TW201219776A - Microfluidic device with conductivity sensor - Google Patents

Abstract

A microfluidic device for processing a fluid, the microfluidic device having an inlet for receiving the fluid, functional sections for processing the fluid, a flow-path extending from the inlet into at least some of the functional sections, CMOS circuitry for operative control of the functional sections, and, a conductivity sensor with a first terminal and a second terminal spaced apart along the flow-path, and a first electrode and a second electrode positioned between the first terminal and the second terminal and spaced apart along the flow-path, wherein, the CMOS circuitry is configured to generate a current between the first terminal and the second terminal, and measure a voltage across the first electrode and the second electrode such that conductivity of the fluid in the flow-path is derived from the current and the measured voltage.

Description

201219776 VI. Description of the Invention [Technical Field] The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatment and analysis for molecular diagnostics. [Prior Art] Molecular diagnosis has been used in the field of providing early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes related to health-prone 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 for the binding (hybridization) of a synthetic DNA (oligonucleotide) short sequence 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, determine the type and pathogenicity of the infectious pathogen, or determine the individual's response to the drug. Nucleic Acid-Based Molecular Diagnostic Test 201219776 A The nucleic acid-based assay has four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (arbitrary) 4. Detection of many sample types, such as blood, Urine, sputum and tissue samples are used for genetic 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 often required at the beginning of a nucleic acid assay. Blood is one of the more commonly sought sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombosis promotes agglutination and maintains activity in vitro. To inhibit coagulation, the sample is mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of the 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 differentially in the dissolution solution leaving the remaining intact cellular material which can then be separated from the sample using centrifugation. This provides a concentrated target cell 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 RNA is quite different from the protocol used to extract the gene 201219776 DN A. However, extracting nucleic acids from a target cell typically involves 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 lyophilized 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 (usually ice ethanol or isopropanol) precipitation step, or via a solid phase purification step, in the presence of a high concentration of chaotropic salt, usually in a subsaturated column. , resin or paramagnetic beads, followed by washing and elution with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protein-cleaving proteinase 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, especially if the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target (in the case of a low concentration of detectable levels). The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PC R). The PCR is known in the art and a broad and complete description of such reactions is provided in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2000. PCR is a useful technique for amplifying a target DNA sequence relative to a complex DNA background. If the rna is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Following 201219776, the resulting cDNA was amplified by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is:

1. Primer pair - short single strand DNA having about 10-30 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis DNA thermostable enzyme 3·deoxyribonucleoside Triphosphate (dNTP) - provides nucleotides to the newly synthesized DNA strands - 4 buffers - the best chemical environment for DNA synthesis. PCR usually involves placing these reagents in small tubes containing extracted nucleic acids (~10- 50 microliters). The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocols for each thermal cycle include the denatured phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denatured phase generally involves heating the reaction temperature to 90-95 ° C to denature the DNA strand; in the adhesive phase, the temperature is lowered to ~50-6 CTC for primer adhesion; then the temperature is raised to the optimum in the extended phase. The DNA polymerase activity temperature is 60-72 ° C for primer extension. This method repeats the cycle by about 20-4 times, with the end result being a target sequence between the millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multi-primer PCR, linker-primed PCR, direct PCR, tandem PCR, instant 201219776 PCR, and reverse transcription Enzyme PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate amplicons of different sizes that are specific for different DNA sequences. Additional information can be obtained from a single experiment by targeting multiple genes at once (in other ways, several trials are required). Optimizing the multi-primer set PCR is more difficult because it requires the selection of primers with approximate adhesion temperatures and amplicon of approximate length and base composition to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR, is a method for amplifying nucleic acids of virtually all DNA sequences in a complex DNA mixture without the need for target-specific primers . This method first digests the target DNA population with a suitable restriction endonuclease. The ligated enzyme is then used to ligate a double stranded oligonucleotide linker (also known as a zygote) with a suitable overhanging end to the terminal of the target DNA fragment. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. Thereby, all fragments derived from the DNA of the adjacent linker oligonucleotide can be amplified. Direct PCR describes a system that performs PCR directly on a sample without the need for any nucleic acid extraction (or with minimal nucleic acid extraction). It has long been believed that the PCR reaction is inhibited by many components present in unpurified biological samples, such as the protohemoglobin component in the blood. Traditionally, PCR requires enhanced purification of target nucleic acids prior to preparation of the reaction mixture. However, with appropriate chemical changes and sample concentration, PCR can be performed or direct PCR can be performed with minimal DNA purification. The chemical properties of the PCR used for direct PCR include the addition of buffer strength, the use of high activity and high persistence -9 - 201219776 processivity polymerase and additions to potential polymerase inhibitors. Repeated sequence PCR utilizes two independent nucleic acid amplifications to increase the probability of amplification of the correct amplicon. One type of repeat PCR is nested PCR. Two pairs of PCR primers are used to perform single locus amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. A second pair of primers (nested primers) used in the second amplification binds to the first PCR product and produces a second PCR product (short in the first PCR product) containing the target nucleic acid. The rationale used in this strategy is that if the wrong locus is amplified by mistakes during the first nucleic acid amplification, the probability of re-amplifying the wrong locus by the second pair of primers is very low, thus ensuring specificity. The amount of PCR product was measured in real time using either real-time PCR or quantitative PCR. 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 reference standard in the reaction. This is particularly useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. The reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), followed by PCR amplification of cDNA. RT-PCR is widely used in expression pro filing 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. Thermostatic amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DNA during amplification of anti--10-201219776, thus eliminating the need for complex machinery. The thermostatic nucleic acid amplification method can be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Amplification (Loop-Mediated) Some thermostatic nucleic acid amplification methods of Isothermal Amplification have been described. The constant temperature nucleic acid amplification method does not rely on the continuous heat denaturation of the template DNA to produce a single strand of the molecule as a template for further amplification, but relies on enzymatic cleavage of a DNA molecule such as an internal nuclease which is specifically restricted at a normal temperature, or It is another method of separating DNA strands by enzymes. The strand-substituted amplification (SDA) is dependent on the ability of a particular restriction enzyme to cleave the unmodified strand of the hemi-modified DNA, and the 5,-3' exonuclease-deficient polymerase extends and replaces The ability of downstream stocks. The exponential nucleic acid amplification is then achieved by coupling a sense and an antisense reaction, wherein the strand substitution from the sense reaction is used as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl, and Mlyl) which cleaves DNA on one of the DNA strands without cutting the DNA in a conventional manner is useful for this reaction. SDA has been improved by using a combination of thermostable -11 - 201219776 restriction enzyme (Aval) and thermostable exo-polymerase (Bst polymerase). This combination appears to increase the amplification efficiency of the reaction from 1 08-fold amplification to 1 〇 1 (fold-fold amplification) so that this technique can be used to amplify unique single-copy molecules. Transcription-mediated amplification (TMA) and nucleic acid-dependent sequence amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than the corresponding genomic DN A. This technique uses two primers and two or three enzymes; RNA polymerase, reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RN A polymerase. In the first step of nucleic acid amplification, the primer hybridizes to a target ribosomal RNA (rRNA) at a defined location. The reverse transcriptase produces a DNA copy of the target rRNA by extension from the 3' end of the promoter. If another RNase is present, the RNA in the obtained RNA:DNA double strand is decomposed by the RNase activity of the reverse transcriptase. Next, the second primer binds to the DNA copy. A double-stranded DNA molecule is produced by synthesizing a new DNA strand from the end of this primer by reverse transcriptase. RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon re-enters the process and serves as a template for a new replication cycle. In recombinant enzyme polymerase amplification (RPA), isothermal amplification of a specific DNA fragment is achieved by binding a reverse oligonucleotide primer to a template DNA and then by DNA polymerase extension. Denaturation of the double-stranded DNA (dsDNA) template does not require heat. In contrast, rPA uses a recombinant enzyme-primer complex to scan dsDNA and facilitate stock exchange at homologous sites. The resulting structure is then stabilized via a -12-201219776 single-stranded DNA binding protein that interacts with the substituted template strand, thereby preventing the primer from exiting via branch migration. The recombinant enzyme is unraveled such that the strand-substituted DNA polymerase (such as a large fragment of B. subtilis p〇丨i (Bsu)) is accessible to the 3' end of the oligonucleotide, followed by extension of the primer. The exponential nucleic acid amplification is accomplished by repeating this process. The helicase-dependent amplification (HDA) mimics the in vivo system, which uses DN A helicase to generate a single-strand template for primer hybridization, followed by extension of the primer by DNA polymerase. In the first step of the HD A reaction, the helicase passes through the target DNA, destroying the hydrogen bonds linking the two strands, which are then joined by a single strand of binding protein. Primer adhesion is allowed by exposure of the single-stranded target region by helicase. Then, DNA polymerase uses free deoxyribonucleoside triphosphates (dNTPs) to extend the 3 ends of each primer to create two sets of DN A replicons. . The two sets of replicated dsDN A strands independently enter the next HDA cycle, resulting in amplification of the target sequence exponential nucleic acid. Other DNA-based thermostating techniques include rolling cycle amplification (RCA) in which DNA polymerase continues to extend primers around a circular DNA template, resulting in a long DNA product consisting of a number of replicate copies of the loop. Prior to the end of the reaction, the polymerase produces tens of thousands of copies of the circular template, and the copy strand is ligated to the original target DNA. This allows for spatial resolution of the target and rapid nucleic acid amplification of the signal. A maximum of 1 to 12 template copies can be generated in 1 hour. Branched-type amplification is a variant of RCA that uses a closed circular probe (C-probe) or a padlock probe and a DNA polymerase with high sustained processivity to hold C under constant temperature conditions. - The probe is subjected to exponential amplification. -13- 201219776 Circular Thermostat Amplification (LAMP) provides high selectivity and uses DNA polymerase and four sets of primers designed to recognize a total of six different sequences on the target DNA. The inner primer containing the sense and antisense strand sequences of the target DNA initiates LAMP. Subsequent strand-initiated DNA synthesis initiated by the outer primer will release a single strand of DNA. This single-stranded DNA can serve as a template for DNA synthesis initiated by the second internal primer and the external primer which hybridize to the other end of the target, thereby producing a stem-loop DNA structure. In the subsequent LAMP cycle, an internal primer hybridizes to the loop on the product and begins to replace DN A synthesis, producing the original armor loop DNA and the new armor loop DNA with twice the length of the arm. The cycle reaction continued to accumulate 1 〇 9 copies of the target copy in less than an hour. The final product is an arm ring DN A having a plurality of inverted repeats of the target and a cauliflower-like structure having a plurality of loops formed by alternating inter-segment adhesions in the same strand. After completion of the nucleic acid amplification, the amplified 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 identify the nucleotide composition of the amplicon using DNA hybridization. Gel electrophoresis is the easiest way to check if the expected amplicon is produced during amplification of the nucleic acid. Gel electrophoresis uses an electric field applied to a gel matrix to separate DNA fragments. The negatively charged DNA fragments will pass through the matrix at different rates, depending on their size. After electrophoresis is completed, the fragments in the gel can be stained for visualization. Ethidium bromide is a commonly used dye which exhibits fluorescence under ultraviolet light.

