TW201211534A - Microfluidic device with PCR section and diffusion mixer - Google Patents

Abstract

A microfluidic device having a sample inlet for receiving a sample of biological material having nucleic acid sequences, a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, a reagent reservoir containing a reagent, and, a diffusion mixing section for mixing the nucleic acid sequences with the reagent, the diffusion mixing section having a microchannel defining a tortuous flow-path having a length sufficient for diffusive mixing of the reagent and the sample, wherein during use, the sample flows from the sample inlet to the PCR section via the diffusion mixing section.

Description

201211534 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatments and analysis for molecular diagnostics. [Prior Art] 0 Molecular diagnosis has been used to provide an area for early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the occurrence and improvement of ineffective health care Patient outcome, φ improved disease management and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobases allows for the binding (hybridization) of the synthetic DN(oligonucleotide) short sequences 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 the individual will get in the future, to determine the type of the infectious pathogen and the pathogen, or to determine the individual's response to the drug. Nucleic Acid-Based Molecular Diagnostic Test 201211534 The nucleic acid-based assay has four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine , sputum and tissue samples are used for genetic analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required at the 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 the cellular material but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by differentially lysing the red blood cells in the lysate, 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 diagnostic analysis to be performed. For example, the protocol used to extract viral RN A is quite different from the protocol used to extract the genome 201211534 DNA. However, self-targeting cell extraction of nucleic acids 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 done using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The precipitation step followed by alcohol (usually ice ethanol or isopropanol) Purification of the nucleic acid, either via a solid phase purification step, prior to washing in the presence of a high concentration of chaotropic salt, usually on a cerium oxide matrix, resin or paramagnetic beads in a fractionation column, followed by low ions The strength buffer is eluted. Any step prior to precipitation of the nucleic acid is the addition of a protein-cutting enzyme to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to disrupt thermal lysis of the cell membrane. The target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogen source. Nucleic acid amplification provides the ability to selectively amplify φ (i.e., replicate) a particular target (in the case of a low concentration in terms of detectability). The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The PCR is known in the art and provides a comprehensive and comprehensible description of such reactions in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008. PCR is a useful technique for amplifying target DNA sequences relative to complex DNA backgrounds. If RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, 201211534 was used to amplify the obtained cDNA by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is:

1. Primer pair - a short single strand of DN A having approximately 10-30 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis DNA thermostable enzyme 3. Deoxyribose nucleus Glycoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - the best chemical environment for DNA synthesis. PCR typically involves placing these reagents in small tubes containing extracted nucleic acids (~1 〇·50 microliters). The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocols for each thermal cycle include the denaturing phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension phases are combined. The denatured phase generally comprises heating the reaction temperature to 90-95 ° C to denature the DNA strand; in the adhesive phase, the temperature is lowered to ~50-60 ° C for adhesion of the primer; then in the extended phase, the temperature is raised to The optimal DN A polymerase activity temperature is 60-72 ° C for extension of the primer. This method repeats the cycle approximately 20-4 times, with the end result being a target sequence between the millions of copies of the primer. Variants of many standard PC R protocols for molecular diagnostics have been developed, including, for example, multiple primer set PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR, and Reverse transcriptase PCI^ 201211534 Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single experiment by sub-targeting multiple genes (in other ways, several trials are required). It is more difficult to optimize multi-primer PCR because it requires the selection of primers with approximate adhesion temperature 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. Using a zymase enzyme, a double-stranded oligonucleotide linker (also known as a zygote) having a suitable overhanging end is then ligated to the terminal of the target DN A fragment. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. By this, all fragments of the DN A source adjacent to the 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 PCR reactions are inhibited by many components present in unpurified biological samples, such as the protohemoglobin component in the blood. Traditionally, PCR requires enhanced purification of target nucleic acids prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentration, PCR can be performed or direct PCR can be performed with minimal DNA purification. Modification of PCR chemistries for direct PCR includes polymerases that potentiate buffer strength, quotient activity and progression (p r 〇 c e s s i v i t y), and additives that chelate with potential polymerase inhibitors. -9- 201211534 Tandem repeat PCR utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. One type of tandem repeat PCR is nested PCR in which two pairs of PCR bows are used to perform single locus amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. The second pair of primers (nested primers) used in the second amplification binds to the first PCR product and produces a second PCR product (short in the first PCR product) containing the target nucleic acid. The rationale used in this strategy is that if the wrong locus is amplified 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 low, thus ensuring specificity. Instant PCR or quantitative PCR was used to measure the amount of PCR product in real time. The initial amount of nucleic acid in a sample can be determined by using a fluorophore containing a probe or fluorescent dye and a reference standard in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. » Reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), followed by PCR amplification of cDNA. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. Thermostatic amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DN A during the amplification reaction, thus eliminating the need for complex machinery. The constant temperature nucleic acid amplification method can be performed at the original position or outside the laboratory environment. -10- 201211534 is easy to operate. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification The method of addition has been described. The thermostatic nucleic acid amplification method does not rely on the continuous heat denaturation of the template DN A to produce a single-stranded molecule as a template for further amplification, but relies on an enzymatic cleavage such as a DNA molecule that specifically limits an endonuclease at a normal temperature, Or other methods of using enzymes to separate DNA strands. The ability of strand-substituted amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi-modified DNA, and the ability to rely on 5'-3' exonuclease-deficient polymerases to extend And replace the downstream stocks. An exponential nucleic acid amplification is then achieved by a coupling sense and an anti-sense reaction, wherein the strands from the sense reaction are substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) which produces a nick on one of the DN A strands without cutting DN A in a usual manner is useful for this reaction. SDA has been improved by the use of a combination of a thermostable restriction enzyme ΜναΙ and a thermostable exo-polymerase (5W polymerase). This combination appears to increase the amplification efficiency of the reaction from 108-fold amplification to 101-fold amplification, such that this -11 - 201211534 technique can be used to amplify unique single-copy molecules. Transcription-mediated amplification (TMA) and nucleic acid-dependent sequence amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcription enzyme, 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 extension from the 3' 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 is bound to the DN A copy. A double-stranded DNA molecule is produced by synthesizing a new DNA strand from the end of the primer by a reverse transcriptase. The RNA polymerase recognizes the promoter in the DN A template and initiates transcription. Each newly synthesized RN A amplicon is re-entered and used as a template for new replication. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a specific DNA fragment is achieved by binding the opposite oligonucleotide to template DN A and extending it by DN A polymerase. Denaturation of double-stranded DNA (dsDNA) templates does not require heat. In contrast, RPA utilizes recombinant enzyme-primer mismatches to scan dsDNA and promote share exchange at cognate locations. The resulting structure is stabilized by the interaction of a single strand of DNA-binding protein with a substituted template strand, thereby preventing the primer from being released due to branch migration. The recombinant enzyme decomposes off the 3' end of the oligonucleotide that is accessible to the DNA polymerase (such as a large fragment of Sacί·//«5· Pol I (551/)), and the primer then begins to extend. This step is repeated by loop to achieve exponential nucleic acid amplification. -12- 201211534 Helicase amplification (HD A) mimics the in vivo system, using DNA helicase in an in vivo system to generate a single strand template for primer hybridization followed by extension of the primer with DN A polymerase. In the first step of the HD A reaction, the helicase traverses the target DNA, destroying the hydrogen bonds that bind the two strands, which are then bound by a single binding protein. The single-strand target area exposed by the helicase allows the primer to adhere. DN A polymerase uses free deoxyribonucleoside triphosphate (dNTP) to subsequently extend the 3' end of each primer to produce two DNA copies. The two replicated dsDNA strands independently enter the next HDA cycle, resulting in exponential nucleic acid amplification of the target sequence. Other DNA-based thermostating techniques include rolling cycle amplification (RCA), in which a DNA polymerase continuously extends a primer around a circular DNA template to produce a long DNA product consisting of a number of circular repeat copies. By terminating the reaction, the polymerase produces thousands of copies of the circular template with a copy strand tethered to the original target DN A . This results in a spatial resolution of the target and rapid nucleic acid amplification of the signal. A template of up to 1 copy can be produced in one hour. φ Branched amplification is a variant of RCA and utilizes a closed circular probe (C-probe) or a latching probe and a highly progressive DNA polymerase for exponential amplification at ambient temperature C-probe. Circular thermostat amplification (LAMP) provides high selectivity and utilizes a DNA polymerase and a primer set containing four specially designed primers that identify a total of six different sequences on the target DN A. The intron starting LAMP containing the sense strand of the target DN A and the antisense strand sequence. The subsequent strand-initiated DNA synthesis triggered by the exogenous primer releases a single strand of DNA. This serves as a template for DNA synthesis initiated by the second internal and external primers, and the second inner and outer primers hybridize to the other end of the target -13-201211534 to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the 'inner primer hybridizes to the loop on the product and initiates the replacement DNA synthesis, producing the original stem-loop DNA and the new stem-loop DNA with twice the stem length. The reaction was continued for one hour and accumulated to 109 copies of the target. The final product is a stem-loop DNA having several inverted repeat targets and a cauliflower-like structure having a plurality of loops (formed by alternately opposing reverse repeat targets in the same strand), after completion of nucleic acid amplification, The amplified product is analyzed to determine whether the expected amplicon is produced (the method for analyzing the product of the amplification amount of the target nucleic acid is to simply measure the size of the amplicon by colloidal electrophoresis, and to identify the amplicon nucleoside using DN A hybridization. Acid composition. Colloidal electrophoresis is one of the simplest ways to check whether a nucleic acid amplification step produces the expected amplicon. Colloidal electrophoresis uses an electric field applied to a colloidal matrix to separate DNA fragments. Negatively charged DNA fragments will be at different rates ( Depending on its size, it moves through the matrix. After the electrophoresis is completed, the fragments in the colloid can be stained to make it visible. The fluorinated phenanthrene ingots which are fluorescent under UV light are the most commonly used dyes. Size markers (DNA ladders) are compared to determine the size of the fragment, and the DN A size marker contains a DNA fragment of known size that is run along with the amplicon. The oligonucleotide primer binds to a specific position adjacent to the target DNA, and the size of the amplified product can be predicted and detected using a band of a known size on the colloid. To confirm whether or not the amplicon is produced When an amplicon is used, it is often hybridized with an amplicon using a DN A probe. -14- 201211534 DN A hybridization means the formation of double-stranded DNA by complementary base pairing. The DN for positive recognition of a specific amplification product A hybridization requires the use of a DN A probe of approximately 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DN A sequence, hybridization will result in favorable temperature, pH and ion concentration conditions. If the hybridization occurs, it indicates that the gene of interest or the DN A sequence appears in the original sample. Optical detection is the most common method for detecting hybridization. The labeled amplicon or φ is a probe to emit fluorescence or electrophoresis. Chemiluminescence and luminescence. These methods induce different states of the excited state of the light-generating portion, but both of them also enable covalent labeling of nucleotide strands. In electrochemiluminescence (ECL), when excited by current, Produced by luminophore molecules or mismatches Light. When fluorescing, illuminating with the excitation light that causes the emission. The illuminating source is used to detect the fluorescent light, and the illuminating source provides the excitation light having the wavelength absorbed by the fluorescent molecules and the detecting unit. The detecting unit includes a photo sensor (such as A photomultiplier tube or a charge coupled device (CCD) array) to detect the transmitted signal φ number and to prevent excitation light from being included in the photosensor output mechanism (such as a wavelength-select filter). Responding to the excitation light, the camping light molecule The Stokes-shifted light is emitted, and the emitted light is collected by the detecting unit. The Stokes converts the frequency difference or the wavelength difference between the emitted light and the absorbed excitation light. The detector detects ECL emissions, and the light sensor is sensitive to the emission wavelength of the ECL type used. For example, a transition metal coordination complex emits light of a visible wavelength, and thus a conventional photodiode and C CD are used as photosensors. The advantage of ECL is that if the ambient light is excluded, the ECL emission can be -15-201211534 to detect the only light present in the system, thus increasing sensitivity. The microarray enables hundreds of thousands of DN A hybridization experiments to be performed simultaneously. Microarrays are useful molecular diagnostic tools for screening thousands of genetic diseases or for detecting the presence of several infectious pathogens in a single test. The microarray consists of a number of different DN A probes that are attached to the substrate and are spotted. The target DNA (amplicon) is first labeled with fluorescent or luminescent molecules (during or after nucleic acid amplification) and then applied to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment where hybridization occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. Not being strict enough may result in highly non-specific binding. Excessive rigor may result in inability to properly combine to cause reduced sensitivity. Hybridization is identified by detecting light emission from the labeled amplicon that hybridizes to the complementary probe. Fluorescence from the microarray is detected using a microarray scanner, typically a computer-controlled, inverting scanning fluorescent conjugated focus microscope that typically uses a laser and photosensor that excites the fluorescent dye ( Such as a photomultiplier tube or CCD) to detect the emitted signal. The fluorescent molecules emit light converted by Stokes (as described above), and the light is collected by the detecting unit. The emitted fluorescence must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. A conjugate focal configuration is often used in microarray scanners to remove out-of-focus information by conjugated focal pinholes located in the image plane. This makes it possible to detect only the focused portion of the light. Light that is prevented above or below the focal plane of the object enters the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons of -16-201211534 into electrical energy, which is then converted into a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. More information on fluorescent probes can be found below: I http : //www.premierbiosoft.com/tech_notes/FRET_probe.html and http: //www.invitrogen.com/site/us/en/home/References /Molecular-Probes-The-

Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-

Energy-Transfer-FRET.html In-situ Care Molecular Diagnostics Despite the advantages of molecular diagnostic tests, this type of test in clinical tests has grown less than expected and still only accounts for a small portion of the implementation of laboratory medicine. This φ is mainly attributed to the complexity and cost associated with nucleic acid testing compared to experiments based on non-amino acid methods. The broad applicability of molecular diagnostic tests to clinical treatment is closely related to the rapid and automated analysis that can significantly reduce costs, from the beginning (sample processing) to the end (resulting in results), and the development of instruments that do not require extensive human intervention. For physicians' clinics, proximity or user-based hospitals, home-based in-home healthcare technologies offer the following benefits: • Rapid results to enable rapid treatment and improved care quality -17- 201211534 • Achieved by testing very small samples The ability to test defects. • Reduce clinical workload. • Reduce laboratory workload and increase work efficiency by reducing management efforts. • Improve the cost per patient by reducing hospital stays, getting results from first visits, and simplifying the handling, storage, and delivery of samples 〇 • Promoting clinical management decisions such as vaccination control and antibiotic use. Molecular Diagnostics Based on On-Wafer Laboratory (LOC) Based on methods for providing automated and accelerated molecular diagnostic analysis for molecular diagnostic systems for fluid technology. The shorter detection time is mainly due to the use of very small amounts of automation, built-in and low overhead cascades in the diagnostic method steps in the microfluidic device. The use of nanoliters and micro-upgrades also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are a common form of microfluidic devices. The on-wafer laboratory device has an MST structure in the MST layer to integrate fluid processing into a single support substrate (typically germanium). The use of the VLSI (Ultra Large Integrated Circuit) technology in the semiconductor industry makes the unit cost of each LOC device very low. However, controlling the flow of fluid through the LOC device, adding reagents, controlling reaction conditions, etc. requires a large volume of external piping and Electronic device. Connecting LOC devices to these external devices actually limits the use of LOC devices for molecular diagnostics to inspection processing. The cost of external devices and their operational complexity preclude the use of LOC-based molecular diagnostics as a local care Practical options for processing. -18- 201211534 In view of the above, there is a need for a molecular diagnostic system based on LOC devices for in situ care. SUMMARY OF THE INVENTION Various aspects of the present invention will be described in the following paragraphs. GVA001.1 This aspect of the invention provides a microfluidic device comprising: a φ channel having an inlet, an outlet, and a meniscus holder between the inlet and the outlet such that the liquid flowing from the inlet toward the outlet stops a meniscus holder for forming a liquid meniscus; and an actuator valve having a movable member for contacting the liquid and a thermal expansion actuator for displacing the movable member for the liquid A pulse is generated to dislodge the meniscus, so that the flow of liquid to the outlet is resumed. GVA001.2 Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action φ. GVA001.3 Preferably, the movable member is configured for movement between a static position and an actuated position (moving from a static position), and the meniscus holder is configured to secure the meniscus to the hole Stop the mouth of the liquid flow. GVA001.4 Preferably, the movable member at least partially defines the aperture. GVA001.5 Preferably, the thermal actuator has an impedance element for causing differential thermal expansion to move the movable member. -19- 201211534 GVA001.6 Preferably, the actuator valve reciprocates the movable member between the static and moving positions until the passage immediately downstream of the orifice is sufficiently squeezed to cause the capillary to reestablish the liquid in the flow direction Preferably, the microfluidic device of claim 6 further comprises a support substrate for the channel and the CMOS circuit between the channel and the support substrate for the operative control actuator valve; And at least one sensor responsive to liquid flow, wherein at least one of the sensors feedback controls the CMOS circuit for operative control of the actuator valve; wherein the at least one sensor is a liquid sensor for The presence or absence of liquid at one of the sensing channels. GVA001.8 Preferably, the orifice is a nozzle in the movable member 〇GVA001.9. Preferably, the movable member is a cantilever structure having a nozzle at the free end and an impedance element between the nozzle and the fixed end. It can be manufactured in large quantities. The inexpensive microfluidic device receives the liquid for processing and/or analysis (providing the necessary liquid propulsion via capillary action and providing the necessary valve function via a reliable, easy-to-manufacture thermal-bend-actuated pressure pulse valve) ). The hot bend actuated pressure pulse valve exhibits inherently superior quality of the microfluidic device technology and eliminates the problems of such techniques. GVA002.1 This aspect of the invention provides a microfluidic device comprising: -20-201211534 channel having an inlet, an outlet and a meniscus holder between the inlet and the outlet such that the inlet flows toward the outlet The liquid stops at the meniscus holder of the liquid forming the meniscus; and the actuator valve has a movable member for contacting the liquid, and for displacing the movable member from the static position to the actuated position The actuator, in the actuating position, the meniscus extends to contact the surface downstream of the meniscus holder such that fluid flow to the outlet is resumed. GVA002.2 Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action. GVA002.3 Preferably, the movable member is configured for movement between a static position and an actuated position (moving from a static position), and the meniscus holder is configured to secure the meniscus to the hole Stop the mouth of the liquid flow. GVA002.4 Preferably, the moveable member at least partially defines the aperture. GVA002.5 Preferably, the thermal actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA002.6 Preferably, the opposing side walls of the channel converge to a narrow portion immediately downstream of the movable member such that when the movable member is moved to the actuated position, the meniscus contacts the narrow portion. GVA002.7 Preferably, the microfluidic device of claim 6 further comprises a support substrate for the channel and the CMOS circuit between the channel and the support substrate for the operative control actuator valve. GVA002.8 Preferably, at least one sensor is responsive to liquid flow - 21 - 201211534, wherein at least one of the sensors feedback controls the CMOS circuit for operative control of the actuator valve. GVA002.9 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at one of the channels. GVA002.1 0 Preferably, the orifice is a nozzle in the movable member 〇GVA002.il Preferably, the movable member is a cantilever structure having a nozzle at the free end and having a resistive element between the nozzle and the fixed end大量 A mass-manufactured and inexpensive microfluidic device receives liquid for processing and/or analysis (providing the necessary liquid propulsion via capillary action and providing the necessary valve function via a reliable, easy-to-manufacture thermal bending actuated surface tension valve) The hot bend actuated surface tension valve exhibits inherently superior quality of the microfluidic device technology and eliminates the problems of such techniques. GVA004.1 This aspect of the invention provides a microfluidic device comprising: an inlet for receiving flow through Liquid of the microfluidic device: an outlet downstream of the inlet; and an error-tolerant multiple valve assembly having a plurality of flow paths extending from the inlet to the outlet and a plurality of valves respectively disposed along each flow path. GVA004.2 is preferred Ground, each flow path is configured for a capillary action drive flow of liquid to the outlet, and each valve train is configured to stop flow to the outlet GVA004.3 Preferably, the microfluidic device also has sensors in the respective flow paths of 201211534, the sensors corresponding to the contact with the liquid. GVA004.4 Preferably, the valve system is bent and actuated Valves each having a moveable member configured to move between a static position and an actuated position (moving from a static position); and an orifice at least partially defined by the moveable member' The orifice is configured to stop the capillary action drive flow by fixing the meniscus to the orifice, wherein in use, the movable member moves to the actuating position to release the meniscus from the orifice, thereby restoring flow to the orifice Capillary action drive flow. GVA004.5 Preferably, the bending actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA004.6 Preferably, the movable member is a cantilever structure having a free end The nozzle has an impedance element between the nozzle and the fixed end 〇GVA004.7. Preferably, the valve is a thermally actuated valve, each of which has an orifice for securing the meniscus to stop capillary action a flow orifice φ and a valve heater for heating the meniscus such that the meniscus is released from the orifice to restore the capillary action drive flow toward the outlet. GVA004.8 Preferably, the valve heater is wound around the orifice GVA004.9 Preferably, each flow path has an upstream valve and a downstream valve, and the sensor is located between the upstream valve and the downstream valve" GVA004.1 0 Preferably, the microfluidic device also has a flow path Preferably, the microfluidic device also has a CMOS circuit that is coupled to the sensor to operatively control the valve and detect an error including any upstream -23-201211534 valve failure to stop the flow of liquid. .12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) portion for amplifying the nucleic acid sequence by a denaturation temperature, a binding temperature and a primer extension temperature of the mixed reagent of the thermocycling nucleic acid sequence and the PCR: wherein, the error The multi-valve assembly is tolerated, and during the thermal cycle, the nucleic acid sequence and the mixed reagent of the PCR are maintained in the PCR section. GVA004.1 3 Preferably, the microfluidic device also has a probe array to hybridize with the target nucleic acid sequence to form a probe-target hybrid; the probe array is located downstream of the error-tolerant multiple valve assembly, such that the multiple valves are mis-tolerant The opening of the assembly allows the amplicon from the PCR section to contact the probe. GVA004.1 4 Preferably, the PCR section is configured to generate sufficient amplicons for thermal cycling with more than 1000 probes over 10 minutes. GVA004.1 5 Preferably, the thermal cycle time of the PCR section is between 0.45 seconds and 1.5 seconds. GVA004.1 6 Preferably, the microfluidic device also has a temperature sensor, wherein the PCR portion has at least one heater for thermally cycling the nucleic acid sequence and PCR mixing, and the temperature sensor and the at least one heater are connected to the CMOS circuit Control at least one force heater with feedback. GVA004.1 7 Preferably, the CMOS circuit turns on the fault tolerance valve assembly after a predetermined number of thermal cycles. GVA004.1 8 Preferably, the PCR section has a plurality of elongated PCR chambers having a longitudinal extent that is much larger than the transverse dimension, and a plurality of heaters each extending longitudinally and parallel to the longitudinal extension of the PCR chamber.

