 013.3 Preferably, the hybridization chamber has a light window on the opposite side of the inner surface, the probe being attached to the inner surface to expose the probe to the excitation light. Preferably, the microfluidic device also has a hybridization chamber array containing different probes for hybridization with the respective target nucleic acid sequences, and a corresponding array of the detection photodiodes and the triggering photodiodes. GAS013.5 Preferably, the detection photodiode system is larger than the photoluminescent diode. GAS013.6 Preferably, the microfluidic device also has: a support substrate; a microsystem technology (MST) layer having a hybrid chamber array; and a CMOS circuit interposed between the MST layer and the support substrate, the CMOS circuit The array of the detection photodiode and the trigger photodiode are included; wherein the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 01 3.7 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence. -55-201219115 GAS013.8 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the hybridization chamber by capillary action. GAS 013.9 Preferably, the microfluidic device also has a pad for electrically connecting to an external device, wherein the CMOS circuit is configured to generate a signal indicative of hybridization of the FRET probe to the target sequence and provide the signal To the pad for transmission to the external device.

GAS013.10 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS013.il Preferably, the FRET probes have a fluorophore and a quencher, respectively, which have a fluorescence lifetime greater than 100 nanoseconds. GAS013.12 Preferably, the fluorophore is a transition metal-ligand complex. GAS013.13 Preferably, the fluorophore is a lanthanide metal-ligand complex.

GAS013.14 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS013.15 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS013.16 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS013.17 Preferably, each of the hybridization chambers has hybridization heaters for providing hybridization heat energy, each of the hybridization heaters being controlled by the CMOS circuit. Preferably, the microfluidic device also has a fluid flow path from the PCR portion through the hybridization chamber to the end liquid sensor. The hybridization chamber is disposed along both sides of the fluid flow path.

GAS01 3.19 Preferably, the fluid flow path is configured to draw fluid from the PCR portion to the liquid end point sensor by capillary action, and each of the hybrid chambers is configured to be filled with the fluid flow by capillary action The fluid of the circuit is such that during use, the CMOS circuit activates the hybrid heater by reacting an output from the liquid endpoint sensor indicating that the fluid has reached the output of the liquid endpoint sensor. GAS013.20 Preferably, the PCR portion has an active valve for retaining the nucleic acid sequence and the primer, dNTP, polymerase and buffer complex when the elongated heater is heated to amplify the nucleic acid sequence The liquid in the PCR section. The sensor triggers the light sensor to provide the correct sensing time, improving the signal to noise ratio and sensitivity. This time delayed fluorescence detection eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. GAS014.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a hybridization chamber containing a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid The probe-target hybridization system is configured to generate a fluorescent signal in response to the excitation light; and a CMOS circuit between the support substrate and the hybridization chamber, the CMOS circuit having an output for returning the fluorescent signal Signal light-57-201219115 an electric diode, and a triggering photodiode for returning an excitation light to produce an output; wherein the CMOS circuit is configured to display the excitation light when the trigger photodiode is The photodiode is activated after deactivation. GAS 014.2 Preferably, the CMOS circuit provides a time delay between deactivation of the excitation light and activation of the photodiode. GAS014.3 Preferably, the microfluidic device also has the hybridization chamber

In the array, each hybridization chamber contains a probe and a corresponding photodiode array. GAS 014.4 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS014.5 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS 014.6 Preferably, the fluorophore is a transition metal-ligand complex. GAS014.7 Preferably, the luminescent group is a lanthanide metal-ligand error

Compound. GAS 014.8 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 014.9 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS014.10 Preferably, the microfluidic device also has a polymerase linkage reaction (PCR) portion for amplifying a target nucleic acid sequence from a biological sample. GAS014.il Preferably, each of the hybridization chambers has a light window for exposing the -58-201219115 FRET probe to the excitation light.

GAS014.12 Preferably, the CMOS circuit has a memory for storing identification data of the FRET probe. GAS014.13 Preferably, the probe array has more than one probe. GAS014.14 Preferably, the time delay is between 1 and 1 millisecond.

GAS014.15 Preferably, the microfluidic device also has: at least one calibration source configured to generate a calibration emission; a calibration photodiode for sensing the calibration emission; wherein the CMOS circuit has a differential circuit The subtracted output of the calibrated photodiode is subtracted from the detected photodiode output. GAS014.16 Preferably, the microfluidic device also has: a plurality of calibration sources distributed throughout the array of hybridization chambers; wherein the calibration source is a calibration probe that does not have a fluorophore. GAS014.17 Preferably, the microfluidic device also has a plurality of calibration chambers containing the calibration source, the calibration chambers being distributed throughout the array of hybridization chambers, wherein when used, from the detection photodiodes The output of either is compared to the output from the calibrated photodiode closest to the detection photodiode. GAS014.18 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns. GAS014.19 Preferably, each of the calibration chambers is surrounded by a three by three hybrid chamber block. -59- 201219115 GAS014.20 Preferably, the quenching agent does not have a natural emission light that is supposed to illuminate the pupil. Each light sensor is optimally sensed by its dedicated proximity trigger light sensor sensing trigger to improve the signal to noise ratio and sensitivity. This time delayed fluorescence detection eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight.

GAS 0 1 5 . 1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a probe array each having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybridization The probe-target hybridization system is configured to generate a fluorescent signal in response to the excitation light; and a CMOS circuit on the substrate having a photodiode array for sensing the deactivation of the excitation light And generating an output signal in response to a fluorescent signal from a probe-target hybrid within the probe array.

GAS 015.2 Preferably, the microfluidic device also has an array of hybridization chambers, each of the photodiodes corresponding to one of the hybridization chambers. GAS 015.3 Preferably, the volume of the test hybridization chamber is less than 900,000 cubic microns each. GAS 015.4 Preferably, the CMOS circuit provides a time delay between sensing the deactivation of the laser and sensing the fluorescent signal. GAS 015.5 Preferably, the photodiode is at a distance of less than 249 microns from the probe in the corresponding hybridization chamber. GAS015.6 Preferably, the probe is a fluorescent resonance energy transfer -60-201219115 (FRET) probe. GAS015.7 Preferably, the FRET probes have a fluorescent krypton and a quenching agent, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS 015.8 Preferably, the fluorophore is a transition metal-ligand complex. GAS 015.9 Preferably, the fluorophore is a lanthanide metal-ligand complex.

Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS015.il Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS015.12 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying a target nucleic acid sequence from a biological sample. GAS015.13 Preferably, each of the hybridization chambers has a light window for exposing the FRET probe to the excitation light.

GAS015.14 Preferably, the CMOS circuit has a memory for storing identification data of the FRET probe. GAS015.15 Preferably, the probe array has more than one probe. GAS015.16 Preferably, the time delay is between 100 picoseconds and 10 milliseconds. GAS015.17 Preferably, the microfluidic device also has: a plurality of calibration sources distributed throughout the array of hybridization chambers, wherein the -61 - 201219115 CMOS is configured to calibrate the photodiode using the calibration source The output. GAS01 5. 1 8 Preferably, the calibration source is a calibration probe that does not have a fluorophore.

GAS015.19 Preferably, the microfluidic device also has a plurality of calibration chambers containing the calibration source, the calibration chambers being distributed throughout the array of hybridization chambers, wherein when used, from the photodiode The output of one is compared to the output from the calibrated photodiode closest to the photodiode. GAS 015.20 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns.

Each light sensor is optimally sensed by its dedicated proximity trigger light sensor sensing trigger to improve the signal to noise ratio and sensitivity. This time delayed fluorescence detection eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. The use of the hybrid to detect the light sensor to trigger the photodetection period provides the benefit of most of the sensor-triggered light detection while allowing the use of larger hybridization detection light sensors to increase sensitivity. GAS016.1 This aspect of the invention provides a microfluidic device comprising: a support substrate: a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe - The target hybridization system is configured to generate a fluorescent signal in response to the excitation light; and a CMOS circuit on the substrate, the CMOS circuit having a light sensor for generating an output signal in response to the fluorescent signal of -62-201219115; The CMOS circuit is configured to initiate a time delay until the excitation light is deactivated until the photosensor is activated. GAS 016.2 Preferably the CMOS circuit is configured to control the excitation light such that deactivation of the excitation light triggers the CMOS circuit to induce a time delay prior to activation of the light sensor.

GAS 016.3 Preferably, the CMOS circuit has a trigger photodiode for echoing the excitation light to generate an output, such that the CMOS circuit induces the excitation photodiode when the excitation photon has been deactivated time delay. GAS 016.4 Preferably, the light sensor is a photodiode and the spacing between the probe and the photodiode is less than 249 microns. GAS 016.5 Preferably, the microfluidic device also has a probe array and a corresponding photodiode array. GAS 016.6 Preferably, the microfluidic device also has an excitation sensing φ photodiode configured to indicate when the excitation light is deactivated. GAS016.7 Preferably, the microfluidic device also has an array of hybridization chambers, each of the photodiodes corresponding to one of the hybridization chambers, and the excitation photodiode is also corresponding to one of the hybridization chambers, The hybridization chamber corresponding to the excitation photodiode does not include a fluorescent reporter. GAS 016.8 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 016.9 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. -63- 201219115 GAS016.10 Preferably, the fluorophore is a transition metal-ligand complex. GAS016.il Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS016.12 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate.

GAS016.13 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS016.14 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion that is used to amplify a target nucleic acid sequence from a biological sample.

GAS 016.15 Preferably, the CMOS circuit has a memory for storing the identification data of the FRET probe. GAS 016.16 Preferably, the probe array has more than one probe.

GAS01 6.17 Preferably, the time delay is between 1 and 1 millisecond. GAS01 6.18 Preferably, the microfluidic device also has a cover overlying the PCR portion, the cap defining a plurality of reagent reservoirs containing reagents for addition to the target nucleic acid sequence. GAS016.19 Preferably, the reagent in the reagent reservoir is selected from the group consisting of: anticoagulants, lysing reagents, restriction enzymes, buffers, dNTPs and primers, and polymerases. This time-delayed fluorescence detection eliminates the need for any wavelength-dependent filter components -64-201219115, making the design inexpensive, small, and lightweight. Reducing the area of the trigger circuit with a delay triggering the light detection period allows for the use of larger hybridization detection light sensors to increase sensitivity. GAS017.1 Aspects of the invention provide a microfluidic device for detecting a target nucleic acid sequence, the microfluidic device comprising: a probe for hybridizing with the target nucleic acid sequence to form a probe-target hybrid, the probe Having a fluorophore to generate fluorescence emission in response to excitation light

a photodiode for detecting the fluorescent emission; a shunt transistor interposed between the photodiode and the voltage source; and a CMOS circuit for controlling the shunt transistor to remove the photodiode A carrier generated by a photon that absorbs excitation light in a polar body. GAS 017.2 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS 01 7.3 Preferably, the microfluidic device also has a transmission transistor between the anode and the output electrode of the optical φ electric diode, wherein the transmission electro-crystal system is configured to be activated when the excitation light is activated Live and activate after the excitation light is deactivated. GAS017.4 Preferably, the microfluidic device also has a reset transistor for removing a carrier wave that the transmission transistor leaks during excitation by the excitation light, the reset crystal system being configured to be excited It is turned on when the light is activated and turned off when the excitation light is deactivated. GAS017.5 Preferably, the microfluidic device also has a supporting substrate Between the supporting substrate and the probe for controlling the shunt transistor, the transmitting transistor and the The transistor is reset, wherein the photodiode system is shunted into the CMOS circuit and the probe is a fluorescence resonance energy transfer (FRET) probe.

Preferably, the microfluidic device also has an array of FRET probes and a photodiode array corresponding to each of the FRET probes, each FRET probe being located in a respective hybridization chamber and configured for use Hybridizing with different target nucleic acid sequences, the hybridization chambers each have a light window to expose the FRET probe to the excitation light.

GAS0 17.7 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence prior to hybridization to the FRET probe. GAS 017.8 preferably 'the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MS T) layer' having a plurality of MST layers configured to attract the fluid through the PCR portion by capillary action and into the hybridization Microchannels in the room. GAS017.9 Preferably, the CMOS circuit has an external

The device is electrically connected to the pad and configured to convert the output from the photodiode to display a signal that the fret probe hybridizes to the target nucleic acid sequence and provide the signal to the pad for transmission to the external device .

GAS 017.10 Preferably, the CMOS circuit has a memory for storing the identification data of the FRET probe. GAS 017.il Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished. GAS017.12 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. -66 - 201219115 GAS01 7.1 3 Preferably, the fluorophore is a transition metal-ligand complex. GAS 017.14 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS017.15 Preferably, the fluorophore is selected from the group consisting of: a ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS01 7. 16 Preferably, the quenching agent does not have a response light

The natural emission of light. GAS 017.17 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS 017.18 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns. GAS 017.19 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS 017.20 Preferably, the probe mass in each of the hybridization chambers is less than 60 picograms. The need for a light sensor with a controllable shunt eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. GAS018.1 Aspects of the invention provide a microfluidic device for detecting a target nucleic acid sequence, the microfluidic device comprising: a probe for hybridizing with the target nucleic acid sequence to form a probe-target hybrid, the probe a photodiode having a fluorophore in response to the excitation light to generate a fluorescent emission for detecting the fluorescent emission; and -67-201219115 a CMOS circuit for activating the photodiode; wherein the fluorescent group is fluorescent The lifetime is greater than 1 nanosecond. GAS018.2 Preferably, the microfluidic device according to claim 1 further comprises a shunt transistor interposed between the photodiode and the voltage source to remove the excitation light in the photodiode. A carrier generated by the photon, wherein the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated.

GAS018.3 Preferably, the microfluidic device also has a transmission transistor interposed between the anode and the output electrode of the photodiode, wherein the transmission electro-crystal system is configured to deactivate when the excitation light is activated and Activated after the excitation light is deactivated. GAS 018.4 Preferably, the microfluidic device also has a reset transistor for removing a carrier wave that the transmission transistor leaks during excitation by the excitation light, the reset crystal system being configured to be excited It is turned on when the light is activated and turned off when the excitation light is deactivated.

Preferably, the microfluidic device also has a support substrate between the support substrate and the probe for controlling the shunt transistor, the transfer transistor and the reset transistor, The photodiode system is incorporated into the CMOS circuit, and the probe is a fluorescence resonance energy transfer (FRET) probe.

GAS018.6 Preferably, the microfluidic device also has an array of FRET probes and corresponding photodiode arrays, each FRET probe being located in a respective hybridization chamber and configured for use with different target nucleic acid sequences Hybridization, the hybridization chamber has a light window to expose the FRET probe to the excitation light -68-201219115 GAS01 8.7. Preferably, the microfluidic device also has a polymerase chain reaction (PCR) portion, which is used for The target nucleic acid sequence is amplified prior to hybridization of the FRET probe. GAS018.8 Preferably, the PCR portion and the hybridization chamber are entangled into a microsystem technology (MS T) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room.

GAS018.9 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the light sensor to display that the FRET probe hybridizes to the target nucleic acid sequence Signal and provide the signal to the pad for transmission to the external device

GAS018.10 Preferably, the CMOS circuit has a memory for storing identification data of the FRET probe. GAS018.il Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished. GAS018.12 Preferably, the FRET probes each have a quenching agent for quenching the fluorescent emission from the fluorophore. GAS018.13 Preferably, the fluorophore is a transition metal-ligand complex. GAS018.14 Preferably, the fluorophore is a lanthanide metal-ligand complex. Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. -69- 201219115 GAS 0 1 8 . 1 6 Preferably, the extinguishing agent does not have a natural emission light that is supposed to illuminate the excitation light. G A S 0 1 8 .1 7 Preferably, the C Μ 0 S circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS018.18 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms.

GAS018.19 Preferably, the probe mass in each of the hybridization chambers is less than 60 picograms. GAS01 8.20 Preferably, the probe mass in each of the hybridization chambers is less than 12 picograms. The need for a light-sensing eagle with a controllable shunt eliminates the need for any wavelength-dependent filter assembly, making the design inexpensive, small, and lightweight. Low shunt resistance improves signal-to-noise ratio and sensitivity. GAS019.1 This aspect of the invention provides a microfluidic device comprising:

a probe for hybridization with a target nucleic acid sequence; a photodiode for detecting fluorescence emission generated by the hybridization of the probe in response to excitation light; and between the photodiode anode and the voltage source a shunt transistor, the shunt cell system configured to remove a carrier generated by photons that absorb excitation light in the photodiode; wherein a periphery of the photodiode and a periphery of the shunt transistor are continuous with each other by. GAS019.2 Preferably, the shunt cell system is configured to be at the -70-

201219115 Turns on when the excitation light is activated and when the excitation light is deactivated. GAS019.3 Preferably, the microfluidic device also has a transmission transistor between the anode and the output electrode of the dielectric diode, the transmission crystal system configured to deactivate and deactivate the excitation light when activated After activation. GAS019.4 Preferably, the microfluidic device also has a carrier transistor that is leaked during excitation by the excitation light, the reset transistor system being configured to activate the excitation light and the excitation light Close when you go to work. GAS 01 9.5 Preferably, the microfluidic device also has a CMOS circuit between the interposer and the support substrate, and the CMOS circuit uses the shunt transistor, the transfer transistor and the reset transistor, and the sensor system thereof The CMOS circuit is incorporated and the probe is a fluorescent co-transfer (FRET) probe. GAS019.6 Preferably, the microfluidic device also has an array of the φ probes, each FRET probe is located in a respective hybridization chamber for hybridization with different target nucleic acid sequences, and the hybrid chambers are respectively The FRET probe is exposed to excitation light. GAS 019.7 Preferably, the microfluidic device also has a poly-purine reaction (PCR) moiety for the nucleic acid sequence in the fluid that is hybridized to the FRET probe. GAS019.8 Preferably, the PCR portion and the hybridization system {System Technology (MST) layer, the MST layer having a plurality of channels configured to attract the fluid through the PCR portion and into the hybridization chamber by the action of E 'The light is transmitted: illuminate•Remove, reset to turn on the probe to control the light energy FRET is equipped with light' enzymatically amplified into microcapillary-71 - 201219115 GAS019.9 Preferably, the CMOS circuit has An external device electrically connected to the pad and configured to convert an output from the photosensor to display a signal that the fret probe hybridizes to the target nucleic acid sequence and provide the signal to the pad for transmission to the external device . GAS019.10 Preferably, the CMOS circuit has a body of information for storing identification data of the FRET probe. GAS019.il Preferably, the CMOS circuit is configured to

The light sensor is activated after a delay of the excitation light. Preferably, the FRET probes have a fluorophore and a quencher, respectively, which have a fluorescence lifetime greater than 100 nanoseconds. GAS019.13 Preferably, the fluorophore is a transition metal-ligand complex. GAS019.14 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS019.15 Preferably, the fluorophore is selected from the group consisting of:

Ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 019.16 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS019.17 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS 019.18 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS 019.19 Preferably, the probe mass in each of the hybridization chambers is less than 60 picograms. -72- 201219115 GAS019.20 Preferably, the probe mass in each of the hybridization chambers is less than 12 picograms. The need for a light sensor with a controllable shunt eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. The shunt around the photodetector has low resistance, thus improving signal to noise ratio and sensitivity. GAS020.1 provides a microfluidic device in accordance with this aspect of the invention.

a probe for hybridizing with a target nucleic acid sequence to form a probe-target hybrid having a photodiode for detecting an active region of the fluorescent emission generated by the probe-target hybrid in response to excitation light; a shunting transistor between the photodiode anode and a voltage source, the shunt cell system configured to remove a carrier generated by photons that absorb excitation light in the photodiode; wherein the shunt cell system Included in the active zone. GAS020.2 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS020.3 Preferably, the microfluidic device also has a transmission transistor interposed between the anode and the output electrode of the photodiode, wherein the transmission electro-crystal system is configured to be turned on and activated when the excitation light is activated The excitation light is deactivated and then turned off. GAS020.4 Preferably, the microfluidic device also has a reset-73-201219115 transistor for removing the carrier that the transmission transistor leaks during excitation by the excitation light, the resetted crystal system is configured It is turned off when the excitation light is activated and when the excitation light is deactivated. GAS020.5 Preferably, the microfluidic device also has a CMOS circuit between the probe and the support substrate. The CMOS circuit is used to control the shunt transistor, the transfer transistor and the reset transistor. Wherein the light sensor is incorporated into the CMOS circuit, and the probe is a fluorescence resonance energy transfer (FRET) probe.

GAS020.6 Preferably, the microfluidic device also has an array of the FRET probes, each of the FRET probes being located in a respective hybridization chamber and configured for hybridization with different target nucleic acid sequences. A window to expose the FRET probe to the excitation light. GAS020.7 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS020.8 Preferably, the PCR portion and the hybridization chamber system are intrusive

A system technology (MST) layer having a plurality of channels configured to attract the fluid through the PCR portion and into the hybridization chamber by capillary action. GAS020.9 Preferably, the CMOS circuit has a pad for electrically connecting to an external device and the CMOS circuit is configured to convert an output from the light sensor to show that the FRET probe hybridizes to the target nucleic acid sequence The signal is provided and the signal is provided to the pad for transmission to the external device GAS020.1. Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. -74- 201219115 GAS020.1 1 Preferably, the CMOS circuit is configured to initiate the light sensor after a delay after the excitation light is extinguished. Preferably, the FRET probes have a fluorophore and a quencher, respectively, which have a fluorescence lifetime greater than 1 nanosecond. GAS020.1 3 Preferably, the fluorophore is a transition metal-ligand complex.

GAS 020.14 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS020.1 5 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS020.1 6 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS020.1 7 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS020.1 8 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS 020.19 Preferably, the probe mass in each of the hybridization chambers is less than 60 picograms. GAS020.20 Preferably, the probe mass in each of the hybridization chambers is less than 12 picograms. The need for a light sensor with a controllable shunt eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. The shunt inside the photodetector has low resistance, thus improving signal to noise ratio and sensitivity. -75-201219115 GAS021.1 This aspect of the invention provides a microfluidic device comprising: a support substrate: comprising a probe a hybridization chamber, the probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to excitation light; and a photodiode , having an active region and an optical axis extending perpendicular to the active region and passing through the hybridization chamber:

The distance between the active region and the probe-target hybrid is less than 249 microns. GAS021.2 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS021.3 Preferably, the probe mass in each of the hybridization chambers is less than 60 picograms. GAS021.4 Preferably, the probe quality system in each of the hybridization chambers

Less than 12 picograms. GAS021.5 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the photodiode system is a component of the CMOS circuit: wherein the CMOS circuit is configured to trigger when the excitation light is deactivated The time is delayed until the photodiode is activated. GAS021.6 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The photon produced by -76- 201219115. GAS021.7 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS021.8 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe.

GAS021.9 Preferably, the microfluidic device also has a hybrid chamber array and a photodiode array comprising different types of fret probes configured to hybridize to different target nucleic acid sequences, each of the hybrid chambers having One of the respective photodiodes. GAS021.10 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS021.il Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room. GAS021.12 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and is configured to convert an output from the photodiode to display a signal that the FRET probe hybridizes to the target nucleic acid sequence And providing the signal to the pad for transmission to the external device. GAS 02 1.13 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS021.14 Preferably, the CMOS circuit is configured to initiate the light sensor after a delay after the excitation light is extinguished. GAS021.15 Preferably, the FRET probe has a fluorophore -77-201219115 and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS021.16 Preferably, the fluorophore is a transition metal-ligand complex. GAS021.17 Preferably, the luminescent group is a lanthanide metal-ligand complex. Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate.

GAS021.19 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS02 1.20 Preferably, the C Ο Ο S circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. This non-imaging optics provides a comprehensive solution that is both mass-produced and inexpensive, with a low number of system components representing a lightweight, highly mobile system. This non-imaging optics provides improved read sensitivity and eliminates the need for optical components in the light collecting component string due to large angle light collection.

GAS022.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a hybridization chamber containing a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid And generating a fluorescent signal in response to the excitation light; and a photodiode having an active region and an optical axis extending perpendicular to the active region and passing through the hybridization chamber; wherein the hybridization chamber has an active region parallel to the photodiode The bottom of the configuration is -78-201219115. The surface of the bottom surface has a center of mass and the active zone is contained in a pyramid where the centroid is located at the apex, and the apex angle is less than 173. . GAS022.2 Preferably, the distance between the active region and the bottom surface is less than 249 microns. GAS022.3 Preferably, the photodiode has a field of view such that a fluorescent signal from the probe-target hybrid is incident on the active region. GAS022.4 Preferably, the microfluidic device also has the support

A CMOS circuit on a substrate, the photodiode system being a component of the CMOS circuit: wherein the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS 022.5 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 022.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS022.7 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS022.8 Preferably, the microfluidic device also has a hybrid chamber array and a photodiode array comprising different types of FRET probes configured to hybridize to different target nucleic acid sequences, each of the hybrid chambers having One of the respective photodiodes. GAS022.9 Preferably, the microfluidic device also has a polymerase-79-201219115 lock reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer, the MST layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room.

GAS022.il preferably, the CMOS circuit has a pad for electrically connecting to an external device, and is configured to convert an output from the photodiode to display a signal that the fret probe hybridizes to the target nucleic acid sequence And providing the signal to the pad for transmission to the external device. GAS022.1 2 Preferably, the CMOS circuit has a memory for storing the identification data of the FRET probe. GAS 022.1 3 Preferably, the CMOS circuit is configured to delay the activation of the excitation light after it is extinguished. Diode. GAS022.1 4 Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 〇〇 nanosecond.

GAS 022.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS022.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS022.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 022.1 8 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS022.1 9 Preferably, the quencher does not have a natural emission of light that should be excited by -80-201219115. GAS022.20 Preferably, the C Μ O S circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. This high-angle emission light collection provides a comprehensive solution that is both mass-produced and inexpensive, and its low system components represent a lightweight, highly mobile system. This large angle of emitted light collection increases the sensitivity of reading and eliminates the need to use optical components in the string of light collecting elements.

GAS023.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; an inlet for receiving a biological sample containing the target nucleic acid sequence; and a nucleic acid sequence for hybridizing to the target nucleic acid sequence to form a probe-target hybrid and a probe that generates a fluorescent signal in response to the excitation light; and a light sensor for sensing the fluorescent signal; wherein, when used, the biological sample is added to the inlet to prevent subsequent addition to the The inlet fluid is in fluid communication with the probe. GAS 023.2 Preferably, the microfluidic device also has a CMOS circuit formed on the support substrate, wherein the photo sensor is incorporated into a photodiode that is a component of the CMOS circuit. GAS 023.3 Preferably, the CMOS circuit is configured to deactivate the excitation light and then activate the photodiode after a time delay. Preferably, the distance between the probe and the photodiode is less than 249 microns. GAS023.5 Preferably, the microfluidic device also has a shunt transistor between the opto-81 - 201219115 diode anode and a voltage source, the shunt cell system being configured to be removed in the photodiode The carrier generated by the absorption of photons of the excitation light. GAS 023.6 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS023.7 Preferably, the microfluidic device also has a probe array that is a fluorescence resonance energy transfer (FRET) probe.

GAS 023.8 Preferably, the microfluidic device also has a hybrid chamber array and a photodiode array comprising different types of FRET probes configured to hybridize to different target nucleic acid sequences, each of the hybrid chambers having a respective One of the photodiodes. GAS 023.9 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS023.1 0 Preferably, the PCR part and the hybridization system are intrusive

A system technology (MST) layer having a plurality of channels configured to attract the sample through the PCR portion and into the hybridization chamber by capillary action. GAS023.il preferably, the CMOS circuit has a pad for electrically connecting to an external device, and is configured to convert an output from the photodiode to display a signal that the FRET probe hybridizes to the target nucleic acid sequence And providing the signal to the pad for transmission to the external device. GAS023.1 2 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 02 3.1 3 Preferably, the CMOS circuit is configured to initiate the photodiode after a delay after the -82-201219115 excitation light is extinguished. GAS023.1 4 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS023.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS023.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex.

GAS023.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS023.1 8 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS 023.1 9 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS023.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight microfluidic device accepts a biological sample, uses its integrated imaging sensor to hybridize the probe to identify the nucleic acid sequence of the sample, and provides an electronic result on the output pad. GAS024.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid and a fluorescent signal in response to the excitation light: And a light sensor located adjacent to the array of probes; wherein -83-201219115 some S-Mingle St probes are used for sensing. The photodetector is sensitized to the photon. The probe is preferably a photodiode array located between the probe array and the support substrate. GAS 024.3 Preferably, the light sensor has a field of view such that a fluorescent signal from the probe-target hybrid is incident on the light sensor. GAS024.4 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, wherein the array of photodiodes is registered with an array of the probes, and the photodiode system is a component of the CMOS circuit; Wherein the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS024.5 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS024.6 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS024.7 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS024.8 Preferably, the microfluidic device also has a hybrid chamber array and a photodiode array comprising different types of fret probes configured to hybridize to different target nucleic acid sequences, each of the hybrid chambers having One of the respective photodiodes. GAS024.9 Preferably, the microfluidic device also has a polymerase linked 201219115 lock reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS024.1 0 Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room.

GAS024.il Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence Hybridizing the signal and providing the signal to the pad for transmission to the external device

GAS024.1 2 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS024.1 3 Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished.

GAS024.1 4 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS024.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS024.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS024.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 024.1 8 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. -85- 201219115 GAS024.1 9 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light. GAS024.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts biological samples, uses its integrated imaging sensor to identify the nucleic acid sequence of the sample via probe hybridization, and provides electronic results on the output pads of the sample.

GAS025.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid and generating a fluorescent signal in response to excitation light a probe; and a CMOS circuit on the support substrate for operating and controlling the excitation light. GAS025.2 Preferably, the operational control includes activating and deactivating the

Excitation light, and adjusting the power supplied to the excitation light. GAS025.3 Preferably, the CMOS circuit has a photodiode for sensing the fluorescent signal, and the CMOS circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS025.4 Preferably, the distance between the probe and the photodiode is less than 249 microns. GAS025.5 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The photon produced by -86- 201219115. GAS025.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS025.7 Preferably, the microfluidic device also has a probe array that is a fluorescence resonance energy transfer (FRET) probe. GAS 025.8 Preferably, the microfluidic device also has a hybrid array

A column and a photodiode array comprising different types of FRET probes configured to hybridize to different target nucleic acid sequences, each having one of its respective photodiodes. GAS 025.9 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS 02 5.1 0 Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room. GAS 02 5.il Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target The nucleic acid sequence hybridizes with a signal and provides the signal to a pad for transmission to the external device. GAS025.1 2 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 025.1 3 Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished. Preferably, the FRET probes have a fluorophore and a quencher, respectively, which have a fluorescence lifetime greater than 100 nanoseconds. GAS025.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS025.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS025.1 7 Preferably, the fluorophore is selected from the group consisting of:

Ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 025.1 8 Preferably, the probe mass in each of the hybridization chambers is less than 270 picograms. GAS 025.1 9 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light. GAS 025.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

The integrated LED driver on the LOC device is operated by the universal USB, providing an easy to use, mass-produced, inexpensive and lightweight system with a low component count. GAS026.1 This aspect of the invention provides a on-wafer laboratory (LOC) device comprising: a support substrate; a microsystem technology (MS T) layer provided with a hybridization portion having fluorescence resonance energy transfer ( An array of FRET probes for hybridization to a target nucleic acid sequence in a fluid; and a light sensor for detecting probe hybridization in the probe array. -88- 201219115

GAS 026.2 Preferably, the light sensor is a photodiode array incorporating a CMOS circuit between the MST layer and the support substrate.

GAS 026.3 Preferably, the hybridization portion has a hybridization chamber array containing different FRET probes, each having a light window to expose the FRET probe to excitation light. GAS026.4 Preferably, the LOC device also has a polymerase chain

A reaction (PCR) portion for amplifying a target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS 026.5 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the hybrid portion by capillary action. GAS026.6 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence The signal is hybridized and the signal is provided to the pad for transmission to the external device. GAS 026.7 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 026.8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. GAS026.9 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS 026.1 0 Preferably, the fluorophore is a transition metal-ligand complex. -89- 201219115 GAS026.1 1 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS026.1 2 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 026.1 3 Preferably, the quencher does not have a natural emission of light that should be excited.

GAS 026.1 4 Preferably, the CMOS circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence. GAS 026.1 5 Preferably, the LOC device also has a hybrid heater that is controlled by the CMOS circuit to provide thermal energy for hybridization. GAS 026.1 6 Preferably, the hybridization portion has a fluid flow path from the PCR portion to the end point liquid sensor, the hybridization chamber being disposed along both sides of the fluid flow path.

GAS 026.1 7 Preferably, the fluid flow path is configured to draw fluid from the PCR portion to the liquid endpoint sensor by capillary action, and each of the hybrid chambers is configured to be filled with the fluid from the fluid by capillary action a flow path fluid such that during use, the CMOS circuit reacts from the liquid end point sensor to indicate that the fluid has reached the output of the liquid end point sensor to activate the hybrid heater. GAS026.1 8 preferably, each The volume of the hybridization chamber is less than 9,000 cubic microns. GAS 026.1 9 Preferably, the photodiode is at a distance of less than 249 microns from the FRET probe. -90 - 201219115 GAS026.20 Preferably, the LOC device also has a plurality of reagent reservoirs for processing different reagents required for the fluid, wherein the flow system is attracted to the endpoint sensor from the inlet by capillary action. It is not necessary to add a liquid from a source other than the LOC device.

The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts a biological sample 'using a synthetic imaging sensor to hybridize with a fluorescence resonance energy transfer (FRET) probe to identify the nucleic acid sequence of the sample, and An electronic result is provided at the output pad, and the FRET probe provides a sequence with high specificity and high reliability for detecting the target. GAS027.1 This aspect of the invention provides a LOC device comprising: a support substrate; a stem-loop probe coupled to the primer, having a nucleic acid sequence and a primer, the nucleic acid sequence being matched to the target nucleic acid sequence, and the primer system For extending the target nucleic acid sequence to form a complementary sequence such that during use, the probe nucleic acid sequence that matches the target nucleic acid sequence is affixed to the complementary sequence to alter the fluorescent emission of the probe in response to the excitation light; A CMOS circuit on the support substrate for operating the control of the excitation light. GAS 027.2 Preferably, the operational control includes activating and deactivating the excitation light, and adjusting a power supply to the excitation light. GAS 027.3 Preferably, the CMOS circuit has a photodiode for sensing the fluorescent signal, and the CMOS circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. • 91 - 201219115 GAS027.4 Preferably, the probe is at a distance of less than 249 microns from the photodiode. GAS027.5 Preferably, the LOC device also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 027.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated.

GAS027.7 Preferably, the LOC device also has a fluorophore and a quenching agent, the fluorophore and the quenching agent being configured to be close to each other when the stem ring is closed, when the stem ring is opened Stay away from each other. GAS027.8 Preferably, the LOC device also has the probe array

a hybridization chamber array and a photodiode array, the probe being a fluorescence resonance energy transfer (FRET) probe comprising a different type of FRET probe configured to hybridize to a different nucleic acid sequence, each The hybridization chamber has one of the respective photodiodes. GAS 027.9 Preferably, the LOC device also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS027.1 0 preferably 'the PCR portion and the hybridization chamber are intrusion into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybrid The passage of the room. GAS027.il Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert the output of the photodiode from -92-201219115 to display the fret probe and The target nucleic acid sequence hybridizes to a signal and provides the signal to a pad for transmission to the external device. GAS027.1 2 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 027.1 3 Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished.

GAS027.1 4 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS 027.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS027.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS027.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 027.1 8 Preferably, the quencher does not have a natural emission of light that is supposed to illuminate. GAS 027.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

GAS027.20 Preferably, the LOC device also has a cover overlying the MST layer, the upper cover defining a plurality of reagent reservoirs containing reagents for addition to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts a biological sample, amplifies the nucleic acid target in the sample, and uses its integrated 93-201219115 imaging sensor via a stem-loop probe attached to the primer. Hybridization to identify the nucleic acid sequence of the sample and provide an electronic result at the output pad, and the stem-loop probe attached to the primer provides a large number of optimal parallel amplification reactions, providing high specificity, sensitivity, and reliability. The sequence of the target is detected. GAS028.1 This aspect of the invention provides a LOC device comprising: a support substrate;

a linear probe linked to a primer having a nucleic acid sequence and a primer, the nucleic acid sequence being matched to a target nucleic acid sequence, and the primer is used to extend the target nucleic acid sequence to form a complementary sequence such that during use, the target nucleic acid is matched The nucleic acid sequence of the sequence is bonded to the complementary sequence to change the fluorescent emission from the probe in response to the excitation light; and the CMOS circuit on the support substrate is used to control the excitation light" GAS028.2 Preferably, the operational control includes activating and deactivating the

Excitation light, and adjusting the power supplied to the excitation light. GAS 028.3 Preferably, the CMOS circuit has a photodiode for sensing the fluorescent emission, and the CMOS circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. Preferably, the distance between the probe and the photodiode is less than 249 microns. GAS 02 8.5 Preferably, the LOC device also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The photon produced by -94- 201219115 carrier. GAS 028.6 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS028.7 Preferably, the LOC device also has a fluorophore and a quenching agent which is removed when the nucleic acid sequence that matches the target nucleic acid sequence is bonded to the complementary sequence. GAS028.8 Preferably, the LOC device also has the probe array

a hybridization chamber array and a photodiode array, the probe being a fluorescence resonance energy transfer (FRET) probe comprising a different type of FRET probe configured to hybridize to a different nucleic acid sequence, each The hybridization chamber has one of the respective photodiodes. GAS028.9 Preferably, the LOC device also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS 02 8.1 0 Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room. GAS 02 8.il Preferably, the LOC device also has a pad for electrically connecting to an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and The target nucleic acid sequence hybridizes to a signal and provides the signal to a pad for transmission to the external device. GAS02 8.1 2 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. -95- 201219115 GAS 02 8.1 3 Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished. GAS028.1 4 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS 028.1 5 Preferably, the fluorophore is a transition metal-ligand complex.

GAS 028.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS 028.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 028.1 8 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS 028.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

GAS028.20 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns. The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts a biological sample, amplifies the nucleic acid target in the sample, and uses its integrated imaging sensor to hybridize with a linear probe coupled to the primer to identify the sample. The nucleic acid sequence, and providing an electronic result on the output pad, provides a large number of optimal parallel amplification reactions by the linear probe coupled to the primer, and also provides high specificity, sensitivity and reliability for detecting the target sequence. GAS 03 0.1 This aspect of the invention provides a LOC device comprising: -96-201219115 support substrate; stem-loop probes linked to the primers each having a nucleic acid sequence and a primer' in the nucleic acid sequence and the plurality of target nucleic acid sequences Matching 'and the primer is used to extend the target nucleic acid sequence to form a complementary sequence' such that during use, the nucleic acid sequence that matches the target nucleic acid sequence is affixed to the complementary sequence to change the response light from the probe Fluorescent emission

a polymerase chain reaction (PCR) portion for amplifying a target nucleic acid sequence in the fluid prior to hybridization with the FRET probe; and a CMOS circuit on the support substrate for use in operational control Excitation light. GAS030.2 Preferably, the operational control includes activating and deactivating the excitation light, and adjusting a power supply to the excitation light. GAS 03 0.3 Preferably, the CMOS circuit has a light sensor for sensing the fluorescent signal, and the CMOS circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS030.4 Preferably, the probe is less than 249 microns from the light sensor. GAS030.5 Preferably, the LOC device also has a shunt transistor between each photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS030.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS03 0.7 Preferably, the LOC device also has a fluorophore and a quenching-97-201219115 quenching agent, the fluorophore and the quenching agent being configured to be close to each other when the stem ring is closed, when the stem ring They are far away from each other when they are opened. GAS030.8 Preferably, the LOC device also has an array of hybridization chambers, wherein the probe is a fluorescence resonance energy transfer (FRET) probe, and each of the hybridization chamber arrays is configured to be associated with the target nucleic acid sequence, respectively One of the types of FRET probes hybridized, each of which corresponds to one of the respective photodiodes. GAS030.9 Preferably, each of the hybridization chambers has a volume of less than 9,000

Cubic micrometers. GAS030.1 0 Preferably, the PCR portion and the hybridization chamber are intrusion into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room. GAS030.il Preferably, the LOC device also has an external

a pad electrically connected to the device, wherein the CMOS circuit is configured to convert an output from the photodiode to display a signal that the FRET probe hybridizes to the target nucleic acid sequence and provide the signal to the pad for transmission to The external device. GAS03 0.1 2 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS030.1 3 Preferably, the CMOS circuit is configured to initiate the photodiode after a delay after the excitation light is extinguished. GAS03 0.1 4 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS030.1 5 Preferably, the fluorophore is a transition metal-ligand-98-201219115 compound. GAS030.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS030.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 03 0. 1 8 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light.

GAS03 0.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS 030.20 Preferably, the LOC device also has a hybrid heater that is controlled by the CMOS circuit to provide thermal energy for hybridization. The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts a biological sample, amplifies the nucleic acid target in the sample, and uses its integrated imaging sensor to hybridize with a stem-loop probe attached to the primer to identify the The nucleic acid sequence of the sample, and providing an electronic result on the output pad, the stem-loop probe attached to the primer provides a large number of optimal parallel amplification reactions, and also provides high specificity, sensitivity and reliability for detecting the target sequence. GAS031.1 This aspect of the invention provides a LOC device comprising: a support substrate; a linear probe coupled to the primer, having a nucleic acid sequence and a primer, the nucleic acid sequence being matched to the target nucleic acid sequence, and the primer is used for Extending the target nucleic acid sequence to form a complementary sequence such that, during use, the nucleic acid sequence that matches the nucleic acid sequence of the -99-201219115 target binds to the complementary sequence to alter the fluorescent emission from the probe in response to the excitation light; The CMOS circuit on the support substrate is used to operate and control the excitation light. GAS03 1.2 Preferably, the operational control includes activating and deactivating the excitation light, and adjusting a power supply to the excitation light. GAS03 1.3 Preferably, the CMOS circuit has a photodiode for sensing the fluorescent emission, and the CMOS circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS031.4 Preferably, the distance between the probe and the photodiode is less than 24.9 microns. GAS031.5 Preferably, the LOC device also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 031.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS03 1.7 Preferably, the LOC device also has a fluorophore and a quenching agent which is removed when the nucleic acid sequence that matches the target nucleic acid sequence is bonded to the complementary sequence. GAS03 1.8 Preferably, the LOC device also has the probe array, hybrid chamber array and photodiode array, the probe is a fluorescence resonance energy transfer (FRET) probe, and the hybrid chamber array is configured to be Different types of FRET probes that hybridize to different target nucleic acid sequences, each of which has one of -100 to 201219115 respective photodiodes. GAS 03 1.9 Preferably, the LOC device also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS031.10 Preferably, the PCR portion and the hybridization chamber are infused into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion and into the hybrid chamber by capillary action The channel.

GAS031.il preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid sequence The signal is hybridized and the signal is provided to the pad for transmission to the external device. GAS031.12 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS031.13 Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished. GAS031.14 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS031.15 Preferably, the fluorophore is a transition metal-ligand complex. GAS031.16 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS031.17 Preferably, the fluorophore is selected from the group consisting of: a nail nectar, a cockroach compound or a mis-integration. -101 - 201219115 GAS031 .18 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light. GAS031.19 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS 03 1.20 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns. The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts a biological sample, amplifies the nucleic acid target in the sample, and uses its integrated imaging sensor to hybridize with a linear probe coupled to the primer to identify the sample. The nucleic acid sequence, and providing an electronic result on the output pad, provides a large number of optimal parallel amplification reactions by the linear probe coupled to the primer, and also provides high specificity, sensitivity and reliability for detecting the target sequence. GAS 03 2.1 Aspects of the invention provide a LOC device for amplifying a nucleic acid sequence and detecting a target nucleic acid sequence, the LOC device comprising: a support substrate; a restriction enzyme for digesting the double-stranded nucleic acid sequence at a known restriction site; a linker primer for ligation to the restriction end of the double-stranded nucleic acid sequence; a heater for heating the nucleic acid sequence during a polymerase chain reaction (PCR); for extending the linker primer along the nucleic acid sequence Deoxyribonucleoside triphosphate (dNTP); and a probe for hybridization to a target nucleic acid sequence. GAS032.2 Preferably, the probe is a FRET (Fluorescence Resonance Energy Transfer) probe, and when these probes hybridize into a probe-target hybrid, they will change the fluorescence of the excitation light to -102-201219115 reaction. GAS032.3 Preferably, the LOC device also has photodiodes for each of the FRET probes. GAS032.4 Preferably, the LOC device also has a CMOS circuit on the support substrate for operating the control light GAS 03 2.5. Preferably, the operational control includes activating and deactivating the

Excitation light. GAS 03 2.6 Preferably, the CMOS circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS 03 2.7 Preferably, the FRET probe is less than 249 microns from the photodiode. GAS 03 2.8 Preferably, the LOC device also has a shunt transistor between each photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 032.9 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS 032.1 0 Preferably, the LOC device also has an array of hybridization chambers containing different types of FRET probes configured for hybridization to different target nucleic acid sequences. GAS032.il Preferably, the LOC device also has a polymerase chain reaction (PCR) portion in which the heater is positioned for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. -103- 201219115 GAS 03 2.1 2 Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action And enter the passage of the hybrid chamber. GAS03 2.1 3 Preferably, the LOC device also has a pad for electrically connecting to an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid The sequence hybridizes the signal and provides the signal to the pad for transmission to the external device. GAS032.1 4 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS032.1 5 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS032.1 6 Preferably, the fluorophore is a transition metal-ligand complex. GAS032.1 7 Preferably, the fluorophore is a lanthanide metal-ligand complex. Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 032.1 9 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS032.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight LOC device accepts a biological sample to amplify a nucleic acid target in the sample, and uses the integrated-104-201219115 imaging sensor to identify the nucleic acid of the sample via probe hybridization. The sequence 'and its output pad provides an electronic result from the ability of the adaptor primer to provide genomic-level amplification and analysis. GAS 03 3.1 This aspect of the invention provides a microfluidic device comprising: a support substrate;

An inlet for receiving a biological sample containing the target nucleic acid sequence; a reagent reservoir containing reagents for addition to the biological sample; and a hybridization chamber containing the probe, the probe having a nucleic acid sequence for use with the target Hybridizing to form a nucleic acid sequence of a probe-target hybrid; wherein the microsystem technology reagent reservoir has a volume of less than 1, 〇〇〇, 〇〇〇, 000 cubic microns, and the hybridization chamber has a volume of less than 900,000 cubic microns GAS033.2 Preferably, the reagent reservoir has a volume of less than 300,000,000 cubic microns and the hybridization chamber has a volume of less than 200,000 cubic microns. GAS 03 3.3 Preferably, the reagent reservoir has a volume of less than 70,000,000 cubic microns and the hybridization chamber has a volume of less than 40,000 cubic microns. GAS 03 3.4 Preferably, the reagent reservoir has a volume of less than 20,000,000 cubic microns and the hybridization chamber has a volume of less than 9000 cubic microns. GAS03 3.5 Preferably, the probe-target hybridization system is configured to emit a fluorescent signal in response to exposure to excitation light. -105- 201219115 GAS03 3.6 Preferably, the microfluidic device also has a photodiode for sensing the fluorescent reaction. GAS03 3.7 Preferably, the microfluidic device also has a CMOS circuit on the support substrate for operationally controlling the excitation light, wherein the photodiode system breaks into the CMOS circuit. GAS 033.8 Preferably, the operational control includes activating and deactivating the excitation light, and adjusting a power supply to the excitation light.

GAS033.9 Preferably, the probe is at a distance of less than 249 microns from the photodiode. GAS03 3.1 0 Preferably, the microfluidic device also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS033.il Preferably, the microfluidic device also has a different

An array of hybridization chambers of probes of the type (the probes are fluorescent resonance energy transfer (FRET) probes configured to hybridize to different target nucleic acid sequences), and an array of photodiodes (to enable each of the hybridization chambers) One of the respective photodiodes). GAS 03 3.1 2 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS 03 3.1 3 Preferably, the PCR portion and the hybridization chamber are incorporated into a microsystem technology (MST) layer having a plurality of configured to attract the fluid through the PCR portion by capillary action and into the hybridization The passage of the room. -106- 201219115 GAS033.1 4 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe a signal that hybridizes to the target nucleic acid sequence and provides the signal to the pad for transmission to the external device

GAS 033.1 5 Preferably, the CMOS circuit is configured to delay the photodiode after the excitation light is extinguished.

GAS03 3.1 6 Preferably, the FRET probes respectively have a fluorophore and a quencher, and the fluorescence lifetime of the fluorophore is greater than 〇〇 nanosecond. GAS03 3.1 7 Preferably, the fluorophore is a transition metal-ligand complex. GAS03 3.1 8 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS03 3.1 9 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS03 3.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The low volume hybridization chamber and reagent reservoir represent, to some extent, a low probe and reagent volume, thereby providing a low probe and reagent cost and an inexpensive detection system. GAS034.1 This aspect of the invention provides a on-wafer laboratory (LOC) device for genetic analysis of a nucleic acid sequence extracted from a biological material, the LOC device comprising: a probe for use with the nucleic acid sequence The target nucleic acid sequence hybridizes -107-201219115 to form a probe-target hybrid, each of which has a reporter fluorophore for emitting a fluorescent signal in response to excitation light; configured to emit fire forever a positive control probe for the light signal; and a negative control probe configured to never emit a fluorescent signal; wherein, when used, a fluorescent signal from the negative control probe is detected to indicate an abnormality; and no The fluorescent signal of the positive control probe indicates an abnormality. GAS034.2 preferably 'the LOC device also has an array of detection photodiodes for sensing the fluorescence signals from the hybrids of the probes, respectively; positive control photodiode system for detection A fluorescent signal of the reporter fluorophore from the positive control probe; and a negative control photodiode system for exposure to the negative control probe. GAS03 4.3 Preferably, the negative control probe does not have a reporter fluorescence GAS034.4. Preferably, the LOC device also has an array of hybridization chambers, each of the hybridization chambers containing at least one of the probes, including the positive control probe. Needle and the negative control probe. GAS03 4.5 Preferably, the LOC device also has: a support substrate; a hybridization portion; a microsystem technology (MS T) layer having the hybridization chamber array; and a CMOS circuit interposed between the MST layer and the support substrate, The CMOS circuit has an array of the detection photodiode, the positive control photodiode-108-201219115 body and the negative control photodiode; wherein the probe in the probe array is a fluorescence resonance energy transfer (FRET) probe. GAS034.6 Preferably, the LOC device also has a nucleic acid amplification unit for amplifying the nucleic acid sequence. GAS034.7 Preferably, the MST layer has multiple MST channels,

The MST channels are configured to draw fluid through the nucleic acid amplification portion and into the hybridization portion by capillary action. GAS 034.8 Preferably, the LOC device also has a pad for electrically connecting to an external device, wherein the CMOS circuit is configured to generate a signal indicative of hybridization of the FRET probe to the target nucleic acid sequence and provide the signal to A pad is provided for transmission to the external device. GAS034.9 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 03 4.10 Preferably, the FRET probes have a fluorophore φ and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS034.il Preferably, the fluorophore is a transition metal-ligand complex. GAS034.1 2 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS034.1 3 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 034.1 4 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. -109- 201219115 GAS 034.1 5 Preferably, the C Μ O S circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence. GAS 034.1 6 Preferably, each of the hybridization chambers has hybridization heaters for providing hybridization heat energy, each of the hybridization heaters being controlled by the CMOS circuit. GAS034.1 7 Preferably, the hybridization portion has a fluid flow path from the nucleic acid amplification portion to the liquid sensor, and the hybridization chamber is disposed along both sides of the fluid flow path. GAS 03 4.1 8 Preferably, the fluid flow path is configured to attract the fluid from the nucleic acid amplification portion to the liquid sensor by capillary action, and each of the hybridization chambers is configured to be filled by capillary action The fluid from the fluid flow path causes the CMOS circuit to activate the hybridization heater during operation, in response to an output from the liquid sensor indicating that the fluid has reached the output of the liquid sensor. GAS 03 4.1 9 Preferably, the hybrid heater has a ring shape and extends around a window of light in each of the hybridization chambers, the position of the light window in the hybrid chamber allowing the excitation light to be incident on the probe. Preferably, the spacing between the hybrid photodiode and the probe is less than 249 microns. The hybridization array provides analysis of the target via hybridization and utilizes the control probe to improve the reliability of the analytical results. GAS03 5.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; -110-201219115 an inlet for receiving a biological sample containing the target nucleic acid sequence; and an array of probes, each probe having A nucleic acid sequence that hybridizes to the target nucleic acid sequence to form a probe-target hybrid; wherein the array of probes comprises a control probe for hybridization to a known target sequence that is always present in the biological sample.