The size of the fragment was determined by comparison to a DNA size marker (DNA-14-201219776 ladder) containing a fragment of the known size and swimming side by side with the amplicon on the gel. Since the oligonucleotide primer binds to a specific site adjacent to the target DNA, the size of the amplification product can be predicted and detected based on the known size of the gel. In order to determine the characteristics of the amplicon, or if several amplicons have been generated, a DNA probe that hybridizes to the amplicon is usually employed. DNA hybridization refers to the formation of double stranded DNA by complementary base pairing. A DNA hybridization technique for clearly identifying a particular amplification product requires the use of a DNA probe of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DNA sequence, hybridization will occur under conditions of temperature, pH and ion concentration. If hybridization occurs, the desired gene or DNA sequence is present in the original sample. Optical detection is the most common method of detecting hybridization. Either the amplicon or probe is labeled to emit light by fluorescence or electrochemiluminescence. The difference in these processes lies in the manner in which the excited state of the photopolymerizable group is produced, but both processes can be used to covalently label the nucleotide strands. In electrochemiluminescence (ECL), light is generated by luminophore molecules or complexes when stimulated by electrical current. In fluorescent light, it causes light to be emitted by excitation light that causes luminescence. Fluorescence is detected using illumination sources and detection units that provide excitation light at wavelengths absorbed by the fluorescent molecules. The detection unit includes a photosensor that detects a transmitted signal (such as a photomultiplier tube or a charge coupled device (CCD) array) and a mechanism that prevents excitation light from being included in the output of the photosensor (such as a wavelength selective filter) ). The fluorescent molecules emit Stokes shifted light to respond to the excitation light, which is then collected by the detection unit -15-201219776. The Stokes shift is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. The E C L emission system is detected using a light sensor that is sensitive to the emission wavelength of the E C L species used. For example, the transition metal-ligand complex emits light of visible wavelengths, so conventional photodiodes and CCDs can be used as photosensors. One of the advantages of ECL is that if the ambient light is shielded, the ECL's emitted light is the only light in the detection system, thus increasing sensitivity. Microarrays allow hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are powerful and powerful tools for molecular diagnostics that can screen thousands of genetic diseases or detect the presence of numerous infectious pathogens in a single experiment. Microarrays consist of a number of different DNA probes that are fixed at the point on the substrate. 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 DNA to the probe microarray. The microarray is incubated 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 cleaned with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The rigor of hybridization and cleaning is critical. Insufficient rigor may result in a highly non-specific combination. Excessive rigor may result in an inability to properly combine, resulting in reduced sensitivity. Hybridization is identified by detecting the light emitted by the labeled amplicons of the hybrid formation of the complementary probe. The fluorescence from the microarray is detected by a microarray scanner, which is usually a computer-controlled inverted scanning fluorescent conjugated focus microscope. The display-16-201219776 micromirror usually uses a laser to excite the fluorescent dye. And a light sensor (such as a photomultiplier tube or CCD) detects the transmitted signal. Fluorescent molecules emit Stokes bits (as described above), which are collected by the detection unit. The emitted fluorescent light must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. In microarray scanners, a conjugate focal configuration of a conjugated focal hole aperture mounted on the image side is typically used to eliminate out-of-focus (out-〇f-f〇CUS) information. This device allows only the light of the focus 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 the detector and then converted into digital signals. 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. For more information on fluorescent probes please see: http://www. Premierbiosoft. Com/tech_notes/FRET_probe. Html and 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-Point Care (P0INT-0F-CARE) Molecular Diagnostics Although molecular diagnostic tests offer many benefits, the growth of such tests in clinical laboratories is still slower than expected and is not the mainstream of laboratory medical tests -17-201219776. This is mainly because nucleic acid tests lead to higher complexity and cost than tests that do not involve nucleic acid methods. The extensive use of molecular diagnostic tests in clinical settings is closely related to the development of instrumentation. 'The instrument must be able to significantly reduce costs, provide rapid (automatic) analysis from initial (sample processing) to final (resulting), without 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-focused home environment. This technology offers many benefits including: • Quick results for immediate treatment and improved care quality • Very small amount The number of laboratories in the sample test 値• Reduce the clinical workload • Reduce the workload of the laboratory and reduce the administrative work to improve the efficiency of the office. • By reducing the number of hospital stays, outpatients can be diagnosed at the initial diagnosis and reduced sample processing, Storage and delivery to improve the cost per patient • Helps clinical management decisions such as infection control and antibiotic use. Laboratory wafer (LOC)-based molecular diagnostics provide micro-fluid technology-based molecular diagnostic systems that can be automated and accelerated. A device for molecular diagnostic analysis. The shorter detection time is mainly due to the fact that the sample body required is less active, automated and within the low overhead of the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. Laboratory wafer (L0C) devices are a common form of microfluidic device. The LOC device has an MST structure within the MST layer for integrating fluid processing onto a single support substrate (typically helium). The unit cost of each LOC device is very low by manufacturing a VLSI (very large integrated circuit) lithographic technique for the semiconductor industry. However, large external piping and electronic devices are required to control the flow of fluid through the L 0 C device, to add reagents, to control reaction conditions, and the like. The connection of LOC devices to these external devices greatly limits the molecular diagnostic use of the l〇C device in a laboratory environment. Cost of external instruments and their operational complexity Eliminate L0C-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 L0C device that can be used for fixed-point care. SUMMARY OF THE INVENTION Various aspects of the present invention are now described in the following paragraphs. GSE001. 1 This aspect of the invention provides a microfluidic device for treating a fluid, the microfluidic device comprising: an inlet for receiving a fluid; a functional region for treating the fluid; a flow path extending from the inlet into the at least one functional region; a sensor having a conductive element located adjacent the fluid flowing along the flow path: -19-201219776 A CMOS circuit configured to measure a temperature of a conductive element; wherein the CMOS circuit is configured to provide a conductive element The predetermined current is measured and the resistance of the conductive element is measured, so that the flow rate is derived from the current, temperature sense, and the resistance, and the flow rate is based on the flow velocity and the flow path cross section of the flow direction and the flow direction. Export. GSE001. Preferably, the electrically conductive element is a heater element supported on the inner surface of the flow path. GSE001. Preferably, the electrically conductive element has a curved configuration. GSE001. 4 Preferably, the flow path is defined by the microchannel. The cross-sectional area of the microchannel with the flow transverse direction is less than 100,000 square micrometers. GSE001. Preferably, one of the functional regions is a polymerase chain reaction (PCR) region, and the fluid is a biological sample containing a target nucleic acid sequence. The PCR region is configured to perform a thermal cycle of the sample to The target nucleic acid sequence is amplified and the microchannel defines a flow path through the PCR region. GSE001. Preferably, the microfluidic device also has at least one elongated heater element for heating the target nucleic acid sequence within the microchannel. GSE001. Preferably, the microfluidic device also has a support substrate and a microsystem technology (MST) layer incorporated into the functional area, wherein the CMOS circuit has digital memory for storing data and operational information for operation during processing and analysis of the sample. Control the ribbon. GSE001. Preferably, the microfluidic device also has a plurality of reagent libraries containing reagents for processing the sample, wherein the data stored in the digital memory -20-201219776 is related to the reagent characteristics. GSE001. Preferably, the data stored in the digital unit is a unique identifier of the microfluidic device. GSE001. Preferably, the operational information stored in the digital memory is related to the time and duration of the thermal cycle. GSE001. Il preferably, the functional region comprises a growing region upstream of the PCR region, and one of the reagent libraries is a restriction enzyme library having a sample and a restriction enzyme for restriction enzyme digestion of the target nucleic acid sequence The temperature of the mixture is maintained at the incubation temperature of the heater. GSE001. Preferably, the microfluidic device also has an array of probes for hybridizing to a target nucleic acid sequence in an amplicon from the PCR region. GSE001. Preferably, the data stored in the digital memory includes probe characteristic data identifying the probe at each of the points within the probe array. GSE001. Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each probe-target hybrid being configured to respond to input A photon is emitted, and the C Μ 0 S circuit incorporates a photosensor for sensing photons emitted by the probe-target hybrid. GSE00 1. Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output. GSE001. Preferably, the microfluidic device also has an array of hybridization chambers for housing the probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. -21 - 201219776 GSE001. Preferably, the photosensor is an array of photodiodes that are registered with the hybridization chamber. GSE001. Preferably, the CMOS circuit has bond pads and is configured to transmit hybridization data to an external device. GSE00 1. Preferably, the sample is taken from the patient and the CMOS circuit is configured to download patient data via the bond pad and store the patient's data in digital memory. GSE001. Preferably, the PCR zone has an active valve for retaining liquid in the PCR zone during thermal cycling and allowing liquid to flow to the hybridization chamber in response to activation signals from the CMOS circuitry. As far as the operation of the microfluidic device is concerned, the microfluidic device can process and/or analyze the flow system in a large and inexpensive manner and the fluid flow rate is monitored by the easily manufactured hot wire flow sensor and controlled in an optimal manner. . GSE002. 1 This aspect of the invention provides a microfluidic device for treating a fluid, the microfluidic device comprising: an inlet for receiving a fluid; a functional region for processing a fluid; a flow path extending from the inlet into at least some of the functional regions; Circuitry for controlling the functional area; and a liquid sensor having an electrode positioned in contact with a fluid flowing along the flow path; wherein the circuit is configured to provide a voltage between the electrodes, Thus, a current above a predetermined threshold 为 is indicative of the presence of liquid at the electrode. -22- 201219776 GSE002. 2 Preferably, the microfluidic device also has a temperature sensor ' for sensing the temperature of the fluid in the flow path and a flow rate sensor having a heater element supported on the inner surface of the flow path, wherein the circuit Is configured to receive a temperature sensor output, provide a predetermined current through the heater element and measure a resistance of the conductive element to derive a flow rate from the current, temperature sensor output, and resistance, and the flow rate is used The flow velocity and the flow path cross section on the heater element and the flow transverse direction are derived. GSE002. Preferably, the heater element has a curved configuration. GSE002. Preferably, the flow path is defined by a microchannel having a cross-sectional area transverse to the flow direction of less than 100,000 square microns. GSE002. Preferably, one of the functional regions is a polymerase chain reaction (PCR) region, and the fluid is a biological sample containing a nucleic acid sequence configured to perform thermal cycling of the sample to sequence the nucleic acid sequence Amplification, the microchannel defines a flow path through the PCR region. GSE002. Preferably, the microfluidic device also has at least one elongated heater element for heating the nucleic acid sequence within the microchannel. GSE002. Preferably, the microfluidic device also has a support substrate and a microsystem technology (MST) layer incorporated in the functional area, wherein the circuit is a CMOS circuit having digital memory for storing data and operating information for processing and The functional area is controlled during the analysis of the sample. GSE002. Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample, wherein the data stored in the digits is related to reagent characteristics. -23- 201219776 GSE002. Preferably, the data stored in the digital memory is a unique identifier of the microfluidic device. GSE002. Preferably, the operational information stored in the digital memory is related to the time and duration of the thermal cycle. GSE002. Il preferably, the functional region comprises a growing region upstream of the PCR region, and one of the reagent libraries is a restriction enzyme library having a mixture for the sample and the restriction enzyme during restriction enzyme digestion of the nucleic acid sequence. The heater is maintained at the incubation temperature. GSE002. Preferably, the microfluidic device also has an array of probes for hybridizing to a target nucleic acid sequence in an amplicon from the PCR region. GSE002. Preferably, the data stored in the digital memory includes probe characteristic data identifying the probe at each of the points within the probe array. GSE002. Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each probe-target hybrid being configured to respond to input The photons are emitted, and the CMOS circuit incorporates a photosensor for sensing photons emitted by the probe-target hybrid. GSE002. Preferably, the data stored in the digital body comprises hybridization data generated from the output of the photosensor. GSE002. Preferably, the microfluidic device also has an array of hybridization chambers for receiving probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. GSE002. Preferably, the photosensor is a photodiode array that is registered with the hybridization chamber -24-201219776. GSE002. Preferably, the CMOS circuit has a bond pad and is configured to transmit hybridization data to an external device. GSE002. Preferably, the sample is taken from the patient and the CMOS circuit is configured to download patient data via the bond pad and store the patient's data in a digital body. GSE002. Preferably, the PCR zone has an active valve for retaining liquid in the PCR zone during thermal cycling and allowing liquid to flow to the hybridization chamber in response to activation signals from the CMOS circuitry. From the point of view of the operation of the microfluidic device, the microfluidic device can process and/or analyze the flow system and the specified location in the flow system in a large and inexpensive manner by the easy-to-manufacture liquid sensor and in the most ideal manner. Control related. GSE003. 1 This aspect of the invention provides a microfluidic device for treating a fluid, the microfluidic device comprising: an inlet for receiving a fluid; a functional region for processing a fluid; a flow path extending from the inlet into at least some of the functional regions: a circuit for controlling the functional area; and a capillary leading edge advancement sensor having a plurality of liquid sensors spaced along the flow path, each liquid sensor having a position along and along the flow path An electrode at a location where the flowing fluid contacts; wherein the circuit is configured to provide a voltage between the electrodes such that a current above a predetermined threshold 为 is indicative of the presence of liquid at the electrode and is used to derive the liquid flow - 25, 2012, 1977 The speed of the edge. GSE003. 2 Preferably, the microfluidic device also has a temperature sensor for sensing the temperature of the fluid in the flow path, and a flow rate sensor having a heater element supported on the inner surface of the flow path, wherein A circuit is configured to receive a temperature sensor output, provide a predetermined current through the heater element, and measure a resistance _ of the conductive element, thereby deriving a flow rate from the current, temperature sensor output, and resistance, and the flow rate The flow velocity and the cross section of the flow path on the heater element and the flow direction are derived. GSE003. Preferably, the heater element has a curved configuration. GSE003. Preferably, the flow path is defined by a microchannel having a cross-sectional area transverse to the flow direction of less than 100,000 square microns. GSE003. Preferably, one of the functional regions is a polymerase chain reaction (PCR) region, and the fluid is a biological sample containing a nucleic acid sequence configured to perform thermal cycling of the sample to sequence the nucleic acid sequence Amplification, the microchannel defines a flow path through the PCR region. GSE003. Preferably, the microfluidic device also has at least one elongated heater element for heating the nucleic acid sequence within the microchannel. GSE003. Preferably, the microfluidic device also has a support substrate and a microsystem technology (MST) layer incorporated in the functional area, wherein the circuit is a CMOS circuit having digital memory for storing data and operating information for processing and The functional area is controlled during the analysis of the sample. GSE003. Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample, wherein the data stored in the digits -26-201219776 is related to reagent characteristics. GSE003. Preferably, the data stored in the digital memory is a unique identifier of the microfluidic device. GSE003. Preferably, the operational information stored in the digital body is related to the time and duration of the thermal cycle. GSE0 03. Il preferably, the functional region comprises a growing region upstream of the PCR region, and one of the reagent libraries is a restriction enzyme library having a mixture for the sample and the restriction enzyme during restriction enzyme digestion of the nucleic acid sequence. The heater is maintained at the incubation temperature. GSE003. Preferably, the microfluidic device also has an array of probes for hybridizing to a target nucleic acid sequence in an amplicon from the PCR region. GSE003. Preferably, the data stored in the digital memory includes probe characteristic data identifying the probe at each of the points within the probe array. GSE003. Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each probe-target hybrid being configured to respond to an input The photons are emitted while the CM Ο S circuit incorporates a photosensor for sensing photons emitted by the probe-target hybrid. GSE003. Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output. GSE003. Preferably, the microfluidic device also has an array of hybridization chambers for housing the probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. -27- 201219776 GSE003. Preferably, the photosensor is an array of photodiodes that are registered with the hybridization chamber. GSE003. Preferably, the CMOS circuit has a bond pad and is configured to transmit hybridization data to an external device. GSE003. Preferably, the sample is taken from the patient and the CMOS circuit is configured to download patient data via the bond pad and store the patient's data in digital memory. GSE003. Preferably, the PCR zone has an active valve for retaining liquid in the PCR zone during thermal cycling and allowing liquid to flow to the hybridization chamber in response to activation signals from the CMOS circuit. The microfluidic device can process and/or analyze the flow system in a large and inexpensive manner and the fluid flow rate is monitored by the easy-to-manufacture capillary leading edge velocity sensor and in an optimal manner. GSE004. 1 This aspect of the invention provides a microfluidic device for treating a fluid, the microfluidic device comprising: an inlet for receiving a fluid; a functional region for treating the fluid; a flow path extending from the inlet into at least some of the functional regions; a CMOS circuit operating the control function area; and a conductive sensor having a first terminal and a second terminal spaced along the flow path and disposed between the first terminal and the second terminal along the flow path a first electrode and a second electrode separated by: wherein the CMOS circuit is configured to generate a current between -28-201219776 of the first terminal and the second terminal, and measure a voltage passing through the first electrode and the second electrode, thereby The conductivity of the fluid in the flow path is derived from the current and the measured voltage. GSE004. 2 Preferably, the microfluidic device also has a temperature sensor for sensing the temperature of the fluid in the flow path, and a flow rate sensor having a heater element supported on the inner surface of the flow path, wherein the circuit Configuring to receive a temperature sensor output, providing a predetermined current through the heater element and measuring the resistance of the conductive element, thereby deriving the flow rate from the current, temperature sensor output, and resistance, and using the flow rate The flow velocity and the flow path cross section on the heater element and the flow transverse direction are derived. GSE004. Preferably, the heater element has a curved configuration. GSE004. Preferably, the flow path is defined by a microchannel having a cross-sectional area transverse to the flow direction of less than 100,000 square microns. GSE004. Preferably, one of the functional regions is a polymerase chain reaction (PCR) region, and the fluid is a biological sample containing a nucleic acid sequence configured to perform thermal cycling of the sample to sequence the nucleic acid sequence Amplification, the microchannel defines a flow path through the PCR region. GSE004. Preferably, the microfluidic device also has at least one elongated heater element for heating the nucleic acid sequence within the microchannel. GSE004. Preferably, the microfluidic device also has a support substrate and a microsystem technology (MST) layer incorporated into the functional area, wherein the CMOS circuit has digital memory for storing data and operational information for operation during processing and analysis of the sample. Control the ribbon. -29- 201219776 GSE004. Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample, wherein the data stored in the digits is related to reagent characteristics. GSE004. Preferably, the data stored in the digital memory is a unique identifier of the microfluidic device. GSE004. Preferably, the operational information stored in the digital memory is related to the time and duration of the thermal cycle. GSE004. Il preferably, the functional region comprises a growing region upstream of the PCR region, and one of the reagent libraries is a restriction enzyme library having a mixture for the sample and the restriction enzyme during restriction enzyme digestion of the nucleic acid sequence. The heater is maintained at a temperature at the incubation temperature. GSE004. Preferably, the microfluidic device also has an array of probes for hybridizing to a target nucleic acid sequence in an amplicon from the PCR region. GSE004. Preferably, the data stored in the digital memory includes probe characteristic data identifying the probe at each of the points within the probe array. GSE004. Preferably, each probe is configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each probe-target hybrid being configured to respond to input The photons are emitted, and the CMOS circuit incorporates a photosensor for sensing photons emitted by the probe-target hybrid. GSE004. Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output. GSE004. Preferably, the microfluidic device also has an array of hybridization chambers for receiving probes -30-201219776 such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. GSE004. Preferably, the photosensor is an array of photodiodes that are registered with the hybridization chamber. GSE004. Preferably, the CMOS circuit has a bond pad and is configured to transmit hybridization data to an external device. GSE004. Preferably, the sample is taken from the patient and the CMOS circuit is configured to download patient data via the bond pad and store the patient's data in digital memory. GSE004. Preferably, the PCR zone has an active valve for retaining liquid in the PCR zone during thermal cycling and allowing liquid to flow to the hybridization chamber in response to activation signals from the CMOS circuitry. In terms of the operation of the microfluidic device, the microfluidic device can process and/or analyze the flow system and the conductivity of the fluid mixture in a large amount and inexpensively by the easy-to-manufacture conductive sensor and in an optimal manner. Control fluid flow rate related. [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED SYSTEM STATEMENT This brief description identifies the main components of the molecular diagnostic system incorporating the system of the present invention. The details of the structure and operation of the system are described in the following patent specification. Referring to Figures 1, 2, 3, 12 and 121, the system has the following top-31 - 201219776 layer components: Test modules 10 and 1 have the dimensions of a typical USB memory key and are very inexpensive to manufacture. Test modules 1 and 1 each comprise a microfluidic device, typically in the form of a pre-packaged on-wafer laboratory (LOC) device 30 and typically have more than 1000 probes for molecular diagnostic detection (see Figures 1 and 1 2 0)). The test module 10 shown in Figure 1 uses a fluorescence-based detection technique to identify the target molecules, and the test module 1 1 shown in Figure 20 is used. Detection technology based on electrochemiluminescence. The LOC device 30 has an integrated light sensor 44 (described in detail below) for detecting fluorescence or electrochemiluminescence. Both test modules 1 and 11 use standard Micro-USB plugs 14 for power, data and control, both with a printed circuit board (PCB) 57 and both with external power supply capacitors 32 and inductors Device 15. Both test modules 10 and 11 are used in a single use for mass production and sale of ready-to-use sterile packaging. The outer casing 13 has a large container 24 for receiving a biological sample and a removable sterile sealing tape 22 (a low viscosity adhesive is preferred) to cover the large container prior to use. A membrane gasket 408 with a membrane shield 410 forms part of the outer casing 13 to reduce moisture loss in the test module while providing pressure relief from small air pressure fluctuations, and the membrane shield 410 protects the membrane gasket 408 Not damaged. The test module reader 12 provides test module 10 or 11 power via a Micro-USB port 16. The test module reader 12 can take a variety of different forms, and the choice of these forms will be described later. The reader 丨 2 shown in Figures 1, 3 - 32 - 201219776 and 120 is in the form of a smart phone system. A block diagram of this reader 12 is shown in FIG. The processor 42 runs the application software from the program memory 43. The processor 42 is also coupled to a display screen 18 and a user interface (ui) touch screen 17 and button 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 location data of the epidemiological database. Alternatively, the location data can be manually entered via the touch screen 17 or button 19. The data store 27 holds genetic and diagnostic information, test results, patient information, analysis and probe data for identifying the position of each probe and its array. Data memory 27 and program memory 43 share a common memory facility. The application software installed in the test module reader 12 provides analysis of results and other test and diagnostic information. For diagnostic testing, the test module 1 〇 (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 peeled back and the biological sample (in liquid form) is loaded into the large sample container 24. Pressing the start button 20 initiates the test via the application software. The sample flows into the LOC device 30, and the on-board assay extracts, cultures, and amplifies the sample nucleic acid (target) and hybridizes it to a pre-synthesized hybrid-reactive oligonucleotide probe. . In the case of a test module 1 (which uses a fluorescence-based detection method), the probe is fluorescently labeled and the LEDs 26 stored in the housing 13 provide the necessary excitation light to induce fluorescence. Emission from hybridized probes (see Figures 1 and 2). The LOC device 30 is loaded with an ECL probe (as described above) in the test mode 1 (which uses electrochemiluminescence (EC L) detection - 33 - 201219776), and the LED 26 is not necessary to generate a fluorescent emission. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 121). The emitted light (fluorescent or luminescent) is detected using a light sensor 44 integrated into the CMOS circuitry of each LOC device. The detected signal is amplified and converted to a digital output, which is then analyzed by test module reader 12. The reader then displays the results. This data can be saved and/or uploaded to a web server containing patient records. Test Module Reader 1 2 Remove the test module 1 〇 or 1 1 and dispose of it appropriately. Figures 1, 3 and 120 show a test module reader 12 in the form of a handset/smartphone 28. In other forms, the test module reader is a laptop/notebook 101, a dedicated reader 103, an e-book reader 107, a tablet 109, or a desktop computer 105 for use in a hospital, private clinic, or laboratory. (See Figure 122). The reader can be connected to a range of other applications, such as medical records, billing, online databases, and multi-user environments. It can also be connected to a range of nearby or remote peripherals, such as printers and patient smart cards. Referring to FIG. 23, the data generated by the test module 1 can be updated by the reader 12 and the network 125 to update the epidemiological data managed on the epidemiological data host system 1U, and managed on the genetic data host system 113. Genetic data, electronic health records managed on an electronic health record (EHR) host system 115, electronic medical records managed on an electronic medical record (EMR) host system 121, and personal health records (PHR) master-34 - 201219776 Machine system 1 2 3 Managed personal health records. Conversely, epidemiological data managed on the epidemiological data host system 111, genetic data managed on the genetic data host system 113, electronic health records managed on the electronic health record (EHR) host system 115, The electronic medical record managed on the electronic medical record (EMR) host system 121 and the personal health record managed on the personal health record (PHR) host system 123 can update the LOC 30 of the test module 1 via the network 125 and the reader 12. Digital memory in the middle. Referring back to Figures 1, 2, 120 and 121, the reader 12 uses the battery power in the mobile phone configuration. The mobile phone reader contains all pre-downloaded test and diagnostic information. Data can also be downloaded or updated via a number of wireless or contact interfaces to interface with peripheral devices, computers or online servers. Mi cro - U S B埠1 6 can be used to connect to a computer or mains to charge the battery. Figure 77 shows a system of test modules 1 for testing that requires only positive or negative results for a particular target, such as testing whether an individual is exposed to, for example, a Η 1N 1 influenza A virus infection. It is sufficient to simply configure the USB power/indicator-definition module 47. No other readers or application software is required. The indicator on the USB power/indicator-defining module 47 signals a positive or negative result. This configuration is ideal for group screening. Other items provided with the system may include test tubes containing reagents for pre-processing certain samples, as well as spatula and blood collection needles for collecting samples. Figure 77 shows the test module system incorporating the spring-loaded retractable lancet -35-201219776 3 90 and the lancet release button 3 92 for convenient use. Satellite phones are available for use in remote locations. Test Module Electronics Figures 2 and 1 2 1 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 provide the entire test module 10 or 1 1 (including photosensor 44, temperature sensor 170, liquid sensor 1 74, and various heaters 152, 154, 182, 234, along with associated drivers 37 and 39, as well as recorders 3 5 and 4 1 ) control and record billion. Only the LED 26 (in the case of the test module 10) 'the external power supply capacitor 32 and the Micro-USB plug 14 are external to the LOC device 30. The LOC device 30 includes a bond pad for connecting these external components. RAM 3 8 and Program and Data Flash Memory 40 have application software and diagnostic and test information for more than 1 000 probes (flash/secure storage, eg via encryption). There is no LED 26 in the case of a test module 1 1 configured for ECL detection (see Figures 120 and 121). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 carries an electrochemical luminescence probe, and each of the hybridization chambers has a pair of ECL excitation electrodes 860 and 870. Many types of test modules 10 are manufactured in a variety of test formats for ready-to-use use. The difference between this test format is the on-machine detection of reagents and probes. -36- 201219776 Some examples of infectious diseases that are rapidly identified by this system include: • Influenza-flu viruses A, B, C, Isavirus, Thogotovirus • Pneumonia-Respiratory Syndrome (RSV) , adenovirus, interstitial pneumonia virus, pneumococci, staphylococcus aureus • tuberculosis - mycobacterium tuberculosis, mycobacterium bovis, mycobacteria, mycobacteria, mycobacteria, mycobacteria, falciparum Insects, Toxoplasma and other protozoan parasites • Typhoid-S. typhimurium serotype • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever - flavivirus • Hepatitis (A to E) • Nosocomial infections - For example: Clostridium difficile, vancomycin-resistant Enterococcus and methicillin-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Cytomegalovirus (CMV) • Epstein-Barr virus (EBV) • Brain Inflammation - Japanese encephalitis virus, Chandipura virus • Pertussis-B. pertussis • Paralytic-paramyxovirus • Meningitis - Streptococcus pneumoniae and Neisseria meningitidis •疽-Bacillus anthracis-37- 201219776 Some examples of hereditary diseases that are rapidly identified by this system include: • Cystic fibrosis • Hemophilia • Sickle cell disease • Tay-Sachs disease • Hemochromatosis (haemochromatosis) • cerebral arterial disease • Crohn's disease • polycystic kidney disease • congenital heart disease • Rett syndrome is identified by this diagnostic system. A small number of cancers include: • Ovary • Colon cancer • Multiple endocrine tumors • Retinoblastoma • Turcot Syndrome The above list is not exhaustive and the diagnostic system can be configured to utilize nucleic acid and proteomic analysis to detect a wider variety of diseases and conditions. Detailed structure of system components LOC device LOC device 3 is the center of the diagnostic system. It uses a microfluidic platform to rapidly perform four major steps in nucleic acid-based molecular diagnostic analysis. 38·201219776, ie, preparing samples, extracting nucleic acids, amplifying nucleic acids, and detecting. The LOC device also has other uses, which will be described in detail later. As mentioned above, test modules 1 〇 and 1 1 can be implemented in many different configurations to detect different targets. Similarly, the LOC device 30 has many different targets for the desired target system. One form of LOC device 30 is the LOC device 301, which is used to fluorescently detect a target nucleic acid sequence in a pathogen of a whole blood sample. For purposes of illustration, the structure and operation of the LOC device 301 will now be described in detail with reference to Figures 4 through 26 and 27 through 57. FIG. 4 is a schematic image of the structure of the LOC device 301. For the sake of convenience, the processing stages shown in Fig. 4 are denoted by reference numerals corresponding to the functional areas of the LOC device 301 performing the processing stage. It also refers to the stages of processing associated with the major steps of the nucleic acid-based molecular diagnostic assay: sample input and preparation 28, extraction 290, incubation 291, amplification 292, and detection 294. Each of the reservoirs, chambers, valves, and other components of the LOC device 301 will be described in more detail later. Figure 5 is a perspective view of the LOC device 301. It is manufactured using high-capacity CMOS and MST (microsystem technology) manufacturing techniques. The hierarchical structure of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) of Fig. 12. The LOC device 301 has a germanium substrate 84 supporting a CMOS + MST chip 48 comprising a CMOS circuit 86 and an MST layer 87 overlying the MST layer 87 with a cap 46. In this patent specification, the term "MST layer" refers to the collective name of the structure and hierarchy of the sample treated with various reagents. Accordingly, these configurations and components are configured to define a flow path in a particular size that is uniquely sized to support the flow of liquid having a physical property similar to that of the sample -39 - 201219776 during capillary processing. In view of this, the MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also be fabricated with dimensions that are applicable to structures driven by capillary action and that can handle very small volumes of structures and components. The particular system described in this patent specification shows the MST layer as a structure and components that are supported on CMOS circuitry 86, but does not include portions of cover 46. However, those skilled in the art will appreciate that the MST layer does not need to have an underlying CMOS or a true overlay cover for processing samples. The overall dimensions of the LOC device shown in the following figures are 1 760 microns x 5 8 24 microns. Of course, the LOC devices that are manufactured for use in different applications can have different sizes. Fig. 6 shows the appearance of the MST layer 87 having the cover portion superimposed thereon. The illustrations AA to AD, AG, and AH shown in Fig. 6 are magnified in Figures 13, 14, 35, 56, 55, and 67, respectively, and are described in detail below for a comprehensive understanding of the structures in the LOC device 301. Figures 7 through 10 show the appearance of the cover 46 independently, while Figure 11 shows the structure of the CMOS + MST device 48 independently. Thin Layer Structures Figures 12 and 22 are schematic diagrams showing the thin layer structure of the CMOS+ MST device 48, the cover 46, and the fluid interaction therebetween. These figures are not intended to be used for illustration. Fig. 22 is a schematic cross-sectional view through the sample inlet 68, and Fig. 22 is a schematic cross-sectional view through the reservoir 54. As best shown in Figure 12 of the -40-201219776, the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86 (which operates the useful components within the MST layer 87 described above). Passivation layer 88 seals and protects CMOS layer 86 from the fluid flowing through MST layer 87. Fluid flows through the shroud channel 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 100, respectively (see, for example, Figures 7 and 16). The cell transport system occurs in a larger channel 94 assembled in the cap 46, while the biochemical process is performed in the smaller MST channel 90. The cell transport channel is sized to transport cells in the sample to a predetermined location in the MST channel 90. Transporting cells larger than 20 microns (e.g., some white blood cells) requires channel sizes greater than about 20 microns, and therefore cross-sectional areas with flow transverse directions need to be greater than 400 square microns. MST channels, especially those that do not require transport of cells in the LOC, can be significantly smaller. It will be appreciated that the cover channel 94 and the MS T channel 90 are generic names, and in particular the MS T channel 90 may also be referred to as, for example, a heated microchannel or a dialysis M S T channel in view of its particular function. The M S T channel 90 is formed by etching through the MST channel layer 100 located on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is surrounded by a roof layer 66 that forms the top of the CMOS + MST device 48 (associated with the orientation shown in the figures). Although sometimes shown in a separate hierarchy, the cover channel layer 80 and the reservoir layer 78 are formed from a single piece of material. Of course, the piece of material may also be a non-single piece. The sheet of material is etched from both sides to form a cap channel layer 80 in which the cap channel 94 is etched and a reservoir layer 78 in which the reservoirs S4, 56, -41 - 201219776 58, 60 and 62 are etched. Storage is formed by micromolding. The engravers are used to create channels that flow laterally to 200,000 microns and as small as 8 square microns. There may be some suitable range of choices for flow and flow in different locations of the LOC device. When the channel contains a large number of samples, the cross-sectional area is suitable to be at most (for example, the cross-sectional area of a mixed channel containing a small amount of liquid or no large cells in a 200 micron track in a layer of 〇〇 〇〇 micron thickness is preferably very small. A lower gasket 64 surrounds the cover channel 94, enclosing the reservoirs 54, 56, 58, 60, and 62» the five reservoirs 54, 56, 58, 60, and 62 dedicated reagents. In the system described herein, The storage reagent, but other reagents can be easily replaced: ♦ Depot 54: Anticoagulant, optional debuffer • Repository 5 6 : Lysis reagent • Repository 58: Restriction enzymes, ligation PCR initiated at the connector (see Figure 76, excerpt 丨al. , Human Molecular Genetics 2, Garland London, 1999)). Depot 60: The amplification mixture (dNTPs, and: reservoir and cap channeling and micromolding technique 2) can have a large cross-sectional area with a large cross-sectional area containing a large number of samples or a wide channel of 20,000 square microns. In the case of a general-purpose, the pre-packaged detection reservoir in the horizontally-highly sealed gasket 82 contains pre-assembled erythrocyte lyase and linker (used by T.  