201211534 GVA004. Preferably, a plurality of elongated addition operations are performed. GVA004. Preferably, the microfluidic device also has an array for detecting probe hybridization within the probe array. A large-volume and inexpensive microfluidic device can be used for receiving; f-analyzed liquids (providing the necessary liquids via capillary action, reliable, easy-to-manufacture, error-tolerant multiple valve assemblies provide the necessary error tolerance for multiple valve assemblies via individual valve error tolerance Provides the necessary reliability for metering, fabrication and operation; and demonstrates the inherently good quality of the technology and eliminates the problems of these technologies. 1 The present invention is directed to a test module for analyzing, comprising: a housing for carrying a hand; an inlet for receiving a liquid containing a nucleic acid sequence; an outlet downstream of the inlet; and a fault tolerance multiple valve assembly having A plurality of flow paths extending from the inlet and a plurality of valves GVA013 respectively disposed along each flow path. Preferably, each flow path drives the flow through the capillary action of the liquid at the outlet, and the flow of each valve through the outlet until it is opened. GVA013. 3 Preferably, the test module also has a sensor in the flow path, and the sensor corresponds to the contact with the liquid GVA013. 4 Preferably, the valve-bend actuated valve heat exchanger is cooled and/or advanced by a unique optical diode, and the microfluidic device is provided by the desired valve function. The retanning for the gene to the outlet for the flow direction configuration to stop at each of the rafts, each having a movable member -25-201211534, the movable member being configured for the static position and the actuated position ( Movement between the movements from the static position; and an aperture defined at least in part by the movable member, the aperture being configured to stop the capillary action drive flow by fixing the meniscus to the aperture, wherein, in use, The moveable member moves to the actuated position to release the meniscus from the orifice such that the capillary action drive flow to the outlet is resumed. GVA013. Preferably, the bend actuated valve has an impedance element for causing differential thermal expansion to move the movable member. GVA013. 6 Preferably, the movable member is a cantilever structure, • has a nozzle at the free end and has an impedance element between the nozzle and the fixed end 〇 GVA013. Preferably, the valves are thermally actuated valves, each having an orifice, the orifice for holding the meniscus to stop the capillary action drive flow, and the valve heater for heating the meniscus to make the meniscus The surface is released from the orifice and resumes the capillary action driving flow toward the outlet" GVA013. 8 Preferably, the valve heater extends around the periphery of the orifice. GVA013. Preferably, each flow path has an upstream valve and a downstream valve, and the sensor is located between the upstream valve and the downstream valve. GVA013. 10 Preferably, the test module also has two of the flow paths. GVA013. Il. Preferably, the test module also has a CMOS circuit electrically coupled to the sensor to operatively control the valve and detect an error including any upstream valve failure to stop the flow of liquid. GVA013. 12 Preferably, the test module also has a polymerase chain reverse -26-201211534 (PCR) part to amplify the nucleic acid sequence by the denaturation temperature, the bonding temperature and the primer extension temperature of the mixed reagent of the thermal cycle nucleic acid sequence and the PCR. Wherein, the error tolerance multiple valve assembly maintains the nucleic acid sequence and the mixed reagent of the PCR in the PCR portion during thermal cycling. GVA013. Preferably, the test module also has a probe array to hybridize with the target nucleic acid sequence to form a probe-target hybrid; the φ probe array is located downstream of the error-tolerant multiple valve assembly, such that the multiple valve components are mis-tolerant Turning on allows the amplicon from the PCR section to contact the probe. GVA013. Preferably, the PCR moiety is configured to generate a sufficient amplicon for thermal cycling over 10 minutes with more than 1000 probes. GVA013. 15 Preferably, the thermal cycle time of the PCR section is between 0. 45 seconds and 1. Between 5 seconds. GVA013. Preferably, the test module also has a temperature sensor, wherein the PCR portion has at least one heater mixed with a thermal cycle nucleic acid sequence and φ PCR, and the temperature sensor and at least one heater are connected to the CMOS circuit for feedback. Control at least one heater. GVA013. Preferably, the CMOS circuit turns on the fault tolerant valve assembly after a predetermined number of thermal cycles. GVA013. Preferably, the PCR portion has a plurality of elongated PCR chambers each having a longitudinal extension that is much larger than the transverse dimension, and a plurality of heaters each extending longitudinally and parallel to the longitudinal direction of the PCR chamber. GVA013. Preferably, the plurality of elongated heaters are operated independently. -27- 201211534 Diode array biological samples withstand multiple pieces through a necessary temperament and are exempted from microfluids for flow to the liquid level fixed calibrator (stopped for use in liquid flow. The corresponding multi-valve detector corresponding to GVA013 . Preferably, the test module also has an optical train to detect probe hybridization within the probe array. A mass-manufactured and inexpensive genetic analysis test module is received to analyze its nucleic acid content (providing the necessary valve function via a reliable, easy-to-manufacture error valve assembly). The error tolerance multi-valve valve mismatch and valve design, fabrication and operation provide reliability; and the operation shows the inherent advantages of microfluidic device technology. GVA017. 1 This aspect of the invention provides a fault tolerant multiple valve assembly for a device comprising: an inlet for receiving a liquid flowing through the microfluidic device: an outlet downstream of the inlet; a plurality of streams extending from the inlet to the outlet Road; and, a plurality of valves respectively arranged along each flow path" GVA0 17. 2 Preferably, each flow path is driven by a capillary action of the liquid configured to exit, and each valve has a bender, the meniscus holder being configured to cause liquid to flow toward the meniscus E toward the outlet ) forms a meniscus. GVA017. Preferably, each valve has an actuator' meniscus holder moving the meniscus such that the GVA017 is restored toward the outlet. 4 Preferably, the third component of the scope of the patent application further comprises sensors respectively located in the respective flow paths, which are in contact with the liquid. GV AO 1 7. 5 Preferably, the sensors in each flow path are located between a plurality of valves in each of the flows 201211534, and the indication of liquid contact from any of the sensors is used to indicate all upstream of the sensor The meniscus holder in the valve has failed. GVA017. Preferably, the flow path is a passage through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action. GVA017. Preferably, each valve has a movable member to contact the φ liquid, and the actuator is a thermal expansion actuator to displace the movable member to cause pulsation in the liquid to move the meniscus from the meniscus holder, such that Restore the flow of liquid to the outlet. GVA017. Preferably, the movable member is configured for movement between the static position and the actuated position (moving from the static position), and the meniscus holder is configured to secure the meniscus to the orifice Stop the orifice of the liquid flow. GVA017. Preferably, the movable member at least partially defines an orifice. GVA017. Preferably, the thermal actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA017. Preferably, the orifice is a nozzle and the movable member is a cantilevered structure having a nozzle at the free end and an impedance element between the nozzle and the fixed end. GVA017. 12 Preferably, each valve has a movable member to contact the liquid, and the actuator is a thermal expansion actuator to displace the movable member from the static position to the actuated position such that the meniscus extends to the meniscus holder -29- 201211534 Surface contact downstream to restore liquid flow towards the outlet. G V AO 1 7 · 1 3 Preferably, the opposite side walls of the passage converge to a narrow portion immediately downstream of the movable member such that when the movable member is moved to the actuating position, the meniscus contacts the narrow portion. GVA017. Preferably, the moveable member at least partially defines the aperture. GVA017. Preferably, the thermal expansion actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA017. Preferably, the orifice is a nozzle and the movable member is a cantilevered structure having a nozzle at the free end and an impedance element between the nozzle and the fixed end. GVA017. Preferably, each valve valve heater heats the meniscus such that the meniscus is released from the meniscus retainer to restore fluid flow toward the outlet. GVA017. 18 Preferably, the meniscus holder is an orifice, and the valve heater extends around the periphery of the orifice" GVA017. Preferably, the valve heater is configured to cause the liquid to boil at the meniscus retainer and release the meniscus from the meniscus retainer. GVA017. Preferably, the microfluidic device also has a CMOS circuit for operatively controlling the valves and detecting an error including a failure of the upstream valve to stop the flow of the liquid, wherein the valves are supported on the CMOS circuit and each has a direct extension into the CMOS circuit. Electrical contact of the metal layer. The error-tolerant multiple valve assembly provides the reliability necessary for microfluidic valve operation through individual valve error tolerance and valve design, fabrication, and operation; and -30-201211534 demonstrates the inherently superior quality of microfluidic device technology and is exempt These technical issues are oriented. GVA018. 1 This aspect of the invention provides a fault tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising: an inlet for receiving a liquid flowing through the microfluidic device; an outlet downstream of the inlet; extending from the inlet to the outlet a plurality of flow paths; ^ a plurality of valves respectively disposed along each flow path; and a sensor respectively located in each flow path, the sensor corresponding to contact with the liquid. GVA018. 2 Preferably, each flow path is configured for a capillary action drive flow of liquid to the outlet, and each valve has a meniscus holder configured to secure the liquid to the meniscus A meniscus is formed at the device (stopping the flow of liquid toward the outlet). GVA018. Preferably, each valve has an actuator for moving the meniscus from the φ meniscus holder such that the flow of liquid toward the outlet is restored. GVA018. 4 Preferably, the sensors in each flow path are located between a plurality of valves in each flow path, and the indication of liquid contact from any of the sensors is used to represent all valves upstream of the sensor The meniscus holder in the middle has failed. GVA018. Preferably, the valve has a movable member to contact the liquid, and the thermal expansion actuator is adapted to displace the movable member to cause pulsation in the liquid to move the meniscus such that the flow of liquid to the outlet is resumed. GVA018. Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action -31 - 201211534. GVA018. Preferably, the movable member is configured for movement between the static position and the actuated position (moving from the static position), and the meniscus holder is configured to secure the meniscus to the orifice Stop the flow of liquids" GVA018. Preferably, the movable member at least partially defines the aperture. GVA018. Preferably, the thermal actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA018. Preferably, the actuator valve reciprocates the movable member between the static and moving positions until the passage adjacent the orifice is sufficiently squeezed to cause the capillary action to reestablish the flow of liquid in the flow direction 〇 GVA018. Il preferably, the movable member is a cantilever structure having a nozzle at the free end and an impedance element between the nozzle and the fixed end 〇 GVA018. Preferably, each valve has a movable member to contact the liquid, and the actuator is adapted to displace the movable member from the static position to the actuating position 'in the actuated position' such that the meniscus extends in the actuated position with the meniscus Surface contact downstream of the face holder to restore liquid flow towards the outlet, GVA0 1 8.  Preferably, the opposite side walls of the channel converge to a narrow portion immediately downstream of the movable member such that when the movable member is moved to the actuated position, the meniscus contacts the narrow portion. GVA018. Preferably, the meniscus holder is an orifice and the 201211534 valve heater extends around the orifice. GVA018. Preferably, the valve heater is configured to cause the liquid to boil at the meniscus retainer and release the meniscus from the meniscus retainer. GVA018. Preferably, each flow path has an upstream valve and a downstream valve, and the sensor is located between the upstream valve and the downstream valve. GVA018. Preferably, the multiple valve assembly also has both of the flow paths. GVA01 8. Preferably, the microfluidic device also has a CMOS circuit for operatively controlling the valves and detecting an error including a failure of the upstream valve to stop the flow of the liquid, wherein the valves are supported on the CMOS circuit and each has a direct extension into the CMOS circuit. Electrical contact of the metal layer. Wrong resistance. Reliability required for microfluidic valves to be provided by multiple valve assemblies via individual valve error tolerance, optimal control via liquid detector sensor feedback, and valve design, fabrication, and operation; and operation exhibits microfluidic devices The inherent quality of technology and the elimination of the problems of these technologies. GVA019. 1 This aspect of the invention provides a fault tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising: an inlet for receiving a liquid flowing through the microfluidic device; an outlet downstream of the inlet; extending from the inlet to the outlet a plurality of flow paths; and a plurality of valves respectively disposed along each of the flow paths; wherein each valve has a movable member to contact the liquid, and the thermal expansion actuator moves to move the movable member to cause pulsation in the liquid The meniscus, which restores the flow of liquid to the outlet. -33- 201211534 GVA0 19. 2 Preferably, each flow path is configured to drive a wicking drive flow of liquid to the outlet, and each valve has a meniscus holder configured to cause the liquid to be in the meniscus The retainer (stops the flow of liquid towards the outlet) forms a meniscus" GVA019. Preferably, the flow path is a passage through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action. GVA0 19. 4 Preferably, the movable member is configured for movement between the static position and the actuated position (moving from the static position), and the meniscus holder is configured to secure the meniscus to the orifice Stop the orifice of the liquid flow. GVA019. Preferably, the movable member at least partially defines the aperture. GVA019. Preferably, the thermal actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA019. Preferably, the actuator valve reciprocates the movable member between the static and moving positions until the passage immediately downstream of the orifice is sufficiently squeezed to cause the capillary action to reestablish the flow of liquid in the flow direction 〇 GVA0I9. Preferably, the movable member is a cantilever structure having a nozzle at the free end and an impedance element between the nozzle and the fixed end 〇 GVA0 1 9. Preferably, the multiple valve assembly also has sensors located in each of the flow paths, the sensors corresponding to contact with the liquid. -34- 201211534 GVA019. Preferably, the sensors in each flow path are located between a plurality of valves in each flow path, and the indication of liquid contact from any of the sensors is used to indicate all valves upstream of the sensor The meniscus holder in the middle has failed. GVA019. Il Preferably, each flow path has an upstream valve and a downstream valve, and the sensor is located between the upstream valve and the downstream valve. GVA019. Preferably, the multiple valve assembly also has both of the flow paths. GVA019. Preferably, the microfluidic device also has a CMOS circuit for operatively controlling the valves and detecting an error including a failure of the upstream valve to stop the flow of the liquid, wherein the valves are supported on the CMOS circuit and each has a direct extension into the CMOS circuit. Electrical contact of the metal layer. Error Tolerance Multiple Thermal Bend Actuated Pressure Pulse Valve Assembly provides the reliability necessary for microfluidic valve operation through individual valve error tolerance and valve design, fabrication, and operation; and operation exhibits inherent superiority of microfluidic device technology φ Quality and exempt from the problems of these technologies. GVA020. 1 This aspect of the invention provides a fault tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising: an inlet for receiving a liquid flowing through the microfluidic device; an outlet downstream of the inlet; extending from the inlet to the outlet a plurality of flow paths; and a plurality of valves respectively disposed along each flow path; wherein each valve has a meniscus holder and a meniscus repositioning mechanism, and the meniscus holder is configured to form a stop The meniscus-35-201211534 face of the liquid flow toward the outlet, and the meniscus repositioning mechanism are configured to deform the meniscus at the meniscus fixation such that the meniscus contacts the surface downstream of the meniscus fixture and Restore the liquid flow towards the outlet. GVA020. 2 Preferably, the surface downstream of the meniscus holder is configured to stop the capillary action drive flow toward the outlet, and the meniscus repositioning mechanism has a movable member to extend the meniscus from the meniscus holder to the surface . GVA020. Preferably, the meniscus repositioning mechanism has a thermal expansion actuator to displace the movable member. GVA020. Preferably, the flow path is a passage through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action. GVA020. 5 Preferably, the movable member is configured for movement between the static position and the actuated position (moving from the static position), and the meniscus holder is configured to secure the meniscus to the orifice Stop the orifice of the liquid flow. GVA020. Preferably, the movable member at least partially defines the aperture. GVA020. Preferably, the thermal actuator has an impedance element for causing differential thermal expansion to move the movable member. GVA020. Preferably, the movable member is a cantilever structure having a nozzle at the free end and an impedance element GVA020 between the nozzle and the fixed end. Preferably, the opposing side walls of the channel converge to a narrow portion downstream of the 201211534 movable member such that when the movable member is moved to the actuated position, the meniscus contacts the narrow portion. GVA020. Preferably, the multiple valve assembly also has sensors respectively located in each flow path, the sensors corresponding to contact with the liquid. GVA020. Il preferably, the sensors in each flow path are located between a plurality of valves in each flow path, and the indication of liquid contact from any of the sensors is used to represent all valves upstream of the sensor The meniscus in the fixed φ ' has failed. GVA020. Preferably, each flow path has an upstream valve and a downstream valve, and the sensor is located between the upstream valve and the downstream valve. GVA020. Preferably, the multiple valve assembly also has both of the flow paths. GVA020. Preferably, the microfluidic device also has a CMOS circuit for operatively controlling the valves and detecting an error including a failure of the upstream valve to stop the flow of the liquid, wherein the valves are supported on the CMOS circuit and each have a direct extension φ to the CMOS circuit Electrical contact of the metal layer inside. Error Tolerance Multiple Thermal Bend Actuated Surface Tension Valve Assembly provides the reliability necessary for microfluidic valve operation through individual valve error tolerance and valve design, fabrication, and operation; and operation exhibits inherently superior quality of microfluidic device technology and The problem of exempting these technologies is facing. GVA02 1. 1 This aspect of the invention provides a fault tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising: an inlet 'for receiving liquid flowing through the microfluidic device; an outlet downstream of the inlet; -37- 201211534 from the inlet a plurality of flow paths extending to the outlet; and a plurality of valves respectively disposed along each flow path; wherein each of the valves has a meniscus holder for fixing the meniscus to stop the liquid flow, and for heating the bend The liquid level valve heater causes the meniscus to be released from the meniscus holder to restore fluid flow toward the outlet. GVA021. 2 Preferably, the meniscus holder is an orifice and a valve heater extending around the periphery of the orifice. GVA021. Preferably, the valve heater is configured to cause the liquid to boil at the meniscus holder and release the meniscus from the meniscus holder. GVA021. Preferably, the flow path is a passage through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action. GVA02 1. 5 Preferably, the multiple valve assembly also has sensors respectively located in each flow path, and the sensor corresponds to contact with the liquid" GVA021. Preferably, the sensors in each flow path are located between a plurality of valves in each flow path, and the indication of liquid contact from any of the sensors is used to indicate all valves upstream of the sensor The meniscus holder in the middle has failed. GVA021. 7 Preferably, each flow path has an upstream valve and a downstream valve, and the sensor is located between the upstream valve and the downstream valve. GVA02 1. Preferably, the multiple valve assembly also has both of the flow paths. OVA021. Preferably, the microfluidic device also has a CMOS circuit for operatively controlling the valves and detecting an error including an upstream valve failure to stop -38-201211534 liquid flow 'where the valves are supported on the CMOS circuit and each has a direct extension to Electrical contact of the metal layer within the CMOS circuit. The error-tolerant multiple boiling pilot valve assembly provides the reliability necessary for microfluidic valve operation through individual valve error tolerance and valve design, fabrication, and operation; and operation exhibits inherently superior quality of microfluidic device technology and eliminates such techniques The problem is oriented. GMI00 1. 1 This invention provides a microfluidic device, the φ comprising: a sample inlet for receiving a sample of a biological material having a nucleic acid sequence » a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence; a reagent storage tank; and a mixing portion for mixing the nucleic acid sequence and the reagent: wherein, in use, the sample flows from the sample inlet to the PCR portion via the mixing portion. GMI00 1. 2 Preferably, the reagent storage tank has a surface tension φ valve with an orifice, and the surface tension valve is configured to fix the meniscus of the reagent, so that the reagent is held in the reagent storage tank until contact with the sample flow to remove the meniscus so that the reagent Flowing out of the reagent storage tank. GMI001. Preferably, the microfluidic device also has a culture portion downstream of the mixing portion, the culture portion being configured to maintain a mixture of the sample and the restriction enzyme in a culture temperature for limiting shear of the nucleic acid sequence. GMI001. Preferably, the mixing portion is a microchannel defining a winding flow path, and the winding flow path has a sufficient length for diffusion mixing to limit the enzyme to the sample. GMI0 0 1. 5 Preferably, the microchannel has a 蜿蜒 structure - 39 - 201211534 GMI001. Preferably, the cross-sectional area across the flow path is between 20,000 square microns and 8 square microns. GMI001. Preferably, the microfluidic device also has a lysis portion upstream of the mixing portion, the lysis portion being configured to lyse cells within the sample to release the genetic material therein. GMI0 0 1. Preferably, the culture portion has a culture heater configured to heat the nucleic acid sequence and to limit the enzyme to the culture temperature. GMI001. Preferably, the microfluidic device also has a support substrate and a microsystem technology (MST) layer 〇 GMI001 in which a lysis portion, a culture portion, and a PC R portion are formed. Preferably, the microfluidic device also has a CMOS circuit and at least one temperature sensor, the CMOS circuit is located between the support substrate and the MST layer, and the temperature sensor is configured to feedback control the culture heater. GMI001. Il Preferably, the lysis section has an active valve at the downstream end to hold the liquid for a predetermined period of time. GMI001. Preferably, the outlet valve is a boiling pilot valve having a meniscus holder for holding the liquid in the lysis section, the boiling pilot valve having a valve heater for boiling the liquid so that the meniscus is bent The level holder releases and resumes the capillary action drive flow out of the lysis section. GMI001. Preferably, the meniscus holder is an orifice and the valve heater is adjacent to the periphery of the orifice. GMI001. Preferably, the microfluidic device also has a cover covering the MST layer, wherein the cover has a restriction enzyme storage tank, a plurality of PCR reagent storage tanks, and a mixing portion in which 201211534 is formed. GMI001. Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, and the dialysis portion is configured to divide cells larger than a predetermined threshold into a partial sample, and the cells are only cells having less than a predetermined threshold The remainder of the sample is processed separately. GMI001. Preferably, the nucleic acid sequence is derived from a cell that is less than a predetermined threshold. φ GMI001. Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, and the microchannel has a crucible structure formed by a series of wide tortuous lines, and each of the wide tortuous lines forms an elongated PCR chamber. One of the channel departments. GMI001. 18 Preferably, each channel portion has a plurality of heaters 〇 GMI001. Preferably, the microfluidic device also has a hybridization section having a probe array φ for hybridization with a target nucleic acid sequence in the sample; and a photosensor for detecting the probe in the probe array Hybrid. GMI00 1. Preferably, the PCR portion has a thermal cycle time of less than 30 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device receives the fluid-containing sample, then uses the microfluidic mixer, adds and mixes the necessary reagents into the fluid or From the mixture of fluids. The microfluidic coupler is a reliable and easy to manufacture mixer that is integrated into the device, providing a low volume, microfluidic, reliable, easy to assemble and inexpensive system. GMI002. 1 This invention provides a microfluidic device comprising: a sample inlet for receiving a sample of a biological material having a nucleic acid sequence » a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence; a reagent containing a reagent a storage tank; and, a diffusion mixing portion for mixing the nucleic acid sequence and the reagent, the diffusion mixing portion having a microchannel defining a tortuous flow path, the winding flow path having a sufficient length for diffusing the mixed reagent and the sample; wherein, when in use, The sample flows from the sample inlet to the PCR portion via the diffusion mixing portion. GMI002. 2 Preferably, the reagent storage tank has a surface tension valve with an orifice, and the surface tension valve is configured to fix the meniscus of the reagent, so that the reagent is held in the reagent storage tank until contact with the sample flow to remove the meniscus so that the reagent is self-reagent The reagent storage tank flows out. GMI002. Preferably, the microfluidic device also has a culture portion downstream of the mixing portion, the culture portion being configured to maintain a mixture of the sample and the restriction enzyme in a culture temperature for limiting shear of the nucleic acid sequence. GMI002. 4 Preferably, the microchannel has a 蜿蜒 structure. GMI002. Preferably, the cross-sectional area across the flow path is between 8 square microns and 20,000 square microns. GMI002. Preferably, the microfluidic device also has a lysis portion upstream of the mixing portion, the lysis portion being configured to lyse cells within the sample to release the genetic material therein. -42- 201211534 GMI002. Preferably, the microfluidic device also has an anticoagulant storage tank upstream of the lysis unit, wherein the sample is whole blood and the anticoagulant storage tank has a surface tension valve with an orifice, and the surface tension valve is configured to fix the anticoagulant. The meniscus is held while the anticoagulant is held until it contacts the blood to remove the meniscus to add anticoagulant to the blood. GMI002. Preferably, the culture portion has a culture heater, and the culture heater is configured to heat the nucleic acid sequence and limit the enzyme to the culture temperature φ GMI002. Preferably, the microfluidic device also has a support substrate and a microsystem technology (MST) layer 〇 GMI002 in which a lysis portion, a culture portion, and a PCR portion are formed. Preferably, the microfluidic device also has a CMOS circuit and at least one temperature sensor, the CMOS circuit is located between the support substrate and the MST layer, and the temperature sensor is configured to feedback control the culture heater. GMI002. Il Preferably, the lysis section has an active valve φ at the downstream end to hold the liquid for a predetermined period of time. GMI002. 1 2 Preferably, the outlet valve is a boiling pilot valve having a meniscus holder for holding the liquid in the lysis section, the boiling pilot valve having a valve heater for boiling the liquid so that the meniscus is self-contained The meniscus retainer releases and resumes the capillary action drive flow out of the lysis section. GMI002. Preferably, the meniscus holder is an orifice and the valve heater is adjacent to the periphery of the orifice. GMI002. Preferably, the microfluidic device also has a cover covering the MST layer, wherein the cover has a restriction enzyme storage tank, a plurality of PCR reagent storage tanks, and a mixing portion in which -43-201211534 is formed. GMI002. Preferably, the microfluidic device also has a dialysis unit, wherein the biological material comprises cells of different sizes, and the dialysis portion is configured to divide cells larger than a predetermined threshold into a partial sample, and the cells are only cells having less than a predetermined threshold The remainder of the sample is processed separately. GMI002. Preferably, the nucleic acid sequence is derived from a cell that is less than a predetermined threshold. GMI002. Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, the microchannel having a 蜿蜒 structure formed by a series of wide tortuous lines, each of the broad tortuous lines forming an elongated PCR The passage of one of the rooms. GMI002. 1 8 Preferably, each channel portion has a plurality of heaters GMI002. Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridizing to a target nucleic acid sequence in the sample; and a photosensor for detecting the probe in the probe array Hybrid. GMI002. 20 Preferably, the thermal cycle time of the PCR section is less than 30 seconds. The easy-to-use, mass-produced and inexpensive microfluidic device receives the sample containing the nucleic acid, then uses the diffusion mixer, adds and mixes the necessary reagents into the sample, or From the mixture of samples 'then use the PCR chamber of the device to amplify the nucleic acid target in the sample. The diffusion mixer is a reliable and easy to manufacture mixer and is integrated into the package -44-201211534, which provides a low-component microfluidic system that is reliable, easy to assemble and inexpensive. The microfluidic PCR chamber is an easy-to-manufacture PCR circulator and is integrated into the device, thus providing a low-component microfluidic system that is easy to assemble and inexpensive. ^ [Embodiment] ^ General This general specification indicates the main components of the molecular diagnostic system including the specific embodiment of the present invention. The details of the system structure and operation are discussed in the following description. Referring to Figures 1, 2, 3, 128 and 129, the system has the following most important components: The test modules 10 and 11 are of the size of a conventional USB flash drive and can be manufactured inexpensively. Test modules 1 and 11 each contain a microfluidic device, which is typically in the form of a φ laboratory (LOC) device 30 on the wafer and preloaded with reagents, and typically has more than 1000 probes for molecular diagnostic analysis (see Figures 1 and 128). The test module 1 shown in Figure 1 uses fluorescence-based detection techniques to identify target molecules, while the test module 11 in Figure 128 uses electrochemiluminescence-based detection techniques. The LOC device 30 has an integrated photosensor 44 (described in detail below) for fluorescence or electrochemiluminescence detection. Test modules 1() and η both use standard micro-USB connectors 14 for power, data, and control, each having a printed circuit board (PCB) 5, 7 and an externally powered capacitor 32 and inductor 15. Test modules 10 and 11 are for the purpose of mass production only -45-201211534 and are distributed in aseptic packaging for use. The outer casing 13 has a large container 24 for receiving biological samples and a removable sterile sealing strip 22 which preferably has a low viscosity adhesive to cover the large container prior to use. The membrane seal 408 having the membrane shield 410 forms part of the outer casing 13 to reduce the moisture resistance in the test module, while the pressure relief is provided by the small pressure fluctuation. The membrane shield 410 protects the membrane seal 408 from damage. The test module reader 12 supplies power to the test module 1 or 11 via the micro-USB port 16. The test module reader 12 can be in many different forms, and its selection system is described later. The version of the reader 12 shown in Figures 1, 3 and 128 is a specific embodiment of a smart phone. A block diagram of the reader 12 is shown in FIG. The processor 42 executes application software from the program storage 43. The processor 42 is also interfaced with a display screen 18 and a user interface (UI) touch screen 17 and 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 with location data. Alternatively, the position data can be input by the touch screen 17 or the button 19 person. The data store 27 holds genetic and diagnostic information, test results, patient information, analysis and probe data for identifying each probe, and its array position. The data storage 27 and the program storage 43 can be shared by a common memory device. The application software installed in the test module reader 12 provides results analysis and additional test and diagnostic information. To perform a diagnostic test, the test module 1 (or test module 1 1) is inserted into the micro-USB port 16 on the test module reader 12. The sterile sealing strip 22 is turned up and the biological sample (in liquid form) is loaded into the large sample container 24 -46-201211534. The start button 20 is pressed to initiate the test by applying the software. The sample flows into the LOC device 30 and is analyzed for extraction, culture, amplification, and hybridization with the sample nucleic acid (target) with a pre-synthesized hybrid-reactive oligonucleotide probe. In the case of the test module 1 ( (which uses fluorescence-based detection), the probe is fluorescently labeled and the LED 26 placed in the housing 13 provides the necessary excitation light to induce fluorescence emission from the hybridized probe. (See Figures 1 and 2). In test module 1 1 (which uses electrochemiluminescence (ECL) based detection), LOC device 30 carries a φ ECL probe (as described above) and LED 26 is not necessary to generate photoluminescent fluorescing. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 129). The light sensor 44 integrated with the CMOS circuitry on each LOC device is used to detect the emission (fluorescence or photoluminescence). Amplify the detected signal and convert it into a digital output analyzed by the test module reader 12. The reader then displays the result, can store the data locally and/or upload the data to a web server containing the patient record. . The test module reader 1 2 removes the test module 1 〇 or 1 1 and φ treats them appropriately. 1, 3 and 128 show a test module reader 配置 2 configured as a mobile phone/smart phone 28. In other forms, the test module reader is a laptop/notebook computer 1, a dedicated reader 103, an e-book reader 107, a tablet 109 or a table used in a hospital, private clinic or laboratory. The computer is 1〇5 (see Figure 130). The reader can interface with additional applications such as patient records, accounting, online databases and multi-user environments. It can also interface with some local or remote peripherals, such as printers and patient smart cards. -47 - 201211534 Referring to Figure 131, through the reader 12 and the network 125, the data generated by the test module 10 can be used to update the epidemiological database maintained by the host system for epidemiological data ill for genetics. The genetic database maintained by the host system of the data 113, the electronic health record maintained by the host system for the electronic health record (EHR), and the host system for the electronic medical record (EMR) 121 are retained. Electronic medical records, as well as personal health records maintained by the host system for personal health records (PHR) 123. Conversely, epidemiological data held by the host system for epidemiological data 111, genetic data held by the host system for genetic data 113, and electronic use are maintained via the network 1 25 and the reader 12. An electronic health record maintained by the host system of the Health Record (EHR) 15 , an electronic medical record held by the host system for the Electronic Medical Record (EMR) 121, and a personal health record (PHR) The personal health record maintained by the host system of 23 can be used to update the digital memory in the LOC 30 of the test module 10. Referring again to Figures 1, 2, 128 and 129, in the mobile telephone configuration, the reader 12 uses battery power. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be loaded or uploaded via some network or contact interface to enable communication with peripheral devices, computers or online servers. Set the Micro-USB port 16 to connect the computer or main power supply to recharge the battery. Figure 79 shows a specific embodiment of the test module 10 which is used for tests that only require the presence or absence of a particular target, such as whether the test individual is infected with, for example, influenza A virus Η 1 N 1 . It is only suitable as a built-in module 47 for U S B power/indicators. No other readers are required or should be used -48 - 201211534 with software. The indicator 45 on the module 47 of the USB power/indicator only shows a positive or negative result. This configuration is ideal for a large number of screenings. Additional items supplied to the system may include test tubes containing reagents for pre-treating a particular sample, and a tongue and lancet containing sample collection. For convenience, the test module of the embodiment shown in Fig. 79 includes a spring-loaded retractable lancet 3 90 and a lancet release button 3 92. Satellite phones can be used in remote areas. Test Module Electronics Figures 2 and 129 are block diagrams of the electronic components of test modules 10 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a clock 33, a power conditioner 31, a RAM 38, and a program and data flash memory 40. These are provided for test module 10 or 1 1 including photosensor 44, temperature sensor 170, liquid sensor 174 and various heaters 152, 154, 182, 234 as a whole with φ and associated driver 3 7 and 3 9 and the control and memory of the registers 3 5 and 4 1 . Only LED 26 (in the case of test module 10), external power supply capacitor 32 and micro-USB connector 14 are external to LOC device 30. The LOC device 30 includes an adhesive pad for attachment to these external components. RAM 3 8 and program and data flash memory 40 have application software and diagnostic and experimental information for more than 1 000 probes (flash/safe storage, such as via encryption). In the case of the test module 11 configured for ECL detection, there is no LED 26 (see Figures 128 and 129). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 is loaded with an electrochemiluminescent probe and hybridization -49-201211534 chamber, each having an ECL excitation electrode pair 860 and 870. Many types of test modules have been manufactured in some experimental forms, which are ready for ready-to-use users. The test format differs in the reagents and probes of the on board assay. Some examples of infectious diseases that are rapidly identified by this system include: • Influenza and influenza viruses A, B, C, infectious salmon anemia virus, tocovirus • pneumonia-respiratory fusion virus (RSV) 'gland Virus, interstitial pneumonia φ poison, pneumococci, Staphylococcus aureus • tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium vaccae and Mycobacterium vaccae • Plasmodium falciparum Insects, toxoplasma and other parasitic protozoa • Typhoid- typhoid bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever - yellow fever virus • Hepatitis (A to E) • Iatrogenic infections - For example, Clostridium pneumoniae, vancomycin-resistant enterococci, and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) • Encephalitis - Japan Encephalitis virus, Zhangdipula virus • Pertussis-pertussis-50- 201211534 • Measles-paramyxovirus • Meningitis - Streptococcus pneumoniae and meningitis • Anthracnose - Anthrax </ br> <br><br><br><br>: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black Mongolian idiots • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic kidney disease Diseases • Congenital Heart Disease • A few options for cancer identified by the diagnostic system include: • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinoblastoma • Turcot syndrome The above list is not Exhaustive, and diagnostic systems can be configured to detect many different diseases and conditions using nucleic acid and proteomic analysis. Detailed Structure of System Components -51 - 201211534 LOC Device The LOC device 30 is the center of the diagnostic system. It uses a microfluidic platform to rapidly perform the four main steps of nucleic acid-based molecular diagnostic analysis, namely sample preparation, nucleic acid extraction, nucleic acid amplification and detection. The LOC device also has an alternative use and will be described in more detail below. As discussed above, test modules 10 and 11 can take many different configurations to detect different targets. Similarly, the LOC device 30 has many different embodiments for targeting the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a pathogen of a whole blood sample. For purposes of explanation, the structure and operation of the LOC device 301 are described in detail with reference to Figures 4 through 26 and 27 through 57. 4 is a schematic diagram showing the structure of the LOC device 301. For the sake of convenience, the processing stages shown in Fig. 4 are represented by the component symbols corresponding to the functional portions of the LOC device 301 that implements the processing stage. The processing stages associated with the major steps of each nucleic acid-based molecular diagnostic assay also represent: sample input and preparation 288, extraction 290, culture 291, amplification 292, and detection of various reservoirs, chambers, 294, L0C devices 301, Valves and other components will be described more closely below. FIG. 5 is a perspective view of the LOC device 301. It is manufactured using high volume CMOS and MST (microsystem technology) manufacturing techniques. The layered construction of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) of Figure 12. The LOC device 301 has a germanium substrate 84 supporting a COMS + MST wafer 48, including a CMOS circuit 86 and an MST layer 87. Cover 46 covers MST layer 87. For the purposes of this patent specification, the term "MST layer" relates to a collection of structures and layers of a sample treated with different reagents. Accordingly, these structures and components are configured to define a flow path having a characteristic rule of -52 - 201211534 inches that supports a capillary action driven liquid flow having physical properties similar to the physical properties of the sample during processing. Accordingly, MS T layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and assemblies that are sized for capillary action and that are processed to very small volumes. The particular embodiment described in this specification shows that the MST layer is a structure and active component supported on CMOS circuitry 86, but excludes the features of cover 46. However, those skilled in the art will understand that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 microns χ 58 24 microns. Of course, LOC devices made for different applications can have different sizes. Figure 6 shows the features of the MST layer 87 overlaid with the cover features. The inserts AA to AD, AG and AH shown in Fig. 6 are individually enlarged in Figs. 13, 14, 35 '56, 5 5 and 63, and the full φ of each structure in the LOC device 301 is described in detail below. . When Figure 11 shows the CMOS + MST device 48 structure independently, Figures 7 through 10 show the features of the cover 46 independently. Layered Structure Figures 12 and 22 are schematic views of the layered configuration of the CMOS + MST device 48, the cover 46, and the fluid interaction therebetween. The drawings are not drawn to scale for the purpose of illustration. Figure 12 is a cross-sectional view through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12, CMOS + MST device 48 has a germanium substrate 84 that supports CMOS circuitry 86»passivation layer 88 that operates the active components in the -53-201211534 MST layer 87 described above to seal and protect CMOS layer 86 from Fluid flows through the MST layer 87. The fluid flows through both the cap layer 46 and the MST channel layer 100 and the MST channel 90 (see, for example, Figures 7 and 16). When biochemical treatment is performed on the smaller MST channel 90, cell transport occurs in the cover. The larger passage 94 made in 46. The cell delivery channel is sized to deliver the cells in the sample to a predetermined location in the MST channel 90. Cells that deliver a size greater than 20 microns (e.g., certain white blood cells) require channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. The MST channel, particularly at locations in the LOC that do not require delivery of cells, can be significantly smaller. It will be understood that the cover channel 94 and the MST channel 90 are common references and that the particular MST channel 90 can also be due to its specific function ( For example) heated microchannels or dialyzed MST channels. The MST channel 90 is formed by etching through the MST channel layer 100 deposited on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is surrounded by a top layer 66 that forms the top of the CMOS + MST device 48 (relative to the orientation shown in the figure). Although sometimes shown as a separate layer, the cover channel layer 80 and the sump layer 78 are formed from a single piece of material. Of course, the sheet of material can also be non-unitary. The sheets of material are etched from both sides to form a cover channel layer 80 and a sump layer 78, and a cover channel 94 is etched in the cover channel layer 80, and sumpes 54, 56, 58' 60 and 62 are etched in the sump layer 78. Alternatively, the sump and cover channel are formed by micro-forming. Both etching and microforming techniques are used to fabricate channels having a cross-flow of up to 20, 〇〇〇 square microns and as small as 8 square microns. Appropriate choices for the section of the passage through the passage of the fluid are available at different locations in the LOC unit. A large number of samples or samples having a large component are accommodated in the channel, and a cross-sectional area of up to 20,000 square micrometers (e.g., a 200 micrometer wide channel in a layer of 100 micrometers thick) is suitable. A small amount of liquid or a mixture free of large cells is contained in the channel, preferably a very small cross-sectional area across the fluid. The lower seal 64 surrounds the lid passage 94 and the upper seal layer 82 surrounds the sump 54, 56, 580, 60 and 6 2 °. The five sump 54, 56, 58, 60 and 62 are preloaded with reagents for specific analysis. In the embodiments described herein, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant, the selectivity of which includes red blood cell lysis buffer • storage tank 5 6 : lysis reagent • Slot 58: Restriction enzymes, ligases, and junctions (for junction initiation PCR) (see Figure 78, excerpt from T.  