GAS 03 5.2 Preferably, the microfluidic device also has a light sensor located adjacent to the probe array, wherein the probe is configured to generate a fluorescent signal in response to the excitation light to cause the light sensor to sense Which probes in the probe array produce the fluorescent signal. GAS 03 5.3 Preferably, the light sensor is a charge coupled device (CCD) array between the probe array and the support substrate. GAS035.4 Preferably, the light sensor is configured to register a photodiode array of the probe array on the support substrate. GAS035.5 Preferably, the photodiode array is less than 249 microns from the probe array. GAS035.6 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the array of photodiodes being a component of the CMOS circuit, wherein when used, the CMOS circuit may not be sensed because The fluorescence signal of the control probe causes an error signal. GAS03 5.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GA0 GAS035.8 Preferably, the microfluidic device also has Photoelectric-111 - 201219115 A shunt transistor between a diode anode and a voltage source, the shunt cell system configured to remove a carrier generated in a photodiode by photons that absorb excitation light. GAS03 5.9 Preferably, the shunt cell system is configured to be turned on when the excitation light is activated and turned off when the excitation light is deactivated. GAS03 5.1 0 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS03 5.il Preferably, the microfluidic device also has an array of hybridization chambers containing different types of FRET probes configured for hybridization to different target nucleic acid sequences. GAS03 5.1 2 Preferably, the microfluidic device also has a polymerase linkage reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS03 5.1 3 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 03 5.1 4 Preferably, the FRET probes each have a fluorophore and a quencher having a fluorescence lifetime greater than 100 nanoseconds. GAS03 5.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS03 5.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS 03 5.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS03 5.1 8 Preferably, the quencher does not have a natural emission of light that should be excited by the excitation light -112-201219115. GAS 03 5.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence." The hybridization array provides analysis of the target via hybridization and utilizes the control The needle improves the reliability of the analysis. GAS03 6.1 This aspect of the invention provides a microfluidic device comprising:

a support substrate; an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe, each probe having a nucleic acid sequence, a fluorophore, and a quenching for hybridizing with the target nucleic acid sequence to form a probe-target hybrid a quenching agent, the fluorophore and the quenching agent are configured to cause the fluorophore to emit a fluorescent signal in response to the excitation light, and the quenching agent quenches the fluorescent signal when the probe is unhybridized, However, it is not possible to quench the fluorescent signal from the probe-target hybrid; and a control probe having a fluorophore but no quenching agent; wherein the control probe should always emit light to emit a fluorescent signal. GAS 036.2 Preferably, the microfluidic device also has a light sensor located adjacent to the probe for sensing that the probes should be excited to generate light to produce a fluorescent signal. GAS 03 6.3 Preferably, the light sensor is a charge coupled device (CCD) array between the probe and the support substrate. GAS 03 6.4 preferably the optical sensor registers a photodiode array of the probe on the support substrate. GAS 03 6.5 Preferably, the photodiode array is less than 249 microns from the probe -113 - 201219115. GAS 03 6.6 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the array of photodiodes being a component of the CMOS circuit, wherein when used, the CMOS circuit may not be sensed because The fluorescence signal of the control probe causes an error signal. GAS 03 6.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS 03 6.8 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS036.9 Preferably, the shunt cell system is configured to be turned on when the excitation light is activated and turned off when the excitation light is deactivated. GAS 036.1 0 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 03 6.1 1 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of FRET probes configured for hybridization to different target nucleic acid sequences. GAS 03 6.1 2 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS 03 6.1 3 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. -114- 201219115 GAS036.14 Preferably, the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS036.15 Preferably, the fluorophore is a transition metal-ligand complex. GAS03 6.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS03 6.1 7 Preferably, the fluorophore is selected from the group consisting of:

Ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 03 6.1 8 Preferably, the quencher does not have a natural emission of light that is supposed to illuminate. GAS03 6.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The hybridization array provides analysis of the target via hybridization and utilizes the control probe to improve the reliability of the analytical results. GAS 03 7.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe, each probe having a nucleic acid sequence for use with the target Hybridizing to form a nucleic acid sequence, a fluorophore, and a quencher of the probe-target hybrid, the fluorophore and the quencher being configured to cause the fluorophore to emit a fluorescent signal in response to the excitation light' and when The quencher quenches the fluorescent signal when the probe is not hybridized' but does not quench the fluorescent signal from the probe-target hybrid; and the reporter having the fluorophore is placed in The probe is exposed to the position of the excitation light at the same time; 115-201219115; wherein the report should always emit light and emit a fluorescent signal. GAS037.2 Preferably, the microfluidic device also has a light sensor located adjacent to the probe and the reporter for sensing that the probes should be excited to generate light and generate a fluorescent signal and sensing therefrom. Reporter Fluorescent Signals GAS03 7.3 Preferably, the light sensor is a charge coupled device (CCD) array between the probe and the support substrate. GAS03 7.4 Preferably, the light sensor registers the probe and the photodiode array of the reporter on the support substrate. GAS 03 7.5 Preferably, the photodiode array is less than 249 microns from the probe and the reporter. GAS037.6 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the array of photodiodes being a component of the CMOS circuit, wherein when used, the CMOS circuit may not be sensed because The report's fluorescent signal causes an error signal. GAS037.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. 刖° GAS 03 7.8 Preferably, the microfluidic device There is also a shunt transistor between each photodiode anode and a voltage source that is configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS037.9 Preferably, the shunt cell system is configured to be at -116-

201219115 Turns on when the excitation light is activated and when the excitation light is deactivated. GAS037.10 Preferably, the probe is a fluorescence resonance energy (FRET) probe. GAS037.il Preferably, the microfluidic device also has an array of !, the hybridization chamber containing different types of FRET probes configured for hybridization to different targets. GAS037.1 2 Preferably, the microfluidic device also has a 3-lock reaction (PCR) portion for the nucleic acid sequence of the target in the body before hybridization to the probe. GAS037.1 3 Preferably, the CMOS circuit has a memory? The memory of the identification data of the FRET probe type. GAS03 7.1 4 Preferably, the fluorescence lifetime of the fluorophore is nanoseconds. GAS 03 7.1 5 Preferably, the fluorophore is a transition metal compound. GAS 03 7.1 6 Preferably, the fluorophore is a lanthanide metal compound. GAS037.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS03 7.1 8 Preferably, the quencher does not have a natural emission of light that is responsive. GAS03 7.1 9 Preferably, the CMOS circuit is configured to provide an analysis of the target by hybridization of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. Increasing the flow of the difference is < 100 ligand mismatching the excitation light to control the degree. The -117-201219115 control probe was used to improve the reliability of the analysis results. GAS038.1 This aspect of the invention provides a wafer on-lab (LOC) device for genetic analysis comprising: a support substrate; an inlet for receiving a biological sample containing the target nucleic acid sequence; an array of probes Each probe has a nucleic acid sequence for hybridizing to the target nucleic acid sequence to form a probe-target hybrid; and a control probe designed to complement a sequence known to be absent from the biological sample. GAS03 8.2 Preferably, the LOC device also has a light sensor located adjacent to the probe array, wherein the probe is configured to generate a fluorescent signal in response to the excitation light to cause the light sensor to sense the probe Which probes in the array of needles produce the fluorescent signal. GAS 03 8.3 Preferably, the light sensor is a charge coupled device (CCD) array between the probe array and the support substrate and the control probe and the support substrate. GAS03 8.4 Preferably, the light sensor is configured to register a photodiode array of the probe array on the support substrate. GAS03 8.5 Preferably, the photodiode array is less than 249 microns from the probe array. GAS03 8.6 Preferably, the LOC device also has a CMOS circuit on the support substrate, the array of photodiodes being a component of the CMOS circuit, wherein when in use, the CMOS circuit responds to the control probe A fluorescent signal causes an error signal. -118-201219115 GAS 03 8.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS03 8.8 Preferably, the LOC device also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove photons that absorb excitation light in the photodiode The generated carrier.

GAS03 8.9 Preferably, the shunt cell system is configured to be turned on when the excitation light is activated and turned off when the excitation light is deactivated. GAS 03 8.1 0 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 03 8.il Preferably, the LOC device also has an array of hybridization chambers containing different types of FRET probes configured for hybridization with different target nucleic acid sequences. GAS 03 8.12 Preferably, the LOC device also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS 03 8.1 3 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS03 8.1 4 Preferably, the FRET probes have a fluorophore and a quencher, respectively, which have a fluorescence lifetime greater than 100 nanoseconds. GAS03 8.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS038.16 Preferably * the camp light group is a steel-based metal-ligand wrong -119-201219115 compound. G A S 03 8.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS03 8.1 8 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS03 8.1 9 Preferably the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The hybridization array provides analysis of the target via hybridization and utilizes the control probe to improve the reliability of the analytical results. GAS03 9.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe, each probe having for hybridizing to the target nucleic acid sequence To form a nucleic acid sequence of a probe-target hybrid, a fluorophore, and a quencher, the fluorophore and the quencher being configured to cause the fluorophore to emit a fluorescent signal in response to the excitation light, and when the probe The quencher quenches the fluorescent signal when the needle is not hybridized, but does not quench the fluorescent signal from the probe-target hybrid: and the control probe without the fluorescent group, so that the contrast probe The needle will never return to the excitation light and emit a fluorescent signal. GAS03 9.2 Preferably, the microfluidic device also has a light sensor located adjacent to the probe for sensing that the probe should be excited to generate light to produce a fluorescent signal. GAS03 9.3 Preferably, the light sensor is a charge coupled device (CCD) array between the probe and the -120-201219115 support substrate. GAS039.4 Preferably, the light sensor registers a photodiode array of the probe on the support substrate. GAS 03 9.5 Preferably, the photodiode array is less than 249 microns from the probe. GAS03 9.6 Preferably, the microfluidic device also has the support

A CMOS circuit on the substrate, the array of photodiodes being a component of the CMOS circuit, wherein when in use, the CMOS circuit generates an error signal in response to sensing a fluorescent signal from the control probe. GAS039.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GA0 GAS03 9.8 Preferably, the microfluidic device also has A shunt transistor between the photodiode anode and the voltage source, the shunt cell system configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS039.9 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and to turn off when the excitation light is deactivated. GAS 039.1 0 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 039.il Preferably, the microfluidic device also has an array of hybridization chambers containing different types of fret probes configured for hybridization to different target nucleic acid sequences. GAS039.1 2 Preferably, the microfluidic device also has a polymerase-121 - 201219115 lock reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS 03 9.1 3 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS039.14 Preferably, the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS039.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS 03 9.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS 03 9.1 7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 03 9.1 8 Preferably, the quencher does not have natural emission light that is responsive to the excitation light. GAS 03 9.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The hybridization array provides analysis of the target via hybridization and utilizes the control probe to improve the reliability of the analytical results. GAS040.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; an inlet for receiving a biological sample comprising a target nucleic acid sequence; each having a nucleic acid sequence for hybridization to a respective nucleic acid sequence of the target a -122-201219115 probe for forming a probe-target hybrid and a fluorescent signal in response to excitation light; and a light sensor corresponding to each of the probes; and a control light sensor not corresponding to any probe, The control light sensor is prevented from sensing the fluorescent signal from the corresponding probe-target hybrid during proper operation of the microfluidic device. Preferably, the light sensor is a photodiode having a spacing of less than 249 microns from the probe, and the contrast light sensor is in contrast to the photoelectric

Diode. GAS040.3 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the photodiode system is a component of the CMOS circuit, wherein when used, the CMOS circuit responds to sensing from the control photodiode The fluorescent signal of the polar body causes an error signal. GAS040.4 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS040.5 Preferably, the microfluidic device also has A shunt transistor between each of the photodiode anode and the voltage source, the shunt cell system configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS040.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS040.7 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS040.8 Preferably, the microfluidic device also has an array of the hybridization chamber -123-201219115, the hybridization chamber containing different types of FRET probes configured for hybridization to different target nucleic acid sequences. GAS040.9 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS040.1 0 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS040.1 1 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS040.1 2 Preferably, the fluorophore is a transition metal-ligand complex. GAS〇4〇.13 Preferably, the fluorophore is a lanthanide metal-ligand complex. Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS040.1 5 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light. GAS040.1 6 Preferably the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. GAS040.1 7 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns. The hybridization array provides analysis of the target via hybridization and utilizes the control probe to improve the reliability of the analytical results. GAS04 1 .1

Aspects of the present invention provide a microfluidic device for detecting a molecule of -124 to 201219115 of a sample, the microfluidic device comprising: a probe for reacting with the target molecule to form a probe-target complex The pin-and-label composite system is configured to emit photons when excited to detect photodiodes of photons emitted by the probe-target complex and to calibrate photodiodes for exposure to a calibration source to generate a calibration signal Body; when used,

The output from the photodiode for detecting photons emitted by the probe-target complex is calibrated by the calibration signal. GAS041.2 Preferably, the microfluidic device also has an array of hybridization chambers for the probe, wherein the target molecularly-labeled nucleic acid sequence hybridizes with a complementary oligonucleotide in the probe to form a probe The target hybrid is such that the probe-target hybrid emits a fluorescent signal when exposed to excitation light. GAS041.3 Preferably, the calibration source is a calibration chamber that does not contain a reporter fluorophore, the photodiode system is located in registration with the hybrid chamber and the calibration photodiode system is registered with the calibration chamber . GAS041.4 Preferably, during use, the output from any of the photodiode arrays is compared to the output from the calibrated photodiode closest to the photodiode. GAS041.5 Preferably, each of the calibration chambers is surrounded by a three by three hybrid cell block. GAS041.6 Preferably, the microfluidic device also has: a support substrate; 125-201219115 having the hybrid chamber array and the microsystem technology (MST) layer of the plurality of calibration chambers; and the MST layer and the support The CMOS circuit between the substrates _ CMOS circuit has the photodiode and the calibrated photodiode; wherein the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 04 1.7 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence. GAS 04 1.8 Preferably, the MST layer has a plurality of MST channels. The MST channels are configured to draw fluid through the PCR portion and into the hybrid portion by capillary action. GAS041.9 Preferably, the microfluidic device also has a pad for electrically connecting to an external device, wherein the CMOS circuit is configured to generate a signal indicative of hybridization of the FRET probe to the target nucleic acid sequence, and to provide the Signal to the pad for transmission to the external device. GAS041.10 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS041.il Preferably, the FRET probes have a fluorophore and a quencher, respectively, which have a fluorescence lifetime greater than 100 nanoseconds. GAS041.12 Preferably, the fluorophore is a transition metal-ligand complex. GAS041.13 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS041.14 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. -126- 201219115 GAS041.1 5 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light. GAS041.16 Preferably the CMOS circuit is configured to control the temperature of the hybridization portion during hybridization of the probe to the target nucleic acid sequence. GAS041.17 Preferably, the microfluidic device also has hybrid heaters for providing hybridization heat, each of which is controlled by the CMOS circuit.

GAS041.18 Preferably, the hybridization portion has a fluid flow path from the PCR portion to the liquid sensor, and the hybridization chamber is disposed along both sides of the fluid flow path. GAS041.19 Preferably, the fluid flow path is configured to attract the fluid from the PCR portion to the liquid sensor by capillary action, and each of the hybrid chambers is configured to be filled with the fluid from the fluid by capillary action The flow path fluid is such that during use, the CMOS circuit reacts the output from the liquid sensor to indicate that the fluid has reached the output of the liquid sensor to activate the miscellaneous heater. GAS041.20 Preferably, the spacing between the photodiode and the probe is less than 249 microns. The hybrid array provides analysis of the target via hybridization and utilizes the calibrated light sensor to improve the reliability, sensitivity, and dynamic range of the analytical results. GAS042.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; 127-201219115 an inlet for receiving a biological sample containing a target nucleic acid sequence; a hybridization chamber containing a probe, each of the probes A nucleic acid sequence having a hybrid for hybridizing to the target nucleic acid sequence to form a probe-target; and a calibration chamber containing a control probe that is incapable of hybridizing to any of the sequences in the biological sample. GAS042.2 Preferably, the microfluidic device also has a plurality of the hybridization chambers and a light sensor located adjacent to the hybridization chambers, wherein the hybridization chamber and the control chamber have a light window to expose the probe to the excitation light, The probe is configured to generate a fluorescent signal in response to the excitation light to cause the light sensor to sense which probes within the probe array produce the fluorescent signal. GAS042.3 Preferably, the light sensor is a charge coupled device (CCD) array between the hybridization chamber and the support substrate. GAS042.4 Preferably, the light sensor is configured to register a photodiode array of the hybridization chambers on the support substrate. Preferably, the photodiode array is less than 249 microns from the probe. * GAS042.6 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the array of photodiodes A component of the CMOS circuit, wherein when in use, the CMOS circuit responds to a fluorescent signal from the control probe to cause an error signal. GAS042.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GA0 GAS042.8 Preferably, the microfluidic device also has The opto-128-201219115 is a shunt transistor between the anode of the diode and the voltage source, the shunt cell system being configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS042.9 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS042.1 0 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe.

GAS042.il Preferably, the microfluidic device also has an array of hybridization chambers containing different types of FRET probes configured for hybridization to different target nucleic acid sequences. GAS042.12 Preferably, the microfluidic device also has a polymerase interlocking reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS042.1 3 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS042.1 4 Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS042.1 5 Preferably, the fluorophore is a transition metal-ligand complex. GAS042.1 6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS042.1 7 Preferably, the fluorophore is selected from the group consisting of: a nail nectar, a bismuth melamine or a erbium melamine. GAS 042.1 8 Preferably, the quencher does not have a natural emission of light that should be excited by -129-201219115. GAS042.1 9 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The hybridization array provides analysis of the target via hybridization and utilizes the calibration probe to improve the reliability, sensitivity, and dynamic range of the analytical results. GAS043.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; an inlet for receiving a biological sample containing the labeled nucleic acid sequence; a hybridization chamber containing the probe, each probe having The target nucleic acid sequence hybridizes to form a nucleic acid sequence of a probe-target hybrid, a fluorophore, and a quencher, the fluorophore and the quencher being configured to cause the fluorophore to emit a fluorescent signal in response to the excitation light And the quencher quenches the fluorescent signal when the probe is not hybridized, but does not quench the fluorescent signal from the probe-target hybrid: and the calibration chamber that does not contain the fluorophore. GAS043.2 Preferably, the microfluidic device also has a light sensor located adjacent to the hybridization chamber and the calibration chamber for sensing which probes should be excited to generate light to generate the fluorescent signal, wherein the hybridization The chamber and the control chamber have a light window to expose the probe to the excitation light. GAS 043.3 Preferably, the light sensor is located in the hybridization chamber and a charge coupled device (CCD) array 〇GAS043.4 between the calibration chamber and the support substrate. Preferably, the light sensor is registered with the light sensor. Hybridization chamber and -130-201219115 The photodiode array of the calibration chamber on the support substrate. GAS043.5 Preferably, the photodiode array is less than 249 microns from the probe. GAS 043.6 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the array of photodiodes being a component of the CMOS circuit, wherein when used, the CMOS circuit responds to sensing from the control The fluorescent signal of the probe causes an error signal"

GAS043.7 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS 043.8 preferably, the microfluidic device also has A shunt transistor between the photodiode anode and the voltage source, the shunt cell system configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS043.9 Preferably, the shunt cell system is configured to turn on when the φ excitation light is activated and to turn off when the excitation light is deactivated. GAS043.1 0 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS043.il Preferably, the hybridization chamber contains different types of FRET probes, each configured for hybridization to a different nucleic acid sequence. GAS043.1 2 Preferably the microfluidic device also has a polymerase interlocking reaction (PCR) portion that is used to amplify the target nucleic acid sequence in the fluid prior to hybridization to the probe. GAS043 1 3 Preferably, the CMOS circuit has a memory for storing the identification data of the different -131 - 201219115 FRET probe type. GAS043.1 4 nanoseconds. Preferably, the fluorescent lifetime of the fluorophore is greater than 100 GAS043.1 5 compound. Preferably, the luminescent group is a transition metal-ligand error GAS 043.1 6 compound. Preferably, the luminescent group is a lanthanide metal-ligand error GAS043.1. Preferably, the luminescent group is selected from the group consisting of: ruthenium chelate, manganese chelate or ruthenium chelate. The natural emission of GAS043.18. Preferably, the quencher does not have a response light that is preferably GAS043.1. Preferably, the C Μ 0 S circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The hybridization array provides analysis of the target via hybridization and utilizes the calibration probe to improve the reliability, sensitivity, and dynamic range of the analytical results. GAS045.1 comprising: a support substrate; this aspect of the invention provides a microfluidic device, an inlet for receiving a biological sample containing a target nucleic acid sequence; each having a nucleic acid sequence for hybridization to a respective nucleic acid sequence of the target a probe for forming a probe-target hybrid and emitting a fluorescent signal in response to the excitation light; and a light sensor corresponding to each of the probes; and a calibration light sensor not corresponding to any probe to enable calibration from Light-132- 201219115 The output of the sensor is subtracted from the outputs from the light sensor in a differential circuit. Preferably, the light sensor is a photodiode spaced less than 249 microns from the probe, and the calibrated light sensor calibrates the photodiode. GAS 045.3 Preferably, the microfluidic device also has a CMOS circuit on the support substrate, the photodiode system is a component of the CMOS circuit

GAS045.4 Preferably, the CMOS circuit is configured to trigger a time delay until the photodiode is activated when the excitation light is deactivated. GAS045.5 Preferably, the microfluidic device also has A shunt transistor between each of the photodiode anode and the voltage source, the shunt cell system configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS045.6 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS045.7 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 045.8 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of fret probes configured for hybridization to different target nucleic acid sequences. GAS045.9 Preferably, the microfluidic device also has a polymerase linkage reaction (PCR) portion for amplifying the target nucleic acid sequence in the stream-133-201219115 prior to hybridization to the probe. GAS045.1 0 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS045.1 2 Preferably, the fluorophore is a transition metal-ligand complex.

GAS045.1 3 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS 045.14 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS045.1 5 Preferably, the quenching agent does not have a natural emission light that is responsive to the excitation light. GAS 045.1 6 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

GAS 045.1 7 Preferably, each of the hybridization chambers has a volume of less than 9,000 cubic microns. The hybrid array provides analysis of the target via hybridization and utilizes the calibration circuit to improve the reliability, sensitivity, and dynamic range of the analytical results. GAS046.1 This aspect of the invention provides a microfluidic test module comprising: an outer casing having an inlet for receiving a biological sample containing the target nucleic acid sequence; and a hybridization chamber mounted in the outer casing, The hybridization chamber contains a probe, and the probe-134-201219115 needle has a nucleic acid sequence for hybridizing with the target nucleic acid sequence to form a probe-target hybrid; wherein the hybridization chamber has a volume of less than 900,000 cubic microns. GAS046.2 Preferably, the hybridization chamber has a volume of less than 200,000 cubic microns. GAS046.3 Preferably, the volume of the hybridization chamber is less than 40,000 cubic microns.

GAS046.4 Preferably, the hybridization chamber has a volume of less than 9000 cubic microns. GAS046.5 Preferably, the probe-target hybridization system is configured to emit a fluorescent signal in response to exposure to excitation light and the hybridization chamber has a light window for exposing the probe to the excitation light. GAS046.6 Preferably, the microfluidic test module also has a light source for generating the excitation light and a photodiode GAS046.7 for sensing the fluorescence reaction. Preferably, the light source is externally mounted. The shell activates the LED, which is used to emit light to penetrate the light window. GAS046.8 Preferably, the microfluidic test module also has a control circuit for operating the control LED. GAS046.9 Preferably, the control circuit controls activation and deactivation of the excitation LED and regulates a power supply to the excitation light. GAS 046.10 Preferably, the control circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS046.il Preferably, the microfluidic test module also has a shunt transistor between the -135-201219115 photodiode anode and a voltage source, the shunt cell system being configured to remove the photodiode The carrier generated by the photon of the excitation light. GAS046.1 2 Preferably, the microfluidic test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) a probe), and an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes).

GAS046.1 3 Preferably, the microfluidic test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe.

GAS046.1 4 Preferably, the microfluidic test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Multiple channels. GAS 046.1 5 Preferably, the microfluidic test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and The target nucleic acid sequence hybridizes to a signal and provides the signal to the electrical connector for transmission to the external device. GAS 046.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. -136-201219115 GAS 04 6.17 Preferably, the CMOS circuit comprises an array of the photodiodes, the array of photodiodes being 249 microns from the FRET probe. GAS046.1 8 Preferably, the FRET probes respectively have a quenching agent having a fluorescence lifetime greater than 100 nanoseconds. GAS046.1 9 Preferably, the fluorophore is a transition metal-complex.

GAS046.20 Preferably, the CMOS circuit is configured to provide a low probe to the low probe body to some extent during the hybridization of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. Cost and inexpensive inspection system. GAS〇47· 1 This aspect of the invention provides a microfluidic set comprising: an outer casing having an inlet for receiving a biological sample containing the target nucleic acid;

a hybridization chamber mounted in the housing, the hybridization chamber retaining a nucleic acid sequence containing one volume of 'the probes respectively having a hybrid for interfacing with the target nucleic acid to form a probe-oxime; wherein each of the hybridization chambers The probe mass is less than 270 picograms. Preferably, the probe in each of the hybridization chambers is less than 60 picograms. GAS047.3 is less than 12 picograms. Preferably, in each of the hybridization chambers, the probe GAS047.4 preferably, in each of the hybridization chambers, the probe electrode is separated from the small fluorophore ligand to control f-convergence, and the test mode sequence is entered. The sequence of the heterogeneous mass of the probe is the quality system -137-201219115 is less than 2.7 picograms. GAS047.5 Preferably, the probe-target hybridization system is configured to emit a fluorescent signal in response to exposure to excitation light and the hybridization chamber has a light window for exposing the probe to the excitation light. GAS047.6 Preferably, the microfluidic test module also has a light source for generating the excitation light and a photodiode 〇GAS047.7 for sensing the fluorescence reaction. Preferably, the light source is mounted on An excitation LED of the outer casing is used to emit light to penetrate the light window. GAS047.8 Preferably, the microfluidic test module also has a control circuit for operating the control LED. GAS047.9 Preferably, the control circuit controls activation and deactivation of the excitation LED and regulates a power supply to the excitation light. GAS 047.1 0 Preferably, the control circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS 047.il Preferably, the microfluidic test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to remove the photodiode in the photodiode The carrier generated by the photons that excite the excitation light. GAS047.1 2 Preferably, the microfluidic test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) The probes' and the array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). -138- 201219115 GAS047.1 3 Preferably, the microfluidic test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization with the FRET probe. . GAS 047.1 4 Preferably, the microfluidic test module is also installed

a microfluidic device in the outer casing adjacent to the excitation LED. The microfluidic device has a support substrate, a CMOS circuit on the support substrate, and a microsystem technology (MST) layer on the CMOS circuit, wherein the PCR portion and The hybridization chamber is incorporated into the MST layer, the MST layer having a plurality of channels configured to attract the fluid through the PCR portion and into the hybridization chamber by capillary action. GAS047.1 5 Preferably, the microfluidic test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe A signal that hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS047.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS047.1 7 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS047.1 8 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 〇〇 nanosecond. GAS 047.19 Preferably, the fluorophore is a transition metal-ligand complex. GAS 047.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during the hybridization of the probe to the target nucleic acid sequence of -139-201219115. This low probe volume represents a low probe cost, which in turn allows for this inexpensive detection system. GAS048.1 Aspects of the invention provide a test module for detecting hybridization of a probe to a target nucleic acid sequence, the test module comprising: a container for receiving a biological sample having a labeled nucleic acid sequence; Is used to hybridize to the target nucleic acid sequence: an excitation light emitting diode (LED) for illuminating the probe array, having a CMOS circuit for sensing a fluorescent light emitting sensor, the fluorescent The light emission system is from any probe in the array that has hybridized to the target nucleic acid sequence, the CMOS circuit being configured to use an output from the light sensor to generate a signal indicating that the probe has hybridized; wherein the probe The array and the CMOS circuit are integrated into a wafer-on-lab (LOC) device included in the test module. GAS048.2 Preferably, the test module also has a nucleic acid amplification section for amplifying the target nucleic acid sequence in the biological sample. GAS 048.3 Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GAS048.4 Preferably, the light sensor is an array of photodiodes and the probe is a fluorescence resonance energy transfer (FRET) probe. GAS048.5 Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 〇〇 nanosecond. GAS048.6 Preferably, the fluorophore is a transition metal-ligand-350-201219115 compound. GAS048.7 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS048.8 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS048.9 Preferably, the quencher does not have a natural emission of light that should be excited to emit the LED.

GAS 048.1 0 Preferably, the excitation LED has an emission wavelength that substantially conforms to the absorption wavelength of the fluorophore. GAS048.il Preferably, the test module also has a USB (Universal Serial Bus) connector for transmitting the signal to an external device, wherein when used, the circuit, the nucleic acid amplification portion and the excitation LED The external device is powered by the USB connector. GAS048.12 Preferably, the test module also has a housing for securely holding the USB connector and the container, the container being in fluid communication with the sample inlet on the LOC device. GAS048.1 3 Preferably, the LOC device has a support substrate and a micro-system technology (MST) layer provided with a probe array, the CMOS circuit being located between the MS T layer and the support substrate, and the CMOS circuit Have this light induction

GAS048.14 Preferably, the MST layer has an array of hybridization chambers containing the FRET probes, each having a light window to expose the FRET probe to the excitation LED. GAS 048.1 5 Preferably, the MST layer has a plurality of MST channels, -141 - 201219115. The MST channels are configured to attract the fluid through the nucleic acid amplification portion and into the hybridization chamber by capillary action. GAS048.1 6 preferably 'the LOC device has a pad for electrically connecting to an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target nucleic acid The signal of sequence hybridization. GAS 04 8.17 Preferably, the CMOS circuit has a memory for storing identification data of different F R E T probe types. GAS 04 8.1 8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation LED is extinguished. GAS048.1 9 Preferably, one of the MST channels defines a fluid flow path from the nucleic acid amplification portion to the liquid sensor, the hybridization chamber being disposed along both sides of the fluid flow path such that When the fluid is drawn to the liquid sensor by capillary action, the flow containing the target nucleic acid sequence is filled into the hybrid chambers. GAS048.20 Preferably, the test module also has at least one valve for preventing the biological sample from entering the nucleic acid amplification portion before the USB connector is inserted into the external device. The integrated image sensor with excitation led eliminates the need for expensive external imaging systems, providing a comprehensive solution that is both mass-produced and inexpensive, with a low system component representing a lightweight, highly mobile system. The integrated image sensor has the benefit of increased read sensitivity due to large angle light collection and eliminates the need to use optical components in the string of light collecting elements. GAS049.1 Aspects of the invention provide a test module for detecting hybridization of a probe to a nucleic acid sequence of 201219115, the test module comprising: a housing configured for hand movement, the housing having a label for receiving a container for a biological sample of a nucleic acid sequence; a probe array for hybridizing to the target nucleic acid sequence; an excitation light source for illuminating the probe array: and a light sensor for sensing from the array Fluorescence emission of any probe that has hybridized to the target nucleic acid sequence.

GAS 049.2 Preferably, the test module also has circuitry for using the output from the light sensor to produce a signal indicating that the probe has been hybridized. GAS 049.3 Preferably, the circuitry is configured to transmit the signal to an external device. GAS049.4 Preferably, the circuit has a USB (Universal Serial Bus) connector for transmitting the signal to an external device. GAS049.5 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the biological sample. GAS049.6 Preferably, the light sensor is an array of photodiodes and the probe is a fluorescence resonance energy transfer (FRET) probe. GAS049.7 Preferably, the FRET probe has a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 〇 nanosecond. GAS 049.8 Preferably, the fluorophore is a transition metal-ligand complex. GAS049.9 Preferably, the fluorophore is a lanthanide metal-ligand wrong -143-201219115 compound. Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 049.il Preferably, the quencher does not have a natural emission of light that should be emitted by the excitation source. GAS049.1 2 Preferably, the excitation source is a light emitting diode (LED) having an emission wavelength substantially conforming to the absorption wavelength of the fluorophore. GAS049.1 3 Preferably, during use, the circuit, the PCR portion, and the excitation source are powered by the external device via the USB connector. GAS049.1 4 Preferably, the PCR portion, the probe array and the optical sensor are provided by a wafer-on-lab (LOC) device mounted in the housing. GAS049.1 5 Preferably, the LOC device has a support substrate and a microsystem technology (MS T) layer provided with a probe array, and the circuit has a CMOS circuit between the M ST layer and the support substrate. GAS 049.1 6 Preferably, the array of photodiodes is incorporated in the CMOS circuit. GAS049.1 7 Preferably, the MST layer has an array of hybridization chambers for containing the FRET probes, each having a light window to expose the FRET probe to the excitation LED. GAS 04 9.18 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the hybridization chamber by capillary action. GAS 049.1 9 preferably 'the LOC device has a pad for electrically connecting to an external package -144 - 201219115, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe A signal that the needle hybridizes to the target nucleic acid sequence. GAS 049.20 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types.

The easy-to-use, mass-produced, inexpensive, lightweight, and portable test module accepts a biological sample, and uses its integrated imaging sensor and excitation source to hybridize the nucleic acid sequence of the sample via fluorescent probe hybridization, and Provide electronic results at the output of the other. GAS050.1 Aspects of the invention provide a test module for detecting hybridization of a probe to a target nucleic acid sequence, the test module comprising: a container for receiving a biological sample having a labeled nucleic acid sequence; Is for hybridizing to the target nucleic acid sequence; an excitation light source for illuminating the probe array; and a light sensor circuit for sensing that the nucleic acid sequence from the array has been hybridized to the target nucleic acid sequence Fluorescence emission of any of the probes; wherein the circuitry is configured to use an output from the light sensor to produce a signal indicative of the hybridization of the probes and to transmit the signal to an external device. GAS050.2 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the biological sample. GAS050.3 Preferably, the light sensor is an array of photodiodes and the probe is a fluorescence resonance energy transfer (FRET) probe. -145- 201219115 GAS050.4 Preferably, the FRET probes respectively have a fluorophore and a quencher having a fluorescence lifetime greater than 100 nanoseconds. GAS050.5 Preferably, the fluorophore is a transition metal ligand complex. GAS050.6 Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS050.7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 05 0.8 Preferably, the quencher does not have a natural emission of light that should be emitted by the excitation source. GAS 05 0.9 Preferably, the excitation source is a light-emitting diode (LED) having an emission wavelength substantially conforming to the absorption wavelength of the fluorophore. GAS050.1 0 Preferably, the test module also has a universal serial bus (USB) connector, wherein when used, the circuit, the PCR portion and the excitation light source are powered by the external device via the USB connector . GAS050.il Preferably, the circuit includes a CMOS circuit constructed on a wafer-on-a-lab (LOC) device mounted in the test module, the LOC device having a sample inlet in fluid communication with the container and having the PCR And the probe array, and the CMOS circuit is provided with the light sensor. GAS050.1 2 Preferably, the test module also has a housing for securely holding the USB connector. GAS 05 0.1 3 Preferably, the LOC device has a support substrate and a microsystem technology (MST) layer provided with the PCR portion and the probe array, and the CMOS circuit is located between the MST layer and the support substrate. -146-201219115 GAS050.14 Preferably, the MST layer has an array of hybridization chambers containing the FRET probes, each having a light window to expose the FRET probe to the excitation LED. GAS 050.1 5 Preferably, the MST layer has a plurality of MST channels configured to attract the fluid through the PCR portion and into the hybridization chamber by capillary action.

GAS050.1 6 Preferably, the CMOS circuit has a pad for electrically connecting to the external device via the USB connector, wherein the CMOS circuit is configured to use an output from the photodiode to generate the FRET display A signal that the probe hybridizes to the target nucleic acid sequence 歹IJ. GAS 050.1 7 Preferably, the CMOS circuit has a memory for storing identification data of different FRET probe types. GAS 050.1 8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. GAS050.1 9 Preferably, one of the MST channels defines a fluid flow path from the PCR portion to the liquid sensor, the hybrid chamber being disposed along both sides of the fluid flow path such that the fluid Upon attraction by the capillary action to the liquid sensor, the fluid containing the target nucleic acid sequence is loaded into the hybridization chambers. GAS05 0.20 Preferably, the test module also has at least one valve for preventing entry of the biological sample into the PCR portion prior to insertion of the USB connector into the external device. The easy-to-use, mass-produced, inexpensive, lightweight, and portable test module accepts biological samples, using its integrated imaging sensor and excitation-147-201219115 source to identify the sample via fluorescent probe hybridization. The nucleic acid sequence, and at its output, provides an electronic result that is used to supply the module's power and messaging requirements. GAS054.1 This aspect of the invention provides a test module comprising: an outer casing having an inlet for receiving a biological sample containing the nucleic acid sequence of the target; a reagent reservoir containing for addition to the biological sample And a hybridization chamber comprising a probe having a nucleic acid sequence for hybridizing with the target nucleic acid sequence to form a probe-target hybrid; wherein the reagent reservoir has a volume of less than 20,000,000 cubic microns and The hybridization chamber has a volume of less than 9,000 cubic microns. GAS 054.2 Preferably, the probe-target hybridization system is configured to emit a fluorescent signal in response to exposure to excitation light. GAS 054.3 Preferably, the test module also has a photodiode for sensing the fluorescent reaction. GAS 054.4 Preferably, the test module also has a light source for generating excitation light and the hybridization chamber has a light window for exposing the probe to the excitation light. GAS 054.5 Preferably, the light source is mounted to the excitation LED of the housing for emitting light to penetrate the light window. GAS 054.6 Preferably, the test module also has a control circuit for operating the control LED. GAS 054.7 Preferably, the control circuit is configured to deactivate the -148-201219115 excitation light and subsequently activate the photodiode after a time delay. GAS054.8 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 054.9 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated.

GAS 054.1 0 Preferably, the distance between the probe and the photodiode is less than 249 microns. GAS 054.1 1 Preferably, the test module also has an array of hybridization chambers containing probes of different types (the probes are fluorescent resonance energy transfer (FRET) probes configured to hybridize to different target nucleic acid sequences And an array of photodiodes (so that each of the hybridization chambers has its own photodiode). GAS 054.1 2 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the fluid prior to hybridization to the FRET probe. GAS 054.1 3 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a micro on the CMOS circuit a system technology (MST) layer, wherein the PCR portion and the hybridization chamber are incorporated into the MST layer, the MST layer having a plurality of channels configured to attract the fluid through the PCR portion and into the hybridization chamber by capillary action . GAS054.1 4 Preferably, the test module also has an electrical connector for communicating with an external -149-201219115 device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET A signal that the probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS 054.1 5 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 054.1 6 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS 054.1 7 Preferably, the fluorescent lanthanide is a transition metal-ligand complex. GAS054.1 8 compound. Preferably, the luminophore is a natural emission of a steel-based metal-ligand error GAS054.1 9 . Preferably, the quencher does not have a response light GAS 054.20. Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The low volume hybridization chamber and reagent reservoir represent, to some extent, a low probe and reagent volume, thereby providing a low probe and reagent cost and an inexpensive detection system. GAS 05 5.1 includes: This aspect of the invention provides a test module that is sized for a hand-held housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; and a probe in the housing It is used to hybridize with the target nucleic acid sequence -150-201219115 to form a probe-target hybrid, each having a fluorophore to cause the probe-target hybrid to emit a fluorescent signal in response to exposure to excitation light. Wherein the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS 055.2 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns.

GAS05 5.3 Preferably, the hybridization chamber has a light window for exposing the probe to the excitation light. GAS 05 5.4 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent reaction. GAS 05 5.5 Preferably, the light source is mounted to the excitation LED of the housing for emitting light to penetrate the light window. GAS 055.6 Preferably, the test module also has a control circuit for operating the control LED. GAS 055.7 Preferably, the control circuit controls activation and deactivation of the excitation LED and regulates a power supply to the excitation light. GAS 05 5.8 Preferably, the control circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS055.9. The test module of claim 8, wherein the control circuit further comprises a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured A carrier generated by absorbing photons of the excitation light in the photodiode is removed. GAS05 5.1 0 Preferably, the shunt cell system is configured to be turned on when the -151 - 201219115 excitation light is activated and turned off when the excitation light is deactivated. GAS 05 5.1 1 Preferably, the test module also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (fret) probes. And an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). GAS 055.1 2 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS055.1 3 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and on the CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber are incorporated into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Channels. GAS055.1 4 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and The target nucleic acid sequence hybridizes to a signal and provides the signal to the electrical connector for transmission to the external device. GAS05 5.1 5 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 05 5.1 6 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being at a distance from the F R E τ probe - 152 - 201219115 at 249 microns. GAS 05 5.1 7 Preferably, the fluorophore is a transition metal-ligand complex. GAS05 5.1 8 Preferably, the fluorophore is a lanthanide metal-ligand complex.

GAS055.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorophore when the probe is in a non-hybrid configuration. Natural light emitted by light. GAS 05 5.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The use of long-life fluorescent probes with time-delayed fluorescence detection improves the sensitivity and reliability of the test system and eliminates the need for any wavelength-dependent filter components, making the design inexpensive, small, and lightweight. GAS 05 6.1 This aspect of the invention provides a test module comprising: a housing sized for hand-held movement, the housing having an inlet for receiving a biological sample containing the labeled nucleic acid sequence; and a probe in the housing, Is used to hybridize with the target nucleic acid sequence to form a probe-target hybrid having a fluorophore to cause the probe-target hybrid to emit a fluorescent signal in response to exposure to excitation light; Light group transition metal-ligand complex. GAS 05 6.2 Preferably, the fluorophore is a ruthenium chelate. GAS 056.3 Preferably, the test module also has a hybridization chamber mounted in the shell - 153 - 201219115 for containing a probe, wherein the hybrid chamber has a volume of less than 9,000 cubic microns. GAS05 6.4 Preferably, the hybridization chamber has a light window for exposing the probe to the excitation light. GAS 056.5 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescence reaction. GAS 056.6 Preferably, the light source is mounted to an excitation LED of the housing for emitting light to penetrate the light window. GAS05 6.7 Preferably, the test module also has a control circuit for operating the control LED. GAS 056.8 Preferably, the control circuit controls the activation and deactivation of the excitation LED and regulates the power supply to the excitation light. GAS 056.9 Preferably, the control circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS056.1 0 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove excitation due to absorption in the photodiode The carrier generated by the photon of light. GAS 056.il Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS05 6.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes) And an array of photodiodes (so that each of the hybridization chambers corresponds to a respective photodiode of -154 to 201219115). GAS 056.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe.

GAS056.1 4 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a CMOS circuit thereon a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber system are interposed into the MST layer, the MST layer having a plurality of MST layers configured to attract the fluid through the PCR portion and into the hybrid chamber by capillary action aisle. GAS05 6.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target A signal that hybridizes to the nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS 056.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 056.1 7 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS05 6.1 8 Preferably, the fluorescent lifetime of the fluorophore is greater than 1 奈 nanosecond. GAS056.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescence-155-201219115 signal from the fluorophore when the probe is in a non-hybrid configuration, the quencher is not It has a natural emission of light that should be excited. GAS056.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The use of a long-lived transition metal-ligand complex fluorescent probe with time-delayed fluorescence detection improves the sensitivity and reliability of the test system and eliminates the need for any wavelength-dependent filter components, making the design less expensive Small and lightweight. GAS05 7.1 This aspect of the invention provides a test module comprising: a housing sized for handheld movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; and a probe in the housing Used to hybridize to the target nucleic acid sequence to form a probe-target hybrid having a fluorophore to cause the probe-target hybrid to emit a fluorescent signal in response to exposure to excitation light; wherein the fluorescent light The group is a lanthanide metal-ligand complex. GAS 057.2 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS05 7.3 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the volume of the hybridization chamber is less than 9,000 cubic microns. GAS0 5 7.4 Preferably, the hybridization chamber has The probe is exposed to the light window of the excitation light. GAS057.5 Preferably, the test module also has a light source for -156-201219115 to generate the excitation light and a photodiode for sensing the fluorescence reaction. GAS 057.6 Preferably, the light source is mounted to an excitation LED of the housing for emitting light to penetrate the light window. GAS 05 7.7 Preferably, the test module also has a control circuit for operating the control LED. GAS 05 7.8 Preferably, the control circuit controls the activation and deactivation of the excitation LED and regulates the power supply to the excitation light.

GAS 057.9 Preferably, the control circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay. GAS 05 7.1 0 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove excitation due to absorption in the photodiode The carrier generated by the photon of light. GAS 05 7.il Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS 057.1 2 Preferably, the test module also has an array of hybridization chambers containing probes of different types (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes) And an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). GAS 05 7.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS057.14 Preferably, the test module also has a microfluidic device mounted on the outer casing -157-201219115 adjacent to the excitation led. The microfluidic device has a support substrate, a CMOS circuit on the support substrate, and a microsystem technology (MST) layer on a CMOS circuit wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion by capillary action and into the hybrid Multiple passages in the room. GAS057.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and The target nucleic acid sequence hybridizes to a signal and provides the signal to the electrical connector for transmission to the external device. GAS 05 7.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 05 7.1 7 Preferably, the CMOS circuit comprises an array of photodiodes having an array of photodiodes spaced from the FRET probe by less than 249 microns. GAS05 7.1 8 Preferably, the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS05 7.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorophore when the probe is in a non-hybrid configuration. The quenching agent does not have a response light. The natural emission of light. GAS057.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The use of long-lived lanthanide metal-ligand complex fluorescent probes with time-delayed fluorescence detection improves the sensitivity and reliability of the test system, and -158-201219115 eliminates the need for any wavelength-dependent filter components, This design is inexpensive, small, and lightweight. GAS 05 8.1 This aspect of the invention provides a test module comprising: an outer casing having an inlet for receiving a biological sample containing the nucleic acid sequence of the target;

a probe in the outer shell for hybridizing to the target nucleic acid sequence to form a probe-target hybrid having a fluorophore, respectively, such that the probe-target hybrid is responsive to exposure to excitation light A fluorescent signal is emitted; wherein the probe is suspended in a fluid. GAS 05 8.2 Preferably, the quality of the probe is less than 270 picograms GAS 05 8.3 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybrid chamber The volume is less than 9,000 cubic microns. GAS05 8.4 Preferably, the hybridization chamber has a light window for exposing the probe to the excitation light. GAS 05 8.5 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescence reaction. GAS 05 8.6 Preferably, the light source is mounted on the outer casing. The LED is activated for emitting light to penetrate the light window. GAS 058.7 Preferably, the test module also has a control circuit for operating the control LED. -159- 201219115 GAS058.8 Preferably, the control circuit controls activation and deactivation of the excitation LED and regulates a power supply to the excitation light. GAS 058.9 Preferably, the control circuit is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.

GAS058.1 0 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove excitation due to absorption in the photodiode The carrier generated by the photon of light. GAS058.il Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS 05 8.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes. And an array of photodiodes (so that each of the hybridization chambers corresponds to a respective photodiode).

GAS 05 8.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS058.1 4 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a CMOS circuit thereon a microsystem technology (MST) layer wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a plurality of MST layers configured to attract the fluid through the PCR portion and into the hybrid chamber by capillary action aisle. -160-

201219115 GAS 05 8 . 1 5 Preferably, the test module also has an electrical connector for communicating with the device, wherein the CMOS circuit is configured to output the FRET probe from the output of the photodiode The target acid sequence hybridizes to the signal and provides the signal to the electrical connector for supply to the external device. GAS 058.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the FRET probe type. GAS 05 8.1 7 Preferably, the CMOS circuit comprises an array of photoconductors, the array of photodiodes being at a distance of 249 microns from the FRET probe. GAS 05 8.1 8 Preferably, the fluorescent lifetime of the fluorophore is greater than nanoseconds. GAS058.1 9 Preferably, the FRET probe has a quenching agent. When the probe system is in a non-hybrid configuration, the fluorescent material from the fluorescent group does not have a natural emission light corresponding to the excitation light. . Preferably, the CMOS circuit is configured to detect the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The suspended probe has a large excitation and emission depth, so readable sensitivity Sex. The suspended probe can be relatively easily and inexpensively. GAS059.1 This aspect of the invention provides a test module comprising: a housing sized for hand-held movement, the housing having a biological sample for receiving the target nucleic acid sequence The externally-converted nuclear transport has a different dipole from the small ^100 to suppress the number, and the control 〇 adds a spot, which contains -161 - 201219115 probe in the outer shell, the probe has a nucleic acid sequence for use with the target The nucleic acid sequence hybridizes to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to the excitation light; and a control circuit having light for returning the fluorescent signal to produce an output signal Inductor: wherein the control circuit is configured to delay a time before the excitation light is deactivated until the photosensor is activated. GAS059.2 Preferably, the control circuit has a trigger light sensor for responding to the excitation light to generate an output such that the control circuit initiates the time delay when the trigger light sensor indicates that the excitation light has been deactivated . GAS 059.3 Preferably, the probes each have a fluorophore such that the probe-target hybrid emits a fluorescent signal in response to exposure to excitation light, and the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS 059.4 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns. GAS 059.5 Preferably, the hybridization chamber has a light window for exposing the probe to the excitation light. GAS 059.6 Preferably, the test module also has an excitation LED mounted to the housing for generating the excitation light. GAS 059.7 Preferably, the control circuit controls the activation and deactivation of the excitation LED and regulates the power supply to the excitation light. GAS059.8 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, and the shunt cell system is configured to be removed in the photodiode by -162-201219115. The carrier generated by the absorption of photons of the excitation light. GAS 059.9 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS059.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS059.il Preferably, the luminescent group is a lanthanide metal-ligand

Compound. GAS059.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes. And an array of photodiodes (so that each of the hybridization chambers corresponds to a respective photodiode). GAS05 9.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS059.14 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and on the CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Channels. GAS 05 9.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert -163-201219115 output from the photodiode to display the FRET A signal that the probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS059.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS059.1 7 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS05 9.1 8 Preferably, the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS 059.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorophore when the probe is in a non-hybrid configuration, the quenching agent does not have a response light The natural emission of light. GAS059.20 Preferably, the C Μ Ο S circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. This time delayed fluorescence detection eliminates the need for any wavelength dependent filter components, making the design inexpensive, small, and lightweight. GAS060.1 This aspect of the invention provides a test module comprising: a housing sized for handheld movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; and a probe in the housing, The probe has a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to excitation light; and -164-201219115 control circuit, There is a light sensor for returning a fluorescent signal to produce an output signal; wherein the control circuit is configured to delay the excitation light by a deactivation time until the light sensor is activated. GAS060.2 Preferably, the control circuit has a trigger light sensor for responding to the excitation light to generate an output such that the control circuit induces the time delay when the trigger light sensor indicates that the excitation light has been deactivated .

GAS060.3 Preferably, the probes each have a fluorophore such that the probe-target hybrid emits a fluorescent signal in response to exposure to excitation light, and the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS060.4 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns. GAS 060.5 Preferably, the hybridization chamber has a light window for exposing the probe to the excitation light. GAS060.6 Preferably, the test module also has an excitation LED mounted to the housing for generating the excitation light. GAS060.7 Preferably, the control circuit controls the activation and deactivation of the excitation LED and regulates the power supply to the excitation light. GAS060.8 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS060.9 Preferably, the shunt cell system is configured to be turned on when the -165-201219115 excitation light is activated and turned off when the excitation light is deactivated. GAS060.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS060.il Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS060.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes. And an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). GAS060.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS060.1 4 Preferably, the test module also has a microfluidic device mounted on the outer casing adjacent to the excitation LED. The microfluidic device has a support substrate, a CMOS circuit on the support substrate, and a CMOS circuit thereon. a microsystem technology (MS T) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Multiple channels. GAS 060.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe and the target A signal that hybridizes to the nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. -166- 201219115 GAS060.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS060.1 7 Preferably, the CMOS circuit includes an array of the photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS060.1 8 Preferably, the fluorescent lifetime of the fluorophore is greater than 1 奈 nanosecond.