Stachan et Science, NY and Primers, Buffers) -42- 201219776 . Repository 62: DNA polymerase. Cover 46 and CMOS + MST layer 48 are in fluid communication via corresponding openings in lower seal 64 and roof layer 66. These openings flow from the MST channel 90 into the cap channel 94 or from the cap channel 94 to the MST channel 90 depending on the flow system, and are referred to as the ingestion port 96 and the down channel 92. LOC device operation is referred to below in the analysis of the blood sample. The pathogen DNA is used to describe the method of operation of the LOC device 301 step by step. Of course, other types of biological or non-biological fluids can also be analyzed using reagents, test protocols, variants of the LOC device, and appropriate settings or combinations of detection systems. Referring back to Figure 4, the analysis of biological samples involves five main steps, including inputting and preparing samples 28 8 , extracting nucleic acids 290, culturing nucleic acids 291, amplifying nucleic acids 292, and detecting and analyzing 2 9 4 * sample input and preparation steps 288 involves mixing the blood with the anticoagulant 116 and then separating the pathogen from the white blood cells and red blood cells by the pathogen dialysis zone 70. As best shown in Figures 7 and 12, blood samples enter the device via sample inlet 68. The capillary action draws the blood sample into the reservoir 54 along the cover channel 94. When the blood flow opens the surface tension valve 1 18 of the reservoir 54, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). The anticoagulant prevents the formation of blood clots that may block the flow. As best shown in Fig. 22, the anticoagulant 116 is aspirated from the reservoir 54 by capillary action and enters the MST channel via the descending channel 92 -43 - 201219776 90. The downcomer 92 has a capillary initiation features (CIF) 102 to shape the geometry of the meniscus such that it does not rest at the edge of the downcomer 92. The venting opening 122 in the upper gasket 82 allows air to replace the anticoagulant 116 when the anticoagulant 116 is aspirated from the reservoir 54. The MST channel 90 shown in Fig. 22 is part of the surface tension valve 118. The anticoagulant 1 16 breaks into the surface tension valve 1 18 and secures the meniscus 120 to the anchor 98 of the meniscus of the ingestion hole 96. Prior to use, the meniscus 1 20 is still secured to the ingestion aperture 96 so that the anticoagulant does not flow into the mask passage 94. As blood flows through the cap channel 94 to the ingestion port 96, the meniscus 120 is removed and the anticoagulant is drawn into the fluid stream. Figures 15 through 21 show an inset AE which is part of the insert AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 118 has three spaced apart MST channels 90 extending between respective corresponding downcomers 92 and intake apertures 96. The number of MST channels 90 in the surface tension valve can be varied to vary the flow rate of reagent into the sample mixture. When the sample mixture and reagent mixture are mixed together by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample stream. Thus, the surface tension valves of each reservoir are configured to meet the desired reagent concentration. The blood enters the pathogen dialysis zone 70 (see Figures 4 and 15), wherein the target cell line is concentrated from the sample using an array of apertures (the size of which is based on a predetermined threshold). Cells smaller than the threshold 通过 cells that pass through the small holes cannot pass through the small holes. The unwanted cells (which may be larger cells trapped by the small well array 1 64 or smaller cells that pass through the small holes) are redirected to waste-44-201219776 unit 76, while the target cells continue to be part of the analysis . In the pathogen dialysis zone 70 described herein, pathogens from whole blood samples are concentrated for microbial DNA analysis. The aperture array is formed by a plurality of apertures 1 64 having a diameter of 3 microns, the fluid input to the input flow in the cover channel 94 is coupled to the target channel 74» the 3 micron diameter aperture 1 64 and the standard The dialysis absorbing wells 1 68 of 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 enough to pass through a small hole 1 64 having a diameter of 3 microns and into the target channel 74 via the dialysis MST channel 204. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 in the cover 46, which will be directed to the waste reservoir 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate specific pathogens or other target cells, such as white blood cells, for human DNA analysis. A more detailed description of the dialysis zone and dialysis variants is provided below. Referring again to Figures 6 and 7, the flow is drawn through the target passage 74 into the surface tension valve 128 of the cracking reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending between the lysis reagent reservoir 56 and the target channel 74. When the meniscus is unfastened 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 (where the surface tension valve 118 has three MST channels 90) (hypothesis) The physical properties of the fluid are approximately equal). Therefore, the proportion of the lysis reagent in the sample mixture is greater than the ratio of the anticoagulant. The lysis reagent and target cells are mixed by diffusion in the standard channel 74 of the chemical cleavage zone 130. The boiling start valve 1 2 6 stops the flow until -45-201219776 enough time for diffusion and lysis to release the genetic material from the target cells (see Figures 6 and 7). The construction and operation of the boiling start valve are described in more detail below with reference to Figures 31 and 32. The Applicant has also identified other types of active valves that may be used herein to replace the boiling start valve (as opposed to passive valves, such as surface tension valve H8). The design of these alternative valves is also described later. When the boiling start valve 126 is opened, the lysed cells flow into the mixing zone 133 for restriction enzyme digestion and linker ligation. Referring to Fig. 3, when the liquid flow releases the meniscus from the surface tension valve 132 at the beginning of the mixing zone 133, the restriction enzyme, linker and ligase are released from the reservoir 58. The mixture flows through the length of the mixing zone 133 for diffusion mixing. At the end of the mixing zone 133 is a descending lane 134 which is directed into the cultivating chamber inlet channel 133 of the incubation zone 114 (see Figure 13). The chamber inlet channel 133 delivers the mixture into the curved structure of the heated microchannel 210, which provides an incubation chamber for holding the sample during restriction enzyme ligation and ligation of the linker (see Figures 13 and 14). . Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in Figure AB of Figure 6. Each of the patterns is shown in sequence to form layers of the CMOS + MST layer 48 and the cover 46. The illustration AB shows the end of the incubation zone 1 1 4 and the beginning of the amplification zone 11 2 . As best shown in Figures 14 and 23, the stream breaks into the microchannel 210 of the incubation zone 114 until it reaches the boiling point activation valve 106 where it circulates and diffuses. As described above, the microchannel 210 upstream of the boiling start valve 106 becomes an incubation chamber containing the sample, restriction enzyme, ligase, and linker. -46- 201219776 The heater 1 54 is then activated and held at a constant temperature for a specified period of time for restriction enzyme digestion and linker engagement. Those skilled in the art will recognize that this incubation step 291 (see Figure 4) is optional and is only required for some types of nucleic acid amplification analysis. In addition, in some cases, a heating step may be required at the end of the incubation period to allow the temperature to break above the incubation temperature. The temperature of the breakthrough causes the restriction enzyme and ligase to deactivate before entering the amplification zone 112. Deactivation of restriction enzymes and ligases is particularly suitable when amplified using thermostatic nucleic acids. After incubation, the boiling start valve 106 is activated (opened) and the flow is returned to the amplification zone 112. Referring to Figures 31 and 32, the mixture breaks into the curved configuration of the heated microchannel 158 (which forms one or more expansion chambers) until it reaches the boiling point activation valve 108. As best shown in the schematic cross-sectional view of Figure 30, the amplification mix (dNTPs, primers, buffers) is released from the reservoir 60 'polymerase and then released from the reservoir 62 into the ligation chamber and the amplification zone (respectively In the intermediate MST channel 212 of 114 and 112). Figures 35 through 51 show the layers of the LOC device 3〇1 in the illustration AC of Figure 6. Each of the figures shows the layers that form the structure of the CMOS + MST device 48 and the cover 46. The illustration AC is the end of the amplification zone 112 and the beginning of the hybridization and detection zone 52. The incubated sample, amplification mixture, and polymerase flow through microchannel 158 to boiling point activation valve 108. After sufficient time for diffusion mixing, the heater 1 5 4 in the microchannel 1 58 is activated to initiate thermal cycling or isothermal amplification. The amplification mixture is subjected to a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target -47- «et·201219776 DNA. After the nucleic acid amplification process, the boiling start valve 1〇8 opens the hybrid and detection zone 52 and flows back. The operation of the boiling start valve will be described in more detail below. As shown in Figure 52, the hybridization and detection zone 52 has a hybrid chamber array 110» Figures 52, 53, 54 and 56 showing detailed hybridization chambers. Array 110 and individual hybridization chambers 180. At the entrance to hybridization chamber 180 is a diffusion barrier 175 that prevents diffusion of the target nucleic acid, probe strands, and hybridized probes between hybridization chambers 180 during hybridization, thereby preventing false hybridization detection results. The diffusion barrier 175 exhibits a sufficiently long flow path length to prevent the target sequence and probe from diffusing out of one chamber and contaminating another chamber during the time when the probe hybridizes with the nucleic acid and detects the signal, thereby avoiding erroneous result. Another mechanism to prevent erroneous readings is to have equal probes in many hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 184 corresponding to the hybridization chamber 180 containing the equivalent probe. In the process of the single result. The abnormal results can be ignored or weighted differently. A CMOS controlled heater 182 can provide the thermal energy required for hybridization (described in detail below). After the heater is activated, hybridization occurs between the complementary target-probe sequences. The LED driver 29 in the CMOS circuit 86 transmits a signal to the LED 26 located in the test module 1 to emit light. These probes produce fluorescence only when hybridization occurs, thereby avoiding the washing and drying steps typically required to remove unbound strands. Hybridization forces the arm-and-loop structure of the FRET probe 186 to open, which causes the fluorophore to emit fluorescing energy in response to the LED excitation light, as discussed in more detail below. Fluorescence is detected by photodiode 184 of -48 - 201219776 in CMOS circuit 86 under each hybrid cell 180 (see hybridization chamber described below). All of the hybrid chamber's Optoelectronics II 84 and associated electronics together form a photosensor 44 (see set of drawings). In other systems, the photosensor can be a charge coupled device column (C C D array). The amplification and conversion detected from the photodiode 128 is further described in detail below by the digital output detection method analyzed by the test module reader 12. Other Details of the LOC Device Modularized Design The LOC device 301 has a number of functional areas including reagent reservoirs 56, 58, 60 and 62, dialysis zone 70, lysis zone 130, incubation zone and amplification zone 11 2. Valve type, plus Humidifier and humidity sensor. In other systems of the device, these functional areas may be omitted, and the functional area may be added' or the functional area may be used for the above alternative purposes. For example, the incubation zone 1 14 can serve as the first addition zone 112' of the tandem amplification analysis system. The chemical lysis reagent library 56 can be used to add the primer-amplification mixture, dNTPs and buffer, while the reagent library 58 is coupled with the reverse transcription. Enzymes and / or polymerases. If the sample requires chemistry, a chemical lysing agent may also be added to the reservoir together with the amplification mixture' or the sample may be heated in the incubation zone for a predetermined thermal cracking. In some systems, if chemical cleavage is required and a mixture of primers, dNTPs, and buffers is required to separate from the chemical cleavage agent, additional reservoirs can be immediately included upstream of reservoir 58 with mixtures, dNTPs, and buffers. Polar body | 68 array signal. The 54 '114 LOC extra-expansion is added to the cleavage repository time. In the case of -49-201219776 In some cases, it may be necessary to omit a step, such as the incubation step 2 9 1 . In this case, an LO C device may be specially fabricated to omit the reagent reservoir 58 and the incubation zone 1 1 4, or the reservoir may not be loaded with reagents at all, or the active valve (if present) will not be active. The reagent is dispensed into the sample stream, which then becomes a channel at all to transport the sample from the lysis zone 130 into the amplification zone 112. The heater can operate independently, so when the reaction requires heat (such as thermal cracking), arranging the heater to not operate at this step ensures that thermal cracking does not occur in LOC units that do not require thermal cracking. The dialysis zone 70 can be located at the beginning of the fluid system within the microfluidic device as shown in Figure 4, or can be located anywhere within the microfluidic device. For example, in some cases, it may be beneficial to perform dialysis to remove cell debris after the amplification phase 292 prior to the hybridization and detection step 294. In addition, one or more dialysis zones can be included at any location throughout the LOC device. Similarly, additional amplification regions 1 1 2 may be included to allow multiple targets to be amplified simultaneously or sequentially, and then detected in the hybridization chamber array 110 with a particular nucleic acid probe. To analyze a sample of similar whole blood in which dialysis is not required, the dialysis zone 70 can be completely omitted from the sample input and preparation zone 28 of the LOC design. In some cases, there is no need to omit the dialysis zone 70 from the LOC device, even though the analysis does not require dialysis. If the presence of the dialysis zone does not have a geometrical barrier to the analysis, the dialysis zone 70 can still be used in the sample input and preparation zones of the LOC without loss of the desired function. In addition, the detection zone 294 may comprise a protein body cell array that is identical to the hybridization chamber array, but is designed to bind or hybridize to the probe target protein present in the non-amplified sample, rather than Designed from -50 to 201219776 A nucleic acid probe that hybridizes to a target nucleic acid sequence. It will be appreciated that the LOC devices used in this diagnostic system are manufactured to have different functional regions depending on the particular LOC application. The vast majority of functional areas are common to many LOC devices, and the addition of additional LOC devices for new applications is a matter of consolidating the appropriate functional areas from the large number of functional area options used in existing devices. Only a few LOC devices are shown in this description and some are shown to illustrate the design flexibility of the LOC devices used in this system. Those skilled in the art will readily recognize that the LOC installation shown in this description is not an exhaustive list and that many additional LOC designs are a combination of functional zones. Sample type LOC variants can accept and analyze sample nucleic acid or protein inclusions in a variety of liquid forms including, but not limited to, blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord, sweat, Chest effusion, tears, pericardial effusion, ascites fluid sample and dip sample. Amplicon obtained from macroscopic nucleic acid amplification is analyzed using a LOC device; in this case, all reagent libraries will be or be configured to not release their contents, and the dialysis, lysis, incubation and zones will only be used The sample is transported from sample inlet 68 to hybridization chamber 180 for detection of nucleic acids as described above. For some sample types, the pre-treatment step is a necessary group of roots and the LOC combination is intended to be placed in a suitable type of liquid and blood, and the ring can also be expanded by amplification, for example -51 - 201219776 For example, semen may need to be liquefied first and mucus may need to be taken to reduce its viscosity before entering the LOC device. Sample Input Referring to Figures 1 and 12, the sample is first added to the eyepiece container 24. The large container 24 is a truncated circular effect that feeds the sample into the inlet 68 of the LOC unit 301 into a 64 micron wide 60 micron deep mask passage 94 which is drawn by capillary action to the anticoagulant reservoir 54. The reagent library uses an analysis system for microfluidic devices (such as LOC with a small volume of reagents to allow the reagent library to contain a small volume of reagents required for biochemical treatment. This volume is easily less than meters, in most cases less than 3 The billion cubic micron is 70 million cubic micrometers and is less than 20 million cubic micrometers in the case shown in the drawing. The dialysis zone refers to the maps of the 15th, 21st, 33th and 34th pathogens. Target cells. Small pore cells in the form of a plurality of 3 micron diameter wells as previously described in 6 6 are filtered from the sample body. When the sample flows through 3 micrometers, the microbial pathogen enters a series of enzymes through the pores. At ί: the large cone of the module 1 ,, which is made up of the hair. From here, it also borrows all the reagents required by 301), 1 billion cubic micrometers, usually a small LOC device 30 1 I In the description of zone 70, the target diameter aperture 164 is analyzed in the roof layer 164. The channel - channel-52-201219776 2 04 is recirculated into the target channel 7 4 via the 16 micron dialysis inlet 168 (see section 3). 3 and 3 4)). The remaining sample (red blood cells, etc.) remains in the mask channel 94. Downstream of the pathogen dialysis zone 70, the cap passage 94 becomes a waste channel 72' which is directed to the waste reservoir 76. In terms of the type of biological sample from which a large amount of waste is generated, the foam insertion member or other porous member 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 pathogen dialysis zone 70 relies entirely on the capillary action of the fluid sample to function. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis zone 70 has a capillary action initiation characteristic device (CIF) 166 (see Figure 33) such that fluid is drawn into the underlying dialysis MST channel 204. The first ingestion port 198 of the target channel 74 also has a CIF 202 (see Figure 15) to avoid complete immobilization of the meniscus when the flow passes through the dialysis uptake port 168. The small component dialysis zone 682 shown in the schematic of Fig. 81 may have a structure similar to the pathogen dialysis zone 70. The small component dialysis zone is sized to allow small target cells or molecules to enter the target channel by making the pore size (and shaped if necessary) to continue the size of the assay to any small target cell or molecule. Separated from the sample. Larger sized cells or molecules are moved to waste repository 766. Thus, LOC device 30 (see Figures 1 and 120) is not limited to the isolation of pathogens having a size of less than 3 microns, but can also be used to isolate cells or molecules of any desired size. Lysis zone -53- 201219776 Referring again to Figures 7, 11, and 13, the genetic material in the sample is released from the cell by a chemical cleavage process. As described above, the lysis reagent from the lysis library 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 128 of the lysis reservoir 56. However, some diagnostic assays are more suitable for thermal cracking processes, even combining the target cells for chemical and thermal lysis. LOC device 301 achieves this by providing heated microchannels 210 of incubation zone 114. The sample stream is drawn into the incubation zone 1 14 and stopped at the boiling start valve 106. The incubation microchannel 210 heats the sample at a temperature that can destroy the cell membrane. In some thermal cracking applications, the enzymatic reaction in the chemical cracking zone 130 is not necessary and the thermal cracking reaction completely replaces the enzyme catalyzed reaction in the chemical cracking zone 130. Boiling Start Valve As described above, the LOC unit 301 has three boiling start valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independently shown boiling start valve 108 located at the heated microchannel 158 terminal of the amplification zone 112. The sample stream 1 1 9 is drawn by capillary action to the heated microchannel 1 58 until the boiling point activation valve 108 is reached. The sample flow is preceded by an anchor 98 that is secured to the meniscus of the valve inlet 146. The geometry of the meniscus anchor 98 stops the meniscus from advancing to contain the capillary flow. As shown in Figures 3 j and 32, the meniscus anchor 98 is an aperture provided from the MST channel 90 to the intake opening of the shroud channel 94. The surface of the meniscus 丨 2〇 -54- 201219776 The force keeps the valve closed. A ring heater 152 is located around the valve inlet 146. The ring heater 125 is controlled by C Μ Ο S via the boiling start valve heater joint 1 5 3 . To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater connector 153. The ring heater 125 heats the liquid sample 119 until it boils. Boiling causes the meniscus 120 to no longer be secured to the valve inlet 146 and causes the shroud passage 94 to begin to wet. Once the cover channel 94 begins to wet, the capillary flow is restored. The fluid sample 1 19 breaks into the cap passage 94 and flows through the valve down channel 150 to the valve outlet 14 4 where the flow driven by capillary action continues along the amplifying zone outlet channel 160 into the hybridization and detection Measuring area 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 10 are single-use devices, it is not necessary to reclose the valve. Incubation Zone and Nucleic Acid Amplification Zone Figures 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubation zone 114 and the amplification zone 112. The incubation zone 114 has a single, heated incubation microchannel 210 that is a curved microchannel etched in the MST channel layer 100 from the downcomer opening 134 to the boiling activation valve 106 (see pages 1 3 and 14). Figure). Controlling the temperature of the incubation zone 14 14 results in a higher efficiency of the enzyme catalytic reaction. Similarly, the amplification zone I12 has a heated -55-201219776 amplification microchannel 158 from the boiling start valve 1〇6 to the curved configuration of the boiling start valve 108 (see Figures 6 and 14). These valves arrest the flow to retain the target cells in the heated incubation or amplification microchannel 210 or 158 as mixing, incubation and nucleic acid amplification occurs. The curved pattern of the microchannel also promotes (to some extent) the mixing of the target cells with the reagents. Heater 1 54 is used to heat the sample cells and reagents in the incubation zone 1 14 and the amplification zone 1 1 2, and the heater 1 54 is controlled by the CMOS circuit 86 using pulse width modulation (PWM). Each of the tortuous portions of the heated cultivating microchannel 210 and the augmented microchannel 158 has three twistable portions that are operable separately and between their respective heater joints 156 (which provide two dimensional control of the input heat flux density). Extended heater 1 54 (see Figure 1 4). As best shown in FIG. 51, the heater 154 is supported on the roof 66 and embedded in the lower gasket 64. The heater material is TiAl, but many other conductive metals are also suitable. The long heater 154 is parallel to the longitudinal direction of each of the channel regions forming the wide tortuous portion of the curved shape. In the expansion zone 112, each of the wide tortuous portions can be operated in the form of separate PCR chambers by controlling individual heaters. The small volume of amplicons required for an analytical system using a microfluidic device, such as LOC device 301, allows a small volume of amplification mixture to be amplified in the amplification zone 112. This volume is easily less than 4 〇〇 升 'in most cases less than 17 〇 liters 'usually less than 70 liters' and in the case of L0C device 301 is 2 liters to 30 nanoliters. Increased heating rate and preferred diffusion mixing -56-201219776 A small cross section of each channel zone increases the heating rate of the augmented fluid mixture. All fluids are kept within a short distance from the heater 1 54. Reducing the cross-section of the channel (i.e., the cross-section of the augmented microchannel 158) by at least 100,000 square microns results in a heating rate that is significantly higher than that of a more "large gauge" instrument. The etch fabrication technique allows the amplifying microchannel 158 to have a cross-sectional area transverse to the flow path of less than 16,000 square microns, resulting in a substantially higher heating rate. It is easy to use etching technology.  The ground reaches a size of about 1 micron. If the number of amplicon required is small (e.g., in the case of LOC device 301), the cross-sectional area can be reduced to less than 2,500 square microns. Diagnostic analysis performed with a probe of 1000 to 2000 in the LOC device and it is necessary to "sample in, answer out" in less than 1 minute, and the cross-sectional area of the flow transverse direction is between 1 square micron and 400 square micron. . The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate of more than 80 Kelvin (K) per second, in most cases at a rate greater than 100 K per second. Typically, the heater element heats the nucleic acid sequence at a rate of more than 1,000 K per second and in many cases the heater element heats the nucleic acid sequence at a rate of more than 10,000 K per second. Typically, the heater element heats the nucleic acid sequence at a rate exceeding 每秒, ΟΟΟΚ, exceeding 1,000,000 K per second, in accordance with the requirements of the analytical system. 000.  000Κ , more than 20,000,000 每秒 per second, more than 40. 000.  000 Κ, more than 80,000,000 每秒 per second, more than 1 60,000,000 每秒 per second. Small cross-sectional channels also facilitate the diffusion of any reagent and sample fluid -57-201219776. In the liquid before the diffusion mixing is completed. The concentration decreases with distance from the interface. The microchannels having a very small cross section transverse to the flow direction are used to keep the two fluids flowing close to the interface to diffuse the mixture more rapidly. Reducing the channel section by at least 100,000 square microns results in a significantly higher mixing ratio compared to more "large size" instruments. Etch fabrication techniques allow the cross-sectional area of the microchannel to be transverse to the flow path to be less than 16,000 square microns, resulting in a substantially higher mixing ratio. If a small volume is required (as in the case of the LOC device 310), the cross-sectional area can be reduced to less than 2500 square microns. Diagnostic analysis is performed in a LOC device with a probe from 1 〇〇〇 to 2000 and requires a cross-sectional area between 1 and 10 square microns in the transverse direction of the flow when the sample is taken in less than 1 minute. That is enough. Short Thermal Cycle Time Keep the sample mixture close to the heater and use very small amounts of fluid to allow thermal cycling to proceed quickly during nucleic acid amplification. In the case of a target sequence of up to 150 base pairs (bp) long, 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, mostly less than 4 seconds. For a sequence of up to 150 base pairs long, the LOC device 30 with some of the most common diagnostic assays has a thermal cycle time of zero. 45 seconds to 1 . 5 seconds. This rate of thermal cycling allows the test module to be much less! Complete the nucleic acid amplification process in minutes; usually less than 220 seconds. For most analyses, 'from the sample fluid entering the sample inlet, the amplified region will produce enough amplicons in the period -58-201219776 to 80 seconds." For many analyses, it occurs within 30 seconds. Enough amplicon. The amplicon is sent to the hybridization and detection zone 52 via the boiling start valve 108 upon completion of a predetermined number of amplification cycles. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chambers 180 in hybridization chamber array 110. The hybridization and detection zone 52 has a 24 χ 45 array of hybridization chambers 180, each having a hybrid reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. The incorporated photodiode 1 84 is used to detect fluorescence produced by hybridization of the target nucleic acid sequence or protein to the FRET probe 186. Each photodiode 184 is independently controlled by a CMOS circuit 86. Any material between the FRET probe 186 and the photodiode 184 must be transparent to the emitted light. Thus, wall region 97 between probe 186 and photodiode 184 is also optically permeable to the emitted light. In the LOC device 301, the wall region 97 is a thin layer (about 0. 5 microns) cerium oxide. Direct incorporation of photodiode 1 84 under each hybridization chamber 1 80 allows the probe-target hybrid to be very small in size but still produces detectable fluorescent signals (see Figure 54). A small amount allows for a small volume of hybridization chamber. The amount of probe required for the detectable amount of the probe-target hybrid is easily less than 270 picograms (equivalent to 900,000 cubic micrometers) before hybridization, and in most cases less than 60 picograms. (equivalent to 200,000 cubic micrometers), usually less than 12 picograms (equivalent to 40,000 cubic micrometers), less than 2. in the case of the LOC device 301 shown in the drawing -59-201219776. 7 picograms (the corresponding chamber has a volume of 9000 cubic microns). Of course, the smaller size of the hybridization chamber allows for higher density chambers, so there are more probes on the LOC device. In the LOC device 301, the hybridization zone has more than 1 000 cells in an area of 1 500 microns x 1500 microns (i.e., less than 225 0 square microns per cell). The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of requiring a small number of probes in each chamber is that only a very small number of probes need to be dispensed into each chamber during manufacture of the LOC device. The system of the LOC device according to the present invention can be spotted using a smear or less probe solution volume. After nucleic acid amplification, the boiling activation valve 108 is activated and the amplicon flows along the flow path 176 and into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates that the heater 182 can be activated when the amplicons are inserted into the hybridization chamber 180. After sufficient hybridization time, LED 26 (see Figure 2) is activated. The openings in each of the hybridization chambers 180 provide an optical window 136 for the FRET probe 186 to contact the excitation radiation (see Figures 52, 54 and 56). The LED 26 is illuminated for a sufficient amount of time to induce a high intensity fluorescent signal from the probe. The photodiode 1 8 4 is absent during excitation. After a process controlled delay of 300 (see Figure 2), the photodiode 184 is activated and the fluorescent emission is detected in the absence of excitation light. The incident light (see Fig. 54) on the photosensitive region 185 of the photodiode 1 84 is converted into a photocurrent, which is then measured by a C Μ O S circuit 86. The hybridization chambers 180 are each loaded with a probe for detecting a single target nucleic acid sequence - 60-201219776. If desired, each hybridization chamber 180 can be loaded with probes to detect more than 1000 different targets. Alternatively, many or all of the hybridization chambers can be loaded with the same probe to repeatedly detect the same target nucleic acid. Copying the probes throughout the hybridization chamber array 110 in this manner increases confidence in the results obtained and, if desired, can be combined by the photodiode of those hybridization chambers to provide a single result. Those skilled in the art will recognize that there may be from 1 to over 1000 different probes on the hybrid chamber array 110 in accordance with the detection specification. Humidifier and Humidity Sensor The illustration AG in Fig. 6 indicates the position of the humidifier 196. The humidifier prevents reagents and probes from evaporating during LOC unit 101 operation. As best shown in the enlarged view of Fig. 55, reservoir 188 connects three evaporators 190 in fluid operation. The reservoir 188 has a full-scale biological water and is sealed during the manufacturing process. As best shown in Figures 5 and 74, water is drawn into the three downcomers 194 and enters the respective intake ports 193 of one of the evaporators 190 along the respective water supply passages 192 by capillary action. The meniscus is fixed at each intake port 193 to retain water. The evaporator has a ring heater 191 surrounding the intake port 193. The ring heater 191 is connected to the CMOS circuit 86 by a conductive post 376 that reaches the top metal layer 195 (see Figure 37). When activated, the ring heater 191 heats the water, causing the water to evaporate and damp the surrounding equipment. The position of the humidity sensor 23 2 is also shown in FIG. However, as best shown in the enlarged view of the illustration AH of Fig. 67, the humidity sensor -61 - 201219776 has a capacitive comb structure. The etched first electrode 296 and the etched second electrode 298 face each other such that their comb teeth are staggered with each other. The opposing electrode forms a capacitor having a current capacity that can be monitored by CMOS circuit 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, so that the capacitance also increases. The humidity sensor 23 2 is adjacent to the hybridization chamber array 1 1 , where humidity measurement is most important to slow the evaporation of the solution containing the exposed probe. The feedback sensor incorporates temperature and liquid sensors throughout the LOC device 301 to provide feedback and diagnostics during device operation. Referring to Figure 35, nine temperature sensors 170 are distributed throughout the amplification zone. 1 2 in. Similarly, the incubation zone 1 14 also has nine temperature sensors 1 70. Each of these sensors uses a bipolar transistor (BJTs) 2 X 2 array to monitor fluid temperature and provide CMOS circuit 86 feedback. The CMOS circuit 86 is used to precisely control the thermal cycling during the nucleic acid amplification process as well as any heating during the thermal cracking and incubation. In the hybridization chamber 180, the CMOS circuit 86 uses the hybridization heater 182 as a temperature sensor (see Figure 5-6). The resistance of the hybrid heater 108 is temperature dependent and the CMOS circuit 86 uses this to derive temperature readings for each of the hybrid chambers 180. The LOC device 301 also has a plurality of MST channel liquid sensors 174 and a cap channel liquid sensor 208. Figure 35 shows a row of MST channel fluids - 62 - 201219776 body sensor 174 at one end of every other tortuous portion of the heated microchannel 158. As best shown in FIG. 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the circuit between the electrodes to indicate their presence at the sensor. Figure 25 shows an enlarged perspective view of the cap channel liquid sensor 208. The TiAl electrode pairs 218 and 220 are placed on the roof layer 66. Between electrodes 218 and 22 0 is a gap 222 to keep the circuit open in the absence of liquid. The circuit is turned off when the liquid is present, and the CMOS circuit 86 uses the feedback to monitor the flow. Independence of gravity The test module 10 is independent of direction. It does not need to be fixed on a flat, stable surface to operate. Fluid flow driven by capillary action into the auxiliary instrument in the absence of an external conduit means allows the module to be truly portable and only need to be inserted into a portable handheld reader such as a mobile phone. Having a gravity-independent operation means that the test module is actually completely independent of acceleration. It is resistant to shock and vibration and can operate on a moving vehicle or when the mobile phone is carried everywhere.