Stachan et al. , Human Molecular Genetics 2, Garland Science, NY and London, 1 999)) • Slot 60: Amplification mixture (deoxyribonucleoside triphosphate (dNTP), bow |, buffer), and • Storage tank 62: DNA Polymerase. Cover 46 and CMOS + MST layer 48 are in fluid communication via respective openings in lower seal 64 and top layer 66. The upper conduit 96 and the lower conduit 92 are representative of whether the fluid flows from the MS T passage 90 to the cover passage 94 or vice versa. -55- 201211534 LOC Device Operation The operation of LOC device 301 is described step by step with reference to the analysis of pathogenic DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using a suitable kit or reagent, test protocol, L Ο C variant, and a combination of detection systems. Referring to Figure 4, analyzing a biological sample involves five major steps, including: sample input and preparation 288, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis. 294 〇 sample input and preparation steps 2 8 8 mixed blood The pathogen is separated from the white blood cells and red blood cells by the anticoagulant 1 16 and then by the pathogen dialysis section 7 . As best shown in Figures 7 and 12, blood samples enter the device via sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood stream opens its surface tension valve 118, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). The anticoagulant prevents the formation of blood clots that can block flow. As best shown in Figure 22, the anticoagulant 1 16 is withdrawn from the reservoir 54 by capillary action and enters the M ST channel 90 via the lower conduit 92. The lower duct 92 has a capillary action initiation feature (CIF) 102 to form a meniscus geometry such that it is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent 122 in the upper seal 82 allows air to replace the anticoagulant 1 16 . The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 is filled with a surface tension valve 118 and secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. Prior to use, the meniscus 120 remains solid at 201211534 and is positioned in the upper conduit 96 such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the lid channel 94 to the upper tube 96, the meniscus 120 is removed and the anticoagulant is drawn into the fluid. Figures 15 through 21 show the insert AE which is part of the insert AA shown in Figure 13. As shown in Figures 15, 16 and 17, surface tension valve 118 has three separate MST passages 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can vary φ to change the flow rate of the reagent entering the sample mixture. When the sample mixture and reagents are mixed by diffusion, the flow rate away from the sump determines the concentration of the reagent in the sample stream. Therefore, the surface tension valve of each sump is configured to meet the desired reagent concentration. The blood is passed through the pathogen dialysis section 7 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of orifices 1 64 of a predetermined size. Cells smaller than the valve stomata pass through the orifice, while large cells cannot pass through the orifice. While the target cells continue to be part of the analysis, the undesired cells φ are reintroduced into the waste unit 76. Undesired cells are large cells blocked by an array of orifices 164 or small cells that pass through the orifice. In the pathogen dialysis section 70 described herein, the pathogen from the whole blood sample is concentrated for microbial DN A analysis. The array of orifices is formed by fluidly communicating the input in the lid channel 94 to a plurality of 3 micron diameter orifices 164 of the target channel 74. The 3 micron diameter orifice 164 and the dialysis suction orifice 168 for the target passage 74 are connected by a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The pathogen is small enough to pass through the dialysis MST channel 204 through the 3 micron diameter orifice 164 and to fill the target channel 74. Cells larger than 3 microns, such as red-57-201211534 blood cells and white blood cells, remain in the waste channel 72 of the lid 46, which leads to the waste reservoir 76 (see Figure 7). Other orifice shapes, sizes, and aspect ratios can be used to isolate specific pathogens or other target cells, such as white blood cells for human DNA analysis. More detailed details of the dialysis section and dialysis variants are provided later. Referring again to Figures 6 and 7, fluid is drawn through target channel 74 to surface tension valve 128 in 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 removed from the sample stream, the flow rate of all seven MST channels 90 will be greater than the flow rate of the anticoagulant reservoir 54, wherein the surface tension valve 118 has three MS T channels 90 (assuming the physical properties of the fluid are Roughly equal). Thus the proportion of lysing reagent in the sample mixture is greater than the ratio of anticoagulant. The lysis reagent and the target cells are mixed by diffusion in the target channel 74 in the chemical lysis unit 130. Boiling the pilot valve 126 stops the flow until diffusion and lysis are performed for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). Referring to Figures 31 and 32, the construction and operation of the boiling pilot valve will be described in detail below. Other active valve types (as opposed to passive valves, such as surface tension valve 18) have also been developed by the applicant, which can be used in place of the boiling pilot valve. These alternative valve designs are also described below. When the boiling pilot valve 1 26 is turned on, the lysed cells flow into the mixing portion 131 to pre-amplify restriction digestion and linker ligation. Referring to Fig. 13, when the fluid is removed from the meniscus on the surface of the surface portion of the mixing portion 131, the restriction enzyme, the linker and the ligase are released from the reservoir 58. For diffusion mixing, the mixture flows through the length of the mixing portion 131. At the end of the mixing portion 131 is a lower duct 134 leading to the incubator inlet passage 133 of the culture portion 114 (see Fig. 13). The incubator inlet channel 133 feeds the mixture into the crucible structure of the heated microchannel 210, which provides a culture chamber for retaining the sample during restriction enzyme cleavage and junctional conjugation (see Figures 13 and 14). Figure 23 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the insert AB of FIG. The figures show successive additions to form layers of the CMOS + MST layer 48 and cover 46 structures. The insert AB shows the end of the culture portion 114 and the start of the amplification portion 112. As best shown in Figures 14 and 23, the fluid fills the microchannel 210 of the culture portion 14 until it reaches the boiling priming valve 106 where the fluid stops when diffusion occurs. As discussed above, the microchannel 210 upstream of the boiling pilot valve 106 becomes a culture chamber containing samples, restriction enzymes, binding enzymes, and linkers. The heater 154 is then activated and maintained at a steady temperature to allow the restriction enzyme to shear and the junction to engage for a specific period of time. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is arbitrary and is only required for some types of nucleic acid amplification assays. Further, in some instances, it may be desirable to have a heating step at the end of the incubation period to increase the temperature above the culture temperature. Before entering the amplification section 112, the temperature is increased to inactivate the restriction enzyme and the ligase. Limiting the inactivation of enzymes and ligases has a specific effect when amplified with isothermal acid. After the incubation, the boiling pilot valve 1〇6 is activated (opened) and the fluid re-enters the amplifying portion 112. Referring to Figures 31 and 32, the mixture is filled with the structure of the microchannels ι58-59-201211534 until it reaches the boiling pilot valve 108, which forms one or more amplification chambers. As best shown in the cross-sectional view of Fig. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and the polymerase is then released from the storage tank 62 into the junction culture section and the amplification section (114 and 112, respectively). The middle MST channel 2 1 2 . Figures 35 through 51 show the layers of the LOC device 301 in the insert AC of Figure 6. The figures show successive layers of layers forming the CMOS + MST device 48 and cover 46 structures. The insert AC shows the end of the amplification section 1 12 and the initiation of the hybridization and detection section 52. The cultured sample, amplification mixture, and polymerase flow through the microchannel 15 8 to the boiling pilot valve 108. After diffusion mixing for a sufficient time, the heater 154 in the microchannel 158 is activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify a sufficient target DN Α. After the nucleic acid amplification procedure, the boiling pilot valve 108 is opened and the fluid re-enters the hybridization and detection portion 52. The operation of the boiling pilot valve is described in more detail below. As shown in Fig. 52, the hybridization and detection section 52 has an array 1 10 of hybridization chambers. Figures 52, 53, 54, and 56 show the hybridization chamber array 110 and the individual hybrid chambers 180 in detail. The entrance to hybridization chamber 180 is a diffusion barrier 175 that prevents the target nucleic acid, probe strands, and hybridization probes from diffusing between hybridization chambers 180 during hybridization to prevent erroneous hybridization assay results. The flow path of the diffusion barrier 175 is long enough to prevent the target sequence and probe from diffusing out of one chamber and contaminating the other during the time the probe and nucleic acid hybridize and detect the signal, thus avoiding erroneous results. Another mechanism to prevent erroneous reading is to have the same probe of -60-201211534 in some hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 184 corresponding to the hybridization chamber 180 containing the same probe. The results of the exceptions in the derived single results can be ignored or given different weights. The thermal energy required for hybridization is provided by a CMOS controlled heater 182 (described in more detail below). Hybridization occurs between the complementary target probe sequences after the heater is activated. The LED driver 29 in the CMOS circuit 86 transmits a message to cause the LEDs 26 located in the test module 10 to illuminate. These probes only fluoresce when φ occurs, thus eliminating the cleaning and drying steps that are often required to remove unbound strands. The stem and loop structure of the hybrid forced FRET probe 186 is opened, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as detailed below. Fluorescence is detected by photodiode 184 located in CMOS circuit 86 under each hybridization chamber 180 (see the description of the hybridization chamber below). The photodiode 184 and associated electronics for all of the hybridization chambers together form a photosensor 44 (see Figure 73). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected by the photodiode 184 is φ large and converted into a digital output that can be analyzed by the test module reader 12. Further details of the detection method are described below. Other Detailed Description of LOC Device The modular design LOC device 301 has a number of functional components including reagent reservoirs 54, 56, 58, 60 and 62, dialysis section 70, lysis section 130, culture section 114 and amplification section 112, and valves. Type, humidifier and humidity sensor. In the LOC device of other specific embodiments, these functional portions may be omitted, but additional functional components or functional components different from those of the above-described devices may be added. For example, the culture portion 1 14 can be used as the first amplification portion 11 2 of the tandem repeat amplification analysis system, and the lysis reagent storage tank 56 can be used to add the first amplification mixture of the primer, the dNTP, and the buffer. A reagent reservoir 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 56, or alternatively, the sample may be heated for a predetermined period of time to cause thermal lysis in the culture. In some embodiments, if chemical lysis is desired and the chemical lysis reagent is mixed therewith, additional reservoirs can be combined upstream of the sump for mixing the primers, dNTPs, and buffers. In some cases, the steps such as the culturing step 291 are omitted. In this case, the LOC device can be specially manufactured to avoid the reagent storage tank 58 and the culture portion 11 4 or the storage tank can carry only the reagent, or when the active valve is present, it is not activated to dispense the reagent into the sample stream, and the culture is carried out. The portion is simply a channel for transferring the sample from the lysis unit 130 to the amplification unit 112. The heaters operate independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the fluid system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after the amplification phase 292, prior to the hybridization and detection step 294, dialysis is performed to remove cell debris. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplification moieties can be combined to enable simultaneous amplification of multiple targets prior to detection or -62-201211534 in a hybrid chamber array 110 using a particular nucleic acid probe. To analyze, for example, a sample of whole blood in which dialysis is not required, the dialysis portion 70 is simply omitted from the sample input and preparation portion 28 of the LOC design. In some cases, even if the analysis does not require dialysis, it is not necessary for the LOC device to omit the dialysis section 70. If the presence of the dialysis section does not cause geometrical obstruction, the sample input and preparation section can still have the LOC of the dialysis section 70 without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to a protein present in the non-amplified sample, rather than being designed to A nucleic acid probe that hybridizes to a target nucleic acid sequence. It will be appreciated that the LOC devices manufactured for use with this diagnostic system are different from the combinations of functional components selected for the particular LOC application. The vast majority of functional units are common to many LOC devices, and the design of additional LOC devices for new applications has functional components that are appropriately combined in the configuration of the large functional options used in existing LOC devices. Only a few LOC devices are shown in this description, and some others are shown to illustrate the design flexibility of the LOC devices manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices shown herein are not exhaustive, and that many additional LOC designs are related to the combination of appropriate functional components. Sample Type LOC Variants can accept and analyze various samples in liquid form. Types of nucleic acid or protein content, including but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, cord blood-63-201211534, breast milk, sweat, pleural effusion, tears , pericardial fluid, peritoneal fluid, environmental water samples and dip samples. Amplicon derived from meganucleic acid amplification can also be analyzed using a LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, and only use dialysis, lysis, The culture and amplification section delivers the sample from the sample inlet 68 to the hybridization chamber for nucleic acid detection 180, as described above. For some sample types, a pre-treatment step is required, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and possibly pre-treat the mucus with enzyme to reduce stickiness. Sample Input A sample is added to the large container 24 of the test module 10 with reference to Figures 1 and 12'. The large container 24 is a truncated cone 'which is fed into the inlet 68 of the LOC unit 301 by capillary action. Here, it flows into the 64 μm wide 60 μηι deep cover channel 94 and is also attracted to the anticoagulant reservoir 54 by capillary action. Reagent Tank The small amount of reagent required for the analysis system using a microfluidic device such as the LOC device 310 allows the reagent reservoir to contain all of the necessary reagents for biochemical treatment, and each reagent reservoir is in a small volume. This volume is indeed less than 〗 〖〇〇〇, 〇〇〇, 〇〇〇 cubic micron' in most cases is less than 300, 〇〇〇, 〇〇〇 cubic micron 'normal less than 70,000,000 cubic microns, and In the case of the LOC device 301 shown in the formula, it is less than 2 Å, 〇〇〇, 〇〇〇 cubic micrometers. 201211534 Dialysis Department Referring to Figures 15 to 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate pathogen target cells from the sample. As previously described, the top layer 66 has a plurality of orifices 316 of a diameter of 3 microns that filter the target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice 164, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via a 16 [mu]π dialyzed extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) of φ is retained in the cover channel 94. Downstream of the pathogen dialysis section 70, the cover channel 94 becomes a waste channel 72 to the waste reservoir 76. Foam inserts or other porous elements 49 in the outer casing 13 of the test module 10 are configured to be in fluid communication with the waste reservoir 76 (see Figure 1) for a biological sample type that produces a substantial amount of waste. The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Figure 33) such that fluid is drawn down into the dialysis MST channel 204 below φ. The first extraction aperture 198 for the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus over the dialysis extraction aperture 168. The small component dialysis section 682, which is schematically shown in Fig. 99, may have a structure similar to the pathogen dialysis section 70. The small component dialysis section separates any small target cells or molecules from the sample by sizing (and shaping, if necessary) to allow the small target cells or molecules to pass to the target channel and continue the analysis of the orifice. Large size cells or molecules are removed to the waste reservoir 766. Thus, LOC device 30 (see Figures 1 and 128) is not limited to isolation of pathogens having sizes less than 3 μπι -65 - 201211534, but can be used to isolate cells or molecules of any desired size. Lysis section Referring again to Figures 7, 11, and 13, the genetic material in the sample by chemical lysis is released from the cell. As described above, the lysis reagent from the lysis tank 56 is mixed with the sample flowing in the target channel 74 downstream of the surface tension valve 128 of the lysis tank 56. However, some diagnostic assays preferably use hot lysis treatment, Or even a combination of chemical and thermal lysis of target cells. The LOC device 301 houses the heated microchannels 210 of the culture portion 114. The sample stream is flooded with the culture portion 114 and stopped at the boiling pilot valve 106. The culture microchannel 210 heats the sample to a temperature at which the cell membrane ruptures. In some hot lysis applications, the enzyme reaction is not required in the chemical lysis unit 130, and the hot lysis completely replaces the enzyme reaction in the chemical lysis unit 130. Boiling Pilot Valve As discussed above, the LOC device 301 has three boiling pilot valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling pilot valve 108 on the heated microchannel 158 side of the amplifying portion 112. By capillary action, the sample stream 1 19 is attracted along the heated microchannel 15 8 until it reaches the boiling pilot valve 108. The meniscus 120 at the leading edge of the sample stream is secured to the meniscus holder 98 of the valve inlet 146. The meniscus holder 98 causes the meniscus to stop moving forward to prevent capillary flow. As shown in Figures 31 and 3, the meniscus holder 98 is a conduit on the orifice provided by the opening of the pipe from the MST passage 90 to the -66-201211534 of the cover passage 94. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 152 is located around the valve inlet 146. The ring heater 152 is controlled by cM〇S via boiling of the valve heater contact I. To open the valve, the CMOS circuit 86 sends an electrical pulse to the valve heater contact 153. The ring heater 1 52 is electrically resistively heated. Until the liquid sample n9 boils, the boiling causes the meniscus 120 to be removed from the valve inlet 146 and begin to wet the lid channel 94. Once the lid passage 94 is wetted, the capillary action is restored. The fluid sample 119 is filled with the passage 94. And flowing through the lower valve line 15 to the valve outlet ι48, wherein the capillary-driven liquid flow advances along the amplification outlet channel 160 into the hybridization and detection portion 52. The liquid sensor 174 is placed for diagnostic purposes. Before and after the valve, it will be understood that once the boiling pilot valve is opened, it is impossible to close it again. However, since the LOC device 301 and the test module 10 are single-purpose devices, it is not necessary to close the valve. Nucleic Acid Amplification Section Figures 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51 show the culture section 114 and the amplification section 112. The culture section 114 has a single, heated culture microchannel 2 1 0, which is etched and becomes The conduit opening 134 to the 蜿蜒 pattern in the MST channel layer 100 of the boiling pilot valve 106 (see Figures 13 and 14). Controlling the temperature of the culture portion 141 enables a more efficient enzyme reaction. Similarly, amplification The portion 2 has a heated microchannel 158 (see FIGS. 6 and 14) heated from the boiling pilot valve 1〇6 to the boiling pilot valve 108 (see FIGS. 6 and 14), mixed, cultured, and nucleic acid amplified. Upon occurrence, the valves stop flow to retain the target cells in the heated culture or amplification microchannels 210 or 158. The microchannel channel pattern also promotes (to some extent) the target cells to mix with the reagents. In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated via a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. The heated culture microchannel 210 and the expansion Each of the turns of the microchannel 158 has three independently operable heaters 15 (extending between the individual heater contacts 156 (see Figure 14)) which provide input heat flux density Two-dimensional control. As best shown in Figure 51, the heater 154 is supported by The top layer 66 is embedded in the lower seal 64. The heater material is TiAl, but many other conductive metals are also suitable. The elongated heater 154 is parallel to the longitudinal length of each channel portion forming a wide meandering meandering flow. In the amplification section 112, each wide stream of music can be manipulated as an independent PCR chamber via individual heater control. Using a microfluidic device, such as the LOC device 301, the small volume of amplification required by the analysis system The subsequence allows amplification of the small volume of the amplification mixture in the amplification section 1 1 2 . This volume is less than 400 nanoliters, in most cases less than 170 nanoliters, typically less than 70 nanoliters, and in the case of LOC device 301, this volume is between 2 nanoliters and 30 nanoliters. . Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a relatively short distance from the heater 1 54. The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 100,000 square microns, while the 201211534 has a significantly higher heating rate than the "large scale" device. The lithography manufacturing technique allows the amplifying microchannel 158 to have a cross-section that provides a higher heating rate substantially less than 16,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small amount of amplicons are required (as is the case in the L〇C device 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1,000 to 2,000 probes on a LOC device and "sample entry, answer out" within 1 minute, the cross-sectional area of the appropriate φ across the fluid is 400 square microns and 1 square. Between microns. The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate of greater than 80 absolute temperatures (K) per second, in most cases at a rate of greater than 100 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than one, 〇〇〇 K per second, and in many cases, the heater element heats the nucleic acid sequence at a rate greater than 1 每秒, 〇〇〇 K per second. Typically, based on the requirements of the analytical system, the heater elements are greater than 100,000 κ per second, greater than 1,000,000 K per second, greater than 10,000,000 K per second, greater than φ 20,000,000 K per second, greater than 40,000,000 K per second, per second. The nucleic acid sequence is heated at a rate greater than 80,000,000 K and greater than 1 60,000, 0 〇〇 K per second. A small cross-sectional area channel is also beneficial for the diffusive mixing of any reagent with the sample fluid. The diffusion of one liquid to another is most pronounced near the interface between the two liquids before the diffusion mixing is completed. The density of the phenomenon decreases with distance from the interface. A microchannel with a relatively small cross-sectional area across the direction of the fluid is used while maintaining the flow of the two fluids at the interface for rapid diffusion mixing. Reducing the channel cross-section to less than 100,000 square microns results in a significantly higher diffusion rate than the "large gauge -69-201211534 mode" device. The lithography manufacturing technique allows the microchannels to have cross sections that traverse less than 1 6,000 square microns and substantially provide a higher mixing rate. If only a very small amount of amplicons are required (as is the case in LOC unit 3〇1), the cross section can be reduced to less than 2,500 square microns. For the diagnostic analysis required for "sample entry, answer out" in 1 minute on a LOC device, the appropriate cross-sectional area across the fluid is 400 square microns and 1 square micron. between. The short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid, resulting in rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (β卩, denaturing, adhesion, and primer extension) was completed in 30 seconds for a target sequence of up to 150 base pairs (bp) long. In most diagnostic analyses, individual thermal cycle times are less than 11 seconds and most are less than 4 seconds. For a target sequence of up to 1 50 base pairs (bp) long, the thermal cycle time of the LOC device 30 for some of the most common diagnostic analyses is zero. 45 seconds to 1. Between 5 seconds. This rate of thermal cycling allows the test module to perform nucleic acid amplification procedures in far less than 10 minutes; often within 220 seconds. For most analyses, the amplification section produces sufficient amplicons from the sample fluid entering the sample inlet within 80 seconds. For most of the analysis, sufficient amplicons were generated within 30 seconds. Upon completion of the predetermined number of amplification cycles, the amplicon is fed to the hybridization and detection section 52 via the boiling pilot valve 108. Hybridization Chamber - 70 - 201211534 Figures 52, 53, 54, 56 and 57 show hybridization chamber 180 in hybridization chamber array 110. The hybridization and detection unit 52 has a 24 X 45 array 1 10 of hybridization chambers 180 each having a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. Photodiode 184 is incorporated to detect fluorescence from a target nucleic acid sequence or protein that hybridizes to FRET probe 186. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. For the emitted light, any material between the FRET probe 186 and the photodiode 184 must be transparent φ. Therefore, the wall portion 97 between the probe 186 and the photodiode 184 must also be optically transparent to the emitted light. In the LOC device 301, the wall portion 97 is a thin layer of ruthenium dioxide (about 0. 5 microns). Direct intrusion of photodiode 184 beneath each hybridization chamber 180 allows the use of a very small volume of probe-target hybridization while still producing a detectable fluorescent signal (see Figure 54). A small volume of hybridization chamber can be used because of the small amount. Prior to hybridization, the amount of probe required for detectable probe-target hybridization is greater than 270 picograms (corresponding to 900,000 cubic micrometers), and in most φ cases less than 60 picograms (corresponding to Up to 200,000 cubic microns, typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and less than 2. in the case of the LOC device 301 shown in the figures. 7 picograms (corresponding to a chamber with a volume of 9,0 cubic micrometers). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes on the LOC device. In the LOC device 301, the hybridization portion has more than 1,000 in an area of 1,500 micrometers by 1,500 micrometers. Individual chambers (ie, each chamber is less than 2,250 square microns). The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of the small number of probes required by each chamber is that during the manufacture of the LOC device, only -71 - 201211534 is required to configure a very small amount of probe solution into each chamber. A specific embodiment of the LOC device according to the present invention may be configured with a probe solution of 1 nanoliter or less. After nucleic acid amplification, the boiling priming valve 108 is activated and the amplicon flows along the flow path 176 and flows into each hybrid. Room 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates when the hybridization chamber 180 is flooded with the amplicon and the heater 182 can be activated. After sufficient hybridization time, LED 26 is activated (see Figure 2). The opening in each hybrid chamber 180 is provided with an optical window 136 to expose the FRET probe 186 to the excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a sufficiently long period of time to induce a high intensity fluorescent signal from the probe. The photodiode 184 is shorted during excitation. After a preprogrammed delay of 300 (see Figure 2), under no excitation light, The light diode 184 and the detection of fluorescent emission. The incident light on the active region 185 of the photodiode 184 (see Figure 54) is converted to a photocurrent that can be measured using a CMOS circuit 86. Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. If desired, each hybridization chamber 180 can carry probes that detect more than 1,000 different targets. Alternatively, many or all of the hybridization chambers may carry the same probe that repeatedly detects the same target nucleic acid. Copying the probes in this manner in the hybridization chamber array 110 increases the confidence of the results obtained, and if desired, all results can be combined by photodiodes of adjacent hybridization chambers to obtain a single result. Those skilled in the art will appreciate that there may be from 1 to over 10,000 different probes on the hybrid chamber array 110, depending on the analytical details. Humidifier and Humidity Sensor -72- 201211534 The insert AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of evaporation of reagents and probes during operation of the LOC device 301. As best shown in the enlarged view of Fig. 55, the water sump 188 is fluidly coupled to the three evaporators 190. The water storage tank 188 is filled with molecular biological grade water and is sealed during manufacture. As best shown in Figures 55 and 76, by capillary action, water is drawn to the three lower conduits 194 and along the individual water supply passages 192 to the three upper conduits 193 of the evaporator 190. The meniscus is fixed to each of the upper φ pipes 193 to retain water. The evaporator has a ring heater 191 which surrounds the upper pipe 193. With a thermally conductive column 376, a ring heater 191 is coupled to the CMOS circuit 86 to the top metal layer 195 (see Figure 37). At startup, the annular heater 191 heats the water causing the water to evaporate and wet the surrounding equipment. The position of the humidity sensor 232 is also shown in FIG. However, preferably, as shown in the enlarged view of the insert AH shown in Fig. 63, the humidity sensor has a capacitive comb structure. The lithographically etched first electrode 2 96 and the lithographically etched second electrode 298 are opposed to each other such that their teeth are interleaved. The φ opposing electrode forms a capacitor having a capacitance that can be monitored by the CMOS circuit 86. As the humidity increases, the dielectric constant of the air gap between the electrodes increases, causing the capacitance to increase. Humidity sensor 232 abuts hybridization array 1 10 (the most important humidity measurement location) to slow the evaporation of the solution containing the exposed probe. Feedback Sensor The temperature and liquid sensor system is integrated into the LOC unit 3 0 1 to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors - 73 - 201211534 170 are assigned to all of the amplification section 112. Similarly, the culture unit 114 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control the thermal cycling during nucleic acid amplification as well as any lysis during hot lysis and culture. In hybridization chamber 180, CMOS circuit 86 uses hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of the hybrid heater 182 is temperature dependent, and the CMOS circuit 86 uses this to drive the temperature of each hybrid chamber 180. The LOC device 301 also has some MST channel liquid sensors 174 and a lid channel liquid sensor 208 » 35 is shown on the line of the MST channel liquid sensor 174 at one of the ends of each of the heated microchannels 158. Preferably, as shown in FIG. 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the current between the electrodes to indicate their presence at the sensor. Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. Opposite TiAl electrode pairs 218 and 220 are deposited on top layer 66. A gap 222 is provided between the electrodes 218 and 220 to keep the circuit open in the absence of liquid. The circuit is closed when the liquid is present and the CMOS circuit 86 utilizes this feedback to monitor the flow. The GRAVITATIONAL INDEPENDENCE test module 10 is self-directed. It does not need to be fastened to a smooth surface to operate the fluid flow driven by capillary action and the lack of external piping to the auxiliary equipment, making the module truly portable and easy to insert into a similar -74- 201211534 Portable handheld readers, such as mobile phones. The gravity autonomous operation represents that the test module is also acceleration independent of all practical ranges. It is shock and vibration resistant and can be operated on a moving carrier or on a mobile phone that is carried. Valve Selection Thermal Bend Actuated Valve Variant 1 Figures 64 and 65 show a first variant 302 of a hot bend actuated valve that is a heat φ bend actuated pressure pulse valve. Figure 65 is a schematic cross-sectional view taken along line 70-70 of Figure 64. The first variant hot bend actuated valve 312 has a movable member in the form of a CMOS activated thermal bend actuator 404 constructed of TiAl, TiN or a similar electrical resistance heater material. The sample stream enters the valve inlet 146 in the MST channel 90, but stops when the meniscus 120 is secured to the orifice 306. The embodiment shown in Figures 64 and 65 shows an aperture outside the movable member, but it can also be at least partially defined by the movable member. In this state, the valve is closed. CMOS circuit 86 transmits a series of electrical pulses to thermal bending actuator φ 304 to open the valve. The CMOS activated thermal bending actuator 306 is coupled to the cantilever portion 162 of the top layer 66 (which is fixed at the inlet end and free at the orifice end). The differential thermal expansion bends the cantilever portion 1 62 such that it moves rapidly toward the passivation layer 88. The fluid draw prevents the liquid sample 119 from flowing back, while the liquid 1 19 is ejected into the cover channel 94 via the orifice 306. The cantilever portion 162 is moved back and forth to the static and displaced position for a period of time to ensure that the meniscus 120 is released from the aperture 306. The sample 119 is accumulated in the lid passage 94 until its surface is wetted and the capillary action drives the flow back. The sample cartridge fills the passage 94 and then flows through the under-valve conduit 150 to the valve outlet 148. -75- 201211534 Hot Bend Actuated Valve Variant 2 Figures 66 and 67 show a second variation of the hot bend actuated valve 308 which is a thermally flex actuated surface tension valve. Figure 67 is a schematic cross-sectional view along line 72-72 shown in Figure 66. The second variant hot bend actuated valve 308 has a CMOS activated thermal bending actuator 304 comprised of TiAl, TiN or similar resistive heater material. The sample flows along the MST passage 90 and flows into the valve inlet of the valve upper conduit 151. 146. The liquid sample 119 is filled with the passage 94 but stops when the meniscus 120 is fixed to the orifice 306. In this state, the valve is closed. To open the valve, CMOS circuit 86 transmits a series of electrical pulses to thermal bending actuator 304. The CMOS activated thermal bending actuator 308 is coupled to the cantilever portion 162 of the top layer 66 (which is fixed at the inlet end and free at the orifice end). The differential thermal expansion bends the cantilever portion 162 such that the aperture 306 moves toward the passivation layer 88. The orifice 306 draws the meniscus 120 from the lid channel 94 into the MST channel 90 to reestablish the capillary flow. To reestablish the capillary flow, the opposing sidewalls of the channel converge to a narrow portion downstream of the movable member such that when the movable member is moved to the actuated position, the meniscus contacts the narrow portion. The liquid sensor 1 74 is placed before or after the debug valve. Thermal Bend Actuated Valve Variant 3 Figures 68 and 69 show a third variant of the hot bend actuated valve 312, which is a thermal bending actuated surface tension valve, and for the liquid sample to be retained in the cover channel 94. . Figure 69 is a schematic cross-sectional view along line 74-74 shown in Figure 68. The third variant of the hot book actuating valve 312 is similar to the second variant hot bend actuating 201211534 valve 3 08 except that the valve inlet 146 is located in the cover channel 94. The sample flows along the cover channel 94 and flows into the valve inlet 146 that is immediately upstream of the CMOS activated thermal bending actuator 304. The liquid sample 119 is filled with the passage 94 but stops when the meniscus 120 is fixed to the orifice 306. In this state, the valve is closed. To open the valve, CMOS circuit 816 transmits a series of electrical pulses to thermal bending actuator 304. The thermal bending actuator 304 is coupled to the cantilever portion 162 of the top layer 66 (which is fixed at the inlet end and free at the orifice end). The differential thermal expansion bends the cantilever portion 162 such that the aperture 306 moves toward the passivation layer 88. The orifice 306 draws the meniscus 120 from the lid passage 94 into the MST passage 90 to reestablish the capillary flow along the valve outlet 148. The liquid sensor 1 74 is placed behind the debug valve. Error Tolerant Multiple Valve Array Figure 70 shows an error tolerant multiple valve array 314 that can be used in place of any of the aforementioned valve variants 108, 302, 308, and 312. Figure 71 is a cross-sectional view of the error-tolerant multiple valve array 314 taken along line 76-76 of Figure 70. In the valve variant described above, the sample has the risk of not being able to be secured to the orifice 306 (or the valve inlet 146 in the case of a boiling pilot valve) and the capillary flow is easily continued to the valve outlet 14 8 . In fact, the valve cannot be closed. Conversely, the valve cannot be opened (ie, the meniscus is not released when the valve is actuated). In response to this, the error tolerance multiple valve array 314 is error tolerant and allows one or more valve errors. The fault tolerant multiple valve array 314 has four individual valves; a first valve 316, a second valve 318, a third valve 320, and a fourth valve 322. All of the valves are of the second variant 308 of the aforementioned thermal bending actuated valve (Hot Bend Actuator Table 201211534 Face Tension Valve). The first flow path 324 and the second flow path 326 extend between the valve inlet 146 and the valve outlet 148. The first and second valves (316 and 318) are disposed along the first flow path 324 and the third and fourth valves (3 20 and 3 22) are located on the second flow path 326. Liquid sensor 174 is between valve inlet 146 and valve outlet 148, and between two valves in each flow path. The sensor indicates whether the first, second, third or fourth valve has failed. If the first or third valves (316 and 320) are unable to secure the meniscus, the second and fourth valves (318 and 322) stop the sample 119. Similarly, if the meniscus cannot be released during the actuation of the first and third valves, an alternative flow path can be used. The fault tolerant multiple valve array 3 14 is considered to be ineffective only when both of the first or second flow paths (324 or 326) fail (almost impossible). In order to provide greater error tolerance, the number of fluid flow paths of the error-tolerant multi-valve array 31 4 or the number of valves of each flow path can be arbitrarily increased. Boiling Pilot Valve Array Figures 80 and 81 show another variation of the error-tolerant multiple valve array 448, with four individual valves all being boiling pilot valves. Regarding the mechanical error tolerance multiple valve array 314, it has a single valve array inlet 46 and a valve outlet 148. Valve inlet 146 and valve outlet 148 are formed in MST channel layer 100 and each have a respective liquid sensor 174 to provide feedback to CMOS circuit 86. The flow at the valve inlet 146' branches into a first flow path 324 and a second flow path 326, both extending between the valve inlet 146 and the valve outlet 148. The first and second boiling pilot valves (4 12 and 416) are disposed along the first flow path 324, and the third and fourth boiling pilot valves (4 14 and 4 18) are attached to the second flow path 3 26 . -78 - 201211534 The liquid sensor 174 is also placed between the two boiling pilot valves in each flow path. The sensor indicates whether the first, second, third or fourth valve has failed. If the first or third boiling pilot valves (4 12 and 4 14) fail, the sample flow is likely to be limited by the second and fourth boiling pilot valves (4 16 and 4 18). Error Tolerance The multiple valve array 448 is considered to be inactive only when both of the first or second flow paths (324 or 326) fail (almost impossible). To provide greater error tolerance, the number of fluid flow paths for the error-tolerant multiple valve array 448 φ or the number of valves for each flow path can be arbitrarily increased. If no boiling pilot valve fails during valve operation, the flow from valve inlet 146 fixes the meniscus in valve upper conduits 42 8 and 43 0 of first boiling pilot valve 412 and third boiling pilot valve 414, respectively. To turn on the boiling pilot valves 4 12 and 4 14, the CMOS circuit 86 delivers a start pulse to the boiling of the two valves to activate the valve heater contact 153. The heaters 152 of the two boiling pilot valves 412 and 414 heat the liquid of the meniscus to a temperature above boiling. The meniscus of the two valve upper pipes 42 8 and 430 is released due to boiling, so that the capillary action φ turbulent flow respectively covers the cover channels 420 and 422 » the under-valve pipes 432 and 43 4 are configured to not fix the meniscus And the capillary action drive flow is stopped. The flow entering the cover channels 420 and 422 continues into the first flow path MST channel 424 and the second flow path MST channel 426 through the under-valve pipes 432 and 434, respectively. The capillary action drive flow continues to the second and fourth boiling pilot valves. 41 6 and 418. Similarly, the flow fixes the meniscus to the valves 436 and 438 of each of the boiling pilot valves, respectively. The liquid sensor 174 in each of the MST channels 424 and 426 provides feedback to the CMOS circuit 86 which thus calculates the start pulse time for the valve -79 - 201211534 heater 152. As with the first and third boiling pilot valves 412 and 414, the heaters 152 in the second and fourth boiling pilot valves 416 and 418 are actuated to cause the liquid at the meniscus to boil and release the meniscus from the upper conduits 436 and 438. The capillary action drive flow into the cover channels 440 and 442 is followed by a capillary action drive flow through the lower conduits 444 and 44, respectively. The flow along the first flow path 3 24 coincides with the flow along the second flow path 326 coincides with the valve outlet 148 at which the liquid sensor 174 indicates that the CMOS circuit 86 is incorrectly resistant to the valve array 448 being opened. The pop-up valve diagram 122 is a plan view of the electric blast valve 71. The sample flows along the MST passage 90 and into the valve inlet 146 formed by the valve upper conduit 151. The liquid sample 119 is filled with the valve interface chamber 73, and when the meniscus 120 is fixed to the orifice 306, it stops at one side of the top film 75. In this state, the valve is closed. As the CMOS circuit 86 forces current through the resistor 77, it blasts along the respective resistance points of the highest current density 79 to break the top rigid junction 81 connected to the film 75 to fix the perimeter. Since the membrane 75 is unsupported and displaced, the valve opens and the liquid sample 119 flows from the valve inlet 146 to the valve outlet 148 » Hot Bending Actuated Bending and Brake Valves Figure 123 shows the bending of the hot bend actuation and the plan view of the brake valve 83. The sample flows along the MST passage 90 and into the valve inlet 146 formed by the valve upper conduit 151. The liquid sample 119 is filled with the valve interface chamber 73, and when the meniscus 120 is fixed to each of the orifices 306, it stops at one side of the top cantilever portion 162 and connects the film 75 to -80-201211534. In this state, the valve is closed. From the CMOS circuit 86 and advancing to a series of electrical pulses of the thermal bending actuator 404, the differential thermal expansion forces the top cantilever portion 162 (engaged with the thermal bending actuator 304) to bias the passivation layer 88, damaging the connecting film 75 and its periphery. Weak top layer rigid joint 81. As the membrane 75 is unsupported and displaced, the valve opens and the liquid sample 1 19 flows from the valve inlet 146 to the valve outlet 148. φ Double hot bending actuated bending and brake valve Fig. 124 shows the bending of the double thermal bending actuation and the plan view of the brake valve 85. The sample flows along the MST passage 90 and into the valve inlet 146 formed by the valve upper conduit 151. The liquid sample 119 is filled with the valve interface chamber 73, and when the meniscus 120 is fixed to each of the orifices 306, it stops at one side of the two top cantilever portions 162 and connects the film 75. In this state, the valve is closed. Upon receipt of the electrical pulse from CMOS circuit 86, the two thermal bending actuators 304 are joined to the top cantilever portion 162 due to differential thermal expansion and are biased toward the passivation layer 88, thereby breaking the rigid junction 81 of the φ film 75 with its periphery. As the membrane 75 is unsupported and displaced, the valve opens and the liquid sample 119 flows from the valve inlet 14 6 to the valve outlet 148. Viscous Valve Figure 125 shows a plan view of the viscous valve 89. The sample flows along the MST passage 90 and into the valve inlet 146 formed by the valve upper conduit 151. The liquid sample 119 is filled with the valve interface chamber 73, and when the meniscus 120 is fixed to each of the orifices 306, it stops at one side of the top cantilever portion 162 and connects the membrane 75. In this state, the valve is closed. From the CMOS circuit 86 and advancing to the thermal bending actuators 3 04 -81 - 201211534, a series of electrical pulses, with differential thermal expansion, forces the top cantilever portion 162 (engaged with the thermal bending actuator 304) to bias the passivation layer 88, forcing the film 75 Contact with the passivation layer 88. Due to the viscous bond between the membrane 75 and the passivation layer 88, the return force is restored and the deformation of the flexible coupling 91 allows for a restorative displacement of the cantilever portion 162, the valve is statically maintained open to allow the liquid sample 119 to flow from the valve inlet 146 to the valve. Exit 148. Viscous Valve Variant Figure 126 shows a plan view of the viscous valve variant 93. The sample flows along the MST passage 90 and into the valve inlet 146 formed by the valve upper conduit 151. The liquid sample 119 is filled with the valve interface chamber 73, and when the meniscus 120 is fixed to each of the orifices 306, it stops at one side of the top cantilever portion 162. In this state, the valve is closed. A series of electrical pulses from CMOS circuit 86 and advanced to thermal bending actuator 304 are used to force differential thermal expansion to force top cantilever portion 162 (engaged with thermal bending actuator 304) to bias and contact passivation layer 88. Due to the viscous relationship between the cantilever portion 162 and the passivation layer 88, the restoring force is offset and the cantilever portion 162 is sufficiently long to make its mechanical flexibility allow the restorative displacement valve of the thermal bending actuator 300 to be statically maintained open. Liquid sample 119 flows from valve inlet 146 to valve outlet 148. BUBBLE BREAK VALVE A plan view of the defoaming valve 95 is shown in FIG. The sample flows along the MST channel 90 and flows into the valve inlet 146 formed by the valve upper conduit 15丨. The liquid sample 119 is filled with the valve interface chamber 73, and when the meniscus 120 is fixed to the orifice 306, it stops at one side of the top film 75. In this state, the valve is closed. The ring heater 152 is resistively energized by electrical pulses from the CMOS circuit 86 until a vapor bubble is created in the liquid sample to force the film 75 to rotate and break the top rigid joint 81. As the membrane 75 is unsupported and displaced, the valve opens and the liquid sample 119 flows from the valve inlet 146 to the valve outlet 148. Other Valve Variants Any of the valve variants described above can be used to form a valve array. In addition, the valve array can include different types of valves. Dialysis variants White blood cell targets The dialysis design in the LOC device 301 described above targets the pathogen. Figure 72 is a schematic cross-sectional view of a dialysis section 328 designed for human DNA analysis to concentrate white blood cells from a blood sample. In addition to restricting the white blood cells from the apertures of the 7-5 micron diameter apertures 165 from the cover channel 94 to the dialysis MST channel 204, it will be understood that the structure is substantially identical to that of the pathogen φ target dialysis section 70 described above. In the case where the sample to be analyzed is a blood sample and there is hemoglobin from the red blood cells to interfere with the subsequent reaction steps, the addition of red blood cell lysis buffer and anticoagulant to the storage tank 54 (see Figure 22) will ensure that most of the dissolution is ensured. The red blood cells (and hemoglobin) of the cells will be removed from the sample during the dialysis step. The commonly used red blood cell lysis buffer is 0. 15M NH4CL, 10mM KHC03' O. lmM EDTA, pH 7. 2-7. 4. However, those skilled in the art will appreciate that any buffer that can use effective lysed red blood cells 〇 leukocyte dialysis unit 328, downstream of the cover channel 94, becomes the target channel 74-83-201211534, making leukocyte splicing a part of the analysis. Again, in this case, the dialysis suction port 168 leads to the waste channel 72 to remove all of the smaller cells and components in the sample. It should be noted that this dialysis variant only reduces the concentration of undesired samples in the target channel 74. Figure 100 shows schematically a large component dialysis section 686 which also separates any large target components from the sample. For further analysis, the orifices in the dialysis section are fabricated in a manner that is sized and shaped to block large target components of interest in the target channel. And in the above-described leukocyte dialysis section, most (but not all) of smaller sized cells, organisms or molecules flow into the waste storage tank 768. Therefore, other embodiments of the LOC device are not limited to separation sizes greater than 7. 5 micron white blood cells, but can be used to isolate cells, organisms or molecules of any desired size.

