TW201209159A - Genetic analysis LOC with non-specific nucleic acid amplification section and subsequent specific amplification of particular sequences in a separate section - Google Patents

Abstract

A lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device having a sample inlet for receiving the sample, a plurality of reagent reservoirs containing dNTP's, primers, polymerase and buffer solution for addition to the sample, a first nucleic acid amplification section for thermal control of the sample to amplify the target nucleic acid sequences, and, a second nucleic acid amplification section for thermal control of amplicon from the first nucleic acid amplification section to further amplify the target nucleic acid sequences.

Description

201209159 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatment and analysis for molecular diagnostics. [Prior Art] Molecular diagnostics have been used in the field of providing early detection of disease prior to 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 DNA and nucleic 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 the synthetic DNA (oligonucleotide) short sequence to the particular nucleic acid sequence used for the nucleic acid assay. 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. -5- 201209159 Nucleic Acid-Based Molecular Diagnostic Test A A 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, For example, blood, urine, sputum and tissue samples are used for genetic analysis. Diagnostic tests determine the type of sample required 'because 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 outset. Blood is one of the more frequently requested sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombotic cells promote agglutination and maintain activity in vitro. To inhibit coacervation, 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 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 dip (d i f f e r e n t i a 11 y ) red blood cells dissolved 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 is extracted. The exact procedure used to extract nucleic acids depends on the sample and the diagnosis to be performed -6 ** 201209159 'Analysis. For example, the protocol used to extract viral RNA is quite different from the protocol used to extract genomic DNA. 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 precipitation with an alcohol (usually ice ethanol or isopropanol) or via a solid phase purification step prior to washing in the presence of a high concentration of chaotropic salt, usually in a fractionation column. The ruthenium oxide matrix, resin or paramagnetic beads are then eluted 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. 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 increase (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 (PCR). The PCR is known in the art and a comprehensive comprehensible 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 RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Then, the obtained cDNA is amplified by PCR. The target (in the middle of the phase to the top of the primer 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 - a short single strand of DNA having approximately 10-30 nucleotides complementary to the flanking target sequence 2. DNA polymerase-synthesis of DNA thermostable enzymes 3. Deoxyribonucleoside III Phosphoric acid (dNTP) - provides nucleotides integrated into the newly formed DNA strands. 4. Buffer - the best chemical environment for DN A synthesis. PCR usually involves placing these reagents in a tube containing extracted nucleic acids (~10-5). 0 microliters). The tube is placed in the thermal cycler of the polymerase chain reactor; an instrument that subjects the reaction to different temperatures at a series of unequal amounts. The standard protocol for each thermal cycle includes the denaturing phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the extension of the primer. In addition to this three-step procedure, a two-step thermal procedure can be employed for adhesion and extension. The denatured phase generally involves heating the reaction temperature by 90-95 °C to denature the DNA strand; in the adhesive phase, the temperature is lowered by ~50-60 °C for adhesion of the primer: then the temperature rise is best in the extended phase. The DNA polymerase activity temperature is 60-72 ° C for extension of the primer. This method repeats the cycle by about 20 to 40 times. The final result is to generate a target sequence between millions of copies. A number of standard PCR protocols for molecular diagnostics have been developed, including, for example, multiple primer set PCR, linker initiation (linker-201209159 primed) PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons that are specific to different DNA sequences. By indexing multiple genes at once, additional information can be obtained from a single experiment (with other The method requires several trials). Optimizing multiple primer set PCR is more difficult because it requires the selection of primers with approximate adhesion temperatures and amplicon with 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 enabling nucleic acid amplification of virtually all DNA sequences in complex DNA mixtures without the need for target-specific Sexual introduction. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). Using a ligase enzyme, a double stranded oligonucleotide linker (also known as a zygote) with a suitable overhanging end is then ligated to the terminal of the target DN A fragment. Nucleic acid amplification is then carried out using an oligonucleotide primer having specificity 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 any nucleic acid extraction (or 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 the target nucleic acid prior to preparation of the reaction mixture. However, using appropriate changes in chemical properties and sample concentration, DNA purification can be minimized while 201209159 is capable of binding to the correct PCR single nucleic acid primer second gene low. By standard clinic, no. This is followed by (ex-recording PCR or direct PCR. PCR chemical adjustments for direct PCR include potentiation of buffer strength, use of high activity and high sustained synthesis (pτ 〇cessi Vity) polymerase and inhibition with potential polymerases Honey additive.

The repeat sequence PC R utilizes two independent nucleic acid amplifications to increase the probability of amplifying the amplicon. Repetitive Sequences One type of PCR is nested, in which two pairs of PCR primers are used to perform amplification of the locus for separate nucleic acid amplification. The first pair of primers hybridize to the sequence of the outer region of the target nucleic acid sequence. The second pair of primers (nested) used in the second amplification binds to the first PCR product and produces a PCR product containing the target nucleic acid (short than the first PCR product). The strategy is based on the fact that if the error is amplified due to a mistake during the first nucleic acid amplification, the probability of re-amplifying the wrong locus by the second pair of primers is very high, thus ensuring specificity. Real-time measurement of the amount of PCR product by real-time PCR or quantitative PCR The initial content of the nucleic acid in the sample can be determined using a fluorescent group containing a probe or a fluorescent dye and a reference in the reaction. This is particularly useful for sub-sections where treatment options may depend on the pathogen contained in the sample and there is a pan-transcriptase PCR (RT-PCR) system for amplifying DNA from RNA to record the enzyme for reverse transcription of RN A into a complementary DN A The enzyme (cDN A ) is extravagantly amplified by PCR. RT-PCR is widely used in expression profiling to determine the expression of a gene or to recognize the sequence of rnA transfection (including transcription initiation and termination sites). It is also used to amplify -10- 201209159 RNA viruses, such as human immunodeficiency virus or hepatitis C virus.

Constant temperature amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DNA during the amplification reaction, and thus does not require complicated 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 Some thermostated nucleic acids such as Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification Amplification methods 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 extension and replaces the downstream The ability of the stock. The exponential nucleic acid amplification is then achieved by a reaction between the sense and the antisense, wherein the strand substitution from the sense reaction serves as a template for the antisense reaction. Using a nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) that produces a nick on one of the DNA strands of -11 - 201209159 DNA, which is not cut in a conventional manner, is useful for this reaction. SDA has been improved by using a combination of a thermostable restriction enzyme (d v α 1 ) and a thermostable exo-polymerase (polymerase). This combination appears to increase the amplification efficiency of the reaction from 1 〇 8 fold amplification to 1 〇 1 倍 amplification, so that this technique can be used to amplify unique single copy molecules. Transcription-mediated amplification (ΤΜΑ) and lysin nucleic acid sequence amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than the corresponding genomic DN Α. This technique uses two primers and two or three enzymes, RN Α polymerase, reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to a target ribosomal RNA (rRNA) at a defined position. The reverse transcriptase produces a DNA copy of the target rRNA by extending from the 31 end of the promoter primer. 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 the primer by reverse transcriptase. RNA polymerase recognizes the promoter in the DNA template and initiates transcription. Each newly synthesized RNA amplicon is re-entered and used 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. Conversely, RPA uses recombinant enzymes

201209159 Subcomplex to scan dsDNA and promote strands at homologous sites. The resulting structure is then stabilized by a single strand of DN A binding interacting with the substituted fj, thereby preventing the primer from exiting via branch migration. Recombinant enzymes are unraveled such that the stock replaces 〇ΝΑ (such as a large fragment of B. subtilis Poll (5^)) which can be the 3' end of g-glycoside, followed by extension of the primer. The exponential nucleic acid amplification is repeated in this process to complete. The lysed enzyme amplification (HDA) mimics the in vivo system, which uses a helicase to generate a single-strand template for primer hybridization, followed by a polymerase extension primer. In the first step of the HD A reaction, passing along the target DNA disrupts the hydrogen bond linking the two strands, which are linked by a single strand of binding protein. The single-strand target zone is allowed to adhere by the helicase to allow the primer to adhere. Then, the DNA polymerase extends the 3' end of each primer using free deoxyglycoside triphosphate (dNTPs) to produce a second replicon. The two sets of replicated dsDN A strands enter the next loop independently, resulting in amplification of the target sequence exponential nucleic acid. Other DNA-based thermostating techniques include rolling cycle RCA, in which DNA polymerase continues to surround the circular DNA template, producing long DNA consisting of many repeating copies of the loop. The polymerase is produced thousands of times before the end of the reaction. The annular mold is 10,000 parts, and the copy of the copy is linked to the original target DNA. The spatial resolution of the target and the rapid nucleic acid amplification of the signal are performed as such. 1 to 12 copies of the template can be produced. Branched amplification is an RCA variant that is replaced with a closed circular probe (C-probe) or padlock i. By the board stocks (branch polymerase 丨 near oligonuclear ^ via reuse of DNA by DN A helicase 丨 再 再 ^ 露 可 ribonucleoside DNA HDA cycle: amplification (i extension of the product; One probe within hours can be allowed and -13-201209159 DNA polymerase with high sustained processivity to exponentially expand the C-probe under constant temperature conditions.

Circular Thermostat Amplification (LAMP) provides high selectivity and uses DNA polymerase and four sets of primers specifically designed to recognize six different sequences on the target DNA. An intron containing the meaning of the target DNA and the antisense strand sequence initiates LAMP. Subsequent replacement of DNA synthesis by a strand initiated by an external primer will release a single strand of DNA. This single-stranded DNA can serve as a template for DNA synthesis initiated by the second primer and the foreign 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 initiates the replacement of 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 loop DNA having a plurality of inverted repeaters of the target and a cauliflower-like structure having a plurality of loops formed by overlapping the target repeaters in the same strand.

After completion of nucleic acid amplification, the amplification product must be analyzed to determine whether or not the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product can be carried out by simply measuring the size of the amplicon by gel electrophoresis to 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 dyeing, and its -14-201209159 exhibits fluorescence under ultraviolet light. The size of the fragment is determined by comparison to a DNA ladder containing DNA fragments of known size and migrating side by side with the amplicons 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. To determine the identity of the amplicon, or if several amplicons have been generated, a DNA probe that hybridizes to the amplicon is typically 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 light-generating portion of the excited state is produced, but both processes can be used to covalently label nucleotide strands. In electrochemiluminescence (ECL), a light system is produced by a luminophore molecule or complex when stimulated by an electric current. In fluorescent light, it emits light by excitation light that causes light emission. 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 light sensor for detecting a transmitted signal (such as a photomultiplier tube or a charge coupled device (CCD) configuration) and a mechanism for preventing excitation light from being included in the output of the light sensor (the -15-201209159 Wavelength selective filter). The fluorescent molecules emit Stokes shifted light to respond to the excitation light, which is then collected by the detection unit. The Stokes shift is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. The ECL emission system is detected using a light sensor that is sensitive to the emission wavelength of the ECL species used. For example, the transition metal-ligand complex emits light of a visible wavelength, so conventional light diodes 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 DN A hybridization experiments to be performed simultaneously. Microarrays are powerful molecular diagnostic tools that screen thousands of genetic diseases or detect the presence of numerous infectious pathogens in a single experiment. The microarray consists of a number of different DN A probes that are fixed at points on the substrate. The target DNA (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 heat-controlled, humid environment for hours or days to allow hybridization between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. After cleaning, the surface of the microarray is dried with a gas stream (usually nitrogen). The rigor of hybridization and cleaning is critical. Insufficient rigor may result in a highly non-specific combination. A rigorous pass may result in an inability to properly combine' resulting in reduced sensitivity. Hybridization is identified by detecting light emitted by the labeled amplicon of the hybrid with the complementary probe. -16- 201209159 'The fluorescence from the microarray is detected by a microarray scanner, which is usually a computer-controlled inverted scanning fluorescent conjugated focus microscope, which typically uses a laser to excite fluorescent dyes and A light sensor, such as a photomultiplier tube or CCD, detects the transmitted signal. The fluorescent molecules emit Stokes displaced light (as described above), which is collected by the detection unit. The emitted fluorescent light must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. In a microarray scanner, a conjugate focal configuration of a conjugated focal hole aperture mounted on the image side is typically used to eliminate out-of-focus information. This device allows only the light of the focused portion to be detected. Light from above and below the focal plane of the target cannot enter the detector, thus increasing the signal-to-noise ratio. The detected fluorescent photons are converted into electrical energy by 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, it is possible to simultaneously identify and analyze the target sequence of the hybrid probe. More information about the fluorescent probe can be found at: http:/ /www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/Molecul ar-Probes-The-Handbook/Technical-Notes-and-Product-

