TW201211532A - LOC device with parallel incubation and parallel DNA and RNA amplification functionality - Google Patents

Abstract

A lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device having an inlet for receiving the sample containing genetic material including DNA and RNA, a supporting substrate, a plurality of reagent reservoirs, a first incubation section, the first incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material, a second incubation section, the second incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material in parallel with the first incubation section, a first nucleic acid amplification section downstream of the first incubation section for amplifying at least some of the genetic material, and, a second nucleic acid amplification section downstream of the second incubation section for amplifying at least some of the genetic material in parallel with the first nucleic acid amplification section, wherein, the first incubation section, the second incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.

Description

201211532 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 the processing and analysis of microfluidics and biochemicals for molecular diagnostics. [Prior Art] φ molecular diagnostics have been used to provide an early indication of disease detection before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the 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 DNA and nucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobase allows for the binding (hybridization) of the synthetic DNA (oligonucleotide) short sequence to the particular nucleic acid sequence used for the nucleic acid assay. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that the individual will get in the future, to determine the type and pathogenicity of the infectious pathogen, or to determine the individual's response to the drug. 201211532 Nucleic Acid-Based Molecular Diagnostic Tests Nucleic acid-based assays have 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 beginning of a nucleic acid assay. 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 lysis solution, leaving the remaining intact cellular material, which can then be separated from the sample using centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. The exact protocol used to extract the nucleic acid depends on the sample and the diagnostic bridge to be implemented by 201211532. For example, the protocol used to extract viral RNA is quite different from the protocol used to extract genomic DNA. However, self-targeting cell extraction of nucleic acids typically involves a cell lysis step followed by 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 cell* followed by a high concentration of chaotropic salt prior to washing. In the presence of a low-ion ion, usually in a cerium oxide matrix, resin or paramagnetic beads in a fractionation column, in an alcohol (usually ice ethanol or isopropanol) precipitation step, or via a solid phase purification step, followed by a low ion The strength buffer is eluted. Any step prior to precipitation of the nucleic acid is the addition of a protein-cleaving proteinase to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to disrupt thermal lysis of the cell membrane. Target DNA or RNA can be present in the extracted material φ in very small amounts, especially if the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target (in the case of a low concentration 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 a target DNA sequence relative to a complex DNA background. If RNA is to be amplified (by PCR), it is first necessary to transcribe 201211532 into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the obtained cdNA was amplified by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is:

1. Primer pair - a short single strand of DNA having about 10-30 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis DNA thermostable enzyme 3. Deoxyribonucleoside Triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - the best chemical environment for DNA synthesis. PCR generally involves placing these reagents in small tubes containing extracted nucleic acids (~10 -50 microliters). The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. Standard procedures for each thermal cycle include the denatured phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denatured phase generally involves heating the reaction temperature to 90-95 ° C to denature the DNA strand; in the adhesive phase, the temperature is lowered to ~50-60 °C for adhesion of the primer; then in the extended phase, the temperature is applied. Warm up to an optimal DNA polymerase activity temperature of 60-72 ° C for extension of the primer. This method repeats the cycle for about 20-40 times, with the end result being a target sequence between the millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multiple primer set PCR, linker-primed PCR, direct PCR, tandem PCR, instant 201211532 PCR, and anti- Transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single trial by targeting multiple genes at once (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 targets - Specific primers. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). Using a zymase enzyme, a double stranded oligonucleotide linker (also known as an adaptor) with a suitable overhanging end is then ligated to the terminal of the target DNA fragment. Nucleic acid amplification is next carried out using oligonucleotide primers specific for the linker sequence. Thereby, all fragments of the DNA 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, P c R requires enhanced purification of the target nucleic acid prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentration, PCR can be performed to minimize PCR purification or direct PCR. PCR chemistry for direct PCR The adjustments for 201211532 include enhancement of buffer strength, use of high activity and processivity polymerases, and additions to potential polymerase inhibitors. The repeat sequence P C R utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. One type of repeat sequence PCR is nested PCR in which two pairs of PCR primers are used to perform single locus amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. 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. The amount of PCR product was measured in real time using either real-time PCR or quantitative PCR. The initial amount of nucleic acid in the sample can be determined by using a probe containing a fluorophore or a fluorescent dye and a set of reference standards 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. The reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), and then cDNA is amplified by PCR. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to amplify RN A viruses, such as human immunodeficiency virus or hepatitis C virus. -10- 201211532 Constant temperature amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DNA during the amplification reaction, and thus does not require complicated machinery. The thermostatic nucleic acid amplification method can be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase P o 1 ymerase Amp 1 Ificatiοn), Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification Some constant temperature nucleic acid amplification methods have been described. The thermostatic nucleic acid amplification method does not rely on the continuous heat denaturation of the template DNA to produce a single-stranded molecule as a template for further amplification, but relies on an enzymatic cleavage such as DMA at a normal temperature by specifically limiting the internal nuclease. 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 5'-3' exonuclease-deficient polymerases to extend and replace Downstream stocks. An exponential nucleic acid amplification is then achieved by a reaction between the sense and the antisense, wherein the strand of the sense reaction is 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 using a conventional method of cutting DN A is useful for this -11 - 201211532 reaction. SDA has been improved by the combination of heat stable restriction enzyme W να 1 ) and thermostable exo-polymerase polymerase. This combination appears to increase the amplification efficiency of the reaction from 1 08-fold amplification to 1 〇 1 ^ amplification, so that this technique can be used to amplify unique single-copy molecules. Transcription-mediated amplification (ΤΜΑ) and nucleic acid sequence-dependent amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, an RNA polymerase, a reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have Rnase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to the target ribosomal RNA (rRNA) at a defined location. The reverse transcriptase produces a DNA copy of the target rRNA by extension from the 3' end of the promoter scorpion. 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 DNA copy. A double-stranded DNA molecule is produced by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon re-enters the process and serves as a template for new replication. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a specific DNA fragment is achieved by binding a relative oligonucleotide primer to the template DNA and extending it by a DNA polymerase. Denatured double-stranded DNA (dsDNA) templates do not require heat. In contrast, RPA uses recombinant enzyme-primer complexes to scan dsDNA and promote share exchange at cognate locations. The structure of the 201211532 obtained is stabilized by the interaction of the single-stranded DNA-binding protein with the substituted template strand, thus preventing the primer from being released due to branch migration. The recombinant enzyme decomposes off the 3' end of the oligonucleotide which is adjacent to the DNA polymerase (such as a large fragment of Pol I (hw)), and the primer then begins to extend. This step is repeated by cycling to achieve exponential nucleic acid amplification. Helicase amplification (HDA) mimics the in vivo system, using DNA helicase in an in vivo system to generate a single strand template for primer hybridization and then extending the primer with a DNA polymerase. In the first step of the HDA reaction, the helicase traverses the target DNA, destroying the hydrogen bonds that bind the two strands, which are then bound by a single strand of binding protein. The single-strand target region 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 create two DNA replicas. 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 DNA polymerases continue to extend the primer around a circular DNA template to produce a long DNA product consisting of a number of circular repeats. In response, the polymerase produces thousands of copies of a circular template with a copy strand tethered to the original target DNA. This results in a rapid nucleic acid amplification of the spatial resolution of the target and the signal. A template of up to 1012 copies can be produced in one hour. Branched amplification is a variant of RCA and uses a closed circular probe (C-probe) or a latching probe and a highly progressive DNA polymerase to exponentially amplify C at ambient temperature - Probe. Circular Amplified Amplification (LAMP) provides high selectivity and utilizes a sputum polymerase and a primer set containing four specially designed primers. The primer set identifies a total of six different sequences on the standard -13-201211532 DNA. The inner primer, which contains the sense strand of the target DNA and the antisense strand sequence, initiates the LAMP. The subsequent strand-derived DNA synthesis initiated by the external 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 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 of DN A synthesis, yielding the original stem-loop DN A and the new stem-loop DNA with twice the stem length. The cycle was continued for less than one hour to accumulate 109 copies of the target. The final product is stem-loop DNA with several inverted repeat targets, and a broccoli-like structure with a plurality of loops (formed alternately with inverted repeat targets in the same strand). After completion of the nucleic acid amplification, the amplified product must be analyzed to determine whether the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product is to simply measure the size of the amplicon by colloidal electrophoresis and use DNA hybridization to identify the nucleotide composition of the amplicon. Colloidal electrophoresis is one of the simplest ways to check the nucleic acid amplification step to produce the desired amplicon. Colloidal electrophoresis utilizes an electric field applied to a colloidal matrix to separate DNA fragments. Negatively charged DNA fragments will move through the matrix at different rates (mainly depending on their size). After the electrophoresis is completed, the fragments in the colloid can be made visible. Bromination of honey under UV light Ethylene phenanthrene ingot is the most commonly used dye. The size of the fragment is determined by comparison with a DNA size marker (DNA ladder) which contains a DNA fragment of a known size which is run along with the amplicon. Due to the integration of the oligonucleotide primer junction 201211532 to a specific location adjacent to the target DNA, the size of the amplified product can be predicted and detected as a band of known size on the colloid. In order to confirm whether an amplicon or a plurality of amplicons are generated, a DNA probe is often used to hybridize with an amplicon. DNA hybridization means the formation of double-stranded DNA by complementary base pairing. DNA hybridization for the positive recognition of a particular amplification product requires the use of a DNA probe of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DNA sequence, the hybridization will be at a favorable temperature, pH and

Occurs under W ion concentration conditions. If hybridization occurs, the gene or DNA sequence of interest is present in the original sample. Optical detection is the most common method of detecting hybridization. The amplicon or probe is labeled to emit light via fluorescing or electrochemiluminescence. These methods have different ways of inducing the excited state of the luminescent moiety, but both are equally capable of covalent labeling of the nucleoside stock. In electrochemiluminescence (ECL), when excited by an electric current, light is generated by a luminophore molecule or a complex. When the fluorescent light is emitted, the excitation light that causes the φ emission emits light. The illuminating source is used to detect fluorescence, and the illuminating source provides excitation light having a wavelength absorbed by the fluorescent molecules and a detecting unit. The detection unit includes a photosensor (such as a photomultiplier tube or a charge coupled device (CCD) array) to detect the emitted signal and a mechanism (such as a wavelength-select filter) that prevents excitation light from being included in the output of the photosensor. Back stress illuminates, the fluorescent molecules emit Stokes-shifted light, and the emitted light is collected by the detection unit. The Tox shift is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. -15- 201211532 The light sensor is used to detect the E C L emission, and the light sensor is sensitive to the emission wavelength of the E C L type used. For example, transition metal coordination complexes emit light of visible wavelengths, thus using conventional photodiodes and .CCDs as photosensors. The advantage of ECL is that if ambient light is excluded, the ECL emission can be the only light present in the detection system, thus increasing sensitivity. The microarray allowed hundreds of thousands of D N A hybridization experiments to be performed simultaneously. Microarrays are useful molecular diagnostic tools that can screen thousands of genetic diseases or detect the presence of several infectious pathogens in a single assay. The microarray consists of a number of different DNA probes that are fixed 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. Less stringent may result in highly non-specific binding. Excessive rigor may result in inability to properly combine and result in reduced sensitivity. Hybridization is identified by detecting the light emission from the labeled amplicon that hybridizes with 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 transmitted signal. The fluorescent molecules emit light that is down-converted to 201211532 (as described above), and the light is collected by the detection 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. Prevents light above or below the focus plane of the object from entering the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons 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 hybridized target sequence can be simultaneously identified and analyzed. Find out more about fluorescent probes here: 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-Hi 幽ights/Fluorescence-Resonance-Energy-Transfer-FRET.html In situ Medical Molecular Diagnostics Although molecular diagnostic tests offer advantages, this type of test in clinical tests The growth was not as good as expected and still only a small part of the implementation of laboratory medicine. This is mainly due 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 associated with rapid and automated analysis that can significantly reduce costs, provide initial (sample processing) to final -17-201211532 (results), and instruments that do not require extensive human manipulation. Development is closely related. For physicians' clinics, proximity or user-based hospitals, home-based healthcare technologies offer the following benefits: • Quick results to quickly promote treatment and improve care. • Ability to test sputum by testing a very small number of samples. • Reduce clinical effort. • Reduce lab workload and increase work efficiency by reducing management effort. • Improve the cost per patient by reducing hospital stays, getting results from outpatient visits at the first visit, and simplifying the handling, storage and delivery of samples. • Promote clinical management decisions such as infection control and antibiotic use. On-wafer laboratory-based molecular diagnostics Molecular diagnostic systems based on microfluidics provide devices that automate and accelerate molecular diagnostic analysis. The shorter detection time is primarily due to the fact that the required sample volume is less active, automated, and low-cost built-in cascaded diagnostic method steps within the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. The on-wafer laboratory (LOC) device is a common form of microfluidic device. The LOC device has an MST structure within the MST layer for integrating fluid processing onto a single support substrate (typically helium). 201211532 The VLSI (Very Large Integral Circuit) lithographic printing technology of the semiconductor industry makes the unit cost of each LOC device very low. However, controlling the flow of fluid through the LOC unit, adding reagents, controlling reaction conditions, and the like requires large external piping and electronics. Connecting the LOC devices to these external devices greatly limits the molecular diagnostic use of the LOC devices in a laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as a practical option in a local healthcare setting. φ 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 invention are now described in the following paragraphs. GPC03 7.1 Aspects of the invention provide a on-wafer laboratory (LOC) device for amplifying a nucleic acid sequence, the LOC device comprising: an inlet for receiving a sample comprising genetic material; a φ support substrate; a plurality of reagent reservoirs; The nucleic acid amplification unit is a nucleic acid sequence amplified in the genetic material; wherein the nucleic acid amplification unit is supported on the support substrate. Preferably, the GPC03 7.2 is a polymerase chain reaction (PCR) unit. GPC03 7.3 Preferably, the LOC device also has a CMOS (Complementary Metal-Oxide-Semiconductor) circuit, a temperature sensor, and a micro-system technology -19 - 201211532 (MST) layer of the PCR unit. The CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit is configured to use the temperature sensor output to feedback control the PCR portion. GPC037.4 Preferably, the PCR portion has a PCR microchannel in which the thermal cycle genetic material, the PCR microchannel defines a flow path having a cross-section across the flow path of less than 100,000 square microns. GPC03 7.5 Preferably, the PCR microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GPC03 7.6 Preferably, the PCR microchannel has a cross-section across the flow path of less than 2,500 square microns. GPC03 7.7 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GPC03 7.8 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a 蜿蜒 structure. GPC037.9 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling. GPC03 7.1 0 Preferably, the 'active valve train' has a boiling initiation valve configured to hold a meniscus holder that blocks the meniscus of the liquid drive flow of the liquid, and to boil the liquid to relieve the bend from the meniscus holder The liquid level causes it to restore the capillary drive flow to the heater. GPC037.il preferably 'the LOC device has a hybridization portion downstream of the PCR portion, the PCR portion has a probe array for hybridization with the target nucleic acid sequence to form a probe-target hybrid and for detecting the probe-target Light sensor in the hybrid -20- • V . • V .201211532. GPC037.12 Preferably, the nuclear 1 acid amplification unit is a thermostatic nucleic acid amplification unit. GPC03 7.1 3 Preferably, the LOC device also has a hybridization portion downstream of the thermostatic nucleic acid amplification portion, and the thermostatic nucleic acid amplification portion has a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid, And a light sensor for detecting the probe-target hybrid. GPC03 7.1 4 Preferably, the hybridization portion has an array of hybridization chambers for containing probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. GPC03 7.1 5 Preferably, the photosensor is an array of photodiodes arranged in alignment with the hybridization chamber array. GPC037.1 6 Preferably, the thermostated nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a cross section having a cross-flow path of less than 1 〇〇, 〇〇〇 square Micron circulation path. GPC037.1 7 Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GPC03 7.1 8 Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 2,500 square microns. GPC03 7.1 9 Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path between 1 square micron and 400 square micrometers. GPC037.20 Preferably, the reagent reservoirs each have a surface tension valve to retain the reagent therein. The surface tension valve has a meniscus holder for fixing the meniscus of the reagent until the contact with the sample stream removes the bend. Level 21 - 201211532 to allow reagents to flow from the reagent reservoir. An easy to use, mass-produced, and inexpensive LOC device receives a biological sample comprising nucleic acid, and then amplifies the nucleic acid target in the sample using a nucleic acid amplification portion of the LOC device and using a reagent stored in a reagent reservoir of the LOC device. The Nucleic Acid Amplification section provides a capillary action advancement of the amplification mixture to simplify the design of the analytical system, further increasing reliability and reducing the cost of the analytical system. Amplification of the target nucleic acid sequence increases the sensitivity of the analytical system and the signal _ noise ratio. The reagent reservoir is integrated with the LOC device and meets all reagent requirements for analysis. The reagent reservoir provides low system component count and simple manufacturing procedures, resulting in an inexpensive analytical system. GPC03 8.1 Aspects of the invention provide a microfluidic device for amplifying a nucleic acid sequence, the microfluidic device comprising: an inlet for receiving a sample comprising genetic material; a plurality of reagent reservoirs containing reagents for addition to the sample; The nucleic acid amplification unit amplifies the nucleic acid sequence in the genetic material. GPC03 8.2 Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GPC03 8.3 Preferably, the microfluidic device also has a CMOS circuit and a temperature sensor, the CMOS circuit being configured to use a temperature sensor output to feedback control the PCR portion. GPC03 8.4 Preferably, the PCR portion has a PCR microchannel in which the thermal cycle genetic material, the PCR microchannel defines a flow path having a cross-section across the flow path -22-201211532 at 100,000 square microns. GPC03 8.5 Preferably, the PCR microchannel has a cross-section across the flow path of less than 1,600 square microns. Preferably, the PCR microchannel has a cross-section across the flow path of less than 2,500 square microns. GPC03 8.7 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel.