Nucleic Acid Amplification Variants Direct PCR Traditionally, PCR requires extensive purification of target DNA prior to preparation of the reaction mixture. However, by appropriately changing the chemical and sample concentrations, nucleic acid amplification can be performed using a minimum amount of DNA purification, or direct amplification can be performed. When nucleic acid amplification is performed by PCR, this method is called direct PCR. When nucleic acid amplification is carried out at a normal temperature controlled in a LOC apparatus, this method is direct constant temperature amplification. Direct nucleic acid amplification techniques have considerable advantages when used in LOC devices, especially with regard to the simplification of the desired fluid design. Direct PCR_ is an amplification technique for direct isothermal amplification that includes increasing buffer strength, using highly active and highly progressive polymerases, and adding to potential polymerase inhibitors -84-201211534. It is also important to dilute the inhibitor in the sample. To exploit direct nucleic acid amplification techniques, LOC devices are designed to incorporate two additional features. The first feature is a reagent reservoir (eg, sump 58 in Figure 8) that is appropriately sized to supply a sufficient amount of amplification reaction mix or diluent such that the final concentration of sample components that may affect the amplification chemistry is sufficient Low to successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LOC structure, such as the pathogen dialysis section 70 of Fig. 4, is used. In this particular embodiment (further illustrated in Figure 6), the concentration of the pathogen sufficient to enter the amplification portion 2 92 is effectively concentrated upstream of the sample extraction portion 290 and the larger cells are discharged to the waste container 76. Dialysis department. In another embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of channel φ to adjust the mixing ratio between the sample and the amplified mixture. For example, to ensure that the dilution of the inhibitor associated with the sample via a single mixing step is in the range of preferably 5 to 20 times, the length and cross section of the sample and reagent channels are designed such that the sample channel upstream of the mixing start position The composition of the flow group is 4 to 1 times higher than that of the channel through which the reagent mixture flows. The flow group resistance in the microchannel is easily controlled by controlling the design. For a constant cross-sectional area, the flow resistance of the microchannel increases linearly with the length of the channel. It is important for the hybrid design that the flow group resistance in the microchannel depends more on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannel of the square section -85-201211534 is inversely proportional to the cube of the smallest vertical dimension. Reverse Transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RNA, such as from RNA virus or messenger RNA, RNA must be reversed as complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction (one-step RT-PCR) can be carried out in the same chamber as the PCR, or it can be a separate initial reaction (two-step RT-PCR). In the LOC variant described herein, a reverse transcription step can be performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the stylized heater 154 to successively perform the nucleic acid amplification step, and simply perform a step. RT-PCR. The buffer, the primer, the dNTP, and the reverse transcriptase are stored and distributed by the reagent storage tank 58, and the culture unit 14 is used for the reverse transcription step, and then amplified in an ordinary manner in the amplification unit 112. The two-step RT-PCR is simply done. Constant temperature nucleic acid amplification For some applications, the preferred nucleic acid amplification method is constant temperature nucleic acid amplification, so that it is not necessary to repeatedly circulate the reaction components at various temperature cycles, but the amplification portion is maintained at normal temperature, usually about 37 ° C. To 41 ° C. Some methods for thermostatic nucleic acid amplification have been described, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), uncoupling thermostated DNA Amplification (HDA), rolling cycle amplification (RCA), branched amplification (RAM), and circular thermostat amplification (LAMP), and any or other isothermal amplification methods of this type can be used specifically for LOC loading 201211534 is set forth in the specific embodiment. To perform a constant temperature nucleic acid amplification, reagent reservoirs 60 and 62 adjacent to the amplification section will carry appropriate reagents for a particular constant temperature method rather than carrying PCR amplification and polymerase. For example, for SDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reagent reservoir 62 contains appropriate endonucleases and exo-DNA polymerases. For RPA, reagent reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins, and reagent reservoir 62 φ contains strand-substituted DNA polymerase, such as. Similarly, for HDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reservoir 62 contains the appropriate DNA polymerase and helicase (rather than using heat) to unwind the double stranded DNA. Those skilled in the art will appreciate that the necessary reagents can be dispensed into two reagent reservoirs in any manner suitable for nucleic acid amplification. For amplification of viral nucleic acids, such as HIV or hepatitis C virus, NASBA or TMA is appropriate because it does not require transcription of the RNA to cDN A first. In this example, the reagent reservoir 60 is filled with amplification buffer, φ primer and dNTP, and the reagent reservoir 62 is filled with RNA polymerase 'reverse enzyme and any RNase Η. For some thermostatic nucleic acid amplification types, an initial denaturation cycle must be employed to separate the two-strand DN Α template before maintaining the temperature of the thermostated nucleic acid amplification for the reaction to continue. Since the temperature of the mixing in the amplification section 112 can be tightly controlled by the heater 154 in the amplification microchannel 158, this denaturation cycle can be easily accomplished in all of the specific embodiments of the LOC device described herein (see Figure 14). 〇 Constant temperature nucleic acid amplification is more tolerant to potential inhibitors in the sample -87-201211534 and is therefore generally suitable for direct nucleic acid amplification of the desired sample. Thus, constant temperature nucleic acid amplification is particularly useful for LOC variant XLIII 673, LOC variant XLIV 6 7 4 and L Ο C variant X L V 11 677, respectively, shown in Figures 101, 102 and 103. Direct thermostatic amplification can also be combined with one or more preamplification dialysis steps 70, 686 or 682 as shown in Figures 101 and 103 and/or a pre-hybridization dialysis step 682 as shown in Figure 1-2. Partial concentration of the target cells in the sample is facilitated prior to nucleic acid amplification, respectively, or unwanted cell debris is removed before the sample enters the hybridization chamber array 110. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be used. Thermostatic nucleic acid amplification can also be performed in parallel amplification sections, such as those described in Figures 96, 97, and 98. Multiplex and some thermostatic nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify rna. Additional Details of the Fluorescence Detection System Figures 58 and 59 show hybridization-reactive FRET probes 236. These are often referred to as molecular beacons and are stem and/or loop probes produced by single-stranded nucleic acids and fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 23 6 prior to hybridization to the target nucleic acid sequence 238. The probe has a ring 240, a stem 242, a fluorophore 246 at the 51 end, and a quencher 248 at the T end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are bonded together to form stem 242. In the absence of a complementary target sequence, as shown in Figure 58, the probe remains closed. The stems 242 maintain the fluorophore-quenching agents relatively close to each other such that a large amount of resonant energy can be transmitted between each other, while 201211534 substantially eliminates the ability of the fluorophore to emit fluorophores when illuminated with excitation light 244. Figure 59 shows a FRET probe 23 6 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 23 8 , the stem-and-loop structure is disrupted, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to fluoresce. The fluorescent emission 250 is optically detected as an indicator that the probe has hybridized. The probe hybridizes to the complementary target with extremely high specificity, since the stem φ system of the probe is designed to be stable to the probe-target helix with a single non-complementary nucleotide. Due to the relatively strong double-stranded DNA, the probe-target helix and the stem helix cannot coexist on the stereo" primer-joined probe-coupled stem-and-loop probe and primer-linked linear probe, also known as As a scorpion-type probe, it is a substitute for molecular beacons and can be used for both real-time and quantitative nucleic acid amplification of LOC devices. Timely amplification can be directly implemented in the hybrid chamber of the LOC. The advantage of using a primer-ligated probe is that the probe element is physically linked to the primer, so that only a single hybridization is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an immediate and efficient response and produces a stronger signal 'shorter response time and better recognition when using separate primers and probes. During manufacture, the probe (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 180 without the need for a separate amplification portion on the LOC device. Alternatively, the amplification portion is not used or used for other reactions. -89- 201211534 Primer-Linked Linear Probes Figures 104 and 105 show the primer-linked linear probe 692 during the first round of nucleic acid amplification and the hybridization configuration during subsequent nucleic acid amplification, respectively. Referring to Figure 104, the primer-coupled probe 692 has a double stem section 242. Wherein the strand-binding primer-coupled probe sequence 696 is homologous to the region on the target nucleic acid 696 and is labeled 5' to its fluorophore 246, and its 31-terminal to oligo via the amplification blocker 694 Nucleotide primer 700. The other 3' end of the stem 242 is labeled with a quencher portion 248. After completion of the first round of nucleic acid amplification, using the currently complementary sequence 698, the probe can surround and hybridize to the extended strand. During the first round of nucleic acid amplification, oligonucleotide primer 700 is attached to target DN A 238 (Fig. 104) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the reading of the polymerase by passing and copying the probe region 696. Upon subsequent denaturation, the hybridized extended oligonucleotide primer 700/template and the double stranded stem 242 of the primer-linked linear probe are separated, thereby releasing the quencher 248. Once the temperature for the adhesion and extension steps is reduced, the primer-linked probe sequence 696 of the primer-linked linear probe is crimped and hybridized to the amplified complementary sequence 698 on the extended strand, and the detected fluorescent indicator Target DNA is present. The unextended primer-linked linear probe retains its double stem and the fluorescence remains quenched. This test method is particularly suitable for rapid detection systems because it relies on a single molecular process. Primer-Linked Stem-and-Ring Probes Figures 106A through 106F show the operation of the primer-coupled stem-and-loop probe 704. Referring to Figure 106A, the primer-linked stem-and-loop probe 76 has a loop 240 complementary to the 201211534 double stranded DNA and a loop 240 of the combined probe sequence. The 5' end of one of the stem strands 708 is labeled with a fluorophore 246. The other strand 710 is labeled with a 3' end quencher 248 and the other strand 710 carries both an amplification blocker 6 94 and an oligonucleotide primer 700. In the initial denaturing phase (see Figure 106B), the strands of the target nucleic acid 238 and the primer-ligated stem 242 separate the stem-and-loop probe 704. When the temperature is cooled for the adhesive phase (see Figure 106C), the oligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 hybridizes to the target nucleic acid sequence 23 8 . Between the extension period φ (see Figure 106D), the complementary 706 of the target nucleic acid sequence 23 is synthesized to form a DN A-share amplification blocker 694 containing both the probe sequence 704 and the amplified product to prevent polymerase The pass and copy probe area 704 is read. After denaturation, the probe sequence of the primer-coupled stem-and-loop probe loop segment 240 (see Figure 106F) is adhered to the complementary sequence 706 on the extended strand when the probe is subsequently attached. This configuration allows the fluorophore 246 to be at a great distance from the quencher 24 8 resulting in a significant increase in fluorescence emission. φ Control Probes The hybridization chamber array 110 includes a number of hybridization chambers 180 with positive and negative control probes for analytical quality control. Figures 118 and 119 illustrate a negative control probe without fluorophore 796, and Figures 120 and 121 depict a positive control probe without quencher 798. The positive and negative control probes have a stem-and-loop structure as described above for the FRET probe. However, whether the probe hybridizes to an open configuration or remains closed, the fluorescent signal 250 will always be emitted from the positive control probe 79 8 and the negative control probe 796 will never emit the fluorescent signal 250. Referring to Figures 118 and 119, the negative control probe 796 does not have a fluorophore (and can be -91 - 201211534 with or without quencher 248) » thus, regardless of the target nucleic acid sequence 238 hybridizes to the probe (see Figure 11) Alternatively, the probe maintains its stem-and-loop configuration (see Figure 1 18), and the response to excitation light 244 can be ignored. Alternatively, the negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing loop 240, a probe sequence will be obtained that will not hybridize to any of the nucleic acid sequences in the sample of interest, the stem 242 of the probe molecule will rehybridize with itself, and the fluorophore and quencher will Stay in close proximity and will not emit visible fluorescence. This negative control signal corresponds to a low order emission from the hybridization chamber 180 where the probe is not hybridized but the quencher does not quench all of the emission from the reporter. Conversely, a positive control probe 798 without quenching agent was constructed, as shown in Figures 20 and 121. Back stress luminescence 244, regardless of whether the positive control probe 798 hybridizes to the target nucleic acid sequence 23 8 , no material quenches the fluorescent emission 25 from the fluorophore 246. Figure 5 2 shows the possible distribution of positive and negative control probes (3 78 and 380, respectively) in the hybrid chamber array 1 10 . Control probes 3 78 and 380 are placed in hybridization chamber 180 and positioned across the line of hybridization chamber array 1 1 0. However, the configuration of the control probes within the array is arbitrary (as in the hybrid chamber array 110 configuration). The fluorophore design requires a fluorophore with a long fluorescence lifetime to allow the excitation light to have sufficient time to decay to a higher level. The intensity of the fluorescent emission at the time of the photosensor 44 is low, thereby increasing the sufficient signal-to-noise ratio. Moreover, the longer fluorescent lifetime -92 - 201211534 represents a larger integrated fluorescence count. The fluorescence lifetime of the fluorophore 246 (see Figure 59) is greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and in most cases greater than 400 nanoseconds. Metal-coordination complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal 'chemical and photochemical stability, these characteristics Both are advantageous characteristics related to the requirements of the fluorescence detection system. The particularly complex metal-coordination complex based on the transition metal ion ruthenium (Ru(II)) is ginseng (2,2'-bipyridyl) ruthenium (II) ([Ru(bpy) 3] 2 + ), the lifetime of which is about 1μ5. This complex is available from Biosearch Technologies under the trade name Pulsar 650. Table 1: Photophysical properties of Pulsar 65 0 (钌 chelate)