GAS060.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorescent group when the probe is in a non-hybrid configuration, the quenching agent does not have a response Natural light emitted by light. GAS 06 0.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The controlled exposure of the fluorophore to the excitation light eliminates the need for any wavelength dependent filter assembly, making the design inexpensive, small, and lightweight. GAS 06 1.1. This aspect of the invention provides a single use test module comprising: a housing sized for hand movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to excitation light; and light sensing To detect the fluorescent signal. GAS061.2 Preferably, the single-use test module also has a control circuit configured to delay the excitation light by a deactivation time until the photosensor -167-201219115 is activated. GAS061.3 Preferably, the control circuit has a trigger light sensor for responding to the excitation light to generate an output such that the control circuit initiates the time delay when the trigger light sensor indicates that the excitation light has been deactivated . Preferably, the probe has a fluorophore to cause the probe-target hybrid to emit a fluorescent signal in response to exposure to the excitation light, and the fluorescence lifetime of the fluorophore is greater than 100 nanoseconds. GAS 61 .5 Preferably, the single use test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybrid chamber has a volume of less than 9,000 cubic microns. GAS 061.6 Preferably, the hybridization chamber has a light window for exposing the probe to the excitation light. GAS061.7 Preferably, the single use test module also has an excitation LED mounted to the housing for generating the excitation light. GAS061.8 Preferably, the control circuit controls the activation and deactivation of the excitation LED and regulates the power supply to the excitation light. GAS061.9 Preferably, the single-use test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to be removed in the photodiode The carrier generated by the absorption of photons of the excitation light. GAS061.10 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS061.il Preferably, the fluorophore is a transition metal-ligand complex. Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS061.13 Preferably, the single-use test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) a probe) and an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes).

GAS061.14 Preferably, the single-use test module also has a microfluidic device mounted on the outer casing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and A microsystem technology (MST) layer on a CMOS circuit, wherein the hybridization chamber is interposed into the MST layer, the MST layer having a plurality of channels configured to attract the fluid from the inlet to the hybridization chamber by capillary action. GAS061.15 Preferably, the single-use test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe A signal that hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS061.16 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS061.17 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS061.18 Preferably, the fluorescence lifetime of the fluorophore is greater than 100-169-201219115 nanoseconds. GAS061.19 Preferably, the FRET probe has a quenching agent to inhibit a fluorescent signal from the fluorescent group when the probe is in a non-hybrid configuration, the quenching agent does not have a natural response to the excitation light. Emitting light. GAS061.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, uses its integrated imaging sensor to identify the nucleic acid sequence of the sample via probe hybridization, and provides an electronic result at the output of the sample. GAS 062.1 Aspects of the invention provide a single-use test module for detecting hybridization of a probe to a nucleic acid sequence, the test module comprising a sample input port for receiving a biological sample having a labeled nucleic acid sequence;

a polymerase chain reaction (PC R) portion for amplifying a target nucleic acid sequence in the biological sample; a probe array for hybridizing to a target nucleic acid sequence in the biological sample; and having a light sensor a circuit for sensing hybridization between the probe and a target nucleic acid sequence in the biological sample; wherein the circuitry is configured to use an output from the light sensor to produce a display that the probes have hybridized Signal. GAS062.2 Preferably, the test module also has an excitation source for illuminating the -170-201219115 probe array, wherein the light sensor is configured to sense any nucleic acid sequence from the array that has been associated with the target Fluorescence emission of hybrid probes. GAS 062.3 Preferably, the test module also has a USB (Universal Serial Bus) connector for transmitting the signal to an external device. GAS062.4 Preferably, the spacing between the light sensor and the probe is less than 24.9 microns.

GAS062.5 Preferably, the light sensor is an array of photodiodes locating the probe and the probe is a fluorescence resonance energy transfer (FRET) probe. GAS062.6 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 100 nanoseconds. GAS062.7 Preferably, the fluorophore is selected from the group consisting of: ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS062.8 Preferably, the quencher does not have a natural emission of light that should be emitted by the excitation source. GAS062.9 Preferably, the excitation source is a light emitting diode (LED) having an emission wavelength substantially conforming to the absorption wavelength of the fluorophore. GAS062.10 Preferably, the test module also has a dialysis section, wherein the biological sample comprises cells of different sizes, and the dialysis section is configured to separate a sample larger than a predetermined valve to a part of the sample system. It is treated separately from the remaining sample containing only cells smaller than the predetermined valve. GAS062.il Preferably, the circuit includes a CMOS circuit constructed on a wafer-on-a-lab (LOC) device mounted in the test module, the -171 - 201219115 LOC device having a sample inlet and having the dialysis portion. GAS062.1 2 Preferably, the test module also has a housing for securely holding the USB connector and having a fluid communication with the sample inlet on the LOC device. GAS062.1 3 Preferably, the LOC device has a support substrate and a micro system technology (MST) layer with a probe array, the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit has The light sensor. GAS062.1 4 Preferably, the MST layer has an array of hybridization chambers containing the FRET probes, each having a light window to expose the FRET probe to the excitation LED. GAS 062.1 5 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid and a plurality of MST channels configured to attract the fluid through capillary action The PCR section also enters the hybridization chamber. GAS062.1 6 Preferably, the CMOS circuit has a pad for electrically connecting to the external device via the USB connector, wherein the CMOS circuit is configured to use an output from the photodiode to generate the FRET display A signal that the probe hybridizes to the target nucleic acid sequence 歹IJ. GAS 062.1 7 Preferably, the CMOS circuit has a memory for storing identification data of different FRET probe types. GAS 062.1 8 Preferably, the CMOS circuit is configured to activate the photodiode after a predetermined delay after the excitation light is extinguished. GAS062.1 9 Preferably, one of the MST channels is defined as a fluid flow path from the PCR portion to the liquid sensor from -172 to 201219115, the hybrid chamber being disposed along both sides of the fluid flow path. The fluid containing the target nucleic acid sequence is loaded into the hybridization chamber when the fluid is drawn to the liquid sensor by capillary action. GAS062.20 Preferably, the test module also has at least one valve for preventing entry of the biological sample into the PCR portion prior to insertion of the USB connector into the external device.

The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, amplifies the nucleic acid target in the sample, and uses the integrated imaging sensor to identify the nucleic acid sequence of the sample via probe hybridization. And provide electronic results at the output of the other. GAS 063.1 This aspect of the invention provides a single use test module comprising: a housing sized for hand movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; in the housing A probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to excitation light; and an excitation source. GAS063.2 Preferably, the excitation source is a rare gas flash tube mounted in the housing. GAS063.3 Preferably, the single-use test module also has an optical filter, wherein the rare gas flash tube illuminates the probe through the optical filter. - 173 - 2012 19115 GAS 063.4 Preferably, the excitation light source is mounted to the housing for generating an excitation LED of the excitation light. GAS063.5 Preferably, the single-use test module also has a light sensor to detect the fluorescent signal. GAS 063.6 Preferably, the single use test module also has a control circuit configured to delay the excitation light until the photosensor is activated.

GAS063.7 Preferably, the control circuit has a trigger light sensor 'for returning the excitation light to generate an output, so that the control circuit causes the time when the trigger light sensor indicates that the excitation light has been deactivated Delayed GAS063.8 Preferably, the probes respectively have a fluorophore such that the probe-target hybrid emits a fluorescent signal in response to exposure to the excitation light, and the fluorescence lifetime of the fluorophore is greater than 100 nanoseconds . GAS063.9 Preferably, the single-use test module also has

Mounted in the housing for a hybridization chamber containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns. GAS063.1 0 Preferably, the control circuit controls the activation and deactivation of the excitation light and regulates the power supply to the excitation light. GAS063.il Preferably, the single-use test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt cell system being configured to be removed in the photodiode The carrier generated by the absorption of photons of the excitation light. GAS063.1 2 Preferably, the shunt cell system is configured to be turned on when the excitation light is activated and turned off when the excitation light is deactivated. -174- 201219115 GAS063.1 3 Preferably, the fluorophore is a transition metal-ligand complex. GAS063.1 4 Preferably, the fluorophore is a lanthanide metal-ligand complex.

GAS063.1 5 Preferably, the single-use test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer ( FRET) probes, and arrays of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). GAS 063. 16 Preferably, the single-use test module also has a microfluidic device mounted on the outer casing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a microsystem technology (MST) layer on the CMOS circuit, wherein the hybridization chamber is interposed into the MS T layer, the MST layer having a configuration to attract the fluid from the inlet to the hybrid chamber by capillary action Channels. GAS063.17 Preferably, the single-use test module also has an electrical connector for communicating with an external device, wherein the CMOS circuit is configured to convert an output from the photodiode to display the FRET probe A signal that hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS063.1 8 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS 063.1 9 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being at a distance from the FRET probe -175 - 201219115 at 249 microns. GAS063.20 Preferably, the FRET probe is from the camp signal when the probe is in a non-hybrid configuration, the quencher does not have the ease of use of the excitation light, can be mass produced, and is inexpensive. The biological sample is accepted and the nucleic acid sequence of the sample is identified by probe hybridization using one of its integrated imaging to provide an electronic result. GAS 065.1 This aspect of the invention provides: a housing sized to be hand-held, an inlet to a biological sample of the nucleic acid sequence of the housing; a probe in the housing, the probe having a nuclear nucleic acid sequence hybridized to form a probe The needle-target hybridization system is configured to generate an excitation LED for generating the excitation light in response to the excitation light, wherein all of the probes are illuminated simultaneously. GAS065.2 Preferably, the test module causes the excitation LED to be deactivated to initiate a time delay to the previous circuit. GAS065.3 Preferably, the circuit has an output that is generated by echoing the excitation light to cause the electrical device to display that the excitation light has been deactivated. GAS 065.4 preferably, the probe has quenching The agent naturally emits light with a majority of the fluorescent light of the opaque group. A lightweight genetic test mode sensor and an excitation source and a test module at the output thereof, having a signal for accepting an acid sequence for use with the target, the probe-target; and the LED system is located The housing also has a trigger light sensor configured to activate the light sensor to induce delay in the trigger light. A fluorophore is provided to cause the -176-201219115 probe-target hybrid to emit a fluorescent signal in response to exposure to excitation light, and the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS 065.5 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns. GAS065.6 Preferably, the hybridization chamber has a light window for simultaneously exposing the probe to the excitation LED.

GAS065.7 Preferably, the circuit controls activation and deactivation of the excitation LED and regulates the supply of power to the excitation light. GAS065.8 Preferably, the test module also has a shunt transistor between the anode of the photodiode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 065.9 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS065.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS 065.il Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS065.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes. And an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). -177- 201219115 GAS 065.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS065.14 Preferably, the test module also has a microfluidic device mounted on the outer casing adjacent to the excitation LED, the microfluidic device having a CMOS circuit supporting the substrate 'on the support substrate, and on the CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber system are interposed into the MST layer, the MST layer having a plurality of MST layers configured to attract the fluid through the PCR portion and into the hybrid chamber by capillary action aisle. GAS065.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the circuit has the CMOS circuit and is configured to convert an output from the photodiode to display the A signal that the FRET probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS065.1 6 Preferably, the CMOS circuit has a memory for storing the different types of FRET probes. GAS 065.1 7 Preferably, the CMOS circuit comprises an array of photodiodes having an array of photodiodes spaced from the FRET probe by less than 249 microns. GAS065.1 8 Preferably, the test module also has a humidifier for controlling the humidity within the housing. GAS065.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorophore when the probe is in a non-hybrid configuration, the quenching agent does not have a response Natural light emitted by light. -178-201219115 GAS065.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, and uses its integrated imaging sensor to excite the LED to hybridize the probe to identify the nucleic acid sequence of the sample, and at the output of the sample. Provide electronic results. The use of the LED without the optical component string provides a low component count module that is cheaper and easier to manufacture.

GAS066.1 This aspect of the invention provides a test module comprising: a housing sized for hand movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe in the housing, the The probe has a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to the excitation light; and an excitation light for generating the excitation light And a lens positioned in the housing for guiding light from the excitation light to simultaneously illuminate the probe. GAS 066.2 Preferably, the test module also has circuitry configured to delay the time before the excitation LED is deactivated until the photosensor is activated. GAS 066.3 Preferably, the circuit has a trigger light sensor for responding to the excitation light to produce an output such that the circuit causes the time delay when the trigger light sensor indicates that the excitation light has been deactivated. Preferably, the probe has a fluorophore to cause the -179-201219115 probe-target hybrid to emit a fluorescent signal in response to exposure to the excitation light, and the fluorescence lifetime of the fluorophore is greater than 10 0 nanoseconds. GAS066.5 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns. GAS066.6 Preferably, the hybridization chamber has a light window for simultaneously exposing the probe to the excitation light. GAS 066.7 Preferably, the excitation light is LED and the circuit controls activation and deactivation of the excitation LED, and adjusts the power supply to the excitation light GAS 066.8. Preferably, the test module also has the photodiode A shunt transistor between the body anode and the voltage source, the shunt cell system configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS066.9 Preferably, the shunt cell system is configured to turn on when the excitation light is activated and when the excitation light is deactivated. GAS066.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS066.il Preferably, the fluorophore is a lanthanide metal-ligand complex. GAS066.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes (the probes are fluorescent resonance energy transfer (FRET) probes configured to hybridize to different target nucleic acid sequences And an array of photodiodes (so that each of the hybridization chambers corresponds to a respective photodiode of -180 to 201219115). GAS066.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe.

GAS066.1 4 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Multiple channels. GAS066.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the circuit has the CMOS circuit and is configured to convert an output from the photodiode to display the A signal that the FRET probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS066.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS066.1 7 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS066.1 8 Preferably, the test module also has a humidifier for controlling the humidity within the housing. GAS066.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescence-181 - 201219115 signal from the fluorophore when the probe is in a non-hybrid configuration, the quenching agent is not It has a natural emission of light that should be excited. GAS 066.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, and uses its integrated imaging sensor and a lens-excited LED to hybridize the nucleic acid sequence of the sample via probe hybridization, and The output of the other provides electronic results. The lens-attached LED is used to improve the distribution of the excitation light, thereby improving the sensitivity and reliability of the detection system. GAS067.1 This aspect of the invention provides a test module comprising: a size suitable for handheld movement a housing having an inlet for receiving a biological sample containing a target nucleic acid sequence; a probe in the housing having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, The probe-target hybridization system is configured to generate a fluorescent signal in response to the excitation light; to generate excitation light for the excitation light; and a crucible disposed in the outer casing for diverting light from the excitation light for simultaneous illumination The probe. GAS067.2 Preferably, the test module also has circuitry configured to delay the time until the excitation of the LED is activated until the excitation of the LED is deactivated. GAS067.3 Preferably, the circuit has a trigger light sensor for generating an output in response to the excitation light, so that the circuit is induced at the trigger light.

The 201219115 shows that the excitation light has been deactivated to cause this time delay. GAS067.4 Preferably, the probe has a fluorophore probe-target hybrid, respectively, in response to exposure to excitation light and emits a fluorescent signal. The fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS067.5 Preferably, the test module also has a hybridization chamber that is mounted for use with a probe, wherein the body of the hybridization chamber is 9,000 cubic microns. GAS067.6 Preferably, the hybridization chamber has a light window for simultaneous probes to the excitation light. GAS067.7 Preferably, the excitation light is LED and the activation of the LED is activated and deactivated, and the adjustment is supplied to the excitation light GAS067.8. Preferably, the test module also has an anode at the anode A shunt transistor between the voltage sources, the shunt cell configuration being configured to remove a carrier of photons in the photodiode due to absorption of excitation light. GAS067.9 Preferably, the shunt cell crystal system is configured to be turned on when excitation light is activated and turned off when the excitation light is deactivated. GAS067.1 0 Preferably, the fluorophore is a transition metal complex. GAS067.il Preferably, the fluorophore is a lanthanide metal complex. GAS067.1 2 Preferably, the test module also has an array of hybridization chambers containing probes of the type (the probes are configured to not have the number, and the shell product is less than the exposure control) The power photoelectron system is produced by a fluorescence resonance energy transfer (FRET) probe hybridized with a different type of ligand mismatched with the -183-201219115 nucleic acid sequence, and an array of photodiodes (so that Each of the hybridization chambers corresponds to the respective photodiode. GAS 067.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS067.1 4 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and on the CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Channels. GAS 067.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the circuit has the CMOS circuit and is configured to convert an output from the photodiode to display the FRET A signal that the probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS 067.16 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS067.1 7 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being less than 249 microns from the FRET probe. GAS067.1 8 Preferably, the test module also has a humidifier for controlling the humidity within the housing. • 184-201219115 GAS067.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorophore when the probe is in a non-hybrid configuration, the quenching agent is not It has a natural emission of light that should be excited. GAS 067.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, and uses its integrated imaging sensor and an excitation LED with a lens and a chirp to identify the nucleic acid sequence of the sample via probe hybridization. And provide electronic results at the output of the other. The distribution of the excitation light is improved by the LED with the lens and the sputum, thereby further improving the sensitivity and reliability of the detection system. Intrusion in the string of optical elements increases the compactness of the test module. GAS 06 8.1 This aspect of the invention provides a test module comprising: a housing sized for hand-held movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe in the housing a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system configured to generate a fluorescent signal in response to excitation light; for generating the excitation light Excitation light; and a mirror positioned in the housing for diverting light from the excitation light to simultaneously illuminate the probe. GAS068.2 Preferably, the test module also has a circuit configured to delay the time until the excitation LED is deactivated until the optical sensor is activated - 185 - 201219115" GAS068.3 preferably, The circuit has a trigger light sensor for responding to the excitation light to produce an output such that the circuit initiates the time delay when the trigger light sensor indicates that the excitation light has been deactivated. Preferably, the probe has a fluorophore to cause the probe-target hybrid to emit a fluorescent signal in response to exposure to the excitation light, and the fluorescence lifetime of the fluorophore is greater than 1 nanosecond. . GAS 068.5 Preferably, the test module also has a hybridization chamber mounted in the housing for containing a probe, wherein the hybridization chamber has a volume of less than 9,000 cubic microns. GAS068.6 Preferably, the hybridization chamber has a light window for simultaneously exposing the probe to the excitation light. GAS068.7 Preferably, the excitation light is LED and the circuit controls the activation and deactivation of the excitation LED, and the power supply to the excitation light is adjusted. GAS068.8 Preferably, the test module also has A shunt transistor between the photodiode anode and the voltage source 'The shunt cell system is configured to remove a carrier generated in the photodiode by photons that absorb the excitation light. GAS068.9 Preferably, the shunt cell system is configured to turn off when the excitation light is activated and when the excitation light is deactivated. GAS068.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS068.il Preferably, the fluorophore is a lanthanide metal-ligand-186-201219115 compound. GAS 068.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes) And an array of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes).

GAS068.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe. GAS068.1 4 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation LED, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and on the CMOS circuit a microsystem technology (MS T) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Multiple channels. GAS068.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the circuit has the CMOS circuit and is configured to convert an output from the photodiode to display the A signal that the FRET probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS068.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS068.1 7 Preferably, the CMOS circuit comprises an array of photodiodes, the array of photodiodes being at a distance from the FRET probe - 187 - 201219115 at 249 microns. GAS068.1 8 Preferably, the test module also has a humidifier for controlling the humidity within the housing. GAS068.1 9 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorophore when the probe is in a non-hybrid configuration, the quenching agent does not have a response Natural light emitted by light.

GAS 068.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence. The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, and uses its integrated imaging sensor and an excitation LED with a lens and a mirror to hybridize the nucleic acid sequence of the sample via probe hybridization. And provide electronic results at the output of the other. The distribution of the excitation light is improved by the LED with the lens and the mirror, thereby further improving the sensitivity and reliability of the detection system. Plunging the mirror into the string of optical elements increases the compactness of the test module.

GAS069.1 This aspect of the invention provides a test module comprising: a housing sized for hand-held movement, the housing having an inlet for receiving a biological sample containing the target nucleic acid sequence; a probe in the housing, For hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybridization system is configured to generate a fluorescent signal in response to excitation light; a laser located in the housing for generating the excitation light. GAS069.2 Preferably, the test module also has a mirror for -188-201219115 to divert light from the laser to simultaneously illuminate the probes. GAS 069.3 Preferably, the test module also has turns for diverting light from the laser to illuminate the probes. GAS069.4 Preferably, the test module also has circuitry configured to delay the excitation of the excitation laser before the photosensor is activated.

GAS069.5 Preferably, the circuit controls the activation and deactivation of the excitation light and regulates the power supply to the excitation light. GAS069.6 Preferably, the probes each have a fluorophore such that the probe-target hybrid emits a fluorescent signal in response to exposure to the excitation light, and the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. GAS069.7 Preferably, the test module also has an array of hybridization chambers, each of the hybridization chambers containing some of the probes, each of the hybridization chambers having a light window for exposing the probes to the excitation light . GAS 069.8 Preferably, the test module also has a shunt transistor between each photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 069.9 Preferably, the shunt cell system is configured to activate upon activation of the laser and deactivate when the laser is deactivated. GAS069.1 0 Preferably, the fluorophore is a transition metal-ligand complex. GAS 069.il Preferably, the fluorophore is a lanthanide metal-ligand complex. -189- 201219115 GAS069.1 2 Preferably, the test module also has an array of hybridization chambers containing different types of probes (the probes are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer ( FRET) probes, and arrays of photodiodes (so that each of the hybridization chambers corresponds to one of the respective photodiodes). GAS069.1 3 Preferably, the test module also has a polymerase chain reaction (PCR) portion for amplifying the target nucleic acid sequence in the sample prior to hybridization to the FRET probe.

GAS069.1 4 Preferably, the test module also has a microfluidic device mounted on the housing adjacent to the excitation laser, the microfluidic device having a support substrate, a CMOS circuit on the support substrate, and a CMOS circuit a microsystem technology (MST) layer, wherein the PCR portion and the hybridization chamber are interposed into the MST layer, the MST layer having a configuration to attract the fluid through the PCR portion and into the hybrid chamber by capillary action Multiple channels.

GAS069.1 5 Preferably, the test module also has an electrical connector for communicating with an external device, wherein the circuit has the CMOS circuit and is configured to convert an output from the photodiode to display the A signal that the FRET probe hybridizes to the target nucleic acid sequence and provides the signal to the electrical connector for transmission to the external device. GAS069.1 6 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GAS069.1 7 Preferably, the CMOS circuit comprises an array of photodiodes having an array of photodiodes spaced from the FRET probe by less than 249 microns. -190- 201219115 GAS069.1 8 Preferably, the test module also has a humidifier for controlling the humidity within the housing. GAS069.19 Preferably, the FRET probe has a quenching agent to inhibit most of the fluorescent signal from the fluorescent group when the probe is in a non-hybrid configuration, the quenching agent does not have a response light The natural emission of light. GAS 069.20 Preferably, the CMOS circuit is configured to control the temperature of the hybridization chamber during hybridization of the probe to the target nucleic acid sequence.

The easy-to-use, mass-produced, inexpensive and lightweight genetic test module accepts a biological sample, and uses its integrated imaging sensor and excitation laser to identify the nucleic acid sequence of the sample via probe hybridization, and outputs it at the other end.埠 Provide electronic results. The use of the laser as an excitation source increases the intensity and wavelength specificity of the excitation light, thereby further improving the sensitivity and reliability of the detection system. GAS 147.1 This aspect of the invention provides a test module for analyzing a sample fluid containing a target molecule, the test module comprising: a housing provided with a container for receiving the sample fluid; for reacting with the target molecule To form a probe of the probe-target complex, the probe-target complex is configured to emit photons when excited: and a light sensor for detecting photons emitted by the probe-target complex. GAS 147.2 Preferably, the test module also has a microfluidic device. The microfluidic device is provided with a sample inlet in fluid communication with the container, the microfluidic device having the probe and the light sensor. -191 - 201219115 GAS147.3 Preferably, the target molecule is labeled with a nucleic acid sequence and the probe is configured to form a probe-target hybrid ' to cause the light sensor to detect a probe within the probe array - the target hybrid. GAS 147.4 Preferably, the microfluidic device has a CMOS circuit with the photosensor and the probe is a fluorescence resonance energy transfer (FRET) probe. GAS 147.5 Preferably, the test module also has a polymerase chain reaction (PCR) portion and an array of hybridization chambers containing the FRET probe for amplifying the fluid prior to hybridization with the FRET probe. The subject nucleic acid sequences, each of the hybridization chambers having a light window to expose the FRET probe to the excitation light. GAS 147.6 Preferably, the light sensor is an array of photodiodes respectively registered with each of the hybrid chambers. GAS 147.7 Preferably, the CMOS circuit has an external

a pad electrically connected to the device, and a cell for storing identification data identifying the type of each fret probe, the CMOS circuit being configured to use the output from the photodiode to produce a probe-target hybridization a signal of the FRET probe of the body and providing the signal to the pad for transmission to the external device 〇GAS 147.8. Preferably, the CMOS circuit is configured to activate the photocell after a predetermined delay after the excitation light is extinguished Diode. GAS 147.9 Preferably, the FRET probes have a fluorophore and a quencher, respectively, and the fluorophore has a fluorescence lifetime greater than 1 〇〇 nanosecond. GAS147.10 Preferably, the luminescent group transition metal-ligand error -192-

201219115 Compound. GAS 147.11 Preferably, the fluorophore is a laminate. GAS 147.12 Preferably, the fluorophore is selected from the group consisting of ruthenium chelate, ruthenium chelate or ruthenium chelate. GAS 147.1 3 Preferably, the quencher does not have natural emission light. GAS 147.14 Preferably, the CMOS circuit hybridizes the probe to the target nucleic acid sequence during the hybrid GAS 147.1. Preferably, the test module is also controlled by the CMOS circuit. GAS 147.16 Preferably, the hybridization portion has a fluid flow path for a point liquid sensor, the hybridization chamber being disposed along two sides. Preferably, the fluid flow path acts to draw fluid from the PCR portion to the liquid, and the hybrid chamber is configured to be filled with fluid by capillary action such that the CMOS circuit is reversed during use. The sensor indicates that the fluid has reached the liquid to activate the hybrid heater. GAS 147.18 Preferably, each of the hybridization chambers is two cubic microns. GAS 147.19 Preferably, the photodiode lanthanide metal-ligand is: The responsive excitation light is configured to control the temperature of the intersection. Having a hybridization heater providing heat for hybridization from the PCR portion to the end of the fluid flow path configured by the burr sensor, and each of the fluid flow paths from the liquid destination sensor has an output L volume less than 9,000 I is less than 249 microns from the FRET probe -193-201219115. GAS 147.20 Preferably, the microfluidic device has a sample inlet of a fluid sample and a plurality of reagent reservoirs for treating the fluid sample, wherein the fluid sample is attracted to the endpoint sensor by capillary action, and is not required Add liquid from the source of the micro. The light sensor offers a comprehensive solution for low-volume system components that are mass-produced, inexpensive, and lightweight. The optical sensing group is increased in read sensitivity due to large angle light collection. The integrated image sensor eliminates the need to use the light collecting element string. GRR001.1 This aspect of the invention provides a microfluidic device for use and analysis, the microfluidic device comprising: a support substrate; a microsystem technology (MST) layer supported by the substrate, having an inlet for receiving a biochemical liquid; And a reagent reservoir containing the reagent for the chemical analysis of the biochemical liquid; wherein the reagent reservoir is provided with an outlet provided with a surface tension valve, and the meniscus retains the reagent in the reservoir Inside the biochemical fluid except the meniscus of the reagent. GRR001.2 Preferably, the MST layer has a flow path from the surface tension valve, the flow path being configured to flow from the inlet to the surface tension valve by directing the biochemical liquid. The benefit of accepting the different tests from the inlet fluid device and having a height shifter into the mold. The optical component is biochemically treated to the biochemical liquid to extend to the capillary action by the contact of the reagent body. 194-201219115 GRR001.3 Preferably, the volume of the reagent reservoir is less than 1,000,000,000 cubic micrometers. ° GRR001.4 Preferably, the reagent reservoir is supported by the MST layer such that the MST layer is interposed between the reagent reservoir and the support substrate. GRR001.5 Preferably, the reagent reservoir is defined in an upper cover comprising a layer of material having a cavity to define the reagent reservoir. GRR001.6 Preferably, the upper cover defines a plurality of reagent reservoirs for use

It contains all the reagents needed for biochemical treatment and analysis. GRR001.7 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR001.8 Preferably, the flow path extends through the MST layer and the upper cover. GRR001.9 Preferably, the surface tension valve has a meniscus anchor, the meniscus anchor configured to secure a meniscus of the reagent such that the meniscus contact flows through the flow path The biochemical liquid. GRR001.10 Preferably, the biochemical liquid system biological sample and the biochemical treatment gene genetic test comprises the target nucleic acid sequence, the MST layer having hybridization for hybridizing with the target nucleic acid sequence to form a probe-target hybrid Probe. GRR001.il Preferably, the microfluidic device also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses The fluorescent signal produces an output indicative of hybridization of the probe to the target nucleic acid sequence. GRR001.12 Preferably, the microfluidic device also has a hybridization chamber containing the probe-195-201219115 needle, the hybridization chamber having a light window for exposing the probe to the excitation light. GRR001.13 Preferably, the MST layer has a polymerase chain reaction (PCR) portion which is used to amplify an oligonucleotide in a sample of the biological material. GRR001.14 Preferably, the reagent reservoir contains one or more of the following reagents: lysis reagent, anticoagulant, polymerase, dNTP, buffer, primer or ligase. GRR001.15 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR001.16 Preferably, the CMOS circuit controls activation and deactivation of an external source of light that is configured to generate the excitation light. GRR001.17 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes, And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. GRR001.18 Preferably, the flow path is configured to attract the sample from the inlet to all of the hybridization chambers by capillary action. GRR001.19 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GRR001.20 Preferably, the photodiode array is less than 249 microns from the -196-201219115 FRET probe. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a biochemical sample 'Processing and analyzing the sample using a reagent stored in a reagent reservoir of the device'. The reagent is added to the biochemical by a surface tension activation valve as needed mixture. The surface tension actuating valve is highly reliable and easy to manufacture, thereby providing a highly reliable, mass-produced, portable and inexpensive inspection system.

Integrating the reagent reservoir into the device provides self-sufficient reagent storage' which in turn provides a low system component count and a simple manufacturing process resulting in an inexpensive detection system. GRR002.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising: a support substrate; a microsystem technology (MST) layer supported by the substrate, the MST layer having And a plurality of reagent reservoirs containing all reagents required for the biochemical treatment; wherein each of the reagent reservoirs has an outlet provided with a surface tension valve to The reagent remains in the reservoir until the reagent meniscus is removed upon contact with the biochemical liquid. GRR002.2 Preferably, the MST layer has a flow path extending from the inlet to a surface tension valve of each of the reagent reservoirs, the flow path configured to attract the biochemical liquid from the inlet to the inlet by capillary action The surface tension valve-197-201219115 GRR002.3 Preferably, the volume of the reagent reservoirs is less than 1,000,000,000,000 cubic microns, respectively. GRR002.4 Preferably, the reagent reservoirs are supported by the MST layer, such that the MS T layer is interposed between the reagent reservoirs and the support substrate 〇GRR002.5. Preferably, the reagent reservoirs The device is defined in the upper cover and the upper cover includes a layer of material having a cavity to define the reagent reservoirs. GRR002.6 Preferably, the MST layer has a heater for treating the biochemical liquid. GRR002.7 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR002.8 Preferably, the flow path extends through the MST layer and the upper cover. GRR002.9 Preferably, the surface tension valve has a meniscus anchor, the meniscus anchor configured to secure a meniscus of the reagent such that the meniscus contact flows through the flow path The biochemical liquid. GRR002.1 0 Preferably, the biochemical liquid system biological sample and the biochemical treatment gene genetic test comprises the target nucleic acid sequence, the MST layer having hybridization with the target nucleic acid sequence to form a probe-target hybridization Body probe. GRR002.il Preferably, the microfluidic device also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses The fluorescent signal produces an output indicative of hybridization of the probe to the target nucleic acid sequence. -198- 201219115 GRR002.1 2 Preferably, the microfluidic device also has a hybridization chamber containing the probe, the hybridization chamber having a light window for exposing the probe to the excitation light. GRR002.1 3 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. GRR002.1 4 Preferably, the reagent reservoir contains reagents having

Lysis reagent, anticoagulant, polymerase, dNTP, primer or ligase GRR002.1 5 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit There is a light sensor and a pad for communicating with an external device. GRR002.1 6 Preferably, the CMOS circuit controls activation and deactivation of an external light source configured to generate the excitation light. GRR002.17 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes, And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. GRR002.1 8 Preferably, the flow path is configured to attract the sample from the inlet to all of the hybridization chambers by capillary action. GRR002.1 9 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. -199- 201219115 GRR002.20 Preferably, the photodiode array is less than 249 microns from the FRET probe.

The easy-to-use, mass-produced and inexpensive microfluidic device receives a biochemical sample, and the reagent is treated and analyzed using a reagent stored in a reagent reservoir of the device, the reagent being added to the biochemical by a surface tension activation valve as needed mixture. The surface tension actuating valve is highly reliable and easy to manufacture, thereby providing a highly reliable, mass-produced, portable and inexpensive inspection system. Integrating the reagent reservoir into the device and preserving all reagents for the assay requires a low number of system components and a simple manufacturing process, resulting in an inexpensive inspection system. GRR003.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising: a support substrate;

a microsystem technology (MS T) layer supported by the substrate, the MST layer having an inlet for receiving a biochemical liquid; and a reagent reservoir containing the reagent required for the biochemical treatment; wherein the reagent reservoir is less than a volume 1 000,000,000 cubic microns. GRR003.2 Preferably, the volume of the reagent reservoir is less than 300.000. 000 cubic microns. GRR003.3 Preferably, the volume of the reagent reservoir is less than 70.000. 000 cubic microns. GRR003.4 Preferably, the reagent reservoir has a volume of less than 20,000,000 cubic microns. -200- 201219115 GRR003.5 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing all reagents required for the biochemical treatment, and each of the reagent reservoirs has a surface An outlet of the tension valve to retain the reagent in the reservoir by the meniscus of the reagent until the reagent meniscus is removed upon contact with the biochemical liquid.

GRR003.6 Preferably, the MST layer has a flow path extending from the inlet to a surface tension valve of each of the reagent reservoirs, the flow path configured to attract the biochemical liquid from the inlet to the inlet by capillary action The surface tension valve GRR003.7 preferably, the reagent reservoirs are supported by the MST layer, such that the MST layer is between the reagent reservoirs and the support substrate GRR003.8. The reagent reservoir is defined in the upper lid 'the upper lid contains a layer of material having a cavity to define the reagent reservoirs. GRR003.9 Preferably, the MST layer has a heater for treating the biochemical liquid. GRR003.1 0 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR003.1 1 Preferably, the flow path extends through the MST layer and the upper cover. GRR003.1 2 Preferably, the surface tension valve has a meniscus anchor, the meniscus anchor configured to secure a meniscus of the reagent such that the meniscus contact flows through the flow path The biochemical liquid. GRR003.1 3 Preferably, the biochemical liquid system biological sample and the bio-201 - 201219115 chemical treatment gene diagnostic test, the biological sample containing a target nucleic acid sequence, the MST layer having a hybridization with the target nucleic acid sequence to form Probe-target hybrid probe. GRRG03.14 Preferably, the microfluidic device also has a light sensor' wherein the probe-target hybrids each have a fluorophore for emitting a fluorescent signal in response to the excitation light to cause the light sensor to sense The fluorescent signal produces an output indicative of hybridization of the probe to the target nucleic acid sequence. GRR003.1 5 Preferably, the microfluidic device also has a hybridization chamber containing the probe, the hybridization chamber having a light window for exposing the probe to the excitation light. GRR003.1 6 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. GRR003.1 7 Preferably, the reagent reservoir contains one or more of the following reagents: lysis reagent, anticoagulant, polymerase, dNTP, buffer, primer or ligase. GRR003.1 8 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR003.1 9 Preferably, the CMOS circuit controls activation and deactivation of an external source of light that is configured to generate the excitation light. GRR003.20 Preferably, the microfluidic device also has an array of hybridization chambers containing probes of different types that are configured to hybridize to different nucleic acid sequences of the standard -202-201219115 for fluorescence resonance energy transfer ( FRET) probes, and the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes.

The easy-to-use, mass-produced, inexpensive microfluidic device receives the biochemical sample and processes and analyzes the sample using reagents stored in the reagent reservoir of the device. Integrating the reagent reservoir into the device and preserving all of the reagent requirements for the assay provides a low number of system components and a simple manufacturing process resulting in an inexpensive detection system. The low volume reagent reservoir also represents, to some extent, a low reagent volume, which in turn provides low reagent costs and further reduces the cost of the detection system. GRR004.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising: a support substrate; a microsystem technology (MST) layer supported by the substrate, the MST layer having And a reagent reservoir containing the reagent required for the biochemical treatment; wherein the reagent reservoir contains less than 1,000,000,000 cubic micrometers of reagent. GRR004.2 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing all reagents required for the biochemical treatment, and each of the reagent reservoirs has an outlet provided with a surface tension valve To retain the reagent in the reservoir by the reagent meniscus until the biochemical liquid contacts the meniscus. GRR004.3 Preferably, the MST layer has a flow path extending from the inlet to a surface tension valve of each of the reagent reservoirs, the flow path being configured to attract the biochemical liquid by capillary action from -203 to 201219115 The inlet flows to the surface tension valve 〇GRR004.4. Preferably, the reagent reservoirs are supported by the MST layer. Thus, the MST layer is interposed between the reagent reservoirs and the support substrate. 〇GRR004.5 Preferably, the reagent reservoirs are defined in an upper cover that includes a layer of material having a cavity to define the reagent reservoirs. GRR004.6 Preferably, the MST layer has a heater for treating the biochemical liquid. GRR004.7 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR004.8 Preferably, the flow path extends through the MST layer and the upper cover. GRR004.9 Preferably, the surface tension valve has a meniscus anchor, the meniscus anchor configured to secure a meniscus of the reagent such that the meniscus contact flows through the flow path The biochemical liquid. GRR004.1 0 Preferably, the biochemical liquid system biological sample and the biochemical treatment gene genetic test comprises the target nucleic acid sequence, the MST layer having hybridization with the target nucleic acid sequence to form a probe-target hybridization Body probe. GRR004.il Preferably, the microfluidic device also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses The fluorescent signal produces an output indicative of hybridization of the probe to the target nucleic acid sequence. -204-201219115 GRR004.1 2 Preferably, the microfluidic device also has a hybridization chamber containing the probe, the hybridization chamber having a light window for exposing the probe to the excitation light. GRR004.1 3 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. GRR004.1 4 Preferably, the reagent reservoir contains reagents having

Lysis reagent, anticoagulant, polymerase, dNTP, buffer, primer or ligase. GRR004.1 5 Preferably, the microfluidic device also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR004.1 6 Preferably, the CMOS circuit controls activation and deactivation of an external source of light that is configured to generate the excitation light. GRR004.1 7 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes. And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. GRR004.1 8 Preferably, the flow path is configured to attract the sample from the inlet to all of the hybridization chambers by capillary action. GRR004.19 Preferably, the CMOS circuit has a body of information for storing identification data of the different FRET probe types. -205- 201219115 GRR004.20 Preferably, the photodiode array is less than 249 microns from the FRET probe. The easy-to-use, mass-produced, inexpensive microfluidic device receives the biochemical sample and processes and analyzes the sample using reagents stored in the reagent reservoir of the device. This low reagent volume provides low reagent cost and an inexpensive detection system. GRR005.1 This aspect of the invention provides a LOC device for performing a genetic diagnostic test, the LOC device comprising: a support substrate; a microsystem technology (MST) layer supported by the substrate, the MST layer having an accept An inlet for a biological sample containing the target nucleic acid sequence; a probe for hybridizing with the target nucleic acid sequence to form a probe-target hybrid; a flow path extending from the inlet to the probe; and a reagent storage containing the reagent A device for adding a reagent to the sample at a flow path upstream of the probe. GRR005.2 Preferably, the reagent reservoir has an outlet in fluid communication with the flow path, the outlet being provided with a surface tension valve to retain the reagent in the reservoir by the reagent meniscus until the flow path The reagent meniscus is removed when the sample stream is in contact. GRR005.3 Preferably, the volume of the reagent reservoir is less than 1,000,000,000,000 cubic microns. GRR005.4 Preferably, the volume of the reagent reservoir is less than 300,000,000 cubic microns. -206- 201219115 GRR005.5 Preferably, the volume of the reagent reservoir is less than 70.000. 000 cubic microns. GRR005.6 Preferably, the volume of the reagent reservoir is less than 20.000. 000 cubic microns. GRR005.7 Preferably, the reagent reservoir is supported by the MST layer such that the MST layer is interposed between the reagent reservoir and the support substrate. GRR005.8 Preferably, the reagent reservoir is defined in the upper cover,

The upper cover includes a layer of material having a cavity to define the reagent reservoir. GRR005.9 Preferably, the cap defines a plurality of reagent reservoirs for use in all of the reagents required for the genetic diagnostic test to be performed. GRR005.10 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR005.il Preferably, the flow path extends through the MST layer and the upper cover. GRR005.1 2 Preferably, the reagent reservoir has a vent to the atmosphere, the vent being sized to prevent leakage of the reagent during storage and during operation of the LOC device, and permitting when the reagent exits the outlet Air flows to the reagent reservoir. GRR005.1 3 Preferably, the LOC device also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses The fluorescent signal produces an output indicative of hybridization of the probe to the target nucleic acid sequence. GRR005.1 4 Preferably, the LOC device also has a hybridization chamber containing the probe, the hybridization chamber having a window for exposing the probe to the excitation light - 207 - 201219115. GRR005.1 5 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. GRR005.1 6 Preferably, the reagent reservoir comprises reagents having one or more of the following: lysing reagent, anticoagulant, polymerase, dNTP, primer or ligase 〇GRR005.1 7 Preferably, The LOC device also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR005.1 8 Preferably, the CMOS circuit controls activation and deactivation of an external source of light that is configured to generate the excitation light. GRR005.1 9 Preferably, the LOC device also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes, And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. GRR005.20 Preferably, the flow path is configured to attract the sample from the inlet to all of the hybridization chambers by capillary action. The easy-to-use, mass-produced and inexpensive genetic analysis LOC-device accepts a biological sample and analyzes the nucleic acid sequence of the sample by hybridizing with an oligonucleotide probe using a reagent stored in a reagent reservoir of the LOC device. . Integrating the reagent reservoir into the device provides a low system component count and a simple -208-201219115 manufacturing process, resulting in an inexpensive inspection system. GRR006.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis comprising: a support substrate; a microsystem technology (MST) layer supported by the substrate, the MST layer having To accept the entry of a biological sample containing the indicated nucleic acid sequence:

a probe for hybridizing to the target nucleic acid sequence to form a probe-target hybrid; and a plurality of reagent reservoirs containing all of the test GRR006.2 required to perform the diagnostic test. Preferably, the LOC device There is also a flow path extending from the inlet to the probe, wherein the reagent reservoirs are disposed along the flow path for adding reagent to the sample at a flow path upstream of the probe. GRR006.3 Preferably, the reagent reservoirs each have an outlet in fluid communication with the flow path, the outlet being provided with a surface tension valve to retain the reagent in the reservoir by the reagent meniscus until the flow path The reagent meniscus is removed when the sample stream is in contact. GAS 006.4 Preferably, the volume of the reagent reservoirs is each less than 1,000,000,000 cubic microns.

GRR006.5 Preferably, the reagent reservoirs are each supported by the MST layer such that the MS T layer is interposed between the reagent reservoirs and the support substrate. GRR006.6 Preferably, the reagent reservoirs are defined in the upper lid. The upper lid comprises a layer of material having a cavity to define the reagent reservoirs. -209-201219115 GRR006.7 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR006.8 Preferably, the flow path extends through the MST layer and the upper cover. GRR006.9 Preferably, the reagent reservoirs each have a vent to the atmosphere, the vent being sized to prevent leakage of the reagent during storage and during operation of the LOC device, and when the reagent is added to the reagent The sample is allowed to flow to the reagent reservoir. GRR006.1 0 Preferably, the LOC device also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting light in response to the excitation light. An optical signal such that the light sensor senses the fluorescent signal to produce an output indicative of hybridization of the probe to the target nucleic acid sequence. GRR006.il Preferably, the LOC device also has a hybridization chamber containing the probe, the hybridization chamber having a light window for exposing the probe to the excitation light. GRR006.1 2 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. GRR006.1 3 Preferably, the reagents contained in the reagent reservoirs include: lysing reagent, anticoagulant, polymerase, dNTP, primer or ligase 〇

GRR006.1 4 Preferably, the LOC device also has a CMOS circuit between the MST layer and the support substrate, and the CMOS circuit has the optical-210-201219115 sensor and a bonding pad for communicating with an external device. . GRR006.1 5 Preferably, the CMOS circuit controls activation and deactivation of an external source of light. The external source is configured to generate the excitation light.

GRR006.1 6 Preferably, the LOC device also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (fret) probes, And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. GRR006.1 7 Preferably, the flow path is configured to attract the sample from the inlet to all of the hybridization chambers by capillary action. GRR006.1 8 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GRR006.1 9 Preferably, the photodiode array is less than 249 microns from the FRET probe. GAS006.20 Preferably, the fluorescent lifetime of the fluorophore is greater than 100 nanoseconds. The readily available, mass-produced, and inexpensive genetic analysis LOC device accepts a biological sample and analyzes the nucleic acid sequence of the sample by hybridization with an oligonucleotide probe using reagents stored in a reagent reservoir of the LOC device. Integrating the reagent reservoir to the LOC device and maintaining all of the reagent requirements for the assay provides a low number of system components and a simple manufacturing process resulting in an inexpensive inspection system. GRR007.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis comprising: -211 - 201219115 support substrate; a microsystem technology (MST) layer supported by the substrate, The MST layer has an inlet for accepting a biological sample containing the target nucleic acid sequence; a hybridization portion containing an array of chambers having a probe for hybridizing with the target nucleic acid sequence to form a probe-target hybrid; and reagent storage a reagent for treating the biological sample prior to hybridization; wherein each chamber of the hybridization portion contains less than 2 70 picograms of each probe and the reagent reservoir contains less than 1000,000,000 cubic microns Reagents. GRR007.2 Preferably, each chamber of the hybrid portion contains less than 60 picograms of each probe and the reagent reservoir contains less than 300,000,000 cubic microns of the reagent. GRR007.3 Preferably, each chamber of the hybridization section contains less than 12 picograms of each probe and the reagent reservoir contains less than 70,000,000 cubic micrometers of the reagent. GRR007.4 Preferably, each chamber of the hybridization section contains less than 2.7 picograms of each probe and the reagent reservoir contains less than 20,000,000 cubic micrometers of the reagent. GRR007.5 Preferably, the LOC device also has a plurality of reagent reservoirs containing all reagents required for performing the diagnostic test, wherein each of the reagent reservoirs has an outlet provided with a surface tension valve To retain the reagent in the reservoir by the reagent meniscus until the biological sample contacts the meniscus. GRR007.6 Preferably, the MST layer has a flow path extending from the inlet to a surface tension valve of each of the reagent reservoirs of -212-201219115, the flow path configured to attract the biological sample stream by capillary action To the surface tension valve. GRR007.7 Preferably, the reagent reservoirs are supported by the MST layer, such that the MS T layer is between the reagent reservoirs and the support substrate GRR007.8. Preferably, the reagent reservoirs The system is defined in an upper cover that includes a layer of material having a cavity to define the reagent reservoirs.

GRR007.9 Preferably, the MST layer has a heater for treating the biochemical liquid. GRR007.1 0 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR007.il Preferably, the flow path extends through the MST layer and the upper cover. GRR007.1 2 Preferably, the reagent reservoirs each have a vent to the atmosphere, the vent being sized to prevent leakage of the reagent during storage and during operation of the LOC device, and when the reagent exits the outlet Air is allowed to flow to the reagent reservoir. GRR007.1 3 Preferably, the biochemical liquid system biological sample and the biochemical treatment gene genetic test comprises the target nucleic acid sequence, the MS T layer having hybridization with the target nucleic acid sequence to form a probe-target Probe for hybrids. GRR007.14 Preferably, the LOC device also has a light sensor, wherein the probe-target hybrids each have a fluorophore for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses the light sensor The fluorescent signal is generated to produce -213-201219115 showing the hybridization of the probe to the target nucleic acid sequence. GRR007.1 5 Preferably, the hybridization chambers each have a light window for exposing the probe to the excitation light. GRR007.16 Preferably, the MST layer has a polymerase chain reaction (PCR) portion which is used to amplify an oligonucleotide in a sample of the biological material. GRR007.1 7 Preferably, the reagent reservoir comprises one or more of the following reagents: lysing reagent, anticoagulant, polymerase, dNTP, primer or ligase 〇GRR007.1 8 preferably, The LOC device also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR007.1 9 Preferably, the CMOS circuit controls activation and deactivation of an external source of light that is configured to generate the excitation light. GRR007.20 Preferably, the LOC device also has an array of hybridization chambers containing probes of different types that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes, and The light sensor is an array of photodiodes such that each of the hybrid chambers corresponds to one of the respective photodiodes. The easy-to-use, mass-produced and inexpensive genetic analysis LOC device accepts a biological sample and analyzes it by hybridizing to a low volume oligonucleotide probe using a low volume reagent stored in a reagent reservoir of the LOC device. The nucleic acid sequence of the sample. The low oligonucleotide probe and reagent volume provides low -214-201219115 probe and reagent costs and an inexpensive detection system. GRR008.1 This aspect of the invention provides a test module for biochemical processing and analysis, the test module comprising: a housing sized for hand movement, the housing having a container for receiving biochemical liquid; and a reagent a reagent reservoir for adding to the biochemical liquid for chemical analysis of the biochemical liquid;

The reagent reservoir is provided with an outlet provided with a surface tension valve to retain the reagent in the reservoir by the meniscus of the reagent until the meniscus of the reagent is removed upon contact with the biochemical liquid. GRR 008.2 Preferably, the test module also has a flow path extending from the container to the surface tension valve, the flow path being configured to attract the biochemical liquid to the surface tension valve by capillary action. GRR008.3 Preferably, the reagent reservoir has a volume of less than 1,000,000,000,000 cubic microns. GRR008.4 Preferably, the test module also has a microfluidic device in the housing, the microfluidic device having a support substrate and an MST layer formed on the substrate, wherein the reagent reservoir is comprised of the MST layer Supported, so the MST layer is between the reagent reservoir and the support substrate. GRR008.5 Preferably, the reagent reservoir is defined in an upper cover comprising a layer of material having a cavity to define the reagent reservoir. GRR008.6 Preferably, the upper lid defines a plurality of reagent reservoirs for use in all reagents required for biochemical processing and analysis to be performed. GRR00 8.7 Preferably, at least one of the reagent reservoirs has a plurality of outlets -215 - 201219115 to increase the flow rate of the reagent out of the reservoir. GRR008.8 Preferably, the flow path extends through the MST layer and the upper cover. GRR008.9 Preferably, the reagent reservoir has a vent to the atmosphere, the vent being sized to prevent leakage of the reagent during storage and during operation of the microfluidic device, and permitting the reagent to flow out of the outlet Air flows to the reagent reservoir. GRR008.1 0 Preferably, the biochemical liquid system biological sample and the biochemical treatment gene genetic test comprises the target nucleic acid sequence, the MS T layer having hybridization with the target nucleic acid sequence to form a probe-target Probe for hybrids. GRR008.1 1 Preferably, the test module also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting a fluorescent signal in response to the excitation light to make the light sensor feel The fluorescent signal is measured to produce an output indicative of hybridization of the probe to the target nucleic acid sequence. GRR008.1 2 Preferably, the test module also has a hybridization chamber containing the probe, the hybridization chamber having a light window for exposing the probe to the excitation light. GRR008.1 3 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. GRR008.1 4 Preferably, the reagent reservoir comprises one or more of the following reagents: lysing reagent, anticoagulant, polymerase, dNTP, primer or ligase-216-201219115 GRR008.1 5 The test module also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR008.1 6 Preferably, the CMOS circuit controls activation and deactivation of an external source of light that is configured to generate the excitation light.