Highlights/Fluorescence-Resonance-Energy-Transfer- FRET. Html -17- 201209159 Point-of-care (ΡΟΙΝΤ-OF-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 testing. This is primarily because nucleic acid testing results in higher complexity and cost than tests that do not involve nucleic acid methods. The extensive use of molecular diagnostic testing in clinical settings is closely related to the development of instrumentation, which must significantly reduce costs, provide rapid (automatic) analysis from initial (sample processing) to final (resulting), and does not require The operation of a large human intervention. Point-of-care technology provides care in the physician's office, on the hospital bed side, or even in a consumer-focused home environment. This technology offers many advantages including: • Quick results for immediate treatment and improved care quality • Very small amount The sample test obtains the number of laboratories • Reduces clinical workload • Reduces laboratory workload and reduces office efficiency by reducing administrative work. • By reducing the number of hospital stays, outpatients can be diagnosed at the time of 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 by -18-201209159 is less active and the low-cost built-in cascaded diagnostic method steps in the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. Laboratory chip (LOC) devices are common in the form of microfluidic devices. 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 the VLSI (very large integrated circuit) lithographic technique of the semiconductor industry. However, controlling the flow of fluid through the L 0 C device, adding reagents, controlling reaction conditions, and the like requires a large external hydroelectric engineering device. The connection of L0C devices to these external devices greatly limits the molecular diagnostics of L0C devices for use in a laboratory environment. The cost of external instruments and their operational complexity are excluded. L0C-based molecular diagnostics are the choice 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. GDI010. 1 This aspect of the invention provides a wafer-on-lab (L0C) device for removing cell debris from a biological sample, the LOC device comprising: a dialysis zone having a large component channel, a small component channel, and a channel and a small component An aperture between the channels for fluid communication, the large component channel having an upstream end for receiving a biological sample, the biological sample being a liquid carrying a mixture of cell debris and a target molecule, the small component channel having -19- 201209159 is connected to the downstream end of the hybridization zone, which has a probe array for target molecule reaction to form a probe-target complex; wherein the pore size is such that the target molecule can flow into the small component channel 'But retain cell debris larger than the threshold 値 size in this large component channel. GDI0 1 0. 2 Preferably, the large component channel and the small component channel have a common side wall, and the plurality of small holes are a series of flow ports extending through the common side wall, each flow port having a small passage to the component channel The opening is thickened into a large opening leading to the small component passage. GDI010. Preferably, the LOC device also has a plurality of small component channels each having a common side wall with the plurality of channels and fluidly coupled to the bulk channel by a series of flow ports. GDI010. Preferably, the small openings of each of the flow openings have a height and a width dimension between 1 and 8 microns. GDI010. Preferably, the LOC device also has a waste storage tank, wherein the large component passage has a downstream end connected to the waste storage tank. GDIO10. Preferably, the LOC device also has a cleavage region upstream of the dialysis zone, wherein the target molecule is a target nucleic acid sequence and the cleavage region is configured to lyse cells in the biological sample and release the target therein Nucleic acid sequence. GDIO10. Preferably, the LOC device also has a nucleic acid amplification region for amplifying the target nucleic acid sequence. GDI010. Preferably, the probe is configured to hybridize to the target nucleic acid sequence to form a probe-target hybrid that produces fluorescence upon re-stress luminescence. -20- 201209159 GDIO 1 0. Preferably, the LOC device also has a CMOS circuit using a nucleic acid amplification region, the CMOS circuit also having a photosensor that emits fluorescence from the probe-target hybrid. GDIO 1 0. Preferably, the hybridizing region has an array of hybridization chambers comprising probes hybridized with a nucleic acid sequence. GDIO10. Il Preferably, the photosensor is an array of adjacent photodiodes. GDI010. Preferably, the CMOS circuit has digital memory for data associated with the processing of the fluid, details of the probe and the location of each probe in the array of hybridization chambers. GDIO 1 0.  Preferably, the CMOS circuit has a temperature sensor for sensing the temperature of the hybridization chamber array. GDI010. Preferably, the LOC device also has a circuit controlled heater that utilizes temperature feedback to maintain the probe and target nucleic acid sequence at hybridization temperature GDI010. Preferably, the photodiode is less than 249 microns from the pair of chambers. GDI010. Preferably, the probe is a fluorescent resonance energy FRET probe. GDI010. Preferably, the hybridization chamber has an optical optical window positioned to contact the FRET probe with excitation light. GDI010. Preferably, the FRET probes each have a cluster and a quencher configured to emit a fluorescent signal to the photodiode at the FRET probe-target hybrid for manipulation of the useful In the sense of sharing with the target hybrid chamber in the storage and data including a small amount of hybridization with the CMOS sensor (window, this one fluorescent needle formation should be stimulated - 21 - 201209159 luminescence, the CMOS The circuitry is configured to cause the photodiode to be activated after a predetermined delay time after the excitation light is extinguished, the digital inclusion comprising the pre-arranged delay. GDIO10. Preferably, the CMOS circuit has a bond pad for electrically connecting an external device and is configured to convert an output from the photodiode into a signal representative of the FRET probe that hybridizes to the target nucleic acid sequence and to signal the signal Provided to the bond pad for transmission to an external device. GDIO 10. Preferably, the LOC device also has a plurality of reservoirs for holding the liquid reagent for addition to the sample. The design of this LOC device has the advantage of directly selecting the sample components containing the target. The design of the LOC device has the advantage of enriching the effective target concentration in the portion of the sample to be further processed by the LOC device. The design of this LOC device has the advantage of removing components of the sample that may inhibit subsequent analysis steps. The design of the LOC device has the advantage of removing undesirable components of the treated mixture which may interfere with later target detection. The design of this LOC device has the advantage of removing components of the mixture that may block cells or joints in the LOC device and degrade operation. GDI01 1. 1 This aspect of the invention provides a wafer-on-lab (LOC) device for removing cell debris from a biological sample comprising a target nucleic acid sequence, the LOC device comprising: a dialysis zone having a large component channel, a small component a channel and a plurality of apertures for fluid communication between the large component channel and the small component channel, the large component channel having an upstream end for receiving the biological sample, the biological sample -22-201209159 a liquid of a mixture of cell debris and a target nucleic acid sequence and having a downstream end for ligating a hybridization region with a probe array hybridized to a target nucleic acid sequence to form a probe-target complex Wherein the pores are sized to allow the target nucleic acid sequence to flow into the small component channel, but retain cell fragments that are larger than the threshold size in the large component channel. GDI01 1. 2 Preferably, the large component channel and the small component channel have a common side wall, and the plurality of small holes are a series of flow ports extending through the common side wall, and each flow port has a passage to the large component channel. Small openings and tapering into large openings leading to small component channels. GDI01 1. Preferably, the L 0 C device also has a plurality of small component channels each having a common side wall with the plurality of channel channels and fluidly coupled to the bulk channel via a series of flow ports. GDI01 1. Preferably, the small openings of the respective flow openings have a height and width dimension between 1 and 8 microns. GDI011. Preferably, the LOC unit also has a waste storage tank' wherein the large component passage has a downstream end connected to the waste storage tank. GDI011. Preferably, the LOC device also has a cleavage region upstream of the amplification region, wherein the cleavage region is configured to lyse cells in the biological sample and release the target nucleic acid sequence therein. GDI011. Preferably, the LOC device also has a nucleic acid amplification region for amplifying the target nucleic acid sequence. GDI011. Preferably, the LOC device also has a CMOS circuit for manipulating the nucleic acid amplification region, the CMOS circuit also having light for sensing the fluorescence emitted from the probe-target hybrid by Sense -23-201209159 Sensor. GDI011. Preferably, the hybridizing region has an array of hybridization chambers comprising probes for hybridization to the target nucleic acid sequence. GDI01 1. Preferably, the photosensor is an array of photodiodes adjacent to each of the hybridization chambers. GDI011. Il. Preferably, the CMOS circuit has a digital register for storing data associated with the processing of the fluid, the data including details of the probe and the location of each probe in the array of hybrid chambers. GD 1011. Preferably, the CMOS circuit has at least one temperature sensor for sensing the temperature of the array of hybridization chambers. GDI011. Preferably, the LOC device also has a heater controlled by the CMOS circuit that utilizes feedback from the temperature sensor to maintain the probe and target nucleic acid sequences at the hybridization temperature. GDI01 1. Preferably, the photodiode is less than 249 microns from the corresponding hybridization chamber. GDIO 11. Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GDI011. Preferably, the hybridization chamber has an optical window positioned such that the FRET probe contacts the excitation light. GDI011. Preferably, the FRET probes each have a fluorophore and a quencher configured to emit a fluorescent signal to the photodiode when the FRET probe forms a probe-target hybrid. The CMOS circuit is configured to cause the photodiode to be activated after a predetermined delay time after the excitation light is extinguished, the digital body including the delay of the pre--24-201209159 arrangement. GDI01 1. 18. Preferably, the CMOS circuit has a bond pad for electrically connecting an external device and is configured to convert an output from the photodiode into a signal representative of a FRET probe that hybridizes to the target nucleic acid sequence, and the signal is Provided to the bond pad for transmission to an external device. GDI01 1. Preferably, the LOC device also has a plurality of reservoirs for holding the liquid reagent for addition to the sample. GDI011. Preferably, the reagent reservoirs each have a surface tension valve having a small aperture configured to hold a meniscus in which the liquid reagent is held until removed from contact with the sample. The meniscus and the liquid reagent are added to the sample stream. The design of this L0C device has the advantage of removing sample components that may inhibit later analysis steps. The design of the LOC device has the advantage of removing unwanted components of the treated mixture which may interfere with later target detection. The design of this LOC device has the advantage of removing components of the mixture that may block the cells or joints in the LOC device and degrade the operation. GDI013. 1 This aspect of the invention provides a wafer-on-a-lab (LOC) device for removing cell debris from a biological sample comprising a target nucleic acid sequence, the LOC device comprising: an amplification region for amplifying the nucleic acid a dialysis zone having a large component channel, a small component channel, and a plurality of small holes for fluid communication between the large component channel and the small component channel, the large component channel being configured to After amplifying the nucleic acid in the amplification region, the biological sample is used to receive the liquid sample of the cell debris and the target nucleic acid sequence, and the size of the small hole allows the target nucleic acid sequence to flow into the small component channel. However, cell debris that is larger than the threshold 値 size in the large component channel is retained; and a probe array that is in fluid communication with the small component channel to hybridize with the target nucleic acid sequence to form a probe-target hybrid. GDIO 1 3. 2 Preferably, the large component channel and the small component channel have a common side wall, and the plurality of small holes are a series of flow ports extending through the common side wall, and each flow port has a passage to the large component channel. Small openings and tapering into large openings leading to small component channels. GDIO 1 3. Preferably, the dialysis zone has a plurality of small component channels each sharing a common side wall with the plurality of channels and fluidly coupled to the bulk channel via a series of flow ports. GDI01 3. Preferably, the small openings of the respective flow openings have a height and width dimension between 1 and 8 microns. GDI013. Preferably, the LOC device also has a waste storage tank, wherein the large component passage has a downstream end connected to the waste storage tank. GDI013. Preferably, the LOC device also has a cleavage region upstream of the amplification region, wherein the cleavage region is configured to lyse cells in the biological sample and release the target nucleic acid sequence therein. GDI013. Preferably, the probe-target hybrid produces fluorescence upon response to excitation light. GDI013. Preferably, the LOC device also has a CMOS circuit for manipulating the nucleic acid amplification region, the CMOS circuit also having a photosensor for sensing the fluorescence emitted from the probe-target hybrid by 201209159 . GDI013. Preferably, the hybridizing region has an array of hybridization chambers comprising probes for hybridization to the target nucleic acid sequence. GDIO 1 3 · 1 0 Preferably, the photosensor is an array of photodiodes adjacent to each of the hybridization chambers. GDI013. Il. Preferably, the CMOS circuit has digital memory for storing data associated with the processing of the fluid, the data including details of the probe and the location of each probe in the array of hybrid chambers. GDI013. Preferably, the CMOS circuit has at least one temperature sensor for sensing the temperature of the array of hybridization chambers. GDI013. Preferably, the LOC device also has a heater controlled by the CMOS circuit that utilizes feedback from the temperature sensor to maintain the probe and target nucleic acid sequences at the hybridization temperature. GDI013. Preferably, each photodiode is less than 249 microns from the corresponding hybridization chamber. GDI013. Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GDI013. Preferably, the hybridization chamber has an optical window positioned such that the FRET probe contacts the excitation light. GDI013. Preferably, the FRET probes each have a fluorophore and a quencher configured to emit a fluorescent signal to the photodiode when the FRET probe forms a probe-target hybrid. The CMOS circuit is configured to cause the photodiode to be activated after a predetermined delay time after the excitation light is extinguished, the digital memory including the predetermined delay of -27-201209159. GDIO 1 3. Preferably, the CMOS circuit is coupled to a bond pad of the external device and is configured to exchange FRET from the light to indicate hybridization with the target nucleic acid sequence to provide the signal to the bond pad for transmission to the external device GDIO 1 .  Preferably, the LOC device maintains a liquid reagent for addition to a reservoir in the sample. GDIO13. Preferably, the reagent reservoir is a port valve; wherein the outlet valve is a surface tension valve each having a system configured to retain a liquid reagent therein to remove the meniscus after contact with the sample and to Liquid Test The design of this LOC device has the advantage of directly selecting the package components. The design of this LOC device has the advantage of shifting the undesired components of the present, which is not desired for subsequent target detection. The design of this LOC device has the advantage of being able to inhibit the components of later analysis steps. This metering has the undesired component of removing the undesired component of the processed sample that may interfere with later target detection. The design has the advantage of removing the components of the mixture that may block the L Ο C joint and degrade the operation. GPC027. 1 This aspect of the invention provides a wafer-on-lab (a signal having an output transducing probe for an electrical diode) comprising a sample of a target nucleic acid sequence, and having a plurality of signals for having a corresponding one An orifice, the meniscus of the orifice, until the agent is added to the sample containing the target in the sample stream, except that the treated component may interfere with the advantage of removing the components of the LOC device in the sample. a chamber in the device of the L〇C device or a device for genetic analysis package L0C), the 201209159 L〇C device comprising: a sample inlet for receiving the sample; a plurality of reagent storage tanks for inclusion in the sample Buffer, dNTP, primer, and polymerase to form an amplification mixture; and a nucleic acid amplification region for maintaining the amplification mixture at a predetermined temperature during isothermal amplification of the target nucleic acid sequence. GPC027. Preferably, the nucleic acid amplification region has a plurality of elongated amplification chambers, each chamber having at least one elongated heater extending parallel to the longitudinal extent of the amplification chamber. GPC027. Preferably, the nucleic acid amplification region has a microchannel and the extended amplification chamber is the region of the microchannel. GPC027. Preferably, the microchannel has a curved configuration formed by a series of wide meandering streams, each broad stream being a channel region forming one of the elongated amplification chambers. GPC027. Preferably, the LOC device also has a CMOS circuit coupled to the at least one heater to operate the at least one heater during constant temperature amplification. GPC027. Preferably, a plurality of elongated heaters are provided along each of the wide channel of each meandering stream. GPC027. Preferably, the plurality of elongated heaters are placed end to end along the channel region. GPC027. 8 Preferably, the extension heater operates independently. GPC027. Preferably, the LOC device also has at least one temperature sensor coupled to the CMOS circuit for feedback control of the extended heater. -29- 201209159 GPCO27. Preferably, the nucleic acid amplification region has an active valve for retaining liquid in the nucleic acid amplification region during constant temperature amplification. GPC027. Il preferably, the active valve is a boiling start valve having a meniscus anchor for retaining the liquid in the nucleic acid amplification zone, the boiling start valve also having a valve heater for heating the liquid to boil, Thereby, the meniscus is separated from the meniscus anchor and the liquid flow driven by capillary action is resumed from the nucleic acid amplification zone. GPC027. Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect liquid in the liquid sensor position to feedback control the valve heater. GPC027. Preferably, the meniscus anchor is a small hole and the valve heater is positioned around the aperture. GPC027. Preferably, the LOC device also has a probe array for hybridization to the target nucleic acid sequence to form a probe-target hybrid. GPC027. Preferably, the LOC device also has a photosensor for detecting the probe-target hybrid. GPC027. Preferably, the photosensor is an optical diode that is associated with the array of probes. GPC027. Preferably, the volume of the liquid in the nucleic acid amplification zone is less than 400 nanoliters. GPC027. Preferably, the PCR microchannel has a cross-sectional area transverse to the flow direction of from 1 square micron to 400 square micrometers. GPC027. Preferably, the LOC device also has a dialysis zone upstream of the nucleic acid amplification zone, wherein the sample comprises components of different sizes, and the -30-201209159 zone is configured to set a component less than a predetermined threshold More than a predetermined valve component is separated. GPCO27. Preferably, the nucleic acid sequence is contained in cells and organisms having a size less than a threshold size. This allows for more sensitive and specific detection of target DNA. This LOC has the advantage of not requiring thermal cycling, which simplifies thermal control electronics, allows for uniform thermal control and reduces material degradation in the LOC device. The design of this device will reduce the degree of evaporation during operation and improve the control of the physical and chemical conditions in the device. GPC028. 1 This aspect of the invention provides a wafer-on-lab (LOC) device for a sample containing a target nucleic acid sequence. The LOC device comprises: a sample inlet for receiving the sample; a plurality of reagent storage tanks for inclusion a dNTP, a recombinant enzyme, a DNA polymerase, and a buffer in the sample to form an amplification mix; and a nucleic acid amplification region for use in the recombinant enzyme synthase amplification (RP A ) of the target nucleic acid sequence The amplification mixture is kept under predetermined conditions. GPC028. Preferably, the nucleic acid amplification region has a plurality of extension chambers, each chamber having at least one elongated heater parallel to the longitudinal region of the amplification chamber. GPC028. Preferably, the nucleic acid amplification region has a microchannel or the like to extend the amplification chamber to each region of the microchannel. The pre-device of the 许 LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO LO Preferably, the microchannel has a curved configuration formed by a series of wide meandering flows, each of which is a channel region forming one of the elongated expansion chambers. GPC028. Preferably, the LOC device also has a CMOS circuit coupled to the at least one heater to operate the at least one heater during the RPA. GPC028. Preferably, a plurality of elongated heaters are provided along each of the wide channel of each meandering stream. GPC028. Preferably, the plurality of elongated heaters are placed end to end along the channel region. GPC028. 8 Preferably, the extension heater operates independently. GPC028. Preferably, the LOC device also has at least one temperature sensor coupled to the CMOS circuit for feedback control of the extended heater. GPCO28. Preferably, the nucleic acid amplification region has an active valve for retaining liquid in the nucleic acid amplification region during constant temperature amplification. GPC028. Il preferably, the active valve is a boiling start valve having a meniscus anchor for retaining the liquid in the nucleic acid amplification zone, the boiling start valve also having a valve heater for heating the liquid to boil, Thereby, the meniscus is separated from the meniscus anchor and the liquid flow driven by capillary action is resumed from the nucleic acid amplification zone. GPC028. Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect liquid in the liquid sensor position to feedback control the valve heater. GPC028. Preferably, the meniscus anchor is a small hole and the valve 201209159 door heater is positioned around the aperture. GPC028. Preferably, the LOC device also has a probe array for hybridizing to the target nucleic acid sequence to form a probe-target hybrid. GPC028. Preferably, the LOC device also has a photosensor for detecting the probe-target hybrid. GPC028. Preferably, the photosensor is an optical diode that is associated with the array of probes. GPC028. Preferably, the volume of the liquid in the nucleic acid amplification zone is less than 400 nanoliters. GPC028. Preferably, the PCR microchannel has a cross-sectional area transverse to the flow direction of from 1 square micron to 400 square micrometers. GPC028. Preferably, the LOC device also has a dialysis zone upstream of the nucleic acid amplification zone, wherein the sample comprises components of different sizes, the dialysis zone being configured to set a component less than a predetermined threshold and greater than a predetermined valve The components are separated. GPCO2 8. Preferably, the nucleic acid sequence is contained in cells and organisms that are less than the predetermined threshold. This LOC device has the advantage of not requiring thermal cycling, which simplifies thermal control electronics, allows for more uniform thermal control and reduces material degradation in the LOC device. The design of this LOC device will reduce the extent of evaporation during operation and improve control of the physical and chemical conditions in the LOC device. This LOC device has the advantage of being provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for target DNA sequences. Recombinant enzyme polymerase amplification has the added benefit of amplifying from a single copy of the target -33-201209159 nucleic acid to a detectable level within 15 minutes. This LOC device has the advantage of being less complex in design and manufacture, which will result in a simpler, more reliable manufacturing process. This LOC device has the advantage of using fewer chemical steps during operation, which results in a simpler, more reliable operation. GPC029. 1 This aspect of the invention provides a wafer on-lab (LOC) device for genetically analyzing a sample comprising a target nucleic acid sequence, the LOC device comprising: a sample inlet for receiving the sample; a plurality of reagent reservoirs comprising a dNTP, a primer, a nicking enzyme, a buffer, and a DNA polymerase added to the sample to form an amplification mixture; and a nucleic acid amplification region for maintaining the amplification mixture during constant temperature amplification of the target nucleic acid sequence At a predetermined temperature. GPC029. Preferably, the nucleic acid amplification region has a plurality of elongated amplification chambers, each chamber having at least one elongated heater extending parallel to the longitudinal extent of the amplification chamber. GPC029. Preferably, the nucleic acid amplification region has a microchannel and the extended amplification chamber is a region of the microchannel. GPC029. Preferably, the microchannel has a curved configuration formed by a series of wide meandering flows, each of which is a channel region forming one of the elongated expansion chambers. GPC029. Preferably, the LOC device also has a CMOS 201209159 circuit coupled to the at least one heater for manipulating the sample during constant temperature amplification. GPC029. Preferably, a plurality of elongated heaters are provided along each of the wide channel of each meandering stream. GPC029. Preferably, the plurality of elongated heaters are placed end to end along the channel region. GPC029. 8 Preferably, the extension heater operates independently. GPC029. Preferably, the LOC device also has at least one temperature sensor coupled to the CMOS circuit for feedback control of the extended heater. GPCO29. Preferably, the nucleic acid amplification region has an active valve for retaining liquid in the nucleic acid amplification region during constant temperature amplification. GPC029. Il preferably, the active valve is a boiling start valve having a meniscus anchor for retaining the liquid in the nucleic acid amplification zone, the boiling start valve also having a valve heater for heating the liquid to boil, The meniscus is removed from the meniscus anchor and the flow driven by capillary action is resumed from the nucleic acid amplification zone. GPC029. 1 2 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect liquid in the liquid sensor position to feedback control the valve heater. GPC029. Preferably, the meniscus anchor is a small hole and the valve heater is positioned around the aperture. GPC029. Preferably, the LOC device also has a probe array for hybridization to the target nucleic acid sequence to form a probe-target hybrid. GPC029. Preferably, the LOC device also has a photosensor for detecting the probe-target hybrid. -35- 201209159 GPC029. Preferably, the photosensor is an optical diode that is associated with the array of probes. GPC029. Preferably, the volume of the liquid in the nucleic acid amplification zone is less than 400 nanoliters. GPC029. Preferably, the PCR microchannel has a cross-sectional area transverse to the flow direction of from 1 square micron to 400 square micrometers. GPC029. Preferably, the LOC device also has a dialysis zone upstream of the nucleic acid amplification zone, wherein the sample comprises components of different sizes, the dialysis zone being configured to use a component less than a predetermined threshold 与 and a predetermined valve The components of the cockroach are separated. GPCO29. Preferably, the nucleic acid sequence is contained in cells and organisms that are smaller than the predetermined threshold size. This LOC device has the advantage of not requiring thermal cycling, which simplifies thermal control electronics, allows for more uniform thermal control and reduces material degradation in the L0C device. The design of this LOC device will reduce the extent of evaporation during operation and improve control of the physical and chemical conditions in the LOC device. This LOC device has the advantage of being provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for target DNA sequences. GPCO30. 1 This aspect of the invention provides a wafer on-lab (LOC) device for genetically analyzing a sample comprising a target nucleic acid sequence, the LOC device comprising: a sample inlet for receiving the sample; a plurality of reagent reservoirs comprising Adding dNTPs into the sample, introducing 201209159, reverse transcriptase, RNA polymerase, and buffer to form an amplification mixture: and a nucleic acid amplification region for use in the constant amplification of the target nucleic acid sequence The addition mixture is maintained at a predetermined temperature. GPCO30. Preferably, the nucleic acid amplification region has a plurality of elongated amplification chambers, each chamber having at least one elongated heater extending parallel to the longitudinal extent of the amplification chamber. GPCO30. Preferably, the nucleic acid amplification region has a microchannel and the extended amplification chamber is the region of the microchannel. GPCO30. Preferably, the microchannel has a curved configuration formed by a series of wide meandering streams, each broad stream being a channel region forming one of the elongated amplification chambers. GPCO30. Preferably, the LOC device also has a CMOS circuit that operates the at least one heater connection during constant temperature amplification. GPCO30. Preferably, a plurality of elongated heaters are provided along each of the wide channel of each meandering stream. GPCO30. Preferably, the plurality of elongated heaters are placed end to end along the channel region. GPCO30. 8 Preferably, the extended heater is operated independently GPCO30. Preferably, the LOC device also has at least one temperature sensor coupled to the CMOS circuit for feedback control of the extended heater. GPC030. Preferably, the nucleic acid amplification region has an active valve for retaining liquid in the nucleic acid amplification region during constant temperature amplification. GPCO30. Il preferably, the active valve is a boiling start valve having a meniscus anchor for retaining the liquid in the nucleic acid amplification zone from -37 to 201209159, the boiling start valve also having a boiling boiling liquid for heating The valve heater is such that the meniscus is disengaged from the meniscus anchor and the flow driven by the capillary action is resumed from the nucleic acid amplification zone. GPCO30. Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect liquid in the liquid sensor position to feedback control the valve heater. GPCO30. Preferably, the meniscus anchor is a small aperture and the valve heater is positioned around the aperture. GPCO30. Preferably, the LOC device also has a probe array for hybridization to the target nucleic acid sequence to form a probe-target hybrid. GPCO30. Preferably, the LOC device also has a photosensor for detecting the probe-target hybrid. GPCO30. Preferably, the photo sensor is an optical diode that is associated with the probe array. - GPCO30. Preferably, the volume of the liquid in the nucleic acid amplification zone is less than 400 nanoliters. GPCO30. Preferably, the PCR microchannel has a cross-sectional area transverse to the flow direction of from 1 square micron to 40 square micrometers. GPCO30. Preferably, the LOC device also has a dialysis zone upstream of the nucleic acid amplification zone, wherein the sample comprises components of different sizes, the dialysis zone being configured to use a component less than a predetermined threshold 与 greater than a predetermined valve 値The components are separated. GPC030. Preferably, the nucleic acid sequence is contained in cells and organisms that are smaller than the predetermined size of the 201209159. This LOC device has the advantage of not requiring thermal cycling, which simplifies thermal control electronics, allows for more uniform thermal control and reduces material degradation in the LOC device. The design of this LOC device will reduce the extent of evaporation during operation and improve control of the physical and chemical conditions in the LOC device. This LOC device has the advantage of being provided by sequence-specific amplification, including: sensitivity provided by amplification; extensive dynamic range; and high specificity for target RNA sequences. I GPC031. 1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetically analyzing a nucleic acid sequence in a sample, the LOC device comprising: a sample inlet for receiving the sample; a first polymerase reaction (PCR) region, Is for thermally cycling a first PCR mixture consisting of dNTPs, primers and buffers with a sample and a polymerase to amplify the nucleic acid sequence; and a second PCR region located downstream of the first PCR region for use A second PCR mixture consisting of dNTPs, primers and buffers with a polymerase and at least some of the amplicons from the first PCR region is thermally cycled. GPC031. Preferably, the first PCR region has an active valve for retaining liquid in the first PCR region during amplification of the nucleic acid sequence in the sample. GPC03 1. Preferably, the active valve is a boiling start valve having a meniscus anchor for retaining the liquid in the first PCR zone, the boiling start valve also having a valve heater for heating the liquid to boil From -39 to 201209159, the meniscus is disengaged from the meniscus anchor and the flow driven by capillary action is resumed from the first PCR zone. GPC031. Preferably, the LOC device also has a second PCR mixture reservoir with an outlet valve and a second polymerase reservoir with an outlet valve for adding the PCR mixture and polymerase prior to amplification in the second PCR zone. In the amplicon from the first PCR region. GPC031. Preferably, the outlet valve is a surface tension valve, each of the surface tension valves having an aperture configured to hold a meniscus capable of holding a liquid reagent therein until after contact with the amplicon The meniscus is removed and the PCR mixture and polymerase are added to the amplicon flowing into the second PCR zone. GPC031. Preferably, the second PCR region has an active valve to retain the liquid in the second PCR region when the nucleic acid sequence in the amplicon from the first PCR region is amplified. GPC031. Preferably, the active valve is a boiling start valve having a meniscus anchor for retaining the liquid in the second PCR zone, the boiling start valve also having a valve heater for heating the liquid to boil, Thereby the meniscus is disengaged from the meniscus anchor and the flow driven by capillary action is resumed from the second PCR zone. GPC031. Preferably, the LOC device also has a hybridization region having a probe array for hybridization to a target nucleic acid sequence in the second PCR region amplicon. GPC03 1 . Preferably, the hybridization zone has a photosensor for detecting hybridization of the probes in the array of probes. -40- 201209159 GPCO31. Preferably, the LOC device also has a dialysis zone upstream of the second PCR and hybridization region, wherein the second PCR region comprises a nucleic acid sequence and a cell fragment, such that the dialysis zone is configured to be larger than the nucleic acid sequence. Cell debris. GPC031. Il preferably, the first PCR zone has a PCR microchannel for increasing the sample during the incubation period and at least one extension heater for thermally cycling the nucleic acid sequence therein, the extended heating being aligned with the PCR microchannel a row. GPC031. Preferably, the PCR mixture has a volume of K liters. GPC031. Preferably, the cross-sectional area of the PCR microchannel and the flow transverse direction is from 1 square micron to 400 square micrometers. GPC031. Preferably, the PCR microchannel has an inlet and a PCR outlet, and the PCR microchannel has at least one extended PCR chamber. GPC031. Preferably, the first PCR zone has a plurality of PCR chambers each formed by a respective region of a PCR microchannel having a curved configuration formed by a series of wide meandering flows, each broad meandering being shaped The channel area of an extended PCR chamber. GPC031. 16 Preferably, each channel zone has a plurality of extended twists GPC03 1. Preferably, the plurality of elongated heaters are placed along the end of the zone. GPC03 1. Preferably, the plurality of extended heaters are each expanded under the respective zones to expand in the sampler system 400 to form an extended channel into the heat exchanger channel independent of -41 - 201209159 operation. GPC03 1. Preferably, the LOC device also has at least one temperature sensor for feedback control of the extended heater. GPCO31. Preferably, the first PCR zone has a thermal cycle time of less than 30 seconds. This L0C device has the advantage of being provided by sequence-specific amplification, including: sensitivity provided by amplification; extensive dynamic range; and high specificity for target DNA sequences. Two-stage amplification allows the protocol to be further refined for highly sensitive detection, which is highly tolerant of similar genetic material and chemical contaminants that may interfere with a single-stage amplification protocol. The product from the first amplification stage can also be divided again to perform multiple parallel reactions in the second expansion stage, which can increase the multiplex capacity of the LOC unit. The L 0 C device with parallel reaction sites is designed to perform diagnostic tests in parallel on very small sample volumes, which increases the range and quality of the diagnostic data obtained. GPC033. 1 This aspect of the invention provides a wafer-on-a-lab (L0C) device for genetically analyzing a sample comprising a target nucleic acid sequence. The LOC device comprises: a sample inlet for receiving the sample; a plurality of reagent reservoirs for inclusion a dNTP, a primer, a polymerase, and a buffer into the sample; and a nucleic acid amplification region for thermal control of the sample for performing the first stage amplification of the target nucleic acid sequence, and for subsequent control from the first stage The temperature of the amplified amplicon is subjected to a second stage of amplification to further amplify the -42-201209159 target nucleic acid sequence. GPC033. Preferably, the nucleic acid amplification region is configured to perform whole genome amplification during the one-stage amplification and to amplify the predetermined nucleic acid sequence during the second increase. GPC033. Preferably, the nucleic acid amplification region is subjected to polymerase chain reaction (PCR) amplification and the thermal control comprises circulating the sample. GPC033. Preferably, the nucleic acid amplification region is configured for constant temperature whole genome amplification and the thermal control comprises maintaining the sample at a predetermined level. GPC033. 5 . Preferably, the nucleic acid amplification region has a first nucleic acid amplification region for one-stage amplification, and a second nucleic acid amplification region located downstream of the first nuclear region to perform the first-stage amplification. GPC033. Preferably, the first nucleic acid amplification region is amplified by a first predetermined nucleic acid sequence and the second nucleic acid amplification region is complemented by a second predetermined nucleic acid sequence, the first predetermined nucleic acid sequence being the nucleic acid sequence The son part. GPC033. Preferably, the reagent storage tank comprises a first test and a second reagent storage tank, the first reagent storage tank containing a first dNTP, a damp enzyme and a buffer for being added to the sample before amplification in the first addition zone And the second reagent storage tank contains a second dNTP, a primer, a polymerase, and a buffer added from the first nucleic acid amplification region before amplification in the first amplification region. GPC03 3. Preferably, the reagent storage tanks each have a first stage expansion for the heat for performing the acid acid amplification configuration at a predetermined temperature to expand the predetermined reagent storage tank nucleic acid extension, polydinucleotide A surface tension valve with a small -43-201209159 orifice is configured that is configured to hold the meniscus in which the liquid reagent remains, until the meniscus is removed by contact with the sample. GPC033. Preferably, the first nucleic acid amplification region has a plurality of elongated amplification chambers, each chamber having at least one elongated heater parallel to the longitudinal extension of the amplification chamber. GPCO33. Preferably, the nucleic acid amplification region has microchannels and the extended amplification chambers are regions of the microchannel. GPC033. Il Preferably, the microchannel has a curved configuration formed by a series of wide meandering flows, each of which is a channel region forming one of the elongated expansion chambers. GPC033. Preferably, the LOC device also has a CMOS circuit coupled to the at least one heater for controlling the temperature of the sample during amplification. GPC033. Preferably, the LOC device also has at least one temperature sensor coupled to the C Μ 0 S circuit for feedback control of the at least one heater. GPC03 3 .  Preferably, the LOC device also has a probe array for hybridization to the target nucleic acid sequence to form a probe-target hybrid. GPC033. Preferably, the LOC device also has a photosensor for detecting the probe-target hybrid. GPC03 3 .  Preferably, the photosensor is an optical diode that is associated with the array of probes. GPC033. Preferably, the volume of the liquid in the first nucleic acid amplification zone is less than 400 nanoliters. 201209159 GPC033. Preferably, the PCR microchannel has a cross-sectional area transverse to the flow direction of from 1 square micron to 40 square micrometers. GPC033. Preferably, each channel region has a plurality of heaters, each heater being extended and placed end to end along the channel region, and the CMOS circuit is configured for independent operation of the plurality of extension heaters Each heater. GPCO3 3. Preferably, the LOC device also has a supporting substrate, wherein the CMOS circuit is located between the probe array and the supporting substrate, and the L Ο C device has the advantages provided by sequence specific amplification, including: Increased sensitivity; broad dynamic range; and high specificity for target DNA sequences. Two-stage amplification allows the protocol to be further refined for highly sensitive detection, which is highly tolerant of similar genetic material and chemical contaminants that may interfere with a single-stage amplification protocol. The product from the first amplification stage can also be divided again to perform multiple parallel reactions in the second amplification stage, which can increase the multiplex capacity of the LOC device. LOC devices with parallel reaction sites are designed to perform diagnostic tests in parallel on very small sample volumes, which increases the range and quality of diagnostic data obtained. Non-specific nucleic acid amplification prior to specificity detection or additional amplification allows for greater sensitivity to rare targets. Non-specific amplification prior to more specific steps also increases the versatility of the LOC device platform by reducing the amount of additional bursts required to add new tests to existing diagnostic panels. GPC034. 1 This aspect of the invention provides a wafer-on-lab (L0C) device for genetically analyzing a sample comprising a target nucleic acid sequence of -45-201209159. The LOC device comprises: a sample inlet for receiving the sample; a plurality of reagent storage tanks, a dNTP, a primer, a polymerase, and a buffer for addition to a sample; a first nucleic acid amplification region' for thermal control of the sample to amplify the target nucleic acid sequence; and a second nucleic acid amplification region, It is used to control the temperature of the amplicon from the first nucleic acid amplification region to further amplify the target nucleic acid sequence. GPC034. Preferably, the first nucleic acid amplification region is configured for whole genome amplification and the second nucleic acid amplification region is configured to amplify a predetermined nucleic acid sequence. GPC034. Preferably, the first nucleic acid amplification region is configured for polymerase chain reaction (PCR) amplification and the thermal control comprises thermal cycling of the sample. GPC034. Preferably, the first nucleic acid amplification region is configured for constant temperature whole genome amplification and the thermal control comprises maintaining the sample at a predetermined temperature. GPC034. Preferably, the second nucleic acid amplification region is a PCR nucleic acid amplification region configured to amplify a predetermined nucleic acid sequence. GPC034. Preferably, the first nucleic acid amplification region is configured to amplify a first predetermined nucleic acid sequence and the second nucleic acid amplification region is configured to amplify a second predetermined nucleic acid sequence, the first predetermined nucleic acid sequence being a sub-portion of the second predetermined nucleic acid sequence. -46 - 201209159 GPC034. Preferably, the reagent storage tank comprises a first reagent storage tank and a second reagent storage tank, the first reagent storage tank containing a first dNTP, an introduction, which is added to the sample before amplification in the first nucleic acid amplification region a polymerase and a buffer, and the second reagent storage tank contains a second dNTP, an introduction molecule added to the amplicon from the first nucleic acid amplification region before amplification in the second nucleic acid amplification region , polymerase and buffer. GPC034. Preferably, the reagent reservoirs each have a surface tension valve with an orifice configured to secure a meniscus in which the liquid reagent remains, until the meniscus is removed by contact with the sample. GPC034. Preferably, the first nucleic acid amplification region has a plurality of elongated amplification chambers, each chamber having at least one elongated heater parallel to the longitudinal extension of the amplification chamber. GPC034. Preferably, the nucleic acid amplification region has microchannels and the extended amplification chambers are regions of the microchannel. GPC034. Il Preferably, the microchannel has a curved configuration formed by a series of wide meandering flows, each of which is a channel region forming one of the elongated expansion chambers.