0 GPC03 8. Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a 蜿蜒 structure. GPC03 8. Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling. GPC03 8. Preferably, the active valve has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve meniscus from the meniscus retainer The surface φ restores the heater of the capillary drive flow. GPC03 8. Preferably, the microfluidic device also has a hybridization portion downstream of the PCR portion, the PCR portion having a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid and for detecting the probe-label A light sensor in the target hybrid. GPC03 8. Preferably, the nucleic acid amplification unit is a thermostatic nucleic acid amplification unit. GPC03 8. Preferably, the microfluidic device also has a hybridization section downstream of the constant temperature nucleic acid amplification section, and the thermostatic nucleic acid amplification section has a probe for hybridizing with the target nuclear 23-201211532 acid sequence to form a probe-target hybrid. A needle array, and a light sensor for detecting a probe-target hybrid. GPC038. Preferably, the hybridization portion has an array of hybridization chambers for containing probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. GPC03 8. Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of hybridization chambers. GPC03 8. Preferably, the thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow path having a cross section across the flow path of less than 100,000 square microns. GPC03 8. Preferably, the nucleic acid amplification microchannel has a cross section across the flow path of less than 1 6,000 square microns. GPC03 8. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 2,500 square microns. GPC03 8. Preferably, the nucleic acid amplification microchannel has a cross section across the flow path between 1 square micrometer and 400 square micrometers. GPC03 8. Preferably, the reagent reservoirs each have a surface tension valve for retaining the reagent therein, and the surface tension valve has a meniscus holder that is used to fix the meniscus of the reagent until contact with the sample stream to remove the meniscus To allow reagents to flow out of the reagent reservoir. An easy to use, mass-produced, and inexpensive microfluidic device receives a biological sample comprising a nucleic acid, and then amplifies the nucleic acid in the sample using a nucleic acid amplification portion of the microfluidic device and using a reagent stored in a reagent reservoir of the microfluidic device Target. • 24 - 201211532 The Nucleic Acid Amplification Division provides capillary action advancement of the amplification mixture, simplifies the design of the analytical system, further increases reliability and reduces the cost of the analytical system. Amplification of the target nucleic acid sequence increases the sensitivity and signal-to-noise ratio of the analytical system. The reagent reservoir is integrated with the microfluidic device and meets all reagent requirements for analysis. The reagent reservoir provides low system component count and simple manufacturing procedures, resulting in an inexpensive analytical system. $ GPC039. 1 The aspect of the invention provides a test module for amplifying a nucleic acid sequence, the test module comprising: a housing having a container for receiving a sample containing genetic material; and a plurality of reagent reservoirs for containing the sample to be added to the sample And a reagent for amplifying the nucleic acid sequence in the genetic material. GPC039. 2 Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GPC039. Preferably, the test module also has a CMOS circuit and a temperature φ sensor, and the CMOS circuit is configured to use a temperature sensor output to feedback control the PCR portion. GPC039. Preferably, the PCR portion has a PCR microchannel in which the thermal cycle genetic material, the PCR microchannel defines a flow path having a cross-section across the flow path of less than 100,000 square microns. GPC〇39. Preferably, the PCR microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GPC039. Preferably, the PCR microchannel has a cross-section across the flow path of less than 2,500 square microns. -25- 201211532 GPC039. Preferably, the PCR microchannel has at least one extended heater element extending parallel to the PCR microchannel" GPC039. Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a 蜿蜒 structure. GPC039. Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling. GPC039. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that prevents capillary flow of liquid, and to boil liquid to relieve meniscus from the meniscus retainer The heater that restores the capillary drive flow. GPC03 9. Preferably, the microfluidic device also has a hybridization portion downstream of the PCR portion, the PCR portion having a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid and for detecting the probe-label A light sensor in the target hybrid. GPC039. Preferably, the nucleic acid amplification unit is a thermostatic nucleic acid amplification unit. GPC039. Preferably, the test module also has a hybridization section downstream of the constant temperature nucleic acid amplification section, and the thermostatic nucleic acid amplification section has a probe array for hybridizing with the target nucleic acid sequence to form a probe-target hybrid, and A light sensor for detecting a probe-target hybrid. GPC03 9. Preferably, the hybridization portion has an array of hybridization chambers for containing probes such that the probes within each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. -26- 201211532 GPC039. Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of hybridization chambers. GPC039. Preferably, the thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow path having a cross section across the flow path of less than 100,000 square microns. GPC03 9. Preferably, the nucleic acid amplification microchannel has a cross section across the flow path of less than 1 6,000 square microns. GPC039. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 2,500 square microns. GPC03 9. Preferably, the nucleic acid amplification microchannel has a cross section across the flow path between 1 square micron and 400 square micrometers. GPC03 9. Preferably, the reagent reservoirs each have a surface tension valve for retaining the reagent therein, and the surface tension valve has a meniscus holder that is used to fix the meniscus of the reagent until contact with the sample stream to remove the meniscus To allow reagents to flow out of the reagent reservoir. An easy-to-use, mass-produced, inexpensive, and portable test module receives a sample containing the nucleic acid, and then amplifies the sample using the nucleic acid amplification portion of the module and using reagents stored in the reagent reservoir of the test module Nucleic acid target. The Nucleic Acid Amplification Department provides capillary action advancement of the amplification mixture, simplifies the design of the analysis system, further increases reliability and reduces the cost of the analysis system. Amplification of the target nucleic acid sequence increases the sensitivity of the analytical system and the signal _ noise ratio. The reagent reservoir is integrated with the test module and meets all of the analytical requirements of the analysis. The reagent reservoir provides low system component counts and simple manufacturing procedures, resulting in an inexpensive analytical system. GPC040. 1 Aspects of the invention provide a microfluidic device for amplifying a nucleic acid sequence, the microfluidic device comprising: an inlet for receiving a sample comprising genetic material: a plurality of reagent reservoirs containing reagents for addition to the sample; The nucleic acid amplification unit amplifies the nucleic acid sequence of the genetic material; and the second nucleic acid amplification unit amplifies the nucleic acid sequence in the genetic material, which is parallel to the first nucleic acid amplification unit. GPC040. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) unit and the second nucleic acid amplification unit is a second polymerase chain reaction (PCR) unit. GPC040. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs for binding to the second set of complementary nucleic acid sequences, the first set of complementary The nucleic acid sequence differs from the second set of complementary nucleic acid sequences. GPC040. 4 Preferably, the 'first PCR portion and the second pCr portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type: polymerase type; deoxyribonucleoside triphosphate Concentration; buffer solution; thermal cycle time; thermal cycle repeat; and, -28-201211532 temperature during a specific PCR phase. GPC040. Preferably, the 'microfluidic device also has a first hybridization portion downstream of the first PCR portion, having a first probe array that hybridizes with the first target nucleic acid sequence to form a probe-target hybrid, and a second a second hybridization portion downstream of the PCR portion, having a second probe array for hybridizing with the second target nucleic acid sequence to form a probe-target hybrid, and for detecting the light of the probe-target hybrid Sensor. φ GPC040. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit (GPC040). Preferably, the first thermostated nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second thermostated nucleic acid amplification portion has a second binding to the second set of complementary nucleic acid sequences. A set of primer pairs, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. GPC040. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the lower φ columns: reverse transcriptase type: polymerase type : concentration of deoxyribonucleoside triphosphate; buffer solution; and, temperature during nucleic acid amplification. GPC〇4〇. Preferably, the microfluidic device also has a first hybridization portion downstream of the first thermolabic nucleic acid amplification portion, having a first probe array that hybridizes with the first target nucleic acid sequence to form a probe-target hybrid, And a second constant temperature section -29 - 201211532 downstream of the nucleic acid amplification section, having a second probe array for hybridizing with the second target nucleic acid sequence to form a probe-target hybrid, and A photosensor that detects the probe-target hybrid. GPC040. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system within each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences. GPC040. Il Preferably, the photosensor is an array of photodiodes arranged in alignment with the first and second probe arrays. GPC040. Preferably, the first thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow path diameter of less than 100,000 square micrometers across the cross section of the flow path. GPC040. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GPC040. Preferably, the reagent reservoirs each have a surface tension valve for retaining the reagent therein, and the surface tension valve has a meniscus holder for fixing the meniscus of the reagent until contact with the sample stream to remove the meniscus Face to allow reagents to flow from the reagent reservoir. GPC040. Preferably, the microfluidic device also has a CMOS circuit and a temperature sensor, and the CMOS circuit is configured to use a temperature sensor to output feedback control of the first and second PCR portions. GPC040. Preferably, the first PCR portion has a PCR microchannel in which the genetic material is thermally cycled, and the PCR microchannel defines a flow path having a cross-section across the flow path of less than 100,000 square microns. 201211532 GPC040. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GPC040. Preferably, the first PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a ruthenium structure. GPC040. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during the thermal cycle period φ. GPC040. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve the meniscus from the meniscus holder A heater that restores the capillary drive flow. An easy to use, mass-produced, and inexpensive microfluidic device receives a biological sample comprising a nucleic acid, and then amplifies the nucleic acid in the sample using a reagent of a parallel nucleic acid amplification portion of the device and φ using a reagent reservoir stored in the microfluidic device Target. The Nucleic Acid Amplification Department provides capillary action advancement of the amplification mixture, simplifies the design of the analysis system, further increases reliability and reduces the cost of the analysis system. Amplification of the target nucleic acid sequence increases the sensitivity of the analytical system and the signal _ noise ratio. Further, the parallel amplification chamber allows for different target or target groups to optimize the use of different primer pairs or different primer pair groups' and also uses different optimized amplification parameters, which in turn increases the analysis. Sensitivity, signal-to-noise ratio and reliability. -31 - 201211532 The reagent reservoir is integrated with the microfluidic device and meets all reagent requirements for analysis. The reagent reservoir provides low system component count and simple manufacturing procedures, resulting in an inexpensive analytical system. G P C 0 4 1 .  1 The aspect of the invention provides a test module for amplifying a nucleic acid sequence, the test module comprising: a housing having a container for receiving a sample containing genetic material; a plurality of reagent reservoirs containing reagents for adding to the sample The first nucleic acid amplification unit amplifies the nucleic acid sequence in the genetic material; and the second nucleic acid amplification unit amplifies the nucleic acid sequence in the genetic material, which is parallel to the first nucleic acid amplification unit. GPC041. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) unit and the second nucleic acid amplification unit is a second polymerase chain reaction (PCR) unit. GPC041. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs for binding to the second set of complementary nucleic acid sequences, the first set of complementary The nucleic acid sequence differs from the second set of complementary nucleic acid sequences. GPC041. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type; polymerase type: deoxyribonucleoside triphosphate Concentration; Buffer solution: Thermal cycle time; -32- 201211532 Thermal cycle repetition; and, Temperature during a specific PCR phase. GPC041. Preferably, the test module also has a first hybridization portion downstream of the first PCR portion, having a first probe array that hybridizes with the first target nucleic acid sequence to form a probe-target hybrid, and a second a second hybridization portion downstream of the PCR portion, having a second probe array for hybridization with a second target nucleic acid sequence to form a probe-target hybrid, and for detecting a probe-target φ hybrid Light sensor. GPC041. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. GPC041. Preferably, the first thermostated nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second thermostated nucleic acid amplification portion has a second binding to the second set of complementary nucleic acid sequences. A set of primer pairs, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. GPC041. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature φ nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: a reverse transcriptase type; a polymerase type; Deoxyribonucleoside triphosphate concentration; buffer solution; and, temperature during nucleic acid amplification. GPC04 1. Preferably, the test module also has a first hybridization portion downstream of the first constant temperature nucleic acid amplification portion, which has a hybridization with the first target nucleic acid sequence -33 - 201211532 to form a probe-labeled mutated parent a probe array, and a second hybridization portion downstream of the second thermolabic nucleic acid amplification portion, having a second probe array for hybridizing with the second target nucleic acid sequence to form a probe/target hybrid, and A photosensor for detecting a probe-target hybrid. GPC04 1 .  Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system within each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences. GPC04 1. Il Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of hybridization chambers. GPC041. Preferably, the first thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow path diameter of less than 100,000 square micrometers across the cross section of the flow path. GPC041. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 16,000 square microns. GPC041. Preferably, the reagent reservoirs each have a surface tension valve therein to retain the reagent therein, and the surface tension valve has a meniscus holder for fixing the meniscus of the reagent until contact with the sample stream to remove the meniscus To allow reagents to flow out of the reagent reservoir. GPC04 1. Preferably, the test module also has a CMOS circuit and a temperature sensor, and the CMOS circuit is configured to use the temperature sensor output to feedback control the first and second PCR sections. GPC041. Preferably, the first PCR portion has a PCR microchannel in which the thermal cycle genetic material, the PCR microchannel defines a flow path having a cross-section of less than 1,00,000 square microns across the flow path 201211532. GPC041. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GPC041. Preferably, the first PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a ruthenium structure. φ GPC041. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during thermal cycling. GPC041. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve the meniscus from the meniscus holder A heater that restores the capillary drive flow. An easy-to-use, mass-produced, inexpensive, and portable test module that accepts a sample containing nucleic acid, and then uses the parallel nucleic acid amplification portion of the module and utilizes reagents stored in the reagent reservoir of the test module. Increase the nucleic acid target in the sample. The Nucleic Acid Amplification Department provides capillary action advancement of the amplification mixture, simplifies the design of the analysis system, further increases reliability and reduces the cost of the analysis system. Amplification of the target nucleic acid sequence increases the sensitivity and signal-to-noise ratio of the analytical system. Further, parallel amplification chambers allow different targets or target groups to be optimized for different pairs of primer pairs or different pairs of primer pairs, and different optimized amplification parameters are used, with increased analysis Sensitivity, News -35- 201211532 - noise ratio and reliability. The reagent reservoir is integrated with the test module and meets all of the reagent requirements for analysis. The reagent reservoir provides low system component counts and simple manufacturing procedures, resulting in an inexpensive analytical system. GPC042. 1 Aspects of the invention provide a microfluidic device for amplifying DNA and RNA, the microfluidic device comprising: an inlet for receiving a sample comprising genetic material comprising DN A and RNA; a plurality of reagent reservoirs for inclusion a reagent to the sample; a first nucleic acid amplification unit for amplifying at least some genetic material; and a second nucleic acid amplification unit for amplifying at least some genetic material in parallel with the first nucleic acid amplification portion. GPC042. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second PCR unit, the configuration thereof Used to amplify RNA in genetic material. GPC042. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second PCR portion has a second set of primer pairs for binding to the second set of complementary nucleic acid sequences, DNA The first set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GPC042. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type; polymerase type; -36- 201211532 deoxyribose Nucleoside triphosphate concentration; buffer solution; thermal cycle time; thermal cycle repetition; and, temperature during a specific PCR phase. GPC042. 5 Preferably, the 'microfluidic device also has a photosensor, a first hybridization portion downstream of the first PCR portion, and a second hybridization portion downstream of the second PCR portion, the first hybridization portion having a first target nucleic acid sequence Hybridizing to form a first probe array of probe-target hybrids, and the second hybridization portion has a second probe array for hybridization with a second target nucleic acid sequence to form a probe-target hybrid, wherein The photosensor system is configured to detect probe-target hybrids. GPC042. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit configured Used to amplify RNA in the φ substance of the genetic material. GPC0 42. Preferably, the first thermostatic nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second thermostated nucleic acid amplification portion has a second set of complementary nucleic acids with the RNA. A second set of primer pairs that are sequence-bound, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. GPC042. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters: the amplification parameter is at least one of the following: -37- 201211532 reverse transcriptase type: polymerization Enzyme type: Deoxyribonucleoside triphosphate concentration: buffer solution; and, the temperature during nucleic acid amplification. GPC042. Preferably, the microfluidic device further has a photo sensor, a first hybridization portion downstream of the first thermostatic nucleic acid amplification portion, and a second hybridization portion downstream of the second thermostatic nucleic acid amplification portion, the first hybridization portion having The first target nucleic acid sequence hybridizes to form a first probe array of probe-target hybrids and the second hybridization portion has a first hybridization with a second target nucleic acid sequence to form a probe-target hybrid A two-probe array in which a photosensor is configured to detect a probe-target hybrid. GPC042. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system within each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences, And the second hybridization portion has an array of second hybridization chambers comprising a second probe such that the second probe line within each hybridization chamber is configured to hybridize to one of the second target nucleic acid sequences. GPC042. Il Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of first and second hybrid chambers. GPC042. Preferably, the first thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow path having a cross section of less than 100,000 square micrometers across the flow path. -38- 201211532 GPC042. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GPC042. Preferably, the reagent reservoirs each have a surface tension valve in which the reagent is retained, and the surface tension valve has a meniscus holder that is used to fix the meniscus of the reagent until contact with the sample stream to remove the meniscus Face to allow reagents to flow from the reagent reservoir. GPC042. Preferably, the microfluidic device also has a CMOS circuit and a temperature sensor, the CMOS circuit being configured to use the temperature sensor output to feedback control the first and second PCR portions. GPC042. Preferably, the first PCR portion has a PCR microchannel, wherein during use, the thermal cycle genetic material, the PCR microchannel defines a flow path having a cross-section across the flow path of less than 100,000 square microns. GPC042. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GPC042. Preferably, the first PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a ruthenium structure. GPC042. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during thermal cycling. GPC042. Preferably, the active valve train has a boiling pilot-39-201211532 valve configured to hold a meniscus retainer that blocks the meniscus drive flow of the liquid, and is used to boil the liquid to be fixed from the meniscus The heater removes the meniscus to restore the capillary drive flow to the heater. An easy to use, mass-produced, and inexpensive microfluidic device receives a sample comprising DNA and RNA sequences, and then amplifies the sample using a parallel nucleic acid amplification portion of the device and using reagents stored in a reagent reservoir of the microfluidic device Standard DNA and RNA sequences. The Nucleic Acid Amplification Department provides capillary action advancement of the amplification mixture, simplifies the design of the analysis system, further increases reliability and reduces the cost of the analysis system. Amplification of the target DNA and RNA sequences increases the sensitivity of the analytical system and the signal-to-noise ratio. Further, parallel amplification chambers allow different targets or target groups to be optimized for different pairs of primer pairs or different pairs of primer pairs, and different optimized amplification parameters are used, with increased analysis Sensitivity, signal-to-noise ratio and reliability. The reagent reservoir is integrated with the microfluidic device and meets all reagent requirements for analysis. The reagent reservoir provides low system component count and simple manufacturing procedures, resulting in an inexpensive analytical system. GPC043. 1 Aspects of the invention provide a test module for amplifying DNA and RNA, the test module comprising: a housing having a container for receiving a sample comprising genetic material comprising DNA and RNA: a plurality of reagent reservoirs, for use a reagent for increasing to a sample; a first nucleic acid amplification portion for amplifying at least some genetic material; and a second nucleic acid amplification portion for amplifying at least some genetic material parallel to the first nucleic acid amplification portion . -40- 201211532 GPC043. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second PCR unit, the configuration thereof Used to amplify RNA in genetic material. GPC043. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second PCR portion has a second set of primer pairs for binding to the second set of complementary nucleic acid sequences, DNA The first set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GPC043. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type; polymerase type; deoxyribonucleoside triphosphate Concentration; buffer solution; thermal cycle time; thermal cycle repetition; and, temperature during a particular PCR phase. GPC04 3. Preferably, the test module also has a photo sensor, a first hybridization portion downstream of the first PCR portion, and a second hybridization portion downstream of the second PCR portion, the first hybridization portion having a hybridization with the first target nucleic acid sequence. Forming a first probe array of probe-target hybrids, and the second hybridization portion has a second probe array for hybridizing with the second target nucleic acid sequence to form a probe-target hybrid, wherein the light The sensor system is configured to detect probe-target hybrids. -41 - 201211532 GPC043. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. Used to amplify RNA in genetic material. GPC043. Preferably, the first thermostatic nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second thermostated nucleic acid amplification portion has a second set of complementary nucleic acids with the RNA. A second set of primer pairs that are sequence-bound, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. GPC043. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: a reverse transcriptase type; a polymerase type; Oxyribonucleoside triphosphate concentration; buffer solution; and, temperature during nucleic acid amplification. GPC 043. Preferably, the test module further comprises a photo sensor, a first hybridization section downstream of the first thermostatic nucleic acid amplification section, and a second hybridization section downstream of the second thermostatic nucleic acid amplification section, the first hybridization section having The first target nucleic acid sequence hybridizes to form a first probe array of probe-target hybrids, and the second hybridization portion has a second hybridization nucleic acid sequence for hybridization with a second target nucleic acid sequence to form a probe-target hybrid A two-probe array in which a photosensor is configured to detect a probe-target hybrid. -42- 201211532 GPC043. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first needle such that the first line configuration within each hybridization chamber is hybridized to one of the first target nucleic acid sequences, and the second portion A second hybridization chamber array comprising a second probe is configured such that a second probe line within each chamber is configured to intersect one of the second target nucleic acid sequences. GPC043. 1 1 Preferably, the photo sensor is an array of photodiodes arranged in alignment with the first and the first φ inter-chamber arrays. GPC043. Preferably, the first thermolabic nucleic acid amplification unit has an acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification micropass has a flow path of less than 100,000 square micrometers across the cross section of the flow path. GPC043. Preferably, the nucleic acid amplification microchannel has a transverse cross-section of less than 1 6,000 square microns. GPC043. Preferably, the reagent reservoirs each have a surface φ valve for retaining the reagent therein, and the surface tension valve has a meniscus fixed for fixing the meniscus of the reagent until contact with the sample stream removes the bend to allow the reagent Flowing out of the reagent reservoir. GPC043. Preferably, the test module also has a CMOS and a temperature sensor, and the CMOS circuit is configured to use a temperature sensor to feedback control the first and second PCR sections. GPC043. Preferably, the first PCR portion has a PCR channel, wherein during use, the thermal cycle genetic material, the PCR microchannel has a cross-section of less than 100,000 square micrometers across the flow path - probe hybrid hybrid The heterogeneous heterogeneous nuclear pathway cross-flow tensor, the liquid-level circuit output micro-pass defines the path -43- 201211532. GPC043. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GPC043. Preferably, the first PCR portion has a plurality of elongated PCR chambers each formed by a channel portion of a respective PCR microchannel, the channel portions being parallel and adjacent to each other such that the PCR microchannel has a ruthenium structure. GPC043. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during thermal cycling. GPC043. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve the meniscus from the meniscus holder A heater that restores the capillary drive flow. An easy-to-use, mass-produced, inexpensive, and portable test module that receives a sample containing DNA and RNA sequences, and then uses the parallel nucleic acid amplification portion of the module and utilizes reagents stored in the reagent reservoir of the test module Amplify the target DNA and RNA sequences in the sample. The Nucleic Acid Amplification Department provides capillary action advancement of the amplification mixture, simplifies the design of the analysis system, further increases reliability and reduces the cost of the analysis system. Amplification of the target DNA and RNA sequences increases the sensitivity of the analytical system and the signal-to-noise ratio. Further, the parallel amplification chamber allows for different target or target groups to be optimized using different primer pairs or different primer pair groups, and also using different optimized amplification parameters' with subsequent analysis Spirit -44- 201211532 Sensitivity, signal-noise ratio and reliability. The reagent reservoir is integrated with the test module and meets all of the reagent requirements for analysis. The reagent reservoir provides low system component counts and simple manufacturing procedures, resulting in an inexpensive analytical system. GCF034. 1 Aspects of the invention provide a on-wafer laboratory (LOC) device for genetic analysis of a biological sample, the LOC device comprising: an inlet for receiving a sample comprising genetic material; a φ support substrate; a plurality of reagent reservoirs; The first nucleic acid amplification unit is configured to amplify the nucleic acid sequence in the genetic material; and the second nucleic acid amplification unit is configured to amplify the nucleic acid sequence in the genetic material, which is parallel to the first nucleic acid amplification unit; wherein, the first Both the nucleic acid amplification unit and the second nucleic acid amplification unit are supported on a support substrate. GC?034. Preferably, the first nucleic acid amplification unit is a first polymerase linked to a φ-lock reaction (PCR) unit and the second nucleic acid amplification unit is a second PCR unit. GCF034. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs for binding to the second set of complementary nucleic acid sequences 'the first set of complementary The nucleic acid sequence differs from the second set of complementary nucleic acid sequences. GCF034. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type; polymerase type; -45- 201211532 deoxyribose Nucleoside triphosphate concentration; buffer solution; thermal cycle time; thermal cycle repetition; and, temperature during a specific PCR phase. GCF03 4. 5 Preferably, the 'LOC device also has a photo sensor, a first hybridization portion downstream of the first PCR portion, and a second hybridization portion downstream of the second pCr portion, the first hybridization portion having a hybridization with the first target nucleic acid sequence Forming a first probe array of probe-target hybrids, and the second hybridization portion has a second probe array for hybridizing with the second target nucleic acid sequence to form a probe-target hybrid, wherein the light The sensor system is configured to detect probe-target hybrids. GCF03 4. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. GCF03 4. Preferably, the first thermostated nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second thermostated nucleic acid amplification portion has a second binding to the second set of complementary nucleic acid sequences. The set of primer pairs differs from the first set of complementary nucleic acid sequences to the second set of complementary nucleic acid sequences. GCF034. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: a reverse transcriptase type; a polymerase type; Oxygen ribonucleoside triphosphate concentration; -46- 201211532 buffer solution; and, temperature during nucleic acid amplification. GCF034. 9 Preferably, the 'LOC device also has a photo sensor, a first hybridization portion downstream of the first constant temperature nucleic acid amplification portion, and a second hybridization portion downstream of the second thermostatic nucleic acid amplification portion, wherein the first hybridization portion has The first target nucleic acid sequence hybridizes to form a first probe array of the probe-target hybrid, and the second hybridization portion has a first hybridization to the second target nucleic acid sequence to form a probe-target hybrid A two-probe array in which a photosensor is configured to detect a probe-target hybrid. GCF034. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system in each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences, And the second hybridization portion has a second hybridization chamber array comprising a second probe such that the second probe line within each hybridization chamber is configured to hybridize to one of the second target nucleic acid sequences. GCF034. Il Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of first and second hybrid chambers. GCF034-1 2 Preferably, the first thermostatic nucleic acid amplification portion has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow having a cross section across the flow path of less than 100,000 square micrometers. path. GCF034. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 16,000 square microns. GCF034. Preferably, the reagent reservoirs each have a surface tension -47 - 201211532 valve to retain the reagent therein, and the surface tension valve has a meniscus holder for fixing the meniscus of the reagent until contact with the sample stream The meniscus is removed to allow reagents to flow out of the reagent reservoir. GCF034. Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the first and second PCR sections, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit The system is configured to feedback control the first and second PCR sections using a temperature sensor output. GCF034. Preferably, the first PCR portion has a PCR microchannel, wherein when used, the thermal cycle sample, the PCR microchannel defines a flow path having a cross-sectional cross-section of less than 100,000 square microns" GCF034. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GCF034. Preferably, the first PCR portion has a plurality of extended PCR chambers each formed by a channel portion of a respective PCR microchannel having a 蜿蜒 structure formed by a series of wide meandering flows, width Each of the meanders is a channel portion that forms one of the extended PCR chambers. GCF034. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during thermal cycling. GCF034. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve the meniscus from the meniscus holder A heater that restores the capillary drive flow. -48- 201211532 Easy-to-use, mass-produced and inexpensive LOC devices pass through it. The sample inlet receives the biological sample and the parallel expansion of the target gene sequence in the sample is performed using reagents stored in the reagent reservoir of the LOC device. Amplification of the target nucleic acid sequence increases the sensitivity and signal-to-noise ratio of the analytical system. Further, parallel amplification chambers allow different targets or target groups to be optimized using different primer pairs or different primer pair groups, and also use different optimized amplification parameters, which in turn increases Analysis sensitivity, signal-to-noise ratio and reliability. The reagent reservoir is combined with the LOC device and meets all of the reagent requirements for analysis. The reagent reservoir provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical system. GCF0 3 5. 1 Aspects of the invention provide a on-wafer laboratory (LOC) device for genetic analysis of a biological sample, the LOC device comprising: an inlet for receiving a sample comprising genetic material: φ a support substrate; a plurality of reagent reservoirs; a first culture portion, the first culture portion is in fluid communication with one of the reagent reservoirs, the reagent reservoir includes an enzyme for reacting with the genetic material enzyme; to amplify the nucleic acid sequence in the genetic material; and, the second culture portion The second culture part is in fluid communication with one of the reagent reservoirs, and the reagent reservoir includes an enzyme for reacting with the genetic material enzyme, the second culture portion is parallel to the first culture portion; the first nucleic acid downstream of the first culture portion An amplification unit for amplifying a nucleic acid sequence of -49-201211532 in the genetic material; and a second nucleic acid amplification portion downstream of the second culture portion for amplifying the nucleic acid sequence in the genetic material, and the second nucleic acid amplification The portion is parallel to the first nucleic acid amplification unit; wherein the first culture unit, the first culture unit, the first nucleic acid amplification unit, and the second nucleic acid amplification unit are supported on the support substrate. GCF035. Preferably, the first nucleic acid amplification unit is a polymerase-binding reaction (PCR) unit and the second nucleic acid amplification unit is a second PCR unit. GCF03 5. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs for binding to the second set of complementary nucleic acid sequences, the first set of complementary The nucleic acid sequence differs from the second set of complementary nucleic acid sequences. GCF035. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type; polymerase type; deoxyribonucleoside triphosphate Concentration; buffer solution; thermal cycle time; thermal cycle repetition; and, temperature during a particular PCR phase. GCF03 5. Preferably, the LOC device also has a photo sensor, a first hybridization portion downstream of the first PCR portion, and a second hybridization portion downstream of the second PCR portion, the first hybridization portion having a hybridization with the first target nucleic acid sequence To form a first probe array of probe-50-201211532 needle-target hybrids, and the second hybridization portion has a second probe for hybridization with the second target nucleic acid sequence to form a probe-target hybrid An array wherein the photosensor is configured to detect a probe-target hybrid. GCF03 5. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. GCF03 5. Preferably, the first thermostated nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences, and the second thermostated nucleic acid amplification portion has a second binding to the second set of complementary nucleic acid sequences. Group primer pair, the first set of complementary nucleic acid sequences are different from the second set of complementary nucleic acid sequences" GCF03 5. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: a reverse transcriptase type; a polymerase type; Oxygen ribonucleoside triphosphate concentration; buffer solution; and, temperature during nucleic acid amplification ◊ GCF03 5. Preferably, the LOC device further has a photo sensor, a first hybridization portion downstream of the first thermostatic nucleic acid amplification portion, and a second hybridization portion downstream of the second thermostatic nucleic acid amplification portion, the first hybridization portion having A target nucleic acid sequence hybridizes to form a first probe array of probe-target hybrids, and a second hybridization portion has a second for hybridization with a second target nucleic acid sequence to form a probe-target hybrid Probe array, wherein the photosensor system is configured to detect -51 - 201211532 probe-target hybrids" GCF035. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system in each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences And the second hybridization portion has a second hybridization chamber array comprising a second probe such that the second probe line within each hybridization chamber is configured to hybridize to one of the second target nucleic acid sequences. GCF03 5. Il Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of first and second hybrid chambers. GCF03 5. Preferably, the first thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the sample at the reaction temperature, and the nucleic acid amplification microchannel defines a flow path diameter of less than 100,000 square micrometers across the cross section of the flow path. GCF03 5. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GCF03 5. Preferably, the reagent reservoirs each have a surface tension valve for retaining the reagent therein, and the surface tension valve has a meniscus holder for fixing the meniscus of the reagent until contact with the sample stream to remove the meniscus surface. GCF03 5. Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the first and second PCR sections, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit The system is configured to feedback control the first and second PCR sections using a temperature sensor output. 201211532 GCF03 5. Preferably, the first PCR portion has a PCR microchannel, wherein, when in use, the thermal cycle sample, the PCR microchannel defines a flow path having a cross-section across the flow path of less than 100,000 square microns. GCF03 5. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GCF03 5. Preferably, the first PCR portion has a plurality of extended PCR chambers, each formed by a channel portion of a respective PCR microchannel, and the φ PCR microchannel has a 蜿蜒 structure formed by a series of wide meandering flows. Each of the wide meanders is a channel portion that forms one of the extended PCR chambers. GCF03 5. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during thermal cycling. GCF03 5. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve the meniscus from the meniscus holder Make it a heater that restores the capillary drive flow. The easy-to-use, mass-produced and inexpensive LOC device receives the biological sample through its sample inlet, performs parallel processing of the sample in the parallel culture portion, and uses the reagent stored in the reagent reservoir of the L0C device in the parallel nucleic acid amplification unit. The target gene sequences in the sample were amplified in parallel. In the culture department, genetic material is processed in various ways, such as nucleic acid restriction and conjugation of aptamer primers, to provide the most suitable or necessary conditions for the next analysis stage, to increase the information content of the analysis results, and to increase the analysis system. Sensitivity and signal - noise ratio and reliability. Further, the -53-201211532 parallel culture chamber allows for different nucleic acid stencils or template groups to optimize for different enzyme reactions, resulting in improved assay diversity. Amplification of the target nucleic acid sequence increases the sensitivity and signal-to-noise ratio of the analytical system. Further, parallel amplification chambers allow different targets or target groups to be optimized for different pairs of primer pairs or different pairs of primer pairs, and different optimized amplification parameters are used, with increased analysis Sensitivity, signal-to-noise ratio and reliability. The reagent reservoir is combined with the LOC device and meets all reagent requirements for analysis. The reagent reservoir provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical system. GCF03 6. 1 Aspects of the invention provide a on-wafer laboratory (LOC) device for genetic analysis of a biological sample, the LOC device comprising: an inlet for receiving a sample comprising genetic material comprising DNA and RNA; a support substrate; a reagent reservoir; a first nucleic acid amplification unit for amplifying at least some genetic material: and a second nucleic acid amplification unit for amplifying at least some genetic material, and the second nucleic acid amplification unit and the first nucleic acid amplification unit Parallel; wherein the first nucleic acid amplification portion and the second nucleic acid amplification portion are both supported on the support substrate" GCF03 6. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second PCR unit configured to be used for Amplify the RNA of 201211532 in the genetic material. GCF03 6. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of interdigitated nucleic acid sequences in the DNA. And the second PCR portion has a second set of primer pairs for binding to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. GCF036. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: φ reverse transcriptase type; polymerase type; deoxyribonucleoside III Phosphoric acid concentration; buffer solution; thermal cycle time; thermal cycle repetition; and, temperature during a specific PCR phase. GCF03 6. Preferably, the LOC device also has a photo sensor, a first hybridization portion downstream of the first φ PCR portion, and a second hybridization portion downstream of the second PCR portion, the first hybridization portion having a first target nucleic acid sequence Hybridizing to form a first probe array of probe-target hybrids, and the second hybridization portion has a second probe library for hybridizing with the second target nucleic acid sequence to form a probe/target hybrid, The photosensor is configured to detect a probe-target hybrid. GCF036. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit configured to be configured to Amplify RNA from -55 to 201211532 in genetic material. GCF036. Preferably, the first thermostated nucleic acid amplification section has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second thermolabic nucleic acid amplification section has a complement to the second set of RNA. A second set of primer pairs to which the nucleic acid sequence is affixed, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. GCF036. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: a reverse transcriptase type; a polymerase type; Oxyribonucleoside triphosphate concentration: a buffer solution; and, the temperature during nucleic acid amplification. GCF036. Preferably, the LOC device further has a photo sensor, a first hybridization portion downstream of the first thermostatic nucleic acid amplification portion, and a second hybridization portion downstream of the second thermostatic nucleic acid amplification portion, the first hybridization portion having A target nucleic acid sequence hybridizes to form a first probe array of probe-target hybrids, and a second hybridization portion has a second for hybridization with a second target nucleic acid sequence to form a probe-target hybrid A probe array in which a photosensor is configured to detect a probe-target hybrid. GCF03 6. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system within each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences, And the second hybridization section -56-201211532 has a second hybridization chamber array comprising a second probe such that the second probe line within each hybridization chamber is configured to hybridize to one of the second target nucleic acid sequences. GCF036. Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of first and second hybrid chambers. GCF036. Preferably, the first thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the reaction temperature of the sample, and the nucleic acid amplification microchannel defines a flow path diameter of less than 100,000 square micrometers across the cross section of the flow path. GCF036. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 16,000 square microns. GCF036. Preferably, the reagent reservoirs each have a surface tension valve for retaining the reagent therein, and the surface tension valve has a meniscus holder for fixing the meniscus of the reagent until contact with the sample stream to remove the meniscus surface. GCF03 6. Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the first and second PCR sections, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit The system is configured to feedback control the first and second PCR sections using a temperature sensor output. GCF03 6. Preferably, the first PCR portion has a PCR microchannel, wherein when in use, the thermal cycle sample, the PCR microchannel defines a flow path having a cross-sectional cross-section of less than 100, 〇〇〇 square microns. GCF03 6. Preferably, the PCR microchannel has at least one parallel heater element extending from -57 to 201211532 to the PCR microchannel. GCF036. Preferably, the first PCR portion has a plurality of extended PCR chambers each formed by a channel portion of a respective PCR microchannel having a 蜿蜒 structure formed by a series of wide meandering flows, width Each of the meanders is a channel portion that forms one of the extended PCR chambers. GCF036. Preferably, the first PCR portion has an active valve for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit to allow liquid to flow to the first hybridization chamber array during thermal cycling. GCF036. Preferably, the active valve system has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid to relieve the meniscus from the meniscus holder A heater that restores the capillary drive flow. The easy-to-use, mass-produced and inexpensive LOC device receives a biological sample through its sample inlet, and uses the reagent stored in the reagent reservoir of the LOC device to perform DNA and RNA of the target gene sequence in the sample in the parallel nucleic acid amplification portion. Parallel processing. Amplification of the target nucleic acid sequence increases the sensitivity and signal-to-noise ratio of the analytical system. Further, the parallel amplification chamber allows for different target or target groups to be optimized using different primer pairs or different primer pair groups, and also using different optimized amplification parameters' with subsequent analysis Sensitivity, signal-to-noise ratio and reliability. The reagent reservoir is combined with the LOC device and meets all reagent requirements for analysis. The reagent reservoir provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical system. -58- 201211532 GCF0 3 7. 1 Aspects of the invention provide a on-wafer laboratory (L〇C) device for genetic analysis of a biological sample, the L〇C device comprising: an inlet for receiving a sample comprising genetic material comprising DNA and RNA; a plurality of reagent reservoirs; a first culture portion, the first culture portion is fluidly connected to one of the reagent reservoirs, and the reagent reservoir includes an enzyme for reacting with genetic material enzymes; The second culture unit is in fluid communication with one of the reagent reservoirs, and the reagent reservoir includes an enzyme for reacting with the genetic material enzyme. The second culture portion is parallel to the first culture portion; the first nucleic acid amplification downstream of the first culture portion a portion for amplifying at least some genetic material; and a second nucleic acid amplification portion downstream of the second culture portion for at least some of the amplified genetic material, the second nucleic acid amplification portion being parallel to the first nucleic acid amplification portion In the φ, the first culture part, the second culture part, the first nucleic acid amplification part, and the second nucleic acid amplification part are all supported on the support substrate. GCF03 7. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second PCR unit configured to be used for Amplify RNA in genetic material. GCF03 7. Preferably, the first PCR portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second PCR portion has a second set of primer pairs for binding to the RNA in -59-201211532 The second set of complementary nucleic acid sequences 'the first set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF03 7. Preferably, the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: reverse transcriptase type; polymerase type; deoxyribonucleoside triphosphate Concentration; Buffer solution: Thermal cycle time; Thermal cycle repeat: and, during the specific PCR phase. GCF03 7. Preferably, the LOC device also has a photo sensor, a first hybridization portion downstream of the first PCR portion, and a second hybridization portion downstream of the second PCR portion, the first hybridization portion having a hybridization with the first target nucleic acid sequence Forming a first probe array of probe-target hybrids, and the second hybridization portion has a second probe array for hybridizing with the second target nucleic acid sequence to form a probe-target hybrid, wherein the light The sensor system is configured to detect probe-target hybrids. GCF03 7. Preferably, the first nucleic acid amplification unit is a first constant temperature nucleic acid amplification unit configured to amplify DNA in the genetic material, and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit configured to be configured to Amplify RNA in genetic material. GCF037. Preferably, the first thermostatic nucleic acid amplification portion has a first set of primer pairs for binding to the first set of complementary nucleic acid sequences in the DNA, and the second constant temperature nucleic acid amplification portion has the second and the second RNA in the 201211532 The second set of primer pairs to which the complementary nucleic acid sequences are bound differs from the first set of complementary nucleic acid sequences to the second set of complementary nucleic acid sequences. GCF03 7. Preferably, the first constant temperature nucleic acid amplification unit and the second constant temperature nucleic acid amplification unit are configured to operate with different amplification parameters, and the amplification parameter is at least one of the following: a reverse transcriptase type; a φ polymerase type; Deoxyribonucleoside triphosphate concentration; buffer solution; and, temperature during nucleic acid amplification. GCF03 7. Preferably, the LOC device further has a photo sensor, a first hybridization portion downstream of the first thermostatic nucleic acid amplification portion, and a second hybridization portion downstream of the second thermostatic nucleic acid amplification portion, the first hybridization portion having A target nucleic acid sequence hybridizes to form a first probe array of probe-target hybrids, and a second φ hybridization portion has a hybridization for hybridization with a second target nucleic acid sequence to form a probe-target hybrid A two-probe array in which a photosensor is configured to detect a probe-target hybrid. GCF037. Preferably, the first hybridization portion has an array of first hybridization chambers comprising a first probe such that the first probe system within each hybridization chamber is configured to hybridize to one of the first target nucleic acid sequences, And the second hybridization portion has a second hybridization chamber array comprising a second probe such that the second probe line within each hybridization chamber is configured to hybridize to one of the second target nucleic acid sequences. 201211532 GCF037. Preferably, the photosensor is an array of photodiodes arranged in alignment with the array of first and second hybrid chambers. GCF03 7.  Preferably, the first thermolabic nucleic acid amplification section has a nucleic acid amplification microchannel to maintain the reaction temperature of the sample, and the nucleic acid amplification microchannel defines a flow path diameter of less than 100,000 square micrometers across the cross section of the flow path. GCF03 7. Preferably, the nucleic acid amplification microchannel has a cross-section across the flow path of less than 1 6,000 square microns. GCF03 7. Preferably, the reagent reservoirs each have a surface tension valve for retaining the reagent therein, and the surface tension valve has a meniscus holder for fixing the meniscus of the reagent until contact with the sample stream to remove the meniscus surface. GCF037. Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the first and second PCR sections, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit The system is configured to feedback control the first and second PCR sections using a temperature sensor output. GCF037. Preferably, the first PCR portion has a PCR microchannel, wherein when used, the thermal cycle sample, the PCR microchannel defines a flow path having a cross-section across the flow path of less than 100,0 square micrometers. . GCF037. Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GCF03 7. Preferably, the first PCR portion has a plurality of extended PCR chambers, each formed by a channel portion of a respective PCR microchannel.