GRR008.1 7 Preferably, the test module also has an array of hybridization chambers containing probes of different types that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes. And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. GRR008.1 8 Preferably, the flow path is configured to attract the sample from the container to all of the hybridization chambers by capillary action. GRR008.19 Preferably, the CMOS circuit has a memory for storing identification data of the different FRET probe types. GRR008.20 Preferably, the photodiode array is less than 249 microns from the FRET probe. The easy-to-use, mass-produced, inexpensive, and portable microfluidic test module accepts a biochemical sample and processes and analyzes the sample using a reagent stored in a reagent reservoir of the module, optionally by surface tension A start valve is added to the biochemical mixture. The surface tension actuating valve is highly reliable and easy to manufacture' which in turn provides a highly reliable, mass produceable, expandable and inexpensive inspection system. GRR009.1 This aspect of the invention provides a test module for biochemical treatment - 217 - 201219115 and analysis, the test module comprising: a housing sized for hand movement, the housing having a container for receiving biochemical liquid; And a reagent reservoir containing a reagent for adding to the biochemical liquid for chemical analysis of the biochemical liquid; wherein the reagent reservoir has a volume of less than 1,000,000,000,000 cubic microns. GRR009.2 Preferably, the volume of the reagent reservoir is less than 300.000.000 cubic microns. GRR009.3 Preferably, the volume of the reagent reservoir is less than 70.000. 000 cubic microns. GRR009.4 Preferably, the volume of the reagent reservoir is less than 20.000. 000 cubic microns. GRR009.5 Preferably, the reagent reservoir is provided with an outlet having a surface tension valve to retain the reagent in the reservoir by the meniscus of the reagent until the chemical meniscus contacts the chemical meniscus. GRR 009.6 Preferably, the test module also has a flow path extending from the container to the surface tension valve, the flow path being configured to attract the biochemical liquid to the surface tension valve by capillary action. GRR009.7 Preferably, the test module also has a microfluidic device in the housing, the microfluidic device having a support substrate and an MST layer formed on the substrate, wherein the reagent reservoir is comprised of the MST layer Supported, so the MST layer is between the reagent reservoir and the support substrate. GRR009.8 Preferably, the reagent reservoir is defined in an upper cover comprising a layer of material having a cavity to define the reagent reservoir. -218- 201219115 GRR009.9 Preferably, the upper lid defines a plurality of reagent reservoirs for containing all of the reagents required for biochemical processing and analysis to be performed. GRR009.1 0 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR009.il Preferably, the flow path extends through the MST layer and the upper cover.

GRR009.1 2 Preferably, the reagent reservoir has a vent to the atmosphere, the vent being sized to prevent leakage of the reagent during storage and during operation of the microfluidic device, and when the reagent exits the outlet Air is allowed to flow to the reagent reservoir. GRR009.1 3 Preferably, the biochemical liquid system biological sample and the biochemical treatment gene genetic test comprises the target nucleic acid sequence, the MS T layer having hybridization with the target nucleic acid sequence to form a probe-target Probe for hybrids. GRR009.14 Preferably, the test module also has a light sensor, wherein the probe-target hybrids each have a fluorescent group for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses The fluorescent signal is generated to produce an output indicating that the probe hybridizes to the target nucleic acid sequence. GRR009.15 Preferably, the test module also has a hybridization chamber containing the probe, and the hybridization chamber has a feature for exposing the probe. A light window to the excitation light" GRR009.1 6 Preferably, the MST layer has a polymerase chain reaction (PCR) portion for amplifying an oligonucleotide in a sample of the biological material. -219- 201219115 GRR009.1 7 Preferably, the reagent reservoir contains one or more of the following reagents: lysing reagent, anticoagulant 'polymerase, dNTP, primer or ligase 〇GRR009.1 8 Preferably, the test module also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit having the light sensor and a pad for communicating with an external device. GRR009.1 9 preferably The CMOS circuit controls activation and deactivation of an external light source configured to generate the excitation light. GRR009.20 Preferably, the test module also has an array of hybridization chambers containing probes of different types that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes, And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. The easy-to-use, mass-produced, inexpensive, and portable microfluidic test module accepts biochemical samples and processes and analyzes the sample using reagents stored in the reagent reservoir of the module. The low volume reagent reservoir provides a low reagent volume to some extent, thereby providing low reagent cost and an inexpensive detection system. GRR010.1 This aspect of the invention provides a test module for performing a genetic diagnosis test, the test module comprising: a housing sized to be hand-held, the housing having a container for a biological sample, the biological sample containing the target nucleic acid sequence An array of chambers comprising a probe for hybridizing to the target nucleic acid sequence to form a probe-target hybrid of -220-201219115; a flow path extending from the inlet to the probe; and a reagent containing the reagent A reservoir is used to add a reagent to the sample at a flow path upstream of the probe; wherein the chamber contains less than 270 picograms of probe, respectively, and the reagent reservoir has a volume of less than 1,000,000,000,000 cubic microns.

Preferably, the chamber contains less than 60 picograms of probes, respectively, and the volume of the reagent reservoir is less than 3 inches, 〇〇〇, 〇〇〇 cubic micrometers. GRR010.3 Preferably, the chamber contains less than 12 picograms of probes, respectively, and the volume of the reagent reservoir is less than 7 inches, 〇〇〇, 〇〇〇 cubic micrometers. GRR010.4 Preferably, the chamber contains less than 2.7 picograms of probe, respectively, and the volume of the reagent reservoir is less than 20,000,000 cubic microns. GRR010.5 Preferably, the reagent reservoir is provided with an outlet provided with a surface tension valve to retain the reagent in the reservoir by the meniscus of the reagent until the biological meniscus contacts the biological meniscus. GRR 010.6 Preferably, the test module also has a flow path extending from the container to the surface tension valve, the flow path being configured to attract the biological sample to the surface tension valve by capillary action. GRR010.7 Preferably, the test module also has a microfluidic device in the housing, the microfluidic device having a support substrate and an MST layer formed on the substrate, wherein the reagent reservoir is comprised of the MST layer Supported, so the MST layer is between the reagent reservoir and the support substrate. GRR010.8 Preferably, the reagent reservoir is defined in an upper cover that includes a layer of material having a cavity to define the reagent reservoir. -221 · 201219115 GRR010.9 Preferably, the cap defines a plurality of reagent reservoirs for use in all reagents required for the genetic analysis to be performed. GRR 010.10 Preferably, at least one of the reagent reservoirs has a plurality of outlets to increase the flow rate of the reagent out of the reservoir. GRR0 10.il Preferably, the flow path extends through the MST layer and the upper cover.

GRR0 10.12 Preferably, the reagent reservoir has a vent to the atmosphere, the vent being sized to prevent leakage of the reagent during storage and during operation of the microfluidic device, and to allow air when the reagent exits the outlet Flow to the reagent reservoir. GRR010.13 Preferably, the test module also has a light sensor, wherein the probe-target hybrid has a fluorophore for emitting a fluorescent signal in response to the excitation light, so that the light sensor senses The fluorescent signal produces an output indicative of hybridization of the probe to the target nucleic acid sequence.

GRR0 1 〇· 1 4 Preferably, the test module also has an LED for generating the excitation light. GRR010.15 Preferably, the chambers are respectively hybridization chambers having a light window for exposing the probe to the excitation light. GRR010.16 Preferably, the MST layer has a polymerase chain reaction (PCR) portion which is used to amplify an oligonucleotide in a sample of the biological material. GRR010.17 Preferably, the reagent reservoir comprises one or more of the following reagents: lysing reagent, anticoagulant, polymerase, dNTP, primer or ligase-222-201219115 GRR010.18 Preferably, The test module also has a CMOS circuit between the MST layer and the support substrate, the CMOS circuit has the photo sensor and a pad for communicating with an external device. GRR010.19 Preferably, the CMOS circuit The activation and deactivation of an external light source is controlled, the external light source being configured to generate the excitation light.

GRR010.20 Preferably, the test module also has an array of hybridization chambers containing different types of probes that are configured to hybridize to different target nucleic acid sequences for fluorescence resonance energy transfer (FRET) probes, And the light sensor is an array of photodiodes such that each of the hybridization chambers corresponds to one of the respective photodiodes. The easy-to-use, mass-produced, inexpensive, and portable genetic test module accepts biological samples and utilizes low volume reagents stored in reagent reservoirs of the module via low volume oligonucleotides Needle hybridization is performed to analyze the nucleic acid sequence of the sample. The low oligonucleotide probe and reagent volume provides low probe and reagent cost and an inexpensive detection system. GVA005.1 This aspect of the invention provides a microfluidic device for processing a fluid sample, the microfluidic device comprising: a reservoir for containing a reagent: and a surface tension valve having a bore configured to Forming the agent to form a meniscus such that the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus to cause the reagent to flow out of the reagent reservoir

GVA005.2 Preferably, the reservoir has a venting opening as an air inlet when the reagent exits the reagent reservoir from -223 to 201219115. GVA005.3 Preferably, the microfluidic device also has a support substrate»Microsystem Technology (MS T) layer on the support substrate; and an upper cover covering the MST layer, the upper cover having a plurality of upper covers and M ST a fluid connection between the layers for fluid to flow from the MS T layer to the upper cover and fluid flow from the upper cover to the MS T layer; wherein at least the fluid connection between the upper cover and the MS T layer One is the surface tension valve. GVA005.4 Preferably, the upper cover has a cover channel connecting at least two of the fluid connectors, the upper cover channel being configured to attract fluid flow between the fluid connectors by capillary action. GVA005.5 Preferably, the reagent reservoir is in fluid communication with the upper lid passageway via at least two of the MST layer and the fluid connection members, one of the fluid connection members being the surface tension valve. GVA005.6 Preferably, the cap passage and the reagent reservoir are formed in a layer of a single material. GVA005.7 Preferably, the upper cover passage is formed on a surface of the upper cover such that an outer surface of the MST layer closes the upper cover passage. GVA005.8 Preferably, the upper cover has an outer surface opposite the surface, the outer surface having a sample inlet for receiving the fluid sample and feeding the fluid sample to the upper cover channel. GVA005.9 Preferably, the fluid sample comprises a biological sample having cells of different sizes, and at least one of the fluid connectors is an array of holes -224 - 201219115, the size of the hole being prevented from being greater than a predetermined valve The cells pass. GVA005.1 0 Preferably, the array of holes is part of a dialysis section, the dialysis section being configured to separate a sample of cells larger than a predetermined valve to a portion, the sample of the portion and k containing less than the predetermined valve The remaining samples of the cells of the sputum are treated separately. GVA005.1 1 Preferably, the biological sample is blood and the system is configured such that cells smaller than the predetermined valve include pathogens.

GVA005.1 2 Preferably, the reagent in the reservoir is an anticoagulant and the cap is configured to mix the blood with the anticoagulant prior to entering the dialysis section. GVA005.1 3 Preferably, the microfluidic device also has a lysis unit in fluid communication with a lysis reagent reservoir containing a lysis reagent, the lysis unit being configured to dissolve the pathogen and release the genetic material therein . GVA005.14 Preferably, the microfluidic device also has a nucleic acid amplification unit for amplifying the nucleic acid in the fluid; wherein the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion, and the upper cover has PCR A reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplification of the nucleic acid sequence. GVA005.1 5 Preferably, the cap has a polymerase reservoir containing a polymerase to mix the polymerase with the fluid prior to amplification of the nucleic acid sequence. GVA005.16 Preferably, the microfluidic device also has the branch

Holding a CMOS circuit between the substrate and the MST layer for operation control of the PCR

-225-201219115 GVA005.1 7 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridization with a target nucleic acid sequence amplified by the PCR portion. GVA005.1 8 Preferably, the _ column has more than 1 000 probes. GVA005.1 9 Preferably the microfluidic device also has a photodiode array for detecting probe hybridization within the probe array. GVA005.20 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the sample. The easy-to-use, mass-produced, inexpensive microfluidic device accepts liquid for treatment and analysis using reagents stored in a reagent reservoir of the device, which reagent is added to the liquid as needed by a surface tension valve. The surface tension valve is highly reliable and easy to manufacture, providing this highly reliable, mass-produced, retractable and inexpensive inspection system. Integrating the reagent reservoir into the device provides self-sufficient reagent storage, which in turn provides a low system component count and a simple manufacturing process resulting in an inexpensive detection system. GVA007.1 This aspect of the invention provides a microfluidic device for testing a fluid, the microfluidic device comprising: an inlet for receiving the fluid; a reservoir containing the reagent; a flow path extending from the inlet; a valve assembly a body for establishing a fluid connection between the flow path and the reservoir, the valve assembly having a plurality of outlet valves and a plurality of passages from the reservoir to the flow path: wherein, when in use, - 226 - 201219115 some of the outlet valves open to allow the reagent to flow through the valve assembly to the flow path to combine with the fluid from the inlet to produce a combined flow of reagents in the combined flow The ratio is determined by the number of open outlet valves. GVA007.2 Preferably, the valve assembly has one of the outlet valves in each of the channels. GVA007.3 Preferably, the outlet valve is provided with a surface tension of the hole

a valve configured to cause the agent to form a meniscus' such that the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir Device. GVA007.4 Preferably, there are more than one of these outlet valves in each of the channels. GVA007.5 Preferably, the reservoir has a venting opening as an air inlet when the reagent exits the reagent reservoir. GVA007.6 Preferably, the microfluidic device of claim 5 further comprises a plurality of the reservoirs - each of the reservoirs comprising a different reagent and a plurality of the respective between the reservoir and the flow path The valve assembly 'the ratio of any of these different reagents in the combined flow is related to the number of outlet valves that are opened in the corresponding valve block. GVA007.7 Preferably, the microfluidic device of claim 5 further comprises a support substrate; a microsystem technology (MST) layer on the support substrate: and a cover over the MST layer, the upper cover having a plurality of a fluid connection between the upper cover and the MST layer for fluid to flow from the MST layer to the upper cover-227-201219115 and fluid flow from the upper cover to the MST layer; and between the upper cover and the MS T layer At least one of the fluid connectors is the surface tension valve that is part of the valve assembly. GVA007.8 Preferably, the flow path extends through the MST layer and the upper cover connects at least some of the fluid connections of the fluid connection, the flow path configured to attract between the fluid connections by capillary action Fluid flow. GVA007.9 Preferably, the fluid comprises biological samples having cells of different sizes, and at least one of the fluid connectors is an array of holes that are sized to prevent passage of cells larger than the predetermined valve. GVA007.1 0 Preferably, the array of holes is part of a dialysis section configured to separate a sample of cells larger than a predetermined valve to a portion of the sample system and containing only less than the predetermined valve The remaining samples of the cells are treated separately. GVA007.il Preferably, the biological sample is blood and the hole is configured to cause cells smaller than the predetermined valve to include a pathogen. GVA007.12 Preferably, one of the reagent reservoirs is an anticoagulant reservoir, the anticoagulant reservoir and the flow path are via a surface of a valve assembly corresponding to the anticoagulant reservoir The tension valve remains in fluid communication such that the anticoagulant mixes with the blood before it enters the dialysis section. GVA007.1 3 Preferably, the microfluidic device of claim 12 further comprises a lysing portion in fluid communication with the flow path, the lysing portion being configured to dissolve the pathogen and release the genetic material therein. GVA007.1 4 Preferably, the microfluid-228-201219115 body device according to claim 13 further comprises a nucleic acid amplification portion for amplifying the nucleic acid in the fluid; wherein the nucleic acid amplification portion A polymerase chain reaction (PCR) portion, and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplification of the nucleic acid sequence. GVA007.1 5 Preferably, the cap has a polymerase reservoir containing a polymerase to polymerize the polymerase with the fluid prior to amplification of the nucleic acid sequence.

GVA007.1 6 Preferably, the microfluidic device of claim 15 further comprises a CMOS circuit between the support substrate and the MST layer for operational control of the PCR portion. GVA007.1 7 Preferably, the microfluidic device of claim 16 further comprises a hybridization portion having a probe array for hybridization with the target nucleic acid sequence amplified by the PCR portion. GVA007.1 8 Preferably, the array has more than 1 000 probes. GVA007.1 9 Preferably, the microfluidic φ body device of claim 18 wherein the probes are fluorescent resonance energy transfer (FRET) probes, and the CMOS circuit further comprises for detecting the probe A photodiode array of probe hybrids within the array of needles. GVA007.20 Preferably, the microfluidic device of claim 19 further comprises a plurality of heaters for controlling the temperature of the sample. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a liquid sample' to be processed and analyzed using reagents stored in a reagent reservoir of the device, the reagent being added to the liquid as desired by a plurality of valves. The necessary mixing ratio between the reagent and other liquid components is determined by the number of valves opened in the -229-201219115, so that it is easy to manufacture and reliably achieve the difficult to control targets in this microfluidic environment. GVA012.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis comprising: a support substrate; a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid a channel for directing the flow of a liquid containing the target nucleic acid sequence, the channel having an inlet, an outlet, and a meniscus anchor between the inlet and the outlet to stop fluid flow from the inlet to the outlet Forming a meniscus at the meniscus anchor; and activating valve provided with a valve heater for heating the meniscus to cause the meniscus anchor to release the meniscus The liquid resumes flow to the outlet 〇GVA012.2. Preferably, the meniscus anchor is bored and the valve heater extends adjacent the edge of the bore. GVA012.3 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to release a meniscus from the meniscus anchor 〇GVA012.4. Preferably, the LOC The device also has a CMOS circuit between the channel and the support substrate for operational control of the activation valve 〇GVA012.5. Preferably, the LOC device also has at least one sensor responsive to the flow of liquid, wherein The at least one sensor provides an anti-230-201219115 feed to the CMOS circuit for operational control of the start-up valve. GVA 012.6 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a location in the channel. GVA012.7 Preferably, the LOC device also has a polymerase chain

a reaction (PCR) portion for heating a circulating nucleic acid sequence and a PCR reagent mixture to achieve a denaturation temperature, an adhesion temperature, and a primer extension temperature, wherein the activation valve is for retaining the nucleic acid sequence and PCR during the heating cycle The reagent mixture is in the PCR portion outlet valve of the PCR portion, and the PCR portion outlet valve is configured to open in response to the activation signal to allow the amplicon to flow to the probe array. GVA012.8 Preferably, the PCR moiety is configured to produce an amplicon sufficient to hybridize to more than 100 probes within 10 minutes of the sample entering the sample inlet. GVA012.9 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds. GVA012.10 Preferably, the LOC device also has a temperature sensor, and wherein the PCR portion has at least one heater for heating and circulating the nucleic acid sequence and the PCR mixture, the temperature sensor and the at least one heating The device is coupled to the CMOS circuit for feedback control of the at least one heater GVA012.il. Preferably, the CMOS circuit activates the PCR outlet valve after a predetermined number of thermal cycles. GVA012.12 Preferably, the PCR portion has a plurality of elongated PCR chambers and a plurality of heaters, the longitudinal length of the elongated PCR chamber being much larger than the sides 231 - 201219115, and the heaters are elongated And parallel to the longitudinal length of the PCR chambers. GVA012.13 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel" GVA012.14 Preferably, the microchannel is configured to lend A liquid containing the nucleic acid sequence is attracted by capillary action from the PCR inlet to the PCR outlet. GVA 012.15 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GVA 012.16 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GVA012.17 Preferably, the plurality of elongate heaters are independently operable. GVA012.18 Preferably, the LOC device also has a photodiode array for detecting probe hybridization within the probe array. The mass-produced and inexpensive genetic analysis LOC device accepts a biological sample and analyzes the nucleic acid content via hybridization with an array of nucleic acid probes, the function of which is provided by a reliable, easy-to-manufacture boiling start valve. The boiling start valve utilizes the inherent positive characteristics of the microfluidic device technology and avoids the disadvantages of such techniques. GVA016.1 This aspect of the invention provides a microfluidic valve that includes: an inlet for receiving a flow of liquid; • 232-201219115 an outlet for outputting the flow of liquid; a bend between the inlet and the outlet a level anchor 'to stop fluid flowing from the inlet to the outlet at the meniscus anchor to form a meniscus; and a valve heater for heating the meniscus to make the meniscus The face anchor releases the meniscus to return the fluid to the outlet. GVA016.2 Preferably, the meniscus anchor is a hole and the valve

The heater extends near the edge of the hole. GVA01 6.3 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to release a meniscus from the meniscus anchor GVA 016.4. Preferably, the microfluidic valve There is also a single flow path from the inlet to the outlet. GVA016.5 Preferably, the microfluidic valve also has: a channel formed in the support substrate for at least partially defining the flow path; a CMOS circuit between the channel and the support substrate for operation Controlling the activation valve; and at least one sensor responsive to the flow of liquid, wherein the at least one sensor provides feedback to the CMOS circuit for operational control of the heater. GVA 016.6 Preferably, the microfluidic valve also has a top layer enclosing the passage, wherein the bore is formed in the top layer and the heater is supported by a top layer other than the passage. -233-201219115 GVA016.7 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a location in the channel. GVA 016.8 Preferably, the top layer has a thickness of less than 5 microns. GVA 016.9 Preferably, the heater has a thickness of less than 4 microns. GVA016.10 Preferably, the channel is defined by the upper cover channel

Covered by the cover, the upper cover channel defines a flow path downstream of the hole such that the meniscus is released to fill the upper cover channel until the capillary drive flow resumes to the exit. GVA016.il preferably, the passageway is covered by an upper cover defining a top cover passage, the upper cover passage defining an upstream flow path immediately adjacent the aperture such that release of the meniscus starts from the passage to the upper cover passage toward the The outlet flow GVA016.12 preferably has a cross-sectional area perpendicular to the flow direction of the channel of less than 100,000 square microns.

GVA016.13 Preferably, the channel has a cross-sectional area perpendicular to the fluid of less than 1 6,000 square microns. GVA 016.14 Preferably, the channel has a cross-sectional area perpendicular to the fluid of less than 2,500 square microns. GVA016.15 Preferably, the channel has a cross-sectional area perpendicular to the fluid of from 1 square micron to 400 square micrometers. GVA016.16 Preferably, the heater material is titanium nitride. The boiling start valve provides a microfluidic valve reliably and manufacturably, utilizing the inherent positive characteristics of the microfluidic device technology and avoiding the disadvantages of such techniques. -234- 201219115 GHU0 01.1 This aspect of the invention provides a microfluidic device for analyzing a fluid sample containing a target molecule, the microfluidic device comprising: a support substrate; on the support substrate for processing the fluid sample a microsystem technology (MST) layer having a probe array configured to bind to the target molecule to form a probe-target complex;

A humidifier is configured to heat water to increase local humidity in the area containing the MST layer. GHU00 1.2 Preferably, the humidifier has a water reservoir and an evaporator to expose water supplied by the water reservoir to a region containing the MST layer and to increase the water vapor pressure of the region. GHU00 1.3 Preferably, the evaporator has a bore configured to retain water using a meniscus formed in the bore, and the evaporator also has a heater adjacent the bore for raising at the bore The temperature of the water. GHU001.4 Preferably, the heater is annular and located in the hole

GHU001.5 Preferably, the evaporator has a supply passage from the reservoir to the bore, the supply passage being configured to draw water to the orifice by capillary action. GHU001.6 Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters. GHU001.7 Preferably, the microfluidic device also has an upper cover covering the MST layer, the upper cover having a plurality of fluid connections between the upper cover and the MST layer for fluid to flow from the MST layer to the upper cover and the fluid From the upper -235 - 201219115 cover flows to the MST layer. GHU001.8 Preferably, the water reservoir is formed on an upper cover, the supply passage is formed in the MST layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole is formed in the upper cover The supply channel is connected to the aperture via the other of the liquid connections. GHU001.9 Preferably, the upper cover has a plurality of reagent reservoirs of different reagents. GHU001.10 Preferably, the microfluidic device also has a cover channel for fluid flow, at least some of the reservoirs being in fluid communication with the upper cover channel via a corresponding surface tension valve, the surface tension valves being configured to The reagent is retained by the surface tension of the meniscus of the reagent until the fluid in the upper cover passage reaches the surface tension valve. GHU001.il Preferably, the surface tension valves respectively have an MST channel in the MST layer, a fluid connection to the reagent reservoir, and another fluid connection to the upper cover channel to cause the bend The liquid level is formed at a fluid connection that is connected to the upper cover passage. GHU 001.12 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of MST channels and a plurality of corresponding fluid connections to the upper cover. GHU001.13 Preferably, the upper cover channel and the receptacles are formed in a layer of a single material. GHU001.14 Preferably, the upper cover channel is formed on a surface of the single layer such that an outer surface of the MST layer closes the upper cover channel. GHU001. 1 5 Preferably, the upper cover has an outer surface of -236-201219115 opposite the surface, the outer surface having an inlet for receiving fluid and feeding the fluid to the upper cover passage. GHU001.16 Preferably, the fluid sample is a biological sample, and at least one of the fluid connectors is an array of holes that are sized and configured to filter out cells larger than the valve. GHU001.17 Preferably the upper cover has a waste receptacle for collecting cells larger than the valve.

GHU 001.18 Preferably, the microfluidic device also has a dialysis section, wherein the biological sample is blood, and the array of holes is part of the dialysis section to remove red blood cells from the fluid sample at the time of use. GHU001.19 Preferably, the microfluidic device also has a nucleic acid amplification portion, wherein the target molecular marker nucleic acid sequence, and the nucleic acid amplification portion is configured to amplify the target nucleic acid sequence in the fluid sample GHU001.20 Preferably, the nucleic acid amplification unit is in the MST layer, one of the reagent reservoirs is a dNTP, a primer and a buffer reservoir, and the other reagent reservoir is a polymerase reservoir. The dNTP, primer and buffer reservoir and the polymerase reservoir are directly coupled to the nucleic acid amplification portion by at least one fluid link configured to form a meniscus for respectively The dNTP, primer and buffer and the polymerase are retained. The mass-produced and inexpensive microfluidic device processes and/or analyzes the fluid, which utilizes an integrated humidifier to prevent any dehumidification of the fluid that can interfere with any aspect of the treatment or analysis. GHU002.1 This aspect of the invention provides a microfluidic test module for analyzing genetic material, the test module comprising: -237-201219115 a housing configured for hand movement; a probe array located in the housing The probe is configured to hybridize to a target nucleic acid sequence to form a probe-target hybrid; wherein the shell has a membrane seal for reducing dewetting within the shell and providing a pressure release when the small gas pressure changes. GHU002.2 Preferably, the microfluidic test module also has a humidifier to increase the humidity in the housing, wherein the probe array is attached to a microfluidic device mounted in the housing, the microfluidic device having a pass A flow path to the probe and a reagent for processing the genetic material, the flow path configured to fluidly connect the genetic material to the reagent and the probe. GHU002.3 Preferably, the microfluidic device has a humidity sensor for sensing humidity within the housing, and the humidity sensor produces an output for controlling the humidifier. GHU002.4 Preferably, the microfluidic device has a support substrate, a microsystem technology (MS T) layer configured to process the genetic material, and a CMOS circuit between the support substrate and the MST layer, The MST layer contains the flow path and the probe array, the CMOS circuit being coupled to the humidity sensor and configured for operational control of the humidifier. GHU002.5 Preferably, the microfluidic device has a nucleic acid amplification portion for amplifying a nucleic acid sequence in the genetic material, and the reagent comprises the following one or more: polymerase, restriction enzyme, dNTP in buffer And primers, lysis reagents and anticoagulants. GHU002.6 Preferably, the humidifier has a water reservoir and an evaporator - 238-201219115, the evaporator having a hole configured to retain water using a meniscus formed in the hole, and the evaporator There is also a heater adjacent the aperture for increasing the temperature of the water at the aperture. GHU002.7 Preferably, the heater is annular and located adjacent the aperture. GHU002.8 Preferably, the evaporator has a self-storage device connected to the

A supply passage for the bore, the supply passage being configured to draw water to the orifice by capillary action. GHU002.9 Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters. GHU002.10 Preferably, the microfluidic test module also has an upper cover covering the MST layer, the upper cover having a plurality of fluid connections between the upper cover and the MST layer for fluid to flow from the MST layer to the upper cover And fluid flows from the upper cover to the MST layer. GHU002.il Preferably, the water reservoir is formed in an upper cover formed in the MS T layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole is attached to the MST The layer is formed such that the supply channel is connected to the aperture via the other of the liquid connections. GHU002.1 2 Preferably, the upper cover has a plurality of reagent reservoirs for processing the reagents of the genetic material. GHU002.1 3 Preferably, the microfluidic test module also has a cover channel for fluid flow, at least some of the reagent reservoirs are in fluid communication with the upper cover channel via a corresponding surface tension valve, the surface tension valve system The reagent is configured to retain the reagent by the surface tension of the reagent meniscus until the fluid in the upper-239-201219115 cover channel reaches the surface tension valve. GHU002.1 4 Preferably, the surface tension valves respectively have an MST channel in the MST layer, a fluid connection to the reagent reservoir, and another fluid connection to the upper cover channel to enable The meniscus is formed at a fluid connection connected to the upper cover channel. GHU002.1 5 Preferably, the surface tension valve for one of the reagent reservoirs and the surface tension valve for the other of the reagent reservoirs have a different number of MST channels and are connected to the upper cover Fluid connection. GHU002.16 Preferably, the microfluidic device has a probe array for hybridization with a target nucleic acid sequence amplified via the nucleic acid amplification portion. GHU002.1 7 Preferably, the microfluidic test module also has an array of photodiodes for sensing the hybridization of any probe in the array, GHU002.1. Preferably, the microfluid The test module also has an excitation source for illuminating the array of probes. GHU002.1 9 Preferably, the CMOS circuit has an array of photodiodes and is configured to generate a signal indicating that the probe has hybridized. GHU002.20 Preferably, the microfluidic test module also has The electrical connector is for transmitting signals from the CMOS circuit to an external device. The easy-to-use, mass-produced, inexpensive, and portable microfluidic test module processes and/or analyzes fluids that utilize membrane seals to prevent any dehumidification of the fluid that can interfere with any aspect of the treatment or analysis. . GHU0 03.1 This aspect of the invention provides a test module for analyzing genetic material, the test module comprising: 240 - 201219115 configured for hand-held moving shell; probe array located in the shell, the probe The needle system is configured to hybridize to the target nucleic acid sequence to form a probe-target hybrid; and a humidifier for increasing the humidity within the shell. GHU003.2 preferably 'the probe array is attached to a microfluidic device mounted within the housing, the microfluidic device having a flow path to the probe and reagents for processing the genetic material, the flow path System configured for fluids

The genetic material is linked to the reagent and the probe. GHU003.3 Preferably the microfluidic device has a humidity sensor for sensing humidity within the housing, and the humidity sensor produces an output for controlling the humidifier. GHU003.4 Preferably, the microfluidic device has a support substrate,

a microsystem technology (MS T) layer configured to process the genetic material, and a CMOS circuit between the support substrate and the MST layer, the MST layer including the flow path and the probe array, the CMOS circuit A humidity sensor is coupled to the humidity sensor and configured to operate to control the humidifier. GHU003.5 Preferably, the microfluidic device has a nucleic acid amplification portion for amplifying a nucleic acid sequence in the genetic material, and the reagent comprises one or more of the following: a polymerase, a restriction enzyme, in a buffer dNTPs and primers, lysis reagents and anticoagulants. GHU003.6 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having a hole configured to retain water using a meniscus formed in the hole, and the evaporator is also adjacent to the The heater of the hole is used to increase the temperature of the water at the hole for -241 - 201219115. GHU003.7 Preferably, the heater is annular and located adjacent the aperture. GHU003.8 Preferably, the evaporator has a supply passage from the reservoir to the orifice, the supply passage being configured to draw water to the orifice by capillary action. GHU003.9 Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters. GHU003.1 0 Preferably, the test module also has an upper cover covering the MST layer, the upper cover having a plurality of fluid connections between the upper cover and the MS T layer for fluid to flow from the MST layer to the upper cover And fluid flows from the upper cover to the MST layer. GHU003.il Preferably, the water reservoir is formed on an upper cover formed in the MST layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole is attached to the MST The layer is formed such that the supply channel is connected to the aperture via the other of the liquid connections. GHU003.1 2 Preferably, the upper cover has a plurality of reagent receptacles for processing the reagents of the genetic material. GHU003.1 3 Preferably, the test module also has a cover channel for fluid flow, at least some of the reagent reservoirs are in fluid communication with the upper cover channel via corresponding surface tension valves, the surface tension valves are configured The reagent is retained by the surface tension of the meniscus of the reagent until the fluid in the upper cover passage reaches the surface tension valve. GHU003.14 Preferably, the surface tension valves respectively have an MST channel in the -242-201219115 MST layer, a fluid connection to the reagent reservoir, and another fluid connection to the upper cover channel So that the meniscus is formed at the fluid connection to the upper cover channel. GHU003.1 5 Preferably, the surface tension valve for one of the reagent reservoirs has a different number of MST channels and is attached to the upper surface of the surface tension valve for the other of the reagent reservoirs Fluid connection.

GHU003.1 6 Preferably, the microfluidic device has a probe array for hybridization to a target nucleic acid sequence amplified by the nucleic acid amplification portion. GHU003.1 7 Preferably, the test module also has an array of photodiodes for sensing the hybridization of any of the probes in the array. GHU003.1 8 Preferably, the test module also has an excitation source for illuminating the array of probes. GHU003.1 9 Preferably, the CMOS circuit has an array of photodiodes and is configured to produce a signal indicating that the probe has hybridized. GHU003.20 Preferably, the test module also has an electrical connector for transmitting signals from the CMOS circuit to an external device. The mass-produced, inexpensive, and portable genetic test module accepts a biological sample to analyze the nucleic acid content thereof, which utilizes an integrated humidifier to prevent any dehumidification of the fluid that can interfere with any aspect of the genetic analysis. . GHU004.1 This aspect of the invention provides a test module for processing a genetic material in a biological sample, the test module comprising: a shell; a microfluidic device mounted in the shell, the microfluidic device having a reagent For treating the genetic material; -243-201219115 a humidifier for increasing the humidity in the shell; a humidity sensor for sensing the humidity in the shell; wherein the humidity sensor is generated for controlling the The output of the humidifier. GHU004.2 Preferably, the humidity sensor and the humidifier are formed in the microfluidic device. GHU004.3 Preferably, the microfluidic device has a support substrate, a microsystem technology (MST) layer configured to process the genetic material, and a CMOS circuit between the support substrate and the MST layer, the CMOS A circuit is coupled to the humidity sensor and configured to operate to control the humidifier. GHU004.4 Preferably, the microfluidic device has a nucleic acid amplification portion for amplifying a nucleic acid sequence in the genetic material, and the reagent comprises one or more of the following: a polymerase, a restriction enzyme, a dNTP in a buffer And an introduction, lysis reagent and anticoagulant" GHU004.5 Preferably, the humidifier has a water reservoir and an evaporator GHU004.6. Preferably, the evaporator has a hole, the hole being configured to be utilized The meniscus formed by the aperture retains water and the evaporator also has a heater adjacent the aperture for increasing the temperature of the water at the aperture. GHU004.7 Preferably, the heater is annular and located adjacent the aperture. GHU004.8 Preferably, the evaporator has a supply passage from the reservoir to the orifice, the supply passage being configured to draw -244-201219115 water to the orifice by capillary action. GHU004.9 Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters.

GHU004.1 0 Preferably, the microfluidic device has an upper cover covering the MST layer, the upper cover having a plurality of fluid connections for fluidly connecting the upper cover to the MST layer.

Preferably, the water reservoir is formed in an upper cover formed in the MST layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole is formed in the upper cover The supply channel is connected to the aperture via the other of the liquid connections. GHU004.12 Preferably, the upper cover has a plurality of reagent reservoirs for processing the reagents of the genetic material. GHU004.1 3 Preferably, the upper cover has a cover passage for fluid flow, and at least some of the reagent reservoirs are in fluid communication with the upper cover passage via a corresponding surface tension valve, the surface tension valves being configured to The surface tension of the meniscus of the reagent retains the reagent until the fluid in the upper cap passage reaches the surface tension valve and the meniscus is removed. GHU004.1 4 Preferably, the surface tension valves respectively have an MST channel in the MST layer, a fluid connection to the reagent reservoir, and another fluid connection to the upper cover channel such that The meniscus is formed at a fluid connection connected to the upper cover channel. GHU004.1 5 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of MST channels and a plurality of corresponding fluid connections to the upper cover. -245-201219115 GHU004.16 Preferably, the microfluidic device has an array of probes for hybridizing with a target nucleic acid sequence in an amplicon from the nucleic acid amplification portion to cause the probe to bind to the target nucleic acid sequence Hybridization forms a probe-target hybrid. GHU004.1 7 Preferably, the probe-target hybrid emits a fluorescent signal in response to the excitation light, and the microfluidic device has an array of photodiodes for sensing the hybrid from the probe-target Fluorescent signal. GHU004.1 8 Preferably, the test module also has an excitation source for illuminating the array of probes. GHU004.19 Preferably, the CMOS circuit has an array of photodiodes to produce a signal indicating that the probe has hybridized. GHU004.20 Preferably, the test module also has an electrical connector for transmitting signals from the CMOS circuit to an external device. The mass-produced, inexpensive, and portable genetic test module accepts a biological sample to analyze the nucleic acid content thereof, which utilizes an integrated humidifier to prevent any dehumidification of the fluid that can interfere with any aspect of the genetic analysis. The humidifier is preferably controlled via a feedback control system. GHU006.1 This aspect of the invention provides a humidity sensor comprising: a pair of electrodes that are close to each other and define an air gap therebetween, such that the electrodes provide a capacitor with air as the dielectric material; And a circuit for sensing a capacitance connected to the electrodes to cause a change in capacitance due to a change in the dielectric constant of the air in the air gap due to a change in humidity: wherein -246 - 201219115 the circuit should be equal to the electrode The capacitance is sensed to produce a signal indicative of the humidity in the air. GHU006.2 Preferably, the electrodes each have a comb-like configuration with their respective teeth extending toward each other and staggered with each other to provide the air gap with a curved shape. GHU006.3 Preferably, the electrodes are defined in a layer of electrically conductive material supported by the substrate and etched by photolithography to provide the comb structures.

GHU006.4 Preferably, the electrically conductive material is titanium nitride and the substrate is on a planar surface on a LOC (lab on wafer) device. GHU006.5 Preferably, the circuit is a CMOS circuit on the LOC device. The easy to manufacture humidity sensor is used in a closed loop humidity control system to prevent any dehumidification of the fluid that can interfere with any aspect of the fluid treatment or analysis. GHU007.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a microsystem technology (MST) layer for processing a fluid sample on the support substrate; and for sensing the inclusion of the MST Humidity sensor for humidity in the region of the layer; wherein the humidity sensor produces an output for controlling the humidity of the region containing the MS T layer. GHU007.2 Preferably, the microfluidic device also has a humidifier, -247-201219115, which is configured to heat water to increase the local humidity of the region containing the MST layer. GHU007.3 Preferably, the microfluidic device also has reagents for treating a target nucleic acid sequence in the fluid sample, wherein the MS T layer has a hybridization configured to hybridize to the target nucleic acid sequence to form a probe-target hybrid. The probe array of the body. GHU007.4 Preferably, the microfluidic device also has a CMOS circuit between the support substrate and the MST layer, the CMOS circuit configured to receive an output signal from the humidity sensor to control the increase Wet device. GHU007.5 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having a bore configured to retain water using a meniscus formed in the bore, and the evaporator is also proximate to the A heater for the hole is used to increase the temperature of the water at the hole. GHU007.6 Preferably, the heater is annular and located adjacent the aperture. GHU007.7 Preferably, the evaporator has a supply passage from the reservoir to the bore, the supply passage being configured to draw water to the orifice by capillary action. GHU007.8 Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters. GHU007.9 Preferably, the microfluidic device also has an upper cover covering the MS T layer, the upper cover having a plurality of fluid connections for fluidly connecting the upper cover to the MST layer. -248-201219115 GHU007.10 Preferably, the water reservoir is formed in an upper cover formed in the MST layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole system An upper cover is formed to connect the supply passage to the hole via the other of the liquid connections. GHU007.il Preferably, the upper cap has a plurality of reagent reservoirs for processing the reagents of the target nucleic acid sequence.

GHU007.12 Preferably, the microfluidic device also has a cover channel for fluid flow, at least some of the reagent reservoirs are in fluid communication with the upper cover channel via a corresponding surface tension valve, the surface tension valves being configured The reagent is retained by the surface tension of the meniscus of the reagent until the fluid in the upper lid passage removes the meniscus. GHU007.1 3 Preferably, the surface tension valves respectively have an MST channel in the MST layer, a fluid connection to the reagent reservoir, and another fluid connection to the upper cover channel such that The meniscus is formed at a fluid connection connected to the upper cover channel. GHU007.14 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of MST channels and a fluid connection member GHU007.1 connected to the upper cover. Preferably, the reagents comprise one or the following Multiple: Polymerase, restriction enzymes, dNTPs and primers in buffer, lysis reagents and anticoagulants. GHU007.1 6 Preferably, the microfluidic device also has an array of photodiodes positioned in registration with each of the probes, wherein, in use, the probes emit fluorescent signals in response to the excitation light to Hybridization to the target nucleic acid-249-201219115 sequence is shown, the photodiode systems being configured to sense hybridization of any of the probe arrays. GHU007.1 7 Preferably, the probes are fluorescent resonance energy transfer probes, each of which has a fluorophore and a quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds. GHU007.1 8 Preferably, the photodiode is less than 249 microns from the array of probes. GHU007.1 9 Preferably, the CMOS circuit has an array of photodiodes and is configured to produce a signal indicating that the probe has hybridized. GHU007.20 Preferably, the microfluidic device also has a pad for communicating with an external device. The mass-produced and inexpensive microfluidic device processes and/or analyzes fluids that utilize an integrated humidifier to prevent any dehumidification of the fluid that can interfere with any aspect of the treatment or analysis, the humidifier being optimal It is controlled by a feedback control system using the humidity sensor. GHU008.1 This aspect of the invention provides a test module for analyzing a sample containing a nucleic acid sequence, the test module comprising: a housing configured for hand movement; an array of probes located in the housing The needle system is configured to hybridize to a target nucleic acid sequence in the sample to form a probe-target hybrid; a humidity sensor for sensing humidity within the shell; wherein the humidity sensor produces an output for the Humidity control inside the shell. GHU008.2 Preferably, the test module also has a humidifier configured to heat water to wet the interior of the housing. -250-201219115 GHU008.3 Preferably, the test module also has a microfluidic device mounted in the housing, wherein the microfluidic device has the probe array, reagents for processing the target nucleic acid sequence, and A flow path for fluidly connecting the sample to the reagent and the probe. GHU008.4 Preferably, the test module is also configured to be used

A microsystem technology (MST) layer for processing the target nucleic acid sequence, and a cover over the MST layer, the upper cover having a plurality of fluid connections for fluidly connecting the upper cover to the MST layer. GHU008.5 Preferably, the upper cap has a plurality of reagent reservoirs for processing the reagents of the target nucleic acid sequence. GHU008.6 Preferably, the microfluidic device has a support substrate and a CMOS circuit between the support substrate and the MST layer, the CMOS circuit being coupled to the humidity sensor and configured for operational control of the increase wet

6. The test module test module of claim 5, wherein the microfluidic device has a nucleic acid amplification portion for amplifying a target nucleic acid sequence in the sample, and the reagent comprises one or more of the following: Enzymes, restriction enzymes, dNTPs and primers in buffers, lysis reagents and anticoagulants. GHU008.7 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having a bore configured to retain water using a meniscus formed in the bore, and the evaporator also has a proximity A heater for the hole is used to increase the temperature of the water at the hole. GHU008.8 Preferably, the heater is annular and located near the aperture -251 - 201219115. GHU008.9 Preferably, the evaporator has a supply passage from the reservoir to the bore, the supply passage being configured to draw water to the orifice by capillary action. GHU008.1 0 Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters.

GHU008.il Preferably, the water reservoir is formed on an upper cover formed in the MST layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole is formed in the upper cover So that the supply channel is connected to the hole via the other of the liquid connections. GHU008.1 2 Preferably, the test module also has a cover channel for fluid flow, at least some of the reagent reservoirs are in fluid communication with the upper cover channel via a corresponding surface tension valve, the surface tension valves are configured The reagent is retained by the surface tension of the meniscus of the reagent until the fluid in the upper lid passage removes the meniscus.

GHU008.1 3 Preferably, the surface tension valves respectively have an MST channel in the MST layer, a fluid connection to the reagent reservoir, and another fluid connection to the upper cover channel such that The meniscus is formed at a fluid connection connected to the upper cover channel. GHU008.1 4 Preferably, the surface tension valve for one of the reagent reservoirs and the surface tension valve for the other of the reagent reservoirs have a different number of MST channels and are connected to the upper cover Fluid connection. GHU008.1 5 Preferably, the probes emit a fluorescent signal in response to the excitation light to indicate hybridization with the target nucleic acid sequence, and the CMOS circuit has -252-201219115 for sensing any hybridization in the probe array. An array of photodiodes. GHU008.16 Preferably, the CMOS circuit is configured to initiate the light sensor after a delay after the excitation light is extinguished. GHU008.1 7 Preferably, the test module also has an excitation source for illuminating the array of probes.

GHU008.1 8 Preferably, the probes are fluorescent resonance energy transfer probes, each having a fluorophore and a quencher, each having a fluorescence lifetime greater than 100 nanoseconds. GHU 008.1 9 Preferably, the CMOS circuit has an array of photodiodes and is configured to produce a signal indicating that the probe has hybridized. GHU008.20 Preferably, the test module also has an electrical connector for transmitting signals from the CMOS circuit to an external device. The mass-produced, inexpensive, and portable genetic test module accepts a biological sample to analyze the nucleic acid content thereof, which utilizes an integrated humidifier to prevent any dehumidification of the fluid that can interfere with any aspect of the genetic analysis. The φ humidifier is preferably controlled via a feedback control system utilizing the humidity sensor. GDI001.1 This aspect of the invention provides a microfluidic device for dialysis of biological material, the microfluidic device comprising: a sample inlet for receiving a sample of biological material having components of different sizes; a dialysis portion of a small component and a larger component, the smaller component being less than a predetermined size valve, and the larger component being greater than the predetermined size valve; and for separately processing the smaller component and/or the comparison Large component microsystem - 253 - 201219115 Technology (MST) layer. GDI001.2 Preferably, the microfluidic device also has a first channel and a second channel and a plurality of holes sized to correspond to a predetermined size, wherein the first and second channels are in fluid communication via the plurality of holes, And the inlet is in fluid communication with the first passage such that only the smaller component flows to the second passage. GDI001.3 Preferably, the first and second channels are configured to

The sample accepted at the inlet is filled by capillary action. GDI001.4 Preferably, the smaller component comprises a target, such that the processing of the smaller component by the MST layer comprises the detection of the target GDI001.5, preferably the target cell of the target, and the The MST layer has a lysing portion attached to the target channel for lysing the labeled cells to release the target nucleic acid sequence therein. GDI001.6 preferably, the labeled nucleic acid sequence of the target and

The MST layer has a nucleic acid amplification section for amplifying the target nucleic acid sequence. GDI001.7 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridization with the target nucleic acid sequence amplified by the nucleic acid amplification portion. GDI001.8 Preferably, the probes are configured to form a probe-target hybrid with the target nucleic acid sequence, the probe-target hybrid emitting fluorescence in response to the excitation light. GDI001.9 Preferably, the microfluidic device also has a CMOS circuit for operationally controlling the nucleic acid amplification portion, the CMOS circuit also having a photo-254-201219115 sensor for sensing hybridization from the probe-target Fluorescent emission of the body. GDI001.10 Preferably, the hybridization portion has an array of hybridization chambers containing probes for hybridization with the target nucleic acid sequence. GDI001.il Preferably, the light sensor is adjacent to each of the hybrids A photodiode array of chambers.

GDI 001.12 Preferably, the CMOS circuit has digital memory for storing data relating to the processing of the fluid, the data including the probe details and the location of each of the probes in the array of hybrid chambers. GDI001.13 Preferably, the CMOS circuit has at least one temperature sensor for sensing the temperature of the hybridization chamber array. GDI001.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuit using feedback from the temperature sensor for maintaining the hybridization temperature of the probe and the target nucleic acid sequence. GDI001.15 Preferably, the photodiode is less than 249 microns from the corresponding hybridization chamber.

GDI001.1 6 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GDI001.17 Preferably, the hybridization chamber has a light window positioned to expose the FRET probe to the excitation light. GDI001.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured such that when the FRET probe has formed a probe-target hybrid, the fluorophore is returned The light should be excited to emit a fluorescent signal to the photodiode, the CMOS circuit being configured to activate the photodiode after a predetermined delay after the excitation light is extinguished, the digital memory -255 - 201219115 including the pre- Programming delay. GDI001.19 Preferably, the CMOS circuit has a pad for electrically connecting to an external device, and is configured to convert an output from the photodiode to display a signal that the FRET probe hybridizes to the target nucleic acid sequence And providing the signal to the pad for transmission to the external device. GDI001.20 Preferably, the microfluidic device also has a plurality of reservoirs for containing liquid reagents for addition to the sample. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section to separate cells of different sizes, and separately processes the nucleic acid contents of the cells separated according to their size. The dialysis function extracts additional information from the sample and increases the sensitivity, signal to noise ratio, and dynamic range of the detection system. Integrating the dialysis system into the device provides a low number of system components and a simple manufacturing process, resulting in an inexpensive inspection system. GPC001.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a sample of a biological material having a nucleic acid sequence; a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence The PCR portion has at least one elongate PCR chamber having a longitudinal length that is much larger than the lateral dimension; and at least one elongated heater element for heating the nucleic acid sequence within the elongate PCR chamber; wherein the elongated heater The elements extend parallel to the longitudinal length of the PCR chamber. - 256 - 201219115 GPC001.2 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel. GPC001.3 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GPC001.4 Preferably, the PCR portion has a plurality of elongated PCR chambers

And the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GPC001.5 preferably has a plurality of elongated heaters along each of the channel portions of each of the wide bends. GPC001.6 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GPC001.7 Preferably, each of the plurality of elongated heaters is independently operable. GPC001.8 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongated heater. GPC001.9 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. GPC00 1.10 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater, which is used for The liquid is boiled to release the meniscus from the meniscus anchor to recover the capillary drive and flow out of the -257-201219115 PCRg [5. GPC001.1 1 Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole. GPC001.12 Preferably, the elongate heater is placed over the top layer defining the top of the microchannel. GPC001.13 Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, the dialysis portion configured to concentrate a sample of a cell smaller than a predetermined valve to a portion of the sample system The treatment is performed separately from the remaining sample containing only cells larger than the predetermined valve 〇GPC001.14 Preferably, the nucleic acid 'sequence is from cells smaller than the predetermined valve. Preferably, at least one of the channel portions has a liquid sensor at a proximal end, the liquid sensor being configured to detect liquid at the liquid detector location to feedback control the heater. GPC001.16 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve provided with a hole configured to cause the reagent to form a meniscus, thus The meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir 〇GPC001.17. Preferably, the reservoir has a vent to As an air inlet when the reagent flows out of the reagent reservoir. GPC001.18 Preferably, the microfluidic device also has a hybridization section, -258-201219115, the hybridization section has a probe array for hybridization with a target nucleic acid sequence in the sample; and for detecting a probe array A sensor array of needle hybridization. GPC001.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds. GPC001.20 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds.

The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR chamber is in the form of an elongated geometry that provides rapid temperature cycling of the mixture and advances the mixture by capillary action. This rapid temperature cycling capability increases the speed of this detection. This capillary action advancement simplifies the design of the test system, further improving the reliability of the test system and reducing its cost. GPC002.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a sample inlet for accepting a sample of biological material having a nucleic acid sequence; a polymerase chain reaction for amplifying the nucleic acid sequence ( a PCR unit having a PCR chamber; and a heater element for heating the nucleic acid sequence in the PCR chamber; wherein the PCR chamber is between the heater element and the support substrate ^ -259 - 201219115 GPC002 .2 Preferably, the PCR portion has a microchannel containing a pCr inlet and a PCR outlet, and the PCR chamber is part of the microchannel. GPC002.3 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GPC002.4 Preferably, the PCR portion has a plurality of PCR chambers, and the microchannels have a curved configuration formed by a series of wide curves, each of the wide curve channel portions forming one of the PCR chambers By. GPC002.5 preferably has a plurality of such heater elements in each of the channel portions. GPC002.6 Preferably, the plurality of heater elements are elongated, the positions of which are aligned with the longitudinal axis of the channel portion and are disposed end-to-end along the channel portion 〇GPC002.7. Preferably, each of the plurality of lengths The heater elements are independently operable. GPC002.8 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heater element. GPC002.9 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. GPC002.10 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater, The liquid is boiled to release the bend-260-201219115 level from the meniscus anchor to recover the capillary drive and exit the PCR section. GPC002.il Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole. GPC002.1 2 Preferably, the elongate heater is supported by a top layer enclosing the microchannel.