Parameter symbol 値 unit absorption wavelength λ abs 460 nm emission wavelength λ em 650 nm absorption coefficient E 1 4800 fluorescence lifetime Tf 1.0 μδ quantum yield H 1 (deoxygenated) N/A lanthanide metal-coordination complex , ruthenium chelate, has been successfully shown as a fluorescent reporter in the FRET probe system and has a long lifetime of 1 600 μ3. -93- 201211534 Table 2: Photophysical properties of ruthenium chelate

Parameter symbol 値 unit absorption wavelength λ abs 330-350 nm emission wavelength λ em 548 nm absorption coefficient E 1 3 8 00 Uabs, and ligand dependent, up to 30000 @ λβ = 340 nm) M-'cm'1 camp Light lifetime Xf 1600 (hybridized probe) Ms Quantum yield H 1 (coordination dependent) The fluorescence detection system used by the N/A LOC device 301 does not utilize filtering to remove unwanted background fluorescence. It is therefore advantageous if the quencher 248 has no natural emission to increase the signal-to-noise ratio. Without natural emission, the quencher 24 8 does not contribute to background fluorescence. High quenching efficiency is also important, which results in no fluorescence before hybridization occurs. Black Hole Cleaner (BHQ), available from Biosearch Technologies, Inc. of Novato, Calif., does not have natural emissions and has high quenching efficiency, as well as suitable quenchers for use in systems. The maximum absorption enthalpy of BHQ-1 occurs at 534 nm and the quenching range is 480-580 nm, making it a suitable quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 5 79 nm and the quenching range is 560-670 nm, making it a suitable quencher for Pulsar 65 0 . Iowa Black FQ and RQ from Integrated DNA Technologies, Coralville, Iowa, are suitable alternative quenchers with little or no background emission. The Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorption enthalpy at 531 nm and is therefore a suitable quencher for the Tb-chelating fluorophore at -94-201211534. Iowa Black RQ has a maximum absorption enthalpy at 656 nm and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 650. In the specific embodiments described herein, quenching agent 248 is a functional portion that is initially attached to the probe, but in other embodiments, the quenching agent can be a separate molecule that is free of solution. Excitation Source In the specific embodiment based on the fluorescence detection described herein, the LED is selected to replace the excitation source of the laser diode, high power lamp or laser because of low power consumption, low cost, and small size. Referring to Figure 107, LEDs 26 are disposed directly on hybrid array 110 on the exterior surface of LOC device 301. Opposite to the array of hybridization chambers 1 1 is a photosensor 44 consisting of an array of photodiodes 184 from each chamber for detecting fluorescent signals (see Figures 53, 54 and φ 73). Figures 108, 109 and 110 illustrate other specific embodiments for exposing the probe to excitation light. In the LOC device 30 shown in FIG. 108, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 onto the hybridization cell array 110. The LED 26 is pulsed and the fluorescent emission is detected by the photo sensor 44. In the LOC device 30 shown in FIG. 109, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first aperture 712, and the second optical prism 714 onto the hybridization chamber array 110. The LED 26 is pulsed and the fluorescent emission is detected by the light -95-201211534 sensor 44. Similarly, the LOC device 30 shown in FIG. 110, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 onto the hybridization chamber array 110. The fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 is dependent on the choice of fluorescent dye. Philips LXK2-PR1 4-R00 is a suitable source of excitation for Pulsar 650 dyes. SET UVT0P3 3 5T039BL LED is a suitable excitation source for the ruthenium chelate label. Table 3: Philips LXK2-PR1 4-R00 LED Specifications