GPC034.12 Preferably, the LOC device also has a CMOS circuit coupled to the at least one heater that controls the temperature of the sample during amplification. GPC034.13 Preferably, the LOC device also has at least one temperature sensor coupled to the C Μ 0 S circuit for feedback control of the at least one heater. GPC034.14 Preferably, the L〇C device also has a probe array for hybridization to the -47-201209159 target nucleic acid sequence to form a probe-target hybrid. GPC034.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrid. GPC034.1 6 Preferably the photosensor is an optical diode that is associated with the probe array. GPC034.1 7 Preferably, the volume of the liquid in the first nucleic acid amplification zone is less than 400 nanoliters. GPC034.1 8 preferably has a cross-sectional area of the PCR microchannel that is transversely tangential to the flow direction from 1 square micron to 400 square micrometers. GPC034.1 9 preferably, each of the channel regions has a plurality of heaters, each heater being extended and placed end to end along the channel region, and the CMOS circuit is configured to operate the plurality of extensions independently Each heater in the heater. GPCO3 4.20 Preferably, the LOC device also has a support substrate, wherein the CMOS circuit is located between the probe array and the support substrate, and the LOC device has the advantages provided by sequence-specific amplification, including: amplification Sensitivity provided; extensive dynamic range; and high specificity for target DNA sequences. Two-stage amplification allows the protocol to be further refined for highly sensitive detection. This highly sensitive detection can tolerate similar genetic material and chemical contaminants that may interfere with a single-stage amplification protocol. The product from the first amplification stage can also be divided again to perform multiple parallel reactions in the second amplification stage. This can increase the multiplex capacity of the LOC device. Performing two amplification stages on separate LO C device modules has the advantage of assisting in the addition of additional reagents between the 201209159 amplification stages. This optimizes the sensitivity and specificity chemistry of the LOC device results. The LOC device is designed with parallel reaction sites so that diagnostic tests can be performed in parallel on very small sample volumes, which increases the range and quality of diagnostic data obtained. Non-specific nucleic acid amplification prior to specificity detection or additional amplification allows for greater sensitivity to rare targets. Non-specific amplification prior to more specific steps also increases the versatility of the LOC device platform by reducing the amount of additional bursts required to add new tests to existing diagnostic panels. [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED SYSTEM STATEMENT This 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, 112 and 113, the system has the following top-level components: Test modules 10 and 1 have typical USB memory The size is very low in manufacturing. Test modules 1 and 1 each contain a microfluidic device 'typically in the form of a pre-packaged on-wafer laboratory (L〇c) device 30 and typically more than one for molecular diagnostic detection Probe (see Figures 1 and 1 12). The test module 1 shown in Figure i is a fluorescent-based detection technique for identifying target molecules, and -49-201209159 is shown graphically in the test in Figure 1 1 2 Module 1 1 uses electrochemiluminescence-based detection technology. The LOC device 30 has a photosensor 44 (described in detail below) for detecting the integration of fluorescence or electrochemiluminescence. Both test modules 1 and 使用 use standard Micro-USB plugs 14 for power, data and control, both of which have printed circuit boards (PCBs) 57 and both have external power supply capacitors 3 2 and Inductor 15. Both test modules 1 and 1 are used for large-scale production and sale of ready-to-use aseptic 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 40 8 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 is 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 12 shown in Figures 1, 3 and 112 is in the form of a smart phone system. A block diagram of this reader 12 is shown in Figure 3. The processor 42 runs application software from the program storage 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 epidemiological database -50- 201209159 location data. Alternatively, the location data can be manually entered via 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. The data storage 27 and the program storage 43 can 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 (or test module 11) 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 LED 26 stored in the housing 13 provides the necessary excitation light to induce fluorescence. Emission from hybridized probes (see Figures 1 and 2). In the test module 11 (which uses electrochemiluminescence (ECL) detection), the LOC device 30 is loaded with an ECL probe (as described above), and the LED 26 is not necessary to generate a fluorescent emission. Conversely, electrodes 8 60 and 8 70 provide an excitation current (see Figure 1 13). 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 a test module reader 12. The reader then displays the results. -51 - 201209159 These 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 3, 112 and 112 show a test module reader 12 configured as a mobile/smartphone 28 format. 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 for use in a hospital, private clinic or laboratory. 〇 5 (see picture 1 14). 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. 1 15 , the data generated by the test module 10 can update the epidemiological data managed on the epidemiological data host system 11 1 via the reader 1 2 and the network 1 2 5 in the genetic data. Genetic data managed on the host system 1 1 3, electronic health records managed on an electronic health record (EHR) host system Π 5, electronic medical records managed on an electronic medical record (EMR) host system 121, and in personal health Record (PHR) personal health records managed on the host system 1 2 3 . Conversely, epidemiological data managed on the epidemiological data host system 111, genetic data managed on the genetic data host system 113, and electronic health managed on the electronic health record (ERR) host system 115 Recorded, electronic medical records managed on an electronic medical record (EMR) host system 1 21 and personal health records managed on a personal health record (PHR) host system 1 23 may be via network 1 2 5 and reader 1 2 Update the digital memory in test module 1 l l 0 C 3 0 201209159. Referring back to Figures 1, 2, 112 and 113, 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. The Micro-USB埠16 can be used to connect to a computer or mains to charge the battery. Figure 7 shows a system of test modules 10 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 Η1N1 influenza A virus infection. It is sufficient to simply set the U SB power/indicator-definition module 4 7 . No other readers or application software is required. The indicator 45 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. Fig. 70 shows a test module system incorporating a spring-loaded retractable lancet 3 90 and a lancet release button 3 92 for convenient use. Satellite phones are available for use in remote locations. Test Module Electronics Sections 2 and 1 1 3 are block diagrams of the electronic components in Test Modules 1 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 29-53-201209159, 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 1 52 , 1 5 4, 1 8 2, 2 3 4 , along with associated drivers 3 7 and 3 9, and recorders 35 and 41) control and memory. Only LED 26 (in the case of test module 10), external power supply capacitor 32 and Micro-USB plug 14 are external to LOC device 30. The LOC device 30 includes a bond pad for connecting these external components. RAM3 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). In the case of the test module 11 configured for ECL detection, there is no LED 26 (see Figures 112 and 113). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 carries an electrochemiluminescent 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. Some examples of infectious diseases that are rapidly identified by this system include: • Influenza-flu viruses A, B, C, Isavirus, Tohogotovirus • Pneumonia-Respiratory Syndrome (RSV), Adenovirus, Interstitial pneumonia virus, Streptococcus pneumoniae, Staphylococcus aureus • Tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium vaccae and Mycobacterium vaccae 201209159 • Plasmodium falciparum, arch Insects and other protozoan parasites • Typhoid-S. typhimurium serotype • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever – flavivirus • Hepatitis (A to E) • Nosocomial infections – eg difficulties Clostridium, vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus • herpes simplex virus (HSV) • cytomegalovirus (CMV) • Epstein-Barr virus (EBV) • encephalitis - Japan Encephalitis virus, Chandipura virus • Hundred cough - Bordetella pertussis • Measles - paramyxovirus • Meningitis - Streptococcus pneumoniae and Neisseria meningitidis • Some examples of hereditary diseases that are rapidly identified by this system include: • Cystic fibrosis • Hemophilia • Sickle cell disease • Tay-Sachs disease • Haemochromatosis • Cerebral artery disease -55- 201209159 • 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 L0C Device The L0C device 30 is the center of the diagnostic system. It uses a microfluidic platform to rapidly perform the four major steps of nucleic acid-based molecular diagnostic analysis—that is, preparing samples, extracting nucleic acids, amplifying nucleic acids, and detecting. The l〇C 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 a number of different targets for the desired target system. An L0C device 30 is in the form of a L0C device 301 for use in fluorescence detection of a target nucleic acid sequence in a pathogen of a whole blood sample. For the sake of explanation, the structure and operation of the LOC device 301 are described in detail with reference to Figures 4 to 26 and 27 to 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 points to the processing stages associated with the major steps of the nucleic acid-based molecular diagnostic assay: sample input and preparation 288, extraction 29, incubation 291, amplification 292, and detection 294. Each of the sump, chamber, valve 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 310 is illustrated in a partial cross-sectional view (not to scale) of FIG. The LOC device 301 has a germanium substrate 84 supporting a CMOS + MST chip 48, which includes a CMOS circuit 86 and an MST layer 87 overlying the MST layer 87 with a cover 46. In this patent specification, the term "MST layer" is used in various The reagent handles the collection name of the construction and hierarchy of the sample. ^ Accordingly, these configurations and components are configured to define a flow path in a unique size that will support the flow of liquid having a physical property similar to the sample driven by capillary action during 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 and components that are driven by capillary action and that can handle very small volumes. The MST layer shown by the particular system described in this patent specification is a structure and a usable component such as supported on CMOS circuit 86, but does not include portions of cover 46. However, those skilled in the art will recognize that the white 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 5824 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 the first, fourth, third, fifth, fifth, and fifth, respectively, and are described in detail below for a comprehensive understanding of the LOC device. Each structure in 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 sketches 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. 2 is a schematic cross-sectional view through the sample inlet 68, and Fig. 22 is a schematic cross-sectional view through the sump 54. As best shown in FIG. 12, 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 M S T layer 87. The fluid flows through the cap channel 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 1 (see, for example, Figures 7 and 16). The cell transporter occurs in the larger channel 94 assembled in the cap 46, -58-201209159 and 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 a channel size greater than about 20 microns, and therefore requires a cross-sectional area that is transverse to the flow direction to be greater than 400 square microns. The location of the MST channel, especially in the LOC, which does not require transport of cells can be significantly smaller. It is known that the cover channel 94 and the MST channel 90 are generic names 'especially the MST channel 90 can also be referred to in view of its specific function ( For example) heated microchannels or dialyzed MST channels. The MST channel 90 is formed by etching through the M S T channel layer 100 located in 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 spaced layers, the cover channel layer 80 and the sump layer 78 are formed from a single piece of material. Of course, the piece of material may also be a 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 sump layer 78 in which the sump 54' 56, 58, 60 and 62 are etched. Alternatively, the sump and the cover channel are formed by micromolding. Both etching and micromolding techniques are used to fabricate channels having cross-sectional areas that are transversely tangential to flow direction as large as 200,000 square microns and as small as 8 square microns. In different locations of the LOC device, there may be some suitable range of choice for the cross-sectional area transverse to the flow direction. When the channel contains a large number of samples, the cross-sectional area is suitable for up to 20,000 square microns (for example, a 200-micron wide channel in a 100-micron thick layer). When the channel contains a small amount of liquid or a mixture of large cells, the cross-sectional area transverse to the flow direction is preferably very small. The lower seal 64 encloses the cover passage 94 and the upper sealing layer 82 surrounds the sump 54, 56, 58, 60 and 62. The five reservoirs 54, 56' 58, 60 and 62 are pre-packaged with a detection reagent. In the system described herein, the reservoir is pre-filled with the following reagents, but other reagents can be easily substituted: • Storage tank 54: anticoagulant, optionally containing red blood cell lysis buffer • Storage tank 5 6 : Lysis reagent • Slot 5 8 : Restriction enzymes, ligases and linkers (PCR for connector initiation (see Figure 69) excerpted from T. Stachan et al, Human Molecular Genetics 2, Garland Science, NY and London , 1999)) • Storage tank 60: amplification mixture (dNTPs, primers, buffer) and • storage tank 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 are referred to as an upper duct opening 96 and a lower duct □ 92 depending on the flow system flowing from the MST passage 90 into the shroud passage 94 or from the shroud passage 94 into the MST passage 90. -60- 201209159 LOC Device Operation The method of operation of the LOC device 301 is described step by step with reference to analyzing the disease-causing gene in the blood sample. 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 major steps, including input and preparation of samples 28 8 , extraction of nucleic acids 290 , incubation of nucleic acids 291 , amplification of nucleic acids 292 , and detection and analysis 2 94 . The sample input and preparation step 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 sump 54 along the cover channel 94. When the blood flow opens the surface tension valve 1 18 of the sump 54, the anticoagulant is released from the sump 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 sump 54 by capillary action and enters the MST channel 90 via the lower conduit opening 92. The lower duct opening 92 has a capillary initiation feature (CIF) 102 to shape the geometry of the meniscus such that it does not rest at the edge of the lower duct opening 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 sump 54. The MST channel 90 shown in Fig. 22 is part of the surface tension valve 118. The anti-coagulant 116 breaks into the surface tension valve 118 and secures the meniscus -61 - 201209159 120 to the anchor 98 of the meniscus of the upper conduit port 96. Prior to use, the meniscus 120 is still secured to the upper conduit port 96 so that the anticoagulant does not flow into the cap passage 94. As the blood flows through the cap passage 94 to the upper port 96, the meniscus 110 is removed and the anticoagulant is drawn into the 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 conduit ports 92 and upper conduit ports 96. The number of MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent into the sample mixture. When the sample mixture and reagent mixture are mixed together by diffusion, the flow rate out of the sump determines the concentration of the reagent in the sample stream. Thus, the surface tension valve of each sump is 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 through the small holes) are redirected to the waste unit 76, while the target cells continue to be part of the analysis. In the pathogen dialysis zone 7 described herein, pathogens from whole blood samples are concentrated for microbial DNA analysis. The array of apertures is formed by a plurality of apertures 1 64 having a diameter of 3 microns that are connected to the target channel 74 in fluid operation to connect the input stream in the cover channel 94. The 3 micron diameter aperture 164 and the dialysis absorption aperture 168 of the target channel 74 are connected by a series of dialysis MST channels 204 (best shown in Figures 15 and 21 201209159). The pathogen is small enough to break into the target channel 74 via the dialysis MST channel 204 through a small pore 1 64' M having a diameter of 3 microns. Cells larger than 3 microns (such as red blood cells and white blood cells) remain in the cover 46 Φ 2@ $ lane 72, which will be directed to the waste sump 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate specific stomach or other target cells, such as white blood cells, for human DNA analysis. More detailed instructions for dialysis zones and dialysis variants are provided below. In Figures 6 and 7, the flow is drawn through the target passage 74 into the surface tension valve 128 of the lysis 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 anti-coagulant reservoir 54 (where the surface tension valve 118 has three MST channels 90) (assuming the 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 target channel 74 in the chemical cleavage zone 130. The boiling start valve 126 stops the flow until sufficient time has passed to cause diffusion and lysis to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling start valve are described in 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 in place of boiling start valves (as opposed to passive valves such as surface tension valve 118). 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 -63 - 201209159 1 3 1 for restriction enzyme digestion and linker ligation. Referring to Fig. 13, when the liquid flow releases the meniscus from the surface tension valve 132 at the start of the mixing zone 131, the restriction enzyme, the linker and the ligase are released from the sump 8. The mixture flows through the length of the mixing zone 133 for diffusion mixing. The end of the mixing zone 133 is the lower conduit opening 134 which is directed into the cultivating chamber inlet passage 133 of the incubation zone 114 (see Figure 13). The chamber inlet channel 133 delivers the mixture into the curved configuration of the heated microchannel 210, which provides a chamber for holding the sample during restriction enzyme digestion and linker ligation (see Figures 13 and 14). ). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 30 1 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 1 1 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 start 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. 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 assays. 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 this breakthrough allows the restriction enzyme and ligase to be deactivated before entering the amplification zone 112. Reactivation of restriction endonuclease-64 - 201209159 and ligase is particularly suitable when using thermostatic nucleic acid amplification. 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 Multiple expansion chambers) until the boiling start valve 108 is reached. As best shown in the schematic cross-sectional view of Fig. 30, the amplification mixture (dNTPs, primers, buffer) is released from the sump 60, and the polymerase is then released from the sump 62 into the ligating chamber and the amplification zone (114, respectively) Figures 35 through 51 of the intermediate MST channel 212 of and 112) show the layers of the LOC device 3〇1 in the inset AC of Fig. 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 start valve 108. After sufficient diffusion mixing time, the heater 154^ in the microchannel 158 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 DN A . 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 5, the hybridization and detection zone 52 has a hybridization chamber array 110. Figures 52, 53, 54 and 56 show detailed hybridization chamber arrays 11 and individual hybridization chambers 180. At the entrance of the hybridization chamber 180 is a diffusion barrier 175' which prevents the target nucleic acid, the probe strand and the hybridized probe from diffusing between the hybridizations -65-201209159 chamber during the hybridization to prevent 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 of the hybridization chamber 180 that should contain the equivalent probe. Abnormal results can be ignored or weighted differently during the progression of a single result. 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 L E D 2 6 located in the test module 10 to cause it to emit light. These probes only produce fluorescence 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 LED excitation light, as discussed in more detail below. Fluorescence is detected by photodiode 184 in CMOS circuit 86 under each hybridization chamber 180 (see hybridization chamber described below). The photodiode 184 of all hybridization chambers and associated electronic products together form a photosensor 44 (see Figure 64). In other systems, the photosensor can be an array of charge coupled devices (CCD arrays). The signal detected from the photodiode 1 84 is amplified and converted into a digital output analyzed by the test module reader 12. This detection method is further described in detail later. -66-54, 201209159 Other details of the LOC device are modularized. The LOC device 301 has a number of functional zones including reagent reservoirs 56, 58, 60 and 62, dialysis zone 70, cracking zone 13 , incubation 1 and expansion Zone 1 1 2. Valve type, 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 be used as the first addition zone 112 of the tandem amplification analysis system. The chemical lysis reagent storage tank % can be used to add the first amplification mixture, dN TP s and buffer, while the reagent storage tank is 58 8 The reverse transcriptase and/or polymerase is added. If the sample needs to be resolved, a chemical lysing agent may also be added to 56 with the amplification mixture or 'the sample may be heated in the incubation zone to be scheduled to undergo 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 sump can be immediately placed upstream of sump 58 to mix the dNTPs and buffer. In some cases, it may be necessary to omit a step, such as step 291. In this case, 'a special device can be manufactured with a reagent storage tank 58 and a cultivation zone 1 14 , or the storage tank may be completely sputum reagents' or the active valve (if present) has no active agent distribution In the sample stream, the incubation zone becomes a channel at all to transport the sample cleavage zone 130 into the amplification zone 112. The heater can be operated independently. Therefore, when the reaction requires heat (such as thermal cracking), the heater 1 14 L0C is additionally provided with an expansion of the sump storage time, and the cultivating is omitted. Do not operate at this -67-201209159 step to ensure that thermal cracking does not occur in LOCs that do not require thermal cracking. The dialysis zone 70 can be located at the beginning of the fluid system within the set as shown in Figure 4 or can be located where the microfluidic device is located. For example, in some cases, in the hybridization and detection steps, dialysis may be performed after the amplification phase 2 2 2 to remove cell debris. In addition, dialysis zones can be incorporated anywhere in the entire L Ο C device. Similarly, additional amplification regions 1 1 2 may be included to simultaneously or sequentially expand the targets, which are then detected by acid probes in hybridization array 110. To analyze a similar whole of which no dialysis is required, the dialysis zone 70 can be completely omitted from the sample input zone 288 of the LOC design. In some cases, there is no need to isolate zone 70 from the LOC device, even though the analysis does not require dialysis. If there is no geometrical barrier to the analysis of the dialysis zone, the dialysis zone 70 can still be used for sample input and production at the LOC without losing the required function. In addition, the detection zone 294 may comprise a protein body array array identical to the hybridization chamber array, but the cassette is designed to bind or hybridize to the probe target protein present in the non-expanded sample, rather than the labeled target nucleic acid. A nucleic acid probe that hybridizes to a sequence. It will be appreciated that the LOCs used in this diagnostic system are manufactured with different functionalities depending on the particular LOC application. The vast majority of functional areas are common to many LOC devices. The additional LOC device for new applications is a matter of reconciling the appropriate functionality from the numerous functional area options used in the device. Any one or more of the plurality of specific nuclear blood samples and preparations in the microfluidic device in the device are omitted in the paired area, and the group of the increased sample and the rooted zone are set. LOC Zone Combination -68- 201209159 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 L0C shown in this description is not an exhaustive list and that many additional LOC designs are a combination of functional areas. Sample type LOC variants can accept and analyze sample nucleic acids or protein inclusions in a variety of liquid forms including, but not limited to, blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical breast milk, sweat, chest Fluid, tears, pericardial effusion, abdominal compartment water sample and dip sample. Amplification from macroscopic nucleic acid amplification is analyzed using a LOC device; in this case, all reagent reservoirs are configured or configured to not release their contents, and the dialysis, lysis, and culture zones will only be used to sample The nucleic acid is detected as described above by transport from the sample inlet 68 to the hybridization chamber. For some sample types, a pre-treatment step is required: Semen may need to be liquefied first and the mucus may need to be pre-embedded with an enzyme to reduce its viscosity before entering the LOC device. Sample Input Referring to Figures 1 and 12, the sample is first added to the test module container 24. The large container 24 is a truncated cone that acts to feed the sample into the inlet 68 of the LOC unit 301. The schematic diagram is familiar with the device and the appropriate type of blood and blood, liquid, and ring can also be air cultured and expanded 180, for example, the first treatment of 1 藉 by the hair from here -69 - 201209159 into the 64 micron wide X60 micron deep cover channel 94 where it is also attracted to the anticoagulant reservoir by capillary action. 5 4 ° Reagent storage tank Small volume reagents required for analysis systems using microfluidic devices (such as L0C device 301) The storage tank contains all the reagents required for biochemical treatment, and each reagent storage tank has a small volume. This volume is easily less than 1 billion cubic micrometers, in most cases less than 300 million cubic micrometers 'typically less than 70 million cubic micrometers' and is less than in the case of the LOC device 301 shown in the figures. 20 million cubic microns. Dialysis Zone Referring to Figures 15 to 21, 33 and 34, the design of the pathogen dialysis zone concentrates the pathogenic target cells from the sample. Apertures 164 in the form of a plurality of 3 micron diameter holes in the roof layer 66 as previously described filter the target cells out of the sample body. As the sample flows through the 3 micron diameter orifice 1 64, the microbial pathogen enters a series of dialysis MST channels 204 through the small holes and then flows back into the target channel 74 via the 16 micron dialysis inlet 168 (see Figures 3 3 and 34). . 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 that is directed to the waste sump 76. In terms of the type of biological sample from which a large amount of waste is produced, the foam insertion member or other porous member in the outer casing 13 of the test module 1 is configured to communicate with the waste storage tank 76 (see Fig. 1). -70- 201209159 The pathogen dialysis zone 70 relies entirely on the capillary action of the fluid sample to operate. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis zone 70 has capillary action initiation features (CIFs) 166 (see Figure 33) such that fluid is drawn into the underlying dialysis MST channel 204. The first intake aperture 198 of the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid flow completely from being fixed to the meniscus as it traverses the dialysis uptake aperture 168. The small component dialysis zone 682 shown in the schematic of Figure 85 can 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 storage tank 766. Thus, LOC device 30 (see Figures 1 and 1 1 2) 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 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 sump 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 128 of the lysis sump 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 accomplishes this by providing heated microchannels 210 of incubation zone 114. The sample stream is drawn into the incubation zone 114 and stopped at the boiling start valve 106. The incubation microchannel 210 heats the sample at a temperature that can destroy the cell membrane -71 - 201209159. 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 I 1 9 is drawn by capillary action to the heated microchannel 1 58 until the boiling start valve 1 〇 8 is reached. Prior to the sample flow, the meniscus 120 is secured to the anchor 98 of the meniscus of the valve inlet 146. The geometry of the meniscus anchor 9 8 stops the meniscus from advancing to contain the capillary flow. As shown in Figures 31 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 tension of the meniscus 1 20 keeps the valve closed. Ring heater 152 is attached around valve inlet 146. The ring heater 152 is controlled by C Μ Ο S via the boiling start valve heater connector 153. 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 1 46 ' 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 1 9 is inserted into the cap passage 94 and flows -72- 201209159 through the valve lower pipe port 150 to the valve outlet 148 where the capillary driven by the capillary action continues to enter the hybrid along the expansion zone outlet passage 160. Detection area 52. A liquid sensor 丨 74 is placed in front of and behind the valve for diagnostic purposes. It will be appreciated that once the boiling start valve is opened, it cannot be closed again. 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 lower conduit opening 134 to the boiling start valve 106 (see Figures 1 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 112 has a heated incubation microchannel 158 from the boiling start valve 106 to the boiling activation valve 1〇8 (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. The sample cell and the reagent are heated by the heater 丨 54 in the incubation zone 114 and the amplification zone 1丨2, and the heater 154 is controlled by the CMOS circuit 86 using pulse width modulation (PWM). Each of the bent portions of the heated cultivating micro-pass-73-201209159 channel 210 and the augmented microchannel 158 has three detachable portions that are separately operable and at their respective heater joints 156 (which provide input heat flux density) The two-dimensional control) extends between heaters 1 54 (see Figure 14). As best shown in FIG. 51, the heater 154 is supported on the roofing layer 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 passage regions forming the wide meandering portion of the curved shape. In the amplification zone 112, each of the wide tortuous portions can be operated in the form of separate P C R 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 400 nanoliters, in most cases less than 1 70 nanoliters, typically less than 70 nanoliters' and in the case of LOC device 301 is between 2 nanoliters to 30 nanometers. Rise. The increased heating rate and preferably the diffusion mixing of the small cross-sections of each channel region increases the heating rate of the augmented fluid mixture. All fluids are kept within a short distance from the heater 154. Reducing the cross-section of the channel (i.e., the cross-section of the amplifying microchannel 158) to 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. The size of the body can be easily achieved by etching techniques to be about 1 micron. If the number of amplicon required is small (e.g., -74-201209159 in the case of LOC device 301), the cross-sectional area can be reduced to 2500 square microns. In the LOC device, the diagnostic analysis is performed at 1000 to 2000 and it is necessary to "sample in, and, in response, the cross-sectional area transverse to the flow direction is between 1 square micrometer and square micrometer is sufficient. The heater element in the microchannel 158 heats the nucleic acid sequence at a rate exceeding Kelvin (K), in the case of a number greater than 1 〇〇K per second. Typically, the heater is subjected to a thermal nucleic acid sequence. The rate exceeds 1, 〇〇〇K per second and in many cases the heater element heats the nucleic acid sequence at a rate in excess of 10, 〇〇〇K per second, according to the requirements of the analytical system, the heater element heats the nucleic acid at a rate of more than one second. 1〇〇, 〇〇〇Κ, more than Κ, ΟΟΟ, ΟΟΟΚ, sec, 10,000,000 Ι per second, more than 20,000,000 每秒 per second, over 40,000,000 Κ, more than 80,000,000 每秒 per second, more than 1 60,000,000 Κ. Small cross-sectional area is also beneficial Mix any reagent with the sample fluid. Before the diffusion mixing is completed, one liquid diffuses to the other liquid and is used near the interface between the two. The concentration decreases with the interface. The cross-section of the cross-section with the flow direction maintains the two fluids flowing close to the interface for more rapid diffusion. The channel cross-section is reduced by at least 100,000 square microns to achieve a significantly higher mixing ratio compared to more "in" instruments. The cross-sectional area of the etching manufacturing technology channel transverse to the flow path is less than 1 6000 amps, resulting in a substantially higher mixing ratio. If a small volume is required (less to few probes are reported), 400 flat 80 is used to open most components. Medium, the general sequence exceeds the distance per second per second per second in the diffusion of the line and the microchannel is mixed. The large size allows the micro-micron as in the case of -75-201209159 LOC device 3 01, which can be used The area is reduced to less than 2,500 square microns. In the LOC device, a diagnostic analysis is performed with a probe of 1000 to 2000 and it is necessary to "cross the tangential direction" with a flow direction of 1 square micron to 40 0 square micrometer in less than 1 minute. That is enough. Short thermal cycle time