GPC002.1 3 Preferably, the microfluidic device also has a dialysis portion in which the biological material comprises cells of different sizes, the dialysis portion being configured to separate a sample of a cell larger than a predetermined valve to a portion of the sample. GPC002.14 is treated separately from the remaining sample comprising only cells smaller than the predetermined valve. Preferably, the nucleic acid sequence is from cells smaller than the predetermined valve. GPC002.1 5 Preferably, at least one of the channel portions has a liquid sensor near the end, the liquid sensor configured to detect liquid at the liquid detector position to feedback control the heater element. GPC002.1 6 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve having a bore configured to cause the reagent to form a meniscus, The meniscus thus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir GPC002.1. Preferably, the reservoir has a vent. As an air inlet when the reagent flows out of the reagent reservoir. -261 - 201219115 GPC002.1 8 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridization with a target nucleic acid sequence in the sample; and for detecting a probe array Sensor array for probe hybridization. GPC002.1 9 Preferably, the PCR section has a thermal cycle time of less than 4 seconds. GPC002.20 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The heater of the PCR chamber is located above the chamber to maximize heat transfer to the PC R mixture and minimize heat transfer to the substrate. GPC003.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a sample of a biological material having a nucleic acid sequence; a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence The PCR portion has a microchannel and a plurality of elongated heaters configured to have a plurality of mutually parallel, adjacent channel portions, the elongated heaters being end to end along each of the channel portions Mode configuration; wherein each of the heaters is independently operable to control the heat flux intensity from the two-dimensional direction to the PCR portion. GPC003.2 Preferably, the microchannel is configured in a curved configuration having a series of wide bends to form the plurality of mutually parallel, adjacent channel portions of -262-201219115. GPC003.3 Preferably, the microfluidic device also has a support substrate and a CMOS circuit, wherein the CMOS circuit is interposed between the support substrate and the PCR portion for controlling the heaters. GPC003.4 Preferably, the microfluidic device also has at least one sensor, wherein the CMOS circuit uses the sensor to feedback control the heaters.

GPC003.5 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor, wherein the CMOS circuit is responsive to the liquid sensor to control the initial startup of the heater, and the temperature is returned The sensor controls the heater in a subsequent manner. GPC003.6 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence through all of the channel portions by capillary action. GPC003.7 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. GPC003.8 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PC R portion, and the boiling start valve also has a valve heater It is used to boil the liquid to release the meniscus from the meniscus anchor to recover the capillary drive and exit the PCR section. GPC003.9 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. - 263 - 201219115 GPC003.1 0 Preferably, the elongate heater is placed over the top layer defining the top of the microchannel. GPC003.il Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, the dialysis portion being configured to separate a sample of a cell smaller than a predetermined valve to a portion of the sample system The treatment is performed separately from the remaining sample containing cells larger than the predetermined valve.

GPC003.12 Preferably, the nucleic acid sequence is from a cell that is less than the predetermined valve. GPC003.1 3 Preferably, each of the channel portions has a cross-sectional area of from 1 square micrometer to 400 square micrometers. GPC003.1 4 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for use with the CR; and a surface tension valve having a bore configured to cause the reagent to form a meniscus , so the meniscus retains the reagent in the reagent reservoir until

The fluid sample is removed upon contact and the reagent exits the reagent reservoir. 〇 GPC003.1 5 Preferably, the reservoir has a vent to serve as an air inlet when the reagent exits the reagent reservoir. GPC003.1 6 Preferably, the microfluidic device also has a probe array for hybridizing with the target nucleic acid sequence in the sample to form a probe-target hybrid, and for detecting the probe array A light sensor of a needle-target hybrid. GPC003.1 7 Preferably, the CMOS circuit is equipped with the light sensor -264 - 201219115 GPC003.1 8 Preferably, the light sensor is registered with the photodiode array of the probe array. GPC003.1 9 Preferably, the PCR section has a thermal cycle time of less than 4 seconds. GPC003.20 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts

A sample of the nucleic acid is then used to amplify the nucleic acid target in the sample using the PCR chamber of the device. The heat flux intensity entering the PCR chamber is controlled by a two-dimensional orientation which results in a highly uniform two-dimensional temperature distribution that provides uniform amplification characteristics and enhanced amplification specificity. GPC004.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a biological material sample having a nucleic acid sequence

a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence, the PCR portion having at least one heater; and at least one sensor; wherein the at least one sensor is configured to provide feedback for feedback The output of at least one heater. GPC004.2 Preferably, the microfluidic device also has a CMOS circuit 'connected to the at least one heater and the at least one sensor for causing the CMOS circuit to use the feedback for controlling the at least one heater - 265- 201219115. GPC004.3 Preferably, the microfluidic device also has a plurality of the temperature sensors and a plurality of liquid sensors, wherein the PCR portion has a plurality of heaters and a plurality of the liquid sensors to enable the CMOS The circuit responds to the liquid sensor to control the initial activation of the heater and to respond to the temperature sensor to control the heater power. GPC004.4 Preferably, the PCR portion has a plurality of elongated PCR chambers, and each of the heaters is elongated and parallel to the longitudinal axis of the PCR chambers. GPC004.5 Preferably, the PCR portion has a PCR inlet. And a microchannel of the PCR outlet, and the elongated PCR chamber is part of the microchannel. GPC004.6 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GPC004.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of which forms a channel portion of the elongate PCR chamber. GPC004.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GPC004.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GPC004.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GPC004.il preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the primer-266-201219115, dNTP, nucleic acid polymerase and buffer mixture in the elongated heater. The liquid in the PCR portion is retained when the nucleic acid sequence is amplified. GPC004.1 2 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion. The boiling start valve also has a valve heater. It is used to boil the liquid to release the meniscus from the meniscus anchor to recover the capillary drive and exit the PCR section.

GPC004.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole. GPC004.1 4 Preferably, the CMOS circuit activates the valve heater after a predetermined number of thermal cycles. GPC004.1 5 Preferably, the microfluidic device also has a dialysis section in which the biological material comprises cells of different sizes, the dialysis section being configured to separate a sample larger than a predetermined valve to a portion of the sample, the sample of the portion Separate from remaining samples containing only cells smaller than the predetermined valve

GPC004.16 Preferably, the nucleic acid sequence is from a cell that is less than the predetermined valve. GPC004.17 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use, and a surface tension valve having a hole configured to cause the reagent to form a meniscus, The meniscus is allowed to retain the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent exits the reagent reservoir. GPC004.1 8 Preferably, the microfluidic device also has a probe array for hybridizing to the target nucleic acid sequence in the -267-201219115 sample to form a probe-target hybrid, and for detecting the probe A probe-targeted hybrid light sensor in the array. GPC004.1 9 Preferably, the PCR section has a thermal cycle time of less than 30 seconds. GPC004.20 Preferably, the PCR portion has a thermal cycle time of less than 11 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR section is controlled by a feedback control system which results in a faster and more accurate temperature cycle. Faster temperature cycling increases the speed of this test. A more accurate temperature cycling capability provides this enhanced amplification specificity. GPC005.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a sample of a biological material having a nucleic acid sequence; a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence The PC R portion has at least one heater; and at least one temperature sensor; wherein the at least one temperature sensor is configured to provide an output for feedback control of the at least one heater. GPC005.2 Preferably, the microfluidic device also has a CMOS circuit coupled to the at least one heater and the at least one temperature sensor, -268-201219115 to enable the CMOS circuit to use the feedback control for the at least one The output of the heater. GPC005.3 Preferably, the microfluidic device also has a plurality of the temperature sensor and a plurality of liquid sensors, wherein the PCR portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor for control The heater is initially activated and returned to the temperature sensor to control the heater power. GPC005.4 Preferably, the PCR portion has a plurality of elongated PCR chambers

The longitudinal length of each of the elongated PCR chambers is much larger than the lateral dimension of each of the heaters, and each of the heaters is elongated and parallel to the longitudinal length of the PCR chambers. GPC00 5.5 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel.

GPC005.6 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the pCR inlet to the PCR outlet by capillary action. GPC005.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of which forms a channel portion of the elongate PCR chamber. GPC005.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GPC005.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GPC005.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GPC005.il Preferably, the PCR portion is provided with a -269-201219115 active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of primer, dNTP, nucleic acid polymerase and buffer in the elongated heater. The liquid in the PCR portion is retained when the nucleic acid sequence is amplified. GPC005.1 2 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PC R portion, and the boiling start valve also has a valve heater. It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. GPC005.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole. GPC005.1 4 Preferably, the CMOS circuit activates the valve heater after a predetermined number of thermal cycles. GPC005.1 5 Preferably, the microfluidic device also has a dialysis section in which the biological material comprises cells of different sizes, the dialysis section being configured to separate a sample larger than a predetermined valve to a portion of the sample, the sample of the portion The treatment is performed separately from the remaining sample containing only cells smaller than the predetermined valve 〇GPC005.1. Preferably, the nucleic acid sequence is derived from cells smaller than the predetermined valve. GPC005.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve having a hole configured to cause the reagent to form a meniscus, The meniscus thus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir -270-201219115 GPC005.1 8 Preferably, the microfluid The device also has a hybridization portion having a probe array for hybridization with a target nucleic acid sequence in the sample; and a light sensor for detecting probe hybridization in the probe array. GPC005.1 9 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.

GPC005.20 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR section is controlled by a temperature feedback control system which results in a faster and more accurate temperature cycle. Faster temperature cycling increases the speed of this test. A more accurate temperature cycling capability provides this enhanced amplification specificity.

GPC006.1 which comprises: The aspect of the invention provides a microfluidic device, a sample inlet for receiving a polymerase chain reaction (PCR) portion of a sample of biological material having a nucleic acid sequence for amplifying the nucleic acid sequence, The PCR portion has at least one heater for heating the nucleic acid sequence to pass through a denatured phase, a binder phase, and an extension phase of the primer, wherein the PCR portion has a thermal cycle time of less than 30 seconds when used. GPC006.2 Preferably, the PCR portion has a thermal cycle of -271 - 201219115 ring time of less than 11 seconds. GPC006.3 Preferably, the PCR portion has a thermal cycle time of less than 4 seconds. GPC006.4 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds. GPC006.5 Preferably, the microfluidic device also has at least one sensor, and a CMOS circuit coupled to the at least one heater and the at least one sensor, such that the CMOS circuit is used from the at least one sensing The output of the device controls the at least one heater with feedback. GPC006.6 Preferably, the microfluidic device also has a plurality of liquid sensors, wherein the PCR portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor to control initial startup and return of the heater At least one temperature sensor should be used to control the heater power. GPC006.7 Preferably, the PCR portion has a plurality of elongate PCR chambers, and each of the heaters is elongate and parallel to the longitudinal axis of the PC R chambers. GPC006.8 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel. GPC006.9 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GPC006.1 0 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GPC006.il Preferably, the channel portion forming each of the equal width curves is provided. 272-201219115 has a plurality of elongated heaters. GPC006.12 Preferably, the plurality of elongate heaters are disposed end to end along the channel portion. GPC006.1 3 Preferably, each of the plurality of elongate heaters is independently operable. GPC006.14 Preferably, the PCR portion is provided at the PCR outlet

An active valve is used to retain the liquid in the PCR portion when the nucleic acid sequence and the mixture of primers, dNTPs, polymerase, and buffer are heated to circulate the nucleic acid sequence. GPC006.1 5 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater Used to boil the liquid to release the meniscus from the meniscus anchor to recover the capillary drive and flow out of the PCRg|5 ° GPC006.16 Preferably, the meniscus anchor is a hole, and the The valve heater is located adjacent the edge of the hole. GPC006.1 7 Preferably, the CMOS circuit activates the valve heater after a predetermined number of thermal cycles. GPC006.1 8 Preferably, the microfluidic device also has a probe array for hybridization with a target nucleic acid sequence amplified via the PCR portion to form a probe-target hybrid. GPC006.19 Preferably, the probe array has more than one probe and the PCR portion is configured to generate an amplicon sufficient to hybridize to all probes in less than 10 minutes. -273-201219115 GPC006.20 Preferably, the microfluidic device also has a photodiode array for detecting probe-target hybrids within the probe array. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR section has a short heating cycle time, thus increasing the speed of the detection. GPC007.1 This aspect of the invention provides a microfluidic device,

The method comprises: a support substrate; a sample inlet for receiving a biological material sample having a nucleic acid sequence; a microsystem technology (MST) layer having a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence The PCR portion has at least one heater for heating and circulating the nucleic acid sequence to pass through a denatured phase, a binder phase, and an extension phase of the primer;

A CMOS circuit interposed between the MST layer and the support substrate, the CMOS circuit being coupled to the at least one heater for operating to control the at least one heater during the heating cycle. GPC007.2 Preferably, the microfluidic device also has at least one sensor such that the CMOS circuit uses the output from the at least one sensor to feedback control the at least one heater. GPC007.3 Preferably, the microfluidic device also has a plurality of the temperature sensor and a plurality of liquid sensors, wherein the PCR portion has a plurality of heaters and a plurality of the liquid sensors to enable the CMOS circuit The liquid sensing -274 - 201219115 is used to control the initial startup of the heater and to respond to the temperature sensor to control the heater power. GPC007.4 Preferably, the PCR portion has a plurality of elongate PCR chambers, and each of the heaters is elongate and parallel to the longitudinal axis of the PCR chambers. GPC007.5 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel.

GPC007.6 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GPC007.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GPC007.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GPC007.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GPC007.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GPC007.1 1 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the primer, dNTP 'nucleic acid polymerase and buffer mixture in the elongated heater to amplify The nucleic acid sequence retains the liquid in the PCR portion. Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, -275-201219115, the boiling start valve also has a valve heater, It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. GPC007.13 Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole. GPC007.14 Preferably, the CMOS circuit activates the valve heater after a predetermined number of thermal cycles. GPC007.1 5 Preferably, the microfluidic device also has a dialysis portion in which the biological material comprises cells of different sizes, the dialysis portion being configured to separate cells larger than the predetermined valve and less than the predetermined valve Cell 〇GPC007.1 6 Preferably, the nucleic acid sequence is derived from cells smaller than the predetermined valve. GPC007.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use, and a surface tension valve having a hole configured to cause the reagent to form a meniscus, The meniscus is retained in the reagent reservoir until the fluid sample contacts, the meniscus is removed and the reagent exits the reagent reservoir. GPC007.1 8 Preferably, the microfluidic device also has a probe array for hybridizing with the target nucleic acid sequence in the sample to form a probe-target hybrid, and for detecting the probe-target hybrid Light sensor. GPC007.1 9 Preferably, the PCR section has a thermal cycle time of less than 30 seconds. GPC007.20 Preferably, the PCR portion has a thermal cycle of -276 to 201219115 ring time of less than 11 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR section is controlled by a semiconductor on-wafer control system which results in a faster and more accurate temperature cycling. Faster temperature cycling increases the speed of this test. More accurate temperature cycling capability provides this enhanced amplification specificity

GPC008.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a sample inlet for receiving a sample of biological material having a nucleic acid sequence; a microsystem technology (MST) layer having a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence, the PCR portion having at least one heater for heating the nucleic acid sequence to pass through a denatured phase, a binder phase, and an extension phase of the primer; and interposed between the MST layer And a CMOS circuit between the support substrate, the CMOS circuit configured to provide energy to the at least one heater using a pulse width modulation (PWM) signal during the heating cycle. GPC008.2 Preferably, the microfluidic device also has at least one sensor such that the CMOS circuit uses the output from the at least one sensor to feedback control the at least one heater. GPC008.3 Preferably, the PCR portion has a plurality of heaters and one temperature sensor to -277-201219115 and at least one liquid sensor' to cause the CMOS circuit to respond to at least one liquid sensor to control the The initial start of the heater and the return of at least one temperature sensor to control the heater power. GPC008.4 Preferably, the PCR portion has a plurality of elongated PCR chambers, each of the elongated PCR chambers having a longitudinal length that is much larger than the lateral dimension, and each of the heaters is elongated and parallel to the PCR chambers. The longitudinal length. GPC008.5 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PC R chamber is part of the microchannel. GPC008.6 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GPC008.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GPC008.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GPC008.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GPC008.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GPC008.il preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. -278- 201219115 GPC008.12 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has valve heating The device is used to boil the liquid to release the meniscus from the meniscus anchor to recover the capillary drive and flow out of the PCR portion. GPC008.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole.

GPC008.1 4 Preferably, the CMOS circuit activates the valve heater after a predetermined number of thermal cycles. GPC008.1 5 Preferably, the microfluidic device also has a dialysis section in which the biological material comprises cells of different sizes, the dialysis section being configured to separate a sample of a cell larger than a predetermined valve to a portion of the sample GPC008.1 is treated separately from the remaining sample containing only cells smaller than the predetermined valve. Preferably, the nucleic acid sequence is from cells smaller than the predetermined valve. GPC008.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve having a bore configured to cause the reagent to form a meniscus, The meniscus thus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir GPC008.1. Preferably, the microfluidic device also has hybridization. The hybridization portion has a probe array for hybridization with the target nucleic acid sequence -279-201219115 in the sample; and a light sensor for detecting probe hybridization in the probe array. GPC008.1 9 Preferably, during use, the PCR section has a heating cycle time determined by the CMOS circuit. GPC008.20 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 seconds.

The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid, and then uses the PCR chamber of the device to amplify the nucleic acid target in the sample. The PCR portion is controlled by a PWM control system, which results in a faster And a more accurate temperature cycle. The faster temperature cycling capability increases the speed of this test. A more accurate temperature cycling capability provides this enhanced amplification specificity. GPC009.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a biological material sample having a nucleic acid sequence

A PCR portion having a polymerase chain reaction (PCR) microchannel for heating a circulating sample to amplify the nucleic acid sequence, the PCR microchannel defining a flow path of the sample to cause the sample to follow the PCR micro The flow of the channel is driven by capillary action; and the cross section of the PCR microchannel perpendicular to the fluid is less than 100,000 square microns. GPC009.2 Preferably, the PCR microchannel has a cross-sectional area of the fluid that is less than 16,000 square microns. -280- 201219115 GPC009.3 Preferably, the PCR microchannel has a cross-sectional area of the fluid that is less than 2,500 square microns. ' GPC009.4 Preferably, the PCR microchannel has a cross-sectional area of the fluid from 1 square micron to 400 square micrometers perpendicularly. GPC009.5 Preferably, the microfluidic device also has at least one elongated heater element A nucleic acid sequence for heating in the elongate PCR microchannel; and the elongate heater element extends parallel to the PCR microchannel.

GPC009.6 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least a portion of the PCR microchannel forms an elongated PCR chamber. GPC009.7 Preferably, the PCR portion has a plurality of elongated PCR chambers respectively formed by respective portions of the PCR microchannels, the microchannels having a curved configuration formed by a series of wide curves, each of which is wide The curve forms a channel portion of one of the elongate PCR chambers. GPC009.8 Preferably, each of the channel portions has a plurality of the elongated heaters.

GPC009.9 Preferably, the track is configured in an end-to-end manner. GPC009.1 0 Preferably, the plurality of elongate heaters are independently operable along the plurality of elongate heater systems. GPC009.il preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. GPC009.1 2 Preferably, the active valve is provided with a meniscus anchor-281 - 201219115 boiling start valve for retaining liquid in the PCR portion. The boiling start valve also has a valve A heater is used to boil the liquid to release the meniscus from the meniscus anchor to restore capillary drive and flow out of the PCR section. GPC009.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located adjacent the edge of the hole. GPC009.1 4 Preferably, the elongate heater is supported by a wall defining one side of the microchannel. GPC009.1 5 Preferably, the microfluidic device also has a dialysis portion in which the biological material comprises cells of different sizes, the dialysis portion being configured to separate a sample larger than a predetermined valve to a portion of the sample, the sample of the portion The treatment is performed separately from the remaining sample containing only cells smaller than the predetermined valve. 〇GPC009.1 6 Preferably, the nucleic acid sequence is derived from cells smaller than the predetermined valve. GPC009.1 7 Preferably, at least one of the channel portions has a liquid sensor near the end, the liquid sensor configured to detect liquid at the liquid detector position to feedback control the heater ° GPC009.1 8 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR: and a surface tension valve provided with a hole configured to form a meniscus of the reagent Therefore, the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir - 282 - 201219115 GPC009.1 9 Preferably, the reservoir The vent has a vent as an air inlet when the reagent exits the reagent reservoir. GPC009.20 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridization with a target nucleic acid sequence in the sample; and for detecting probe hybridization light in the probe array sensor.

The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR chamber has a small cross-sectional area that provides rapid temperature cycling of the mixture and advances the mixture by capillary action. This fast temperature cycling capability increases the speed of this test. The capillary action advancement simplifies the design of the detection system, further improving the reliability of the detection system and reducing the cost thereof. GPC0 10.1 This aspect of the invention provides a microfluidic device,

a support substrate; a sample inlet for receiving a biological material sample having a nucleic acid sequence; a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence, the PCR portion having a PCR chamber: and for heating the PCR chamber a heater element of a nucleic acid sequence; wherein, when used, the heater element heats the nucleic acid sequence at a rate of more than 80K per second - 283 - 201219115 GPC010.2 Preferably, the heater element is used per second during use The nucleic acid sequence is heated at a rate of more than 100 K. GPC010.3 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 1000 K per second during use. GPC010.4 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 10,000 κ per second during use.

GPC010.5 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 100,000 K per second during use. GPC010.6 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 1000,000 K per second during use. GPC0 1 0.7 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 10,000,000 K per second during use. GPC0 1 0.8 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 20,000,000 K per second during use.

GPC010.9 Preferably, the heater element heats the nucleic acid sequence at a rate of more than 40,000,000 K per second during use. GPC 010.10 Preferably, during use, the heater element heats the nucleic acid sequence at a rate of more than 80,000,000 K per second. GPC010.il Preferably, the heater element heats the nucleic acid sequence at a rate of more than 160,000,000 K per second during use. GPC01 0.1 2 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the PCR chamber is part of the microchannel. GPC010.13 Preferably, the microchannel is configured to be by capillary -284-

201219115 acts to attract liquid containing the nucleic acid sequence from the PCR inlet to the outlet. GPC01 0.14 Preferably, the PCR portion has a plurality of PCR chambers. The microchannels have a curved configuration formed by a series of wide curves, each of the channel portions forming one of the PCR chambers. GPC 010. 15 Preferably, each of the channel portions has a plurality of the adding elements. GPC010.16 Preferably, the plurality of heater elements are longitudinally aligned with the longitudinal axis of the channel portion and along the channel portion in an end-to-end manner GPC010.17 preferably, each of the plurality of elongated heater elements Operate independently. GPC 010.18 Preferably, the microfluidic device also has at least a temperature sensor for feedback control of the elongate heater element. GPC010.19 Preferably, the PCR portion is actively operated at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of dNTP, polymerase and buffer in the elongated heater to amplify the nucleic acid sequence. The liquid in the PCR section. GPC010.20 Preferably, the active valve is provided with a boiling surface anchor boiling start valve for retaining liquid in the PCR portion. The boiling start valve also has a valve heater for boiling liquid The liquid level is released from the meniscus anchor to recover the capillary drive and flow to the PCR section. The easy-to-use, mass-produced and inexpensive microfluidic device is connected to the PCR, and the wide curved heat exchanger is configured to be in a holder with a primer, and the sample containing the nucleic acid containing -285-201219115 is bent out The nucleic acid target in the sample is then amplified using the PCR chamber of the device. The PCR section has a fast rate of temperature change that provides a short heating cycle time and improves overall detection speed. GPC01 1.1 This aspect of the invention provides a microfluidic device comprising: a support substrate; a sample inlet for accepting a sample of biological material having a nucleic acid sequence; a polymerase chain reaction for amplifying the nucleic acid sequence (PCR) a PCR chamber having a PCR chamber; and a heater element for heating the nucleic acid sequence in the PCR chamber; wherein, when used, the PCR portion generates an amplification sufficient to hybridize to the probe array in less than 10 minutes child. GPC011.2 Preferably, when used, the PCR moiety produces an amplicon sufficient to hybridize to the probe array in less than 260 seconds. GPC01 1.3 Preferably, when used, the PCR moiety produces an amplicon sufficient to hybridize to the probe array in less than 80 seconds. GPC011.4 Preferably, when used, the PCR moiety produces an amplicon sufficient to hybridize to the probe array in less than 30 seconds. GPC01 1.5 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the PCR chamber is part of the microchannel. GPC011.6 Preferably, the microchannel is configured to be capillary

The action draws a liquid containing the nucleic acid sequence from the PCR inlet to the PCR -286-201219115 outlet. GPC011.7 Preferably, the PCR portion has a plurality of PCR chambers, and the microchannels have a curved configuration formed by a series of wide curves, each of the wide curve system channel portions forming one of the PCR chambers By. GPC011.8 Preferably, each of the channel portions has a plurality of the heater elements.

GPC011.9 preferably 'the plurality of heater elements are elongated, the position being aligned with the longitudinal axis of the channel portion and the GPC01 1.10 being disposed end to end along the channel portion. Preferably, each of the plurality of elongated heating The components are operable independently. GPC011.il Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongated heater element. GPC01 1.12 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid sequence. The liquid in the PCR section is retained. GPC011.13 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used for retaining liquid in the PCR portion. The boiling start valve also has a valve heater. Boiling the liquid to release the meniscus from the meniscus anchor to recover the capillary drive and exit the PCR section. GPC01 1 .1 4 Preferably, the meniscus anchor is bored and The valve heater is located adjacent the edge of the hole. -287- 201219115 GPC011.15 Preferably, the elongated heater is embedded in a top layer enclosing the microchannel. GPC011.16 Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, the dialysis portion configured to separate a sample larger than a predetermined valve cell to a portion of the sample system of the portion Separate from remaining samples containing only cells smaller than the predetermined valve

GPC01 1.17 Preferably, the nucleic acid sequence is derived from cells smaller than the predetermined valve. GPC011.18 Preferably, each of the channel portions has a liquid sensor proximate one end, the liquid sensor configured to detect liquid at the liquid detector position to feedback control the heater element. GPC011.19 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use;

Having a surface tension _ of the aperture configured to cause the reagent to form a meniscus, such that the meniscus retains the reagent within the reagent reservoir until the fluid sample contacts and removes the meniscus The reagent flows out of the reagent reservoir GPC01 1.20. Preferably, the microfluidic device also has a hybridization portion having the probe array for hybridization with a target nucleic acid sequence in the sample; and for detecting a probe A light sensor that hybridizes to the probe in the array. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid, and then uses the PCR chamber of the device to amplify the -288-201219115 nucleic acid target in the sample. The PCR section is capable of rapid nucleic acid amplification and improves the overall detection speed. GPC012.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a sample of biological material having a nucleic acid sequence;

a PCR portion having a polymerase chain reaction (PCR) microchannel for heating a PCR mixture of the sample, a polymerase, a dNTP, and a buffer solution to amplify the nucleic acid sequence, the PCR microchannel defining a flow path of the sample The flow of the sample along the PCR microchannel is driven by capillary action; wherein the volume of the PCR mixture is less than 400 nanoliters. GPC012.2 Preferably, the PCR mixture has a volume of less than 170 nanoliters. GPC012.3 Preferably, the volume of the PCR mixture is less than 70%

GPC012.4 Preferably, the PCR mixture has a volume of less than 30 nanoliters. GPC012.5 Preferably, the PCR microchannel has a cross-sectional area of the fluid ranging from 400 square microns to 1 square micrometer. GPC012.6 Preferably, the microfluidic device also has at least one elongated heater element for heating the nucleic acid sequence within the elongate PCR microchannel; and the elongate heater element extends parallel to the PCR microchannel . -289- 201219115 GPC012.7 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least a portion of the PCR microchannel forms an elongated PCR chamber. GPC012.8 Preferably, the PCR portion has a plurality of elongated PCR chambers respectively formed by respective portions of the PCR microchannels. The microchannels have a curved configuration formed by a series of wide curves. The curve forms a channel portion of one of the elongate PCR chambers. GPC012.9 Preferably, each of the channel portions has a plurality of the elongated heaters. GPC0 1 2.1 0 Preferably, the plurality of elongated heating agents are disposed in an end-to-end manner along the channel portion. GPC012.il Preferably, each of the plurality of elongated heaters is independently operable. GPC012.12 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heater. GPC012.13 Preferably, the PCR portion has an active valve at the PCR outlet for retaining the liquid in the PCR portion as the elongated heater heats the PCR mixture. GPC012.14 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, and the anchor is used for retaining liquid in the PCR portion. The boiling start valve also has a valve heater. The liquid is boiled to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. GPC012.15 Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, the dialysis portion is configured with -290-201219115 to separate a sample larger than a predetermined valve to a portion of the sample, A portion of the sample is processed separately from the remaining sample comprising only cells smaller than the predetermined valve. GPC012.16 Preferably, the nucleic acid sequence is from a cell that is less than the predetermined valve.

GPC012.17 Preferably, at least one of the channel portions has a liquid sensor at a proximal end, the liquid sensor configured to detect liquid at the liquid detector location to feedback control the heater. GPC012.18 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve provided with a hole configured to cause the reagent to form a meniscus, thus The meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir GPC012.19. Preferably, the microfluidic device also has a hybridization portion. The hybridization portion has a probe array for hybridization with a target nucleic acid sequence in the sample; and a light sensor for detecting probe hybridization in the probe array. GPC012.20 Preferably, the PCR portion has a thermal cycle time of less than 30 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts a sample containing nucleic acid and then amplifies the nucleic acid target in the sample using the PCR chamber of the device. The PCR section provides for capillary action to advance the PCR mixture, which simplifies the design of the detection system, further improving the reliability of the detection system and reducing its cost. The microfluidic device uses a small amount of PCR mixture volume, which provides rapid nucleic acid amplification, increases overall detection speed and also provides low reagent cost. This low reagent cost in turn allows for complete storage of reagents on the wafer, which reduces the number of components and assembly complexity of the inspection system, further reducing the cost of the inspection system. GPC014.1 This aspect of the invention provides a test module for amplifying a nucleic acid sequence in a sample fluid, the test module comprising: a housing provided with a container for receiving the sample fluid: having a polymerase chain reaction ( PCR) A PCR portion of a microchannel for heating a PCR mixture of the sample fluid, polymerase, dNTP, primer, and buffer solution to amplify the nucleic acid sequence; wherein the PCR mixture has a volume of less than 400 nanoliters. GPC014.2 Preferably, the PCR mixture has a volume of less than 170 nanoliters. GPC014.3 Preferably, the PCR mixture has a volume of less than 70 nanoliters. GPC014.4 Preferably, the PCR mixture has a volume of less than 30 nanoliters. GPC014.5 Preferably, the PCR microchannel has a cross-sectional area of the fluid ranging from 400 square microns to 1 square micrometer. GPC014.6 Preferably, the test module also has at least one elongated heater element for heating the nucleic acid sequence in the elongate PCR microchannel; and -292-201219115 the elongated heater element parallel to the PCR Microchannel extension. GPC014.7 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least a portion of the PCR microchannel forms an elongated PCR chamber. GPC014.8 Preferably, the PCR portion has a plurality of elongated PCR chambers respectively formed by respective portions of the PCR microchannels, the microchannels having a curved configuration formed by a series of wide curves, each of which is wide The curve forms a channel portion of one of the elongate PCR chambers.

GPC014.9 Preferably, each of the channel portions has a plurality of the elongated heaters. GPC01 4.1 0 Preferably, the track is configured in an end-to-end manner. GPC014.1 1 Preferably, the plurality of elongate heaters are independently operable along the plurality of elongate heater systems. GPC014.12 Preferably, the test module also has at least one temperature sensor for feedback control of the elongated heater.

GPC014.13 Preferably, the PCR portion has an active valve at the PCR outlet for retaining the liquid in the PCR portion as the elongated heater heats the PCR mixture. GPC0 14.14 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater, which is used for The liquid is boiled to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. GPC014.15 Preferably, the test module also has a dialysis section, wherein the biological material of -293-201219115 comprises cells of different sizes, the dialysis section being configured to separate a sample of cells larger than a predetermined valve to a part of the sample, The portion of the sample is treated separately from the remaining sample containing only cells smaller than the predetermined valve. GPC014.16 Preferably, the nucleic acid sequence is derived from a cell that is less than the predetermined valve. GPC014.17 Preferably, at least one of the channel portions has a liquid sensor at a proximal end, the liquid sensor configured to detect liquid at the liquid detector location to feedback control the heater. GPC014.18 Preferably, the test module also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve provided with a hole configured to cause the reagent to form a meniscus, thus The meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir 〇GPC0 14.19. Preferably, the test module also has a hybridization portion. The hybridization portion has a probe array for hybridization with a target nucleic acid sequence in the sample: and a light sensor for detecting probe hybridization in the probe array. GPC014.20 Preferably, the PCR portion has a thermal cycle time of less than 30 seconds. The easy-to-use, mass-produced, inexpensive, and portable genetic test module accepts a sample containing nucleic acid, and then uses the PCR portion of the module to amplify the nucleic acid target in the sample. The PCR section provides for capillary action to advance the PCR mixture, which simplifies the design of the detection system, further improving the reliability and reducing the cost of the test system. The module uses a small amount of PCR mixture volume, which provides rapid nucleic acid amplification, improves overall detection speed and also provides low reagent costs. This low reagent cost in turn allows for complete storage of the reagents on the wafer, which reduces the number of components and assembly complexity of the inspection system, further reducing the cost of the inspection system. GPC017.1 This aspect of the invention provides a method for genetic analysis

A wafer-on-lab (LOC) device containing a sample of a target nucleic acid sequence, the LOC device comprising: a sample inlet for receiving the sample: a plurality of reagent reservoirs containing a buffer for addition to the sample, dNTPs, arches, and polymerase chain reaction (PCR), which are used to heat cycle the sample, buffer, dNTPs, primers, and polymerase to amplify the target nucleic acid sequence. GPC017.2 Preferably, the PCR portion has a plurality of elongate PCR chambers, each chamber having at least one elongated shape extending parallel to the longitudinal axis of the PCR chambers

Heater. GPC017.3 Preferably, the PCR portion has a microchannel comprising a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel. GPC017.4 Preferably, the PCR portion has a plurality of elongate PCR chambers, and the microchannels have a curved configuration formed by a series of wide curves, each of the wide curves forming in the elongate PCR chamber One of the channel departments. GPC 017.5 Preferably, the LOC device also has a CMOS circuit coupled to the at least one heater for operating the at least one heater during the heating cycle. - 295 - 201219115 GPC017.6 Preferably, each of the channel portions along each of the wide bends has a plurality of elongated heaters" GPC017.7. Preferably, the plurality of elongated heaters are along the channel The department is configured in an end-to-end manner. GPC017.8 Preferably, the elongate heaters are independently operable. GPC017.9 Preferably, the LOC device also has at least one temperature sensor coupled to the CMOS circuit for feedback control of the elongated heater. GPC 017.10 Preferably, the PCR portion has an active valve for retaining the liquid in the PCR portion when the elongated heater heats the PCR mixture to amplify the nucleic acid sequence. GPC017.il Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater, The liquid is boiled to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. GPC 017.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor configured to detect liquid at the liquid detector location to feedback control the valve heater. GPC017.13 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GPC017.14 Preferably, the LOC device also has a probe array for hybridizing to the target nucleic acid sequence to form a probe-target hybrid. -296-201219115 GPC017.15 Preferably, the LOC device also has a hybrid light sensor for detecting the probe-target hybrid" GPC 017.16 Preferably, the light sensor is registered with the probe A photodiode array of needle arrays. GPC017.17 Preferably, the liquid in the PCR portion has a volume of less than 400 nanoliters.

Preferably, the PCR microchannel has a cross-sectional area of the fluid ranging from 400 square microns to 1 square micrometer. GPC017.19 Preferably, the PCR portion has a thermal cycle time of less than 30 seconds. GPC017.20 Preferably, the liquid in the PCR portion has a volume of less than 30 nanoliters. This LOC device has the benefit of less complex design and manufacturing requirements and will therefore result in a simpler, more reliable manufacturing. This LOC device has the benefit of fewer chemical steps during operation, thus resulting in a simpler, more reliable operation. The design of this LOC device will increase the speed of sample analysis. This LOC device has the benefits provided by sequence-specific amplification, including: sensitivity provided by amplification, broad dynamic range, and high specificity for the target DNA sequence. GPC018.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetically analyzing a sample containing a target nucleic acid sequence, the LOC device comprising: a sample inlet for accepting the sample; a reservoir containing a buffer, dNTP, primer, and polymerase for addition to the sample to form a mixture of amplification -297-201219115; and a nucleic acid amplification portion for use in constant temperature amplification of the target nucleic acid sequence The temperature of the amplification mixture is controlled during the period. Preferably, the nucleic acid amplification portion has a plurality of elongate expansion chambers' each chamber having at least one elongate heater extending parallel to the longitudinal axis of the amplification chambers. GPC018.3 Preferably, the nucleic acid amplification portion has an amplification inlet

And amplifying the microchannels of the outlet, and the elongated amplification chamber is a portion of the microchannel GPC018.4. Preferably, the microchannel has a curved configuration formed by a series of wide curves. The tract system forms a channel portion of one of the elongate expansion chambers. GPC018.5 Preferably, the LOC device also has a CMOS circuit coupled to the at least one heater for operating the at least one heater during the heating cycle.

Preferably, GPC 018.6 has a plurality of elongated heaters along each of the channel portions of each of the wide bends. GPC018.7 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GPC018.8 Preferably, the elongate heaters are independently operable. GPC 018.9 Preferably, the LOC device also has at least one temperature sensor coupled to the CMOS circuit for feedback control of the elongated heater. -298-201219115 GPC018.10 Preferably, the nucleic acid amplification portion has an active valve for retaining in the nucleic acid amplification portion when the elongated heater controls the temperature of the amplification mixture to amplify the nucleic acid sequence liquid. GPC018.il Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the nucleic acid amplification portion, and the boiling start valve also has a valve heater. Used to boil the liquid to release the meniscus from the meniscus anchor to restore capillary drive and flow out

The nucleic acid amplification unit. GPC018.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor configured to detect liquid at the liquid detector position to feedback control the valve heater. GPC018.13 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GPC018.14 Preferably, the LOC device also has an array of probes for hybridizing to the target nucleic acid sequence to form a probe-target hybrid. GPC018.15 Preferably, the LOC device also has a light sensor for detecting hybridization of the probe-target hybrid. GPC018.16 Preferably, the light sensor is a photodiode array that registers the probe array. GPC018.17 Preferably, the liquid in the nucleic acid amplification portion has a volume of less than 400 nanoliters. Preferably, the amplified microchannel has a cross-sectional area of the fluid ranging from 40 to 2 square microns to 1 square micrometer. GPC018.19 Preferably, the liquid in the nucleic acid amplification section has a volume of from -299 to 201219115 in a volume of 30 nanoliters. GPC01 8.20 Preferably, the CMOS circuit has pads for communication with external devices. This LOC device has the benefit of less complex design and manufacturing requirements and will therefore result in a simpler, more reliable manufacturing. This LOC device has the benefit of fewer chemical steps during operation, thus resulting in a simpler, more reliable operation. The design of this LOC device will increase the speed of sample analysis. This can result in more sensitive and specific detection of the target DNA. This LOC device has the benefit of not requiring a heating cycle that simplifies the heating control electronics, allows for more uniform temperature control and reduces material degradation of the LOC device. The design of this LOC device will reduce the extent of evaporation during operation, allowing for improved control of the physical and chemical conditions within the LOC device. GPC019.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetically analyzing a whole blood sample containing a target nucleic acid sequence, the LOC device comprising: a sample inlet for receiving the whole blood sample a plurality of reagent reservoirs containing buffers, dNTPs, primers, and polymerases for addition to the whole blood sample; and a polymerase chain reaction (PCR) portion for heating and circulating the whole blood sample, buffering The liquid, dNTP, primer and polymerase are used to amplify the target nucleic acid sequence. GPC 019.2 Preferably, the PCR portion has a plurality of elongate PCR chambers, each chamber having at least one elongate heater extending parallel to the longitudinal axis of the PCR chambers. -300- 201219115 GPC01 9.3 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel. GPC019.4 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GPC019.5 Preferably, the LOC device also has at least one

A heater is connected to the CMOS circuit for operating the at least one heater during the heating cycle. Preferably, GPC 019.6 has a plurality of elongated heaters along each of the channel portions of each of the wide bends. GPC01 9.7 preferably 'the plurality of elongated heaters are disposed end to end along the channel portion. GPC019.8 Preferably, the elongate heaters are independently operable. GPC019.9 Preferably, the LOC device also has at least one temperature sensor coupled to the φ CMOS circuit for feedback control of the elongate heater. GPC019.10 Preferably, the PCR portion has an active valve for retaining the liquid in the PCR portion when the elongated heater heats the PCR mixture to amplify the nucleic acid sequence. GPC019.il Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater, The liquid is boiled to release the meniscus from the meniscus anchor to recover the capillary drive and flow out of the -301 - 201219115 PCR section. GPC019.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor configured to detect liquid at the liquid detector location to feedback control the valve heater. GPC019.13 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GPC019.14 Preferably, the LOC device also has an array of probes for hybridizing to the target nucleic acid sequence to form a probe-target hybrid. GPC019.15 Preferably, the LOC device also has a light sensor for detecting the probe-target hybrid. GPC0 19.16 Preferably, the light sensor is a photodiode array that registers the probe array. GPC019.17 Preferably, the liquid in the PCR portion has a volume of less than 400 nanoliters. GPC019.18 Preferably, the PCR microchannel has a cross-sectional area of the fluid ranging from 400 square microns to 1 square micrometer. GPC019.19 Preferably, the PCR portion has a thermal cycle time of less than 30 seconds. GPC019.20 Preferably, the liquid in the PCR portion has a volume of less than 30 nanoliters. The L0C device is designed to optimize the benefits of processing components in blood samples, including white blood cells, red blood cells, pathogens, and soluble materials. This LOC device has the benefit of less complex design and manufacturing requirements and will result in a simpler, more reliable manufacturing. This LOC device has the benefit of fewer chemical steps than -302-201219115 during operation, thus resulting in a simpler, more reliable operation. The design of this LOC device will increase the speed of sample analysis. This LOC device has the benefit of sequence-specific amplification, including: sensitivity provided by amplification, broad dynamic range, and high specificity for the target DNA sequence.

GPC023.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetically analyzing a sample containing a target nucleic acid sequence, the LOC device comprising: a sample inlet for accepting the sample; a reagent reservoir containing a buffer for adding to the sample to form an amplification mixture, dNTPs, primers, and a polymerase derived from thermophiles; and a nucleic acid amplification unit for use in the nucleic acid sequence of the target Controlling the temperature of the amplification mixture during amplification" GPC023.2 Preferably, the polymerase is derived from a DNA polymerase of Thermus aquaticus. GPC023.3 Preferably, the polymerase is derived from DN A polymerase of Thermoco ecus litorali s. GPC023.4 Preferably, the polymerase is derived from a DNA polymerase of Pyrococcus furiosus. GPC023.5 Preferably, the polymerase is derived from a DNA polymerase of Pyrococcus abyssi. GPC023.6 Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCH) portion, and the amplification mixture is subjected to a heating cycle in the PCR portion to amplify the nucleic acid sequence. -303-201219115 GPC023.7 Preferably, the PCR portion has a plurality of elongate PCR chambers, each chamber having at least one elongate heater extending parallel to the longitudinal axis of the PCR chambers. GPC023.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagent therein, the surface tension valve having a meniscus anchor for forming a meniscus of the reagent until the sample The meniscus is removed upon contact with the fluid to allow the reagent to flow out of the reagent reservoir and mix with the sample. GPC023.9 Preferably, the PCR portion has a microchannel containing a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel. GPC023.1 0 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate PCR chambers. GPC023.il Preferably, the microfluidic device also has a CMOS circuit coupled to the at least one heater for operating the at least one heater during the heating cycle. GPC 023.1 2 Preferably, a plurality of elongated heaters are disposed end to end along the channel portion, and the heaters are independently operable. GPC023.1 3 Preferably, the LOC device also has a support substrate, wherein the CMOS circuit is interposed between the micro channel and the support substrate, and the CMOS circuit has at least one temperature sensor for feedback control Long heater. GPC023.1 4 Preferably, the PCR portion has an active valve for retaining the liquid in the PCR portion when the elongated heater controls the temperature of the amplification mixture to amplify the nucleic acid sequence. -304- 201219115 GPC023.1 5 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used for retaining liquid in the nucleic acid amplification portion, and the boiling start valve is also A valve heater is provided for boiling the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the nucleic acid amplification section. GPC023.1 6 Preferably, the LOC device also has the active

A liquid sensor downstream of the valve, the liquid sensor configured to detect liquid at the liquid detector location to feedback control the valve heater. GPC023.1 7 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GPC023.1 8 Preferably, the LOC device also has a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid, and a light sensor for detecting the probe-target hybrid. GPC023.1 9 Preferably, the amplifying microchannel has a cross-sectional area of the fluid ranging from 400 square microns to 1 square micrometer. GPC023.20 Preferably, the light sensor is a photodiode array that registers the probe array. This LOC device has the benefit of less complex design and manufacturing requirements, thus resulting in a simpler, more reliable manufacturing process. . This LOC device has the benefit of fewer chemical steps during operation, thus resulting in a simpler, more reliable operation. The design of this LOC device will increase the speed of sample analysis. This enzyme amplifies the target DNA more rapidly than other polymerases (higher processing efficiency) GLY003.1 This aspect of the invention provides a microfluidic device for dissolving cells in fluids -305-201219115, The microfluidic device comprises: a support substrate; an inlet for receiving a fluid containing cells; a lysis portion in fluid communication with the inlet, the lysis portion having at least one heater for heating the fluid; and a reservoir containing the lysis reagent And a CMOS circuit interposed between the support substrate and the lysis unit, the CMOS circuit configured to selectively dissolve the cells by thermal lysis in the lysis unit, and utilize the lysis reagent The cells are chemically dissolved, or the cells are chemically and thermally dissolved using both the lysis unit and the lysis reagent. GLY003.2 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence released from the cells, the PCR portion having at least one PCR heating coupled to the CMOS circuit And at least one sensor; wherein the CMOS circuit is configured to feedback control the at least one PCR heater using the sensor. GLY003.3 Preferably, the microfluidic device also has a temperature sensor and a liquid sensor, wherein the PCR portion has a plurality of heaters and the CMOS circuit is configured to respond to the liquid sensor to control the The initial start of the heater and the response to the temperature sensor to control the heater power. GLY003.4 Preferably, the PCR portion has a plurality of elongated PCR chambers having longitudinal lengths that are much larger than their lateral dimensions, and each of the heaters is elongated and parallel to the PCR The longitudinal length of the room. -306-201219115 GLY003.5 Preferably, the PCR portion has a microchannel provided with a PCR inlet and a PCR outlet, and the elongated PCR chamber is part of the microchannel. GLY003.6 Preferably, the microchannel is configured to attract liquid containing the nucleic acid sequence from the PCR inlet to the PCR outlet by capillary action. GLY003.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming in the elongate PCR chamber

One of the channel departments. GLY003.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GLY003.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GLY003.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GLY003.il preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. GLY003.12 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used for retaining liquid in the PCR portion, and the boiling start valve also has a valve heater. The liquid is boiled to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. GLY003.1 3 Preferably, the meniscus anchor is a hole and the valve -307 - 201219115 heater is located adjacent the edge of the hole. GLY003.14 Preferably, the CMOS circuit activates the valve heater after a predetermined number of thermal cycles. GLY003.1 5 Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, the dialysis portion being configured to separate a sample larger than a predetermined valve to a portion of the sample, the sample of the portion Separate from remaining samples containing only cells smaller than the predetermined valve

GLY003.1 6 Preferably, the nucleic acid sequence is derived from cells smaller than the predetermined valve. GLY003.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for PCR use; and a surface tension valve having a bore configured to cause the reagent to form a meniscus, The meniscus thus retains the reagent in the reagent reservoir until

The fluid sample is removed from contact with the meniscus and the reagent flows out of the reagent reservoir 〇GLY003.1. Preferably, the microfluidic device also has a hybridization portion having an array of probes for use with the sample. The target nucleic acid sequences are hybridized to form a probe-target hybrid; and a photodiode array for detecting probe-target hybrids in the probe array. GLY003.1 9 Preferably, the PCR section has a thermal cycle time of less than 4 seconds. GLY003.20 Preferably, the PCR portion has a thermal cycle time of between 0.45 seconds and 1.5 - 308 - 201219115 seconds. The easy-to-use, mass-produced, inexpensive microfluidic device accepts a biochemical sample that utilizes chemical and thermal lysis subunits to solubilize cells or cell organs and process the lysate.

The lysis process is performed by cell extraction analysis and diagnostics in the sample, and subsequent processing and analysis of the targets are provided. Integrating the lysis subunit into the device provides a simple detection step, a low number of system components, and a simple system manufacturing process, resulting in an inexpensive detection system. The choice of chemical or thermal lysis treatment simplifies the need for detection chemistry and provides the ability to handle a wide range of sample types. GLY006.1 This aspect of the invention provides a test module for dissolving cells in a fluid, the test module comprising: a housing provided with a container for receiving a fluid lysing reagent reservoir containing cells a lysing reagent and having a valve; and a lysing portion in fluid communication with the container for accommodating the fluid during lysis of the cells, the lysing portion having a lysis heater for heating in the lysis portion a fluid; wherein the valve is located upstream of the lysis portion and configured to open when the fluid flows into the lysis portion, thereby adding the lysis reagent to the fluid. GLY006.2 Preferably, the valve is provided with a surface tension valve of a bore configured to form a meniscus to retain the lysis reagent therein until the meniscus is removed upon contact of the fluid, The lysis reagent is added to the fluid stream to enter the lysis section. -309-201219115 GLY 006.3 Preferably, the lysis portion has a lysis microchannel configured to attract fluid from the container by capillary action, the lysis portion having an active valve located at a downstream end of the lysis microchannel For retaining the fluid during dissolution of the cells. GLY006.4 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used for retaining fluid in the lysis unit, and the boiling start valve also has a valve heater. It is used to boil the liquid to release the meniscus from the meniscus anchor to recover the capillary drive and exit the lysate. GLY006.5 Preferably, the lytic microchannel has a cross-sectional area of the fluid ranging from 8 square microns to 20,000 square microns. GLY006.6 Preferably, the test module also has a support substrate, wherein the lysis unit and the lysis reagent reservoir are supported by the support substrate and configured as a on-wafer laboratory (LOC) device. GLY006.7 Preferably, the test module also has a CMOS circuit between the support substrate and the lysis unit, the CMOS circuit configured to operate a valve heater that controls an end point downstream of the microchannel And a lysis heater in the lysis unit. GLY006.8 Preferably, the test module also has a polymerase chain reaction (PC R) portion provided with at least one heater for heating the circulating fluid to amplify the nucleic acid sequence released from the cells . GLY006.9 Preferably, the test module also has at least one sensor, wherein the CMOS circuit is configured to use the sensor to feedback control the at least one PCR heater. -310- 201219115 GLY006.1 0 Preferably, the test module also has a plurality of heaters and at least one temperature sensor and at least one liquid sensor to cause the CMOS circuit to respond to at least one liquid sensor To control the initial activation of the heater and to return at least one temperature sensor to control the heater power GLY006.il Preferably, each of the plurality of heaters is independently operable.

GLY006.1 2 Preferably, the PCR portion is configured to have a PCR microchannel having the same cross section as the lysing microchannel, and the PCR portion also has an active valve located at a downstream end of the PCR microchannel to expand the nucleic acid sequence The increase period is used to retain the fluid. GLY006.1 3 Preferably, the test module also has a PCR mixture reservoir and a polymerase reservoir, wherein the PCR mixture reservoir comprises dNTPs, primers and a buffer solution and the polymerase reservoir comprises a polymerase" GLY006.1 Preferably, the test module also has an upper cover, the upper φ cover defines the lysis reagent reservoir, the PCR mixture reservoir and the polymerase reservoir, wherein the lysis unit and the PC R portion are interposed The reservoir is interposed between the reservoir and the support substrate. GLY006.1 5 Preferably, the test module also has a dialysis section, wherein the cells in the fluid are different in size, and the dialysis section is configured to separate a sample larger than a predetermined valve to a part of the sample, the sample of the part It is treated separately from the remaining sample containing only cells smaller than the predetermined valve. GLY006.1 6 Preferably, the nucleic acid sequence is from a cell that is less than the predetermined valve. -311 - 201219115 GLY006.1 7 Preferably, the test module also has an anticoagulant reservoir, wherein the flow system is a whole blood sample such that an anticoagulant is added to the whole blood upstream of the dialysis portion And the dialysis section is configured to concentrate white blood cells to a portion of the whole blood sample. GLY006.1 8 Preferably, the test module also has a hybridization portion downstream of the PCR portion, the hybridization portion having a probe array for hybridization with a predetermined target nucleic acid sequence in an amplicon from the PCR portion; and

A light sensor for detecting probe hybridization in a probe array. GLY006.1 9 Preferably, the probe array is for use in hybridizing to the target nucleic acid sequence to form a probe-target hybrid fluorescent probe, the probe-target hybridization system configured to be exposed to excitation light The photon of the light is emitted. GLY006.20 Preferably, the light sensor is positioned to respectively register the photodiode array of the fluorescent probe.