Parameter Symbol 单位 Unit Wave Length λ e X 460 n m Transmit Frequency V e m 6.52(10)14 Hz Output Power Pi 0.515 (min) (3⁄4 1A W Transmitting Graph Lambertian Data Graph N/A Table 4: SETUVT0P334T039BLLED Specifications

Parameter Symbol 单位 Unit Wavelength λ e 340 n m Transmitting frequency Ve 8.82(10)14 Hz Power Ρι 0.000240 (min) @ 20mA W Pulse forward current I 200 m A Emission pattern Lambertian N/A

Ultraviolet excitation 矽 absorbs a small amount of light in the UV spectrum. Therefore, the use of UV excitation light is advantageous from -96 to 201211534. A UV LED excitation source can be used, but the broad spectrum of LED 26 reduces the effectiveness of this method. For this, a filtered UV LED can be used. Incidentally, the UV laser can be an excitation source unless it is not practical for a particular test module market due to the relatively high cost of the laser. The L E D driver LED driver 29 drives the φ LED 26 at a fixed current for a desired duration. The low power USB 2.0 certified device is available with a minimum operating voltage of 4.4 volts at up to 1 unit load (1 mA). Standard power conditioning circuits are used for this purpose. Photodiode Figure 54 shows photodiode 184 that is integrated into CMOS circuit 86 of LOC device 301. Light diode 184 is part of CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over one of the CCDs. CCD is another sensing technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard processing steps. On-wafer inspection is inexpensive and reduces the size of the array system. The shorter optical path length reduces noise from the surrounding environment to efficiently collect fluorescent signals and reduces the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the fraction of photons colliding with its active region 185. The photonic system is efficiently converted into photoelectrons. For standard enthalpy treatment, the quantum efficiency of visible light is in the range of 0.3 to 0.5 depending on processing parameters such as the number of cover layers and absorption characteristics. -97- 201211534 Photodiode 1 84 The detection valve determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and the number of hybrid chambers 180 in the hybridization and detection section 52 (see Figure 52). The size and number of chambers are technical parameters, limited by the size of the LOC device (1 760 μm x 5824 μm in the example of LOC device 301), and are subject to other functional modules (such as pathogen dialysis). The size of the portion 70 that can be used after the portion 70 and the amplifying portion 1 12) is limited. For standard 矽 processing, photodiode 184 detects a minimum of 5 photons. However, in order to confirm reliable detection, the minimum 値 can be set to 10 photons. Therefore, the quantum efficiency range is from 0.3 to 0.5 (as discussed above), the fluorescence emission from the probe is a minimum of 17 photons, and the 30 photons contain a suitable margin for errors in reliable detection. The non-uniform electrical characteristics of the photodiode 184, autofluorescence, and residual excitation photon flux that are not fully attenuated introduce and shift background noise to the output signal. The background is removed from each output signal using one or more calibration signals. A calibration signal is generated by exposing one or more of the calibration photodiodes 184 in the array to respective calibration sources. A low calibration source is used to determine the negative result of the target 尙 not reacting with the probe. A high calibration source represents a positive result from the probe-target complex. In the specific embodiment 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 - 98-201211534 Contains a probe with a reporter and a quencher configured to expect quenching to occur forever. The output signal from such a calibration chamber 382 is very close to the noise and bias in the output signal from all of the hybrid chambers in the LOC device. The output signal produced by the other hybrid chamber minus the calibration signal 'substantially removes the background and leaves the signal produced by the fluorescent emission (if any signal is produced). Signals generated by ambient light in the area of the array are also removed. φ It is understood that the above negative control group probes with reference to Figs. 118 to 121 can be used in the calibration chamber. However, as shown in Figures 2 and 113, which are enlarged views of the inserts DG and DH of the LOC variant X 728 shown in Figure 111, another option is to fluidly isolate the calibration chamber 382 from the amplicons. When hybridization is prevented by fluid isolation, background noise and bias can be cleared by chambers that are fluidly isolated or by any probe that contains a lack of reporter or any "standard" probe that does have both a reporter and a quencher. Judgment The calibration chamber 382 can provide a high calibration source to generate a high signal to the corresponding light φ diode. The high signal corresponds to all probes in the chamber that has been hybridized. Reporting with no quenching agent or only with a reporter spotting probe will consistently provide a signal that a large number of probes in the hybridization chamber have hybridized within the hybridization chamber. It is also understood that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 for the entire array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by a relatively close calibration chamber 382, the calibration is more accurate. Referring to Figure 56, the hybridization chamber array 1 1 has one calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybrid chambers 180. In this configuration, the hybridization chamber -99-201211534 180 is calibrated by the immediately adjacent calibration chamber 382. Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, Figure 117 shows a differential imager circuit 788 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. The differential imager circuit 788 samples signals from the pixels 790 and the "virtual" pixels 792. In one embodiment, the "virtual" pixel 7 92 is shielded from light illumination, so its output signal provides a dark reference. Alternatively, the "virtual" pixel 792 can be exposed to the laser with the remainder of the array. In a particular embodiment where the "virtual" pixel 792 is light compliant, the signal produced by ambient light in the region of the array is also subtracted. The signal from pixel 790 is weak (eg, close to a dark signal), and it is difficult to distinguish between background and very weak signals without reference to dark signal levels. During use, "read-column" 794 and "start" Read_column_(1" 795 and turn on M4 797 and MD4 80 1 transistors. Turn off switches 8 07 and 809 so that the outputs from pixel 790 and &quot;virtual&quot; pixel 792 are stored in pixel capacitor 803 and virtual pixel capacitor, respectively. On page 05. After the pixel signal is stored, switches 8 07 and 08. 0 are disabled. The "read_row" switch 81 1 and the virtual "read_row" switch 813 are then turned off and switched at the output. The capacitor amplifier 815 amplifies the differential signal 817. The suppression and enabling of the photodiode must suppress the photodiode 184 during the excitation of the LED 26 and the photodiode 184 must be enabled during the fluorescent period. FIG. 74 is a circuit diagram of the single photodiode 184. Figure 75 is a timing diagram of the photodiode control signal. The circuit has a photodiode 184 and six MOS transistors, Mshunt 394, Mtx 3 96, Mreset 3 98, Msf 400, Mread 402, and Mbias 404. -100- 201211534, tl, transistor Mshunt 394 and Mreset 398 are turned on by pulling Mshunt gate 384 and resetting gate 38 8 high. During this period, the excitation photons are generated in photodiode 184. The carrier must be removed when the amount of carrier produced is sufficient to saturate the photodiode 184. During this cycle, due to leakage of the transistor or diffusion of the carrier due to excitation in the substrate Mshunt 394 directly removes the carriers generated in photodiode 184, while Mreset 398 resets any carriers accumulated at node 'NS' 406. After excitation φ, the capture cycle begins at t4. The response from the emission of the fluorophore is captured and integrated into the circuit on node 'NS' 060. This is achieved by dragging the tx gate 386 high, which turns on the transistor Mtx 396 and the transfer photodiode 184 Any accumulated carrier-to-node 'NS' 406 » capture cycle may be as long as a fluorescent emission. The output from all photodiodes 184 in the hybrid cell array 110 is simultaneously captured. End capture cycle t5 and start reading Take the loop between t6 This delay is due to the need to read the respective photodiodes 184 in the hybridization chamber array φ 11 分别 after the capture cycle (see Figure 52). The first photodiode 184 to be read is read. The cycle will have the shortest delay before the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Mread 402 is turned on by dragging the gate 3 93 high. The source-slaffer transistor Msf 400 is used to buffer and read the voltage of the 'NS' node 406. Additional methods for enabling or suppressing photodiodes are discussed below: 1 - Suppression Method - 101 - 201211534 Figures 114, 115 and 116 show possible configurations 778, 780, 782 for Mshunt transistor 394. Mshunt transistor 3 94 has a very high turn-off ratio when the maximum 値 |FCS| = 5 V is enabled during excitation. As shown in FIG. 14, the Mshunt gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in FIG. 115, the Mshunt gate 3 84 can be configured to surround the photodiode 184. A third option is to fabricate the Mshunt gate 3 84 within the photodiode 184, as shown in FIG. According to the third option, the photodiode active area 185 is less. These three configurations 778, 780, and 782 reduce the average path length from all locations in the photodiode 184 to the Mshunt gate 3 84. In FIG. 14, the Mshunt gate 384 is attached to one side of the photodiode 184. This is the simplest configuration to make and the smallest impact on the active region of the photodiode 185. However, any carrier remaining at the far end of the photodiode 184 takes a long time to diffuse through the Mshunt 3 8 » In Figure 115, the Mshunt gate 3 84 surrounds the photodiode 184. This further reduces the average path length of the carriers in the photodiode 184 to the Mshunt gate 384. However, extending the Mshunt gate 384 around the photodiode 184 causes the photodiode active region 185 to be substantially reduced. Configuration 782 in FIG. 116 positions Mshunt gate 3 84 in active region 185. This provides the shortest average path to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active zone 185 is greatest. It also creates a wide leak path. 2. Enabling method a. Trigger photodiode drives shunt transistor with a fixed delay -102- 201211534 b. The flip-flop photodiode drives the parallel transistor with a programmable delay. c. The parallel drive transistor is driven by the LED drive pulse with a fixed delay 〇 d. The parallel transistor is driven with a programmable delay as in 2 c. φ Fig. 77 shows a schematic view of the photodiode 184 and the flip-flop photodiode 187 embedded in the CMOS circuit 86 through the hybridization chamber 180. The small portion of the corner of the photodiode 184 is replaced by the flip-flop photodiode 187. area. The trigger photodiode 187 having a small area is sufficient because the intensity of the excitation light is higher than that of the fluorescent emission. The flip-flop photodiode 187 is sensitive to the excitation light 244. The flip-flop photodiode 187 shows that the excitation light 244 has extinguished and activates the photodiode 184 (see Fig. 2) after a brief delay of At3 00. This delay allows the fluorescent diode 184 to detect the fluorescent emission from the FRET probe φ 1 86 without the excitation light 244. This enables detection and enhancement of signal to noise ratio. Under each hybrid cell 180, both photodiode 184 and flip-flop photodiode 187 are located in CMOS circuit 86. The photodiode array is combined with appropriate electronic components to form a photosensor 44 (see Figure 73). The photodiode 184 is a pn junction made during the fabrication of the CMOS structure without the need for additional masking or steps. During MST fabrication, the dielectric layer (not shown) over the photodiode 184 is arbitrarily thinned using standard MST photolithography techniques to cause more of the phosphor to illuminate the active region 185 of the photodiode 184. The photodiode 184 has a field of view such that a fluorescent signal from the probe/target hybridization within the hybridization chamber 180 is incident on -103 - 201211534 onto the surface of the sensor. The converted fluorescence becomes a photocurrent that can then be measured using the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to the trigger photodiode 187. These choices can be used in combination with 2a and 2b above. Fluorescence Delay Detection The following derivation is a fluorescence delay detection using a long-life fluorophore for the combination of the above LED/fluorescent clusters. After the ideal pulse excitation of the fixed intensity Ie between times h and ί2 shown in Figure 60, the camping intensity is derived as a function of time. Let [S1] (0 at time t equal to the intensity of the excited state, then during and after excitation, the number of excited states per unit volume per unit time is described by the following differential equation: back (7) + on the leg... (1) dt tf hve where c For the molar concentration of the fluorophore, ε is the molar quenching coefficient, ▽6 is the excitation frequency, and h = 6· 62 606896 (1 0 broad and 34 Js is the Planck constant. This differential equation has the general formula: ^ - + p(x)y = q(x) αχ There is a solution: -104- ...(2)201211534 y(x) q{x)dx + k Now use this to solve the equation (1) = +ke -'lrf (3) hve and from (3): then at time η, [51](i〇= 0, k