Keeping the sample mixture close to the heater and using very small amounts of fluid allows the thermal cycle to proceed rapidly 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 target sequence of up to 150 base pairs long, the thermal cycle time of the L Ο C device 30 with some common diagnostic analysis is 〇 · 4 5 seconds to 1.25 seconds. This rate of thermal cycling allows the test module to complete the nucleic acid amplification process in much less than j 〇 minutes; typically less than 220 seconds. For most analyses, starting from the sample fluid entering the sample inlet, the amplified region will produce enough amplicons in + to 80 seconds. For many analyses, enough amplicons are produced in 3 Q seconds. 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 the hybrid chamber array 11 -76 - 201209159. The hybridization and detection zone 52 has a heteropoly 24x45 array 110 each having a hybrid reactive FRET finder element 182 and an integrated photodiode 184. Inclusions 1 84 were used to detect fluorescence from hybridization of the target nucleic acid sequence or protein to 186. Each photodiode 184 is independently controlled by 86. The FRET probe 186 and photodiode 184 material must be permeable to the emitted light. Therefore, the wall region 97 between the photodiodes 184 is permeable to the emitted light. In the LOC device 301, the wall region 97 is 0.5 micron) cerium oxide. The needle-target hybrid that directly incorporates photodiode 184 into each hybridization chamber 180 is very small in size but still produces an optical signal (see Figure 54). A small amount of small-volume hybridization allows for a detectable amount of probe-target hybrid that is easily less than 270 picograms (equivalent to 900,000 cubic micrometers in most cases less than 60 picograms (equivalent to 20 Ten thousand cubic meters are often less than 12 picograms (equivalent to 40,000 cubic micrometers), which in the case of LOC device 301 is less than 2.7 cubic chambers of 9000 cubic micrometers. Of course, hybrid chambers allow for higher density chambers, This is on the LOC device. In the LOC device 301, the hybridization zone has an area of 1500 microns (i.e., less than 225 0 square microns per chamber) of 1 000 chambers. The smaller volume also reduces the reaction time. Faster. Another room that requires a small amount of probes in each chamber. 180 • 1 8 6. Any probe between the heated photodiode FRET probe CMOS circuit is also optically A thin layer (about the next possible detection of the fluorescing hybridization chamber. The amount of probe required), in the vast majority of micrometers, is shown in the figure (corresponding to the smaller size of the probe xl 5 00 micron has more than hybridization and detection points for manufacturing the -77-20120 Only a very small number of probes need to be dispensed into each chamber during the 9159 LOC device. The system of the LOC device according to the present invention can be spotted using a probe solution volume of 1 picolite or less. After nucleic acid amplification, the boiling start valve 108 It is activated and the amplicon flows along the flow path 176 and enters each hybridization chamber 180 (see Figures 5 and 56). The endpoint liquid sensor 178 indicates that amplification occurs in the hybridization chamber 180. When the sub-timer 'the heater 182 can be activated. After enough hybridization time, the LED 26 (see Figure 2) is activated. The opening in each of the hybridization chambers provides an optical window 1 3 6 for FRET Probe 186 is exposed to excitation radiation (see Figures 52, 54 and 56). The LED 26 is illuminated for a time sufficient to induce a high intensity fluorescent signal from the probe. During excitation, the photodiode is absent. After the process control delay 300 (see Figure 2), the photodiode 184 is activated and the fluorescent emission is detected in the absence of excitation light. The incident light on the photosensitive surface of the photodiode 184 is visible (see Figure 5) is converted to photocurrent and measured by CMOS circuit 86. Each of the 180 is loaded with a probe for detecting a single standard nucleic acid sequence. If necessary, each hybridization chamber 180 can be loaded with probes to detect more than 1000 different labels. In addition, many or all of The hybridization chamber can be loaded with the same probe to repeatedly detect the same target nucleic acid. In this way, replicating the probe across the hybridization chamber array 1 10 can increase confidence in the results obtained, and if desired, can be contiguous to those hybridization chambers. The light diodes are combined 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 according to the detection specifications. Humidifier and Humidity Sensor The illustration AG in Fig. 6 indicates the position of the humidifier 1 96. The humidifier prevents reagents and probes from evaporating during the L 0 C device 310 operation. As best shown in the enlarged view of Figure 5, the water sump 1 8 8 is connected to three evaporators 190 in fluid operation. The water storage tank 188 is filled with water and is sealed during the manufacturing process. As best shown in Figures 55 and 67, water is drawn into the three lower conduit openings 1 94 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 that surrounds the intake port 193. The ring heater 191 is connected to the CMOS circuit 86 by a conductive post 386 to the top metal layer 195 (see Figure 37). When activated, the ring heater 191 heats the water, allowing the water to evaporate and damp the surrounding equipment. The position of the humidity sensor 232 is also shown in FIG. However, as best shown in the enlarged view of the illustration AH of Fig. 63, the humidity sensor 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 232 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. -79- 201209159 Feedback Sensors Temperature and liquid sensors are included throughout the LO C unit 310 to provide feedback and diagnostics during device operation. Referring to Figure 35, nine temperature sensors 1 70 are distributed throughout the amplification zone 112. Similarly, the incubation 1 14 also has nine temperature sensors 170. Each of these sensors uses a bipolar transistor (BjTs) 2x2 array to monitor fluid temperature and provide feedback from CMOS circuit 86. The CMOS circuit 86 is used to precisely control the thermal cycling during acid amplification and any of the twisted hybridization chambers 180 during thermal cracking and incubation, and the CMOS circuit 86 uses the hybrid heater 1 as a temperature sensor (see Figure 56). ). The resistance of the hybrid heater 182 is temperature dependent and the CMOS circuit 86 uses this to derive temperature readings for each of the cells 180. The LOC device 301 also has a plurality of MST channel liquid sensors 1 and a cap channel liquid sensor 208. Figure 35 shows a row of MST channel body sensors 174 at one end of every other tortuous portion in the heated channel 158. As best shown in Figure 37, the MST channel body sensor 174 is a pair of electrodes formed by the exposed regions of the top metal 195 in the CMOS structure 86. The circuit between the liquid-off electrodes indicates that it is present at the sensor. Figure 25 shows an enlarged perspective view of the cap channel liquid sensor 208. The pair of TiAl electrode pairs 218 and 220 are placed between the roof layer 66 electrodes 218 and 220 as slits 222 to keep the circuit open when no liquid is present. The liquid is turned off when the liquid is present, and the CM0S circuit 86 monitors the liquid flow by using the feedback of 201209159 with the 微 - - - - 微 微 微 微 微 微 微 微 微 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 2012 Independence of gravity Test module 1 〇 is independent of direction. It does not need to be fixed on the surface to operate. The flow of fluid driven by capillary action into the auxiliary instrument under the conduit means allows the module to be truly inserted into a portable handheld reader such as the portable one. Operation with nothing to do with gravity means that the test module is completely independent of acceleration. It is resistant to shock and vibration and can be operated on the line or when the mobile phone is carried everywhere. Dialysis Variants The dialysis zone is provided with a flow channel to prevent air bubbles from being confined. The following description is referred to as a LOC system of LOC variant view 518 and is shown in Figures 72, 73, 74 and 75. The dialysis variant VD 1518, which is full of fluid sample and does not trap the bubble in the channel, also has an additional material surface layer 594 called interface layer 594 located in the capped MST channel layer 100 of the CMOS+ MST device 48. between. The interfacial layer 5 94 allows for more complex fluid interaction between the MST layers 87 without the size of the plate 84. Referring to Fig. 73, the bypass passage 600 flows through the design sample stream from the interface waste passage 604 to the interface target passage time delay. This time delay allows the fluid sample to flow through the dialysis zone of the vehicle device that is flat and stable and lacks external portable and mobile phones. L 0 C layer. The boundary layer 80 and the agent sump are added to the sputum base. At the time of the flow 602, the MST pass-81 - 201209159 lane 2 04 reaches the dialysis intake port 168, where the meniscus is fixed. By the capillary action initiation feature (CIF) 202 from the bypass channel 600 to the inlet port of the interface target channel 602, the sample stream is drawn into the interface target channel from upstream of all of the dialysis intake ports 168 of the dialysis MST channel 204. 602. When there is no bypass channel 600, the interface target channel 602 still charges from the upstream end, but the meniscus finally reaches and passes through the unfilled intake port belonging to the MST channel, causing the air to be confined to the burr. . The restricted air reduces the flow rate of the sample through the white blood cell dialysis zone. A pre-hybridization filter variant of the LOC device, LOC variant ΧΠ758, uses a small component dialysis zone 682 placed at the exit of the amplification zone 12 (see Figures 96-103). The small component dialysis zone 682 provides a pre-hybridization filtration purification stage 293 (see Figure 96). Prehybridization filters remove cell debris that remains in the sample stream after cell lysis. Hybridization efficiency may be affected by cell debris, so it is advantageous to reduce the concentration of cell debris prior to hybridization. Referring to Figures 101, 102 and 103, the small component dialysis zone 682 has three adjacent channels fabricated in the bottom channel layer 1; the large component channels 760 are flanked by two small component channels 762. A flow port 764 in the form of a series of reverse tapered openings along one of the sides of the large component channel 760 provides a fluid connection to the small component channel 762. In most practical applications, the flow port will be between 1 and 8 microns wide and between 1 and 8 microns high. When the sample flows down the large component channel 760, the small enough particles flow through the small component channel 762 through the inverse tapered opening (eg, the amplicon), while the larger particles (eg, the 201209159 cell debris) remain large The component channel, which ultimately terminates the closed end 766. The smaller particles continue to follow the small component channels to the opposite sides of the chamber array 110, where they follow the path of the array to the respective closed ends 768 (see Figure 103). Upon detection of the small component amplicon, all individual hybridization chambers are inoculated.