Nucleic Acid Amplification Variants Direct PCR Traditionally, PCR requires large-scale purification of target DNA to prepare a reaction mixture. However, by appropriately modifying the chemistry and sample concentration, only minimal DNA purification or direct amplification is required for nucleic acid amplification -63-201219776 is feasible. When the nucleic acid amplification process is pCR, this method is called direct PCR. In a LOC device in which the nucleic acid amplification is carried out under controlled, constant temperature, the method is direct isothermal amplification. The use of direct nucleic acid amplification techniques in LOC devices has considerable advantages' in particular to simplify the fluid design required. Adjustments to the amplification chemistry for direct PCR or direct isothermal amplification include potentiation of buffer strength, use of polymerases with high activity and high sustained synthesis, and additions to potential polymerase inhibitors. It is also important to dilute the inhibitors present in the sample. In order to utilize direct nucleic acid amplification techniques, the LOC device design includes two additional features. The first feature is a library of reagents of appropriate size (eg, reservoir 58 in Figure 8) to provide a sufficient amount of amplification reaction mixture or diluent, which may interfere with the final concentration of the sample components of the amplification chemistry. Low enough to allow successful nucleic acid amplification. The desired dilution of the non-cellular sample components is between 5 and 20 times. Different LOC structures (e.g., pathogen dialysis zone 70 in Figure 4) are used where appropriate to ensure that the concentration of the target nucleic acid sequence is maintained at a sufficiently high concentration for amplification and detection. In this system (further illustrated in Figure 6) is employed upstream of the sample extraction zone 2 90 to effectively concentrate the dialysis zone small enough to enter the pathogen of the amplification zone 292 and drain the larger cells to In the waste container 76. In another system, a dialysis zone is employed to selectively deplete proteins and salts in the plasma while retaining the desired cells. The second LOC structure that supports direct nucleic acid amplification is characterized by a channel aspect ratio to adjust the mixing ratio between the sample and the components of the amplification mixture. For example, to ensure that the inhibitor bound to the sample is in the range of 5 to 20 times the dilution ratio of the -64-201219776 in a single mixing step, the length and cross-section of the sample and reagent channels are designed to allow the sample channel The flow impedance formed by the upstream of the starting mixing position is 4-1 times higher than the flow impedance of the passage through which the reagent mixture flows. Controlling the flow impedance in the microchannel can be easily achieved by designing the control geometry. For a fixed cross section, the flow impedance of the microchannel increases linearly with the length of the channel. For hybrid designs, it is important that the flow impedance in the microchannels is more strongly dependent on the smallest cross-sectional dimension. For example, when the aspect ratios are inconsistent, the flow impedance of a microchannel having a rectangular cross section is inversely proportional to the smallest orthogonal dimension of the cube. Reverse transcriptase PCR (RT-PCR) When the sample or nucleic acid species analyzed or extracted is RNA (such as from RNA virus or messenger RNA), the RNA needs to be reversed into complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction can be carried out in the same reaction chamber as the PCR (single-step RT-PCR), or can be carried out separately in the form of a starting reaction (two-step RT-PCR) » in the LOC variant described herein, The single-step RT-PCR can be carried out simply by adding a reverse transcriptase and a polymerase to the reagent library 62, and formulating the heater 1 54 to perform a reverse transcription step and then performing a nucleic acid amplification step. The two-step RT-PCR can also be easily performed by using a reagent library 58 for storing and dispensing buffers, primers, dNTPs and reverse transcriptase, and a breeding region 114 for the reverse transcription step, and then in the amplification region 112. The way to achieve amplification. Thermostatic Nucleic Acid Amplification-65- 201219776 For some applications, thermostated nucleic acid amplification is a nucleic acid method, which avoids the need to circulate back to the ground through various temperature cycles, but maintains the amplified region at a constant temperature of 37t to 41 °C. The thermostatic nucleic acid amplification methods that have been described are strand-substituted amplification (SDA), transcription-mediated amplification (TMA) sequence amplification (NASBA), and recombinant enzyme polymerase amplification (chase-dependent isothermal DNA amplification (HDA). ), rolling shock (RCA), branched-type amplification (RAM), and circular (LAMP), and in any particular system of LOC devices described in these or other isostatic amplification methods. Amplification zones 2 and 62 will be loaded with appropriate reagent amplification mixtures and polymerases for specific thermostatic methods. For example: in terms of SDA, containing amplification buffers, primers and dNTI»S, and reagent library 62 nickase and Exo-DNA polymerase. In terms of RPA, the assay containing amplification buffer, primer 'dNTPs and recombinant enzyme protein library 62 contains a strand-substituted DNA polymerase, such as Bsu. In HDA, reagent library 60 contains amplification buffers and primers. Reagent library 62 contains the appropriate strands of DNA polymerase and unwinding DNA, rather than using heat. It is familiar with the art that the necessary reagents can be between any reagent pool suitable for the nucleic acid amplification process. HIV or C type Hepatitis virus nucleic acid, NASBA or TMA is suitable, because the preferred components of amplification should be repeated, usually about a variety of types, including, depending on the nucleic acid RPA), and the cycle amplification amplification can be used for the L reagent library 60. The PCR package does not contain 60 capsules, and the reagents are similarly in the form of dNTPs, and the enzymes are used to separate the duplexes from the two viruses. 66- 201219776 RNA is transcribed into cDNA. In this example, reagent library 60 contains amplification buffers, primers, and dNTPs, and reagent library 62 contains RNA polymerase, reverse transcriptase, and selectable 'RNaseHe for some forms of thermostatic nucleic acid amplification. 'It may be necessary to have an initial denaturation cycle to separate the double-stranded DNA template before maintaining the temperature at the temperature for constant temperature nucleic acid amplification. This is easily accomplished in all systems of the LOC devices described herein because the temperature of the mixture in the amplification zone 112 can be carefully controlled by the heaters 1 54 in the amplification microchannels 158 (see section 14). Figure). Thermostatic nucleic acid amplification is more tolerant of potential inhibitors in the sample and, therefore, is generally suitable for use in situations where nucleic acid amplification is required directly from the sample. Therefore, constant temperature nucleic acid amplification can sometimes be used in LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLW077, etc. shown in Figures 82, 83 and 84, respectively. Direct thermostatic amplification may also be combined with one or more preamplification dialysis steps 70, 686 or 682 as shown in Figures 82 and 84 and/or prehybridization dialysis step 682 as indicated in Figure 83, The target cells in the partially concentrated sample are assisted prior to nucleic acid amplification, respectively, or the 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-hybrid dialysis can be used. Thermostatic nucleic acid amplification can also be performed simultaneously in amplification regions as shown graphically in Figures 78, 79 and 80, the diversity of thermostatic nucleic acid amplification and certain methods (such as LAMP) can be compatible with the initial reverse transcription step to expand Increase RNA. -67- 201219776 Other Design Variant Conductivity Sensors Figure 1 18 is a schematic cross-sectional view of a conductivity sensor 8 1 用于 for detecting salts and various targets, which causes, for example, conduction The rate is altered by an enzyme-catalyzed reaction or antibody binding for detection. The conductivity sensor 810 measures the conductivity of the liquid 8 1 2 in the channel 800 by passing a current between the first terminal 82 and the second terminal 808 and measures through the first electrode 804 and the second The voltage of the electrode 806. The first and second terminals 082, 088, and the first and second electrodes 804, 806 are part of the top metal layer 195 of the CMOS circuit 86 exposed through the window in the passivation layer 88. Flow Rate Sensor In addition to the temperature and liquid sensors, the LOC device can also incorporate a CMOS-controlled flow rate sensor 740 as illustrated schematically in Figure 119 and in LOC Variant X 728 (see pages 93-109). Figure). These sensors are used to measure the flow rate in two steps. In the first step, the resistance of the bent heater element 814 is determined by applying a low current and measuring the voltage, and the temperature of the element 814 is determined using a known relationship between the resistance and the temperature of the heater element. At this stage, the heat dissipated in element 814 is minimal and the temperature of the liquid in the channel is equal to the calculated temperature of element 814. In the second step, a higher current is applied to the curved heater element 814, thereby increasing the temperature of the component 814, and some of the heat is lost in the flowing liquid. When a higher current is applied, the new resistance of element 814 can be determined by again measuring the voltage across the element 8 1 4 -68 - 201219776 and the increased temperature is again calculated via CMOS circuit 86. The flow rate of the liquid can be determined using the new temperature of the curved heater element 814 and the temperature of the known sample liquid calculated in the first step. The flow rate of the liquid in the channel can be calculated from the known channel cross-section geometry and flow rate. Capillary leading edge travel velocity sensor The velocity of the meniscus before the front end of the sample stream can be determined by triggering the time delay between the various liquid sensors 1 74. Additional details of the fluorescence detection system Figures 58 and 59 show hybridization-reactive FRET probes 236 » These are commonly referred to as molecular beacons and are generated from single-stranded nucleic acid arm-and-loop probes, which are Fluorescence is produced when the complementary nucleic acids hybridize. Figure 58 shows a single FRET probe 23 6 prior to hybridization to the target nucleic acid sequence 23 8 . The probe has a ring 240, an arm 242, a fluorophore 246 at the 5' end and a quencher 248 at the 3' end. The loop 240 is composed of a sequence complementary to the target nucleic acid sequence 238. The complementary sequences on either side of the probe sequence are bonded together to form an arm 242. As shown in Figure 58, the probe remains closed when the complementary target sequence is lacking. The arm 242 maintains the fluorophore-quenching agent pairs close to each other such that significant resonant energy transfer can occur between them, substantially eliminating the ability of the fluorophore to fluoresce when illuminated by the excitation light 244. Figure 59 shows the FRET probe 23 6 configured for opening or hybridization. -69- 201219776 When the hybrid target nucleic acid sequence 23 8 hybridizes, the arm-and-loop structure is destroyed, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the camping light 246 to emit fluorescence. . The fluorescent emission 250 can be optically detected as an indication that the probe has hybridized. Since the arm helix of the probe is designed to be more stable than the probe/target helix with non-complementary mono-nucleotide, the probe hybridizes to the complementary target with very high specificity. Since the double-stranded DNA is relatively tight, the probe-targeted helix and the arm-helical are spatially impossible to coexist. The probe-and-primer-attached arm-and-loop probe and the linear probe connected to the primer are connected to the primer. Or a scorpion probe can be used in place of molecular beacons and can be used for both real-time and quantitative nucleic acid amplification in LOC devices. Immediate amplification can be performed directly in the hybridization chamber of the LOC device. An advantage of using a probe attached to a primer is that the probe element is actually linked to the primer, so that only a single hybridization is required during nucleic acid amplification without the need to hybridize the primer and probe, respectively. This ensures that the reaction is instantaneously efficient and produces stronger signals, shorter reaction times, and better discrimination than when using separate primers and probes. During manufacture, the probe (along with the polymerase and amplification mixture) will be stored in hybridization chamber 180 and a separate amplification zone will not be required on the LOC device. Alternatively, the amplification zone is left unused or used for other reactions. Linear probes linked to primers Figures 8 and 8 show the primer-ligated linear probe 692 and its hybridization configuration during the first nucleic acid amplification cycle and during the subsequent nucleic acid amplification cycle of -70-201219776, respectively. Referring to Figure 85, the linear probe 692 coupled to the primer has a double-stranded arm segment 242. One of the double strands incorporates a probe-ligated probe sequence 696 which is homologous to a region on the target nucleic acid 696 and whose 5' end is labeled with a fluorophore 246, the 3' end of which is via a amplification block. The fragment 694 is ligated to the oligonucleotide primer 700. The 3' end of the other strand of arm 242 is labeled with a quencher portion 248. Upon completion of the first cycle of nucleic acid amplification, the probe can wrap around and hybridize to an extended strand with sequence 698 (currently complementary). During the first cycle of nucleic acid amplification, the oligonucleotide primer 700 binds to the target DNA 238 (Fig. 85) and is further 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 69 6 . Upon subsequent denaturation, the extended oligonucleotide primer 700/template hybrid is separated and the double-stranded arms 242 of the linear probe attached to the primer are also separated to release the quencher 248. Once the temperature for the adhesion and extension steps is lowered, the primer-ligated probe sequence 696 of the primer-ligated linear probe is coiled and hybridized to the amplified complementary sequence 69 8 on the extended strand, and detected. Fluorescence to the presence indicates that the target DN A is present. A non-extended linear probe attached to the primer retains its double-stranded arm and the fluorescence remains quenched. This detection method is particularly well suited for use in fast detection systems because it relies on a single molecular process. Arm-and-loop probes attached to primers Figures 87A through 87F show the operation of the arm-and-loop probes 7〇4 attached to the primers. Referring to Figure 87A, the primer-linked arm-and-loop probe 201219776 704 has an arm 242 of complementary double-stranded DNA and a loop 240 comprising the probe sequence. The Y end of one of the arms 708 is labeled with a fluorophore 246. The other 3' end of 710 is labeled with quencher 248 and carries both amplification blocker 694 and oligonucleotide primer 700. In the initial denaturation phase (see Figure 87B), the double strands of the target nucleic acid 23 8 are separated, and the arms 242 of the arm-and-loop probe 704 attached to the primer are also. When the temperature of the adhesion phase is cooled (see Figure 87C), the oligonucleotide primer 700 on the primer-linked arm-and-loop probe 704 hybridizes to the target nucleic acid sequence 23 8 . During extension (see Figure 87D), complement 706 of the target nucleic acid sequence 238 is synthesized to form a DNA strand comprising both probe sequence 704 and amplification products. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 704. When the probe is subsequently ligated (continuously after denaturation), the probe sequence of the loop-to-primer-arm-and-loop probe loop 240 (see Figure 87F) is attached to the complementary sequence on the stretched strand. 706. This configuration leaves the fluorophore 246 far away from the quencher 248, resulting in a significant increase in fluorescence emission. Control Probes The hybridization chamber array 1 1 〇 includes some hybridization chambers 180 with positive and negative control probes for analytical quality control. Figures 1 1 4 and 1 1 5 illustrate the negative control probes without fluorophore 796. Figures 1 16 and 7 show sketches of positive control probes without quencher 798. The positive and negative control probes have an arm-and-loop configuration as described above for the FRET probe. However, the fluorescent signal 250 is always emitted from the positive control probe 798 and the negative control probe 796 has not emitted the fluorescent signal 250, regardless of whether the probe hybridizes to open-72-201219776, puts the configuration on or off. In the 114 and 115 maps, the negative control fluorescent probe 796 does not have a fluorophore (and may or may not have a quencher 248). Thus, whether or not the target nucleic acid sequence 298 hybridizes to the probe (see Figure 115), or whether the probe retains its arm-and-loop configuration (see Figure 114), the reaction to excitation light 244 It is trivial. Alternatively, the negative control probe 796 can be designed to remain quenched at all times. For example: Synthetic loop 240, with a probe sequence that does not hybridize to any of the nucleic acid sequences in the sample being examined, the probe molecule 242 will rehybridize itself, the fluorophore and quencher will remain near It will not emit a noticeable fluorescent signal. This negative control signal will correspond to a low amount of emission from hybridization chamber 180 (wherein the probe is not hybridized, but the quencher does not quench all of the emission from the reporter). Conversely, as illustrated by Figures 1 16 and 117, the positive control probe 79 8 is constructed without a quencher. Whether or not the positive control probe 798 hybridizes to the target nucleic acid sequence 23, the fluorescent light 260 emitted by the fluorophore 246 in response to the excitation light 244 is not quenched by anything. Figure 52 shows the distribution of possible positive and negative control primers in the entire f hybridization chamber array 110 (3 7 8 and 380, respectively). The control probes 378 and 380 were placed in a hybridization chamber 180 which was located across the line of the hybridization chamber array 11〇. However, the control probes within the array were arbitrarily arranged (as in the hybrid chamber array 110 configuration). Fluorescent Group Design -73- 201219776 A fluorophore with a long fluorescent lifetime is required in order to allow sufficient time for the intensity of the excitation light to decay below the intensity of the fluorescent emission, at which point the light sensor 44 is enabled, thereby providing sufficient Signal to noise ratio. In addition, longer fluorescent lifetimes translate into larger integrated fluorescence photon counts.萤 Fluorescent Cluster 2 46 (see Figure 59) has a fluorescence lifetime longer than 100 nanoseconds, usually longer than 200 nanoseconds, more often longer than 300 nanoseconds and in most cases longer than 400 nanoseconds. The transition metal or rare earth-based metal-gametide complex has a long lifetime (from hundreds of nanoseconds to milliseconds), sufficient quantum yield, and high thermal, chemical, and photochemical stability, which are necessary for fluorescent detection systems. Conditions are all advantageous properties. A metal-gametide complex based on a transition metal ion ruthenium (Ru ( Ε )) is a tris(2,2'-bipyridyl) nail (Π) ([Ru(bpy)32+) ), which has a lifetime of about 1 microsecond. This complex is commercially available from Biosearch Technologies brand Pulsar 650. Table 1: Light of Pulsar 650 (钌 chelate) £ Kissing property parameter number of symbols 値 unit absorption wavelength ^abs 460 nm emission wavelength λβιη 650 nm extinction coefficient Ε 14800 M'cin'1 egg light lifetime Tf 1.0 μδ quantum capacity Η 1 (deoxygenated) Ν/Α 铽 chelate, a lanthanide metal-gametide complex has been successfully demonstrated as a fluorescent reporter in the FRET probe system, and also has a frequency of 1 600 micro-74-201219776 seconds Long life. Table 2: Photophysical properties of ruthenium chelate