The easy-to-use, mass-produced, inexpensive, and portable genetic test module accepts biochemical samples that utilize chemical and thermal lysis subunits to lyse cells or cell cytometers and process the lysate. The cells in the sample are analyzed and diagnosed, and subsequent processing and analysis of the targets are provided. Integrating this lysis unit into the module provides a simple detection step and low detection system complexity, resulting in an inexpensive detection system. The choice of chemical or thermal lysis treatment simplifies the need for detection chemistry and provides the ability to handle a wide range of sample types. GIN001.1 This aspect of the invention provides a microfluidic device comprising: -312 - 201219115 support substrate; an inlet for receiving a fluid; a microsystem technology (MST) layer for treating the fluid, the MST layer a culture portion having at least one heater for maintaining a predetermined culture temperature of the fluid; and a CMOS circuit interposed between the MS T layer and the support substrate, the CMOS circuit and the at least one heater Connect for operational control

At least one heater. GIN001.2 Preferably, the microfluidic device also has at least one sensor, wherein the CMOS circuit uses the sensor to feedback control the at least one heater. GIN 001.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor, wherein the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor to control the The heater is initially activated and the temperature sensor is returned to subsequently control the heaters. GIN001.4 Preferably, the culture portion has a plurality of elongated chambers having a longitudinal length that is much larger than the lateral dimensions of the elongated chambers, and each of the heaters is elongated and parallel to the chambers Longitudinal length. GIN001.5 Preferably, the culture portion has microchannels provided with a culture inlet and a culture outlet, and the elongated culture chambers are part of the microchannels. GIN001.6 Preferably, the fluid sample is a biological material comprising a nucleic acid sequence, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action. -313-201219115 GIN001.7 Preferably, the microchannel has a winding configuration formed by a series of wide curves, each of which forms a channel portion of one of the elongated growth chambers. GIN001 -8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. G IN 0 0 1 · 9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GIN001.10 Preferably, each of the plurality of elongated heaters is independently operable. GIN001.il preferably, the culture portion has an active valve at the culture outlet for maintaining the mixture of the restriction enzyme and the nucleic acid sequence in the culture portion when the temperature suitable for the restriction of digestion is maintained by the elongated heater liquid. GIN001.12 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining fluid in the culture portion, and the boiling start valve also has a valve heater, It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the culture. GIN001.13 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN001.14 Preferably, the CMOS circuit activates the valve heater after a predetermined incubation time. GIN001.15 Preferably, the microfluidic device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes. The trans-314-201219115 is configured to concentrate cells smaller than a predetermined valve to For a portion of the sample, the portion of the sample is treated separately from the remaining sample containing only cells larger than the predetermined valve. GIN001.16 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by restriction enzymes in the culture section.

GIN001.17 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for use in the culture portion: and a surface tension valve having a bore configured to cause the reagent to form a meniscus Therefore, the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir GIN001.18. Preferably, the microfluidic device also has hybridization The hybridization portion has a probe array for hybridization with a target nucleic acid sequence in the sample; and a light sensor for detecting probe hybridization in the probe array. GIN001.19 Preferably, the microfluidic device also has a nucleic acid amplification unit. GIN001.20 Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion which has a thermal cycle time of from 0.45 seconds to 1.5 seconds. The easy-to-use, mass-produced, inexpensive microfluidic device accepts the fluid' and then uses the culture of the device to culture the fluid for a desired period of time at the desired temperature to effect the desired reaction. The microfluidic device is produced in large quantities and inexpensively using microsystem technology (MST). -315- 201219115 Integrating the culture into the device provides a simple step and low system complexity, which in turn provides an inexpensive system. GIN002.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a fluid sample; a culture portion for maintaining the fluid sample at a culture temperature, the culture portion having at least one heater ;and

At least one sensor: wherein the at least one sensor is configured to provide an output for feedback control of the at least one heater. GIN002.2 Preferably, the microfluidic device also has a CMOS circuit coupled to the at least one heater and the at least one sensor such that the C Μ OS circuit uses the sensor to feedback control the at least one heating Device

GIN002.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor, wherein the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor to control the The heater is initially activated and the temperature sensor is returned to subsequently control the heaters. GIN002.4 Preferably, the culture portion has a plurality of elongated chambers having a longitudinal length that is much larger than the lateral dimensions of the elongated chambers, and each of the heaters is elongated and parallel to the chambers Longitudinal length. GIN002.5 Preferably, the culture portion has a microchannel provided with a culture inlet and a culture outlet, and the elongated culture chamber is a portion of the microchannel. - 316 - 201219115 GIN002.6 Preferably, the fluid sample A biological material comprising a nucleic acid sequence, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action. GIN002.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate culture chambers. GIN002.8 Preferably, the channel portion of each of the equal width curves is formed

Having a plurality of elongated heaters GIN002.9 Preferably, the plurality of elongated heaters are disposed end to end along the passage portion. GIN002.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GIN002.il preferably, the culture portion has an active valve at the culture outlet for maintaining the mixture in the culture portion when the elongated heater maintains a mixture of the restriction enzyme and the nucleic acid sequence at a temperature suitable for restriction of digestion Liquid GIN002.1 2 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining fluid in the culture, the boiling start valve also has a valve heater It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the culture. GIN002.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN002.14 Preferably, the CMOS circuit activates the valve heater after a predetermined incubation time of -317 - 201219115. GIN002.1 5 Preferably, the microfluidic device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes, the dialysis portion configured to concentrate a sample of cells smaller than a predetermined valve to a portion of the sample, The portion of the sample is processed separately from the remaining sample containing only cells larger than the predetermined valve. GIN002.16 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by restriction enzymes in the culture section. GIN002.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing the reagent used in the culture portion; and a surface tension valve provided with a hole configured to cause the reagent to form a meniscus So that the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir 〇GIN002.1 8 preferably, the microfluidic device There is also a hybridization section having a probe array for hybridizing to the target nucleic acid sequence in the sample: and a light sensor for detecting probe hybridization in the probe array. GIN002.1 9 Preferably, the microfluidic device also has a nucleic acid amplification unit, GIN 002.20. Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit having a frequency of between 0.45 seconds and 1.5 seconds. Thermal cycle time. The easy-to-use, mass-produced, inexpensive microfluidic device accepts the fluid, and then the culture portion of the device is used to culture the fluid for a desired period of time from -318 to 201219115 to effect the desired reaction. The culture is controlled by a feedback control system which results in faster and more accurate temperature control. Faster temperature control improves overall processing speed. More accurate temperature cycling capabilities provide improved reaction conditions. Integrating the culture into the device provides a simple step and low system complexity, which in turn provides an inexpensive system. GIN003.1 This aspect of the invention provides a microfluidic device,

The method comprises: a sample inlet for receiving a fluid sample; a culture portion for maintaining the fluid sample at a culture temperature, the culture portion having at least one heater and at least one temperature sensor; wherein the at least one temperature sensing The device is configured to provide an output for feedback control of the at least one heater. GIN003.2 Preferably, the microfluidic device also has a CMOS circuit coupled to the at least one heater and the at least one temperature sensor, such that the CMOS circuit uses the temperature sensor to feedback control the at least one heating Device. GIN003.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor' wherein the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor for control The heaters are initially activated and are returned to the temperature sensor for subsequent control of the heaters. GIN003.4 Preferably, the culture portion has a plurality of elongate chambers having a longitudinal length that is much larger than their lateral dimensions 'and each of the plus-319-201219115 heat exchangers are elongated and parallel to The longitudinal length of the chambers. GIN003.5 Preferably, the culture portion has a microchannel provided with a culture inlet and a culture outlet, and the elongated culture chamber is a portion of the microchannel. GIN003.6 Preferably, the fluid sample contains a nucleic acid sequence Biomaterial, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action. GIN003.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate culture chambers. GIN003.8 Preferably, the channel portion forming each of the equal-width curves has a plurality of elongated heaters" GIN003.9. Preferably, the plurality of elongated heaters are end-to-end along the channel portion. Mode configuration. GIN003.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GIN003.il preferably, the culture portion has an active valve at the culture outlet for retaining the mixture in the culture portion when the elongated heater maintains a mixture of the restriction enzyme and the nucleic acid sequence at a temperature suitable for restriction of digestion liquid. GIN003.12 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining fluid in the culture, and the boiling start valve also has a valve heater, It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the culture. -320-201219115 GIN003.13 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN003.1 4 Preferably, the CMOS circuit activates the valve heater after a predetermined incubation time.

GIN003.1 5 Preferably, the microfluidic device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes, the dialysis portion being configured to concentrate a sample of cells smaller than a predetermined valve to a portion of the sample, The portion of the sample is processed separately from the remaining sample containing only cells larger than the predetermined valve. GIN003.1 6 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by restriction enzymes in the culture section. GIN003.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for use in the culture portion; and a surface tension valve having a hole configured to cause the reagent to form a bend a liquid level, such that the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir GIN003.1 8 preferably 'the microfluidic device There is also a hybridization section having a probe array for hybridization with a target nucleic acid sequence in the sample; and a light sensor for detecting probe hybridization in the probe array. GIN003.1 9 Preferably, the microfluidic device also has a nucleic acid amplification unit. GIN003.20 Preferably, the nucleic acid amplification portion is a polymerase chain reaction block (PCR) portion having a thermal cycle time of between 0.45 seconds and 1.5 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts the fluid, and then the culture portion of the device is used to culture the fluid at a desired temperature for a desired period of time to effect the desired reaction. The culture is controlled by a temperature feedback control system which results in faster and more accurate temperature control. Faster temperature control improves overall processing speed. More accurate temperature cycling capabilities provide improved reaction conditions. Integrating the culture into the device provides a simple step and low system complexity, which in turn provides an inexpensive system. GIN004.1 This aspect of the invention provides a on-wafer laboratory (LOC) device comprising: a support substrate: a sample inlet for receiving a fluid sample; a culture portion in fluid communication with the sample inlet, the culture portion Having at least one heater; and a CMOS circuit interposed between the support substrate and the culture portion; wherein the CMOS circuit is coupled to the at least one heater to maintain the fluid sample at a culture temperature for a culture time. GIN004.2 Preferably, the LOC device also has at least one temperature sensor, wherein the CMOS circuit uses the temperature sensor to feedback control the at least one heater. GIN004.3 Preferably, the LOC device also has at least one temperature sensor and at least one liquid sensor, wherein the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor to control the Wait for the initial start of the "-322- 201219115 heater and return to the temperature sensor for subsequent control of the heaters. GIN004.4 Preferably, the culture portion has a plurality of elongated chambers having a longitudinal length that is much larger than the lateral dimensions of the elongated chambers, and each of the heaters is elongated and parallel to the chambers Longitudinal length. GIN004.5 Preferably, the culture portion has a microchannel provided with a culture inlet and a culture outlet, and the elongated culture chamber is a portion of the microchannel.

GIN004.6 Preferably, the fluid sample is a biological material comprising a nucleic acid sequence, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action. GIN004.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate culture chambers. GIN004.8 Preferably, the channel portion forming each of the equal-width curves has a plurality of elongated heaters" GIN004.9. Preferably, the plurality of elongated heaters are end-to-end along the channel portion. Mode Configuration" GIN004.10 Preferably, each of the plurality of elongated heaters is independently operable. GIN004.1 1 Preferably, the culture portion has an active valve at the culture outlet for maintaining the mixture of the restriction enzyme and the nucleic acid sequence in the culture portion when the temperature suitable for the restriction of digestion is maintained by the elongated heater Liquid 0 GIN004.1 2 Preferably, the active valve is provided with a boiling surface anchor-323-201219115 boiling start valve for retaining fluid in the culture portion, the boiling start The valve also has a valve heater that is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the culture. GIN004.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN004.14 Preferably, the CMOS circuit activates the valve heater after a predetermined time interval. GIN004.1 5 Preferably, the LOC device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes, the dialysis portion configured to concentrate a sample of cells smaller than a predetermined valve to a portion of the sample, A portion of the sample is processed separately from the remaining sample containing only cells larger than the predetermined valve. GIN004.1 6 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by restriction enzymes in the culture section. GIN004.17 Preferably, the LOC device also has a reagent reservoir for containing reagents for use in the culture portion: and a surface tension valve having a bore configured to cause the reagent to form a meniscus, The meniscus thus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir 〇GIN004.1. Preferably, the LOC device also has a hybridization The hybridization section has a probe array-324-201219115 column for hybridizing to the target nucleic acid sequence in the sample: and a light sensor for detecting probe hybridization in the probe array. GIN004.19 Preferably, the LOC device also has a nucleic acid amplification unit GIN004.20. Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) portion having a thermal cycle of between 0.45 seconds and 1.5 seconds. time.

The easy-to-use, mass-produced and inexpensive microfluidic device accepts the fluid, and then the culture portion of the device is used to culture the fluid at a desired temperature for a desired period of time to effect the desired reaction. The culture is controlled by a semiconductor on-wafer control system that results in faster and more accurate temperature control. Faster temperature control improves overall processing speed. More accurate temperature cycling capabilities provide improved reaction conditions. Integrating the culture and its on-wafer semiconductor control system into the device provides simple steps and low system complexity, which in turn provides an inexpensive system.

GIN005.1 Itinerary: The aspect of the invention provides a microfluidic device, a sample inlet for receiving a fluid sample; a culture portion in fluid communication with the sample inlet, the culture portion having at least one heater for The fluid sample is maintained at the culture temperature for a culture period; and the CMOS circuit provides energy to the at least one heater using a pulse width modulation (PWM) signal. GIN005.2 Preferably, the microfluidic device also has at least one -325-201219115 temperature sensor, wherein the CMOS circuit uses the temperature sensor to feedback control the at least one heater. GIN005.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor, wherein the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor for control The heaters are initially activated and are returned to the temperature sensor for subsequent control of the heaters.

GIN005.4 Preferably, the culture portion has a plurality of elongated chambers, the longitudinal lengths of the elongated chambers being much larger than their lateral dimensions, and each of the heaters being elongated and parallel to the chambers Longitudinal length. GIN005.5 Preferably, the culture portion has microchannels provided with a culture inlet and a culture outlet, and the elongated culture chambers are part of the microchannels. GIN005.6 Preferably, the fluid sample is a biological material comprising a nucleic acid sequence, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action.

GIN005.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate culture chambers. GIN005.8 Preferably, the passage portion forming each of the equal width curves has a plurality of elongated heaters. G IN 0 0 5.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GIN005.1 0 Preferably, each of the plurality of elongated heaters is independently operable. -326- 201219115 GIN005.1 1 Preferably, the culture portion has an active valve at the culture outlet for maintaining the mixture of the restriction enzyme and the nucleic acid sequence when the temperature is suitable for limiting the digestion temperature The liquid in the culture section.

GIN005.1 2 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used to retain fluid in the culture, and the boiling start valve also has a valve heater. It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the culture. GIN005.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN005.1 4 Preferably, the CMOS circuit activates the valve heater after a predetermined incubation time. GIN005.1 5 Preferably, the microfluidic device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes, the dialysis portion being configured to concentrate a sample of cells smaller than a predetermined valve to a portion of the sample, The portion of the sample is processed separately from the remaining sample containing only cells larger than the predetermined valve. GIN005.1 6 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by restriction enzymes in the culture section. GIN005.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for use in the culture portion; and a surface tension valve having a bore configured to cause the reagent to form -327 - 201219115 meniscus, so the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent flows out of the reagent reservoir 〇GIN005.1 8 preferably, The microfluidic device also has a hybridization portion having a probe array for hybridization with a target nucleic acid sequence in the sample; and a light sensor for detecting probe hybridization in the probe array. GIN005.1 9 Preferably, the microfluidic device also has a nucleic acid amplification unit. GIN005.20 Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion having a thermal cycle time of from 0.45 seconds to 1.5 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts the fluid, and then the culture portion of the device is used to culture the fluid at a desired temperature for a desired period of time to effect the desired reaction. The culture is controlled by a PWM control system which results in faster and more accurate temperature control. Faster temperature control improves overall processing speed. More accurate temperature cycling capabilities provide improved reaction conditions. Integrating the culture and its PWM control system into the device provides simple steps and low system complexity, which in turn provides an inexpensive system. GIN006.1 This aspect of the invention provides a microfluidic device comprising: a sample inlet for receiving a fluid sample; a culture portion having an elongated culture chamber having a longitudinal length that is much greater than a lateral dimension, and at least one a heater for maintaining the fluid sample at a culture temperature for a culture period; wherein -328-201219115 the at least one heater also extends GIN006.2 parallel to the horizontal axis of the elongated culture chamber. Preferably, the microfluidic device also has a CMOS circuit and at least one temperature sensor 'where the CMOS circuit is coupled to the at least one heater and the at least one temperature sensor' to cause the CMOS circuit to use the temperature sensor to feedback control the at least one heater .

GIN 006.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor 'where the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor to control the The heater is initially activated and the temperature sensor is returned to subsequently control the heaters. GIN006.4 Preferably, the culture portion has a plurality of elongate chambers having a longitudinal length that is much larger than the lateral dimensions of the elongate chambers, and each of the heaters is elongated and parallel to the chambers Longitudinal length. GIN006.5 Preferably, the culture portion has microchannels provided with a culture inlet and a culture outlet, and the elongated culture chambers are part of the microchannels. GIN006.6 Preferably, the fluid sample is a biological material comprising a nucleic acid sequence, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action. GIN006.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves, each of the wide curves forming a channel portion of one of the elongate culture chambers. GIN006.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. -329- 201219115 GIN006.9 Preferably, the plurality of elongated heating agents are disposed in an end-to-end manner along the passage portion. GIN006.1 0 Preferably, each of the plurality of elongated heaters is independently operable. GIN006.il preferably, the culture portion has an active valve at the culture outlet for maintaining the mixture of the restriction enzyme and the nucleic acid sequence in the culture portion when the temperature suitable for the restriction of digestion is maintained by the elongated heater Liquid" GIN006.12 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is for retaining fluid in the culture, the boiling start valve also has a valve heater It is used to boil the liquid so that the meniscus is released from the anchoring point of the meniscus to recover the capillary drive and flow out of the culture. GIN006.1 3 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN006.1 4 Preferably, the CMOS circuit activates the valve heater after a predetermined incubation time. GIN006.1 5 Preferably, the microfluidic device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes, the dialysis portion configured to concentrate a sample of cells smaller than a predetermined valve to a portion of the sample, The portion of the sample is processed separately from the remaining sample containing only cells larger than the predetermined valve. GIN006.1 6 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by the -330-201219115 restriction enzyme in the culture section. GIN006.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for use in the culture portion; and a surface tension valve having a bore configured to cause the reagent to form a meniscus a surface, such that the meniscus retains the reagent in the reagent reservoir until the fluid sample contacts the meniscus and the reagent exits the reagent reservoir

GIN006.1 8 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridization with a target nucleic acid sequence in the sample: and for detecting probe hybridization in the probe array Light sensor. GIN006.19 Preferably, the microfluidic device also has nucleic acid amplification GIN006.20. Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion having a thermal cycle of between 0.45 seconds and 1.5 seconds. time. The easy-to-use, mass-produced and inexpensive microfluidic device accepts the fluid, and then the culture chamber of the device is used to culture the fluid for a desired period of time at the desired temperature to effect the desired reaction. The chamber is in the form of an elongated geometry that provides rapid temperature control of the mixture and advances the mixture by capillary action. Fast temperature control improves overall processing speed. This capillary action advancement simplifies the design of the system, further improving the reliability of the system and reducing its cost. GIN007.1 This aspect of the invention provides a microfluidic device comprising: -331 - 201219115 a support substrate; a sample inlet for receiving a fluid sample; a culture portion having a culture chamber, and at least one for maintaining the The fluid sample is a heater at a culture temperature for a period of time; wherein the culture chamber is interposed between the at least one heater element and the support substrate. GIN007.2 Preferably, the microfluidic device also has a CMOS circuit and at least one temperature sensor, wherein the CMOS circuit is connected to the at least one heater and the at least one temperature sensor to make the CMOS circuit The temperature sensor is used to feedback control the at least one heater. GIN007.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor, wherein the culture portion has a plurality of heaters to cause the CMOS circuit to respond to the liquid sensor to control the The heater is initially activated and the temperature sensor is returned to subsequently control the heaters. GIN007.4 Preferably, the culture portion has a plurality of elongate chambers having a longitudinal length that is much larger than the lateral dimensions of the elongate chambers, and each of the heaters is elongated and parallel to the chambers Longitudinal Length" GIN007.5 Preferably, the culture portion has a microchannel provided with a culture inlet and a culture outlet, and the elongated culture chamber is a portion of the microchannel. GIN007.6 Preferably, the fluid sample is a biological material comprising a nucleic acid sequence, and the microchannel is configured to attract a liquid containing the nucleic acid sequence from the culture inlet to the culture outlet by capillary action. GIN007.7 Preferably, the microchannel has a curved configuration formed by a series of wide curves - 332 - 201219115, each of the wide curves forming a channel portion of one of the elongate culture chambers. GIN007.8 Preferably, the channel portion forming each of the equal width curves has a plurality of elongated heaters. GIN007.9 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GIN007.10 Preferably, each of the plurality of elongated heaters is independently

Operation. GIN007.il preferably, the culture portion has an active valve at the culture outlet for maintaining the mixture of the restriction enzyme and the nucleic acid sequence in the culture portion when the temperature suitable for the restriction of digestion is maintained by the elongated heater liquid

GIN007.1 2 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used to retain fluid in the culture, and the boiling start valve also has a valve heater. It is used to boil the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the culture. GIN007.1 3 Preferably, the heater is located at the edge of the hole. GIN007.14 Preferably, the meniscus anchor is a hole and the valve CMOS circuit activates the valve heater after a predetermined incubation time. GIN007.1 5 Preferably, the microfluidic device also has a dialysis unit, wherein the fluid sample is a biological material comprising cells of different sizes, the dialysis portion being configured to concentrate a sample of cells smaller than a predetermined valve to a portion of the sample - 333-201219115, the sample of this part is treated separately from the remaining sample containing only cells larger than the predetermined valve. GIN007.1 6 Preferably, the dialysis section is located above the culture section, and the cells smaller than the predetermined valve chamber comprise a nucleic acid sequence to be digested by restriction enzymes in the culture section. GIN007.1 7 Preferably, the microfluidic device also has a reagent reservoir for containing reagents for use in the culture section;

a surface tension valve having a bore configured to cause the reagent to form a meniscus such that the meniscus retains the reagent within the reagent reservoir until the fluid sample contacts the meniscus and The reagent flows out of the reagent reservoir GIN007.1. Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridizing with the target nucleic acid sequence in the sample; and for detecting A light sensor that hybridizes to the probe in the array of needles.

GIN007.1 9 Preferably, the microfluidic device also has a nucleic acid amplification unit. GIN007.20 Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion having a thermal cycle time of between 0.45 seconds and 1.5 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device accepts the fluid, and then the culture chamber of the device is used to culture the fluid for a desired period of time at the desired temperature to effect the desired reaction. The heater of the chamber is positioned above the chamber to maximize heat transfer to the culture mixture and minimize heat transfer to the substrate. -334- 201219115 GIN008.1 This aspect of the invention provides a microfluidic device comprising: an inlet for receiving a fluid; a culture portion having a microchannel configured to have a plurality of parallel, adjacent a channel portion, and a plurality of elongated heaters disposed end to end along each of the channel portions;

Each of the heaters is independently operable to control the heat flux intensity from the two-dimensional direction to the cultivating portion. GIN008.2 Preferably, the microchannel is configured in a curved configuration having a series of wide curves to form the plurality of mutually parallel, adjacent channel portions. GIN008.3 Preferably, the microchannel The fluid device also has a support substrate and a CMOS circuit, wherein the CMOS circuit is interposed between the support substrate and the culture portion for controlling the heaters. GIN008.4 Preferably, the microfluidic device also has at least one sensor, wherein the CMOS circuit uses the sensor to feedback control the heaters. GIN008.5 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor, wherein the CMOS circuit corresponds to the liquid sensor to control the initial startup of the heater, and the temperature is returned The sensor controls the heater in a subsequent manner. GIN008.6 Preferably, the fluid sample is a biological material comprising a nucleic acid sequence, and the microchannel is configured to attract liquid containing the nucleic acid sequence through all of the channel portions by capillary action. -335-201219115 GIN008.7 Preferably, the culture portion has an active valve at a downstream end point for retaining the culture portion when the elongated heater maintains a mixture of restriction enzyme and nucleic acid sequence at a temperature suitable for restriction of digestion Liquid in the middle" GIN008.8 Preferably, the active valve is provided with a meniscus anchor

a boiling start valve for retaining fluid in the culture portion, the boiling start valve also having a valve heater for boiling liquid to make the meniscus from the meniscus anchor Released to resume capillary drive and exit the culture. GIN008.9 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GIN008.1 0 Preferably, the CMOS circuit activates the valve heater after a predetermined incubation time. GIN008.il preferably, the microfluidic device also has a dialysis unit,

Wherein the fluid sample is a biomaterial comprising cells of different sizes, the dialysis section being configured to concentrate a sample of cells less than a predetermined valve to a portion of the sample and the remainder of the cell comprising only cells greater than the predetermined valve The sample is treated separately. GIN008.1 2 Preferably, the dialysis section is located upstream of the culture section, and the cells larger than the predetermined valve chamber comprise a nucleic acid sequence to be digested by the restriction enzymes in the culture section. GIN008.1 3 Preferably, the microfluidic device also has a reagent reservoir for containing a restriction enzyme for use in the culture portion and a surface tension valve provided with a hole configured to contain the restriction The solution of the enzyme forms a meniscus such that the meniscus retains the restriction enzyme in the reagent reservoir until the -336 - 201219115 fluid sample contacts the meniscus and the restriction enzyme flows out of the reagent reservoir . GIN008.14 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridizing with the target nucleic acid sequence in the sample to form a probe-target hybrid, and for detecting the probe A light sensor of a needle-target hybrid. GIN008.1 5 Preferably, the microfluidic device also has the same

The nucleic acid amplification department downstream of the cultivating department. GIN008.1 6 Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion having a thermal cycle time of from 0.45 seconds to 1.5 seconds. GIN 00 8.1 7 Preferably, the microfluidic device also has a lysis unit for disrupting the cell membrane and the intracellular membrane of the cells contained in the sample, the lysate being located upstream of the culture portion and downstream of the dialysis portion. GIN008.1 8 Preferably, the fluid sample is blood, and the cells larger than the predetermined valve include white blood cells. GIN00 8.1 9 Preferably, the microfluidic device also has an anticoagulant reservoir for the addition of an anticoagulant to the sample upstream of the dialysis section. GIN008.20 Preferably, the light sensor is positioned to register the array of photodiodes of the probes. The easy-to-use, mass-produced and inexpensive microfluidic device accepts the fluid, and then the culture chamber of the device is used to culture the fluid for a desired period of time at the desired temperature to effect the desired reaction. The heat flux intensity entering the chamber is controlled by a two-dimensional orientation which results in a highly uniform two-dimensional temperature distribution that provides uniform culture characteristics and enhances the reaction results of -337-201219115. GLA001.1 This aspect of the invention provides a test module for processing and analyzing a blood sample, the test module comprising: a housing provided with a container for receiving blood; a functional portion for processing and analyzing the Sample: and a lancet used to collect the blood sample from the patient. GLA001.2 Preferably, the lancet can be retracted into the outer casing but biased

To the extended position, wherein the sharp end of the lancet protrudes from the outer casing. GLA001.3 Preferably, the lancet is fitted with a spring that is held in a retracted position within the housing against the spring offset during use until released by the user" GL A 00 1.4 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the blood sample. GLA001.5 Preferably, one of the reagent reservoirs contains an anticoagulant for addition to a blood sample upstream of the functional portions.

GLA00 1.6 Preferably, one of the functional portions is for separating a cell larger than a predetermined valve to a portion of the blood sample, such that the remaining sample contains cells smaller than the predetermined valve. GLA001.7 Preferably, one of the functional portions is a lysis chamber such that during use, cells in the blood sample are dissolved to release any genetic material therein. GLA001.8 Preferably, one of the reagent reservoirs comprises a lysis reagent for use in dissolving cells in the lysis chamber. GLA001.9 Preferably, the lysis chamber has a heater for dissolving the fine-338-201219115 cells. GLA001.10 Preferably, one of the functional units is a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence from the genetic material.

GLA001.il Preferably, the functional portions include a culture portion located upstream of the PCR portion, and one of the reagent reservoirs is a restriction enzyme reservoir having a heater to maintain the sample and the restriction enzyme The mixture is at a culture temperature during which the restriction enzyme digests the nucleic acid sequence. GLA001.12 Preferably, the test module also has a CMOS circuit and a temperature sensor, wherein the control circuit uses the output of the temperature sensor to feedback control the PCR portion. GLA001.13 Preferably, the test module also has a probe array for hybridization with a target nucleic acid sequence in an amplicon from the PCR portion. GLA001.14 Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence (included in the amplicon), the various probe-target hybridization systems configured to respond The photons are emitted while being input, and the CMOS circuit has a light sensor to sense photons emitted by the probe-target hybrid. GLA001.15 Preferably, the test module also has an array of hybridization chambers for interposing probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. GLA001.16 Preferably, the light sensor is associated with a photodiode array of the hybridization chamber. GLA001.17 Preferably, the control circuit has a digital memory for storing the hybridization data from the light sensor output of -339-201219115 and a data interface for transmitting the hybrid data to the external device. GLA001.18 Preferably, the PCR portion has an active valve for retaining liquid in the PCR portion during thermal cycling and allowing the liquid to flow to the hybridization chamber in response to an activation signal from the control circuit. GLA001.19 Preferably, the active valve is provided with a meniscus anchor and a boiling start valve of the heater, the meniscus anchor configured to form a meniscus to prevent capillary flow of the liquid The heater is used to boil the liquid to release the meniscus from the meniscus anchor to restore the capillary drive flow. GLA001.20 Preferably, the meniscus anchor is a hole having a ring shape and located adjacent to the edge of the hole. The integrated lancet is easy to use, mass-produced, inexpensive, and The portable diagnostic test module carries a blood sample and processes and analyzes the sample. The sterilized integrated lancet eliminates the need for external lancet replenishment and cost, while ensuring the user's sterility and safety. GGA00 1.1 This aspect of the invention provides a test module for analyzing a genetic material in a biological sample, the test module comprising: a housing provided with a container for receiving the biological sample; a functional part, the system For processing and analyzing the biological sample; a controller for operating the functional units: and a flow path from the container to the functional portions; wherein the flow path is configured to be attracted by capillary action to be analyzed Sample -340 - 201219115 The product flows from the container to the functional parts, regardless of the directionality of the module relative to gravity. GGA001.2 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample. GGA001.3 Preferably, one of the functional portions is a nucleic acid amplification portion for amplifying a nucleic acid sequence in the genetic material. GGA001.4 Preferably, the nucleic acid amplification part is polymerase chain reaction

Preferably, the functional portion includes a culture portion located upstream of the PCR portion and one of the reagent reservoirs is a restriction enzyme reservoir having a heater to maintain A culture temperature at which the mixture of the sample and the restriction enzyme is digested by the restriction enzyme during the nucleic acid sequence is included. GGA001.6 Preferably, the test module also has a temperature sensor, wherein the controller uses the output of the temperature sensor to feedback control the PCR portion. GGA001.7 Preferably, the PCR portion has a PCR microchannel for heating to circulate the sample to amplify the nucleic acid sequence, the PCR microchannel defining a portion of the sample flow path and having less than 1 〇〇, 〇〇〇 square The cross-sectional area of the fluid perpendicular to the micrometer. GGA001.8 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least a portion of the PCR microchannel forms an elongated PCR chamber. GGA001.9 Preferably, the test module also has at least one elongated heater element for heating the nucleic acid sequence within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel. -341 - 201219115 GGA001.10 Preferably, the PCR portion has a plurality of elongated PCR chambers respectively formed by respective portions of the PCR microchannels, the microchannels having a curved configuration formed by a series of wide curves Each of the wide curves forms a channel portion of one of the elongate PCR chambers. GGA001.11 Preferably, each of the channel portions has a plurality of the elongated heaters. GGA001.12 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GGA001.13 Preferably, the elongate heaters are independently operable. GGA001.14 Preferably, the test module also has a hybridization portion downstream of the PCR portion, the hybridization portion having a probe array for hybridizing with the target nucleic acid sequence in the sample, and for detecting the array A light sensor that hybridizes to any probe. GGA001.15 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of the primer, the dNTP, the polymerase and the buffer in the elongated heater to amplify the nucleic acid. The liquid in the PCR portion is retained during the sequence. GGA001.16 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, the anchor is used for retaining liquid in the PCR part, and the boiling start valve also has a valve heater. The liquid is boiled to release the bay level from the meniscus anchor to recover the capillary drive and exit the PCR section. GGA001.17 Preferably, the meniscus anchor is a hole and the valve -342-201219115 heater is located at the edge of the hole. GGA001.18 Preferably, the reagent reservoirs each have a surface tension valve having a bore configured to cause the reagent to form a meniscus, such that the meniscus retains the reagent in the reagent reservoir The meniscus is removed until the fluid sample contacts and the reagent flows out of the reagent reservoir. GGA00 1. 1 9 Preferably, the test module also has an on-wafer test

a chamber (LOC) device having a sample inlet in fluid communication with the container, a support substrate, a microsystem technology (MST) layer, a CMOS circuit between the support substrate and the MST layer, and an upper cover, wherein the control The device is inserted into the CMOS circuit, the MST layer has the functional portion, and the upper cover covers the MS T layer and defines the reagent reservoirs. GGA001.20 Preferably, the PCR section is configured to complete the thermal cycling of the sample in less than 30 seconds. The easy-to-use, mass-produced, inexpensive, lightweight, and portable genetic diagnostic test module has a self-contained reagent inventory that accepts biological samples, processes the sample, and analyzes the module using the integrated sensor. The genetic material in the sample, and at the output of the sample, provides an electronic result. The module's fluid propulsion and reagent storage is based purely on capillary action, providing gravity-free operation of the module, such as in space flight. GGA003.1 This aspect of the invention provides a test module for analyzing a genetic material in a biological sample, the test module comprising: a housing provided with a container for receiving the biological sample; For processing and analyzing the biological sample; a controller for operating the functional units: and -343-201219115 a flow path from the container to the functional portions; wherein the flow path is configured to The capillary action attracts the sample to be analyzed from the container to the functional portions, regardless of the directionality of the module. GGA003.2 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample. GGA003.3 Preferably, one of the functional portions is a nucleic acid amplification portion for amplifying a nucleic acid sequence in the genetic material. GGA003.4 Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GGA003.5 Preferably, the functional portions include a culture portion located upstream of the PCR portion and one of the reagent reservoirs is a restriction enzyme reservoir. The culture portion has a heater to maintain the sample and the restriction enzyme. The mixture is at a culture temperature during which the restriction enzyme digests the nucleic acid sequence. GGA003.6 Preferably, the test module also has a temperature sensor, wherein the controller uses the output of the temperature sensor to feedback control the PCR portion. GGA003.7 Preferably, the PCR portion has a PCR microchannel for heating the sample to amplify the sample flow path of the nucleic acid sequence 'the PCR microchannel definition portion and having a vertical flow of less than 100,000 square microns The cross-sectional area. GGA003.8 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least a portion of the PCR microchannel forms an elongated pCR chamber. GGA 0 0 3.9 Preferably, the test module also has at least one elongated heater element for heating the nucleic acid sequence -344 - 201219115 column in the elongated PCR microchannel, the elongated heater element being parallel Pc R microchannel extension. GGA003.1 0 Preferably, the PCR portion has a plurality of elongated PCR chambers formed by respective portions of the PCR microchannels. The microchannels have a curved configuration formed by a series of wide curves. A wide curve forms the channel portion of one of the elongate PCR chambers. GGA003.il Preferably, each of the channel portions has a plurality of the elongated heaters.

GGA003.1 2 Preferably, the plurality of elongated heaters are disposed end to end along the channel portion. GGA003.1 3 Preferably, the elongate heaters are independently operable.

GGA003.14 Preferably, the test module also has a hybridization portion downstream of the PCR portion, the hybridization portion having a probe array for hybridizing with the target nucleic acid sequence in the sample and for detecting the array A light sensor that hybridizes to any probe. GGA003.1 5 Preferably, the PCR portion is provided with an active valve at the PCR outlet for heating and circulating the nucleic acid sequence and the mixture of primer, dNTP, polymerase and buffer in the elongated heater to amplify the The nucleic acid sequence retains the liquid in the PCR portion. GGA003.1 Preferably, the active valve is provided with a boiling start valve of a meniscus anchor, which is used to retain liquid in the PCR portion. The boiling start valve also has a valve heater for boiling the liquid to release the meniscus from the meniscus anchor to resume capillary drive and exit the PCR section. -345- 201219115 GG A003.1 7 Preferably, the meniscus anchor is a hole and the valve heater is located at the edge of the hole. GGA003.1 8 Preferably, the reagent reservoirs each have a surface tension valve having a bore configured to cause the reagent to form a meniscus, such that the meniscus retains the reagent in the reagent reservoir The meniscus is removed within the device until the fluid sample contacts and the reagent exits the reagent reservoir. G G A 0 0 3 . 1 9 Preferably the test module also has an on-wafer experiment

a chamber (LOC) device having a sample inlet 'support substrate, a microsystem technology (MST) layer, a CMOS circuit and an upper cover interposed between the support substrate and the MST layer in fluid communication with the container, wherein the control The device is inserted into the CMOS circuit, the MST layer has the functional portion, and the upper cover covers the MS T layer and defines the reagent reservoirs. GGA003.20 Preferably, the PCR section is configured to complete the thermal cycling of the sample in less than 30 seconds.

The easy-to-use, mass-produced, inexpensive, lightweight, and portable genetic diagnostic test module has a self-contained reagent inventory that accepts biological samples, processes the sample, and analyzes the module using the integrated sensor. The genetic material in the sample 'and at its output 埠 provides an electronic result. The module's fluid propulsion and reagent storage is based purely on capillary action, providing directional independent operation of the module, for example in the case of ocean navigation. GRE006.1 This aspect of the invention provides a microfluidic assembly for detecting a target molecule in a sample fluid and displaying a positive or negative result, the microfluidic collection comprising: a test module comprising: -346- 201219115 A housing with a container for receiving the sample; a functional unit 'which is used to process the sample and detect the target molecule; a communication interface controller for transmitting one or more functions An output signal; and an indicator module detachably coupled to the communication interface, the indicator module having an indicator for providing an indication that the target molecule is present in the fluid sample.

GRE006.2 Preferably, the indicator module has a power supply to operate the test module. GRE006.3 Preferably, the indicator module provides power to the test module via the communication interface. GRE006.4 Preferably, the communication interface is a universal serial bus (USB) connector and the indicator module has a USB. GRE006.5 Preferably, the test module has a controller for operating the functional units and providing the output signals to the communication interface. GRE006.6 Preferably, the test module has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing identification data for the reagents. GRE006.7 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic test module. GRE006.8 Preferably, the sample is a biological sample containing a genetic material, and one of the functional portions is a nucleic acid amplification portion for amplifying a nucleic acid sequence in the genetic material. GRE006.9 Preferably, the nucleic acid amplification part is polymerase linked to the anti-347-201219115 (PCR) part and the data stored in the digital memory includes thermal cycle time and number of cycles. GRE006.1 0 Preferably, the functional portions include a culture portion located upstream of the PCR portion and one of the reagent reservoirs is a restriction enzyme reservoir having a heater to maintain the sample and the restriction enzyme The mixture is at a culture temperature during which the restriction enzyme digests the nucleic acid sequence. GRE006.il Preferably, the microfluidic assembly also has a probe array for hybridization with the target nucleic acid sequence in the amplicon from the PCR portion. GRE006.1 2 Preferably, the data stored in the digital memory includes probe identification data to identify probes at various locations within the array of probes. GRE006.1 3 Preferably, the test module has a light sensor, wherein each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence (included in the amplicon), Various probe-target hybridization systems are configured to emit photons in response to an input, and the light sensor is configured to sense photons emitted by the probe-target hybrid. GRE006.1 4 Preferably, the data stored in the digital memory comprises hybridization data generated from the light sensor output. GRE006.1 5 Preferably, the test module has an array of hybridization chambers for containing probes such that probes within each of the hybridization chambers are configured to hybridize to one of the target nucleic acid sequences. GRE006.1 6 Preferably, the light sensor is a register of the photodiode array of the hybridization chamber. -348-201219115 GRE006.17 Preferably, the controller is configured to transmit the hash material to an external device other than the indicator module. GRE006.1 8 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the dedicated reader and store the patient data in the digital memory.

GRE006.1 9 Preferably, the PCR portion has an active valve for retaining liquid in the PCR portion during thermal cycling and allowing the liquid to flow to the hybridization chamber in response to an activation signal from the controller. GRE006.20 Preferably, the active valve is provided with a meniscus anchor and a boiling start valve of the heater, the meniscus anchor configured to form a meniscus to prevent capillary flow of the liquid The heater is used to boil the liquid to release the meniscus from the meniscus anchor to restore the capillary drive flow. The easy-to-use, mass-produced, inexpensive, lightweight, and portable microfluidic diagnostic test module has a self-contained reagent inventory that accepts samples and processes and analyzes the sample material using the integrated sensor of the module. And provide electronic results at the output of the other. The module's communication interface can be arbitrarily chosen to interface with a very inexpensive and lightweight USB power/indicator module that provides power and minimal user interface support for the module. The on-board digital memory of the module securely stores test result information. The ability to store test results information makes it possible for the diagnostic module to be tested using only the USB power/indicator module, and then analyze the test results with a fully functional reader. The preservation of the test results information will ensure that the information will not be illegally used through illegal channels. The USB Power/Indication -349- 201219115 module eliminates the need for a dedicated, cumbersome and expensive module support system. GSA001.1 This aspect of the invention provides a method of analyzing nucleic acid content in a blood sample, the method comprising the steps of: providing a test module having a housing configured for hand movement, the housing having a container For receiving blood, the test module has a lysis unit installed in the outer casing for dissolving cells and organisms in the blood to release the genetic material therein, and a hybrid portion of the probe array is provided for Hybridization of the target nucleic acid sequence in the genetic material, and circuitry for sensing which probes have hybridized and generating hybridization data; providing a test module reader for reading hybridization data from the test module; Introducing into the container; interfacing the test module with the test module reader: wherein the test module reader analyzes the nucleic acid content from the hybrid data. GSA001.2 Preferably, the test module and the test module reader are configured to interface via an electrical connector, the test module obtaining operational power from the test module reader. GSA001.3 Preferably, the method further comprises: providing a lancet to obtain a drop of blood from the patient. Preferably, the lancet is retractable into the outer casing but is biased toward the extended position, wherein the sharp end of the lancet protrudes from the outer casing. GSA001.5 Preferably the lancet is spring loaded and the lancet is held in a retracted position within the housing against the spring deflection during use until released by the user. -350-201219115 GSA001.6 Preferably, the method also has a plurality of reagent reservoirs containing reagents for processing the blood sample. GSA001.7 Preferably, one of the reagent reservoirs contains an anticoagulant for use in a blood sample that is added downstream of the container. GSA 001.8 Preferably, the method also has a dialysis section for separating a component greater than a predetermined valve cartridge to a portion of the blood sample such that the remaining sample contains less than a component of the predetermined valve cartridge.

GSA001.9 Preferably, one of the reagent reservoirs comprises a lysis reagent for use in dissolving cells in the lysis chamber. GSA001.10 Preferably, the lysis chamber has a heater for dissolving the cells. GSA001.11 Preferably, the method also has a polymerase chain reaction (PCR) portion for amplification from the The nucleic acid sequence of a genetic material. GSA001.12 Preferably, the method also has a culture portion located upstream of the PCR portion and one of the reagent reservoirs is a restriction enzyme reservoir having a heater to maintain the blood sample and the restriction The culture temperature of the mixture of enzymes during digestion of the nucleic acid sequence by the restriction enzyme. GSA001.13 Preferably, the method also has a temperature sensor, and the circuit uses the output of the temperature sensor to feedback control the PCR portion. GSA001.14 Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence (included in an amplicon from the PCR portion), the various probe-target hybridization systems Photons are configured to emit light in response to an input, and the circuit has a light sensor to sense photons emitted by the probe-target hybrid. Preferably, the method also has an array of hybridization chambers for containing probes such that the probes within each of the hybridization chambers are configured to hybridize to one of the target nucleic acid sequences. GSA001.1 6 Preferably, the light sensor is associated with a photodiode array of the hybridization chamber. GSA001.17 Preferably, the circuit has digital memory for storing hybrid data from the output of the light sensor. GSA001.1 8 Preferably, the PCR portion has an active valve for retaining liquid in the PCR portion during thermal cycling and allowing the liquid to flow to the hybridization chamber in response to an activation signal from the circuit. GSA001.19 Preferably, the active valve is provided with a meniscus anchor and a boiling start valve of the heater, the meniscus anchor configured to form a meniscus to prevent capillary flow of the liquid The heater is used to boil the liquid to release the meniscus from the meniscus anchor to restore the capillary drive flow. GSA 001.20 Preferably, the meniscus anchor is a hole having a ring shape and located adjacent the edge of the hole. The easy-to-use, mass-produced and inexpensive HPLC device for analyzing the nucleic acid content of a blood sample receives a blood sample through the sample container, and dissolves the sample cell in the lysis chamber to release the genetic material of the sample. The target gene sequence is amplified and analyzed by hybridization with an oligonucleotide probe and sensing of the integrated imaging array to analyze the nucleic acid sequence of the sample. The lysis process extracts the genetic material from the cells in the sample and provides subsequent processing and analysis of the targets. Integrating the lysis unit into the -352 - 201219115 device provides a simple detection step, low system component count, and simple system manufacturing process, resulting in an inexpensive inspection system. Amplification of the target gene sequence increases the sensitivity and signal to noise ratio of the detection system. The probe hybridization provides analysis of the target via hybridization. The integrated probe hybridization unit provides a comprehensive solution that is easy to use, mass-produced, and inexpensive with a low number of system components.

The integrated image sensor eliminates the need for expensive external imaging systems, provides a comprehensive solution that is both mass-produced and inexpensive, and its low system components represent a lightweight, highly mobile system. The integrated image sensor has the benefit of increased read sensitivity due to large angle light collection and eliminates the need to use optical components in the string of light collecting elements. GSA002.1 This aspect of the invention provides a method of analyzing nucleic acid content in a biological fluid, the method comprising the steps of: providing a test module having a housing configured for hand movement, the housing having a container For receiving the biological fluid, the test module has a hybrid portion provided with a probe array for hybridizing with a target nucleic acid sequence in the biological fluid, and for sensing which probes have hybridized and generating hybridization data. a test module reader for reading hybridization data from the test module; introducing the biological fluid into the container; interfacing the test module with the test module reader; wherein the test module The reader analyzes the nucleic acid content from the hybridization data. - 353 - 201219115 GSA002.2 Preferably, the test module and the test module reader are configured to interface via an electrical connector, the test module obtaining operational power from the test module reader. GSA002.3 Preferably, the biological fluid comprises cells, and the test module has a lysis unit mounted in the housing for dissolving the cells to release the genetic material therein. GSA 02.4 Preferably, the method also has the step of treating the biological fluid prior to introduction into the container. GSA002.5 Preferably, the biological fluid comprises one or more of the following blood, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, cord blood, breast milk, sweat, pleural effusion, tear fluid, pericardial fluid, peritoneal fluid, and expectorant sample. GSA002.6 Preferably, the biological flow system is derived from a polymerase chain reaction (PCR) amplicon. GSA 02.7 Preferably, the method also has a plurality of reagent reservoirs containing reagents for treating the biological fluid. GSA002.8 Preferably, the method also has a dialysis section for separating cells larger than the predetermined valve to a portion of the biological fluid such that the remaining biological fluid contains cells smaller than the predetermined valve" GSA002.9 Preferably, one of the reagent reservoirs comprises a lysis reagent for use in dissolving cells in the lysis chamber. GSA002.1 0 Preferably, the lysis chamber has a heater for dissolving the cells. Preferably, the method also has a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence from the biological fluid. GSA002.1 2 Preferably, the method also has a culture portion located upstream of the PCR portion, and one of the reagent reservoirs is a restriction enzyme reservoir having a heater to maintain the biological fluid and the restriction The culture temperature of the mixture of enzymes during digestion of the nucleic acid sequence by the restriction enzyme.

GSA 02.1 3 Preferably, the method also has a temperature sensor, and the circuit uses the output of the temperature sensor to feedback control the PCR portion. GSA002.1 4 Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence (included in an amplicon from the PCR portion), the various probe-target hybrids The system is configured to emit photons in response to an input, and the circuitry has a light sensor to sense photons emitted by the probe-target hybrid. Preferably, the method also has an array of hybridization chambers for containing probes such that the probes within each of the hybridization chambers are configured to hybridize to one of the target nucleic acid sequences. GSA002.1 6 Preferably, the light sensor is associated with a photodiode array of the hybridization chamber. GSA 002.1 7 Preferably, the circuit has a digital body for storing hybrid data from the output of the light sensor. GSA 002.1 8 Preferably, the PCR portion has an active valve for retaining liquid in the PCR portion during thermal cycling and allowing the liquid to flow to the hybridization chamber in response to an activation signal from the circuit. GSA002.19 Preferably, the active valve is provided with a meniscus anchor-355-201219115 and a boiling start valve of the heater, the meniscus anchor configured to form a meniscus to block the liquid The capillary drive stream is used to boil the liquid to release the meniscus from the meniscus anchor to recover the capillary drive flow. GSA 002.20 Preferably, the meniscus anchor is a hole having a ring shape and located adjacent the edge of the hole. The easy-to-use, mass-produced and inexpensive HPLC device for analyzing the nucleic acid content of the biological fluid sample receives a biological fluid sample through the sample container, and dissolves the sample cell in the lysis chamber to release the sample. The genetic material, the target gene sequence is amplified, and the nucleic acid sequence of the sample is analyzed by hybridization with an oligonucleotide probe and sensing of the integrated imaging array. The lysis process extracts the genetic material from the cells in the sample and provides subsequent processing and analysis of the targets. Integrating the lysis subunit into the device provides a simple detection step, a low number of system components, and a simple system manufacturing process, resulting in an inexpensive detection system. Amplification of the target gene sequence increases the sensitivity and signal to noise ratio of the detection system. The probe hybridization provides analysis of the target via hybridization. The integrated probe hybridization unit provides a comprehensive solution that is easy to use, mass-produced, and inexpensive with a low number of system components. The integrated image sensor eliminates the need for expensive external imaging systems, provides a comprehensive solution that is both mass-produced and inexpensive, and its low system components represent a lightweight, highly mobile system. The integrated image sensor -356 - 201219115 has the benefit of increased read sensitivity and eliminates the need to use optical components in the string of light collecting elements due to large angle light collection. GPK00 1 · 1 This aspect of the invention provides a microfluidic test module comprising: 'a housing for hand movement; and a microfluidic device mounted within the housing for processing a biological sample, the housing providing proximity Micro-environment of a microfluidic device, wherein

The outer casing has a membrane seal of flexible material between the microenvironment and the atmosphere for reducing pressure variations in the microenvironment due to atmospheric pressure fluctuations. GPK001.2 Preferably, the outer casing has a guard that protects the membrane seal from damaging contact. GPK001.3 Preferably, the microfluidic test module also has a sensor and a functional unit to adjust the microenvironment in response to feedback from the sensor.

GPK00 1.4 is preferably within the microfluidic device. GPK00 1 .5 Preferably, the sensor and the functional unit construct the sensor moisture sensor, and the functional unit is a humidifier. GPK00 1.6 Preferably, the microfluidic device is a wafer-on-lab (LOC) device and an upper cover, the LOC device having a support substrate and a micro-system technology (MST) layer on the support substrate, the MST layer having an MST channel And a plurality of fluid connectors for fluid communication with the upper cap and the cap has a sample inlet and an upper cap passage for fluid communication with the fluid connectors. - 357 - 201219115 GPK001.7 Preferably, the humidifier has a water reservoir and an evaporator to expose water supplied from the water reservoir to a region containing the MST layer and to increase the water vapor pressure of the region. GPK001.8 preferably 'the evaporator has a bore configured to retain water using a meniscus formed in the bore, and the evaporator also has a heater adjacent the bore for raising the bore in the bore The temperature of the water. GPK001.9 Preferably, the heater is annular and located adjacent the aperture" GPK001.10 Preferably, the evaporator has a supply passage from the reservoir to the bore, the supply passage being configured to be by capillary The action draws water to the hole. GPK001.il Preferably, the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters. GPK001.12 Preferably, the water reservoir is formed on an upper cover formed in the MST layer such that the water reservoir is connected to the supply passage via one of the plurality of liquid connectors, and the hole is formed in the upper cover The supply channel is connected to the aperture via the other of the liquid connections. GPK001.13 Preferably, the upper cover has a plurality of reagent reservoirs of different reagents. GPK001.14 Preferably, the outer casing has a container for receiving an untreated biological sample through the opening and a cover movable between the open and closed positions, the opening being exposed when the cover is in the open position, when the cover is closed The opening is closed in position, the container being configured to be in fluid communication with the sample inlet to cause the biological sample to flow to the sample inlet by capillary action. - 358 - 201219115 GPK001.15 Preferably, the cover is a sealing tape having a low viscosity adhesive for sealing the opening in the closed position. GPK001.16 Preferably, the MST layer has a heater for heating the fluid within the MS T channel.