Hve substitutes (4) into (3): ...(4) [51](〇/ggC7~y /ggCr,,丨)&quot;factory hve hve at time h,: [51](r2) = ^^ -^^e-(^ ...(5) nve hve in i 2 O, the excited state is exponentially attenuated _ [51](〇= [51](i2&gt;-(,-,j)/^ ... (6) Substituting (5) into (6): [5Ί](〇= ί^-[\ - e-{,^')lTf y^,Tr . hve The intensity of the fluorescence is obtained by the following equation: (6) Description: • (7)

If{t) = -^^-hvfVl (8) -105 201211534 where V/ is the fluorescence frequency, η is the quantum yield, and 丨 is the optical path length. Then from (7): ...(9) Receive) =-[1 _ e-«2-hVr, -C-h)/Tf dt hve Substituting (9) into (8):

If (/) = ΙεεοΙη^~[1 - e~(,^lrf Λβ-^'τί because 』------&gt; 〇〇, I. (/) —&gt; Iesclη — Γ/ Κ Therefore' We can write the following approximation, which describes the attenuation of the fluorescence intensity after a sufficiently long excitation pulse (i2-h &gt;&gt;Tf): for I 八tXeclT^e"" &quot;.(11) In the previous section, we summarize the situation for /2 hills>gt;Tf, and for tth = e'(,&quot;,2)/r/ 〇 From the above equation, we can derive the following formula: rif {t) = rieec^e~{,'hVtf ... (12) where ~(〇= :^ is the number of fluorescent photons per unit area per unit time and is the number of excitation photons per unit area per unit time Therefore, -106- 201211534

CO nfit)=\nf{t)dt (13) h where seven is the number of fluorescent photons per unit area and ί3 is the time point at which the photodiode is turned on. Substituting (12) into (13): co nf = dt ... (1 4) h At present, the number of fluorescent photons reaching the photodiode per unit area per unit time is obtained by: «;(〇=« &gt; ...(15) where nine is the light collection efficiency of the optical system. Substituting (12) into (15) we find that ηί(ί) = φ0ηΐεοΙηβ'(,~,ι)'Τί ...(16) Ground, the number of fluorescent photons reaching the photodiode per unit of fluorescent area will be as follows: 〇〇 = = = and substituting (16) and integrating: h fis ^φ^εο\ητ{βΛΗ~1ινχι Therefore, ns =φ0ή The ideal enthalpy of £εοΙητ/β'&amp;/, Τ^ (17) is that when the photodiode 184 is generated by the fluorescent photon, the electron rate is equal to that generated by the excitation photon in the photodiode 184. Rate, because the excitation photon flux decays more quickly than the fluorescence photon flux decays. 0 The sensor output electron rate per unit of fluorescence area of the fluorescence is: -107- 201211534 έ&gt;(0 = ^Λ( 0 where φ/ is the quantum efficiency of the sensor at the wavelength of the fluorescence. Substituting (17) we get: = (18) Similarly, due to the per unit of excited photons The output electron ratio of the light area is: έ; (0 = ^&gt;-(,-, ι)/Γ* (19) where is the quantum efficiency of the sensor at the excitation wavelength, and Te is relative to the excitation The time constant of the "off" characteristic of the LED. After time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends its decay time, but we assume that this pair has a negligible effect on If(t), so We adopt a conservative approach. Currently, as mentioned earlier, the ideal of ί3 is 当: Therefore, we get from (18) and (19): φ/φ0ήεβ:Ιηβ'{ι,'&quot;ι ), Τ / = φβηβε~{ίι', 2) ι^ and after reforming we get: Ιη(εα!ηί^-) —i~~-(20)

Tf Te from the above two paragraphs 'we get the following two expressions: ns = φ 〇ή Ρ Ρ / ... , , ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...

Ai=_LAi ...(22)

Tf re where Ρ = εεΙη and Δί = ί3-ί2, we also know, in fact, 》27. The ideal time for fluorescence detection and the number of fluorescent photons detected using Philips LXK2-PR14-R00 LED and Pulsar 650 dyes determine the ideal detection time as determined by equation (22): recall the concentration of the amplicon, and assume For all amplicon hybridizations, the fluorescing fluorophore concentration was: c = 2.8 9(10)_6 mol/L. The height of the chamber is the optical path length 1 = 8 (10 plants 6 m. The fluorescent region has been regarded as equivalent to the photodiode region, but the actual fluorescent region is substantially larger than the photodiode region; therefore, it can be assumed that eight =0.5 is the light collection efficiency of the optical system. The characteristics of the photodiode, # = 10 Φβ is the extremely conservative ratio of the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength. With a typical LED attenuation lifetime &amp;= 0.5 nanoseconds and using the Pulsar650 specification, it is possible to determine Δί : F = [1.48(10)6][2.89(10)^][8(10)-6](1) =3.42 (10)-5, ln([3.42(10)-5](10)(0.5)) 1 1- 1(10)-7 ~ 0.5(1 〇r9 =4.34(10)-9 s -109- 201211534 The number of photons detected is determined using equation (21). First, the number of excitation photons emitted per unit time is determined by examining the illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian emission mode, so: fi! =YilQcos(0) ...(23)

The angle between the direction of the axis of the LED and the direction of the axis of the LED is the number of photons emitted per unit time per unit solid angle, and ^. For ^ in the direction of the forward axis. The total number of photons emitted by the LED per unit time is:

ή, = fn, dQ Ω = J^cos do Ω Ω ... (24) Now, ΑΩ = 2π[1 - cos(0 + Δ0)] - 2;r[l - cos(0)] ΔΩ = 2n [cos(9) - cos{9 + Δ^)] 4;rsin (6) cos l&quot;A0, 2 2 + 4^cos(^)sin2 Αθ''

c/Ω = 2; rsin(0)t/0 is substituted into (24): π 2 ή, = j&quot;2;T^丨. Cos(0)sin(0)zhou ο rearranged, we get «/0 =- ... (2 6) π LED output power is 0.515 watts and ve = 6.52 (10) 14 Hz, therefore: -110- -( 27) -(27)201211534 Ρι hve __0.515_ ~ [6.63(10)-34][6.52(10)14] =1.19(10)18 photons/sec brings this 入 into (26) we get: ..._ 1.19 (10) 18 = 3.79 (10) 17 photons / sec / sphericity Referring to Figure 61, the optical center 2H and the lens 254 of the LED 26 are shown schematically. The photodiode is 16 micrometers x 16 micrometers, and for the photodiode in the middle of the array, the solid angle (Ω) of the light cone emitted from the LED 26 to the photodiode 184 is approximately = Ω = sensor area / r2 [ 16 (ΙΟ)-6] [16(10)·6] 2.825(10)-3]2 = 3.21(10)_5 Sphericality It will be understood that the central photodiode 184 of the photodiode array 44 is used for these calculations. use. Sensors located at the edge of the array receive only 2% lower photons for the Lambertian excitation source intensity distribution at the time of the hybridization event. Number of excitation photons emitted per unit time: he = η) Ω ... (28) = [3.79(10)17][3.21(1〇Γ5] =1.22(10)13 Photons/sec Reference equation (29 ): ns =$aneFTfe'^&quot;Tf ' =(0.5)122(10)113.42(10)-11(10)4^-434^^ -111 - 201211534 =208 Photon/Sensor So, use Philips With the LXK2-PR14-R00 LED and the Pulsar 650 fluorophore, we can easily detect any hybridization events that cause these numbers of photons to be excited. The SET LED illumination geometry is shown in Figure 62. Id = 20 mA, LED With minimum optical power output Pl = 240 microwatts, wavelength center at = 340 nm (absorption wavelength of ruthenium chelate). Drive LED at ID = 200 mA, linearly increase output power to Pi = 2.4 mW. Place the optical center 252 of the LED at 17.5 mm from the hybrid array 1 10 and we will focus the output flux on the photon pass in a 2 mm diameter dot with a maximum diameter of 2 mm at the dot of the hybrid array plane. The amount is obtained from Equation 27. P, 2.4(10)~3 ~ [6.63(10)-34][8.82(10)'4] =4.10(10)15 Photon/sec Using Equation 2 8, we get: 4.1 0(10)15 [16(10)-6]2 叩(1〇)3]2 =3.34(10)11 photons/sec Now, return to Equation 22 and use the previously listed Tb chelate properties,

Af_ln[(6.94(10)-5 )(10)(0.5)] 1(10)-3 0.5(10)'9 -112- 201211534 =3·98(10)_9 seconds Now from the equation 2 1 : ns = (0.5)[3.34(10)η][6.94(10)-5][1(10)-3]β-3·98α〇Γ,/Ι(1〇Γ3 = 11,600 photon/sensor The photon theoretical number emitted by the SET LED and the ruthenium chelate system from the hybridization event can be easily detected and well exceeds the lower limit of 30 photons, which is used for the photosensor indicated by the above. The on-wafer detection of the reliable separation of the probes required to detect the maximum spacing between the photodiodes avoids the need for detection by a conjugated focal microscope (see prior art). This deviates from the time and cost associated with conventional detection techniques in relation to the system. Savings are an important factor. Traditional inspections require the use of lens and curved mirror imaging optics. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. Place the photodiode close to non-φ The probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode is 1 micron, the collection angle of the emitted light is as high as 173°. Calculated by considering the light emitted from the probe closest to the center of the surface of the hybridization chamber of the photodiode, the photodiode has a planar active surface region parallel to the surface of the chamber. The light can be absorbed by the photodiode therein. The emission cone is defined as having a transmitting probe at its apex and at the sensor corners around its plane. For a 16 micron X 16 micron sensor, the cone has an apex angle of 170°; The extension of the body is such that the area conforms to the 29 micron X 19.75 micron hybrid cell area, the apex angle is 173° -113 - 201211534. The separation between the chamber surface and the active surface of the photodiode is 1 micron or Smaller is easier to achieve. Applying a non-imaging optical method requires the photodiode 184 to be very close to the hybridization chamber to collect sufficient photons of the fluorescent emission. The maximum spacing between the photodiode and the probe is determined by reference to Figure 5 below. Using the ruthenium chelate fluorophore and the SET UVT0P3 3 5 T039BL LED, we calculated 1,1,600 photons from individual hybridization chambers 180 to 16 micron χ 16 micron photodiodes 184. In implementing this calculation, we It is assumed that the light collecting region of the hybridization chamber 180 has the same bottom area as the photodiode active region 185, and half of the total number of hybrid photons reaches the photodiode 184. That is, the light collection efficiency of the optical system is N = 0.5. More precisely, we It is possible to write A = [(the bottom area of the light collection area of the hybridization chamber) / (photodiode area)] [Ω / 4π], where Ω = solid angle which is opposite to the representative point on the substrate of the hybridization chamber Light diode. For the correct (square) square pyramid geometry: Ω = 4arcsin(a2/(4d〇2 + a2)), where d. = the distance between the chamber and the photodiode, and "the size of the light diode." Each hybrid cell releases 23 200 photons, and the selected photodiode has a lower detection limit of 17 photons, so the minimum optical efficiency required is: φ0 = 1 7/23200 = 7.3 3 X 1 0'4 The bottom area of the light collection region of the hybridization chamber 180 is 29 microns χ 19·75 micro 201211534. The dQ is obtained, and the maximum limit distance between the hybridization chamber and the photodiode 1 84 is dG = 249 μm. In this limitation, the collection cone angle as defined above is only 0.8 ° » It should be noted that this analysis ignores the negligible effect of refraction. LOC Variants The LOC device 301, described and illustrated in detail above, is only one of many possible φ LOC device designs. Some possible combinations will now be set forth in the context of a flow chart (from sample input to detection) and/or display of LOC device variants using different combinations of the various functional components described above. The flow chart is appropriately divided into a sample input and preparation stage 288, an extraction stage 290, a culture stage 291, an amplification stage 292, a pre-hybridization stage 293, and a detection stage 294. For the sake of clarity and conciseness, only a brief description or summary of all LOC variants is shown and no detail configuration is shown. Also for the sake of clarity, smaller functional units, such as liquid sensors and temperature sensors, are not shown, but it should be understood that these functional units have been incorporated into the following LOC device designs. position.