Parameter symbol number 値 unit absorption wavelength. kbs 330-350 nm emission wavelength λεηι 548 nm extinction coefficient Ε 13800 depends on kbs and gametes, up to 30000 @ λβ = 340 nm) M'1 cm·1 fluorescence lifetime Tf 1600 ( Hybrid probe) μδ Quantum capacity Η 1 (depending on gametes) The fluorescence detection system used by the N/A LOC device 301 does not use filters to remove unwanted background fluorescence. Therefore, in order to increase the signal to noise ratio, it is advantageous if the quencher 248 does not have a natural emission. Quencher 248 does not cause background fluorescence when there is no natural emission. High quenching efficiency is also important to prevent fluorescence until hybridization occurs. The Black Hole Quenchers (BHQ), available from Biosearch Technologies, Novato, Calif., have no natural emission and high quenching efficiency and are therefore suitable quenchers for the system. BHQ-1 has a maximum absorption at 5 34 nm and a quenching range of 48 0-580 urn, which makes it a suitable quencher for ruthenium chelate fluorophores. BHQ-2 has maximum absorption at 5 79 nm and a quenching range of 560-670 nm, making it a suitable quencher for Pulsar 650. Iowa Black Quencher (Iowa Black FQ and RQ) (available from Integrated DNA Technologies, Coralville, Iowa) -75- 201219776 is a suitable alternative quencher with little or no background emission. The Iowa Black FQ has a quenching range of 420-620 nm, which has a maximum absorption at 531 nm and is therefore a suitable quencher for the ruthenium chelate fluorophore. Iowa Black RQ has a maximum absorption at 656 rim and a quenching range of 500-700 n m, which makes it an ideal quencher for P u 1 s a r 65 5 0. In the system described herein, the quencher 248 is initially attached to the functional portion of the probe, but in other systems the quencher may be a separate molecule that is free in solution. Excitation source In the fluorescence detection based on the system described here, LED is selected as the excitation source due to low power consumption, low cost and small size, instead of laser diode, high power lamp or laser . Referring to Fig. 88, the LED 26 is placed directly above the hybrid cell array 110 on the outer surface of the LOC device 301. Opposite to the cell of the hybridization chamber array 1 is a photosensor 44 made up of a photodiode array 184 for detecting fluorescent signals from the cells (see Figures 53, 54 and 68). . Other systems for contacting the probe with excitation light are illustrated in Figures 89, 90 and 91. In the LOC device 30 shown in Fig. 89, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 onto the hybrid array 810. The excitation LED 26 is pulsed and the fluorescent emission is detected by the photo sensor 44. In the LOC device 30 shown in FIG. 90, the excitation light 244 generated by the excitation LED 26 is used by the lens 254. The first optical pupil 712 and the -76-201219776 two optical pupils 714 are directed onto the hybridization chamber array 110. The excitation LED 26 is pulsed and the fluorescent emission is detected by the photo sensor 44. Similarly, in the LOC device 30 shown in FIG. 91, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 onto the hybridization chamber array 110. Similarly, the excitation LED 26 is pulsed and the fluorescent emission is detected by the light sensor 44. The excitation wavelength of the LED 26 is dependent on the selected fluorescent dye. Philips LXK2-Prl4-R00 is the appropriate source of excitation for the Pulsar 650 dye. SET UVT0P3 3 5T039BL LED is the appropriate excitation source for the ruthenium chelate label. Table 3: Philips LXK2-Pr 1 4-R00 LED Specifications