The 201211532 PCR microchannel has one of a series of wide curved manifolds for each of the extended PCR chambers. GCF037.1 9 Preferably, the first PCR is used to retain liquid in the first PCR section and respond The activation signal allows the liquid to flow to the first hybridization chamber _ GCF037.20. Preferably, the active valve system prevents the bending of the meniscus of the capillary drive flow of the liquid, and the liquid is used to boil the liquid from the meniscus. A heater that restores the capillary drive flow. The sample inlet, which is easy to use, can be mass-produced, and inexpensive, receives the biological sample, is cultured in parallel, and is amplified by the target DNA in the sample by the parallel nucleic acid amplification section stored in the LOC device.

In the culture department, the genetic material undergoes various nucleic acid restriction and conjugation of various genomic primers, and the most suitable or necessary conditions in the analysis phase of reverse transcription, enhances the tolerance, and increases the sensitivity and sensitivity of the analysis system. Further, the parallel culture chamber allows the different groups to optimize the different enzyme reactions. The amplification of the target nucleic acid sequence increases the analysis of the i-to-noise ratio. Further, the parallel amplification chamber allows one group to optimize the use of different primer pairs or different uses of different optimized amplification parameters, and then the force I ζ structure, the channel portion of the wide bender The part has an active valve from the CMOS circuit during the thermal cycle. A reagent having a boiling indexer configured to fix the artifact holder releases the meniscus to allow the LOC device to perform a sample in the parallel buffer of the sample through which the sample is processed, for example, To provide information on the results of the next analysis, the number / noise ratio and reliable J nucleic acid template or template group: to enhance the diversity of analysis. Flow sensitivity and signal-=the same target or target group: the primer pair group, and also the sensitivity of the analysis, the signal -63- 201211532 - noise ratio and reliability. The reagent reservoir is combined with the LOC device and meets all reagent requirements for analysis. The reagent reservoir provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical system. [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview This general specification indicates the main components of a molecular diagnostic system incorporating a specific embodiment of the present invention. The details of the construction and operation of this system are described in the following description. Referring to Figures 1, 2, 3, 123 and 124, the system has the following most important components: Test modules 10 and 11 are typical USB flash drives and are very inexpensive to manufacture. The test modules 10 and 11 each comprise a microfluidic device, typically in the form of a laboratory on a wafer (LOC) device 30, preloaded with reagents and typically more than one or more probes for diagnostic analysis of the molecule. Needle (see Figures 1 and 1 2 3). When the test module 1 1 in FIG. 1 2 3 uses an electrochemiluminescence-based detection technique, the test module 10 shown in FIG. 1 uses a fluorescence-based detection technique to identify the target. molecule. The LOC device 30 has an integrated light sensor 44 for fluorescence or electrochemiluminescence detection (described in more detail below). Both test modules 10 and 1 1 use standard micro USB connectors 14 for power, data and control, each having a printed circuit board (PCB) 5 7, and each having an externally powered capacitor 32 and an inductor - 64- 201211532 1 5. Both test modules 1 and 11 are for a single use for mass production and are distributed in aseptic packaging for use. The outer casing 13 has a large container 24 for receiving a biological sample 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 guard 410 forms part of the outer casing 13 to reduce the moisture resistance in the test module, while the pressure drop is provided by the small pressure fluctuation. The membrane guard 4 10 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 described in a number of different forms' and its selection. The reader 12 version shown in Figures 1, 3 and 123 is a specific embodiment of a smart phone. A block diagram of the reader 12 is shown in FIG. Processor 42 executes the application software from program storage 43. The processor 42 is also interfaced with a display screen 18 and a user interface (UI) touch screen 1 7 and button 19, a cellular radio 2 1 , a wireless network connection 23 ', and a satellite navigation system 25. Honeycomb radio 21 and wireless φ network connection 2 3 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location information. Alternatively, the location data can be manually entered using the touch screen 17 or button 19. The data store 27 holds genetic and diagnostic information, test results, patient information, analysis and probe data for identifying each probe, and its array position. The data store 27 and the program store 43 can be shared by a common memory device. The application software installed in the test module reader 12 provides results analysis and additional test and diagnostic information. To perform a diagnostic test, the test module 1 (or test module i) is inserted into the micro-USB port 16 on the test module reader 12. The sterile dense -65-201211532 sealing tape 22 is turned up and the biological sample (in liquid form) is loaded into the sample large container 24. Pressing the start button 20 to initiate the test by applying the software" sample flows into the LOC device 30 and is extracted, cultured, amplified, and pre-synthesized hybrid-reactive nucleosides by on-board assay. The acid probe hybridizes to the sample nucleic acid (target). In the case of the test module 10 (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 from the hybridized probe. Launch (see Figures 1 and 2). In test module 11 (which uses electrochemical luminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to produce photoluminescent fluorescing. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 124). A light sensor 44 integrated with a CMOS circuit on each LOC device is used to detect the emission (fluorescent or photoluminescence). The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the result. Data can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 11 is removed from the test module reader 1 2 and processed as appropriate. 1, 3 and 123 show a test module reader 12 configured as a mobile/smartphone 28. In other forms, the test module reader is a laptop/notebook 101, a dedicated reader 103, an e-book reader 107, a tablet computer 109 or used in a hospital, private clinic or laboratory. Desktop computer 05 (see Figure 125). The reader can interface with a number of additional applications, such as patient records, accounting, online databases, and multi-user environments. It can also interface with some local or remote peripherals. -66- 201211532 such as printers and patient smart cards. Referring to FIG. 126, the data generated by the test module 10 through the reader 12 and the network 125 can be used to update the epidemiological database contained in the host system for epidemiological data 111 for genetic data 113. The genetic database contained in the host system, the electronic health record contained in the host system for electronic health record (EHR) 15 , and the host system for electronic medical record (EMR) 12 1 There are electronic medical records, and personal health records contained in the host system for personal health record (PHR) 123. Conversely, the epidemiological data contained in the host system for epidemiological data 111, the genetic data contained in the host system for genetic material 113, and the use of the network 1 25 and the reader 12 are used. An electronic health record contained in the host system of the Electronic Health Record (EHR) 15 , an electronic medical record contained in the host system for the Electronic Medical Record (EMR) 121, and a personal health record (PHR) 123 The personal health record contained in the host system can be used to update the digital memory in test module 10 LOC 30 φ. Referring again to Figures 1 '2' 123 and 124, in the mobile phone configuration, the reader 12 uses battery power. Mobile phone reading. The extractor contains all preloaded test and diagnostic information. Data can also be loaded or updated via some wireless or contact interface to enable communication with peripheral devices, computers or online servers. Set the mini-USB port 16 to connect to the computer or main power supply to recharge the battery. Figure 70 shows a specific embodiment of a test module for testing whether only a specific target is present or not, such as whether the test individual is subjected to -67-201211532, for example, influenza A virus Η 1 N 1 infection. It is only suitable as a built-in module 47 for USB power/indicator only. No other readers or application software is required. The indicator 45 on the module 47 of the USB power/indicator only shows a positive or negative result. This configuration is ideal for large screenings. Additional items supplied to the system may include test tubes containing reagents for pre-treating a particular sample, and a tongue depressor and lancet containing sample collection. For convenience, the test module of the embodiment shown in Fig. 70 includes a spring-loaded retractable lancet 390 and a lancet release button 392. Satellite phones can be used in remote areas. Test Module Electronics Figures 2 and 1 24 are block diagrams of the electronic components of test modules 10 and 11. The CMOS circuit integrated in the on-wafer laboratory device (LOC) 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a timer 33, a power conditioner 31, a RAM 38, and a program and data. Flash memory 40. These provide test patterns for the entire including the photo sensor 44, the temperature sensor 170, the liquid sensor 1 74, and various heaters 1 5 2 ' 1 5 4, 1 8 2, 2 3 4 Group 1 0 or 1 1 and associated drives 3 7 and 3 9 and the registers and registers of registers 3 5 and 4 1 . Only the L E D 2 6 (in the example of the test module 10), the external power supply capacitor 3 2 and the micro USB plug 14 are external to the laboratory device 30 on the wafer. The on-wafer laboratory device 30 includes an adhesive pad for joining to the outer components. The RAM 38 and the program and data flash memory 40 have application software and diagnostic and detection information for one probe (flash/protection - 68-201211532 full storage, for example via encryption). In the example of the test module 11 configured with ECL detection, there is no LED 26 (see Figures 123 and 124). The data is encrypted by the on-chip laboratory device 30 to preserve storage and communicate with external devices. The on-wafer laboratory device 30 is loaded with electrochemiluminescent probes and the hybridization chamber, each having an ECL excitation electrode pair 860 and 870. Many types of test modules 1 are manufactured in some form of test and are ready for ready use. These test forms differ in the onboard analysis of reagents and probes. Some examples of infectious diseases that are quickly identified by this system include: • Influenza-influenza virus A, B, C, infectious salmon anemia virus, toco soil virus • pneumonia-respiratory fusion virus (RSV), gland Virus, interstitial pneumonia virus, pneumococci, Staphylococcus aureus • tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africana, Mycobacterium vaccae and Mycobacterium vaccae • Plasmodium falciparum, Toxoplasma gondii and other parasitic protozoa • Typhoid-cold bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever - flavivirus • Hepatitis (A to E) • Iatrogenic infections – such as refractory spores Bacteria, vancomycin-resistant enterococci, and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) -69 - 201211532 Giant cell virus (CMV) Epstein-Barr virus (EBV) Encephalitis - Japanese encephalitis virus, Zhangdipula virus Baishu cough-pertussis measles-paramyxovirus meningitis-Streptococcus pneumoniae and meningococcus anthracis anthracnose-B. anthracis identified by this system Some examples include: spastic fibrosis, hemophilia, sickle cell anemia, black leukoplakia, hemochromatosis.  Crohn's disease Crohn's disease Polycystic kidney disease Congenital heart disease Leier's disease A few options for cancer identified by this diagnostic system include: Ovarian cancer Colon cancer Multiple endocrine tumors Retinoblastoma • Turk's disease (Turcot) Syndrome) 201211532 The above list is not exhaustive and the diagnostic system can be configured to detect many different diseases and symptoms using nucleic acid and protein analysis. Detailed Structure of System Components LOC Device The LOC device 30 is the center of the diagnostic system. It uses microfluidic platforms to rapidly perform four important steps in nucleic acid-based molecular diagnostic analysis, φ, sample preparation, nucleic acid extraction, nucleic acid amplification, and detection. LOC devices also have alternative uses' and will be described in more detail below. As discussed above, the test modules 1 〇 and π can take many different configurations to detect different targets. Similarly, LOC device 30 has many different specific 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 illustration, the structure and operation of LOC device 301 are described in detail with reference to Figures 4 through 26 and 27 through 57. φ Figure 4 is a schematic diagram of the structure of the LOC device 301. For the sake of convenience, the processing stages shown in Fig. 4 are represented by element symbols corresponding to the functional portions of the LOC device 301 that implements the processing stage. The processing stages associated with the major steps in each of the nucleic acid-based molecular diagnostic assays also represent: sample input and preparation 288, extraction 290, culture 291, amplification 292, and detection 294. The various reservoirs, chambers, valves, and other components of LOC device 301 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 structure of the LOC device -71 - 201211532 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, covering the MST layer 87 with a cover 46. 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 size that supports a capillary action driven liquid flow having physical properties similar to the physical properties of the sample during processing. Accordingly, MST 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 appreciate 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 X 5824 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, 55 and 63, and a sufficient understanding of the respective structures in the LOC device 301 is described in detail below. When Fig. 11 shows the structure of the CMOS + MST device 48 independently, Figs. 7 to 10 independently show the features of the cover 46. -72- 201211532 Layered Structure Figures 12 and 22 are schematic views showing the layered configuration of the CMOS+ MST device 48, the cover 46, and the fluid interaction therebetween. The figures are not drawn to scale for illustrative purposes. Figure 12 is a cross-sectional view through the sample inlet 68 and Figure 22 is a cross-sectional view through the reservoir 54. As best shown in FIG. 12, the φ CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuitry 86 that operates the active components within the MST layer 87 described above. Passivation layer 88 seals and protects CMOS layer 86 from fluid flow through the MST layer 87. Fluid flows through both the cover channel 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 100 (see, for example, Figures 7 and 16). While biochemical treatment is being performed on the smaller MS T channel 90, cell transport occurs in the larger channel 94 made in the cover 46. The cell transport channel is sized to carry the cells in the sample to a predetermined point in the MST channel 90. Transporting cells larger than 20 microns in size (e.g., certain white blood cells) requires channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. In particular, the M S T channel at a position in L Ο C which does not require transport of cells can be significantly small. It will be understood that the cover channel 94 and the MST channel 90 are of the same reference and that the particular MST channel 90 can also be referred to as, for example, a heated microchannel or a dialysis MST channel depending on its particular function. The MST channel 90 is formed by uranium engraving through an M S 通道 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 which 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 8 and the reservoir layer 78 are formed from a single piece of material. Of course, the sheet of material may also be non-uniform. The sheet of material is etched from both sides to form a lid channel layer 8 and a reservoir layer 78' etches the lid channel 94 in the lid channel layer 80, and the reservoirs 54, 56, 58, 60 and 62 are etched in the reservoir layer 78. Additionally, the reservoir and the cover channel are formed by a micro-shaping process. Both etching and microforming techniques are used to fabricate channels having a cross-sectional area across the fluid that is as large as 20,00 square feet and as small as 8 square microns. There are a series of suitable choices for one of the cross-sectional areas of the channel across the fluid at different locations in the LOC device. A large number of samples or samples having a large component are accommodated in the channel, and a cross-sectional area of more than 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 across a very small cross-sectional area of the fluid. A lower seal 64 surrounds the cover passage 94 and the upper seal layer 82 surrounds the reservoirs 54' 56, 58, 60 and 62». The five reservoirs 54, 56, 58, 60 and 62 are preloaded with analytical specific reagents. In the specific embodiment of this description, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Reservoir 54: Anticoagulant with a choice of red blood cell lysis buffer • Reservoir 56: Lysis Reagent - 74 - 201211532 • Reservoir 58: Restriction of enzymes, ligases and linkers (for linker-priming PCR (see Figure 69, from T.  