GPK001.17 Preferably, the MST channels each have a cross-sectional area of from 1 square micron to 400 square micrometers for biochemical treatment of components within the biological sample, and the upper cap channels respectively have greater than 400 square microns The cross-sectional area is for accepting the biological sample and the cells transported within the biological sample to a predetermined location in the MS T channel. GPK001.1 8 Preferably, the housing has a lancet for puncturing a patient's finger to obtain a blood sample for introduction into the container. GPK001.19 Preferably, the lance is movable between a retracted and extended position, and the housing has an offset mechanism to bias the lance toward the extended position and a user-activated catch to The lancet remains in the retracted position until it is activated by the user. GPK001.20 Preferably, the LOC device has a hybridization portion comprising a probe nucleic acid sequence configured to detect hybridization of a target nucleic acid sequence within a cell of the sample fluid with the probe nucleic acid sequence. The easy to use, mass produced, inexpensive and portable microfluidic test module accepts a fluid sample and processes and analyzes the sample. The necessary fluid propulsion on the modular fluid device is provided via capillary action, and the pressure relief within the microenvironment of the module is provided via a flexible membrane covering the pressure relief. This capillary propulsion maintains low system component count, low system complexity, and a simple manufacturing process that further reduces the cost of the system. The flexible film -359 - 201219115 sheet pressure relief system also maintains low system component count, low system complexity and a simple manufacturing process that further reduces the cost of the system. [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION The present general description refers to the main components of the molecular diagnostic system in which the embodiment of the present invention is embodied. The full details of the system structure and operation are discussed in the following description. Referring to Figures 1, 2, 3, 96 and 97, the system has the following most important components: Test Modules 1 and 1 are the size of a conventional U S B flash drive, which can be produced very cheaply. Test modules 10 and 1 each comprise a microfluidic device, typically in the form of a lab-on-lab (LOC) device 30, which preloads reagents and typically more than 1000 probes for molecular diagnostic analysis (see Figure 1). And 96). The test module 10 illustrated in Figure 1 uses a fluorescent substrate detection technique to identify target molecules, whereas the test module 11 of Figure 96 uses an electrochemiluminescence substrate detection technique. The LOC device 30 has an integrated light sensor 44 (described in detail below) for fluorescence or electrochemiluminescence detection. Both the test module 10 and the cymbal use the standard micro-USB connector 14 for power supply, data and control. Both test modules have a printed circuit board (P C B) 5 7 and an external power supply capacitor 3 2 and an inductor 15. Test modules 1 0 and 1 1 are for single use only and are mass produced and distributed for aseptic packaging for immediate use. The outer casing 13 has a large container 24 that accepts biological samples and a removable non-360-201219115 bacteria sealing tape 22, which preferably has a low viscosity adhesive to cover the large container prior to use. A membrane seal 408 having a membrane guard 410 forms part of the outer casing 13 to reduce the reduction in humidity within the test module while providing a pressure relief during small air pressure changes. Membrane guard 410 protects membrane seal 408 from damage.

The test module reader 12 supplies power to the test module 10 or 11 via the micro-USB port 16. The test module reader 12 can be in many different forms, the choice of which is described below. The reader 12 version shown in Figures 1, 3 and 96 is an implementation of a smart phone. The block diagram of the reader 12 is shown in Figure 3 » The processor 42 executes the application software from the program storage 43. The processor 42 is also interfaced with a display screen 18 and a user interface (UI) touch screen 17 and buttons 19, a cellular radio 21, a wireless network connection 23, and a satellite navigation system 25. The cellular radio 21 and the wireless network connection 23 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location information. The location data can optionally be manually entered via touch screen 17 or button 19. The data store 27 stores genetic and diagnostic information, test results, patient information, analysis and probe data for identifying each probe and its array position. The data store 27 and the program store 43 can be shared by a common memory device. The application software installed in the test module reader 12 provides results analysis and other test and diagnostic information. To perform a diagnostic test, the test module 1 (or test module 1 1) is inserted into the micro-USB port 16 on the test module reader 12. The sterile sealing tape 22 is torn back and the biological sample (in liquid form) is loaded into the sample large container 24. Press the start button 20 to start the test via the application software. The sample flows into the -361 - 201219115 LOC device 30 and the device is subjected to on-board analysis to extract, culture, amplify and pre-synthesize the hybrid-reactive oligonucleotide probe with the sample nucleic acid (target) Hybrid. In the case of the test module 10 (which uses detection of a fluorescent substrate), the probes are fluorescently labeled and provide the necessary excitation light by the LSD 26 mounted in the housing 13 to induce the hybrid probe. Light emission (see Figures 1 and 2). In the case of test module 1 (which uses electrochemiluminescence (ECL) detection), LOC device 30 is loaded with an ECL probe (as described above) and LED 26 is not required to produce luminescent emissions. The excitation current is actually provided by electrodes 860 and 870 (see Figure 97). The emission (fluorescence or illumination) is detected by a light sensor 44 integrated into the CMOS circuitry of each LOC device. The detection signal is amplified and converted to a digital output for analysis by the test module reader. The reader then displays the results. This information can be stored locally and/or uploaded to a network server containing patient records. Test module 10 or 11 is removed from test module reader 12 and processed as appropriate. Figures 1, 3 and 96 show a test module reader 12 designed as a mobile phone/smartphone 28. Other forms of test module readers may be laptop/notebook 101, dedicated reader 103, e-book reader 107, tablet 109 or desktop computer 105 for use in hospitals, private clinics or laboratories ( See Figure 9 8). The reader can interface with additional applications such as patient records, accounting, online databases and multi-user environments. It can also interface with some local or remote peripherals, such as printers and patient smart cards. Referring to FIG. 99, the data generated by the test module 1 can be used to update the stream stored by the epidemiological data host system 1 1 through the reader 1 2 and the network 125 -362 - 201219115

The medical database stored in the phylogenetic database, the genetic data host system 113, the electronic health record stored in the electronic health record (EHR) host system 151, and the electronic medical record (EMR) host system 121 are stored. Electronic medical records, as well as personal health records stored by the Personal Health Record (PHR) host system 123. Conversely, the epidemiological data stored in the epidemiological data host system 1 1 1 , the genetic data stored in the genetic data host system 113, and the electronic health stored in the electronic health record (EHR) host system 115 The recorded, electronic medical records stored in the electronic medical record (EMR) host system 121, and the personal health records stored in the personal health record (PHR) host system 123 can be used to access the network 125 and the reader 1 2 to update the digital memory in the LOC 3 0 of the test module 1 Referring to Figures 1, 2, 96 and 97, the reader 12 in the mobile phone configuration uses battery power. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be downloaded or uploaded via some wireless or contact interface to initiate communication with peripheral devices, computers or online servers. The micro-USB port 16 φ is used to connect a computer or main power supply for charging the battery. Figure 70 shows an embodiment of a test module 10 for requiring only positive or negative test results for a particular target, such as for testing whether a person is infected with, for example, influenza A virus H1N1. Only the USB power/indicator module 47 built for a specific purpose is required. No other readers or application software is required. The indicator 45 on the USB power/indicator module 47 only displays a positive or negative result. This configuration is ideal for large screenings. Other items that may be provided by the system may include a test tube containing reagents for pre-treating a particular sample and a spatula and a lancet for sample collection. Figure 70 - 363 - 201219115 shows the implementation of the test module for the convenience of the spring-loaded telescopic lancet 3 90 and the lancet release button 392. The satellite phone can be used in remote areas. Test Module Electronics Figures 2 and 97 are block diagrams of the electronic components in test modules 10 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a clock 33, a power conditioner 31, a RAM 38, and a program and data flash memory 40. These components provide for the entire test module 1 or 11 including the light sensor 44, temperature sensor 170, liquid sensor 174 and various heaters 152, 154, 182, 234 and associated drivers 37 and 39 and temporary storage The control of the devices 35 and 41 is 100 million. Only LED 26 (with test module 10 as an example), external power capacitor 32 and micro USB connector 14 are external to LOC device 30. The LOC device 30 includes pads for connection to these external components. The RAM 38 and the program and data flash memory 40 have application software and diagnostic and test information for more than 1000 probes (flash/security storage, for example via encryption). In the test module 1 1 designed for ECL detection, the test module 1 1 does not contain the LED 26 (see Figures 96 and 97). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 is loaded with electrochemiluminescent probes each having a pair of ECL excitation electrodes 860 and 870. Many types of test modules 10 are manufactured in several test formats for stock use. The difference between the different test formats is the reagents and probes of the onboard assay. -364- 201219115 Some examples of infectious diseases that can be quickly identified using this system include: • Influenza-influenza virus A, B, C 'Isavirus, Toho goto virus • Pneumonia-Respiratory Syndrome Virus (RSV), Adenovirus, Interstitial Pneumonia Virus, Pneumococci, Staphylococcus aureus • Tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium tuberculosis, Carbachis Mycobacterium and voles

• Plasmodium falciparum, Toxoplasma gondii and other parasitic protozoa • Typhoid-cold bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue-yellow fever virus • Hepatitis (A to E) • Medical source Sexual infections - such as Clostridium pneumoniae, vancomycin-resistant enterococci and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Cytomegalovirus (CMV) • Epstein-Barr virus (EBV) • Brain Inflammation - Japanese encephalitis virus, Zhangdipula virus • Pertussis - pertussis • Measles - paramyxovirus • Meningitis - Streptococcus pneumoniae and Neisseria meningitidis Anthracnose - Bacillus anthracis -365 - 201219115 Some examples of genetic diseases identified using this system include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black idiots • Hemochromatosis • Cerebral arterial disease

• Crohn's disease • Polycystic kidney disease • Congenital heart disease • Lay's disease A few examples of cancers identified by this diagnostic system include: • Ovarian cancer • Colon cancer • Multiple endocrine tumors

• Retinal blastoma • Turcot syndrome The above list is incomplete and the diagnostic system can be configured to use nucleic acid and proteomic analysis to detect more diverse diseases and conditions. Detailed structure of system components LOC device L0C device 30 is the core of the diagnostic system. The device rapidly performs the four major steps of molecular diagnostic analysis of nucleic acid substrates on a microfluidic platform, -366 - 201219115

That is, sample preparation, nucleic acid extraction, nucleic acid amplification, and detection. The LOC device is also of selective use and will be described in detail below. As discussed above, test modules 1 and 11 can take many different configurations to detect different targets. Similarly, LOC device 30 can also be directed to the subject matter of interest to create a variety of different embodiments. One form of LOC device 30 is a LOC device 301 for fluorescent detection of the target nucleic acid sequence of a pathogen in a whole blood sample. For purposes of illustration, the structure and operation of LOC device 301 will now be described in detail with reference to Figures 4 through 26 and 27 through 57. 4 is a representative diagram of the structure of the LOC device 301. For the sake of convenience, the processing stages shown in Fig. 4 are denoted by the component symbols corresponding to the functional portions of the LOC device 301 that implements the processing stage. The processing stages associated with each of the major steps in the diagnostic analysis of the nucleic acid substrate are also shown: sample input and preparation 2 8 8 , extraction 290, culture 291, amplification 292, and detection 294 . The various reservoirs, chambers, valves and other components of the LOC device 301 will be described more closely below. Figure 5 is a perspective view of the LOC device 301. The device is fabricated using high volume CMOS and MST (microsystem technology) fabrication techniques. The layered configuration of the LOC device 301 is illustrated in a schematic (not to scale) partial cross-sectional view of FIG. The LOC device 301 has a germanium substrate 84 that supports a COMS + MST wafer 48 that includes a CMOS circuit 86 and an MST layer 87 and has an upper cover 46 that covers the MST layer 87. For the purposes of this patent specification, the term "MST layer" refers to a collection of structures and layers of a sample treated with various reagents. Accordingly, these structures and components are configured to define a flow path having a feature size that will support the physical properties of the sample driven and processed during capillary action -367 - 201219115

Like the flow of liquid. In view of this, the MST layers and components are typically fabricated using surface micromachining techniques and/or stereo micromachining techniques. However, other manufacturing methods can also produce structures and components that are sized for capillary drive flow and for handling very small samples. The particular embodiment described in this specification shows that the MST layer is a structure and active component supported by CMOS circuitry 86, but does not have the features of upper cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the top cover to process the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 microns X 58 24 microns. Of course, LOC devices made for different applications can have different sizes.

Figure 6 shows the features of the MST layer 87 that overlaps the features of the upper cover. The AA to AD, AG, and AH regions shown in Fig. 6 are enlarged in Figs. 13, 14, 35, 56, 55, and 63, respectively, and are described in detail below to fully understand the respective structures in the LOC device 301. Figures 7 through 10 show the features of the upper cover 46 independently. Figure 11 shows the structure of the CMOS + MST device 48 independently. Hierarchical Structure Figures 12 and 22 illustrate the hierarchical structure of the CMOS + MST device 48, the upper cover 46, and the fluid interaction between the two. The figures are not drawn to scale in order to illustrate the zero. Figure 12 is a schematic cross-sectional view through the sample inlet 68, and Figure 22 is a schematic cross-sectional view through the reservoir 54. Figure 12 clearly shows that the 'CMOS + MST device 48 has an active device within the MST layer 87 on which the CMOS circuit 86 of the CMOS circuit 86 is supported. Passivation layer -368- 201219115 88 seals and protects CMOS layer 86 from flowing into the MST layer 87.

The liquid flows through both the upper cap layer 94 and the MST channel layer 100 in the upper cap layer 46 and the MST channel layer 100 (see, for example, Figures 7 and 16). Cell transport occurs in the larger channel 94' made in the upper cover 46 and biochemical treatment takes place in the smaller MST channel 90. The size of the cell delivery channel is designed to deliver cells in the sample to a predetermined location in the MST channel 90. Cells that transport more than 20 microns (such as some white blood cells) require channel sizes greater than 20 microns, so the cross-sectional area of the traverse flow must be greater than 4 square microns. The position of the MST channel, particularly in the LOC where the cells are not required to be transported, can be significantly smaller. It will be understood that the upper cover channel 94 and the MST channel 90 are generically referred to as 'specific MST channels 90 may also be referred to for their particular function ( For example) heating microchannels or dialysis MS T channels. The MST channel 90 is formed by etching a MST channel layer 100 deposited on the passivation layer 88 and patterned by a photoresist. The MST channel 90 is closed by a top layer 66 that forms the top of the CMOS + MST device 48 (shown in the figure direction). Although sometimes shown in separate layers, the upper cover channel layer 80 and the reservoir layer 78 are formed from a single piece of material. Of course, the sheet material may also be non-unitary. Both sides of the sheet material are etched to form an upper cover channel layer 80 and a reservoir layer 78, and an upper cover channel 94 is etched in the upper cover channel layer 80, and reservoirs 54, 56, 58, 60 are etched in the reservoir layer 78. 62. Alternatively, the reservoir and the upper cover channel are formed by micromolding. Both etching and micromolding techniques are used to fabricate channels having a cross-sectional area of up to 20,000 square microns and a minimum of 8 square microns across the fluid. ' -369- 201219115 For different locations in the LOC unit, you can select the appropriate cross-sectional area of the cross-flow fluid. A cross-sectional area of up to 20,000 square microns (e.g., a channel having a width of 200 microns in a layer having a thickness of 100 microns) is appropriate when a large number of samples or samples having a large component are accommodated in the channel. Preferably, the cross-sectional area of the fluid across the fluid is very small when the channel contains a small amount of liquid or a mixture free of large cells.

Lower sealing layer 64 encloses upper cover passage 94, upper sealing layer 8 2 encloses reservoirs 54, 56, 58' 60 and 62°. Five reservoirs 54, 56, 58, 60 and 62 preload reagents of particular analysis. In the embodiments described herein, the reservoirs are preloaded with the following reagents, but can be easily replaced with other reagents: • Reservoir 54: anticoagulant, optionally including red blood cell lysate • reservoir 56: lysis Reagent

• Reservoir 5 8: restriction enzymes, ligases and linkers (for linker initiation PCR (see Figure 69, excerpt from T. Stachan et al., Human Molecular Genetics 2, Garland Science, NY and London, 199 9) 9)). Reservoir 60: amplification mixture (dNTP, primer, buffer), and reservoir 62: DNA polymerase. Upper cover 46 and CMOS + MST layer 48 are in fluid communication via corresponding openings in lower seal 64 and top layer 66. These openings are referred to as ascending port 96 and descending port 92-370-201219115 according to the flow system flowing from the MST channel 90 to the upper cap channel 94 or vice versa. The operation of the LOC device 301 is based on the analysis of pathogenic DNA in the blood sample. The examples are described step by step. Of course, other types of biological or non-biological fluids can also be analyzed using appropriate reagents, test procedures, LOC variants, and combinations or combinations of detection systems. Referring to Figure 4, the analytical biological sample is largely divided into five steps, including sample input and preparation 28, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294.

Sample input and preparation step 28 8 involves mixing the blood with the anticoagulant 1 16 followed by separation of the pathogen from the pathogen dialysis section 70 with white blood cells and red blood cells. As best seen in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action draws the blood sample along the upper cover channel 94 to the reservoir 54. When the sample blood stream opens the surface tension valve 18 of the reservoir 54, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants prevent clot formation from obstructing flow. As best shown in Fig. 22, the anticoagulant 116 is aspirated from the reservoir 54 by capillary action and enters the MST channel 90 via the descending port 92. The drop port 92 has a capillary initiation feature (CIF) 102 to control the geometry of the meniscus such that the meniscus is not fixed at the edge of the drop port 92. The venting opening 122 in the upper sealing layer 82 allows air to replace the anticoagulant 116 that is aspirated from the reservoir 54. The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and secures the meniscus 120 to the meniscus anchor 98 of the riser 96. Prior to use, the meniscus 120 remains fixed at the riser 96 so that the anticoagulant does not flow into the upper lid passage 94. As the blood flows through the upper cover passage 94 to the ascending port 96, the meniscus 120 is removed by the -371 - 201219115 and the anticoagulant is drawn into the flow. Figures 15 through 21 show the AE zone, which is part of the AA zone shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 118 has three separate MST passages 90 extending between the respective lowering port 92 and the rising port 96" MS T channel 90 in the surface tension valve The number can vary to change the flow rate of the reagent into the sample mixture. The concentration of the reagent in the sample stream is determined by the flow rate of the sample mixture and reagents as they flow out of the reservoir. Thus, the surface tension valve of each reservoir is configured to meet the desired reagent concentration. The blood enters the pathogen dialysis section 7 (see Figures 4 and 15), where the target cells are concentrated from the sample using a well array of sizes 1 64 according to a predetermined valve. Cells smaller than the valve pass through the hole, and large cells cannot pass through the hole. The undesired cells are either larger cells that are blocked by the well array 164 or smaller cells that pass through the well, which are transduced to the waste unit 76, but the target cells are still part of the analysis. In the pathogen dialysis section 70 described herein, the pathogen system from the whole blood sample is concentrated for microbial DN A analysis. The array of holes is formed by a plurality of 3 micrometer diameter holes 164 that fluidly communicate with the input flow in the upper cover channel 94 to the target channel 74. The 3 micron diameter aperture 164 and the dialysis riser aperture 168 in the target channel 74 are connected by a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The pathogen is small in volume and can therefore be filled through the 3 micron diameter aperture 164 and filled through the dialysis MST channel 206. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 of the upper cover 46, which leads to the waste receptacle 76 (see Figure 7). -372- 201219115 Other pore shapes, sizes, and aspect ratios can be utilized to isolate specific pathogens or other target cells, such as white blood cells for human DN A analysis. Detailed instructions for the dialysis section and dialysis variants are provided below.

Referring again to Figures 6 and 7, fluid is drawn through the target passage 74 to the surface tension valve 128 of the lysis reagent reservoir 56. The surface tension valve 128 has seven M ST channels 90 that extend between the lysis reagent reservoir 56 and the target channel 74. When the meniscus is removed by the sample stream, the flow rate from all seven MST channels 90 will be greater than the flow rate from the anticoagulant reservoir 54, wherein the surface tension valve 118 of the reservoir 54 has three MST channels 90 (hypothesis) The physical properties of the liquids are approximately equal). Thus the ratio of lytic reagent to sample mixture is greater than the ratio of anticoagulant to sample mixture. The lysis reagent and the target cells are mixed by diffusion in the target channel 74 in the chemical lysis section 130. The boiling start valve 126 stops the flow until sufficient time for diffusion and lysis has occurred to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling start valve are described in detail below with reference to Figs. Other active valve types (as opposed to passive valves such as surface tension valve 118) have also been developed by the applicant, and other types of active valves can be used herein to replace the boiling start valve. These alternative valve designs are also described below. When the boiling start valve 126 is opened, the lysed cells flow into the mixing portion 133 to perform restriction enzyme cleavage and linker ligation before amplification. Referring to Figure 13', when the fluid removes the meniscus of the surface tension valve 132 at the beginning of the mixing portion 131, the restriction enzyme, linker and ligase are released from the reservoir 58. This mixture flows through the length of the mixing portion 131 to be diffused and mixed. The end of the mixing -373-201219115 portion 131 is the lowering port 134 leading to the incubator inlet passage 133 of the culture portion 114 (see Fig. 13). The incubator inlet channel 133 feeds the mixture into a heated microchannel 210 in a crimped configuration that provides a culture chamber for holding the sample during restriction enzyme cleavage and linker ligation (see Figures 13 and 14). 9 Figures 23, 24, 25, 26, 27' 28 and 29 show the layers of the LOC device 301 in the AB region of Figure 6. Each of the figures shows a layer of successive additions to form the structure of the CMOSS + MST layer 48 and the upper cover 46. The AB region shows the end point of the culture portion 114 and the starting point of the amplification portion 1 12 . As best shown in Figures 14 and 23, the fluid fills the microchannel 210 of the culture portion 14 until it reaches the boiling start valve 106 where the fluid stops to allow diffusion to occur. As discussed above, the microchannel 210 in the upstream of the boiling start valve 106 becomes the culture chamber containing the sample, restriction enzyme, ligase, and linker. Heater 154 is then activated and maintains a stable temperature for a specific period of time to allow restriction enzyme cleavage and linker engagement to occur. Those skilled in the art will appreciate that this culturing step 291 (see Figure 4) is optional and is only required for some types of nucleic acid amplification assays. In addition, in some instances, it may be desirable to provide a heating step at the end of the incubation period to cause the temperature to rise above the culture temperature. This temperature rise causes the restriction enzyme and the ligase to deactivate before entering the amplification section 112. Deactivation of restriction enzymes and ligases is particularly important when using isothermal nucleic acid amplification. After the incubation, the boiling start valve 106 is activated (opened) and the fluid continues to enter the amplifying portion 112. Referring to Figures 31 and 32, the mixture is filled with a heated microchannel 1 58 in a twisted configuration until it reaches a boiling start valve 108, which forms one or more amplification chambers. As is clear from the cross-sectional view of Fig. 30, the amplification -374-201219115 mixture (dNTP, primer, buffer) is released from the reservoir 60 and the polymerase is then released from the reservoir 62 into the junction culture section and the amplification section (respectively Intermediate MST channel 212 of 114 and 112).

Figures 35 through 51 show the layers of LOC device 301 in the AC region of Figure 6. The figures show the layers of successive additions to form the structure of the CMOS + MST device 48 and the upper cover 46. The end of the AC region amplification unit 112 and the starting point of the hybridization and detection unit 52. The cultured sample, amplification mixture, and polymerase flow through microchannel 158 to boiling start valve 108. After sufficient time for diffusion mixing, the heaters 15 in the microchannels 158 are activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification procedure, the boiling start valve 108 is opened and the fluid continues to enter the hybridization and detection portion 52. The operation of the boiling start valve is described in detail below. As shown in Fig. 52, the hybridization and detection unit 52 has a hybridization chamber array 1 10 . Figures 52, 53, 54, and 56 show hybrid chamber array 110 and single hybridization chamber 180 in detail. A diffusion barrier 175 is provided at the entrance to the hybridization chamber 180 to prevent diffusion of the labeled nucleic acid, probe strands, and hybridized probes between hybridization chambers during hybridization to prevent erroneous hybridization assay results. The diffusion barrier 175 represents a flow path of sufficient length to prevent the target sequence and probe from diffusing out of one chamber and contaminating another chamber during the time required for the probe and nucleic acid hybridization and signal to be detected, thus avoiding erroneous results. Another mechanism to prevent erroneous results is to include the same probe in multiple hybridization chambers. CMOS circuit 86 derives a single result from photodiode 184 corresponding to hybridization chamber 180 containing the same probe. Abnormal when deducing a single result

S -375- 201219115 Results can be ignored or given different weights.

The thermal energy required for hybridization is provided by a CMOS controlled heater 182 (described in more detail below). After activation of the heater, hybridization occurs between complementary target-probe sequences. The LED driver 29 in the CMOS circuit 86 transmits a message to the LED 26 located in the test module 1 to illuminate it. These probes only fluoresce when hybridization occurs, so the cleaning and drying steps typically required to remove unbound strands are omitted. The stem loop structure of the hybrid forced FRET probe 186 is opened, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as described in detail below. Fluorescence is detected by photodiodes 184 located in CMOS circuitry 86 below each hybridization chamber 180 (see description of hybridization chambers below). Photodiode 184 and associated electronic devices for all hybridization chambers together form a light sensor 44 (see Figure 64). In other embodiments, the light sensor can be an array of charge coupled devices (CCD arrays). The detection signal from the photodiode 1 84 is amplified and converted to a digital output for analysis by the test module reader 12. Further details of the detection method are described below. Other Detailed Description of the LOC Device The modular design LOC device 301 has a number of functional components (including reagent reservoirs 54, 56, 58, 60 and 62, dialysis section 70, lysis section 130, culture section 114, and amplification section 1 1 2), valve type, humidifier and humidity sensor. In other embodiments of the LOC device, these functional portions may be omitted, but additional functional portions may be added or the functional portions may be used for different purposes than those described above. -376- 201219115

For example, the culture portion 114 can be used as the first amplification portion 112 of the tandem amplification analysis system, and the chemical lysis reagent reservoir 56 is used to add the first amplification primer, dNTP, and buffer. The mixture, and reagent reservoir 58 are used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with the amplification mixture) may also be added to the reservoir 56, or alternatively, the sample may be heated for a predetermined period of time to cause thermal lysis in the culture section. . In some embodiments, if chemical lysis is desired and it is desirable to separate the chemical lysis reagent from the primer, dNTP, and buffer mixture, an additional reservoir can be inserted upstream of the reservoir 58 immediately adjacent to the mixture. In some cases it may be desirable to omit steps, such as cultivation step 291. In this case, the LOC device may be specially manufactured to avoid the reagent reservoir 58 and the culture portion 114, or may simply not be loaded with the reagent, or if an active valve is present, the active valve is not activated to release the reagent to In the sample stream, the culture portion becomes a channel for transferring only the sample from the lysis unit 130 to the amplification portion φ Π 2 . The heaters are independently operable, so when the reaction is dependent on heating, such as a hot lysis reaction, setting the heater off during this step ensures that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the fluid system within the microfluidic device as shown in Figure 4, or can be located at any other location within the microfluidic device. For example, in some cases, it may be advantageous to dialyze after the amplification phase 2 9 2 to remove cell debris prior to hybridization and detection step 2 94. Alternatively, two or more dialysis sections can be inserted at any location in the LOC device. Similarly, it is possible to inject additional amplifications 112 such that the multiple targets can be amplified simultaneously or sequentially prior to detection in the hybridization chamber array no with the special -377-201219115 nucleic acid probe. Since the dialysis is not required for analyzing a sample such as whole blood, the dialysis unit 70 is simply omitted from the sample input and preparation unit 288 of the LOC design. In some cases, it is not necessary to omit the dialysis section 70 of the LOC device, even if the analysis does not require dialysis. If the presence of the dialysis section does not cause a geometric obstruction to the analysis', the LOC for the dialysis section 70 can still be used for the sample input and preparation section without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to the protein of the sample target present in the non-amplified sample, rather than a design. A nucleic acid probe that hybridizes to a target nucleic acid sequence. It will be appreciated that the LOC devices manufactured for use with this diagnostic system are different combinations of functional components selected for a particular LOC application. The vast majority of functional units are the same for many LOC devices, and the design of additional LOC devices for new applications relates to the combination of appropriate functional components from the various functional options used in existing LOC devices. Only a few LOC devices are shown in this description. 'More LOC devices are illustrated in a diagram to illustrate the design flexibility of the LOC device manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices described in this specification are not exhaustive, and that many additional 1"0^^ designs are suitable for the assembly of appropriate functional combinations. Sample type LOC variants are acceptable and analytically liquid. Form of sample type -378- 201219115

Nucleic acid or protein content, including but not limited to blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, cord blood, breast milk, sweat, pleural effusion, tears, pericardial fluid, peritoneal fluid , environmental water samples and beverage samples. Amplicon obtained from macroscopic nucleic acid amplification can also be analyzed using the LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, and the dialysis, lysis The culture and amplification section will only be used to deliver the sample from the sample inlet 68 to the hybridization chamber 180 for nucleic acid detection as described above. Some sample types require a pre-treatment step prior to input into the LOC device. For example, semen may require liquefaction and mucus may require enzyme pretreatment to reduce viscosity. Sample Input Referring to Figures 1 and 12, the sample is added to the large container 24 of the test module 1 . The large container 24 is a truncated cone that is fed by capillary action into the inlet 68 of the φ LOC device 301. The sample is here flowed into a 64/m width x 60 from the m depth over the lid channel 94 and is also attracted to the anticoagulant reservoir 54 by capillary action. Reagent Reservoirs A small amount of reagents required for the analysis system of a microfluidic device, such as LOC device 301, is used to allow the reagent reservoirs to contain all of the reagents required for biochemical treatment in each reagent reservoir having a small volume. This volume must be less than 1, 〇〇〇, 〇〇〇, 〇〇〇 cubic micron, mostly less than 3 〇〇, 〇〇〇, 〇〇〇 cubic micron, -379- 201219115 usually less than 70, 〇〇〇, 〇〇 〇 cubic micron, and in the illustrated LOC device 301 is less than 20, 〇〇〇, 〇〇〇 cubic micron. Dialysis Section Referring to Figures 15 through 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate cells from the pathogen of the sample. As previously described, a plurality of apertures 164 having a 3 micron diameter opening in the top layer 66 filter the target cells from the sample body. As the sample flows through a 3 micron diameter hole 1 64, the microbial pathogen enters a series of dialysis MST channels 204 through the opening and flows back into the standard channel 74 via the 16 #m dialysis rising hole 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) remains in the upper cover channel 94. Downstream of the pathogen dialysis section 70, the upper cover passage 94 becomes a waste passage 72 to the waste reservoir 76. For a biological sample type that produces a large amount of waste, a foam insert or other porous element 49 in the outer casing 13 of the test module 10 is configured to be in fluid communication with the waste reservoir 76 (see Figure 1). The function of the pathogen dialysis section 70 is entirely dependent on the capillary action of the fluid sample. A 3 micron diameter hole 164 at the upstream end of the pathogen dialysis section 70 has a capillary initiation feature (CIF) 166 (see Figure 33) to allow fluid to be drawn downward into the dialysis MST channel 204 below it. The first rising aperture 198 of the target channel 74 also has a C IF 202 (see Figure 15) to prevent fluid from easily forming a meniscus in the dialysis rising aperture 168. The small component dialysis section 682 shown in Fig. 74 may have a structure similar to that of the pathogen analysing section 70. The small component dialysis section separates cells or molecules from any small label of the sample by the size (and shape if necessary) of the well, -380-201219115. The well is adapted to allow the small cells or molecules to pass through the target channel and continue further. analysis. Large size cells or molecules are removed to the waste reservoir 76 6 . Therefore, the LOC device 30 (see Figs. 1 and 96) is not limited to isolation of pathogens having a size of less than 3 // m, and can also be used to separate cells of any desired size or molecular lysis.

Referring again to Figures 7, 11, and 13, the genetic material in the sample is released from the cell by chemical lysis. As described above, the lysing reagent from the lysis reagent reservoir 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 128 of the lysis reagent reservoir 56. However, some diagnostic assays are more suitable for use with hot lysis, or even a combination of chemical and thermal lysis of the target cells. The LOC device 301 fills the heating microchannel 210» of the mixing portion 114 with the sample stream to fill the culture portion 11 and stops at the boiling start valve 106. The culture microchannel 210 heats the sample to a temperature at which the cell membrane ruptures. The enzyme reaction in the chemical lysis unit 130 is not required in some hot lysis applications, and the enzyme reaction in the chemical lysis unit 130 is completely replaced by the hot lysis. The boiling start valve is as described above, and the LOC device 301 has three boiling points. Valves 126, 106 and 108 are activated. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view showing the boiling start valve 108 shown separately at the end of the heating microchannel 158 of the amplifying portion 112. The sample stream 1 19 is attracted through the heated microchannel by capillary action -381 - 201219115

1 5 8 until the boiling start valve 1 0 8 is reached. The meniscus 120 is fixed to the meniscus anchor 98 of the valve inlet 146 prior to the sample flow. The geometry of the meniscus retainer 98 stops the advancing meniscus to prevent capillary flow. As shown in Figures 31 and 32, the meniscus anchor 98 is provided by a hole provided from the MST passage 90 to the raised opening of the upper cover passage 94. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 152 is located around the valve inlet 146. The ring heater 152 is controlled by C Μ Ο S via the boiling start valve heater contact 153.

To open the valve, the CMOS circuit 86 delivers an electrical pulse to the valve heater contact 153. The ring heater 152 is heated by electrical resistance until the liquid sample 119 is boiled. This boiling removes the meniscus 120 from the valve inlet 146 and begins to wet the upper cover passage 94. Once the upper cover passage 94 is wetted, the capillary action is restored. The fluid sample Π 9 fills the upper lid passage 94 and flows through the valve lowering port 150 to the valve outlet 148 where the capillary driven liquid flow continues to advance along the amplifying portion outlet passage 160 to the hybridization and detection portion 52. Liquid sensor 174 is placed before and after the valve for diagnostic purposes. It will be appreciated that once the boiling start valve is opened, it can no longer be closed. However, since the LOC device 301 and the test module 1 are single-use devices, there is no need to close the valve. Culture section and nucleic acid amplification section Figures 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51 show the culture section Π 4 and the amplification section 1 1 2 . The culture portion 141 has a single strip of heated culture microchannels 210 which are etched into a meandering pattern in the MST channel layer 1〇〇, -382-201219115 starting at the lowering port 134 and finally boiling the activation valve 106 (see Figure 13). And 14). Controlling the temperature of the culture portion 114 allows the enzyme reaction to occur with higher efficiency. Similarly, the amplifying portion 112 has a heating amplifying microchannel 158 (see Figs. 6 and 14) which is a bouncing structure that starts from the boiling start valve 106 to the boiling start valve 1〇8. When mixing, culture, and nucleic acid amplification occurs, the valves stop the flow to retain the labeled cells in the heated culture or amplification microchannel 210 or 158. The curved pattern of the microchannels also promotes (to some extent) the target cells and reagents

In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated by a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each curved corner of the heated culture microchannel 210 and the expanded microchannel 158 has three independently operable heaters 15 4 extending between the individual heater contacts 156 (see Figure 14)), which provides two-dimensional control of the input heat flux density. As best shown in Figure 51, the heater 154 is supported by the top layer 66 and embedded in the lower sealing layer 64. The material of the φ heat exchanger is TiAl, but many other conductive metals are also suitable. The elongated heater 154 is parallel to the longitudinal length of each of the channel portions forming the wide curve of the curved structure. In the amplification section 112, each wide curve can be controlled via an individual heater as a separate PCR chamber. The use of a small volume of amplicons required for the analysis system of a microfluidic device, such as LOC device 301, allows for the use of a small volume of amplification mixture in amplification portion 112 for amplification. The volume must be less than 400 nanoliters, mostly less than 17 nanoliters, usually less than 70 nanoliters, and the LOC device 301 is used as an example of between 2 nanoliters and 30 nanoliters. *-383-201219115 heating rate increase and comparison The small cross-sectional area of each of the channels of the diffusion mixing increases the heating rate of the amplification fluid mixture. The distance between all fluids and heaters 154 is quite short. Reducing the cross-sectional area of the channel (i.e., the cross-section of the augmented microchannel 158) to less than 100,000 square microns provides a significantly higher heating rate than a "large scale" device. The lithography technique allows the amplifying microchannel 158 to have a cross-sectional area across the flow path of less than 16,000 square microns, which provides a substantially higher heating rate. Dimensional features of 1 micron size are readily available using lithography manufacturing techniques. If only a very small amount of amplicons are required (in the case of LOC device 301), the cross-sectional area can be reduced to less than 2,500 square microns. For a diagnostic analysis performed on a LOC device with 1, 〇〇〇 to 2,000 probes and requiring "input of the sample, yielding results" within 1 minute, a cross between 400 square microns and 1 square micron The cross-sectional area of the fluid is appropriate. The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate of greater than 80 absolute temperatures (K) per second, in most cases at a rate of greater than 100 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than 1 〇〇〇 κ per second, and the heater element often heats the nucleic acid sequence at a rate greater than 1 0000 K per second. Typically, the heater element is greater than 100,000 K per second, greater than 1,000,000 K per second, greater than 10, 〇〇〇, 000 K per second, greater than 20,000,000 K per second, and greater than 40 per second, depending on the needs of the analytical system. The nucleic acid sequence is heated at a rate of 000,000 K, greater than 80 per second, 〇〇〇, 〇〇〇K, and greater than 1600,000,000 K per second. Small cross-sectional area channel is also beneficial for the diffusion of any reagent and sample fluid -384 - 201219115

mixing. The phenomenon of one liquid diffusing to another liquid is most pronounced near the interface between the two liquids before diffusion mixing is completed. The concentration decreases as the distance from the interface increases. The use of microchannels having a relatively small cross-sectional area across the fluid direction causes the two fluids to flow closer to the interface for faster diffusion mixing. Reducing the cross-sectional area of the channel to less than 100,000 square microns provides a significantly higher mixing rate than "large scale" equipment. The lithography manufacturing technique allows the microchannel to cross the flow path with a cross-sectional area of less than 16,000 square microns, which provides a significantly higher mixing rate. If only a very small volume (in terms of LOC device 301) is required, the cross-sectional area can be reduced to less than 2500 square microns. Cross-fluid section between 400 square microns and 1 square micron for diagnostic analysis on a LOC device with 1000 to 2000 probes and requiring "input sample, result" in 1 minute The area is appropriate. The rapid thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid to perform a rapid thermal cycle during nucleic acid amplification. For sequences of up to 150 base pairs (bp) in length, each thermal cycle (i.e., denaturation, adhesion, and primer extension) is completed in less than 30 seconds. In most diagnostic analyses, the individual thermal cycle time is less than 11 seconds and most are less than 4 seconds. For the LOC device 30, which performs some of the most common diagnostic analyses, the thermal cycle time for a sequence of up to 150 base pairs (bp) is between 0. 45 seconds and 1.5 seconds. This rate of thermal cycling allows the test module to complete the nucleic acid amplification process in as little as 10 minutes; usually in less than 220 seconds. -385- 201219115 For most analyses, the amplification produces enough amplicons in less than 80 seconds after the sample fluid enters the sample inlet. Many assays produce enough amplicons in 3 seconds. When a predetermined number of amplification cycles are completed, the amplicon is fed to the hybridization and detection portion 52 via the boiling start valve 108. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show the hybrid chamber 180 in the hybrid chamber array 11A. The hybridization and detection section 52 has a 24x45 array 110 of hybridization chambers 180 each having a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. The photodiode 184 is introgressed to detect fluorescence produced by hybridization of the target nucleic acid sequence or protein to the FRET probe 186. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. Any substance between the FRET probe 186 and the photodiode 184 must be transparent to the emitted light. Therefore, the wall portion 97 between the probe 186 and the photodiode 184 can also be optically penetrated by the emitted light. In LOC device 301, wall portion 97 is a thin layer of hafnium oxide (about 0.5 microns). Direct injection of photodiode 184 beneath each hybridization chamber 180 allows for the detection of a very small probe-target hybrid volume but still produces a detectable fluorescent signal (see Figure 5 4). This small amount allows the use of a small volume of hybridization chamber. The amount of detectable probe-target hybrid requires a pre-hybridization probe that is less than 270 picograms (corresponding to 900,000 cubic micrometers), and in most cases less than 60 picograms (corresponding to 200,000 cubic micrometers) ''usually less than 12 picograms (corresponding to 40,000 cubic micrometers), and is less than 2.7 picograms (corresponding to a chamber volume of 9,000 cubic micrometers) as an example of the LOC device 301-386-201219115 shown in the accompanying drawings. Of course, reducing the size of the hybridization chamber allows for a higher density chamber' thus allowing the LOC device to have more probes. In the LOC device 301, the hybrid portion has more than one, 〇〇〇 chambers (i.e., less than 2,250 square microns per chamber) in an area of 1,500 micrometers by 1,50 micrometers. Smaller volumes also reduce reaction time, so hybridization and detection can be faster. Another advantage of requiring a small number of probes in each chamber is that only a very small amount of probe is required to be dissolved during the manufacture of the LOC device.

Liquid into each room. Embodiments of the LOC device of the present invention can be spotted using a probe solution having a volume of 1 picoliter or less. After nucleic acid amplification, the boiling start valve 108 is activated and the amplicon flows along the flow path 176 and flows into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 displays the point at which the hybridization chamber 180 is filled with the amplicon and the heater can be activated. After a sufficient hybridization time, LED 26 (see Figure 2) is activated. The openings in each of the hybridization chambers 180 are provided with light windows 136 to expose the FRET probes 186 to φ excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a time long enough to induce a high intensity fluorescent signal from the probe. Photodiode 184 is shorted during excitation. After a preprogrammed delay 300 (see Figure 2), the photodiode 184 is enabled and detects fluorescent emissions in the absence of excitation light. The incident light on the active region 185 (see Fig. 54) of the photodiode 184 is converted into a photocurrent that can then be measured by the CMOS circuit 86. The hybridization chambers 180 each carry a probe for detecting a single target nucleic acid sequence. If desired, each hybridization chamber 180 can carry probes that detect more than 1,000 different targets. Alternatively, many or all of the hybridization chambers may carry the same probe to repeat -387 - 201219115 to detect the same target nucleic acid. Copying the probes in the hybridization chamber array 110 in this manner results in increased confidence in the results obtained, if desired to provide a single result by combining the results of the photodiodes adjacent to the hybridization chambers. Those skilled in the art will appreciate that there may be from 1 to more than 10,000 different probes on the hybrid chamber array 110, depending on the analytical specification. Humidifier and Humidity Sensor Figure 6 AG Region Indication The position of the humidifier 196. The humidifier prevents reagents and probes from evaporating during operation of the LOC device 301. Preferably, as shown in the enlarged view of Figure 55, the reservoir 188 is in fluid communication with the three evaporators 190. The water reservoir 1 88 contains molecular biological grade water and is sealed during manufacture. Preferably, as shown in Figures 55 and 67, water is drawn by capillary action to the three descending ports 194 and along the individual water supply passages 192 to the three riser ports 193 of the evaporator 190. The meniscus is fixed to each of the risers 193 to retain water. The evaporator has a ring heater 119 which surrounds the riser 1 93. The ring heater 191 is connected to the top metal layer 195 of the CMOS circuit 86 by a conductive post 376 (see Fig. 37). At startup, the ring heater 191 heats the water to evaporate and wet the surrounding equipment. Figure 6 also shows the location of the humidity sensor 232. However, as best shown in the enlarged view of the AH zone of Figure 63, the humidity sensor has a capacitive comb structure. The lithographically etched first electrode 296 and the lithographically etched second electrode 298 are opposed to each other such that their teeth are interleaved. The opposing electrode forms a capacitor having a capacitance that can be monitored by CMOS circuit 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, resulting in an increase in capacitance -388- 201219115. The humidity sensor 232 is adjacent to the array of hybridization chambers 1 1 where the humidity measurement is important to slow the evaporation of the solution containing the exposed probe. Feedback sensor

Temperature and liquid sensors are inserted into the LOC device 301 to provide feedback and diagnostics during operation of the device. Referring to Fig. 35, nine temperature sensors 170 are distributed throughout the amplifying portion 112. Similarly, the culture unit 114 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control thermal cycling during nucleic acid amplification as well as thermal lysis and any heating during incubation. In hybridization chamber 180, CMOS circuit 86 uses hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of the hybrid heater 182 is temperature dependent and the CMOS circuit 86 utilizes this to derive temperature readings for each of the hybrid chambers 180. The LOC device 301 also has a number of MST channel liquid sensors 174 and an upper cover channel liquid sensor 208. Figure 35 shows a row of MST channel liquid sensors 174 at one end of each corner of the heated microchannel 158. Preferably, as shown in FIG. 37, the MST channel liquid sensor 174 is formed by a pair of electrodes formed by exposed regions of the top metal layer 195 of the CMOS structure 86. The liquid seals the current between the electrodes to indicate their presence at the sensor. Figure 25 shows an enlarged perspective view of the upper cover channel liquid sensor 208. Relative TiAl electrode pairs 218 and 220 are deposited on top layer 66. Between the electrodes 2 18 and 220 is a gap 222 which is used to keep the circuit open in the absence of liquid - 389 - 201219115. The circuit is turned off when the liquid is present and the CMOS circuit 86 utilizes this feedback to monitor the flow. Non-gravity dependent test module 1 is non-directional dependent. These modules do not need to be fixed to a smooth surface to operate. The fluid flow driven by the capillary action and the external tubing that does not have to be connected to the auxiliary device make the module truly portable and can be easily inserted into a similar portable handheld reader, such as a mobile phone. Operation with non-gravity dependence means that these test modules also contribute to all practical applications. They can withstand shock and vibration, they can operate on moving vehicles, or when the mobile phone is moving.

Nucleic Acid Amplification Variants Direct PCR customary 'PCR requires extensive purification of the target DNA prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentrations, it is possible to perform nucleic acid amplification or direct amplification with minimal DNA purification. When the nucleic acid amplification program is PCR, this method is called direct PCR. When the nucleic acid amplification is carried out in a controlled constant temperature LOC unit, the method is directly thermostated. Direct nucleic acid amplification techniques have significant advantages when used in LOC devices, especially with regard to simplifying the fluid design required. Adjustments to the amplification chemistry of direct PCR or direct isothermal amplification include increasing buffer strength, using highly active and processivity polymerases and additives chelated with latent polymerase inhibitors. The inhibitor in the diluted sample is also -390- 201219115 important.

To take advantage of direct nucleic acid amplification techniques, the LOC device design incorporates two additional features. The first feature is a reagent reservoir (e.g., reservoir 58 in Figure 8) that is suitably sized to supply a sufficient amount of amplification reaction mixture or diluent to cause the sample to interfere with amplification chemistry The final concentration of the ingredients is low enough to allow for successful nucleic acid amplification. The desired dilution factor of the non-cellular sample component is between 5 and 20 times. Different LOC structures are used where appropriate to confirm that a sufficiently high target nucleic acid sequence concentration is maintained for amplification and detection, such as the pathogen dialysis section 70 of Figure 4. In this embodiment (further illustrated in Figure 6), a concentration that effectively concentrates the pathogens that are small enough to enter the amplification section 292 and discharges the larger cells to the dialysis section of the waste container 76 is used upstream of the sample extraction section 290. . In another embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is the channel aspect ratio design to adjust the mixing ratio between the sample and the amplified mixed components. For example, to ensure that the inhibitor associated with the sample is diluted to a preferred range of 5 to 20 times via a single mixing step, the length and cross-section of the sample and reagent channels are designed to be at the beginning of the mixing The upstream sample channel has a flow impedance that is 4 to 19 times higher than the flow impedance of the channel through which the reagent mixture flows. The flow impedance of the microchannel can be easily controlled by controlling the design geometry. In terms of a fixed cross-sectional area, the flow impedance of the microchannel increases linearly with the length of the channel. It is important for the hybrid design that the flow impedance in the microchannel is most dependent on the smallest cross-sectional area size. For example, when the -391 - 201219115 aspect ratio is extremely non-uniform, the flow of the microchannel with a square cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse Transcriptase PCR (RT-PCR) When the sample nucleic acid type analyzed or extracted is RN A, n is like! & From RNA viruses or messenger RNA, RNA must first be reverse transcribed into complementary DNA (cDNA) before PCR amplification. The reverse transcription reaction can be carried out in the same chamber as the PCR (one-step RT-PCR), or it can be a separate initial reaction (two-step RT-PCR). In the LOC variant described herein, a one-step RT-PCR can be performed by simply adding a reverse transcriptase to a reagent reservoir 62 containing a polymerase and staging the heater 154 to perform a reverse transcription step before performing the nucleic acid The amplification step is carried out. The two-step RT-PCR can also use the reagent reservoir 58 to store and distribute the buffer, primer, dNTP and reverse transcriptase, and use the culture unit 1 to perform the reverse transcription step, followed by the amplification step II2. Amplification in the usual manner is simply accomplished. Thermostatic Nucleic Acid Amplification For some applications, thermostatic nucleic acid amplification is a preferred method of nucleic acid amplification, so that the reaction component does not need to be subjected to repeated cycles of different temperatures, but the amplification is maintained at typically about 37 °C. To the constant temperature of 4 plant C. Some thermostatic nucleic acid amplification methods have been described, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence basal amplification (NASBA), recombinase polymerase amplification (RPA), helicase Dependent thermostatic DNA amplification (HDA), rolling cycle amplification (RCA), branched-type amplification (RAM), and circular mediation constant-392-201219115 temperature amplification (LAMP), any of these methods or others The isothermal amplification method can be used in a particular embodiment of the LOC device described herein.

In order to perform a thermostatic nucleic acid amplification, the reagent reservoirs 60 and 62 adjacent to the amplification section will carry appropriate reagents for a particular constant temperature method rather than carrying a PCR amplification mixture and a polymerase. For example, in the case of SDA, the reagent reservoir contains an amplification buffer, an primer, and dNTP, and the reagent reservoir 62 contains an appropriate endonuclease and an exo-DNA polymerase. In the case of RPA, the reagent reservoir 60 contains amplification buffer, primer, dNTP, and recombinase protein, and the reagent reservoir 62 contains a strand-substituted DNA polymerase such as Bsu. Similarly, in the case of HD A, the reagent reservoir 60 contains amplification buffer, primers and dNTPs, and the reagent reservoir 62 contains appropriate DNA polymerase and helicase to unwind the double DN A instead of using heat. . Those skilled in the art will appreciate that the necessary reagents can be dispensed into the two reagent reservoirs in any manner suitable for the nucleic acid amplification process. To amplify viral nucleic acids from RN A viruses such as HI V or hepatitis C virus, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDN A first. In this example, the reagent reservoir 60 is filled with an amplification buffer, an primer, and dNTP, and the reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase. Some forms of thermostatic nucleic acid amplification must have an initial denaturation cycle to separate the double-stranded DN Α template before maintaining the temperature suitable for constant temperature nucleic acid amplification for the reaction to proceed. This can be easily achieved in embodiments of all of the LOC devices described herein because the temperature of the mixing in the amplification section 112 can be carefully controlled by the heater 154 in the amplification microchannel 158 (see Figure 14). . Thermostatic nucleic acid amplification is more tolerant to potential inhibitors in the sample -393-201219115 and is therefore generally suitable for situations where direct nucleic acid amplification from the sample is desired. Therefore, thermostatic nucleic acid amplification is sometimes used for LOC variant XLIII 673, LOC variant XLIV 674, and LOC variant XLVII 677, etc., shown in Figures 75, 76 and 77, respectively. Direct thermostatic amplification can also be combined with one or more pre-amplification dialysis steps 70, 686 or 682 as shown in Figures 75 and 77, and/or a pre-hybridization dialysis step 682 as shown in Figure 76, respectively. Part of the target cells in the sample are concentrated prior to nucleic acid amplification, or unwanted cell debris is removed before the sample enters the hybridization chamber array 110. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybridization dialysis can be used. Thermostatic nucleic acid amplification can also be performed in parallel amplifications such as those shown in Figures 71, 72, and 73. Multiplex and some methods of constant temperature nucleic acid amplification such as LAMP are compatible with the initial reverse transcription step to amplify RN A . Additional details of the fluorescence detection system Figures 5 and 59 show hybridization-reactive FRET probes 23 6 . These probes, often referred to as molecular beacons, are stem-loop probes produced from single-stranded nucleic acids that fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization to the underlying nucleic acid sequence 23 8 . The probe has a ring 240, a stem 2 42 , a fluorophore 2M at the 5' end, and a quencher 248 at the V end. The loop 240 consists of a sequence complementary to the underlying nucleic acid sequence 238. The complementary sequences on both sides of the probe sequence are affixed together to form stem 242. When the complementary target sequence is absent, the probe remains closed as shown in Figure 58. The stem 242 maintains the fluorophore-quenching agent pairs close to each other such that significant resonance energy transfer can occur between them, substantially eliminating the fluorophore-394-201219115 emitting fluorescence when illuminated by the excitation light 244 Ability. Figure 59 shows FRET probe 236 in an open or hybridized configuration. When hybridized to the complementary target nucleic acid sequence 2W, the stem-loop structure is destroyed, and the fluorophore and the quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to emit fluorescence. The fluorescent emission 25 0 is optically detected as an indicator that the probe has hybridized.