Parameter Symbol Number 单位 Unit Wavelength λεχ 460 nm Transmit frequency Vem 6.52(10)14 Hz Output power Pl 0.515 (minutes) @ ΙΑ W Radiation pattern Lambertian variation profile N/A Table 4 : SET UVT0P3 34T039BL LED Specifications

Number of parameter symbols 値 Unit wavelength λβ 340 nm Transmit frequency Ve 8.82(10)14 Hz Output power Pi 0.000240 (minutes) @ 20mA W Pulsed forward current I 200 mA Radiation pattern Lambertian N/A -77- 201219776 Ultraviolet excitation does not absorb light in the ultraviolet spectrum. Therefore, it is advantageous to use ultraviolet light to excite light. A UV 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. Alternatively, a UV laser can be used as an excitation source unless the relatively high cost of the laser is impractical for a particular test module market. LED Driver The time required for the LED driver 29 to drive the LED 26 at a constant current. The lower power USB 2.0-certifiable unit can draw up to 1 unit load (l00mA) with a minimum operating voltage of 4.4V. A standard power conditioning circuit can be used for this purpose. Photodiode Figure 54 shows the photodiode 184 integrated into the CMOS circuit 86 of the LOC device 301. The photodiode 184 is fabricated as part of the CMOS circuit 86 without additional shielding or steps. Alternative sensing techniques that can be integrated on the same chip using non-standard processing steps, or fabricated on adjacent chips, have significant advantages over CCDs for CMOS photodiodes. The cost of on-chip detection is low and the size of the detection system is reduced. The shorter optical path length reduces noise from the surrounding environment to efficiently collect fluorescent signals without the need for a combination of conventional optical lenses and filters. The quantum efficiency of the photodiode 184 is a photon that strikes its photosensitive surface -78-201219776 185, and is effectively converted into a photo-electron portion. In terms of standard processing procedures, the quantum efficiency is 0.3 to 0.5 in terms of visible light depending on the method parameters such as the number of cladding layers and the absorption properties. The detection threshold of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection threshold also determines the size of the photodiode 180, thereby determining the number of hybridization chambers 180 in the hybridization and detection zones 52 (see Figure 52). The size and number of chambers is limited by the size of the LOC device (in the case of LOC device 301, which is 1760 microns X 5824 microns) and is incorporated into other functional modules (such as pathogen dialysis zone 70 and amplification zone 1). 12) Technical parameters of the available space. In terms of standard 矽 processing, photodiode 1 84 detects a minimum of 5 photons. However, to ensure reliable detection, it can be set to a minimum of 1 photon. Therefore, when the quantum efficiency ranges from 0.3 to 0.5 (as described above), the fluorescent emission from the probe should be a minimum of 17 photons, but for reliable detection, 30 photons will incorporate the appropriate margin of error. Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, autofluorescence, and residual excitation photon fluxes that are not completely degraded 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 contacting one or more of the calibrated photodiodes 184 in the array with respective calibration sources. A low calibration source was used to determine the negative result in which the target did not react with the probe. The high calibration source is an indication of the positive result produced from the probe-target complex. In the system described in -79-201219776, the low calibration source is provided by a calibration chamber 382 in the hybrid chamber array n〇, which: does not contain any probes; includes a probe that does not have a fluorescent reporter Or, a probe having a reporter and a quencher configured to be expected to quench forever. The output signal from such a calibration chamber 382 is very similar to the noise and offset in the output signals from all of the hybrid chambers of the LOC device. Subtracting the calibration signal from the output signal produced by the other hybrid chamber substantially removes the background and leaves the signal produced by the fluorescent emission (if any). The signal generated from the ambient light in the area of the cell array is also subtracted. Referring to Figures 1 14 to 1 1 7 it will be appreciated that the negative control probe described above can be used in a calibration chamber. However, as shown in Figures 105 and 106 (which is an enlarged view of the illustrations DG and DH of the LOC variant X 728 shown in Figure 98), another option is to fluidly isolate the calibration chamber 382 from the amplicon. The background noise and offset can be emptied by the chamber that isolates the fluid, or by a probe containing no reporter, or even any hybridization due to fluid isolation that has a reporter and quencher. "Probe to determine. The calibration chamber 382 can provide a high calibration source to produce a high signal in the corresponding photodiode. This high signal corresponds to all probes that have been hybridized in the chamber. A spotted probe with a reporter and no quencher, or with only a reporter will continue to provide a signal similar to the hybridization chamber in which most of the probes have hybridized. It is also known to use a calibration chamber 382 instead of a control probe or with a control probe. -80 - 201219776 There may be any number and arrangement of calibration chambers 382 throughout the array of hybrid chambers. However, if the photodiode 184 is calibrated with a very similar calibration chamber 382, the calibration is more accurate. Referring to Figure 56, each of the eight hybridization chambers 180 has one calibration chamber 382. That is, the calibration chamber 382 is tethered to the center of every 3x3 hybridization chamber 180. In this configuration, the hybrid chamber 180 is calibrated by the calibration chamber 382 in the immediate vicinity. Figure 113 shows a differential imager circuit 788 for subtracting the signal from the photodiode 184 corresponding to the calibration chamber 382, which is the result of the excitation light, from the fluorescent signal from the surrounding hybridization chamber 180. go with. The differential imager circuit 788 takes sample signals from pixel 790 and "false" pixels 729. In a system, the "false" pixel 792 is protected from light, so its output signal provides a dark reference. Alternatively, the "dummy" pixel 792 can be exposed to the excitation light with the remaining array. The signal generated from the ambient light of the cell array region is also subtracted from the system in which the "false" pixel 792 is open to light. The signal from pixel 790 is small (i.e., close to the dark signal), and it is difficult to distinguish between the background and the very small signal without reference to the dark level. During use, 'read one row' 794 and "read_row_d" 795 are activated, and M4 797 and MD4 8 0 1 transistors are turned on. Switches 807 and 809 are turned off' thereby storing the output signals from pixel 790 and "false" pixel 792 in pixel capacitor 803 and dummy pixel capacitor 805, respectively. After the pixel signal has been stored, switches 807 and 809 do not function. Then, the "read_col" switch 811 and the dummy "read_C0l" switch 813 are turned off. The converted capacitor amplifier 815 at the output amplifies the differential signal 8 1 7 . -81 - 201219776 Containing and enabling photodiodes The photodiode 184 needs to be suppressed during excitation by LED 26, which is enabled during the fluorescent period. Fig. 69 is a circuit diagram of a single photodiode 184, and Fig. 70 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 400 'Mread 402 and Mbias 404. At the beginning of the excitation period t], the transistors Mshunt 394 and Mreset 398 are turned on by pulling open the Mshunt gate 384 and resetting the gate 388 high. During this time, the excitation photons generate a carrier in the photodiode 184. These carriers must be removed because the amount of carrier generated may be sufficient to saturate the photodiode 1 84. During this cycle, Mshunt 394 directly removes the carrier generated in photodiode 184, while Mreset 3 98 resets any accumulation at node 'NS' 406 due to leakage in the transistor or diffusion of the carrier due to excitation to the substrate. a. After the excitation, the cycle is captured at t4. During this cycle, the reaction from the fluorophore emission is captured and integrated on the node 'NS' 406 in the circuit. This can be achieved by pulling up the tx gate 386, since pulling up the tx gate 3 86 can open the transistor Mtx 396 and transfer any carrier accumulated on the photodiode 184 to the node 'NS' 460. The capture cycle can be as long as the fluorophore emission. The output from all of the photodiodes 184 in the hybrid cell array 110 is simultaneously captured. There is a time 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 hybrid array -82 - 201219776 column 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read has the shortest delay before the read cycle, and the last photodiode 184 has the longest delay before the read cycle. During the read cycle, the transistor Mread 402 is turned on by pulling up the read gate 3 93. The source tracker transistor Msf 400 is used to buffer and read the 'NS' node 406 voltage. Other options for enabling or suppressing photodiodes are discussed below: 1. Containment Methods Figures 1 10, 1 1 1 and 1 12 show three possible configurations of Mshunt transistors 3 94 778, 780, 782. The Mshunt transistor 394 has a high turn-off ratio at maximum I VGS 丨 = 5 V, which is enabled during the excitation period. As shown in Fig. 10, the Mshunt gate 3 84 is disposed at the edge of the photodiode 184. Alternatively, as shown in FIG. 111, Mshunt gate 3 84 may be configured to surround the photodiode 184. A third option is to place the Mshunt gate 384 within the photodiode 184 as shown in FIG. In this third option, the photodiode photosensitive surface 1 8 5 will be reduced. These three configurations 778, 780, and 782 reduce the average path length from all locations of the photodiode 184 to the Mshunt gate 3 84. In Fig. 110, the Mshunt gate 3 84 is attached to one side of the photodiode 184. This configuration is the easiest to manufacture and has the least impact on the photodiode photosurface 185. However, any carrier that continues to stay on the far side of the photodiode 184 will conduct longer through the Mshunt gate 3 84. In Figure 11, the Mshunt transistor 384 surrounds the photodiode -83-201219776 1 84. This further reduces the average path length of the carrier in the photodiode 1 84 to the Mshun gate 3 84. However, extending the Mshunt gate 3 84 to the vicinity of the periphery of the photodiode 184 reduces the photosensitive surface 185 of the photodiode. Configuration 782 in Fig. 112 places Mshunt gate 3 84 within photosensitive surface 185. This provides the shortest average path length to the Mshunt Gate 3 84, so the transition time is the shortest. However, the impact on the photosensitive surface 185 is the greatest. It also brings a wide leak path. 2. Enable method a. Trigger the photodiode to drive the shunt transistor for a fixed delay time. b. Trigger the photodiode to drive the shunt transistor with a programmed delay time. c. The shunt transistor is driven from the LED drive pulse with a fixed delay time. d. The shunt transistor is driven in the manner of 2c but has a programmed delay time. Figure 75 is a schematic cross-sectional view showing the photodiode 184 and the trigger photodiode 187 embedded in the CMOS circuit 86 through the hybridization chamber 180. A small area in the corner of the photodiode 184 is replaced by a photodiode 187. Triggering the photodiode 187 with a small area is sufficient because the intensity of the excitation light will be high compared to the fluorescence emission. The triggering photodiode 187 is sensitive to the excitation light 244. The trigger photodiode 187 indicates that the excitation light 244 has extinguished and activates the photodiode 184 after a short delay time Δt3 00 (see Figure 2). This delay allows the fluorescent -84 - 201219776 photodiode 184 to detect fluorescent emissions from the FRET probe 1 86 in the absence of excitation light 244. This can detect and improve the signal to noise ratio. Both photodiode 184 and trigger photodiode 187 are located in CMOS circuitry 86 of each hybrid cell 180. The array of photodiodes is combined with appropriate electrons to form photosensor 44 (see Figure 68). The photodiode 184 is a pn junction diode fabricated without additional shielding or steps in the fabrication of the CMOS structure. During the MST fabrication, an insulating layer (not shown) on the photodiode 184 can be selectively thinned using standard MST photolithography to allow more phosphor to illuminate the photoreceptor surface 185 of the photodiode 184. . Photodiode 184 has a field of view such that a fluorescent signal from the probe-target hybrid within the hybrid chamber 180 is incident on the face of the sensor. This fluorescent light is converted into a photocurrent, which is then measured using a CMOS circuit 86. Additionally, one or more of the hybridization chambers 180 can be dedicated to only one of the triggering photodiodes 187. These options can be used in combination with the above 2a and 2b. Fluorescence Delay Detection The following derivation clarifies the use of long-lived fluorophores for the above LED/fluorescent combination to delay detection of fluorescence. The fluorescence intensity is derived as shown in Fig. 60. Between time t i and t2, after excitation with the ideal fixed intensity Ie pulse, it is derived as a function of time. Let [S 1 ] ( t ) be equal to the density of the excited state at time t, then the number of excited states per unit time during and after the excitation is described by the following differential equation: -1 (1) 〇+迎I 丛...(!) dt xF hve where C is the molar concentration of the fluorophore, ε is the molar extinction coefficient, Ve is the excitation frequency and h= 6.626068 96( 1 0)·34 Js The PUnk constant. This differential equation has the following general form: ^-+ρ(Φ = Φ) It has the answer: \pU)^ q(x)dx+k jpMdx ...(2) Now use this formula to solve式))..SCT, hVe .(3) Now, at time ti' [SI] (ti) =0, and from (3

I l,lTf //IS hv. k =——:~-e' f ...(4) [sim at time t2 : [•SI](6)= —----e hve hve ...(5) t^t2, the excited state is exponentially decayed and this can be described by: [51](〇= [51](/2)e-{,-,j)/r/ (6) -86- 201219776 will (5 Substituting (6): [SmJ-^[\-e(t^), riV{, 'h), tf (7) hye The fluorescence intensity is generated by the following formula: I (t) = ~^-sl^thvfni (8) f dx f where Vf is the fluorescence frequency, η is the quantum yield and 1 is the optical path length. Now, from (7): = >-(call)~ ...(9)

Dt hve L Substituting (9) into (8): IAt) = /e£c/7—[1 -e'<,2_,l)/r/ }e~(,~,lVTf ...(10 ) ίΐΖίί — 〇〇, in terms of " β, therefore, we can write the following approximation equation, which describes the case where the fluorescence intensity decays after a sufficiently long excitation pulse (t2- tl >> Tf): = /#/7 〆 〆 "2)" ', 对叫丽(9) In the previous paragraph, we conclude that in the case of t2- ti >> τ f, IAt) = IeScln^-e- {,-hVTf . For θί2. From the above formula, we can derive the following points: nf(t) = nesc^eH'~'l), Tf (12) where nr (0 - τ V/ is per unit area The number of fluorescent lights in time, and -87- 201219776 is the number of excited photons per unit time per unit area.

GO = ... (13) where ~ is the number of fluorescent photons per unit area and t3 is the instant of opening of the photodiode. Substituting (1 2 ) into (1 3 ): 〇〇

Hf = dt (14) Now, the number of fluorescent photons reaching the photodiode per unit time is ~ ~(7), which is generated by the following formula: 研)=\_ (15) & is the light collection efficiency of the optical system. Substituting (1 2 ) into (1 5 ), we find that ηί(ή = φΰη\εαΙηβ-υ-'^ (16) Similarly, the number of fluorescent photons reaching the photodiode per unit of fluorescence area ~, will be as follows: 〇〇ns = [ns{t)dt h and substituted (1 6 ) and integrated:

Hs ^Φ^εαΙητ^'^'11 Therefore, the ideal 値 of ns = φ^ή^Ιητfe u, Xf ... (17) \ t3 is the ratio of the electrons generated by the photodiode 184 caused by the fluorescent photons and excited by When photons cause the ratio of electrons generated by photodiode 184 to be equal, the rate at which the flux of the excited photons decays far exceeds the decay rate of photons in the fluorescent-88-201219776. The output electron ratio of the sensor per unit of fluorescence area caused by fluorescence is · έ} (0 = ^(0 which is divided into the quantum efficiency of the sensor at the wavelength of the fluorescence. Substituting (1 7 ),得: έ> (0 = 卜'2)/Γ/ (18) Similarly, the sensor output electron ratio per unit of fluorescence area due to excitation photons is: ·έ; (0 = ^&gt ;'(,', ί)/Γ* -(19) where & is the quantum efficiency of the sensor at the excitation wavelength, and % is the time constant corresponding to the "off" characteristic of the excited LED. After time t2, LED attenuated photon flux will increase the intensity of the fluorescent signal and prolong its decay time, but we assume that this has a negligible effect on 1Kt). Therefore, we take a conservative approach. Now, as mentioned earlier, the ideal of t3 is when:

Ef(t3) = eM Therefore, from (1 8 ) and (19), we can get: φ, φ^εαίηβ^-1^ = φ; η^~^ When rearranging again, we find:

r/ From the previous two sections, we have the following two working equations: -89 - 201219776 ns =φ0ήιΡτ/β~&,>Τ/ ...(21) Δ/= \ Φ\~^ ... (22 )

Tf, _F = £C^77 and ^ = 6 , 1N mn , .. where . We also know that when implemented, w>. . The optimum time for fluorescence detection and the number of photons that were detected using Philips LXK2-PR14-R00 L E D and P u 1 s a r 65 5 dye were determined as follows. The optimal detection time is determined using equation (22): Review the concentration of the amplicon, and assuming that all amplicons are hybridized, the concentration of the luminescent luminophore is: c = 2.89 (10) Mohr / liter chamber Height is the length of the light path 1 = 8 ( 10 ) '6m » We make the fluorescent area equal to our photodiode area 'but our actual fluorescent area is substantially larger than our photodiode area; therefore, we can The approximate assumption is that the light collection efficiency of our optical system is nine=Q·5. From the characteristics of the photodiode, the ratio of the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency at the excitation wavelength is t = 10 ♦e is a very conservative number 値. With a typical LED decay life of ~ = 0.5 nanoseconds and using the Pulsar 650 specification, At: F = [1.48(10)6 ][2.89(10)-6 ][8(10)'6 ](1) = 3.42(10)-5 -90 - 201219776 ^ ln([3.42(10)-5](10)(0.5)) i i-1 (10)-6 ~ 0.5(10)-9 =4.34(10) The number of photons detected by '9s' is determined using equation (2 1 ). First, the number of excitation photons emitted per unit time is determined by inspection through the illumination geometry. The Philips LXK2-PR14-R00 LED has a Lambertian radiation pattern, so: fi, = nl0 cos(0) ... (23) where Π/ is offset from the LED forward axial angle at each unit solid angle The number of emitted photons per unit time, and Η«• is the valve in the forward axial direction & The total number of photons emitted by the LED per unit time is: ή, = j ή, άΩ Ω

=J w)0 cos(0)JQ Ω (24) Now, ΔΩ = 2ττ[1 — cos(0 + Δ 0)] — 2;τ[1 - cos(0)] △Ω = 2;r[ Cos(0) - cos(0 + A0)] 4^sin(0)cos^^jsini-^j + 4^cos(0)sin2f·^ άΩ.-2π$ιη(θ)άθ Substitute this into (24 ): η 2 ή, = j*2;^0 cos(0)sin(0)i/0

-91 - 201219776 After rearranging, obtain: H,0=two...(26) π LED output power is 〇.515 w and ve= 6.52(10)14Ηζ, therefore: =_0.515_ ~[6.63(10) -34][6.52(10)14] =1.19(10)18 Photon/s Substituting this ( into (2 6 ), obtain: ...a 1.19(10)18 =3.79(10)17 Photon stealing reference picture 61' The optical center 252 and lens 254 of the LED 26 are shown in schematic form. The photodiode is 16 microns x 16 microns, and for the photodiode in the middle of the array, the solid angle (Ω) of the conical light emitted from the LED 26 to the photodiode 184 is approximately: Ω = sense Area of the device /r2 [16 (10)-6][16(10)'6] 2.825(10)-3f = 3.21(10)-5 sr It is known that the central light of the photodiode array 44. The electric diode 1 8 4 is intended for use in these calculations. In terms of the Lambert source excitation intensity distribution, the sensors at the edge of the array will only receive 2% less photons when hybridized. The number of excited photons emitted per unit time is: -92 - 201219776 he =η,Ω ../28) =[3.79(10)17][3.21(10)'5] =1.22(10)13 Photon/s Now refer to equation (2 9 ): / · τη ~At I τ f ns ns = (0.5)[1.22(10)13 ][3.42(10)'s ][1(10)-6 je"4 34(1 〇r, = 208 photons/sensors.