Stachan et al. , Human Molecular Genetics 2, Garland Science, NY and London, 1 999 excerpt) • Reservoir 60: amplification mix (deoxynucleotide triphosphates (dNTPs), bow 1 buffer, buffer) and • reservoir 62: DNA polymerase φ The lid 46 and the CMOS + MST layer 48 are in fluid communication via respective openings in the lower seal 64 and the top layer 66. The openings represent the upper conduit 96 and the lower conduit 92 depending on whether fluid flows from the MST passage 90 to the cover passage 94 or vice versa. LOC Device Operation The operation of the LOC device 301 is described step by step with reference to the analysis of pathogen DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using kits or combinations of appropriate reagents, assay protocols, LOC variants, and detection systems. Referring to Figure 4, analyzing a biological sample involves five major steps, including: sample input and preparation 28, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294. The sample input and preparation step 28 8 involves mixing the blood with the anticoagulant 116 and then separating the pathogen from the white blood cells and red blood cells by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, the blood sample enters the device via the sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the reservoir 54. When the sample blood flow 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 116 is withdrawn from the reservoir 54 by capillary action and enters the MST channel 90 via the lower conduit 92. The lower duct 92 has a capillary starting configuration feature (CIF) 02 to form the meniscus geometry such that it is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the reservoir 54, the venting opening 222 in the upper seal 82 allows air to replace the anticoagulant 116. The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and is secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. The meniscus 120 remains fixed to the upper conduit 96' prior to use 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, the surface tension valve 118 has three separate M ST channels 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent entering the sample mixture. The concentration of the reagent in the sample stream is determined by the flow rate of the sample mixture as it is mixed by diffusion and the reagents leaving the reservoir. Thus the surface tension valve of each of the reservoirs is configured to meet the desired reagent concentration. -76- 201211532 The blood passes through the pathogen dialysis unit 70 (see Figures 4 and 15: the target target cells are concentrated using a sample of holes 1 64 sized according to a predetermined valve. Cells smaller than the valve pass through the holes, Large pores can pass through the pores. When the target cells continue to be part of the analysis, the unwanted cells are reintroduced into the waste unit 76. The unwanted cells are large cells blocked by the array of holes 1 64, Or passing through the cells. φ is concentrated in the pathogen dialysis section 70 as described herein for microbial DNA analysis from the whole blood-like pathogen. The array of holes communicates with the input stream in the cover channel 94 to the target. The plurality of meter-diameter holes 1 64 of the channel 74 are formed. The 3 micron diameter hole 1 64 and the dialysis suction port 168 of the target channel 74 are connected by a series of dialysis channels 204 (best shown in Figure 15). And 21) the pathogen is so small that the target channel 74 is filled through the 3 micron diameter hole 164 via the dialysis MST channel 204. Cells larger than 3 microns, such as red blood cells and φ balls, remain in the waste channel 72 of the cover 46, The waste channel is stored Other pore shapes, sizes, and aspect ratios can be used to isolate other target cells of a particular pathogen, such as white blood cells for human DNA analysis. More details on the dialysis section and dialysis variants. 6 and 7, the fluid is drawn through the target through S to the surface tension valve 128 in the lysis reagent reservoir 56. The tension valve 128 has seven MST channels 90 extending from the lysis reagent 56 and Between the target channels 74. When the meniscus flows from the sample, the column flows from the cell to the small cell of the cell, 3 is slightly smaller than the MST, and the white blood is discharged to the waste body or the surface of the I 74 surface. The flow rate from all seven of the MST channels 90 will be greater than the flow rate from the anticoagulant reservoir 54 when the storage is removed from -77 to 201211532, wherein the surface tension valve 18 has three MST channels 90 (assuming the The physical properties of the fluid are substantially equal. Therefore, the proportion of the lysing reagent in the sample mixture is greater than the ratio of the anticoagulant. The lysis reagent and the target cell are within the chemical lysis unit 130 By diffusion mixing in the target channel 74. The initiation valve 126 stops the flow until diffusion and lysis occur for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling initiation valve are described in detail with reference to Figures 31 and 32. Other active valve types (as opposed to passive valves such as the surface tension valve 118) have also been developed by the applicant, which can be used in place of the boiling initiation valve. These alternative valve designs are also described below. Upon initiation of the valve 126, the lysed cells flow into the mixing section 131 for pre-amplification restriction digestion and linker ligation. Referring to Figure 13, when the fluid removes the meniscus on the surface tension valve 132 initiated by the mixing portion 131, the restriction enzyme, linker and ligase are released from the reservoir 58. The mixture flows through the length of the mixing portion 131 for diffusion mixing. At the end of the mixing portion 131 is a lower duct 134 (see Fig. 13) leading to the incubator inlet passage 133 of the culture portion 114. The incubator inlet channel 133 feeds the mixture into a heated microchannel 2 10 蜿蜒 configuration that provides a culture chamber that retains the sample during restriction digestion and linker ligation (see Figures 13 and 14). ). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 inserted in the -78-201211532 object AB of Figure 6. The various figures show the continuous addition of the layers forming the CMOS + MST layer 48 and the structure of the disk 46. 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 breaks into the microchannels 210 of the culture portion n4 until the boiling initiation valve 106 is reached, wherein the fluid stops while diffusion occurs. As discussed above, the microchannel 210 upstream of the boiling initiation valve 106 becomes a culture chamber containing the sample, restriction enzymes, ligase φ, and linker. The heater 154 is then activated and maintains a stable temperature for the occurrence of restriction enzyme digestion and linker binding for a specific period of time. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is selected and only requires some type of nucleic acid amplification analysis. Again, 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. This temperature increase before entering the amplification section 112, so that the restriction enzyme and the ligase are not activated. When a constant temperature nucleic acid is used to amplify φ, there is a specific correlation between restriction enzymes and ligase inactivation. After the cultivation, the boiling initiation valve 106 is activated (turned on) and the fluid flows back to the amplification unit 112. Referring to Figures 31 and 32, the mixture fills the crucible structure of the heated microchannel 158 until the boiling initiation valve 1〇8 is reached, which form one or more amplification chambers. As best seen in the cross-sectional view of Figure 30, the amplification mixture (dNTPs, primers, buffer) is released from the reservoir 60 and the polymerase is then released from the reservoir 62 into the culture section and the amplification section (each The intermediate MST channel 212 of 1 14 and 1 12). Figures 35 through 51 show the layers of LOC device -79 - 201211532 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 end of the insert AC-based amplification unit 112 and the start of the hybridization and detection unit 52. The cultured sample, amplification mixture, and polymerase flow through microchannel 1 58 to boiling initiation valve 108. After diffusion mixing for a sufficient period of time, the heater 1 54 in the microchannel 1 58 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 sufficient target DNA. After the nucleic acid amplification procedure, the boiling initiation valve 108 is opened and the fluid re-enters the hybridization and detection portion 52. The operation of the boiling initiation valve is described in more detail below. As shown in Figure 52, the hybridization and detection section 52 has an array of hybridization chambers 110. Figures 5, 5, 3, 5 and 5 show the hybridization chamber array 110 and the individual hybridization chambers 180 in detail. The entrance to the hybridization chamber 180 is a diffusion barrier 175 that prevents diffusion of the target nucleic acid, probe strands, and hybridization probes between the hybridization chambers 180 during hybridization to prevent erroneous hybridization detection results. The path length 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 in some of the hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 184 of the hybridization chamber 180 containing the same probe. The results of exporting anomalies in this single result can be ignored or given a different weight. The thermal energy required to supply hybridization is provided by CMOS controlled heater 182 (described in more detail below). Hybridization occurs between the complementary target probe sequences after activation of the heater. The LE D drive 201211532 29 in the C Μ S circuit 8 6 transmits a message to cause the LED 26 located in the test module 10 to illuminate. These probes only fluoresce when hybridization occurs to avoid the cleaning and drying steps typically required to remove unbound strands. Hybridization forces the stem-and-loop structure of the FRET probe 186 to open, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as detailed below. Fluorescence is detected by photodiode 184 in the CMOS circuit 86 located under each of the hybrid cavities 180 (see hybrid cell description below). The photodiode 184 and associated electronics for all of the hybridization chambers together form the photosensor 44 (see Figure 64). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected by the photodiode 184 is amplified and converted into a digital output that can be analyzed by the test module reader 12. Further details of the detection method are described in the following. Other Detailed Description of the LOC Device The modular design LOC device 301 has a number of functional components including reagent reservoirs 54, 56, 58, 60 and 62, dialysis section 70, and lysis unit. 130, culture unit 114 and amplification unit 112, valve type, humidifier, and humidity sensor. In other embodiments of the LOC device, such functional portions may be omitted, and additional functional portions or functional portions for alternative uses of the devices may be added. For example, the culture portion 1 14 can be used as the first amplification portion 112 of the repetitive sequence amplification analysis system, and the chemical lysis reagent reservoir 56 can be used to add the first amplification mixture of the primer, the dNTP, and the buffer, and use Reagent reservoir 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be lysed, a chemical lysis reagent (along with amplification mix) may be added to the reservoir 56, or alternatively, the sample may be heated in the culture by heating the sample for a predetermined period of time. Hot lysis occurs. In some embodiments, if chemical lysis is required and the chemical lysis reagent is mixed with the mixture, an additional reservoir can be combined upstream of the reservoir 58 for mixing the primer, dNTP, and buffer. In some cases, the steps such as the culturing step 291 are omitted. In this case, the LO C device can be specifically fabricated to avoid the reagent reservoir 58 and the culture portion 114 or the reservoir can be loaded with reagents, or if there is an active valve, it is not activated to dispense the reagent into the sample stream. The culture unit is simply a passage 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 thermal lysis. The dialysis section 70 can be located at the beginning of a fluidic 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 2 94, dialysis is performed to remove the cell debris system. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplifications 1 1 2 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using specific nucleic acid probes in the hybridization chamber array 1 1 . 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 288 of the LOC design. In some cases, even if the analysis does not require dialysis, it is not necessary to omit the dialysis section 70 from the LOC device. If the presence of the 201211532 analytic portion does not cause geometric obstruction, the sample input and preparation portion can still have the LOC of the dialysis portion 7 without losing the required function. Further, the detecting portion 294 can include a protein body chamber array. a probe that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to a sample target protein present in the non-amplified sample, rather than a nucleic acid probe designed to hybridize to the target nucleic acid sequence. needle. It will be appreciated that the LOC device φ manufactured for use in this diagnostic system differs in the combination of functional components selected for the particular LOC application. Most of the functional components are common to many LOC devices, and the design of additional LOC devices for new applications has a combination of appropriate functional components in the bulk of the 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 a variety of nucleic acid or protein contents in liquid form, including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord Blood, breast milk, sweat 'periosteal effusion, tears, pericardial fluid, peritoneal fluid, environmental water samples and beverage 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, dissolution Cells, -83-201211532 Culture and Amplification to transfer samples from sample inlet 68 to hybridization chamber 180 for nucleic acid detection, 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 Referring to Figures 1 and 12, a sample is added to the large container 24 of the test module 1 . The large container 24 is a truncated cone that 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 reservoirs The use of microfluidic devices, such as LOC device 301, requires a small amount of reagent for the analytical system such that the reagent reservoir contains all of the necessary reagents for biochemical treatment, and each reagent reservoir is in a small volume. This volume is indeed less than 1. 000.  000. 000 cubic micrometers, in most cases less than 300. 000.  000 cubic micrometers, typically less than 70,000,000 cubic micrometers, and less than 20. in the case of the LOC device 301 shown in the figures. 000.  000 cubic microns. Dialysis section Referring to Figures 15 to 21, 3 3 and 3 4, the pathogen dialysis section 70 is designed to concentrate the pathogen target cells from the sample. As previously described, the top layer -84 - 201211532 66 is a plurality of orifices of aperture 3 164 having a diameter of 3 microns, filtering the target cells from a large number of samples. As the sample flows through an orifice 164 having a diameter of 3 microns, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via the 16 μιη dialysis extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) 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. The foam insert or other porous element 49 in the outer casing 13 of the test module 1 is configured to be in fluid communication with the waste reservoir 76 for a biological sample φ type that produces a substantial amount of waste (see Figure 1). . The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. A 3 micron diameter orifice 1 64 located at the upstream end of the pathogen dialysis section has a capillary action initiation feature (CIF) 166 (see Figure 33) such that fluid is drawn down into the dialysis MST channel 204 below. The first suction aperture 198 for the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily occluding the meniscus above the dialysis suction port 168. The small component dialysis section 682, which is schematically shown in Fig. 78, may have a structure similar to that of the pathogen dialysis section. The small component dialysis section separates the sample from any small target cells or molecules by size (and shaping, if necessary) suitable for the orifices that allow the small target cells or molecules to pass to the target channel and continue to be further analyzed. Large size cells or molecules are removed to waste reservoir 766. Thus, LOC device 30 (see Figures 1 and 123) is not limited to isolation of pathogens having a size of less than 3 μηη, but can be used to separate cells or molecules of any desired size. -85- 201211532 Lysis Department Referring again to Figures 7, 1 1 and 1 3 ' by chemical lysis, the genetic material in the sample is released from the cell. As noted above, the lysis reagent from lysis reservoir 56 is mixed with the sample stream in target channel 74 downstream of surface tension valve 128 for lysis reservoir 56. However, some diagnostic assays are better suited for thermal lysis, 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 1 14 . The sample stream was flooded with the culture portion 1 14 and stopped at the boiling initiation valve 1 〇6. The culture microchannel 2 10 heats the sample to the 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 Initiating Valve As discussed above, LOC unit 301 has three boiling inducing valves 126, 106 and 1〇8. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling initiation valve 108 at the end of the heated microchannel 158 of the amplification section 112. By capillary action, the sample stream 1 1 9 is drawn along the heated microchannel 158 until it reaches the boiling initiation 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 geometry stops the arm level from moving forward to prevent capillary flow. As shown in Figures 31 and 32, the meniscus holder 98 is an orifice provided by the opening of the pipe from the MST passage 90 to the cover passage 94. The meniscus 丨 2〇 -86- 201211532 The surface tension keeps the valve closed. A ring heater 152 is located around the valve inlet 146. Ring heater 1 52 is controlled by C Μ Ο S via boiling induced valve heater contact 丨53. An electrical pulse is sent to the valve heater contact 1 53 to open the valve 'CMOS circuit 86. The ring heater 1 52 is resistively heated until the liquid sample 119 is boiled. Boiling removes meniscus 120 from valve inlet 146 and begins to wet cover passage 94. Once the lid passage 94 is wetted, the capillary action is restored. The fluid sample 1 19 is filled with the passage 94 and flows through the under-valve conduit 150 to the valve outlet 148, wherein the capillary-driven liquid flow advances along the amplifying portion outlet passage 160 into the hybridization and detection portion 52. The liquid sensor 174 is placed before and after the valve for diagnosis. It will be understood that once the boiling trigger 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. Culture section and nucleic acid amplification section Figs. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51 show the culture unit 1 1 4 and the amplification unit 1 1 2 . The culture portion 141 has a single, heated culture microchannel 210 that is etched to form a ruthenium pattern in the MST channel layer 100 from the lower conduit opening 134 to the boiling initiation valve 106 (see Figure 13 and 1 4). Controlling the temperature of the culture section 1 1 4 enables a more efficient enzyme reaction. Similarly, the amplifying portion 1 1 2 has an amplifying microchannel 158 (see Figs. 6 and 14) which is heated from the boiling inducing valve 106 to the boiling structure of the boiling initiation valve 108. When mixing, culture, and nucleic acid amplification occurs, the valve, such as -87-201211532, stops the flow to retain the target cells in the heated culture or amplification microchannel 210 or 158. The microchannel 蜿蜒 pattern also promotes (to some extent) the target cells to mix with the reagents. The sample cells and reagents in the culture unit 1 14 and the amplification unit 1 1 2 are heated via a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each of the turns of the heated culture microchannel 210 and the amplification microchannel 158 has three independently operable heaters 154 extending between the individual heater contacts 156 (see Figure 14). It provides two-dimensional control of the input heat flux density. As best shown in Figure 51, heater 154 is supported on top layer 66 and buried in 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 of the channel portions forming the meandering meandering. In the amplifying section 1 1 2, each wide zigzag can be operated as an independent PCR chamber via individual heater control. Using a microfluidic device, such as the LOC device 310, the small volume of amplification required for 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, less than 70 nanoliters, and in the case of LOC device 310, this volume is between 2 nanoliters and 30 nanoliters. between. 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 micro-88-201211532 meters, while the "high-scale" equipment has a significantly higher heating rate. The lithography technique allows the amplifying microchannel 158 to have a cross-section that spans less than 16,000 square microns of flow path that substantially provides a higher heating rate. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small amount of amplicons are required (as in the case of L Ο C device 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1 to 2,000 probes on the LOC device and "sample entry, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and 1 Between square microns. The heating element in the amplification microchannel 158 heats the nucleic acid sequence at a rate greater than 80 absolute temperatures (K) per second, in most cases at a rate greater than 100 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than 1000 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 analysis system, the heater elements are greater than 100,000 K per second, greater than 1, per second, 〇〇〇, 〇〇〇K, greater than 10 per second, 〇〇〇, 〇〇〇K, per second. The nucleic acid sequence is heated at a rate greater than 20,000,000 K, greater than 40,000,000 K per second, greater than 80,000,000 K per second, and greater than 160, 〇〇〇, 〇〇〇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. Use a microchannel with a relatively small cross-sectional area across the direction of the fluid while keeping the two fluids flowing through the interface to quickly diffuse the mixture. Reducing the channel cross-section to less than 100,000 square microns results in a significantly higher diffusion rate for "large scale" equipment. The lithography manufacturing technique allows the microchannels to have a cross-section that spans a flow path of substantially less than 16,000 square microns that substantially provides a higher mixing rate. If only a very small amount of amplicons are required (as is the case in LOC unit 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1 to 2,000 probes on the LOC device and "sample entry, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and 1 Between square microns and a short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid, resulting in a rapid thermal cycle during the nucleic acid amplification process. Each thermal cycle (i.e., variability, 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 150 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 thermal cycling allows the test module to complete the nucleic acid amplification process in less than one minute: often within 220 seconds. For most analyses, the augmentation produces sufficient amplicons from the sample fluid entering the sample inlet within 80 seconds. For most analyses, sufficient amplicons are generated within 30 seconds. When the predetermined number of amplification cycles is completed, the -90-201211532 amplicon is fed to the hybridization and detection portion 52 via the boiling initiation valve 1 〇8. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chambers 180 in the hybridization chamber array 110. The hybridization and detection unit 52 has a 24×45 array 1 10 of hybridization chambers 180 each having a hybrid-reactive FRET probe 186 and a heater element 1. 82 and integrated photodiode 184. Photodiode 184 φ was incorporated to detect fluorescence from the target nucleic acid sequence or protein hybridized to the FRET probe 186. Each photodiode 184 is independently controlled by a CMOS circuit 86. For the emitted light, any material between the FRET probe 186 and the photodiode 184 must be transparent. Therefore, the wall portion 97 between the probe 186 and the photodiode 1 84 must also be optically transparent to the emitted light. In the LOC device 301, the wall portion 97 is a thin layer of cerium oxide (about 0. 5 microns). Direct intrusion of photodiode 184 under each hybridization chamber 180 allows for the use of a very small volume of probe-target hybrid, yet still produces detectable fluorescent signals (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 to detect the probe-target hybrid amount is greater than 270 picograms (corresponding to 900,000 cubic micrometers), and in most cases less than 60 picograms (corresponding to Up to 200,000 cubic micrometers, typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and less than 2. in the case of the LO C device 3 0 1 shown in FIG. 7 picograms (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes placed on the LOC device - in the LOC device 301, in an area of 1,500 microns by 1,500 microns, the hybrid has More than one, one chamber (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 for each chamber is that during the manufacture of the LOC device, only a very small amount of probe solution needs to be placed into each chamber. A specific embodiment of the LOC device according to the present invention can be configured using a probe solution having 1 nanoliter or less. After nucleic acid amplification, the boiling initiation valve 108 is activated and the amplicon flows along the flow path 176 and into each of the hybridization chambers 180 (see Figures 52 and 566). The endpoint liquid sensor 178 indicates the point at which the hybridization chamber 1 1000 is charged with the amplicon and the starter heater 182. After sufficient hybridization time, LED 26 is activated (see Figure 2). The opening in each hybridization chamber 180 is provided with an optical window 136 to expose the FRET probe 186 to excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a sufficient 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), the photodiode 184 is enabled and the fluorescent emission is detected under no excitation light. 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 the 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 probe in this manner in the hybrid chamber array 1 1 使得 increases the confidence of the results obtained, with -92-201211532 and, if desired, combining all the results by photodipoles of adjacent hybridization chambers To get a single result. Those skilled in the art will appreciate that depending on the analysis details, there may be from 1 to over 1, different probes on the hybrid chamber array no. Humidifier and Humidity Detector The insert AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of φ evaporation of reagents and probes during operation of the LOC device 301. As best shown in the enlarged view of Fig. 55, the water reservoir 188 is fluidly coupled to the three evaporators 190. The water reservoir 188 is filled with water for molecular biological grades and is sealed during manufacture. As best shown in Figures 55 and 67, by capillary action, water is drawn to the three lower tubes 1 94 and along the individual water supply channels 192 to the three upper tubes 193 of the evaporator 190. The meniscus is fixed to each of the upper ducts 193 to retain water. The evaporator has a ring heater 191 which is connected to the CMOS circuit 86 by a heat transfer column 376, φ ring heater 191, to the top metal layer 195 (see Fig. 37). At startup, the ring heater 191 heats the water causing the water to evaporate and wet the surrounding devices. The position of the humidity sensor 23 2 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 296 and the lithographically etched second electrode 298 are opposed to each other such that their teeth are interleaved. The opposing electrodes form a capacitor having a capacitance that can be monitored by CMOS circuitry 86. As the humidity increases, the dielectric gap between the electrodes increases with the constant -93-201211532, causing the capacitance to increase. The humidity sensor 232 abuts the hybridization chamber array 1 1 〇 (the most important humidity measurement location) to slow the evaporation of the solution containing the exposed probe. The feedback sensor temperature and liquid sensor system is integrated into the LOC device 301 to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors 1 70 are assigned to the amplification unit 1 1 2 as a whole. Similarly, the culture unit 1 14 also has nine temperature sensors I 70 . These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control thermal cycling during the nucleic acid amplification process as well as any thawing during thermal lysis and culture. In hybridization chamber 180, CMOS circuit 86 uses hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of hybrid heater 182 is temperature dependent and CMOS circuit 86 utilizes this to obtain a temperature reading of each hybridization chamber 180. The LOC device 301 also has a number of MST channel liquid sensors 174 and a cover channel liquid sensor 206. Figure 35 shows 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 Figure 37, the M S T channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the circuit between the electrodes to indicate where they are located in the sensor. -94- 201211532 Figure 25 shows an enlarged perspective view of the cover channel liquid sensor 208. Pairs of TiAl electrode pairs 218 and 220 are deposited on top layer 66. A gap 222 is provided between the electrodes 2 18 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 uses this feedback to monitor the flow. The GRAVITATIONAL INDEPENDENCE test module is autonomous. 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 tubing to the auxiliary device make the module truly portable and easily plugged into a similar portable handheld reader, such as a mobile phone. 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 mobile vehicles or on mobile phones. Dialysis Variants A dialysis section having a fluid passageway to avoid trapped bubbles. The following is a specific embodiment of the LOC apparatus of LOC Variant VIII 518 shown in Figures 72, 73, 74 and 75. The LOC device has a dialysis section that is filled with a fluid sample and trapped in a channel without bubbles. LOC Variant VIII 518 also has an additional layer of material, referenced to interface layer 594. Interface layer 594 is disposed between cover channel layer 80 and the MST channel layer 1 of CMOS + MST device 48. The interface layer 594 causes a more complex fluid interconnection between the reagent reservoir and the MST layer 87 without increasing the size of the germanium substrate 84-95-201211532.

Referring to Figure 73, bypass passage 600 is designed to introduce a time delay from the fluid sample from interface waste channel 106 to interface target channel 602. This time delay causes the fluid sample to flow through the dialysis M S T channel 2 0 4 to the dialysis draw 168 of the fixed meniscus. Utilizing the capillary action initiation feature (CIF) 202 of the bypass channel 600 at the upper conduit to the interface target channel 602, from the point upstream of all dialysis suction holes 168 of the dialysis MST channel 204, the sample fluid is filled with the interface target Channel 6 02. Without the bypass channel 6 00, the interface target channel 602 still begins to charge from the upstream end, but finally, the traveling meniscus arrives and passes through the pipe above the unfilled MST channel, leading to the Point to capture the air. The trapped air reduces the sample flow rate through the leukocyte dialysis unit 3 28 . Nucleic acid amplification variant

Many variants of parallel P C R LOC devices have multiple amplifications operating in parallel. For example, the LOC variant VII 492 shown in Figure 71 has parallel amplification sections 112.1 to 112.4 which allow simultaneous multiplex nucleic acid amplification analysis. The LOC variant XI 746 shown in Fig. 106 also has parallel amplifications 112.1 to 112.4, but also has additional parallel cultures lit1 to 14.4 so that the samples can be processed differently before amplification. Other LOC variants, such as LOC XIV 641 shown in Figure 77, show that the number of parallel amplifications can be "X", which is only limited by the size of the LOC device. Larger LOC devices can be made to accommodate a greater number of parallel amplifications. -96 - 201211532 For a specific target size or specific amplification mix, the separate amplification sections can be configured to execute at different cycle times and/or temperatures. When a plurality of amplification sections are performed in parallel, the LOC apparatus can operate a multiplex nucleic acid amplification process or a unipl ex amplification process in each section. In multiplex nucleic acid amplification, more than one pair of primers is used for amplification of more than one marker sequence. A parallel nucleic acid amplification system having "m" chambers can perform η-reamplification, where η = n(l) + η(2) + ··.+ n(i) + .·.+ n(m) And n(i) is the different number of primer pairs used for the multiplex amplification performed in chamber "i", it is known that the SNR (signal-to-noise ratio) in the parallel amplification system is larger than the single chamber system The SNR of the η re-amplification performed in . In the special case of n(i) = 1, the amplification in chamber "i" becomes only a single amplification.