LOC Variant VII Figures 82 to 96 show LOC variant VII 492. Features or structures are denoted by the same element symbols corresponding to the same features or structures in the previous figures. As shown in Figure 96, this variant extracted 290 using parallel nucleic acid amplification, cultured 291, amplified 2 92, and detected 294 human DNA. There are four different amplification sections 112.1 to 112.4 to increase the sensitivity of the analysis and to improve the signal-to-noise ratio of the detected fluorescent light -115-201211534 light. The design also uses the aforementioned white blood cell dialysis section 3 2 8 , lysis reagent 5 6 'chemical lysis section 1 30, restriction enzyme, zygote and linker 58, and culture section 1 14 . However, at the exit of chemical lysis section 130, culture section 114, and amplification sections 112.1 through 112.4, this LOC variant replaces a single active valve with several types of fault-tolerant valve arrays 309, 313, 462, 464, 466, and 468. . The second array of thermal bending actuated valves 308 having a 2x2 array of valve arrays feed different amplicon from each of the amplification sections 112.1 to 112.4 into different hybridization sections 1 1 〇. 1 to 1 1 〇·4, in which Fluorescence is detected by the photo sensor 44. Figure 82 is a perspective view of LOC Variant VII 492 showing via 122 in sample seal layer 82, sample inlet 68 and evaporator 190. The waste sump 76 is desealed so that excess waste can be transferred to the porous member 49 in the test module (see Figure 1). A series of bond pads 104 extend along one edge and the humidity sensor 23 is also exposed to sense the humidity of the microenvironment within the test module. Figure 83 is an exploded perspective view of the cover 46. The upper reservoir 82 is removed to expose the reagent reservoirs (54, 56, 58, 188, 60.1 - 60.4 and 62_1 - 62.4) below the through holes 122. The LOC variant VII 492 has four amplification sections 112 to 112.4, and four amplification mixing reservoirs 60.1 to 60.4 and four polymerase reservoirs 62.1 to 62.4, which provide reagents to individual amplification sections 1 12.1 to 1 12.4 〇 Figure 84 is a bottom side view of the cover 46 showing the structure of the cover channel 94 and the lower portion of the reagent reservoir (60.1, 60.2, ... 62.1, 62.2, etc.). Figure 85 superimposes the features of the cover 46 on the features of the CMOS + MST device 48. Figure 86 - 116 - 201211534 shows the features of the CMOS + MST device 48 separately. Figure 87 shows the cover channel 94, the sump and the valve array assembly separately. The waste channel 72 leads to the bottom side of the waste reservoir 76, while the target channel 74 leads to the chemical lysis unit 130 downstream of the lysis reagent reservoir 56. Figures 88, 89, 90, 91, 92, 93 and 94 are enlarged inserts BA to BG, respectively. Blood samples enter via sample inlet 68. Capillary action draws the sample through the lid channel 94 to the anticoagulant surface tension valve 118 (see Figure 88). The anticoagulant from the φ reservoir 54 is combined with the blood sample and continues to the leukocyte dialysis section 328 (see Figure 87). As best shown in Figure 93, the target channel 74 and the waste cell channel 72 are connected by a series of dialysis MST channels 204 through the MST layer 87. Target channel 74 is coupled to dialysis MST channel 204 via an array of individual 7.5 micron apertures 165. The dialysis MST channel 204 is connected to the waste channel 72 via a dialysis extraction port 168. The dialysis MST channel 204 at the upstream end 502 of the dialysis section does not have a lower conduit. The capillary action causes feature 166 to ensure that the sample stream is not fixed to the array of 7.5 micron orifices, but rather flows through 165 through the φ analysis MST channel 204. Referring again to Figures 85, 87 and 88, a sample stream having a higher target cell concentration flows to the lysis reagent surface tension valve 128. The lysing reagent from the reservoir 56 is combined with the sample and enters the chemical lysis unit 130. The flow stops at the 2x2 error tolerant array 309 of the second type of hot bending actuated valve 308. The mixed lysis reagent is diffused in a pause time programmed CMOS circuit 86 such that a sufficient number of target cells are lysed. After a sufficient time (less than 0.5 seconds), the valve in the fault-tolerant valve array 309 is activated (ie, opened), and the flow continues to the downstream portion of the mixing section 133" -117 - 201211534 when the genetic material is released When it is released, the restriction enzyme, the zygote and the linker from the storage tank 58 are added via the surface tension valve 132. The sample stream continues through the remainder of the mixing section 131 to the lower conduit 134 and into the heated microchannel of the culture section 114 (see Figure 87). The sample stream is flooded with the culture portion 114 until it reaches the 2x2 error-tolerant valve array 3 1 3 . After a sufficient incubation time, the fault tolerance valve array 3 1 3 is activated. The sample stream enters a common supply channel 504 of the amplification section. The common supply channel 504 feeds into the inlets 506, 508, 510, and 512 of the four different amplification sections 112.1 through 112.4 (see Figure 89). Each of the amplification mixing tanks 60.1 to 60.4 has surface tension valves 138 connected to the inlets 506, 508, 510 and 512 of the amplification section, respectively. Similarly, each of the polymerase reservoirs 62.1 through 62.4 has surface tension valves 140 (see Figure 87) that are coupled to the inlets 506, 508, 510, and 512, respectively. By fixing the meniscus, surface tension valves 138 and 140 hold the reagents in their individual reservoirs. As the sample flows down to each inlet, the meniscus is removed and the reagents are combined with the sample (first amplifying the primer, dNTP, and buffer, followed by the polymerase). Each of the four different amplification sections 112.1 to 112.4 is pooled with a sample, amplification mix, and polymerase. The individual flows through each of the amplification sections are stopped at the individual amplification exit error tolerance valve arrays 462, 464, 466, and 468 (see Figure 90). CMOS Controlled Thermal Cycle Amplification of Genetic Material" Referring specifically to Figures 85, 91 and 95, amplification of the outlet valve arrays 462, 464, 466 and 468 is initiated such that the four amplicons pass through different inlets 494, 496, 498, 500 flows into different hybridization chamber arrays 110.1 to 11〇_4. Each of the different hybrid chamber arrays has its own endpoint liquid sensor 178. Feedback from the individual endpoints - 118 - 201211534 The feedback from the detector 178 triggers the hybrid heater 182, and the LED driver 29 in the CMOS circuit indicates that the LED 26 is illuminated. Hybridization with the FRET probe 182 in any of the individual hybridization chambers 18'' is detected by photodiodes 184 underneath their chambers (see Figure 54). Conclusion The device 'system and method described herein promotes molecular diagnostic tests at low cost and high speed φ and in situ care. The above-described system and its components are merely illustrative, and many variations and modifications will be readily apparent to those skilled in the art without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will be described with reference to the accompanying drawings, wherein: FIG. 1 shows a test module and test module configured for fluorescence detection. Figure 2 is a schematic view of the electronic components in the test module configured for fluorescence detection; Figure 3 is a schematic view of the electronic components in the test module reader; Figure 4 is a representation BRIEF DESCRIPTION OF THE DRAWINGS FIG. 5 is a perspective view of a LOC device; FIG. 6 is a plan view of a LOC device having all layer structures and features superposed on each other; -119- 201211534 FIG. 7 is a cover with independent display Figure 8 is a top perspective view of the inner channel and the sump shown in phantom. Figure 9 is an exploded top plan view of the inner channel and the sump shown in phantom. Figure 10 shows the upper channel. A perspective view of the bottom of the configuration cover: Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device independently: Figure 12 is a schematic view of the sample inlet of the LOC device; Figure 13 is the insert AA shown in Figure 6. Magnified view; Figure 14 is Figure 6 is an enlarged view of the insert AE shown in Figure 13; Figure 16 is a partial perspective view showing the laminated structure of the LOC device in the insert AE; 17 is a partial perspective view illustrating the laminated structure of the LOC device in the insert AE; FIG. 18 is a partial perspective view illustrating the laminated structure of the LOC device in the insert AE; FIG. 19 is a view illustrating the insert AE Partial perspective view of the laminated structure of the LOC device: Figure 20 is a partial perspective view showing the laminated structure of the LOC device in the insert AE; Figure 21 is a view showing the laminated structure of the LOC device in the insert AE Figure 12 is a schematic view of the lysing reagent sump shown in Figure 21; Figure 23 is a partial perspective view showing the laminated structure of the LOC device in the insert AB; Figure 24 A partial perspective view of the laminated structure of the LOC device in the insert AB; Figure 2 5' is a partial perspective view of the laminated structure of the L〇c device in the insert AI: Figure 26 is an illustration of the insert Partial perspective view of the laminated structure of the LOC device in AB; Figure 27 is an L〇c device illustrating the insert AB Partial perspective view of the laminated structure of the LOC device in the insert AB; Figure 29 is a partial perspective view of the laminated structure of the LOC device in the insert AB. Figure 30 is a schematic view of the amplification mixing tank and the polymerase storage tank; Figure 31 shows the characteristics of the independent boiling pilot valve; Figure 32 is a diagram of the boiling boiling pilot valve taken along line 33_33 shown in Figure 31. Figure 33 is an enlarged view of the insert AF shown in Figure 15; Figure 34 is a schematic view of the upstream end of the dialysis section taken along line 35-35 shown in Figure 33; Figure 35 is Figure 6. A magnified view of the insert AC shown in Fig. 36 is a further enlarged view showing the amplification portion in the insert AC; -121 - 201211534 Fig. 37 is a further enlarged view showing the amplification portion in the insert AC; A further enlarged view of the amplification portion is shown in the insert AC; Fig. 39 is a further enlarged view of the insert AK shown in Fig. 38; Fig. 40 is a further enlarged view showing the amplification portion in the insert AC; A further enlarged view of the amplification portion is shown in the insert AC; FIG. 42 shows the expansion in the insert AC. Figure 4 is a further enlarged view of the insert A shown in Figure 42; Figure 44 is a further enlarged view showing the amplification portion in the insert AC; Figure 45 is a view showing the enlarged portion of the insert AC; Further enlarged view of the insert AM; Fig. 46 is a further enlarged view showing the amplification portion in the insert AC; Fig. 47 is a further enlarged view of the insert AN shown in Fig. 46; Fig. 48 is the insert AC A further enlarged view showing the amplification section; FIG. 49 is a further enlarged view showing the amplification section in the insert AC; FIG. 50 is a further enlarged view showing the amplification section in the insert AC; Figure 52 is an enlarged plan view of the hybridization section: Figure 53 is a further enlarged view of two independent hybridization chambers; Figure 54 is a schematic diagram of a single hybridization chamber: Figure 55 is an insert AG shown in Figure 6. An enlarged view of the humidifier illustrated in Fig. 56 is an enlarged view of the insert AD shown in Fig. 52; Fig. 57 is an exploded perspective view of the LOC device in the insert AD: Fig. 58 is a FRET probe in a closed configuration Figure: Figure 59 is a diagram of a FRET probe in an open and hybrid configuration; -122- 201211534 60 is a plot of excitation light versus time; FIG. 61 is a diagram of excitation illumination geometry of the hybrid chamber array; FIG. 62 is a diagram of sensor illumination technology LED illumination geometry; FIG. 63 is an insert of FIG. An enlarged plan view of the humidity sensor shown in AH; Figure 64 shows the features of the first variant of the thermal bending actuated valve; φ Figure 65 is the thermal bending actuated valve of the line 70-70 of Figure 64 Figure 66 shows a feature of a second variant of the thermal bending actuated valve; Figure 67 is a schematic illustration of a second variant of the thermally bent actuated valve along line 72-72 of Figure 66; A feature of a third variation of the thermal bending actuated valve is shown; Figure 69 is a schematic illustration of a third variation of the thermal bending actuated valve along line 74-74 of Figure 68; φ Figure 7A shows the fault tolerant valve array Figure 71 is a schematic diagram of an error-tolerant valve array along line 76-76 of Figure 70. Figure 72 is a schematic view of a white blood cell target dialysis section; Figure 73 is a schematic view showing a portion of the photodiode array of a portion of the photosensor _ ; Figure 74 is a circuit diagram of a single photodiode; Figure 75 is the time of the photodiode control signal Figure 76 is an enlarged view of the evaporator shown in the insert AP of Figure 55; -123- 201211534 Figure 77 is a schematic diagram of the hybridization chamber and the detection photodiode and the flip-flop photodiode; Figure 78 is the junction-priming Figure 71 is a schematic representation of a test module with a lancet; Figure 80 shows a boiling pilot valve array; Figure 81 is a perspective view of the boiling pilot valve taken along line 97-97 shown in Figure 80; Figure 82 is a perspective view of the LOC variant VII; Figure 83 is an exploded top view of the cover of the LOC variant VII (with internal channels and sump indicated by dashed lines); Figure 84 is a display for the cover of the LOC variant VII. Bottom view of the cover channel » Fig. 85 is a plan view showing the features and structures of all layers of the LOC variant VII superimposed on each other; Fig. 86 is a plan view showing the structure of the CMOS + MST of the LOC variant VII independently; Fig. 87 is a stand-alone display A plan view of the L0C variant VII of the lid structure, Fig. 88 is an enlarged view of the insert BA shown in Fig. 85; Fig. 89 is an enlarged view of the insert BB shown in Fig. 85; Fig. 90 is a view of Fig. 85. An enlarged view of the insert BC is shown; Fig. 91 is an enlarged view of the insert Bd shown in Fig. 85; Figure 92 is an enlarged view of the insert BE shown in Figure 88; Figure 93 is an enlarged view of the insert bf shown in Figure 92; 201211534 Figure 94 is an enlarged view of the insert BG shown in Figure 85; 95 is an enlarged view of the hybridization and detection portion of LOC variant VII; Figure 96 is a graphical representation of the structure of LOC variant VII; Figure 97 is a graphical representation of the structure of LOC variant VIII; Figure 98 is the structure of LOC variant XIV Figure 99 is a graphical representation of the structure of the LOC variant XLI; Figure 100 is a graphical representation of the structure of the LOC variant XLII; φ Figure 101 is a graphical representation of the structure of the LOC variant XLIII; Figure 102 is a LOC variant Graphical representation of the structure of XLIV; Figure 103 is a graphical representation of the structure of the LOC variant XLVII; Figure 104 is a diagram of the primer-linked linear fluorescent probe during the initial amplification; Figure 05 is the period of the subsequent amplification cycle Diagram of the primer-linked linear fluorescent probe; Figures 106A to 106F graphically illustrate the thermal cycling of the primer-linked fluorescent stem-and-φ loop probe; Figure 107 is related to the hybrid chamber array and the second light The polar body is excited by the LED; Figure 108 is the hybrid array for guiding the light to the LOC device. A summary of the excitation LEDs and optical lenses on the column; Figure 109 is a schematic illustration of the excitation LEDs, optical lenses, and apertures used to direct light to the hybrid chamber array of the LOC device; Figure 110 is for a light guide Brief description of the excitation LED, optical lens, and mirror configuration on the array of hybrid chambers to the LOC device; -125- 201211534 Figure 111 is a plan view showing all features superimposed on each other and showing the position of the insert DA to DK; 112 is an enlarged view of the insert DG shown in FIG. 111; FIG. 113 is an enlarged view of the insert DH shown in FIG. 111; and FIG. 4 shows an embodiment of the parallel transistor for the photodiode; An embodiment showing a parallel transistor for an optical diode; Fig. 116 shows an embodiment of a parallel transistor for a photodiode; Fig. 11 is a circuit diagram of the differential imager; φ Fig. 118 schematically depicts a stem -and-negative control fluorescent probe of the ring structure» Fig. 9 schematically depicts the negative control fluorescent probe of Fig. 1 18 in an open structure; Fig. 120 schematically depicts the stem-and-ring structure Positive Control Fluorescent Probes> Figure 121 is a brief description of the open Figure 120 is a schematic view of the fluorescent probe; # Figure 122 is a plan view of the electric blast valve; Figure 123 is a plan view of the bending and brake valve for thermal bending actuation; Figure 124 is a double thermal bending actuated bending and brake valve Figure 125 is a plan view of a viscous valve; Figure 126 is a plan view of a viscous valve variant; Figure 127 is a plan view of a bubble break valve; Figure 128 shows a configuration for use with EC L detection Test module and test module reader; -126- 201211534 Figure 1 2 9 is a schematic diagram of the electronic components in the test module used with ECL detection; Figure 1 3 0 shows the test module and alternative test Module reader; and Figure 1 3 1 shows the test module and the alternative 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 13 : Case 14 : Micro - USB Connector 15 : Sensor 16 : Micro - USB 埠 17 : Touch Screen 18 : Display Screen 19 : Button 2 〇 : Start Button 2 1 : Honeycomb Radio 22 : Aseptic sealing tape 23: wireless internet connection 24: large Container 25: Satellite navigation system 26: Light-emitting diode-127-201211534 2 7: Data storage 2 8: Telephone 29: LED driver 30: LOC device 3 1 : Power conditioner 32: Capacitor 33: Clock 3 4: Control Φ 35 : register 36 : USB device driver 37 : driver 3 8 : random access memory 3 9 : driver 40 : flash memory 41 : register 42 : processor call 43 : program memory 44 : Light sensor 45: indicator 46: cover 4 7 : module 48 : CMOS + MST device 49 : porous element 5 2 : detecting portion -128 - 201211534 54 : sump 56, 56.1, 56.2, 56.3: sump 5 7 : Printed circuit board 5 8 , 5 8 · 1 , 5 8.2 : • Storage tank 60, 60.1-60.12, 60.X: Storage 6 2 . X : Storage tank 62, 62.1, 62.2 ' 62.3 ' 62.4 ' 6 4 : Lower seal 66 : Top layer 6 8 : sample inlet 70 : dialysis section 72 : waste channel 73 : cover valve interface channel 74 : target channel 75 : membrane 76 : waste reservoir 77 : resistor 78 : sump layer 80 : cover channel layer 8 1 : Rigid link 82: upper sealing layer 84: 矽 substrate 86: CMOS circuit 87: MST layer 8 8: passivation layer - 129 201211534 90 : MST channel 9 1 : flexible coupling 92: Pipe 94: Cover Channel 96: Upper Pipe 97: Wall 98: Meniscus Holder 1 00: MST Channel Layer 101: Laptop/Notebook 102: Capillary Action Start Feature 103: Dedicated Reader 105: Desktop computer 106: boiling start valve 107: e-book reader 108: boiling start valve 109: tablet computer 110, 110.1-110.12, 110.X: hybridization chamber array 1 1 1 : epidemiological data 112, 112.1-112.12 , 112.X: Amplification part 1 1 3 _·Genetic data 1 1 4 · 1 -1 1 4 · 4 : Culture part 1 1 5 : Electronic health record 1 1 6 : Anticoagulant 1 1 8 : Surface Tension valve-130- 201211534 1 1 9 : Liquid sample 1 2 0 : Meniscus 1 2 1 : Electronic medical record 1 2 2 : Ventilation hole 1 2 3 : Personal health record 1 2 5 : Network 126 : Boiling Valves 128, 128.2, 128.3: Surface tension valve 130, 130.1-130.3: Lysis part 1 3 1 : Mixing section 132, 1 32.1, 1 32.3: Surface tension valve 1 3 3 : Incubator inlet passage 134: Lower duct 1 3 6: optical window 138, 138.1, 138.2, 138.X: surface tension valve 140, 140.1, 140.2, 140.X: surface tension valve 1 4 6 : valve inlet 148: valve outlet 150: valve under the pipe 1 5 1 : Valve upper pipe 1 5 2 : Ring heater 1 5 3 : Valve heater contact 1 5 4 : Heater 1 5 6 : Heater contact -131 - 201211534 158 : Micro channel 160 : Outlet channel 162 : Cantilever Part 1 6 4 : orifice 1 6 5 : orifice 166 : capillary action initiation feature 1 6 8 : dialysis extraction orifice 170 : temperature sensor φ 174 : liquid sensor 175 : diffusion barrier 176 : flow path 178 : Liquid sensor 180: hybridization chamber 1 8 2 : heater 184 : photodiode 1 8 5 : active region _ 1 86 : probe 1 87 : photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : Ring heater 192 : Water supply channel 1 93 : Upper pipe 194 : Lower pipe - 132 - 201211534 1 9 5 : Top metal layer 1 9 6 : Humidifier 1 9 8 : Draw hole 202 : Capillary action starting feature 204 : MST Channel 206: Boiling Pilot Valve

207: boiling pilot valve 20 8 : liquid sensor 210 : microchannel 2 1 2 : MST channel 2 1 8 : electrode 220 : electrode 222 : gap 23 2 : humidity sensor 2 3 4 : heater 23 6 : FRET Probe 23 8 : Target nucleic acid sequence 240 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 248 : Quencher 250 : Fluorescence signal 2 5 2 : Optical center - 133 201211534 2 5 4 : Lens 2 8 8: sample input and preparation 2 90: extraction stage 291: culture stage 292: amplification stage 29 3 : pre-hybridization filtration purification stage 294: detection stage 296: first electrode 2 9 8 : second electrode 3 0 0 : Delay 301: LOC device 3 02 : variant 304 : actuator 306 : orifice 3 0 8 : valve 3 0 9 : array 3 12 : valve 3 1 3 : array IJ 3 1 4 : array 3 1 6 : valve 3 1 8 : Valve 3 20 : Valve 3 22 : Valve 324 : Flow path 201211534 326 : Flow path 328 : White blood cell dialysis section 3 76 : Thermal conductivity column 3 78 : Positive control probe 3 80 : Negative control probe 3 82 : Calibration Room 3 8 4 : Noisy

3 8 6 : Gate 3 8 8 : Gate 3 9 0 : Retractable lancet 392 : Lancet release button 3 9 3 : Wide pole 394: MOS transistor 396: MOS transistor 398: MOS transistor 400: MOS transistor 402: MOS transistor 4 0 4 : Μ Ο S transistor 406: node 408: film seal 4 1 0 : membrane guard 412: valve 414: valve 4 1 6 : valve - 135 201211534 4 1 8: Valve 420: Cover channel 422: Cover channel 42 8: Valve upper pipe 4 3 0: Valve upper pipe 43 2: Valve down pipe 43 4: Valve down pipe 43 6 : Valve upper pipe 43 8 : Valve upper pipe 440: Cover channel 442: cover channel 4 4 8 : array 4 6 2 : array 4 6 4: array J 4 6 6 : array 4 6 8 : array 492 : LOC variant 494 : inlet 496 : inlet 498 : inlet 500 : Inlet 506: Inlet 508: Inlet 5 1 0: Inlet - 136 - 201211534 5 1 2 : Inlet 5 1 8 : LOC Variant 5 94: Interfacial layer 600: Bypass channel 602: Interface target channel 604: Scrap channel 63 8 : hot lysate 641 : LOC variant 673 : LOC variant 674 : LOC variant 682 : dialysis section 686 : dialysis step 692 : primer - linked linear probe 694 : amplification blocker 6 9 6 : probe Region 698: Complementary Sequence 700: Oligonucleotide Glycoside primer 704: stem-and-loop probe 706: complementary sequence 708: stem 710: strand 712: first stop 714: second stop 1 7 1 6 : first mirror - 137 201211534 7 1 8 : second mirror 740 : flow rate sensor 746 : LOC variant 758 : LOC variant 7 6 0 : large component channel 7 6 2 : small component channel 764 : opening 766 : blind terminal 768 : blind terminal 7 7 8 : Configuration 780: Configuration 782: Configuration 78 8: Differential Imager Circuitry 790: Pixel 792: Virtual Pixel 7 94: Read_Column 795: Read_Column 796: Negative Control Probe 797: (Crystal) 798: Positive Control Probe 80 1 : (Crystal) 803 : Pixel capacitor 805 : Virtual pixel capacitor 807 : Switch 201211534 8 0 9 : Switch 8 1 1 : Switch 8 1 3 : Switch 815 : Capacitor amplifier 8 1 7 : Differential signal 860 : ECL Excitation electrode 8 70 : ECL excitation electrode

Claims (1)

201211534 VII. Patent Application Range: 1. A microfluidic device comprising: a sample inlet for receiving a sample of a biological material having a nucleic acid sequence » a polymerase chain reaction (PCR) portion for amplifying a nucleic acid sequence; a reagent storage tank; and a diffusion mixing portion for mixing the nucleic acid sequence and the reagent, the diffusion mixing portion having a microchannel defining a tortuous flow path, the winding flow path having a sufficient length for diffusing the mixed reagent and the sample; At the time, the sample flows from the sample inlet to the PCR portion via the diffusion mixing portion. 2. The microfluidic device of claim 1, wherein the reagent storage tank has a surface tension valve having an orifice, and the surface tension valve is configured to fix the meniscus of the reagent, so that the meniscus maintains the reagent in the reagent storage tank until Contact with the sample stream removes the meniscus such that the reagent flows out of the reagent reservoir. 3. The microfluidic device of claim 2, further comprising a culture portion downstream of the mixing portion, the culture portion being configured to maintain a mixture of the sample and the restriction enzyme at a culture temperature limited to shear of the nucleic acid sequence. 4. The microfluidic device of claim 3, wherein the microchannel has a ruthenium structure. 5. The microfluidic device of claim 4, wherein the cross-sectional area across the flow path is between 8 square microns and 20,000 square microns. 6. The microfluidic device of claim 5, further comprising a lysate upstream of the mixing portion, the lysis portion being configured to lyse cells within the sample to release the genetic material therein. -140- 201211534 7. The microfluidic device of claim 6, further comprising an anticoagulant reservoir upstream of the lysis unit, wherein the sample is whole blood and the anticoagulant storage tank has a surface tension valve having an orifice, The surface tension valve is configured to hold the meniscus of the anticoagulant while maintaining the anticoagulant until contact with the blood to remove the meniscus to add anticoagulant to the blood. 8. The microfluidic device of claim 4, wherein the culture portion has a culture heater configured to heat the nucleic acid sequence and φ to limit the enzyme to the culture temperature. 9. The microfluidic device of claim 8, further comprising a support substrate and a microsystem technology (MST) layer having a lysis portion, a culture portion, and a PCR portion formed therein. 10. The microfluidic device of claim 9, further comprising a CMOS circuit and at least one temperature sensor, the CMOS circuit being between the support substrate and the MST layer, and the temperature sensor configured for feedback control Train the heater. Φ 11. The microfluidic device of claim 10, wherein the lysate has an active valve at the downstream end to maintain the liquid for a predetermined period of time. 12. The microfluidic device of claim 11, wherein the outlet valve is a boiling pilot valve having a meniscus holder for holding the liquid in the lysis section, the boiling pilot valve having a boiling fluid for boiling The valve heater 'releases the meniscus from the meniscus holder and resumes the capillary action drive flow out of the lysis section. 13. The microfluidic device of claim 12, wherein the meniscus holder is an orifice and the valve heater is adjacent to the periphery of the orifice. The microfluidic device of claim 13 further comprising a cover covering the MST layer, wherein the cover has a restriction enzyme storage tank, a plurality of PCR reagent storage tanks, and a mixing portion formed therein. 15. The microfluidic device of claim 1, further comprising a dialysis section, wherein the biological material comprises cells of different sizes, and the dialysis section is configured to divide cells larger than a predetermined threshold into a partial sample, the system comprising only The remainder of the sample of cells smaller than the predetermined threshold is processed separately. 16. The microfluidic device of claim 15 wherein the nucleic acid sequence is from a cell that is less than a predetermined threshold. 17. The microfluidic device of claim 7, wherein the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, the microchannel having a series of wide tortuous turns Structure, each wide tortuous line forms a channel portion of one of the elongated PCR chambers. 18. The microfluidic device of claim 8, wherein each channel portion has a plurality of heaters. The microfluidic device of claim 1, further comprising a hybridization portion having a probe array for hybridizing with a target nucleic acid sequence in the sample; and a photo sensor for detecting Probe hybridization within the probe array. 20. The microfluidic device of claim 1, wherein the thermal cycle time of the PCR portion is less than 30 seconds. -142-