Nucleic Acid Amplification Variant Repeat PCRs Figures 78, 79, 80, 81 and 82 (except for other figures) illustrate the LOC loading of the first amplification region 112.1 in which the amplification regions 1 12.1 and 12.2 are continuously operated. A reagent reservoir for amplifying mix 60.1 and 62.1 for polymerase. Each of the extensions added after the initiation zone also includes two reagent reservoirs for amplifying the storage tank 60.2 in the reservoir 62.2 of the polymerase. Repeated PCR analysis can be performed on successive amplified regions, which are used for pre-amplification to increase the sensitivity of subsequent nucleic acid amplification in the 112.2 line of the amplified region. A continuous amplification zone can also be used for nested reactions. In a repetitive PCR for preamplification, the first amplification 1 1 2.1 is used to amplify a nucleic acid sequence amplification of a sample comprising the target sequence, which is not necessarily specific to the target sequence (eg, whole genome amplification) ) does increase the concentration of the target sequence. After pre-amplification, the sample is mixed from the reagents of reservoirs 60.2 and 62.2 and then into the second extension 12.2. The reagents stored in storage tank 60.2 include specific primers that are crossed by a cross to illustrate. Addition of the region and the use of amplification into the PCR extension zone » this, but with the extension zone to only -83- 201209159 amplification of the target sequence in the pre-amplified sample mixture. It should be noted that a similar method in which the PCR in the first or second amplification stage is replaced by a thermostatic technique can also be used to achieve the advantages of pre-amplification. Nested PCR is a special form of repetitive PCR with the added benefit of high target specificity. In nested PCR, the nucleic acid amplification step in the first amplification region 1 1 2.1 uses primers to amplify a sequence larger than the final target sequence to form a portion of the amplification mixing reagent stored in the storage tank 6 0 · 1 ( It is complementary to the region outside the target sequence). The reaction in the first amplification region 1 1 2 · 1 produces an amplicon that is a target sequence plus a flanking region. This amplified mixture was mixed with the reagent from storage tank 60.2 and the polymerization enzyme from 62.2. The reagent stored in sump 60.2 includes a primer complementary to the portion of each end of the target sequence, i.e., a sub-portion of the amplicon from the first amplification stage. When nucleic acid amplification is performed in the second amplification region 1 1 2 · 2, the chance of generating amplification at a position independent of the target is greatly reduced because the concentration of the amplicon from the first amplification stage It is much higher than the concentration of the original sample molecules. The sensitivity and specificity of the nested PCR can also be obtained when the one or two PCR amplification stages are replaced by sequence specific constant temperature amplification techniques. The polymerases are separately stored and independently added to the sample mixture. There will be advantages to be able to use different polymerases for the pre-amplification and final nucleic acid amplification steps. For example: this allows selection of a low error rate (eg, proofreading) polymerase for the preamplification step to avoid creating a target sequence containing a false or false standard sequence while allowing for higher speed or temperature comparisons. The polymerase is tolerated for final amplification. -84-