Therefore, with the Philips LXK2-PR14-R00 LED 650 fluorophore, we can easily detect any hybridization that causes photons to be emitted. The SET LED illumination geometry is shown at 62 id = 20 mA, with the LED having a minimum optical power output centered at λβ = 340 nm (铽 absorption wavelength), ρ^ΜΟμλΥ. The drive LED will linearly increase the output power to ρι=2.4. The optical center 252 of the LED is placed at a distance from the hybridization array 1 1 , and we concentrate this output flux approximately at the maximum diameter of the dot size. Photons in a 2 mm diameter dot at a distance from the hybridization plane are generated by Equation 27. hVe 2.4(10)~3 _ [6.63(10)'34][8.82(10)14] = 4.10(10)15 Photon/s Using Equation 28, obtain: and Pulsar in this number graph. I d = 2 0 m A mW in the chelate. The flux of i 17.5 mm 2 mm is borrowed -93- 201219776 ne = η, Ω 4.10(10)15 3.34(10)1 [16(10)6]2 41(10)3]2 photon/s Now, Review Equation 22 and use the previously listed chelate properties, 1η[(6.94(10)~5)(10)(0·5)] -_1___Ί_ 1(10)'3 ~ 0.5(10)-9 =3.98 (10)_9s Now, from Equation 2 1 : η, = (0.5) [3.34(10)1,][6.94(10)-5][1(10)-3>-39*(,0>', /1(1°)'3 = 11,600 photons/sensors. The theory of the number of photons using the hybridization of the SET LED and the ruthenium chelate system can be easily detected, and at least 3 photons can be used as described above. The reliable detection of the signal by the light sensor requires that the detection exceeds the measurement. The on-wafer detection of the maximum spacing between the probe and the photodiode does not require a confocal microscope (see background of the invention). The departure of traditional detection technology is an important factor saving time and cost. Traditional detection requires that the imaging optics must use a lens or a curved mirror. Through the use of the diagnostic system, the diagnostic system does not require a complicated and cumbersome optical series. Very close to the probe for high collection efficiency The advantage of the material thickness between the probe and the photodiode is measured in micrometers, and the collection angle is up to 173. This angle is considered to be optically detected by considering the system, and the non-imaging light will be photo-electric: The emission is calculated from the light emitted by the probe at the center of mass of the hybridization chamber located at -94-201219776 near the photodiode (which has a planar photosensitive surface region parallel to the face of the hybridization chamber). The angle cone (the light in this cone can be absorbed by the photodiode) is defined by its apex with a launch probe and one of the sensors at its periphery. For a 16 micron χΐ6 micron sensor, The apex angle of the cone is 170°; in the case where the photodiode is expanded to have an area commensurate with the 29 μm X 19.75 μm hybridization chamber, the apex angle is 173. The surface of the hybridization chamber is 1 μm. The separation of the photosensitive surfaces of the smaller or smaller photodiodes can be easily achieved. The use of non-imaging optical systems does require that the photodiodes 1 84 must be very close to the hybridization chamber to collect sufficient fluorescent emission photons. The maximum spacing between the electrical diode and the probe is determined as described below with reference to Figure 54. Using the ruthenium chelate fluorophore and the SET UVT0P335T039BL LED, we calculated that 11600 photons arrived from each of the hybrid chambers 180. 16 micron x 16 micron photodiode 184. In performing this calculation, we assume that the light collection region of our hybridization chamber 180 has the same bottom area as our photodiode photosensitive region 185 and half of the total number of hybrid photons The photodiode 184 is reached. That is, the light collection efficiency of the optical system is φ 〇 = 0.5. More precisely, we can write Φ 〇 = [the bottom area of the light - the collection area of the hybrid chamber) / (photodiode area)] [Ω / 4ττ], where Ω = the representative point at the bottom of the hybridization chamber The solid angle of the opposite direction of the photodiode. In the geometry of the pentagonal pyramid: 1 fi = 4arcsin(a2/(4d〇2 + a2)), where d〇 = the distance between the cell and the photodiode -95-201219776, and a is the photodiode The size of the polar body. Each hybrid cell released 23,200 photons. The detected photodiode of the selected photodiode is 17 photons: therefore, the minimum optical efficiency required is: φ 〇 = 1 7/23200 = 7.33x1 0'4 The bottom area of the light collection region of the hybridization chamber 180 It is 29 microns x 19.75 microns. When answering d〇, the maximum limit distance between the bottom of our hybrid chamber and our photodiode 184 will be dG = 249 microns. In this limitation, the collection cone angle as defined above is only 0.8°. It should be noted that this analysis ignores this negligible refraction. Test module with microfluidic device with dialysis device, LOC and interconnected caps Figure 152 shows a test module 1 1 for analyzing sample fluids containing target molecules. The test module Π includes a housing 13 having a container 24 for receiving sample fluid, a removable sterile sealing tape 22 to cover the container 24, a membrane gasket 408 with a membrane shield 410 prior to use (which forms the housing 1) Part 3, to reduce the loss of moisture in the test module, while protecting the membrane gasket 40 8 with the membrane shield 410 without damaging the pressure, the printed circuit board (PCB) 57, micro Flow device 783, porous element 49, standard Micro-USB plug external power supply capacitor 32 and inductor 15 for power, data and control. The microfluidic device 783 has a dialysis device 784 (which is in fluid communication with the container 24 and configured to separate the target molecule from other components of the sample), and the LOC device for analyzing the target molecule with -96-201219776 78 5 and a cover 51 covering the LOC device 785 and a dialysis device 784 for establishing fluid communication between the LOC device 78 5 and the dialysis device 784. Reagent Loading and Probe Styling System The robots shown in Figures 63 through 66, the droplet ejection system, are filled with reagents and water in reagent libraries 54, 56, 58, 60 and 62 (see Figure 6). The robotic system also spots oligonucleotide FRET probe 186 or ECL probe 237 into hybridization chamber 180. Droplet dispensing technology is an inexpensive spotting technique that delivers droplets of reproducible volume and can dispense small droplets of many different solutions simultaneously. This allows the LO C unit to be mass produced with extremely high production capacity and low cost. The reagent and probe spotting system includes three robotic subsystems: 1. a reagent dispensing robot 256 (see Figure 63) - a microtube 258 (see Figure 64), each having a drop dispenser 262 to The reagents are dispensed into reservoirs 54, 56, 58, 60 and 62 and dispensed into reservoir 188 (see Figure 6). The patterned upper seal 82 is then applied (if necessary) to the cover 46. 2. The ON EC refill robot 2 74 (see Figure 65) - a microtube 25 8 with a droplet dispenser 2 62 that dispenses the probe into an oligonucleotide ejector chip (ONEC) 272 In repository 278 (see Figures 71 and 72). The ONEC repository 278 is fed into the thermal droplet generator array 271. The 〇NEC is then used in the third robotic subsystem, the LOC spotting robot. -97- 201219776 3. LOC spotting robot 289 (shown graphically in Figure 66) - ONEC 272 uses thermal droplet generator 271 to point probes in each hybrid chamber 80 of LOC device 30 (see page 72) Figure). Microtubes The reagent dispensing robot 256 and the ONEC refilling robot 274 both use the microtube 2M as shown in Fig. 64. Probes and reagents are ordered directly from the supplier of the microtube (not shown). A small amount of liquid is transferred from a microtube to a container 259 on each microtube 2 58 to form a small aliquot (usually between 282 microliters and 400 microliters) that can be refrigerated with a microtube until needed. Each microtube 258 has a piezoelectric droplet dispenser 262 and a closed quality assurance chip (i.e., integrated circuit) 266 with flash memory and electronics contacts 264 for power and data transfer. The drop dispenser 262 has a piezoelectric actuator 261 configured to discharge droplets having a volume of 50 picoliters to 150 picoliters to load the reagents reasonably quickly while maintaining an accurate drip position. Probe and Reagent Identification Protocol Quality Assurance Chip 266 (see Figure 64) has digital memory for storing, identifying, and tracking specification data for reagents or oligonucleotide probe solutions characterized in microtubes 285. At the end of the spotting and loading process, the data from each microtube 2 58 is downloaded and stored in the LOC along with other loading and spotting data via a control microprocessor 263 that controls the reagent dispensing robot or probe dispensing robot. The program and data flash of the device 30 is -98-201219776. This data is used for diagnostic information and processing work, quality control, and auditing. Referring to Figure 73, ONEC 272 also has digital memory (such as flash memory 28 1) in the ONEC CMOS structure 285 to store specification data for the oligonucleotides, such as probe characteristics, lot numbers, and the like. Like the LOC device, the ONEC refill robot 274 downloads the specification data from the quality assurance chip 266 on the micro-tube 258 to the ONEC flash. The automatic information transmission will be an error and the use of the wrong micro-tube event. The possibility is minimized and the test module reader 12 or other system components can identify this error when processing diagnostic information. Reagent dispensing robots Figures 63 and 124 show top and side views of a simplified reagent dispensing robot 256. It includes: • Microtubes 25 8, which contain reagents and molecular biology grade water (only some microtubes are shown) • Mechanical/electrical racks 286 (only outlines are shown) that support and provide microtubes 25 8 Electrical Connections • XY A platform 268 that provides a surface-recording camera 270 for detachably securing a partially depth-sawed wafer 260 or other fixed array, such as a detachable PCB wafer 720, that provides feedback to the control microprocessor 263 to map the exact position of the piezoelectric droplet dispenser 262 -99-201219776 using the piezoelectric droplet dispenser 262 on the microtube 258 to dispense the reagent and water directly into the LOC device repository 54, 56, 58, 60 and 6 2 and the humidifier reservoir 1 8 8 . ONEC Refill Robot Figure 65 shows the ONEC refill robot 274. It is similar to the reagent dispenser robot 25 6 and includes: • 1 080 microtubes 258, which contain solutions of oligonucleotide probes (for illustration, not all microtubes are shown) • Mechanical/electrical machines Rack 2 8 6 (showing only outlines) - supporting and providing microtubes 2 5 8 electrical connections • oligonucleotide ejector chip (ONEC) 272 - with 1 080 ONEC depots 2 78, each offering 4 ONEC Individual ejector of thermal droplet generator 271 2 8 7 (see Figures 71 and 72) • XY stage 26 8 : support oligonucleotide ejector chip (ONEC/s) 272 • Recording camera 270, which provides Feedback is given to control microprocessor 263 to map the exact location of the thermal droplet generator 271. Move the ONEC2 7 2 under the mechanical/electrical frame 286. A unique probe solution is dispensed from each microtube 286 into each of the ONEC libraries 278. TheONEC 272 is then used in the probe spotting robot 273 to spot a single drop of probe solution in the hybridization chamber 180 of the LOC device.

ONEC -100- 201219776 The figures 71, 72 and 73 are shown in detail (^ ugly 272. This is used for non-contact spotting on the surface (such as the array of hybrid chambers in any l〇C device) The above-mentioned oligonucleotide spotting device has an overall size of 23,296 micrometers and 1760 micrometers and is fabricated using a large number of effective photolithographic fabrication techniques. Each ONEC has 1 080 etched on a single-chip substrate 2 Depot 278 in reservoir side 277 (see Figure 73). Each ONEC has more than 1 000 depots 278 with full probe detection required for spotting on the LOC devices described herein. This allows for each LOC The spotting process is a single step, meaning that there is no need to use more than one ONEC to spot the LOC configured for each particular analysis. The ONEC repository 278 has a rectangular bottom with a depth of 200 microns (96 microns x 208 microns) Each of the ONEC reservoirs 278 delivers the probe suspension to a respective ejector 287. The liquid suspension of the probes is drawn into the common chamber 282 via a pair of chamber inlets 284 (see Figure 72). The entrance 284 of the chamber is two from the storage 278 to the total The chamber 282 has a diameter of 21 micrometers. One of the four thermal droplet generators 271 generates steam froth by heating the actuator 280 and discharges the probe droplets into the hybrid chamber 180 through the nozzle 283 in the ejector side 279. If there is a droplet generator failure, there are four thermal droplet generators 271 that can be backed up. The LOC probe spotting robot shows the LOC probe spotting robot 289 in Figures 66 and 92. For the sake of clarity. The components of the PCB wafer 720 other than the LOC device 30 are not shown. The following includes the following: -101 - 201219776 • ONEC 272 - an oligonucleotide ejector chip having 1 080 banks 278, each of which is full of probe solution (See Figures 71 and 72) • XY stage 268 that holds the LOC矽 wafer 260 (see Figure 66) or the detachable PCB wafer 720 (see Figure 92) that is partially sawn away. • Recording camera 270, It provides feedback to control microprocessor 263 to map the exact location of the ONEC thermal droplet generator 271. The LOC(R) wafer 260 or detachable PCB wafer 720 is detachably fixed along two orthogonal On the axis conversion platform. The ONEC 2 72 is available The separation mode is supported on a chuck 265 adjacent to the platform, and the ejector 2 87 is facing the platform (see Figure 66). The LOC 矽 wafer 260 or the detachable PCB wafer 720 is controlled by the control processor 263. Moving relative to the ONEC 272. The ejector that is controlled by the controlled processor 263 is spotted for each LOC device hybrid chamber 180. Using a volume of less than 1 〇〇 picoliter reduces reaction time and allows for increased density of the hybrid chamber array. The use of small-volume probe droplets for spotting in the past has been difficult due to the accurate and reliable discharge of very small droplets. Mistakes in the direction of the drop may not be spotted in the correct chamber and may contaminate the adjacent room. The ONEC 2 72 can be driven to produce a series of droplet volumes. For accurate dispensing, the droplets produced by ONEC 2 72 will be less than 100 picoliters. To improve the accuracy of the dispensed probes and reagents (in terms of volume and position on the LOC device), droplets produced by ONEC can be reduced to less than 25 picolitres, preferably no more than 6 picolitres. The 0NEC 272 dispenses the probe solution from -102 to 201219776 to 1080 hybridization chambers 180 with a drop of 0.1 picoliter to a volume of 皮.6 picolitres and a high degree of positioning accuracy. The hybrid chamber array 110 is configured in 24 rows with 45 adjacent cells per row (see Figure 52). The sample flow path 176 extends between every two rows such that the entire array has a generally square shape to be substantially uniformly illuminated by the LEDs 26. Since the hybrid chamber array 1 1 〇 is limited to an area of less than 1 500 μm x 500 μm, the correct rate of the ONEC 272 is naturally high. The recording camera 270 is used via control processor 263 to determine the exact position of the ONEC thermal droplet generator 271, and the droplet generator drive pulse is synchronized with the XY stage 268 via the ONEC bond pad 276. The LOC probe spotting robot 2 73 using the ONEC 272 and the camera 270 can easily spot the probe on a surface (such as the hybridization chamber array 110) at a rate of more than one probe per second; In most cases, it is performed at a speed of more than 140 probes per second. Typically, the array of droplet generators spot the probe on the surface at a rate of more than 20,000 probes per second, and in many cases, the array of droplet generators has 300,000 probes per second. The probe is spotted on the surface at a rate of 1 million probes per second. The droplet generator array fabricated on the germanium substrate by photolithography allows the ONEC 272 to spot the oligonucleotides on the surface at a much greater density than existing probe spotters. ONEC 272 easily spotted at a density of more than one probe per square millimeter. In most cases, the spot density is more than 8 probes per square millimeter. In most cases, the spot density is more than 60 probes per square millimeter, typically, the density is 500 probes per square millimeter to 1500 probes per square millimeter. -103- 201219776 This uses the ONEC 272 as The LOC probe spotting robot 2 73 of the biochemical storage device can easily store biochemical substances on the surface at a rate of more than 100 droplets per second, in most cases with more than 1400 droplets per second. Speed is going on. Typically, the array of droplet generators spot the droplets on the surface at a rate of more than 20,000 droplets per second, in many cases the array of droplet generators has 300,000 droplets per second to The droplets are spotted on the surface at a rate of 1 million droplets per second. The LOC probe spotting robot 273 using the ONEC 272 as a biochemical storage device can easily store biochemical substances on the surface at a density of more than one droplet per square millimeter. In most cases, the spot density exceeds 8 droplets per square millimeter. In most cases, the spot density exceeds 60 droplets per square millimeter, typically from 500 droplets per square millimeter to 1500 droplets per square millimeter. Conclusion The devices, systems, and methods described herein facilitate molecular diagnostic testing at low cost, high speed, and point-of-care. The above-described system and its components are purely for the purpose of illustration and many variations and modifications of the spirit and scope of the invention will be readily apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS A preferred system of the present invention will now be described by reference to the accompanying drawings -104-201219776 wherein: Figure 1 shows a test module and test module reader configured for detecting fluorescence Figure 2 is a general schematic diagram of the electronic components in the test module configured to detect fluorescence; Figure 3 is a general schematic diagram of the electronic components in the test module reader, and Figure 4 is the LOC device. A schematic image of the structure; Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device with functions and structures from all layers superimposed on each other; Figure 7 is a cover with independent display A plan view of the LOC device of the architecture; Figure 8 is a top perspective view of the cover with the inner channel and reservoir shown in phantom; Figure 9 is a top perspective view of the cover with the inner channel and reservoir shown in phantom Figure 10 is a bottom perspective view of the cover showing the configuration of the top channel; Figure 11 is a plan view of the LOC device, showing the configuration of the CMOS + MST device independently; Figure 12 is the LOC device Schematic diagram of the section at the entrance of the sample: Figure 13 is an enlarged view of the illustration AA shown in Figure 6; Figure 14 is an enlarged view of the illustration AB shown in Figure 6; -105- 201219776 Figure 15 is the 13th The enlarged view of the illustration AE shown in the figure; Fig. 16 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AE, and Fig. 17 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AE, Figure 18 is a partial perspective view showing the thin layer structure of the LOC device in the inset AE: Figure 19 is a partial perspective view showing the thin layer structure of the LOC device in the inset AE; Fig. 20 is a view showing the LOC in the illustration AE a partial perspective view of the thin layer structure of the device; Fig. 21 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AE; Fig. 22 is a schematic cross-sectional view of the lysis reagent library shown in Fig. 21, the 23rd The figure is a partial perspective view illustrating the thin layer structure of the LOC device in the inset AB; Fig. 24 is a partial perspective view illustrating the thin layer structure of the LOC device in the inset AB: Fig. 25 is a view illustrating the LOC device in the illustration AI a partial perspective view of a thin layer structure; Figure 26 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AB; Fig. 27 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AB -106-201219776; A thin partial perspective view of the LOC device within; Figure 29 is a partial perspective view of the thin section of the LOC device in the illustration AB; Figure 30 is a snapshot of the amplification mixture reservoir and the polymerase library g I , ρΓ| Fig. 31 shows the characteristics of the boiling start valve independently; Fig. 32 is a schematic sectional view of the starting valve taken through the line 3; 3-33 shown in Fig. 31; Fig. 33 is the view shown in Fig. 15. Fig. 34 is an enlarged view of the upstream end of the analysis zone taken by the line 35-35 shown in Fig. 33; Fig. 35 is an enlarged view of the illustration AC shown in Fig. 6 and Fig. 36 is A further view in the illustration AC of the amplification zone is shown; Figure 37 is a further view showing the illustration AC of the amplification zone; Figure 38 is a view showing the inside of the illustration AC of the amplification zone; The figure shows the enlarged view in the illustration AK shown in Figure 38; Figure 40 shows the amplification The illustration in the AC, the view; the structure of the structure of the surface is indicated by the boiling S; the result of the penetration > step amplification step amplification step amplification step by step amplification -107-201219776 Figure 41 shows the illustration of the amplification zone AC Further enlarged view of the inside, Fig. 42 is a further enlarged view showing the illustration AC of the amplification chamber; Fig. 43 is a further enlarged view of the illustration AL shown in Fig. 42, and Fig. 44 is a view showing the expansion A further enlarged view of the illustration of the increase zone AC,