The probe hybridizes to the complementary target with very high specificity because the stem helix of the probe is designed to be more stable than the probe-targeted helix with a non-complementary single nucleotide. Since the double-stranded DNA is quite robust, the probe-targeted helix and the stem helix cannot coexist in the stereoscopic space. The probe-attached probe and primer-attached stem-loop probe and the primer-connected linear probe (also known as the scorpion-type probe) are alternative molecular beacons that can be used in LOC devices for immediate and quantitative Nucleic acid amplification. Immediate amplification can be performed directly in the hybridization chamber of the LOC device Φ. An advantage of using a probe coupled to a primer is that the probe element is actually linked to the primer, so that only a single hybridization event occurs during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an immediate and efficient response, resulting in a stronger signal, shorter reaction times and better recognition than when using separate primers and probes. The probes (and polymerase and amplification mixture) will be deposited in the hybridization chamber 180 during manufacture without the need to provide separate amplifications on the LOC device. Alternatively, the amplification portion is not used or used for other reactions. -395- 201219115 Linear Probes Linked to Primers Figures 78 and 79 show the linearity of the linear probe 692 linked to the primer during the first round of nucleic acid amplification and the primer-ligated linkage during subsequent nucleic acid amplification. Probe. Referring to Figure 78, the linear probe 692 coupled to the primer has a double stem section 2 42. One of the probes 696 is ligated to the primer 696 which is homologous to the region 696 on the target nucleic acid, and is labeled with fluorophore 246 at the 5' end thereof, and is amplified by the 3' end. The fragment 694 is linked to the oligonucleotide primer 700. The 3' end of the other strand of stem 242 is labeled with a quencher group 2M. After completion of the first round of nucleic acid amplification, the probe can be tucked up and hybridized to an extended strand having sequence 698 (now complementary). During the first round of nucleic acid amplification, the oligonucleotide primer 700 is affixed to the target DNA 23 8 (Fig. 78) and then extended to form a DNA strand containing both the probe sequence and the amplification product. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 696. Upon subsequent denaturation, the extended oligonucleotide primer 70/template hybridization system is isolated, and the double-strand 2 42 of the linear probe linked to the primer is also separated, thereby releasing the quencher 2 4 8 . When the temperature is lowered for the bonding and extension step, the primer-linked probe sequence 696 of the linear probe linked to the primer is rolled up and hybridized with the amplified complementary sequence 698 on the extended strand, and the display can be detected. Fluorescence of the target DNA. A linear probe that is not extended and attached to the primer retains its double stem and the fluorescence remains quenched. This test method is especially suitable for rapid detection systems because it requires only a single molecule. Stem ring probe connected to the primer -396- 201219115

Figures 80A through 80F show the operation of stem loop probe 704 coupled to the primer. Referring to Fig. 80A, the stem loop probe 704 attached to the primer has a stem 242 of complementary double stranded DNA and a loop 240 having a probe sequence. The 5' end of one of the stem strands 708 is labeled with a fluorophore 246. Another 710 line is labeled with a 3' end quencher 248 and carries both amplification blocker 694 and oligonucleotide primer 700. During the initial denaturing phase (see Figure 80B), the strands of the target nucleic acid 23 8 are separated, and the stem 242 of the stem loop probe 704 attached to the primer is also separated. When the temperature is cooled to effect the binding phase (see Figure 80C), the oligonucleotide primer 700 on the stem-loop probe 704 attached to the primer hybridizes to the target nucleic acid sequence 238. During extension (see Figure 80D), the complementary sequence 706 of the target nucleic acid sequence 23 is synthesized to form a DNA strand containing both the probe sequence 704 and the amplification product. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 704. - When the probe is bonded after denaturation, the probe sequence of the loop segment 2 40 of the stem-loop probe attached to the primer (See Fig. 80F) is bonded to the complementary sequence 706 on the extended strand. This configuration causes the fluorophore 246 to be at a great distance from the quencher 248, resulting in significantly enhanced fluorescence emission. Control Probes Hybridization chamber arrays 包括 0 include some hybridization chambers 180 with positive and negative control probes for analysis of quality control. Figures 92 and 93 schematically illustrate negative control probes without Camp No. 796, and Figures 94 and 95 show positive control probes without quencher 798. The positive and negative control probes have a stem-loop structure as described above for the FRET probe. However, regardless of whether the probes hybridize to an open configuration or remain closed, the positive control probe 798 will emit a firefly-397-201219115 light signal 250, and the negative control probe 796 will never emit a fluorescent signal 250. Referring to Figures 92 and 93, the negative control probe 796 does not have a fluorophore (which may or may not have a quencher 248). Thus, regardless of whether the target nucleic acid sequence 238 hybridizes to the probe (see Figure 93) or the probe maintains its stem-loop configuration (see Figure 92), the reaction to excitation light 244 can be ignored. Alternatively, the negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing a loop 240 having a probe sequence that does not hybridize to any of the nucleic acid sequences in the study sample, the stem 242 of the probe molecule will self-hybridize itself to render the fluorophore and quencher Stay close together without emitting visible fluorescent signals. This negative control signal corresponds to a small amount of emission from the hybridization chamber 180, wherein the probes are not hybridized but the quencher does not quench all of the emission from the reporter. Conversely, the positive control without the quencher Needle 798 is constructed as shown in Figures 94 and 95. Regardless of whether the positive control probe 798 is hybridized to the target nucleic acid sequence 238, no material will quench the fluorescent emission 250 from the fluorophore 246 in response to the excitation light 244. Figure 52 shows the possible distribution of positive and negative control probes (378 and 380, respectively) in hybridization chamber array 110. The control probes 3 78 and 380 are placed in a hybridization chamber 180 located on the line across the hybridization chamber array 1 10 . However, the configuration of the control probes within the array is arbitrary (as is the configuration of the hybrid chamber array 1 1 )). Fluorescent group design -398- 201219115 requires a fluorescent group with a long fluorescent lifetime' This is to allow the excitation light to have enough time to decay to a lower intensity than the fluorescent emission when the light sensor 44 is activated. The strength, thereby providing a sufficient signal to noise ratio. Moreover, a longer fluorescent lifetime represents a larger integrated fluorescent photon count. The luminescent group 246 (see Figure 59) has a 'light lifetime' of greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and in most cases greater than 400 nanoseconds.

Metal-ligand complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal, chemical, and photochemical stability. A feature that is advantageous for the needs of fluorescent detection systems. In particular, the metal-ligand complex based on the transition metal ion ruthenium (Ru (II)) is ginseng (2,2,-bipyridyl) ruthenium (II) ([Ru(bpy)3] 2 + ), his life is about lys. This complex is commercially available from Biosearch Technologies under the trade name Pulsar 650.

Table 1: Photophysical properties of Pulsar 650 (钌 chelate)

Parameter symbol 値 unit absorption wavelength Xabs 460 nm emission wavelength ^em 650 nm absorption coefficient E 14800 M'1 cm*1 fluorescence lifetime Tf 1.0 \xs quantum yield Η 1 (deoxygenated) N/A 铽 chelate is A lanthanide metal-ligand complex that has been shown to be successful as a fluorescent reporter for the FRET probe system and has a long lifetime of 1 600 s - 399 - 201219115. Table 2: Photophysical properties of ruthenium chelate parameters Symbol 値 Unit absorption wavelength Xabs 330-350 nm Emission wavelength λβπι 548 nm Absorption coefficient Ε 13800 (with Xabs and ligand dependence, up to 30000 @ λε = 340 nm) M-W1 Fluorescence lifetime Tf 1600 (hybridization probe) μδ Quantum yield Η 1 (ligand dependence) Ν/Α The fluorescence detection system used in the LOC device 301 does not use filters to remove unwanted backgrounds. Fluorescent. Therefore, if the quencher 248 has no natural emission in order to increase the signal to noise ratio, it is advantageous. Quenchant 248 without natural emission will not contribute to background fluorescence. High quenching efficiency is also important, so there is no fluorescence before the hybridization event occurs. The Black Hole Quencher (B HQ), available from Biosearch Technologies, Inc. of Novato, Calif., does not have a natural emission and has high quenching efficiency and is suitable for use in the quenching agent of the system. The maximum absorption enthalpy of BHQ-1 occurs at 543 nm and the quenching range is 4 80-5 80 nm, making it suitable for quenching Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 5 79 nm and the quenching range is 560-670 nm, making it suitable for quenching puisar 650 » Iowa black quenching from Intergrated DNA Technologies, Coralville, Iowa The agent (Iowa Black FQ and RQ) is a suitable alternative quenching-400-201219115 extinguishing agent with little or no background emission. The Iowa Black FQ has a quenching range from 420 to 620 nm and has a maximum absorption enthalpy at 531 nm, making it suitable as a quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of Iowa Black RQ occurs at 656 nm and the quenching range is from 500 to 700 nm, making it an ideal quencher for Pulsar 65 0 .

In the embodiments described herein, the quencher 248 is a functional group that is attached to the probe initially, but in other embodiments, the quencher may be a separate molecule that is free of solution. Excitation source In the implementation of the fluorescence detection as the substrate described in this paper, the LED is selected as the excitation source instead of the laser diode, high power lamp or laser because of the low power consumption, low cost and small size of the LED. . Referring to Figure 81, LEDs 26 are disposed directly over hybrid array array 110 on the exterior surface of LOC device 301. Opposite to the array of hybridization chambers 10 is a light sensor 44 consisting of an array of φ photodiodes 184 for detecting fluorescent signals from the chambers (see Figures 53, 54 and 64) » Figure 82 , 83 and 84 schematically illustrate other embodiments for exposing the probe to excitation light. In LOC device 30, as shown in FIG. 82, excitation light 244 generated by excitation LED 26 is directed by lens 254 onto hybrid array 110. The excitation LED 26 is pulsed and detected by the light sensor 44. The fluorescent emission is in the LOC device 30 as shown in FIG. 83. The excitation light 244 generated by the excitation LED 26 is obtained by the lens 254 and the first optical 稜鏡 712. And the second optical -401 - 201219115 稜鏡714 is directed over the hybridization chamber array 1 10 . The excitation LED 26 is pulsed and the fluorescent sensor 44 detects the fluorescent emission. Similarly, in the LOC device 30 shown in Fig. 84, the excitation light 2 44 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 to the hybridization cell array 110. . Furthermore, the excitation LED 26 is pulsed and the fluorescence emission is detected by the light sensor 44. The excitation wavelength of the LED 26 is dependent on the choice of fluorescent dye. Philips LXK2-PR14-R00 is the appropriate excitation source for the Pulsar 65 0 dye. SET UVT0P3 3 5 T039BL LED is the appropriate excitation source for the ruthenium chelate label.

Table 3: Phili ps LXK2-PR14-R00 LED Specifications Parameter Symbol 値 Cell Wavelength λβχ 460 nm Transmit Frequency Vem 6.52(10)14 Hz Output Power Pl 0.515 (min) @ ΙΑ W Transmit Mode Lambertian Data Graph N/A Table 4: SET UVT0P3 34T039BL LED Specifications

Parameter symbol 値cell wavelength λβ 340 nm emission frequency Ve 8.82(10)14 Hz power Pi 0.000240 (min) @ 20mA W pulse forward current I 200 mA emission mode Lambertian N/A ultraviolet excitation light-402- 201219115 矽absorption Ultraviolet light. Therefore, it is advantageous to use ultraviolet excitation light. An LED excitation source can be used, but the broad spectrum of LED 26 reduces the effectiveness of this method. To solve this problem, a filtered UV LED can be used. Optionally, an ultraviolet laser can be used as an excitation source unless the relatively high cost of the laser is not practical for a particular test module market. LED driver

The time required for the LED driver 29 to drive the LED 26 at a fixed current. The low-power USB 2.0-certified unit can supply up to 1 unit load (1 mA) and a minimum operating voltage of 4.4 volts. Standard power conditioning circuits are used for this purpose. Photodiode Figure 54 shows a photodiode 184 that breaks into the CMOS circuit 86 of the LOC device 301. The photodiode 184 is part of the CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCDs, an alternative sensing technique that can be integrated on the same wafer or fabricated on adjacent wafers using non-standard processing steps. On-wafer inspection is inexpensive and reduces the size of the array system. This shorter optical path length reduces noise from surrounding environments for efficient collection of fluorescent signals and reduces the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the fraction of photons that are effectively converted into photoelectrons in the photons that strike the active region 185. In standard 矽 processing, the quantum efficiency of visible light ranges from 〇·3 to 0.5, depending on the parameters of the -403-201219115, such as the amount of cover layer and absorption characteristics.

The detection valve of the photodiode 184 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and therefore the number of hybridization chambers 1 80 in the hybridization and detection section 52 (see Figure 52). The size and number of such chambers are limited by the size of the LOC device (in the case of LOC device 301, which is 1760 microns X 5 8 24 microns) and incorporated into other functional modules such as the pathogen dialysis unit. And the technical parameters of the size of the available space after the amplification unit 112. The photodiode 18 4 detects a minimum of 5 photons in terms of standard 矽 processing. However, to ensure reliable detection, the minimum 値 can be set to 1 光 photons. Thus, in the quantum efficiency range of 0.3 to 0.5 (as discussed above), the fluorescence emission from the probes should be a minimum of 17 photons, but 30 photons will contain an appropriate margin of error for reliable detection. Calibration room

The heterogeneity of the electrical characteristics of the photodiode 184, the autofluorescence, and the residual excitation photon flux that is not fully attenuated introduce background noise and offset into the output signal. This background is removed from each output signal using one or more calibration signals. The calibration signal is generated by exposing one or more of the calibrated photodiodes 184 in the array to individual calibration sources. The low calibration source is used to determine the negative result of the target 尙 not reacting with the probe. The high calibration source is indicative of a positive result from the probe-target complex. In the embodiment described herein, the low calibration source is provided by a calibration chamber 382 in the hybrid chamber array 110, the calibration chamber 3 82: -404-201219115 does not contain any probes; a probe for a light reporter; or a probe having a reporter and a quencher configured to cause quenching to occur forever.

The output signal from the calibration chamber 382 is very close to the noise and bias in the output signals from all of the hybrid chambers in the LOC device. The output signal from the other cells minus the calibration signal substantially removes the background, leaving the signal generated by the fluorescent emission (if any). Signals generated by ambient light in the area of the self-room array are also removed. It will be appreciated that negative control probes as described above with reference to Figures 92 through 95 can be used in the calibration chamber. However, as shown in Figures 86 and 87 (which is an enlarged view of the DG and DH regions of LOC variant X 728 as described in Figure 85), another option is to make the calibration chamber 382 and the amplicons fluid. isolation. The background noise and bias can be determined by maintaining the fluid isolation chamber clearance or by using a probe containing no reporter or indeed any "standard" probe having both a reporter and a quencher, as the hybrid Blocked by fluid isolation. The calibration chamber 382 can provide a high calibration source to produce a high signal at the corresponding photodiode. This high signal corresponds to all probes that have been hybridized in the chamber. Spotting a probe with a reporter but no quencher, or a probe with only a reporter will consistently provide a signal that approximates that most of the probes in the hybridization chamber have been hybridized. It will also be appreciated that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of the calibration chambers 3 82 throughout the hybrid chamber array is arbitrary. However, if the photodiode 184 is calibrated by a relatively close calibration chamber 382 -405 - 201219115, the calibration will be more accurate. Referring to Figure 56, the hybridization chamber array 110 has one calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 3 82 is placed in the middle of each three-by-three hybrid chamber 180 square. In this configuration, the hybridization chambers 18 are calibrated by the immediately adjacent calibration chamber 382. Figure 91 shows a differential imager circuit 788 for subtracting the signal from the fluorescent signal of the surrounding hybridization chamber 180 corresponding to the photodiode 184 of the calibration chamber 382 due to the excitation light. The differential imager circuit 7 8 8 samples the signal from pixel 790 and "virtual" pixel 792. In one embodiment, the "virtual" pixel 792 is masked to prevent light illumination, so that its output signal provides a dark reference. Alternatively, the "virtual" pixel 792 can be exposed to the excitation light along with other portions of the array. In the embodiment in which the "virtual" pixel 792 is exposed to light, the signal produced by the ambient light in the area of the array of cells is also subtracted. The signal from pixel 790 is very weak (ie, close to the dark signal), and it will be difficult to distinguish between background and very weak signals without reference to the dark signal level. During use, the "read-column" 794 and "read" _Column_d 795 is activated and the M4 797 and MD4 80 1 transistors are turned on. Switches 807 and 809 are turned off to cause outputs from the pixel 790 and "virtual" pixel 792 to be stored in pixel capacitor 803 and virtual pixel capacitor 805, respectively. After the pixel signal is stored, switches 807 and 809 are disabled. The "read-and-row" switch 8 1 1 and the virtual "read_row" switch 8 1 3 are then turned off, and the switched capacitor amplifier 815 at the output amplifies the differential signal 817. Inhibition and Activation of Photodiode -406- 201219115 The photodiode 184 must be suppressed during LED 26 excitation and must be activated during fluorescence. Fig. 65 is a circuit diagram of a single photodiode 184, and Fig. 66 is a timing diagram of a photodiode control signal. The circuit has a photodiode 184 and six MOS transistors Mshunt 394, Mtx 3 96,

Mreset 3 98, Msf 40 0, Mread 402 and Mbias 404. At the beginning of the excitation cycle tl, the transistors Mshunt 394 and Mreset 3 98 are turned on by pulling up the Mshunt gate 3 84 and resetting the gate 3 88. During this period, the excitation photons generate a carrier in the photodiode 184. These carriers must be removed because the amount of carrier generated is sufficient to saturate the photodiode 184. During this cycle, Mshunt 3 94 directly removes the carrier generated in photodiode 184, and Mreset 398 resets any accumulation of node 'NS' 406 due to transistor leakage or carrier diffusion due to excitation in the substrate. Carrier. After excitation, the capture cycle begins at t4. In this cycle, the emission reaction from the fluorophore is captured and integrated into the circuit on node 'NS' 608. This is achieved by pulling up the tx gate 386, which turns on the transistor Mtx 396 and transfers any carrier accumulated on the photodiode 184 to the node 'NS' 406. The length of the capture cycle can be as long as the fluorophore emission. The output from all of the photodiode 184 in the hybrid chamber array 110 is simultaneously captured. There is a delay between the end of the capture cycle t5 and the beginning of the read cycle t6. This delay is due to the need to separately read each photodiode 184 in the hybridization chamber array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, while the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Mread -407 - 201219115 402 is turned on by pulling the read gate 3 93 high. The voltage of the 'NS' node 406 is buffered and read using the source follower transistor Msf 400. Other optional methods for initiating or suppressing the photodiode are discussed below: 1. Inhibition method

Figures 88, 89 and 90 show three possible configurations of Mshunt transistor 3 94 778, 78 0, 782. The Mshunt transistor 3 94 has a very high turn-off ratio at maximum |FCS| = 5 V, which is activated during excitation. As shown in FIG. 88, the Mshunt gate 3 84 is configured at the edge of the photodiode 184. Optionally, as shown in FIG. 89, the Mshunt gate 3 84 is configured to surround the photodiode 184. The third option is to configure the Mshunt gate 3 84 within the photodiode 184 as shown in FIG. In the third option, the active region 185 of the photodiode will be smaller.

These three configurations 778, 780, and 782 reduce the average path length from all locations in the photodiode 184 to the Mshunt gate 3 84. In Fig. 88, the Mshun, the gate 3 84 is located on one side of the photodiode 184. This configuration is the easiest to manufacture and does not occupy the active region of the photodiode 185» However, it is stuck in Any carrier on the far side of the photodiode 184 takes a long time to pass through the Mshunt gate 3 84. In FIG. 89, the Mshunt gate 3 84 surrounds the photodiode 184. This further reduces the average path length of the carrier in photodiode 184 to Mshunt gate 3 84. However, the Mshunt gate 384 extending around the periphery of the photodiode 184 causes a significant reduction in the photodiode active region 185. Configuration 782 in Figure-408-201219115 90 positions Mshunt Gate 3 84 in active area 185. This provides the shortest average path length to Mshunt Gate 3 84, so the transition time is the shortest. However, the encroachment of the active zone 185 is greatest. It also creates a wider leakage path. 2. Starting method a- Trigger the photodiode to drive the shunt transistor with a fixed delay

b. Trigger the photodiode to drive the shunt transistor with a programmable delay. c. The shunt transistor is driven by a LED drive pulse with a fixed delay d. The shunt transistor is driven as a 2c but with a programmable delay. Figure 68 is a schematic cross-sectional view of the hybridization chamber 180 showing the photodiode 184 and the trigger photodiode 187 embedded in the CMOS circuit 86. φ is to trigger the photodiode 187 to replace the small area in the corner of the photodiode 184. "The triggering photodiode 18 7 with a small area is sufficient because the intensity of the excitation light will be higher than that of the fluorescent emission. . The trigger photodiode 187 is sensitive to excitation light 244. The trigger photodiode 187 temporarily stores the illuminator 244 to be extinguished and activates the photodiode U4 after a brief delay of At3 00 (see Fig. 2). This delay allows the fluorescent photodiode 184 to detect fluorescent emissions from the FRET probe 186 when there is no excitation light 244. This makes it possible to detect and improve the signal to noise ratio. Both the photodiode 184 and the trigger photodiode 187 are located at -409-201219115

The hybrid chamber 180 is in the CMOS circuit 86. The array of photodiodes is combined with appropriate electronic components to form a photosensor 44 (see Figure 64). The photodiode 184 is a pn junction made during the fabrication of the CMOS structure and does not require additional masking or steps. During MST fabrication, a dielectric layer (not shown) over photodiode 184 is arbitrarily thinned using standard MS T photolithography techniques to allow more phosphor to illuminate active region 185 of photodiode 184. . The photodiode 184 has a field of view such that a fluorescent signal from the probe-target hybrid within the hybridization chamber 180 is incident on the surface of the sensor. The fluorescent light is converted into a photocurrent which can then be measured using a CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 can be configured to only trigger the photodiode 187. These choices can be used in combination with 2a and 2b above. Fluorescent delay detection

The following derivation illustrates the use of long-lived fluorophores for delayed detection of the above-described combination of LED/fluorescent clusters. The fluorescence intensity is derived from the time function by the ideal pulse of the fixed intensity Ie, which is derived from the time function, as shown in Fig. 60. Let [Sl](t) be equal to the intensity of the excited state at time t, then the number of excited states per unit volume per unit time during and after excitation is described by the following differential equation: Wu 11 (0+ on the leg (1) dt tf hve where c is the molar concentration of the fluorophore, ε is the molar quenching coefficient, v<; is the excitation frequency, and h = 6.62606896(1 0)-34 Js is the Planck constant. The equation has the general formula: -410- 201219115 ^ + p(x)y = q(x) αχ There is a solution: \e^P(x)dx q(x)dx + k ..(2) Now use this Solution (〇' [s\m hv. + ke-"" (3)

k at time~, [51](~) = 〇...(4) hv. and from (3) substituting (4) into (3) ·· hve hve at time t2: ...(5) Hve hve

When the excited state is exponentially decayed and described by equation (6): [guide] = 〆 (called (eight) ... (6) ;) substitution (6): ** ζ t 』 ... (7) The fluorescence intensity is obtained by the following equation : where V/ is the fluorescence frequency, η is the quantum yield, and 1 is the optical path length. So from (7): -411 - ...(9) 201219115 收收)_ IeSC[\ -(f-r,)/^ dt hve Substituting (9) into (8):

If(t) = /e£c/7—[l-e"(,,',,)/r/]e'(,',l)/r/ ^ e When ^ί·->〇〇 , (call) ~

τ! K describes the fluorescence. Therefore, we can write the following approximation, which is attenuated after a sufficiently long excitation pulse 〇2-~>> rf): when Gt2,...(ii) is on In the section, we summarize the case of f when Qt2, I (/^IeSdr^e", From the above equation, we can derive the following expression: rifit) = nesc^e~(,~,l)lTf .. (12) where and · IAt) seven (0 = ^7 is the number of fluorescent photons per unit area per unit time 弋 = g - is the number of excitation photons per unit area per unit time. Therefore,

CQ nf(t)^\rif{t)dt (13) h Dipole opening The center is the number of fluorescent photons per unit area, and ί3 is the time point of photoelectric activation. Substituting (1 2) into (1 3 ): 〇0 nf = jne£c^e'(,~'l),r/dt (14) fluorescing between the current 'arrival photodiodes per unit Area per unit time -412 - 201219115 Photon number % (〇 is obtained by: «, (0 = «/(〇^0 ...(15) where nine is the light collection efficiency of the optical system ❺ will (12) Substituting (15) we found that ^(0 = Φ〇ηΒ£αΙηε'{'', ι)/ι/ ((6) stomach-like ground's photon number per unit of fluorescent area per photon of the photodiode will be as follows : 〇〇φ 圮 and substituting (1 6) and integrating: ^ =Φ〇ηΙ!ε〇Ιητ/β~(ΐ3~'ι)/^ Therefore, ns =φ0ήΐεαΙητ/β'^Ιτ^ ...(17 The ideal enthalpy of h is that when the electron rate generated by the fluorescent photons in the photodiode 184 becomes equal to the electron rate generated by the excited photons in the photodiode 184, the excitation photon flux attenuation ratio Fluorescent photon flux decays more quickly.

The electron rate that the sensor emits per unit of fluorescent area due to fluorescence is: where is the quantum efficiency of the sensor at the wavelength of the fluorescent light. Substituting (17) we get: 4(7)=Lu... (18) Similarly, the electron rate per unit of fluorescent area of the sensor is _·e; (0 = ^«>" M2) / rr - 〇 9) where is the quantum efficiency of the sensor at the excitation wavelength 'and ^ is the time constant of the "cut off" characteristic of the LED that should be excited by -413 - 201219115. After time t2, the attenuated photon flux of the LED will increase the intensity of the fluorescent signal and extend the decay time of the fluorescent signal, but we assume that the effect on If(t) can be ignored' so we take a conservative method. At present, as mentioned before, the ideal system is: Heart (6) = 3⁄4 (6)

Therefore, from (18) and (19) we get: φ/φ0ηΙ!εοΙηβ~^~, ι)ΙΤ/ = and after reforming we get: ln(fc/7—) χ fe— —(20) xf In the above two paragraphs, we get the following two expressions: =^〇/ieFr/e_A, /r/ ...pi) + abalone,

Δ/= \ (22) where corpus = £^/77 and = ί3 - ί2. We also know that i2 »r/ is actually. The ideal time for fluorescence detection and the number of fluorescent photons detected using Philips LXK2-PR14-R00 LED and Pulsar 6 5 0 dye are determined as follows. The ideal detection time is determined using equation (22): recalling the concentration of the amplicon, and assuming that all amplicons are hybridized, the fluorescing fluorophore concentration is: c = 2.8 9(10)·6 mol/ L. The height of the chamber is the optical path length 1 = 8 (1 0 wide and 6 m. The fluorescent area has been regarded as equivalent to the photodiode area, whereas the actual -414-201219115 fluorescent area is substantially larger than the photodiode area. Therefore, it can be assumed that nine = 0.5 is the light collection efficiency of the optical system. From the characteristics of the photodiode, 1 = :0 is extremely conservative, the Φβ quantum efficiency of the photodiode at the fluorescence wavelength The ratio of quantum efficiency to the excitation wavelength at 激发. With a typical LED decay lifetime Γί = 0.5 nanoseconds and using the Pulsar 650 specification, Δί : F = [1.48(10)6 ] [2.89(10)~ 6 ] [8(10)-6 ](1)

Ln([3.42(10)-s](10)(0.5)) 1(10)-6 ~ 0.5(10)-9 =4.34(10)-9 s The number of detected photons is used (21 ) Decided. First, the number of excitation photons emitted per unit time is determined by examining the illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian emission mode, so: n, = 'ril0 cos(0) ...(23) where % is the angle to the direction of the LED's forward axis, 每 per unit solid angle per unit The number of photons emitted at a time, and ^ is the b in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: ή, = Ω = j«; 〇cos(0)i/Q (24) Now, -415- 201219115 △Ω = 2;r[l - cos(^ + Δ0)] - 2;r[l - cos(6)] ΔΩ = 2; r[cos (9)-cos(0 + Δ0)] 4^sin(0)cosf—]siniA0>l J dQ = 2usm( 0)d9 + 4^cos(^)sin2 αθλ Substituting this into (24): η 2 ή' = J*2; 0i; 0cos (6) sin(0)i/0 ο = ^'ιο

Rearranged, we get: «/〇 = ...(26) π The output power of the LED is 0.515 watts and ve = 6.52 (10) 14 Hz, so ή,^-p- ...(27)

Ave __0.515_ = [6.63(10)'34] [6.52(10)14]

= 1.19(10)18 Photon 渺 Bring this 入 into (26) We get: ... 1.19(10)18 一^- =3.79(10)17 Photon 渺 面 参照 Refer to Figure 61, the optical center of LED 26 252 and lens 25 4 are shown schematically. The photodiode system is 16 micrometers χ 16 micrometers and for the photodiode in the middle of the array, the solid angle (Ω) of the light cone emitted from the LED 26 to the photodiode 184 is approximately: -416 - 201219115 Ω = sense Detector area /r2 [16(10)116(10)4] ~[2.825(10)-3 f~~ =3.21(H))·5 Sphericality will understand the central photodiode of photodiode array 44 Body 184 is used for these calculations. Sensors located at the edge of the array receive only less than 2% of the photons when using the hybridization event of the Lambertian excitation source intensity distribution.

Number of excitation photons emitted per unit time: ne = η, Ω ... (28) = [3.79(10), 7] [3.21(l〇r5] = 1.22(10)13 Photon 渺 Reference equation (29 ): ns = (0.5)[1.22(10)13][3.42(10)-5][l(10)_6]e-434(1〇r, /1(10)^ = 208 photon/sensor Therefore, using the Philips LXK2-PR14-R00 LED and the Pulsar 650 fluorophore, we can easily detect any hybridization events that cause these numbers of photons to be emitted. The SET LED illumination geometry is shown in Figure 62 »When ID = 20 At milliamperes, the LED has a minimum optical power output of Pl = 240 μW and a wavelength center at λε = 3 40 nm (absorption wavelength of the ruthenium chelate). Driving the LED at ID = 200 mA will linearly increase the output power To 1 > 1 = 2.4 mW. By placing the optical center 252 of the LED about 17.5 mm from the hybrid array 1 1 , we focus this output flux on a dot size with a maximum diameter of 2 mm. -417- 201219115 The photon flux in the 2 mm diameter point of the hybrid array plane is obtained from Equation 27. ή,10h^e — 2.4(10)~3 A[6.63(10)-34] [8.82(10 ) 14] = 4.10(10)15 Photon 渺Using Equation 28 we get: ne = η, Ω 4.10(10)15

[16(10)-6]3 vs. 1(10)·3]2 3.34(10)11 Photons/sec Now, return to Equation 22 and use the previously listed Tb chelate properties, A< ln[(6.94 (10)-5)(10)(0.5)] _ _1___1_ 1(10)-3 ~ 0.5(10)-9 =3.98(10)·9 seconds

Now self-condition 21:

Ns = (0.5)[3.34(10)n][6.94(10)-5][l(10)-3]e-398(10)',/1(1〇)'J = 1 1,600 photons/sensory The theoretical number of photons emitted by the hybridization event using the SET LED and the ruthenium chelate system can be easily detected and far exceeds the lower limit of 30 photons required for reliable detection of the photosensor as described above. value. Maximum separation between the probe and the photodiode The detection of hybridization on the wafer does not require a conjugated focal microscope (see previous technique -418-201219115

Test). This difference from traditional inspection techniques represents an important factor in saving time and cost. Traditional inspection requires imaging optics that must use lenses and curved mirrors. By employing non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. The placement of the photodiode in close proximity to the probe has the advantage of extremely high collection efficiency: when the material thickness between the probe and the photodiode is 1 micron, the emission angle of the emitted light is as high as 173°. . This angle is calculated by considering the light emitted from the probe closest to the center of the surface of the hybridization chamber of the photodiode, which has a planar active surface region parallel to the surface of the chamber. Light within the cone of light emission can be absorbed by a photodiode defined as having a firing probe at its apex and at the sensor corners around its plane. Taking a 16 micron X 16 micrometer sensor as an example, the apex angle of the pyramid is 170°; in the case where the photodiode is expanded such that its area conforms to the 29 micron x 9.75 micron hybrid cell area, the top The angle is 173°. The interval between the surface of the chamber and the active surface of the photodiode is 1 micron or less, which is easily achieved. The application of non-imaging optical methods does require that the photodiode 184 be placed very close to the hybridization chamber to collect a sufficient amount of fluorescent emission photons. The maximum spacing between the photodiode and the probe is determined as described below with reference to Fig. 54. With J ruthenium chelate fluorophore and SET UVT0P335T039BL LED, we calculated 11600 photons from individual hybridization chambers 180 to 16 micron x 16 micrometers of photodiode 184. In carrying out this calculation, we assume that the light collection region of the hybridization chamber 180 has the same bottom area as the photodiode active region 185, and that half of the total number of hybrid photons reaches the photodiode 184-419-201219115. That is to say, the light collection efficiency of the optical system is ???=0.5. More precisely, we can write out = [(the bottom area of the light collection area of the hybridization chamber) / (photodiode area)] [Ω / 4 π], where Ω = representative on the substrate of the hybridization chamber The solid angle of the optoelectronic diode of the point. In terms of the geometry of the regular square pyramid: Q = 4arcsin(a2/(4d〇2 + a2)), where d〇 = the distance between the chamber and the photodiode, and a is the size of the photodiode . Each hybrid cell released 23,200 photons. The selected photodiode has a detection low limit of 17 photons, so the minimum optical efficiency required is =1 7/23 200=7.3 3 xl0·4 light collection area of the hybridization chamber 180 The bottom area is 29 microns x 19.75 microns. Solving do, the maximum limit distance between the hybridization chamber and the photodiode 184 is "= 249 microns. Under this limitation, the collection cone angle as defined above is only 0.8. This should be noted. The analysis ignores the negligible effect of refraction. Conclusions The devices, systems, and methods described herein facilitate rapid, inexpensive, and molecular diagnostic tests suitable for in situ care. The systems and compositions described above are illustrative only and do not depart from the spirit of the invention. In the scope of the general inventive concept, many variations and modifications will be readily apparent to those skilled in the art. -420- 201219115 [Simplified Description of the Drawings] The preferred embodiment of the present invention will now be referred to the accompanying drawings. For illustrative purposes only, FIG. 1 shows a test module and a test module reader configured for fluorescence detection; and FIG. 2 is an electronic device in a test module configured for fluorescence detection. a schematic diagram of the components;

Figure 3 is a schematic view of the electronic components in the test module reader; Figure 4 is a schematic diagram of the structure of the LOC device; Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device, all layers Figure 7 is a plan view of the LOC device, showing the structure of the upper cover separately; Figure 8 is a top view of the upper cover, showing the internal passage and the reservoir in broken lines; Figure 9 is an exploded top view of the upper cover, The dotted line shows the internal channel and the reservoir; Figure 10 is the bottom view of the upper cover, showing the configuration of the upper channel; Figure 11 is a plan view of the LOC device, which shows the structure of the CMOS + MST device independently; Figure 12 is the sample inlet of the LOC device Figure 13 is an enlarged view of the AA area shown in Figure 6; Figure 14 is an enlarged view of the AB area shown in Figure 6; Figure 15 is an enlarged view of the AE area shown in Figure 13; The perspective view illustrates the layered structure of the LOC device in the AE zone: -421 - 201219115 A partial perspective view of Figure 17 illustrates the layered structure of the LOC device in the AE zone; a partial perspective view of Figure 18 illustrates the LOC device segment in the AE zone Layer structure; part of the perspective view of Figure 19 illustrates the AE area The layered structure of the LOC device; the partial perspective view of Figure 20 illustrates the layered structure of the LOC device in the AE area; the partial perspective view of Figure 21 illustrates the layered structure of the LOC device in the AE area; Figure 22 is a diagram of Figure 2 A cross-sectional view of the lysing reagent reservoir shown; a partial perspective view of Fig. 23 illustrating the layered structure of the LOC device in the AB region; and a partial perspective view of Fig. 24 illustrating the layered structure of the LOC device in the AB region; A partial perspective view of 25 illustrates the layered structure of the LOC device in the AI zone; a partial perspective view of Fig. 26 illustrates the layered structure of the LOC device in the AB zone; and a partial perspective view of Fig. 27 illustrates the LOC device in the AB zone Layered structure; Figure 28 is a partial perspective view showing the layered structure of the LOC device in the AB zone; Figure 29 is a partial perspective view showing the layered structure of the LOC device in the AB zone; A schematic cross-sectional view of the polymerase reservoir; Figure 31 shows the characteristics of the boiling start valve independently; Figure 3 is a schematic cross-sectional view of the boiling start valve taken along line 3 2 - 3 2 shown in Figure 31; Figure 33 Figure 15 is an enlarged view of the AF area; Figure 34 is a schematic cross-sectional view of the upstream end of the dialysis section, along the section Figure 33 is an enlarged view of the AC region shown in Figure 6; Figure 3 is a re-enlarged view of the AC region, which shows the amplification portion; Figure 3 is the AC region Further, the figure shows an amplification section; FIG. 38 is a re-enlarged view of the AC zone, which shows an amplification section; -422-201219115. FIG. 39 is a re-enlarged view of the AK zone shown in FIG. 38; A re-enlarged view of the AC region, which shows an amplification chamber; Figure 41 is a re-enlarged view of the AC region, which shows an amplification portion; Figure 42 is a re-enlarged view of the AC region, which shows an amplification chamber; FIG. 44 is a re-enlarged view of the AC region shown in FIG. 42; FIG. 44 is a re-enlarged view of the AC region, and FIG. 45 is a re-enlarged view of the AM region shown in FIG.

Figure 46 is a re-enlarged view of the AC region, which shows an amplification chamber; Figure 47 is a re-enlarged view of the AN region shown in Figure 46; Figure 4 is a re-enlarged view of the AC region, which shows an amplification chamber; Figure 49 is a re-enlarged view of the AC region, which shows an amplification chamber; Figure 5 is a re-enlarged view of the AC region, which shows an amplification portion; Figure 51 is a schematic cross-sectional view of the amplification portion; Figure 52 is a hybridization portion. Figure 53 is a re-enlarged plan view of two independent hybridization chambers; Figure 54 is a schematic cross-sectional view of a single hybridization chamber; Figure 55 is an enlarged view of the humidifier in the AG region shown in Figure 6; Figure 5 is an exploded perspective view of the LOC device in the AD region: Figure 58 is a diagram of a FRET probe in a closed configuration; Figure 5 is an open and hybridized configuration. Figure 15 is a time-intensity diagram of the excitation light; Figure 6 is a diagram of the excitation illumination geometry of the hybrid chamber array; Figure 62 is a diagram of the sensor electronic technology LED illumination geometry; -423- 201219115 Figure 63 is an enlarged plan view of the humidity sensor shown in the AH area of Figure 6; Figure 64 is a schematic view showing the photodiode array of the light sensor Figure 6 is a circuit diagram of a single photodiode; Figure 6 is a timing diagram of the photodiode control signal; Figure 67 is an enlarged view of the evaporator shown in the AP area of Figure 5; A schematic cross-sectional view of a hybridization chamber for detecting a photodiode and a trigger photodiode;

Figure 69 shows the PCR initiated by the linker; Figure 70 is a representative view of the test module with the lancet; Figure 71 is a representative view of the structure of the LOC variant VII; Figure 72 is a representative of the structure of the LOC variant VIII. Figure 73 is a representative diagram of the structure of the LOC variant XIV; Figure 74 is a representative diagram of the structure of the LOC variant XLI; Figure 75 is a representative diagram of the structure of the LOC variant XLIII: Figure 76 is the structure of the LOC variant XLIV Representative map

Figure 77 is a representation of the structure of the LOC variant XLVII; Figure 78 is a diagram of a linear fluorescent probe coupled to the primer during the first round of amplification, and Figure 7.9 is a linear fluorescence coupled to the primer during the subsequent amplification cycle. Figure 80A to 80F illustrate the heating cycle of the fluorescent stem ring probe connected to the primer; Figure 81 is a schematic diagram of the excitation LED relative to the hybridization chamber array and the photodiode; -424- 201219115 82 is a schematic diagram of an excitation LED and an optical lens for directing illumination to a hybrid cell array of a LOC device; FIG. 83 is a schematic diagram of an excitation LED, an optical lens, and an optical 用于 for directing illumination to a hybrid cell array of a LOC device; 84 is a schematic diagram of an excitation LED, an optical lens, and a mirror arrangement for directing illumination to an array of hybrid chambers of the LOC device;

Figure 8 is a plan view showing that all features overlap each other and indicates the position of the DA to DK zone; Figure 86 is an enlarged view of the DG zone of Figure 85; Figure 87 is an enlarged view of the DH zone shown in Figure 85. Figure 8 shows an embodiment of the shunt transistor of the photodiode; Figure 8 shows an embodiment of the shunt transistor of the photodiode; Figure 90 shows the shunt transistor of the photodiode Figure 91 is a circuit diagram of a differential imager; Figure 92 is a schematic illustration of the stem-loop configuration of the negative control fluorescent probe; Figure 93 is a schematic illustration of the open configuration of the negative control fluorescent probe of Figure 92; The schematic representation of the stem loop configuration of the fluorescent probe is schematically illustrated; Figure 95 is a schematic illustration of the open configuration of the positive control fluorescent probe of Figure 94; Figure 96 shows the test module and test module configured for ECL detection. Group Reader: Figure 97 is a schematic diagram of the electronic components in the test module configured for ECL detection; Figure 98 shows the test module and the selective test module reader; Figure 9 shows the test mode Group and test module readers and storage of various materials -425- 201219115 Library Host system. [Main component symbol description] 1 〇 : Test module 1 1 : Test module 1 2 : Test module reader 13 : Shell

14 : Micro USB connector 1 5 : Inductor 1 6 : Micro USB port 17 : Touch screen 18 : Display screen 19 : Button 20 : Start button

2 1 : Honeycomb radio 22 : Aseptic sealing tape 2 3 : Wireless internet connection 24 : Large container 2 5 : Satellite navigation system 26 : Light-emitting diode 2 7 : Data storage 28 : Mobile phone / smartphone 29 : USB Compatibility LED Driver 30: On-Chip Lab (LOC) Device - 426 - 201219115 3 1 : Power Regulator 32 : Capacitor 33 : Clock 34 : Controller 35 : Register 36 : USB Device Driver 3 7 : Driver

38 : RAM 39 : Drive 40 : Program and Data Flash Memory 41 : Register 42 : Processor 43 : Program Memory 44 : Light Sensor 45 : Indicator 46 : Upper Cover 47 : USB Power / Indicator Module 48 : COMS + MST wafer 49 : porous member 52 : hybridization and detection portion 54 : receptacle 5 6 : receptacle 57 : printed circuit board (PCB) 5 8 : receptacle - 427 - 201219115 60 : receptacle 62 : receptacle 6 4 : Lower sealing layer 66 : Top layer 68 : Sample inlet 70 : Pathogen dialysis section 72 : Waste channel

74: Target channel 76: Waste unit (reservoir) 78: Reservoir layer 80: Upper cover channel layer 8 2: Upper sealing layer 84: 矽 Substrate 86 : CMOS circuit 87 : MST®

8 8 : Passivation layer 90 : MST channel 92 : Drop port 94 : Upper cover channel 96 : Riser 97 : Wall 98 : Meniscus anchor 1 00 : MST channel layer 101 : Laptop / notebook - 428 - 201219115 102 : Capillation start feature (CIF) 103 : Dedicated reader 105 : Desktop computer 106 : Boiling start valve 107 : E-book reader 108 : Boiling start valve 109 : Tablet PC

1 1 〇: hybridization chamber array U 1 : epidemiological data host system 1 12 : amplification unit 1 1 3 : genetic data host system 1 1 4 : culture unit 115: electronic health record (EHR) host system 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Sample flow 1 2 0 : Meniscus 121 : Electronic medical record (EMR) host system 122 : Vent. 123 : Personal health record (PHR) host System 125: Network 126: Boiling Start Valve 1 28: Surface Tension Valve 130: Chemical Lysis Department - 429 - 201219115 1 3 1 : Mixing Part 1 32: Surface Tension Valve 1 3 3 : Incubator Inlet Channel 1 34: Drop Port 136: Light window 146: Valve inlet 1 48: Valve outlet

1 50 : Lowering port 1 5 2 : Heater 153 : Valve heater contact 1 5 4 : Heater 1 5 6 : Heater contact 158 : Micro channel 160: Amplification section outlet channel 164 : Hole array

166: capillary starting feature 168: dialysis rising hole 170: temperature sensor 174: liquid sensor 175: diffusion barrier 1 7 6 : flow path 178: end point liquid sensor 1 80: hybridization chamber 1 8 2 : heating -430- 201219115 1 8 4 : Photodiode 1 8 5 : Active region 186: Oligonucleotide FRET probe 187: Trigger photodiode 1 8 8 : Water reservoir 190 : Evaporator 1 9 1 : Heating Device

192: water supply passage 193: rising port 194: lowering port 195: top metal layer 196: humidifier 198: first rising hole 202: capillary starting feature 204: dialysis MST channel 208: liquid sensor 210: microchannel 212 : Intermediate MST channel 2 1 8 : TiAl electrode 220 : TiAl electrode 222 : Gap 232 : Humidity sensor 2 3 4 : Heater 236 : FRET probe - 431 201219115

23 8 : Target nucleic acid sequence 240 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 2 4 8 : Quencher 2 5 0 : Fluorescence emission 2 5 2 : Optical center 2 5 4 : Lens 28 8 : Sample Input and Preparation 290: Extraction 291: Culture 292: Amplification 294: Detection

2 9 6 : First electrode 298 : Second electrode 3 00 : Preprogrammed delay 301 : On-wafer laboratory (LOC) device 3 76 : Conductive column 3 7 8 : Positive control probe 3 8 0 : Negative control probe 3 82 : Calibration room 3 8 4 : Wide pole 3 8 6 : Interpole - 432- 201219115 3 8 8 : Gate 3 9 0 : Lancet 3 92 : Lancet release button 3 9 3 : Interpolar 394 : Mshunt Transistor 396: MOS transistor

3 9 8 : Μ Ο S transistor 400: MOS transistor 402: MOS transistor 404: MOS transistor 4 0 6 : node 408 : film seal 4 1 0 : film guard

673 : LOC variant XLIII 674 : LOC variant XLIV 677 : LOC variant XLVII 682 : small component dialysis section 6 8 6 : dialysis section 692 : linear probe 694 connected to the primer: amplification blocker 696 : probe Sequence 6 9 8 : Sequence 700: Oligonucleotide primer 704: Stem loop probe linked to primer -433-201219115 706: Complementary sequence 708: Stem 71 0: Another strand 712: First optical 稜鏡7 1 4: second optical 稜鏡 7 1 6 : first mirror 7 1 8 : second mirror 72 8 : LOC variant X 766 : waste receptacle 778 : configuration 780 : configuration 782 : configuration 788 : differential imager Circuit 790: Pixel 7 9 2 : Virtual Pixel 794: Read_Column 795: Read_Column_d 796: Negative Control Probe 797: M4 79 8: Positive Control Probe 801: MD4 8 0 3 : Pixel Capacitor 805 : Virtual Pixel Capacitor 8 0 7 : Switch 201219115 8 〇 9 : Switch 811 : "Read _ Line" Switch 813 : Virtual "Read _ Line" On 8 1 5 · 'Switched Capacitor Amplifier 817 : Differential Signal 860 : ECL Excitation Electrode 870 : ECL excitation electrode

Claims (1)

201219115 VII. Patent Application Range: 1. A microfluidic test module comprising: a housing for hand movement; and a microfluidic device mounted in the housing for processing a biological sample, the housing providing proximity to the microfluidic device a microenvironment; wherein the outer casing has a membrane seal of a flexible substance between the microenvironment and the atmosphere for reducing pressure changes in the microenvironment due to fluctuations in atmospheric pressure. 2. The microfluidic test module of claim 1, wherein the outer casing has a guard for protecting the membrane seal from damaging contact. 3. The microfluidic test module of claim 2, further comprising a sensor and a functional unit to adjust the microenvironment in response to feedback from the sensor. 4. The microfluidic test module of claim 3, wherein the sensor and functional unit are constructed in the microfluidic device. 5. The microfluidic test module of claim 4, wherein the sensor is a humidity sensor and the functional unit is a humidifier. 6. The microfluidic test module of claim 5, wherein the microfluidic device is a lab-on-a-chip (LOC) device and an upper cover, the LOC device having a support substrate and a microsystem technology (MST) layer on the support substrate, the MST layer having an MST channel and a plurality of fluid connections for fluid communication with the upper cover, the upper cover having a sample inlet and an upper cover channel for fluid communication with the fluid connections . 7. The microfluidic test module of claim 6, wherein the humidifier has a water reservoir and an evaporator to expose water supplied from the water reservoir to an area containing the MST layer and to increase the area. Water vapor pressure. 8. The microfluidic test module of claim 6, wherein the evaporator has a hole configured to retain water using a meniscus formed in the hole, and the evaporator also has a hole adjacent to the hole A heater is used to increase the temperature of the water at the orifice. 9. The microfluidic test module of claim 8, wherein the heater is annular and located adjacent the aperture. 10. The microfluidic test module of claim 5, wherein the evaporator has a supply passage from the reservoir to the orifice, the supply passage being configured to draw water to the orifice by capillary action. 11. The microfluidic test module of claim 1, wherein the evaporator has a plurality of supply channels, a plurality of corresponding holes, and a plurality of corresponding heaters. 12. The microfluidic test module of claim 1, wherein the reservoir is formed in an upper cover, the supply passage being formed in the MST layer such that the reservoir is via one of a plurality of fluid connections The supply channel is coupled and the aperture is formed in the upper cover such that the supply channel is coupled to the aperture via the other of the fluid connections. 13. The microfluidic test module of claim 12, wherein the upper cover has a plurality of reagent reservoirs of different reagents. 14. The microfluidic test module of claim 4, wherein the outer casing has a container for receiving an untreated biological sample through the opening and a cover movable between the open and closed positions, wherein the cover is in the open position Opening the opening, -437-201219115 The opening is closed when the lid is in the closed position, the container being configured to be in fluid communication with the sample inlet to cause the biological sample to flow by capillary action to the sample inlet 〇1 5 . The microfluidic test module of claim 14 wherein the cover is a sealing tape having a low viscosity adhesive for sealing the opening in the closed position. 16. The microfluidic test module of claim 9, wherein the MST layer has a heater for heating the fluid within the MST channel. 17. The microfluidic test module of claim 16, wherein the MST channels each have a cross-sectional area of from 1 square micron to 400 square micrometers for biochemical treatment of components in the biological sample, And the upper cover channels each have a cross-sectional area greater than 400 square microns for receiving biological samples and transporting cells within the biological sample to predetermined locations in the MST channel. 18. The microfluidic test module of claim 14, wherein the outer casing has a lancet for puncturing a patient's finger to obtain a blood sample for introduction into the container. 19. The microfluidic test module of claim 18, wherein the lancet is movable between a retracted and extended position, and the housing has an offset mechanism to bias the lance to an extended position, and the user A catch is activated to retain the lance in the retracted position until activated by the user. 20. The microfluidic test module of claim 19, wherein the LOC device has a hybridization portion comprising a probe nucleic acid sequence configured to detect a target nucleic acid sequence within a cell of the sample fluid Hybridization with the probe nucleic acid sequence. -438-