201209159

Direct PCR Traditionally, PCR requires large-scale purification of the target mixture. However, it is feasible to minimize the DNA 在 when performing nucleic acid amplification by appropriately modifying the chemistry and |. When the nucleic acid amplification process is PCR, PCR is carried out. In which the nucleic acid amplification is in a controlled, $LOC device, the method is direct isothermal amplification. The use of direct nucleic acid amplification technology has considerable advantages, especially in fluid design. Adjustments for direct PCR or direct thermostatic loading include the enhancement of buffer strength, the use of high-potency polymerases, and the inhibition of potential polymerase inhibitors in the presence of inhibitors in the sample. In order to take advantage of direct nucleic acid amplification technology, the LOC has two additional features. The first feature is to have a suitable gauge π such as: sump 58 in Figure 8 to provide a sufficient amount of diluent or diluent, such that it may interfere with the amplification chemistry | concentration is low enough to allow for successful nucleic acid amplification. The non-cell ΐ dilution is between 5 and 20 times. When appropriate, the conformation (e.g., pathogen dialysis zone 70 in Figure 4) is maintained at a concentration high enough to maintain a concentration of H acid. In this system (further illustrated in Figure 6), upstream of Zone 2 90, the dialysis zone of pathogens as small as 2 92 can be effectively concentrated and the larger cells are lined up. In another system, the dialysis zone is used to selectively re-prepare the concentration of DNA, so that the method of cultivating or directly amplifying the method is called simplification in the LOC device. Addition of augmentation chemical and high-continuation additives. The dilute device design includes a reagent storage tank (the amplification reaction is mixed with the different L 0 C junction L of the final i sample component of the component to ensure the target core; the amplification and detection system is in the sample extraction to enter the expansion Increase the area to the waste container 76 丨Depleted Blood Award -85- 201209159 Protein and salt while retaining the desired cells. The second LOC structure supporting direct nucleic acid amplification is designed to adjust the sample aspect ratio of the channel. Mixing ratio with the components of the amplification mixture. For example, in order to ensure that the dilution ratio of the inhibitor bound to the sample in a single mixing step is in the range of preferably 5X-20X, the length and cross section of the sample and reagent channels are designed. Thus, the flow impedance formed by the sample channel (upstream of the start of the mixing position) is 4-1 times higher than the flow impedance of the channel through which the reagent mixture flows. The flow impedance in the control microchannel can be easily transmitted through the control geometry. Designed to achieve. 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 microchannel The flow impedance in the middle relies more strongly on the smallest cross-sectional dimension. For example, when the aspect ratio is inconsistent, the flow impedance of the microchannel with 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), RNA must be reverse transcribed into complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction can be performed in the same reaction as PCR. Performing in a chamber (single-step RT-PCR), or in the form of separate initial reactions (two-step RT-PCR). In the LOC variants described herein, single-step RT-PCR can be performed simply via reagents The reverse transcription enzyme and the polymerase are added to the storage tank 62, and the heater 1 54 is programmed to perform the reverse transcription step and then the nucleic acid amplification step. The two-step RT-PCR can also be easily stored and used. The dispensing buffer 201209159 'reagent, dNTPs and reverse transcriptase reagent reservoir 58 and the incubation zone 114 with the step are then achieved in the amplification zone 112 as a normal side. Thermostatic nucleic acid amplification is constant for some applications. Nucleic acid amplification is a nucleic acid amplification method, which avoids the need to circulate the reaction components through various thermal cycles, but maintains the amplification zone at a constant temperature, usually about 4 1 ° C. The thermostated nucleic acid expansion has been described. There are a variety of methods for amplification, including generational amplification (SDA), transcription-mediated amplification (TMA), nuclear-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), and dependent thermostated DNA amplification (HDA). Rolling cycle amplification ('branched amplification (RAM) and circular thermostated amplification (LAMP)) These or other isothermal amplification methods can be used in the specific systems described herein: To perform a thermostatic nucleic acid amplification, the adjacent amplification zone trials 60 and 62 will load the appropriate reagent PCR amplification mixture and polymerase for a particular thermostated method. For example, in the case of SDA, tank 60 contains amplification buffers, primers, and dNTPs, while reagent storage | contains appropriate nicking enzymes and exo-DNA polymerase. In the RPA aspect, the sump 60 contains amplification buffers, primers, dNTPs, and recombinant fermentation broth, while the reagent sump 62 contains a stock-substituted ruthenium polymerase. For example, in the HDA aspect, the reagent storage tank 6 contains amplification buffers and dNTPs. 'And the reagent storage tank 62 contains a suitable DNA polymerization reverse transcription amplification preferably with a repeat of 3 7 ° C. The acid sequence helicase RCA), and the LOC agent storage tank, rather than the reagent storage f 62 package , reagent protein B su. Agent, Enzyme and Solution -87- 201209159 The enzyme is used to unwind the strands of double-stranded DNA instead of using heat. Those skilled in the art will appreciate that the necessary reagents can be separated between two reagent reservoirs in any manner suitable for the nucleic acid amplification process. In order to amplify viral nucleic acids from RNA viruses such as HIV or hepatitis C virus, NASBA or TMA is suitable because it is not necessary to first transcribe RNA into cDNA. In this example, reagent reservoir 60 contains buffers, primers, and dNTPs, and reagent reservoir 62 contains RNA polymerase, reverse transcriptase, and, optionally, RNaseH. 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 1). 4 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, thermostatic nucleic acid amplification is sometimes used in the LOC variants XLIII 6 73, LOC variant XLIV 674 and LOC variant XLVH677 - etc. shown in Figures 86, 87 and 88, respectively. Direct thermostatic amplification may also be combined with one or more of the preamplification dialysis steps 70, 686 or 682 as shown in Figures 86 and 88 and/or the prehybridization dialysis step 6 8 as indicated in Figure 8 2. To assist the target cells in the partially concentrated sample before nucleic acid amplification, respectively, or to remove unwanted cell debris before the sample enters the hybridization chamber array 1 1 0. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be used -88-201209159. Thermostatic nucleic acid amplification can also be performed simultaneously in amplification regions as shown graphically in Figures 71, 76 and 77, 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 RN A. Additional details of the fluorescence detection system Figures 58 and 59 show hybridization-reactive FRET probes 23 6 . These are commonly referred to as molecular beacons and are the production of arm-and-loop probes from single-stranded nucleic acids that produce fluorescence upon hybridization with complementary nucleic acids. Figure 58 shows a single FRET probe 236 before hybridization to the target nucleic acid sequence 23 8 . The probe has a loop 240, an arm 242, a fluorophore 246 at the 51 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 5, the probe remains closed when the complementary target sequence is lacking. The arm 242 maintains the fluorophore-quenching agents in close proximity to one another 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 2 44. Figure 59 shows the FRET probe 23 6 configured for opening or hybridization. Upon hybridization to the complementary target nucleic acid sequence 23 8 , the arm-and-loop structure is broken and the fluorophore and quencher are spatially separated to restore the ability of the fluorophore 246 to fluoresce. 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 probe-targeted helix than the non-complementary single core-89-201209159 Stable 'The probe hybridizes to a complementary target with very high specificity. Since the double-stranded DNA is relatively tight, the probe-target spirochete and the arm spirochete are not spatially coexistent. Control Probes The hybridization chamber array 1 1 〇 includes some hybridization chambers with positive and negative control probes for analysis of quality control 1 80 ° 1 and 1 〇 9 diagrams to illustrate negative control of fluorescing group 796 Pins '110 and 111 are 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 an open configuration or remains off. Referring to Figures 1 and 10, 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 23 8 hybridizes to the probe (see Figure 109), or whether the probe retains its arm-and-loop configuration (see Figure 1-8), the excitation light 244 The response is negligible. Alternatively, the negative control probe 796 can be designed to remain quenched at all times. For example, the synthesis loop 240 has a probe sequence that does not hybridize to any of the nucleic acid sequences in the sample being examined, the arms 242 of the probe molecule will re-hybridize themselves, and 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 (where the probe is not hybridized, but the quencher will not quench all of the emission from the reporter) -90-201209159. Conversely, as illustrated by Figures 1 1 0 and 1 1 1 , the configuration of the male 798 does not have a quencher. Regardless of the positive control probe target nucleic acid sequence 23 8 hybridization, the fluorophore 246 responds to the excitation of the fluorescent light 250 without being quenched by anything. Figure 52 shows the distribution of the potential control probes in the entire hybridization chamber array 110 (3 78 and 3 80 , respectively). 37 8 and 3 80 are placed in a chamber 1 80 spanning the hybrid chamber array 1 10 . However, the control probes in the array are in the configuration of any hybridization chamber array. 1) The fluorophore design requires a fluorophore with a long fluorescence lifetime so that the intensity of sufficient luminescence decays below the intensity of the fluorescing emission. At this time, 44 is enabled to provide sufficient signal to noise ratio. In addition, it is better translated into a larger integrated fluorescent photon count. Fluorescent clusters 24 6 (see Figure 59) have a lifetime longer than 100, usually longer than 200 nanoseconds, more often longer than 300 and in most cases longer than 400 nanoseconds. The transition metal or rare earth-based metal-gametoid lifetime (from hundreds of nanoseconds to milliseconds), sufficient quantum yield, and photochemical stability are all beneficial properties for fluorescent detection systems. . A hybridization arrangement in which the transition metal ion ruthenium (Ru(H)) is used as a primer 798 for positive and negative light on the control probe line (as with time, the photosensor is long). The metal-gametide complex of the luminescence of the luminescence of the luminescence of the celsius and the high-temperature and chemical conditions of the compound is three (2, 2 ^ 联Pyridine) 钌(Π) ([钌(bipyridine)3] 2+ ), which has a lifetime of about 1 microsecond. This complex is commercially available from Biosearch Technologies brand pulsar 65 0. Table 1: Pulsar 65 0 light Physical properties (钌 chelate) Parameter symbol number 値 Unit absorption wavelength Xabs 460 nm Emission wavelength λβιη 650 nm Extinction coefficient Ε 14800 M-'cm*1 Camp light lifetime Tf 1.0 μδ Quantum yield Η 1 (deoxidized) Ν/Α A ruthenium chelate, a lanthanide metal-gametide complex, has been successfully demonstrated as a fluorescent reporter in the FRET probe system and also has a long lifetime of 1 600 microseconds. Table 2: Light of ruthenium chelate Physical property parameter number of symbols 値 unit absorption wavelength ^abs 330-350 nm Wavelength ^em 548 nm extinction coefficient Ε 13800 (depending on the gametes, up to 30000 @, = 340 nm) M-'cm'1 camp light lifetime Tf 1600 (hybrid probe) μδ quantum yield Η 1 (depending on配/Α 201209159 LOC device 3 0 1 The fluorescence detection system used does not use filters to remove unwanted background fluorescence. Therefore, in order to increase the signal-to-noise ratio, if the quencher 248 does not have natural emission Advantageously, the 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) (It can be from Novato, California

Biosearch Technologies has obtained a natural quencher with no natural emission and high quenching efficiency. BHQ-1 has a maximum absorption at 534 nm and a quenching range of 48 0-58 Onm, which makes it a suitable quencher for the ruthenium chelate fluorophore. BHQ-2 has maximum absorption at 579 nm and a quenching range of 560-670 nm, making it a suitable quencher for Pulsar 650. Iowa Black Quenchers (Iowa Black FQ and RQ) (available from Integrated DNA Technologies of Coralville, Iowa) are suitable alternative quenchers with little or no background emission. The Iowa Black FQ has a quenching range of 420-620 nm, which has maximum absorption at 531 nm and is therefore a suitable quencher for the ruthenium chelate fluorophore. Iowa Black RQ has maximum absorption at 656 nm and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 650. In the system described herein, the quencher 248 is initially attached to the functional moiety of the probe' but in other systems the quencher may be a separate molecule that is free in solution. Excitation source-93- 201209159 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 thunder Shoot. Referring to Figure 89, the LED 26 is placed directly over the array of hybrid cells 110 on the outer surface of the LOC device 301. Opposite the chamber of hybrid array 110 is a photosensor 44 made of photodiode array 184 for detecting fluorescent signals from each chamber (see Figures 53, 54 and 64). Other systems for contacting the probe with excitation light are illustrated in Figures 90, 91 and 92. In the LOC device 30 shown in Fig. 90, the excitation light 244 generated by the excitation LED 20 is directed by the lens 254 onto the hybrid cell array IJ 1 10 . The excitation LED 26 is pulsed and the fluorescent emission is detected by the optical sensor 44. 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 optical prism 712 and the second optical prism 714 onto the hybridization cell 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. 92, 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 to 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-PR14-R00 is a suitable source of excitation for Pulsar650 dyes. SET UVT0P3 3 5T039BL LED is a suitable excitation source for the ruthenium chelate label 201209159.

Table 3: Philips LXK2-PR14-R00 LED Specifications

Parameter Symbol Number 单位 Unit Wavelength λεχ 460 nm Transmit frequency Vem 6.52(10)14 Hz Output power Pi 0.515(min) @ ΙΑ W Radiation pattern Lambertian variation profile N/A Table 4 : SET UVT0P334T039BL LED Specifications

Number of parameter symbols 値 Unit wavelength 340 nm Transmit frequency Ve 8:82 (10) 14 Hz Power Pl 0.000240 (minutes) @ 20mA W Pulsed forward current I 200 mA Radiation pattern Lambertian N/A LED driver This LED The time required for driver 29 to drive LED 26 at a constant current. The lower power USB 2.0-certifiable unit draws up to 1 unit load (1 mA) with a minimum operating voltage of 4.4V. Standard power conditioning circuits can be used for this purpose. Photodiode Figure 54 shows the photodiode 184 integrated into the CMOS circuit 86-95-201209159 of the device 301. The photodiode 184 is fabricated as part of a CM0S circuit 816 without additional shadowing 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 such that photons strike the photosensitive surface 185 and are effectively converted into photo-electrons. In the standard process, the quantum efficiency is 0.3 to 0.5 in terms of visible light depending on the method parameters such as the number of cover 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 chirp also determines the size of the photodiode 128, thereby determining the number of hybridization chambers 180 in the hybridization and detection zones 52 (see Figure 52). The size and number of chambers are limited by the size of the LOC device (in the case of LOC device 301, which is 1 760 microns x 5 8 24 microns) and after incorporating other functional modules (such as pathogen dialysis zone 70 and expansion) The technical parameters of the available space of the zone 112). In terms of standard 矽 processing, the photodiode 184 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 a suitable margin of error. -96- 201209159 Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, the autofluorescence, and the remaining excitation photon fluxes that are not fully 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 calibration photodiodes 1 84 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 herein, the low calibration source is provided by a calibration chamber 382 in the hybrid chamber array 110, which: does not contain any probes; includes probes that do not have a fluorescent reporter; or, includes A probe configured to predict the quenching of the reporter and the quencher 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 other hybrid chambers substantially removes the background and leaves the signal produced by the salient light emission (if any). Signals generated from ambient light in the area of the array of the chamber are also subtracted. Referring to Figures 1 0 to 1 1 1 it will be appreciated that the above negative control probe can be used in the calibration chamber. However, as shown in Figures 94 and 95 (which is an enlarged view of the illustrations DG and DH of the LOC variant X 728 shown in Figure 93) 'Another option is to isolate the calibration chamber 3 82 from the amplicon on the fluid. . The background II 移 ® shift can be emptied by the chamber that isolates the fluid, or by a probe containing no reporter of -97-201209159' or even any contamination due to fluid isolation, with a reporter and a quencher The 'normal' probe is used to determine. The calibration chamber 382 can provide a high calibration source to generate 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. 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 107 shows a differential imager circuit 7.8 which is used to correlate the signal from the photodiode 1 84 corresponding to the calibration chamber 382 (this is the result of the excitation light) from the surrounding hybridization chamber. The fluorescent signal of 1 800 is subtracted. The differential imager circuit 788 takes sample signals from pixel 790 and "virtual" pixel 792. The "virtual" pixel 792 is shielded from light in a system so its output signal provides a dark reference. Alternatively, the "virtual" pixel 7 9 2 can be exposed to the excitation light with the remaining array. The signal generated from the ambient light of the array region of the chamber is also subtracted from the "virtual" pixel 7 92 that 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. -98- 201209159 During use, 'read_row' 794 and "read_ are activated, M4 797 and MD4 801 transistors are turned on. 8 09 is turned off, so that the output signals from pixel 790 and "virtual" are stored in pixels respectively. Capacitor 803 and virtual 805. After the pixel signal has been stored, after the switches 8〇7 and 809, the “read_c〇l” switch 811 and the dummy “read_coI are turned off, and the converted capacitor amplifier 8 at the output divides the signal 8 1 7. Contains and enables The photodiode is activated during the excitation of the LED 26 during the period of the photodiode 1 to be activated. Fig. 65 is a single photodiode 184. Figure 66 is a timing diagram of the photodiode control signal. The electrode body 184 and the six MOS electrodes Crystal, Mshunt 3 94 'Mreset 3 98, MSf 400, Mread 402 and Mbias 404. At the beginning of tl, the transistors Mshunt 394 and Mreset 398 are turned on via the Mshunt gate 3 84 and the gate voltage is reset, the excitation photons are The carrier is generated in the photodiode 184. This is removed because the amount of carrier generated may be sufficient to cause the photodiode. During this cycle, Mshunt 3 94 directly removes the carrier generated in the light II, while the Mreset 3 98 resets any electricity. The crystal is accumulated in the node carrier due to the diffusion of the carrier generated by the excitation. After the excitation, the capture cycle starts at t4. The reaction of the fluorophore emission is captured and integrated in the circuit to control _row_d" 7 9 5 Off 807 and ί" pixel 7 9 2 pixel capacitor does not work. However, switch 8 1 3 1 5 amplifies the difference 84, and in the circuit diagram of the firefly, the road has light two, Mtx 396, in the excitation period pole 386 high . During this period, some carriers must be 184 saturated in the body of the polar body 184 or leak in the body; Ν 3,406 during the ring period from ;, NS'406 on -99-201209159. This can be achieved by applying a high voltage to the tx gate 386, since the voltage of the diode 386 can open the transistor M tx 3 9 6 and transfer the carrier on any photodiode 184 to the node 'NS. 406. This capture period can be as long as the fluorophore emission. The output from all of the photodiodes 184 in hybridization 110 is simultaneously captured. There is a delay between the end of the capture cycle t5 and the beginning of the read cycle t6. This delay is due to the need to separately read each of the photodiodes 184 in the array 110 after the capture cycle (see Figure 52). The first taken photodiode 184 has the shortest delay before the reading cycle, while the most dipole 184 has the longest delay before the reading cycle. The transistor Mrea is turned on during the reading cycle by applying a high voltage to the reading gate 3 9 3 (buffering with the source tracker transistor Msf 400 and reading the 'NS' ft voltage. Other alternative ways to enable or suppress the photodiode Discussion 1. Containment methods Figures 104, 105, and 106 show that Mshunt transistor 394 can be configured with 778, 78 0, and 7 82. The Mshunt transistor 394 I VGS | = 5V has a high turn-off ratio during excitation. Startup. As shown in Fig. 104, the Mshunt gate 3 84 is disposed at the edge of the body 184. Alternatively, as shown in Fig. 105, the Msh 3 84 can be configured to surround the photodiode 184. The third option The Mshunt gate 384 is placed in the photodiode 184, as in step 106. Under this third option, the photodiode 1 8 5 is reduced. The hf tx gate accumulates in the array of chambers between the arrays. During the light to be read, the 402 ° i point 406 is as follows: Three can be used at the maximum level of the light dioxin unt smear will be shown in the figure -100- 201209159 These three configurations 778, 780 and 782 are lowered from the light diode 184 The average path length of all locations to the Mshunt gate 384. In Fig. 104, the Mshunt gate 3 84 is on one side of the photodiode 184. This configuration is the easiest to fabricate and has the least impact on the photodiode 185. However, it remains at the far end of the photodiode 1 84. Any carrier on the side will take longer to pass through the Mshunt gate 3 84. In Figure 105, the Mshunt transistor 3 84 surrounds the photodiode 184. This further reduces the carrier in the photodiode 184 to the Mshunt gate The average path length of 3 84. However, extending the Mshunt gate 3 84 to the vicinity of the periphery of the photodiode 184 reduces the photoreceptor surface 185 of the photodiode by more. The configuration 782 in Fig. 106 places the Mshunt gate 3 84 Inside the photosensitive surface 185. This provides the shortest average path length to the Mshunt gate 3 84. Therefore, the transition time is the shortest. However, the impact on the photosensitive surface 185 is the most. It also brings a wider leakage path. Enable Method a. Trigger the photodiode to drive the shunt transistor with 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 galvanic crystal is driven in the manner of 2c but has a programmed delay time. Fig. 68 is a schematic cross-sectional view showing the photodiode 184 and the triggering photodiode 187 embedded in the CMOS circuit 86 through the hybridization chamber U0. A small area in the corner of the photodiode 184 is replaced by a trigger photodiode-101 - 201209159 1 87. Triggering the photodiode 丨87 with a small area is sufficient because the intensity of the excitation light will be high compared to the fluorescent emission. The trigger photodiode 187 is sensitive to the excitation light 2 44 . The trigger photodiode 187 indicates that the illuminator 244 has extinguished and activates the photodiode 184 after a short delay time of ^3 00 (see Figure 2). This delay causes the fluorescent photodiode 184 to detect fluorescent emissions from the FRET probe 186 under the lack of excitation light 244. This will 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 64). The photodiode 184 is a pn junction diode fabricated without additional shielding or steps in the fabrication of the CMOS structure. During MST fabrication, an insulating layer (not shown) on photodiode 180 can be selectively thinned using standard MST photolithography to allow more phosphor to illuminate photosensitive surface 185 of photodiode 184. Photodiode 184 has a field of view such that a fluorescent signal from the probe-target hybrid within hybridization 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 light diodes 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, and is derived as a function of time between time Ο and 〇 by using the ideal fixed intensity Ie pulse -102-201209159. Let [si](() be equal to the density of the excited state at time t, then the number of excited states per unit volume per unit volume during and after excitation is described by the following differential equation: d[s\] dt tf hve Where C is the molar concentration of the fluorophore, ε is the molar extinction coefficient, _Ve is the excitation frequency and h = 6.62606896 (10) _34 JS is the Planck constant. This differential equation has the following general form: y- + p(x)y = q(x) αχ It has the answer:

^P{x)dx q{x)dx + k Now use this formula to solve equation (1) '1>(3) hve now, at time ίι ' [S1] ( (1 ) = 0 ' and from (3 ) ··

Lscrf t ,r

Jc = -"~f-eh'T!...(4) hve Substituting (4) into (3) Just ordering - dividing her -103 ...(5) 201209159 At time:

IeSCT^ leECXf Λ-(/2-/,)/Γ/ -- --—— c hve hve When tg ί 2, the excited state decays exponentially and this can be described by: [51](〇= [51] (/2)β'(,·,ι)/Γ/ (6) Substituting (5) into (6): [51](/) = μ _ e-^lrf y^f (7)

Hye The fluorescence intensity is generated by the following formula: where vf is the fluorescence frequency, η is the quantum yield and 1 is the optical path length. Now, from (7): 4^1](〇__ Iesc dt ~~~h7eV ~ " Substituting (9) into (8):

If (〇= ΙΒεάη^-[\ - e~(,1-,l),T/ ]e-(,Wj)/^ (10) —~— ^ 〇〇If (〇-> ΙεεοΙη —ε~(,~,ϊ)Ιτ, in terms of, Κ Therefore, we can write the following approximation equation, which describes the case where the fluorescence intensity decays after a sufficiently long excitation pulse (〇-h>gt;Tf): Yes, Μ ··_·(11) In the previous paragraph, we conclude that in the case of i2-h>gt; T f, If(t) = Iesc^e-{,~'^ -104- 201209159 From the above formula, we can derive the following: «/(〇= rie£c^e'{,^Vtf ...(12) where...",-7Af) η; (0 — τ v/ is the number of fluorescence per unit time per unit area, and ne = -i- is the number of excitation photons per unit time per unit area. Therefore, 00 nf(t)=\nf(t)dt ...( 13) where \ is the number of fluorescent photons per unit area and ί3 is the instant time when the photodiode is turned on. Substituting (1 2 ) into (1 3 ): 00 nf = ^rieec^e'(l',l) lTf dt (14) h Now, the number of fluorescent photons 'K0' reaching the photodiode per unit time per unit area is generated by: K') = h /(O0〇·,·(15) • where Α is the light collection efficiency of the optical system. Substituting (12) into (1 5 ), we find that ris(t) = φ0ηεε€ΐηβ (,".(16) Similarly, the number of fluorescent photons reaching the photodiode per unit of fluorescence area will be as follows: 〇〇, ί and substituting (1 6 ) and integrating: ns =φϋη€εοΙητ^{Η', ι)ΙΤί Therefore, -105- 201209159 ns =Φ〇ήίεοΙητίβ~&,ΙΤ/ ...(17) /3 The ideal 値 is the ratio of electrons generated by photodiode 184 caused by fluorescent photons and photodiode 184 caused by excited photons When the ratio of generated electrons is equal, the rate at which the excitation photon is attenuated is much faster than the decay rate of the fluorescent photon. The ratio of the sensor output electrons per unit of fluorescence area caused by fluorescence is: β/(ί) = φ / η Μ where is the quantum efficiency of the sensor at the wavelength of the fluorescence. Substituting (1 7 ), you can get: 4 (〇 = MWX... (18) Similarly, the area per unit of fluorescence generated by the excitation photons The sensor output electron ratio is: ee(0 = ^«>"(,",2)/r* -(19) where is the sensor Excitation wavelength quantum efficiency 'and ^ corresponding to an LED excitation "off" time constant of the characteristics. After the time/2, the LED attenuates the photon flux to increase the intensity of the fluorescent signal and prolong its decay time, but we assume that this has a negligible effect on 1<〇. Therefore, we adopt a conservative approach. Now, as mentioned earlier, the ideal of G is: ^(/3) = ^(/3) Therefore, from (18) and (19): & wide, 2) / r / = Wee_ ( ,r'2)/r· -106- 201209159 When rearranging 'we found: ΗεοΙη^1·) ί3 _'2 = jj ...(20)

Tf Te From the previous two sections, we have the following two working equations: Ίή/τ〆...(21) 4〆Δί = -H on...(22) T/ Te where F = £c/t7 and = . It is also known that, when implemented,... t \>> Tf ° The best time for fluorescence detection and the number of fluorescent photons detected using Philips LXK2-PR14-R00 LED and P u 1 sar 6 5 0 dye The optimal detection time is determined using equation (22): Reviewing the concentration of the amplicon and assuming that all amplicons are hybridized, the concentration of the luminescent luminophore is: c = 2.89(10) _6 The height of the moor/liter chamber is the length of the optical path 1 = 8 (10) · 6ιη. We make the area of the fluorescent light equal to the area of our photodiode, but our actual fluorescent area is substantially larger than the area of our photodiode; Therefore, we can roughly assume that the light collection efficiency of our optical system is A = 〇.5. From the characteristics of the photodiode, the quantum efficiency of the photodiode at the fluorescence wavelength is t = 10 at the excitation wavelength. In terms of the ratio of quantum efficiency, nine is a very conservative number 値3 with typical LED decay life S=0·5 and using P Ulsar 650 specification -107- 201209159, can be measured Δί : F = [1.48(10)6][2.89(10)-6][8(10)-6](1) =3.42(10)5 Α. Ιη ([3·42(10)·5](10)(0.5)) Δ/= ϊ---i- 1(10)-factor 0.5(10)-9 =4.34(10)-9 s , each In each of the detected photons, (23), the number of photons emitted by equation (2 1 ) is measured by equation (2 1 ), and the number of excitation photons emitted by the unit is measured by the checksum. ^ ^ Philips LXK2-PR14-R00 LED has Lambertian ( ) radiation mode, so: «/ = «/〇COS(^)

Where S is the number of emitted photons per unit solid angle unit time away from the advancing angle of the LED, and ~ is the forward axis. The total number of photons emitted by the LED per unit time is: ή, = jh]dQ Ω

=Jh;0 cos(0)c/Q Ω (24) Now, ΑΩ = 2π[1 —cos(0 + A0)] — 2;r[l-cos(0)] △Ω = 2;r[ Cos(0) - cos(0 + Δ0)]

/ 4^sin(0)cos V

+ 4^cos(^)sin2 '△θ'l~2~, dQ. = 2ns\n{G)d0 Substituting this into (24) -108- 201209159 ή, = {2^;ocos(0)sin( 0)^ 0 = monument. After rearranging, obtain: 衿 / 〇 = two ... (26) 7 Γ LED output power is 0.515 W and ve = 6.52 (10 Therefore: 4Hz,

• ..(27) __ 0.515 ~[6.63(10)-34][6.52(10)14] = 1.19(10)18 Photon/S Substituting this ( into (2 6 ), obtaining: ...1.19(10)18 n, 〇=~ir~ = 3.79(10)17 Photon/sr Referring to Figure 61, the optical center 252 of the LED 26 is shown in a schematic view. The photodiode is a 16 micron x 16 micron diode in the middle of the array. The solid angle (Ω) of the conical light emitted from the LED 26 to the light is about: Ω = area of the sensor / Γ 2 254 system and the light Body 184 [16 [10) 116(10), 2.825(10)-3]2 = 3.21(10).5 sr It is known that the central light dipole of the photodiode array 44 is intended for use in these calculations. At the Lambert source intensity distribution cube 184, the sensor at the edge of the -109-201209159 array will only receive less than 2% of the number of excitation photons emitted per unit time during hybridization: ne = η, Ω ...(28) =[3.79(10),7][3.21(10)"5] =1.22(10)13 Photon/s Now refer to equation (2 9 ): ns = (0.5)^.22( 10)^1^.42(10)-^^(10)^^-434°°)^1°0^ = 208 Photon/Sensor Therefore, use Philips LXK2-PR14-R00 LED and 65 0 Fluorescent Mission, we can easily detect any hybridization that causes this photon to be emitted. The SET LED illumination geometry is shown in Figure 62. ID = 20 mA, the LED has a minimum optical power output centered at λ6 = 340 nm (铽 chelated wavelength), Ρι = 240μ\ν. In ID: The drive LED will linearly increase the output power to Pl = 2.4m W. The optical center 2 5 2 is placed away from the hybridization chamber array 1 1 0 17.5. We have concentrated this output light flux to a maximum diameter of 2 mm. The photon flux in the 2 mm diameter dot at the far side of the hybridization plane is generated by Equation 27. Photon.

Among the number of Pulsar. In the suction of the object: 200mA will be LED mm, the amount of the dot is borrowed -110- 201209159 ήι =

Pi hve 2.4(10)-3 ~ [6.63(10)-34][8.82(lΟ)14] =4.10(10)15 Photon/s Use equation 2 8 to obtain he = η, Ω 4.10(10)15 [ 16(10)-6]2 π[1(1〇)_3]2 3.34(10)1 丨 Photon/s Now, review Equation 22 and use the previously listed ruthenium chelating properties

At ln[(6.94(10)-s)(10)(0.5)] 1(1〇Γ3 0.5(10)-9 =3.98(10)-9 s Now, from Equation 2 1 : =(0.5)[3.34 (10) π][6.94(10)-5][1(10)-3]£ =11,600 photons/sensors using the theory of SET LED and the number of photons of scorpion scorpion can be very 30 photons. According to the above-mentioned detection by the maximum inter-hybridization between the optical probe and the photodiode (see the background of the invention), this is an important factor for saving time and cost - 3.9_-9/1 (1〇 The hybridization of the Γ3 compound system is detected by Mai Yi, and at least the sensor needs to be reliably detected. The deviation from the detection system needs to be detected by the confocal microscope. Traditional detection requires imaging optics, -111 - 201209159 This imaging optics must use a lens or curved mirror. By using non-imaging optics' the diagnostic system does not require a complex and cumbersome optical series. Place the photodiode very close to the probe It has the advantage of extremely high collection efficiency: when the thickness of the material between the probe and the light-emitting body is 1 micron, the collection angle of the emitted light is up to 173. This angle is calculated by considering light emitted from a probe positioned at the center of mass of the hybridization chamber surface closest to the photodiode having a planar photosensitive surface region parallel to the face of the hybridization chamber. The cone (the light in this cone can be absorbed by the photodiode) is defined by its apex having a transmitting probe and one of the sensors being at the periphery of its plane. For a 16 micron X 1 6 micron sensor, The apex angle of the cone is 17 〇.; the apex angle is 173° when the photodiode is expanded to have an area commensurate with the 29 μm X 19.75 μm hybridization chamber. The separation of the photosensitive surface of a photodiode of 1 micron or less can be easily achieved. The use of a non-imaging optical system does require that the photodiode 184 must be very close to the hybridization chamber to collect sufficient fluorescent emission photons. The maximum spacing between the needles is determined as described below with reference to Figure 5. Using the ruthenium chelate fluorophore and the SET UVT0P3 3 5 T039BL LED, we calculated that 1 1 600 photons were reached from each of the hybrid chambers 1 80 Our 16 micron xl6 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 photoreceptor 185 and that half of the total number of hybrid photons arrives at the photodiode 184. That is, the light collection efficiency of the optical system is = 0.5. -112- 201209159 More precisely, we can write down = [(the bottom area of the light collection area of the hybrid chamber) / (photodiode area)] [ Ω/4π], where Ω = the solid angle of the photodiode at the representative point at the bottom of the hybridization chamber. In terms of the geometry of the pentagonal pyramid: Ω = 4 arcsine (a2/(4dQ2+a2)), where d 〇 = the distance between the chamber and the photodiode, and a is the size of the photodiode. Each hybrid cell released 23,200 photons. The detection threshold of the selected photodiode is 17 photons; therefore, the minimum optical efficiency required is: ^= 17/23200 = 7.33 xl O'4 The bottom area of the light collection region of the hybridization chamber 180 is 29 microns X19. 75 microns. When answering d〇, the maximum limit distance between the bottom of our hybrid chamber and our photodiode 184 will be dQ = 249 microns. In this limitation, the collection cone angle as defined above is only 0.8°. It is important to note that this analysis ignores this negligible refraction. LOC Variants The LOC device 301, described and illustrated above, is just one of many possible LOC device designs. Some possible combinations of LOC device variants using the various combinations of the various functional zones described above from the sample inlet to the detection will now be described and/or illustrated in a flow chart. Where appropriate, the flow chart is divided into a sample input and preparation stage 288, an extraction stage 290, an incubation stage 291, an amplification stage 292, a pre-hybridization stage 293, and a detection stage 294. In all of the brief descriptions or only the LOC variants shown in the schematic form -113-201209159, the complete layout is not shown for clarity and conciseness. In addition, smaller functional units, such as liquid sensors and temperature sensors, have not been shown for clarity, but it is appreciated that these are incorporated into the appropriate locations in the following LOC device designs. The LOC variant X Π LOC variant ΧΠ 758 is shown in Figures 96 to 103. The LOC device extracts 290, incubated 291, expands 292, and detects 294 pathogenic DNA, and uses pre-hybridization purification step 293 to increase hybridization efficiency. A sample (such as whole blood) is added to the sample inlet 68 (see Figure 98) and the capillary action draws the sample to the surface tension valve 1 18. The anticoagulant from the sump 54 is added thereto. The sample continues to flow through the mask channel 94 to the pathogen dialysis zone 70. The dialysis zone 70 has a bypass passage 600 to prevent trapped air bubbles (see Figure 98). After dialysis in the pathogen dialysis zone 70, the red blood cells and white blood cell flow are directed to the waste sump 76, and the pathogens in the sample continue to flow to the surface tension valve 128 where the lysing agent from the sump 56 is added. The sample is drawn into the chemical lysis chamber 130, which is retained by the boiling start valve 206 until the lysing agent has diffused through the sample to release most, if not all, of the pathogen DNA. When the boiling start valve 206 is opened, the sample flows to the surface tension valve 132 where the restriction enzymes, ligase and linker primers from the sump 58 are added. The sample is inoculated into the culture zone Π4 and heated when restriction enzyme digestion of the pathogen DNA and ligation of the linker (see Figure 98-114-201209159). After the restriction enzyme digestion and linker ligation, the boiling start valve 207 is opened for the sample. Flowing into the amplification zone 112. The amplification mixture from the sump 60 is added thereto via a surface tension valve 138 and the polymerase from the sump 62 is added thereto through the surface tension valve 140 as the sample flows into the amplification zone 112. The pathogen DNA is first After amplification via thermal cycling, the boiling start valve 108 opens for the amplicon to flow into the small component dialysis zone 682 where large components are removed (see Figure 98). From Figures 1 01 and 02 Preferably, there is a large component channel 760 between the two small component channels 762 formed in the bottom channel layer 100 of the small component dialysis zone 682 (see Figure 97). The large component channel 760 is A flow port formed by a series of reverse tapered openings 764 (which are smaller at the ends of the large component channels) is connected to the small component channels 762. In most practical applications, the flow port is between 1 and 8 microns wide and 1 to 8 microns high. When the amplicon flows in large Upon component channel 760, a small component (less than the inverse tapered opening 764) begins to diffuse into the small component channel 762. When the liquid stream advances to the downstream end of the small component dialysis zone 682, the large component channel 760 The concentration of the small components is reduced. Another advantage of the micromachined flow port is that the number of flow ports per unit length along the channel is very high, making the separation more efficient. In order to effectively separate the components of the desired size, adjacent The distance between the flow ports is between 1 micrometer and 1 micrometer; in the system shown in Figure 102, the distance between adjacent flow ports is 8 microns. Figure 103 shows the downstream end of the small component dialysis zone 682. The large component passage 760 is transferred into a wide meandering flow ending at the closed end 766. This wide meandering flow provides a waste storage tank. The two small component passages 762 are directed to -115-201209159. The hybrid chamber array 1 1 0 The opposite sides, which here follow the 蜿蜒 path through the array to the respective closed end 768. The small component amplicon is plunged into all individual hybrid chambers prior to the start of the hybridization heater timing, and then probed Needle-target Hybridization detection (as described above) The small component dialysis zone 682 removes cellular debris that may remain in the sample stream after cell lysis. Cell debris may interfere with hybridization efficiency.

LOC Variant XXXIX The LOC variant XXXIX 609 shown in Figure 83 does not lyse cells or use sample dialysis prior to nucleic acid amplification. This LOC device 669 has a contiguous sequence of repeating sequence amplification regions 1 1 2 1 and 1 1 2 · 2 to amplify nucleic acids. The sample is purified by the small component dialysis zone 682 after amplification of the nucleic acid in the nucleic acid amplification zone 1 1 2.2. The small component dialysis zone 682 removes cells, particles and debris larger than a certain size and transfers them to the waste storage tank 766. Smaller sample components, such as dissolved molecules and amplified nucleic acids, flow into the array of hybrid chambers containing sequence-specific probes in each chamber. The light sensor 44 detects light emitted from the probe, and the design of the probe determines the extent to which the light is hybridized. As with all LOC devices, humidifier 1 96 and humidity sensor 232 are used to control evaporation and condensation in LOC variant XXXIX 669, particularly the hybrid chamber array 110.