Figure 45 is a further enlarged view of the illustration AM shown in Figure 44. Figure 46 is a further enlarged view showing the illustration AC of the amplification zone; Figure 47 is an illustration AN shown in Figure 46. Further enlarged view of the inside, Fig. 48 is a further enlarged view showing the illustration AC of the amplification chamber, Fig. 49 is a further enlarged view showing the illustration AC of the amplification chamber, and Fig. 50 is a view showing the amplification A further enlarged view of the area illustration AC, Figure 51 is a schematic cross-sectional view of the amplification zone; Figure 52 is an enlarged plan view of the hybridization zone; Figure 53 is a further enlarged plane of the two hybridization chambers separated View, -108- 201219776 Figure 54 is a schematic cross-sectional view of a single hybridization chamber; Figure 55 is an enlarged view of the humidifier illustrated in the illustration AG shown in Figure 6; Figure 56 is shown in Figure 52. An enlarged view of the illustration AD; Figure 57 is a perspective exploded view of the LOC device in the illustration AD; Figure 58 is an illustration of a FRET probe in a closed configuration; and Figure 59 is an illustration of a FRET probe in an open and hybrid configuration; Figure 60 shows the intensity of the excitation light. Time-lapse graph; Figure 61 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; Figure 63 is a diagram of the sensor electronic technology LED illumination geometry; The plane of the reagent dispensing robot is not intended; Fig. 64 is a perspective view of a reagent microvial with a built-in droplet generator; Fig. 65 is for loading the selected probe into the probe ejection chip Schematic diagram of the oligonucleotide ejection robot; Figure 66 is a schematic plan view of the probe spotting robot for loading the probe into the LOC device on the partially sawn saw wafer; Figure 67 is the first 6 is an enlarged plan view of the humidity sensor shown in the illustration AH in the figure; FIG. 6 is a schematic diagram showing a part of the photodiode array of the photo sensor; -109- 201219776 Fig. 69 is a single Circuit diagram of photodiode: Fig. 70 is a timing diagram of the photodiode control signal; Fig. 71 shows an oligonucleotide discharge chip (ONEC); Fig. 72 shows an illustration from the illustration AO in Fig. 71 The NEEC droplet generator array is shown; Fig. 73 is a schematic cross-sectional view of the droplet generator array taken along line 91-91 shown in Fig. 72; Fig. 74 is the illustration AP in Fig. 55. A magnified view of the evaporator shown in Figure 75; Figure 75 is a schematic cross-sectional view of a hybridization chamber with a detection photodiode and a trigger photodiode: Figure 76 is a diagram of the PCR of the linker priming; A schematic image of a test module with a lancet: Figure 78 is a sketch image of the LOC variant W architecture; Figure 79 is a sketch image of the LOC variant architecture; Figure 80 is a LOC variant XIV architecture Figure 81 is a schematic diagram of the LOC variant XLI architecture; Figure 82 is a schematic illustration of the LOC variant XLIII architecture; Figure 83 is a schematic representation of the LOC variant XLIV architecture: Figure 84 A graphical representation of the LOC variant XLW architecture; Figure 85 is a graphical representation of the linear fluorescent probe attached to the primer during the initial cycle of the amplification reaction: Figure 8 6 is the linearity of the primer connection during the subsequent cycle of the amplification reaction. Fluorescent probe graphic: -110- 201219776 Sections 87A to 87F illustrate the connection with primers Thermal cycling of the fluorescent arm-and-loop probe; Figure 88 is a schematic illustration of the excited LED of the hybrid chamber array and photodiode; Figure 89 is a hybridization chamber for directing light to the LOC device Schematic description of the excitation LED and optical lens on the array; Figure 90 is a schematic illustration of the excitation LED, optical lens and optical 用于 on the array of hybridization chambers for directing light to the LOC device; Figure 91 is for A schematic illustration of the excitation LED, optical lens, and mirror arrangement on the array of hybrid cells that direct light to the LOC device; Figure 92 is a schematic plan view of the probe spotting robot for loading the probe into the separable In the LOC device on the PCB; Fig. 93 is an image of the structure of the LOC variant X; Fig. 94 is a perspective view of the LOC variant X; Fig. 95 is a plan view of the LOC variant X, which independently displays the CMOS + structure of the MST device; Fig. 96 is a perspective view of the bottom side of the cover, the reagent library is shown by a broken line; Fig. 97 is a plan view showing only the appearance of the cover independently; Fig. 98 is a view showing all the superimposed ones Look and display a plan view of the position of the illustration DA to DK; Figure 99 Fig. 100 is an enlarged view of the illustration DB shown in Fig. 98; '101 is an enlarged view of the illustration DC shown in Fig. 98; -111 - 201219776 Figure 102 is an enlarged view of the illustration DD shown in Figure 98; Figure 103 is an enlarged view of the illustration DE shown in Figure 98; Figure 104 is an illustration of the illustration DF shown in Figure 98. Magnified view; Fig. 105 is an enlarged view of the illustration DG shown in Fig. 98; Fig. 106 is an enlarged view of the illustration DH shown in Fig. 98; Fig. 107 is an illustration DJ shown in Fig. 98 Magnified view; Fig. 108 is an enlarged view of the illustration DK shown in Fig. 98; Fig. 109 is an enlarged view of the illustration DL shown in Fig. 98; Fig. 110 shows a diversion for the photodiode A system of transistors; Figure 111 shows a system for a shunt transistor for a photodiode: Figure 112 shows a system for a shunt transistor for a photodiode; Figure 113 shows a differential imaging Circuit diagram of the instrument; Figure 1 1 4 illustrates the arm-and-loop of the fluorescent probe negative control group Figure 1 1 5 shows the open configuration of the fluorescent probe negative control group of Figure 1 1 4: Figure 1 16 shows the arm-and-loop of the fluorescent probe positive control group Configuration; Figure 1 1 7 illustrates the open configuration of the fluorescent probe positive control group of Figure 1 16: Figure 118 is a cross-sectional view of the conductive sensor used in the LOC device -112-201219776 Figure 1 is a diagram illustrating a CMOS-controlled flow rate sensor; Figure 20 shows a test module and test module reader configured for ECL detection; Figure 1 2 1 is a test A general schematic of the electronic components in the module, the test module is configured for ECL detection; Figure 1 22 shows the test module and an alternative test module reader; Figure 1 23 shows the test module and Test module reader and host system for storing various databases; Figure 1 24 is a schematic side view of the reagent spotting robot; Figure 125 is a schematic diagram of an electrochemical-based test module with a multi-device microfluidic device image. [Main component symbol description] 1 〇: Test module 1 1 : Test module 1 2 : Test module reader 1 3 : Case 14: Micro-USB plug 15 : Inductor 16: Micro-USB 埠 1 7 : Touch screen 18: Display screen 19: Button-113-201219776 20: Start button 2 1 : Honeycomb radio 2 2: Aseptic sealing tape 2 3: Wireless network connection 24: Large container 25: Satellite navigation system 26: Light-emitting diode 27 : Data Memory 28: Mobile Phone / Smartphone 29 : LED Driver 30 : LOC Device 31 : Power Conditioner 3 2 : External Power Supply Capacitor 33 : Clock

3 4 : Controller 3 5 : Recorder 36 : USB device driver 3 7 : Driver 38 : RAM 3 9 : Excitation driver 40 : Flash memory 4 1 : Excitation recorder 42 : Processor 43 : Program memory - 114 201219776 44: Light sensor 4 5 : Indicator 46 : Cover 47 : USB power / indicator - Qualification module 48 : CMOS + MST chip 49 : Porous element 51 : Cover 52 : Hybridization and detection zone 5 4 : Repository 5 6 : Repository 57 : Printed circuit board 5 8 : Depot 60 : Depot 62 : Depot 6 4 : Seal 6 6 : Roof layer 6 8 : Sample inlet 7 0 : Dialysis zone 72 : Waste channel 74 : Target channel 76 : Waste repository 7 8 : Depot layer 80 : Cover channel layer 8 2 : Sealing layer - 115 - 201219776 84 : 矽 Substrate 86 : CMOS circuit 87 : MST layer 8 8 : Passivation layer 90 : MST Channel 92: Down channel 94: Cover channel 96: Intake hole 9 7: Wall area 98: Meniscus anchor 1 00: MST channel layer 101: Laptop/notebook 1 0 2: Capillary action start function 103 : Dedicated Reader 105 : Desktop Computer 106 : Boiling Point Start Valve 107 : E-Book Reader 108 : Boiling Point Start Valve 109 : Tablet PC 1 1 〇: Miscellaneous Intersection array 1 1 1 : Epidemiological data host system 1 1 2 : Amplification area 1 1 3 : Genetic data host system 1 1 4 : Cultivation area -116- 201219776 1 1 5 : Electronic health record (EHR) host System 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Sample stream 120 : Meniscus 121 : Electronic medical record (EMR) host system 1 2 2 : Ventilation hole 123 : Personal health record (PHR) Host system 1 2 5 : Network 126 : Boiling start valve 128 : Surface tension valve 1 3 0 : Chemical cracking zone 1 3 1 : Mixing zone 1 3 2 : Surface tension valve 1 3 3 : Incubator inlet channel 134: Down channel 1 36: Optical window 1 4 6 : Valve inlet 1 4 8 : Valve outlet 150 : Down channel 152 : Ring heater 1 5 3 : Heater connector 1 5 4 : Heater 156 : Heater connector -117- 201219776 158 : Micro Channel 160: Amplification zone exit channel

164: small 孑 L 166 : capillary action activation characteristic device 1 6 8 : intake hole 170 : temperature sensor 174 : liquid sensor 175 : diffusion barrier 1 7 6 : flow path 178 : liquid sensor 1 80 : Hybridization chamber 1 8 2 : Heater 1 84 : Photodiode 1 85 : Photosensitive region 186: FRET Probe 187: Trigger photodiode 1 8 8 : Reservoir 190 : Evaporator 1 9 1 : Ring heater 192 : Water supply channel 193: Ingestion port 194: Down channel 195: Top metal layer 1 9 6 : Humidifier -118 - 201219776 1 9 8 : Intake hole 202: Capillary action Starting characteristic device 204: Dialysis MST channel 210: Microchannel 2 1 2 : MST channel 218 : TiAl electrode 220 : TiAl electrode 222 : slit 232 : humidity sensor 2 3 4 : heater 23 6 : FRET probe 23 7 : ECL probe 2 3 8 : target nucleic acid sequence 240 : Ring 242: Double-stranded arm 244: Excitation light 246: Fluorescent group 248: Quencher 2 5 0: Fluorescence emission 254: Lens 2 56: Reagent dispensing robot 2 5 8 : Microtube 259: Container 2 6 0 : L Ο C矽 wafer - 119 201219776 261 : Piezoelectric actuator 262 : Drop dispenser 263 : Control microprocessor 2 6 4 : Electronic connector 265 : Chuck 266 : Quality Assurance Chip 268 : XY Stage 270 : Recording Camera 2 7 1 : Thermal Droplet Generator Array 272 : Oligonucleotide Discharger Chip 273 : LOC Probe Staking Robot 274 : ONEC Refilling Robot 275 : Single Chip矽 Substrate 276: Bonding pad 2 7 7 : Depot side 2 7 8 : Depot 279 : Ejector side 2 8 0 : Actuator 281 : Flash memory 2 82 : Chamber 283 : Nozzle 2 84 : Entrance of the chamber 28 5 : ONEC CMOS Structure 286: Mechanical / Electrical Frame - 120 201219776 2 87 : Ejector 2 8 8 : Input and Preparation of Sample 2 89 : LOC Staking Robot 290 : Extraction of Nucleic Acid 2 9 1 : Nucleation of Nucleic Acid 292 : Amplification of Nucleic Acid 2 94 : Detection and analysis 296 : first · electrode 298 : second electrode 3 00 : programmed delay 301 : LOC device 3 7 6 : conducting column 3 7 8 : positive control probe 3 80 : negative control probe 3 82 : Calibration room 3 84 : Mshunt gate 3 8 6 : t X gate 3 8 8 : Gate 3 9 0 : Blood collection needle 3 9 2 : Blood collection needle release button 3 93 : Reading gate 3 94 : Transistor Mshunt 3 96 : Transistor Mtx 3 98: transistor Mreset 201219776 4 0 0 : transistor M sf 402 : transistor Mread 4 04 : transistor Mbias 4 0 6 : 'NS ' section Point 408: Membrane Seal 410: Membrane Shield

673 : LOC variant XLIII 674 : LOC variant XLIV 677 : LOC variant XLW 6 8 2 : dialysis zone 6 8 6 : dialysis zone 692 : linear probe 694 linked to the primer: amplification blocker 6 9 6 : Probe area

6 9 8 : Preface! J 700: Oligonucleotide primer 704: Arm-and-loop probe 708: Arm strand 710: Arm strand 712: First optical 稜鏡 714: Second optical 稜鏡 7 2 0 : PCB wafer 72 8 : LOC change Body X 740: Flow Rate Sensor - 122- 201219776 766 : Waste Storage 77 8 : Possible Configuration of Mshunt Transistor 394 780 : Possible Configuration of Mshunt Transistor 394 782 : Possible Configuration of Mshunt Transistor 394 7 83 : Micro Flow device 784: dialysis device 7 8 5 : LOC device 78 8 : differential imager circuit 7 9 0 : pixel 792 : "false" pixel 794 : read_row 7 9 5 : read_ro w_d 796 : fluorophore 797 : M4 transistor 798 : quencher 8000: channel one 8 Ο 1 : MD 4 transistor 8 02 : first terminal 8 0 3 : pixel capacitor 804 : first electrode 8 05 : dummy pixel capacitor 8 06 : second electrode 8 〇 7 : Switch 8 08 : Second terminal -123- 201219776 8 0 9 : Switch 8 1 0 : Conductivity sensor 8 1 1 : "read_col" Switch 8 12 : Liquid 813: False "read_col" Switch 8 1 4 : Heating Element 8 1 5 : capacitor amplifier 8 1 7 : differential signal 860 : electrode 8 70 : electrode -124

Claims (1)

201219776 VII. Patent application scope 1. A microfluidic device for treating a fluid, the microfluidic device comprising: an inlet for receiving the fluid; a functional area for processing the fluid; extending from the inlet into at least some of the functional area a flow path for operating a CMO S circuit that controls the functional area; and a conductive sensor having a first terminal and a second terminal separated along the flow path, and located at the first terminal and the second terminal And a first electrode and a second electrode separated along the flow path; wherein the CMOS circuit is configured to generate a current between the first terminal and the second terminal, and measure a voltage passing through the first electrode and the second electrode Thus, the conductivity of the fluid in the flow path is derived from the current and the measured voltage. 2. The microfluidic device of claim 1, further comprising a temperature sensor for sensing a temperature of the fluid in the flow path, and a flow having a heating element supported on an inner surface of the flow path a rate sensor, wherein the circuit is configured to receive a temperature sensor output, provide a predetermined current through the heater element, and measure a resistance of the conductive element from the current, temperature sensor output, and resistance The flow rate is derived and the flow rate is derived using the flow rate and the cross-section of the flow path transverse to the direction of flow at the heater element. 3. The microfluidic device of claim 2, wherein the heater element has a curved configuration. -125-201219776 4. The microfluidic device of claim 3, wherein the path is defined by a microchannel having a cross-section transverse to the flow direction of less than 1 million square microns. 5. The microfluidic device of claim 4, wherein one of the regions is a polymerase chain reaction (PCR) region, and the fluid is a biological sample containing a sequence configured for sample circulation To amplify the nucleic acid sequence, the microchannel defines a flow path through the PCR. 6. The microfluidic device of claim 5, wherein the region further comprises at least one nucleic acid sequence extension heater element for heating the microchannel. 7. The microfluidic device of claim 6 of the patent, further comprising a support substrate and a microsystem technology (MST) layer incorporated in the functional area, the CMOS circuit having a digital body for storing data and operating information to The functional area is controlled during processing and analysis of the sample. 8. The microfluidic device of claim 7, further comprising a plurality of reagent libraries containing reagents for processing the sample, wherein the data in the digits is related to the characteristics of the reagents. 9. The microfluidic device of claim 8, wherein the data in the digital memory is a unique identifier of the microfluidic device. 10. The microfluidic device of claim 3, wherein the operational information in the digital memory is related to the time and duration of the thermal cycle. 1 1. The microfluidic device of claim 10, wherein the PCR column of the hot zone of the functional area of the functional nucleic acid is stored in the step of storing the storage and storage time of the step-by-step package -126-201219776 A culture zone upstream of the PCR zone, and one of the reagent libraries is a restriction enzyme library. The incubation zone has a heater for maintaining the temperature of the mixture of the sample and the restriction enzyme at the incubation temperature during restriction enzyme digestion of the nucleic acid sequence. 12. The microfluidic device of claim 11, further comprising a probe array for hybridizing to a target nucleic acid sequence in an amplicon from the PCR region. I3. The microfluidic device of claim 12, wherein the data stored in the digital memory comprises probe characteristic data identifying probes at respective points in the probe array. 1 4. The microfluidic device of claim 13 wherein each probe is configured to form a probe-target hybrid with each of the complementary target nucleic acid sequences contained in the amplicon, each probe - The target hybrid is configured to emit photons in response to an input, and the CMOS circuit incorporates a photosensor for sensing photons emitted by the probe-target hybrid. 1 5. The microfluidic device of claim 4, wherein the data stored in the digital memory comprises hybridization data generated from a photosensor output. The microfluidic device of claim 5, further comprising an array of hybridization chambers for housing the probe, such that the probes within each hybridization chamber are configured to be one of the target nucleic acid sequences Hybrid. 1 7. The microfluidic device of claim 16, wherein the photosensor is a photodiode array that is registered with the hybridization chamber. A microfluidic device as claimed in claim 16 wherein the CMOS circuit has a bond pad and is configured to transmit the hybrid number -127 - 201219776 data to an external device. The microfluidic device of claim 18, wherein the sample is taken from a patient, and the CMOS circuit is configured to download patient data via the bond pad and store patient data in digital memory in. 20. The microfluidic device of claim 18, wherein the PCR zone has an active valve for retaining liquid in the PCR zone during thermal cycling and allowing liquid to flow to the hybridization chamber in response to activation from the CMOS circuit signal. -128-