The LOC variant XL 670 is illustrated in Figure 84 of the LOC variant XL. The LOC device lyses the cells (lysis reagent reservoir 56 and chemical lysis zone 130) to extract genetic material from the -116-201209159 sample cells and then amplifies the nucleic acid in amplification zone 112. After amplification in the repeat region 1 1 2.1 and 1 1 2 · 2, the sample is purified in the small component dialysis zone 6 82 (where the large component is transferred to the waste storage tank 766) and then into the hybridization chamber array 1 1 0 The hybrid is detected by photosensor 44. 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 embodiment of the present invention will now be described with reference to the accompanying drawings, wherein: FIG. 1 shows a test module and a test module reader configured to detect fluorescence; A general schematic diagram of the electronic components in the test module configured to detect fluorescence; FIG. 3 is a general schematic diagram of the electronic components in the test module reader; FIG. 4 is a schematic diagram of the structure of the LOC device Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device with functions and structures from all layers superimposed on each other -117-201209159; Figure 7 is a cover with independent display A plan view of the LOC device of the hood structure; Figure 8 is a top perspective view of the cover with the inner channel and the sump shown in phantom; Figure 9 is a top perspective exploded view of the cover with the inner channel and the sump shown in phantom Figure 10 is a bottom perspective view of the cover showing the configuration of the top channel; Figure 1 is a plan view of the LOC device, showing the configuration of the CMOS + MST device independently; Figure 12 is the LOC device A schematic view of the section at the entrance of the sample; Fig. 13 is an enlarged view of the illustration AA shown in Fig. 6; Fig. 14 is an enlarged view of the illustration AB shown in Fig. 6; Fig. 15 is an illustration shown in Fig. 13. Figure 16 is a partial perspective view showing the thin layer structure of the LOC device in the inset AE; Figure 17 is a partial perspective view showing the thin layer structure of the LOC device in the inset AE; Figure 18 is a view A partial perspective view of the thin layer structure of the LOC device in the illustration AE; Fig. 19 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AE; Fig. 20 is a view showing the thin layer structure of the LOC device in the illustration AE -118-201209159 partial perspective view; 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 lysing reagent storage tank shown in Fig. 21; 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 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; Figure 27 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AB; Figure 28 is a diagram showing the LOC in the illustration AB A partial perspective view of the thin layer structure of the device; Fig. 29 is a partial perspective view showing the thin layer structure of the LOC device in the illustration AB; Fig. 30 is a schematic cross-sectional view of the amplification mixture storage tank and the polymerase storage tank; The characteristics of the boiling start valve are independently displayed; Fig. 32 is a schematic cross-sectional view of the boiling start valve obtained by the line 3 3 - 3 3 shown in Fig. 31; -119- 201209159 Fig. 33 is the picture shown in Fig. 15. An enlarged view of the illustration AF; Fig. 34 is a schematic cross-sectional view of the upstream end of the dialysis zone obtained by the line 3 5 - 3 5 shown in Fig. 33; Fig. 35 is an enlarged view of the illustration AC shown in Fig. 6; Figure 36 is a further enlarged view showing the illustration AC of the amplification region, Figure 37 is a further enlarged view showing the illustration AC of the amplification region; Figure 38 is a diagram showing the amplification region in the AC. Further enlarged view, Figure 39 is the 38th A further enlarged view of the illustration AK shown in the figure, Fig. 40 is a further enlarged view showing the illustration AC of the amplification chamber; Fig. 41 is a further enlarged view showing the illustration AC of the amplification area, the 42nd The figure shows a further enlarged view in 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 further in the illustration AC of the amplification area Figure 45 is a further enlarged view of the illustration AM shown in Figure 44; -120- 201209159 Figure 46 is a further enlarged view showing the illustration AC of the amplification zone; Figure 47 is the 46th A further enlarged view of the illustration AN shown in the figure; · 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; Figure 50 is a further enlarged view showing the illustration AC of the amplification zone; Figure 51 is a schematic cross-sectional view of the amplification zone; Figure 52 is an enlarged plan view of the hybridization zone; Hybrid A further enlarged plan view of the chamber, Fig. 54 is a schematic cross-sectional view of a single hybridization chamber; Fig. 55 is an enlarged view of the humidifier illustrated in the illustration AG shown in Fig. 6; Fig. 56 is a view of Fig. 52 An enlarged view of the illustrated illustration AD; Figure 57 is a perspective exploded view of the LOC device in the illustration AD; Figure 58 is an illustration of a closed configuration FRET probe; Figure 59 is an open and hybrid configuration of the FRET probe Figure 60; Figure 60 is a graph of the intensity of the excitation light over time; Figure 61 is a diagram of the excitation illumination geometry of the hybrid chamber array; Figure 62 is the sensor electronics technology LED illumination geometry (-121 - 201209159

Diagram of Sensor Electronic Technology LED illumination geometry ; Fig. 63 is an enlarged plan view of the humidity sensor shown in the illustration AH of Fig. 6; Fig. 64 is a view showing a part of the photodiode of the photo sensor Schematic diagram of the array; Fig. 65 is a circuit diagram of a single photodiode; Fig. 66 is a timing diagram of the photodiode control signal; Fig. 67 is an enlarged view of the evaporator shown in Fig. 55 of Fig. 55; Figure 68 is a schematic cross-sectional view of a hybridization chamber having a photodiode and a trigger photodiode; Figure 69 is a graph of PCR driven by a linker; Figure 7 is a schematic image of a test module with a lancet Figure 71 is a schematic image of the LOC variant VD architecture; Figure 72 is a plan view of the LOC variant with features and structures from all layers superimposed on each other; Figure 73 is shown in Figure 72. An enlarged view of the illustration CA; Fig. 74 is a partial perspective view showing the thin layer structure of the LOC variant VDI in the illustration CA shown in Fig. 72; Fig. 75 is an illustration of the illustration CE shown in Fig. 73 Magnified view; Figure 76 shows LOC variant V1D Figure 77 is a schematic diagram of the XC architecture of the LOC variant; Figure 78 is a schematic diagram of the XV architecture of the LOC variant; -122- 201209159 Figure 79 is a variant of the XVin architecture of the L〇C variant Fig. 80 is a schematic diagram of the L〇c variant XXII architecture; Fig. 8 is a schematic diagram of the L〇c variant χχν architecture; Fig. 82 is a diagram of the L0C variant XX architecture Figure 83 is a schematic diagram of the LOC variant XXXIX architecture; Figure 84 is a schematic illustration of the LOC variant XL architecture; Figure 85 is a schematic representation of the LOC variant XLI architecture; Figure 86 is a LOC variant Schematic description of the body XLDI architecture; Figure 87 is a schematic illustration of the LOC variant XLIV architecture; Figure 88 is a schematic illustration of the L〇c variant XLW architecture; Figure 89 is for the hybrid chamber array and photodiode Schematic description of the irritating LED; Figure 90 is a schematic illustration of the excitation LED and optical lens used to direct the light to the array of hybrid chambers of the LOC device; Figure 91 is a diagram for directing the hybridization of light to the LOC device Schematic description of the excitation LED, optical lens and optical prism on the array of chambers; Figure 92 is a diagram of the hybridization chamber array for directing light to the LOC device Schematic description of the excitation LED, optical lens and mirror arrangement; Fig. 93 is a plan view showing all the superimposed appearances and showing the position of the illustrations DA to DK; Fig. 94 is the illustration DG shown in Fig. 93 Magnified view; Fig. 95 is an enlarged view of the illustration DH shown in Fig. 93; Fig. 96 is an image of the structure of the LOC variant ;; Fig. 97 is a perspective view of the LOC variant ;; -123- 201209159 Figure 98 is a plan view of the L0C variant χ π, which shows all the superimposed appearances and shows the position of the illustrations FA to FC; Figure 99 is a plan view of the L0C variant ,, which only shows the cover independently. Figure 1 is a plan view of the LOC variant X ,, which independently shows the structure of the CMOS + MST assembly; Figure 101 is an enlarged view of the illustration FA shown in Figure 98; Figure 102放大An enlarged view of the illustration FB shown in Fig. 98; an enlarged view of the illustration FC shown in Fig. 103 and Fig. 98; Fig. 10 shows a system for the splitting transistor of the photodiode» Figure 105 shows a system for a shunt transistor for an optical diode. Figure 106 shows A system for a shunt transistor for a photodiode » Figure 107 is a circuit diagram of a differential imager: Figure 1 08 illustrates the arm-and-loop configuration of a fluorescent probe negative control; Figure 09 is a schematic diagram showing the open configuration of the fluorescent probe negative control group of Figure 1 08; Figure 1 1 0 is a diagram illustrating the arm-and-loop configuration of the fluorescent probe positive control group; The figure illustrates the open configuration of the fluorescent probe positive control group of Figure 110; -124- 201209159 Figure 112 shows the test module and test module reader configured for ECL detection; 3 is a general schematic diagram of the electronic components in the test module, the test module is configured for ECL detection; Figure 1 14 shows the test module and the alternative test module reader; 1 1 5 The figure shows the test module and the test module reader, and the host system for storing various databases; [Key component symbol description] I 〇: Test module II: Test module 1 2: Test module reader 1 3: Shell 14 : Micro-USB Plug 1 5 : Inductor 16: Micro-USB 璋17: Touch Screen 18: Display Screen 19 : Button : Start button 2 1 : Honeycomb radio 22 : Aseptic sealing tape 23 : Wireless network connection 24 : Large container -125 201209159

2 5 : Satellite navigation system 26 : LED 2 7 : Data storage 28 : Mobile phone / smart phone 29 : LED driver 3 0 : LOC device 3 1 : 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

4 0 : Program and data flash memory 4 1 : Excitation recorder 42 : Processor 4 3 : Program memory 44 : Light sensor 4 5 : Indicator 4 6 : Cover 47 : USB power / indicator - Qualification module 48: CMOS + MST device -126- 201209159 49: porous element 5 1 : cover 5 2 : hybridization and detection zone 5 4 : sump 5 6 : sump 5 7 : printed circuit board 5 8 : sump 60.1: Reagent storage tank 60.2: reagent storage tank 62.1: reagent storage tank 62.2: reagent storage tank 64: gasket 6 6 : roof layer 6 8 : sample inlet 7 0 : dialysis zone 72 : waste channel 74 : target channel 76 : waste storage tank 78 : storage tank layer 80: cover channel layer 82: sealing layer 84: 矽 substrate 86: CMOS circuit 87: MST layer 201209159 8 8 : passivation layer 90: MST channel 92: lower pipe port 94: cover channel 96: upper pipe port 9 7: Wall area 9 8 : Anchor of the meniscus 1 〇〇: bottom channel layer 101: laptop/notebook 102: capillary action activation feature 103: dedicated reader 105: desktop computer 106: boiling start valve 107: electronic Book Reader 1 〇8: Boiling Start Valve 1 0 9 : Tablet PC 1 1 〇: Hybrid Chamber Array 1 1 1 : Epidemiology Host system 1 1 2 1 . Amplification area 1 1 2.2 : Amplification area 113: Genetic data host system 1 1 4 · Cultivation area 1 1 5 : Electronic health record (EHR) host system 1 1 6 : Anticoagulant -128- 201209159 1 1 8 : Surface tension 闘 1 1 9 : Sample flow 1 2 0 : Meniscus 121 : Electronic medical record (EMR) host system 1 2 2 : Ventilation hole 123 : Personal health record (PHR) host system 125: Network 126: Boiling start valve 1 28: Surface tension valve 1 3 0 : Chemical cracking chamber 1 3 1 : Mixing zone 1 3 2 : Surface tension valve 1 3 3 : Incubator inlet channel 134: Lower pipe port 1 36 : Optical window 1 3 8 : Surface tension valve 1 4 0 : Surface tension valve 146 : Valve inlet 1 4 8 : Valve outlet 150 : Lower pipe port 152 : Ring heater 153 : Heater connector 1 5 4 : Heater 1 5 6 : Heater connector -129- 201209159 158 : Microchannel 160: Amplification zone outlet channel 1 6 4 : Small 孑匕 166 : Capillary action activation feature 1 6 8 : Dialysis intake port 1 7 0 : Temperature sensor 4 174 : Liquid sensor 1 7 5 : diffusion barrier 1 76 : flow path 1 7 8 : liquid sensor 1 80 : hybridization chamber 1 8 2 : heater 1 84 : light diode 1 8 5 : Photosensitive surface 186: FRET probe 187 : Trigger light diode 1 8 8 : Water tank 190 : Evaporator 1 9 1 : Ring heater 192 : Water supply channel 1 9 3 · Intake port 1 94 : Lower pipe mouth 195: Top metal layer 1 9 6 : Humidifier -130 - 201209159 1 9 8 : Intake hole 202 : Capillary activation feature 204 : Dialysis MST channel 206 : Boiling start valve 207 : Boiling start valve 210 : Microchannel 2 1 2 : MST channel 218 : TiA1 electrode 220 : TiAl electrode 2 2 2 : slit 2 3 2 : sensor 2 3 4 : heater 23 6 : FRET probe 2 3 8 : target nucleic acid sequence 240 : ring 242 : 244 : 246 : 248 : 250 : Arm Excitation Light Fluorescent Group Quencher Fluorescent Signal Fluorescence Emission 2 5 4 : Lens 2 8 8 : Preparation Stage 290 : Extraction Stage 201209159 2 9 1 : Cultivation Stage 292 : Amplification Stage 293 Pre-hybridization filter purification stage 294: detection stage 296: first electrode 298: second electrode 300: programmed delay 30 1: LOC device 328: white blood cell dialysis zone 3 7 6 : conduction column 3 7 8 : positive control Needle 3 80: Negative control probe 3 82 : Calibration chamber 3 84 : Mshunt gate 3 8 6 : t X gate 3 8 8 : Gate 3 9 0: blood collection needle 392: blood collection needle release button 3 93 : reading gate 3 9 4 : transistor M shunt 3 9 6 : transistor M tx 3 9 8 : transistor M reset 4 0 0 · transistor M sf 4 02: Transistor Mread 201209159 404: Transistor Mbias 4 0 6 : Node 'NS 1 408 : Membrane seal 4 1 0 : Membrane shield 5 1 8 : L Ο C Variant Μ 5 94 : Interfacial layer 600: Bypass aisle

602: Interface Target Channel 604: Interface Waste Channel 669: LOC Variant XXXIX 67 3: LOC Variant XLIII 674: LOC Variant XLIV 677: LOC Variant XLVH 6 8 2: Dialysis Step Small Component Dialysis Zone 6 8 6 : Dialysis step 7 1 2 : first optical prism 7 1 4 : second optical prism 728 : LOC variant X 75 8 : LOC variant X Π 760 : large component channel 762 : small component channel 764 : flow port 766: 760 Closed end 201209159 76 8 : Closed end 778: Possible configuration of Mshunt transistor 394 7 8 0 : M shunt transistor 3 9 4 possible configuration 7 8 2 : Mshunt transistor 3 94 possible configuration 7 8 5 : LOC device 7 8 8 : Differential Imager Circuit 7 9 0 : Pixel 792 : "Virtual" Pixel 794 : Read _ Column 795 : Read _ Column _ Virtual 796 : Fluorescent Cluster 7 9 7 : Μ 4 Transistor 798 : Quenched Agent 8 0 1 : MD 4 transistor 8 〇 3 : pixel capacitor 8 05 : virtual pixel capacitor 8 0 7 : switch 809 : switch 811 : "read_col" switch 813 : virtual "read_col" switch 815 : capacitor amplifier 8 1 7: differential signal 8 6 0 : electrode 8 7 0 : electrode -134-

Claims (1)

201209159 VII. Scope of Application for Patention·· 1 · A wafer-on-lab (LOC) device for genetic analysis of a sample containing the nucleic acid sequence of the standard E. The LOC device comprises: a sample inlet for receiving the sample; a sump comprising dNTPs, primers, polymerases and buffers for addition to the sample; a first nucleic acid amplification region for thermally controlling the sample to amplify the target nucleic acid sequence: and, for thermal control from An amplicon of the first nucleic acid amplification region to further amplify a second nucleic acid amplification region of the target nucleic acid sequence. 2. The LOC device of claim 1, wherein the first nucleic acid amplification region is configured for whole genome amplification and the second nucleic acid amplification region is configured to amplify a predetermined nucleic acid sequence. 3. The LOC device of claim 2, wherein the first nucleic acid amplification region is configured for polymerase chain reaction (PCR) amplification and the thermal control comprises thermal cycling of the sample. 4. The LOC device of claim 2, wherein the first nucleic acid amplification region is configured for constant temperature whole genome amplification and the thermal control comprises maintaining the sample at a predetermined temperature. 5. The LOC device of claim 2, wherein the second nucleic acid amplification region is a PCR amplification region configured to amplify a predetermined nucleic acid sequence. 6. The LOC device of claim 1, wherein the first nucleic acid amplification region is configured to amplify a first predetermined nucleic acid sequence, and the second -135-201209159 nucleic acid amplification region is configured to A second predetermined nucleic acid sequence is amplified, the first predetermined nucleic acid sequence being a sub-portion of the second predetermined nucleic acid sequence. 7. The LOC device of claim 1, wherein the reagent storage tank comprises a first reagent storage tank and a second reagent storage tank. The first reagent storage tank contains a pre-amplification addition in the first nucleic acid amplification region. Entering a first dNTP, a scorpion, a polymerase, and a buffer in the sample, and the second reagent storage tank is configured to be added to the first nucleic acid amplification region before being amplified in the second nucleic acid amplification region The second dNTP, primer, polymerase and buffer of the amplicon. 8. The LO C device of claim 7, wherein the reagent reservoirs each have a surface tension valve with a small aperture configured to hold a meniscus in which the liquid reagent remains therein until The meniscus is removed by contact with the sample. 9. The LOC device of claim 1, wherein the first nucleic acid amplification region has a plurality of elongated amplification chambers, each chamber having at least one elongated heater parallel to the longitudinal extension of the amplification chamber. 10. The LOC device of claim 9, wherein the nucleic acid amplification region has microchannels, and the extended amplification chambers are regions of the microchannel. 1 1 The LOC device of claim 10, wherein the microchannel has a curved configuration formed by a series of wide meandering flows, each of which is a channel region forming one of the elongated amplification chambers. 12. The LOC device of claim 9, further comprising a CMOS circuit coupled to the at least one heater for performing thermal control of the sample during amplification. 13. The LOC device of claim 12, comprising at least one temperature sensor coupled to the CMOS circuit to make the at least one heater. 14. The L0C device of claim 13, comprising a probe array for hybridization to the target nucleic acid sequence, a needle-target hybrid. 15. The L0C device of claim 14, which comprises a photosensor for detecting the probe-target hybrid. 16. The L0C device of claim 15 wherein the sensor is a photodiode that is registered with the probe array. 17. The LOC device of claim 16 is the first nucleic acid extension. The volume of liquid in the zone is less than 400 nanoliters. 18. The L0C device of claim 17 of the patent application, wherein the cross-sectional area of the channel and the flow direction is 1 square micrometer square micrometer. 19. The LOC device of claim 13 wherein the channel region has a plurality of heaters, each heater being extended and placed end to end, and the CMOS circuit is configured to operate independently Each of the plurality of elongated heaters. 20. The LOC device of claim 19, comprising a support substrate, wherein the CM〇S circuit is located between the probe array support substrates. Further feedback control further explores further in the light body array, the micro-to-400 end of each of the channel regions is used for further column alignment with the -137-