Direct P C R 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 of φ, 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 in a LOC apparatus at a controlled normal temperature, the 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. Amplification chemical adjustments for direct PCR or direct isothermal amplification include increased buffer strength, the use of highly active and highly progressive polymerases, and additions to potential polymerase inhibitors. It is also important to dilute the inhibitors present in the sample. -97- 201211532 To exploit direct nucleic acid amplification technology, the LOC device design incorporates two additional features. The first feature is a reagent reservoir (eg, reservoir 58 in Figure 8) that is appropriately sized to supply a sufficient amount of amplification reaction mix or diluent such that it may interfere with the finalization of the sample component of the amplification chemistry The concentration is low enough 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 is used, such as the pathogen dialysis section 70 in Figure 4. In this particular embodiment (further illustrated in Figure 6), the concentration of the pathogen sufficient to enter the amplification section 292 is effectively concentrated upstream of the sample extraction section 2 90 and the larger cells are discharged to the waste reservoir. 76 dialysis department. In another specific 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 the channel 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 preferred range of 5 to 20 times, the length of the sample and reagent channels and the cross-section 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. The flow resistance against a constant cross-sectional area 'microchannel' increases linearly with channel length. It is important for the hybrid design that the flow group resistance in the microchannel is more dependent on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannels of the square cross section is inversely proportional to the cube of the smallest vertical dimension. -98 - 201211532 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 reverse transcribed into 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, the reverse transcription step can be first cycled and the nucleic acid amplification step can be continued by adding φ plus reverse transcriptase and polymerase to reagent reservoir 62 and stylized heater 154, simply A one-step RT-PCR was performed. The buffer, the primer, the dNTP, and the reverse transcriptase are stored and distributed by the reagent reservoir 58, and the culture unit 114 is used for the reverse transcription step, and then amplified in the amplification unit 112 in an ordinary manner. The two-step RT-PCR is simply done. Constant-temperature nucleic acid amplification φ 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. Has been described - some constant temperature nucleic acid amplification methods, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), unwinding thermostat DNA amplification (HDA), rolling cycle amplification (RCA), branched amplification (ram), and circular thermostat amplification (LAMP), and any or other isostatic amplification methods of this can be used in the LOC devices herein. In a particular embodiment. -99-201211532 The reagent reservoirs 60 and 62 for performing the constant temperature nucleic acid amplification' contiguous amplification unit will carry an appropriate reagent for a specific constant temperature method instead of carrying a PCR amplification mix and a polymerase. For example, for s D A , reagent reservoir 6 〇 contains amplification buffer, primer and d N T P, and reagent reservoir 6 2 contains appropriate endonuclease and exo-DNA polymerase. For RPA, reagent reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins' and reagent reservoir 62 containing a stock-substituted DNA polymerase, such as 仏. Similarly, for HDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reagent 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 viruses, such as HIV or hepatitis C virus nucleic acids, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDNA. In this example, reagent reservoir 60 is filled with amplification buffer, primers, and dNTPs, and reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase®. For some types of thermostatic nucleic acid amplification, an initial denaturation cycle must be employed to separate the double-stranded DNA template before maintaining the temperature of the thermostated nucleic acid amplification for continued reaction. 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 apparatus described herein (see Figure 1 4). Thermostatic nucleic acid amplification is more resistant to potential inhibitors in the sample than -100-201211532 and is therefore generally suitable for direct nucleic acid amplification of the desired sample. Thus, thermostatic nucleic acid amplification is particularly useful for LOC Variant XLIII 673, LOC Variant XLIV 674, and LOC Variant XL VII 677, respectively, shown in Figures 79, 80, and 81. Direct thermostatic amplification can also be combined with one or more preamplification dialysis steps 7, 686 or 682 as shown in Figures 79 and 81 and/or a pre-hybridization dialysis step 682 as shown in Figure 80, 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. Constant temperature nucleic acid amplification can also be performed in parallel amplification sections, such as those described in Figures 71, 76 and 77. Multiplex and some constant temperature nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA. Other Design Variant Flow Sensors In addition to temperature and liquid sensors, the LOC device can also incorporate a CMOS controlled flow rate sensor 740, as illustrated in Figure 122 and in LOC Variant X 728 ( See Figures 89 to 105). These sensors are determined by the flow rate in the two steps. In the first step, the temperature of the heater element 814 is determined by applying a low current and measuring the voltage to determine the resistance of the heater element 814, and thus the resistance between the heater element and the temperature is used. The temperature of the element 8 14 is determined by the known relationship. At this stage, the heat dissipated in element 8.1 is minimal and the temperature of the liquid in the channel is equal to the temperature of the calculated element 814. In the second step, a higher current is applied to the -101 - 201211532 heater element such that the temperature of element 814 increases and some of the heat is lost due to the flowing liquid. While applying a higher voltage, the new resistance of element 814 is determined and the increased temperature is again calculated by CMOS circuit 86 by again measuring the voltage across element 814. The flow rate of the liquid is determined using the new temperature of the helium heater element 814 and the known sample liquid temperature as calculated in the first step. The flow rate of the liquid in the channel is calculated from the known channel cross-section geometry and flow velocity. Additional Details of the Fluorescence Detection System Figures 58 and 59 show hybridization-reactive FRET probes 23 6 . These are often referred to as molecular beacons and are stem-and-loop probes produced from single-stranded nucleic acids and fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization to the target nucleic acid sequence 238. The probe has a ring 240, a stem 242, a fluorophore M6 at the 5' end, and a quencher 248 at the 3' end. Loop 240 contains 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 in close proximity to each other such that a large amount of resonant energy can be transmitted between each other and substantially eliminate the ability of the fluorophore to emit fluorophores when illuminated by the excitation light 244. Figure 59 shows a FRET probe 236 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 2 3 8 , the stem-and-loop structure is destroyed, and the fluorophore and the quenching agent are spatially separated, thereby restoring the ability of the fluorophore to be 241. Optically detected ground fluorescence emission of 250 is used as a probe for the 201211532 indicator that has been hybridized. The probe hybridizes to the complementary target with extremely high specificity, because the stem helix of the probe is designed to be a probe with a single non-complementary nucleotide - the target is stable. Due to the relatively strong double-stranded DNA, the stereo probe - The target helix and the stem helix cannot coexist. Primer-linked probe φ primer-linked stem-and-loop probe and primer-linked linear probe, also known as scorpion probe, is a substitute for molecular beacons and can be used for immediate and in LOC devices. Quantitative nucleic acid amplification. Timely amplification can be performed directly in the hybridization chamber of the LOC device. The advantage of using a primer-ligated probe is that the probe element is physically linked to the primer' so only a single hybrid event is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures immediate and efficient response and produces stronger signals, shorter reaction times when using separate primers and probes, and better recognition. During manufacturing, φ probes (mixed with 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. Primer-Linked Linear Probes Figures 82 and 83 show the configuration of the primer-ligated linear probe 692 during the first round of nucleic acid amplification and the hybridization during subsequent nucleic acid amplification, respectively. Referring to Figure 82, the primer-coupled linear probe 692 has a double stem segment 242. One of the probe sequences 696, which binds to the primer, is homologous to the region on the target -103-201211532 target nucleic acid 696 and is labeled with its 5' end by fluorophore 246, and is linked via amplification blocker 694. The 3' end to the oligonucleotide primer 700. The 3' end of the other strand of stem 242 is labeled with quenching portion 248. After completion of the first round of nucleic acid amplification, the probe can be surrounded and hybridized to the extended strand using the currently complementary sequence 69. During the first round of nucleic acid amplification, the oligonucleotide primer 7 is attached to the target DNA 23 8 (Fig. 82) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the polymerase from reading through and copying probe region 696. Upon subsequent variability, the hybridized extended oligonucleotide primer 700/template and the primer-linked linear probe of the double stem 242 are separated, thereby releasing the quencher 248. Once the temperature for the adhesion and extension steps is reduced, the primer-joined probe sequence 6 96 of the primer-joined 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 84A through 84F show the operation of the primer-coupled stem-and-loop probes 704. Referring to Figure 84A, the primer-ligated stem-and-loop probe 704 has a stem 240 that complements the double stranded DNA and a loop 240 of the combined probe sequence. Labeling one of the stems of the stem with a fluorophore 2 4 6 to the 5' end of the stem with 3'-end zen 248 labeled another 710' and the other 710 with an amplification blocker 694 and Both nucleotide primers 700. In the initial denaturing phase (see Figure 84B), the target 201211532 target nucleic acid 23 8 and the primer-ligated stem 242 are separated from the stem-and-loop probe 7 04. When the temperature is cooled for the adhesive phase (see Figure 84C), the oligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 hybridizes to the target nucleic acid sequence 238. During extension (see Figure 84D), the complement 706 of the target nucleic acid sequence 23 is synthesized to form a DNA strand containing both the probe sequence 704 and the amplified product. Amplification blocker 69 4 prevents the reading of polymerase through and copy probe region 704. After denaturation, the probe sequence of the loop-and-loop probe loop segment 240 (see Figure 84F) is adhered to the complementary sequence 706 on the extended strand when the probe is subsequently attached. This configuration causes the fluorophore 246 to be very far from the quencher 248, resulting in a significant increase in fluorescence emission. The control probe hybridization chamber array 110 includes a plurality of hybridization chambers 180 with positive and negative control probes for analytical quality control. FIGS. 118 and 119 illustrate a negative control probe 796 of φ non-fluorescent clusters, And Figures 120 and 121 depict a positive control probe 798 without quenching agent. The positive and negative control probes have a stem-and-loop structure as described above for the FRET probe. However, regardless of whether the probe hybridizes to an open configuration or remains closed, the fluorescent signal 250 will always be emitted from the positive control probe 798 and the negative control probe 796 will never emit the fluorescent signal 250. Referring to Figures 1 18 and 119, the negative control probe 796 has no fluorophore (and may or may not have a quencher 248). Thus, regardless of whether the target nucleic acid sequence 238 hybridizes to the probe (see Figure 119) or the probe maintains its stem-and-loop configuration (see Figure 118), the response to the excitation light 244 can be ignored. Alternatively, the negative control probe 796 can be designed from -105 to 201211532 such that it remains quenched forever. For example, by synthesizing loop 240, a probe sequence that will not hybridize to any of the nucleic acid sequences in the sample being studied will be re-hybridized with the stem 242 of the probe molecule and the fluorophore and quenching The agent will remain 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 is not quenched from all of the emission of the indicator. Conversely, a positive control probe 7 8 8 without quenching agent is constructed as shown in Figures 1 2 0 and 121. Back to the stress luminescence 244, whether or not the positive control probe 798 is hybridized to the target nucleic acid sequence 2 3 8 , the no substance quenches the fluorescent emission 250 from the fluorophore 246. Figure 52 shows a possible distribution of positive and negative control probes (3 78 and 380, respectively) in the hybridization chamber array 110. Control probes 3 78 and 380 are placed in hybridization chamber 180 and positioned to traverse the line of hybridization chamber array 110. However, the configuration of the control probes within the array is arbitrary (like hybridization chamber array 1 1 〇 组态 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 lower intensity than the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal For the noise ratio. Moreover, a longer fluorescence lifetime represents a larger integrated fluorescence count. Fluorescence lifetime 2 46 (see Figure 59) has a fluorescence lifetime greater than 100 nanoseconds, often greater than 200 nanoseconds, more commonly greater than 300 nanoseconds, and greater than 400 nanoseconds in most -106-201211532 cases. . 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. Both are advantageous features related to the needs of fluorescent detection systems. The particularly complex metal-coordination complex based on the transition metal ion ruthenium (Ru(II)) is ginseng (2,2'-bipyridyl) ruthenium (II) φ ([Ru(bpy) ) 3] 2 + ) 'The life of the other is about 1μδ. This complex is commercially available from Biosearch Technologies under the tradename pulSar 6 50. Photophysical properties of tHlsar 650 (钌 integrator)

Parameter symbol 値 unit absorption wavelength ^abs 460 nm emission wavelength λειη 650 nm absorption coefficient Ε 14800 M.W1 fluorescence lifetime Tf 1.0 called quantum yield -------- Η 1 (deoxidized) N/A ϋ ¥ € ® - a ligand complex, a ruthenium chelate, has been successfully shown as a fluorescent indicator in the FRET probe system and has a long lifetime of 16 〇〇μ3. -107- 201211532 Table 2: Photophysical properties of ruthenium chelate

Parameter symbol 値 unit absorption wavelength ^bs 330-350 nm emission wavelength 548 nm absorption coefficient Ε 13800 (hbs, and ligand-dependent, up to 30000 (3⁄4 λ-e = 340 nm) M·1 cm·1 fluorescence Lifetime Xf 1600 (hybridized probe) μδ Quantum yield Η 1 (coordination dependent) N/A LOC device 3 0 1 The fluorescence detection system used does not use filters to remove unwanted background fluorescence. If the quencher 248 has no natural emission to increase the signal-to-noise ratio, it is therefore advantageous. Without natural emission, the quencher 248 does not contribute to background fluorescence. High quenching efficiency is also important, which makes There was no camping before the hybridization. The black hole quencher (BHQ) from Biosearch Technologies, Inc., Novato, Calif., did not have natural emission and high quenching efficiency, and was suitable for the system's suitable quenching agent. The maximum absorption enthalpy of -1 occurs at 5 3 4 nm and the quenching range is 480-5 80 nm, making it a suitable quenching agent for T b - honey glazing. BHQ-2 maximum absorption Helium occurs at 5 79 nm and the quenching range is 560 to 670 nm making it suitable for the Pulsar 650. Quenching agent Iowa Black FQ and RQ from Intergrated DNA Technologies, Coralville, Iowa, are suitable alternative quenchers with little or no background emission. I〇wa Black FQ has a quenching range of 420-620 nm and a maximum -108-201211532 absorption enthalpy at 531 nm and is therefore a suitable quencher for Tb-chelating fluorophores. Iowa Black RQ has a maximum at 656 nm. The absorption enthalpy and quenching range is from 50,000 to 70 nm, making it an ideal quencher for Pulsar 650. In the specific embodiment described herein, the quencher 248 is initially attached to The functional portion of the probe, but in other embodiments, the quencher can be a separate molecule that is free of solution. The excitation source is in the specific embodiment based on the fluorescence detection described herein because of low power consumption Low cost and small size, the LED is selected as an excitation source instead of a laser diode, a high power electric lamp or a laser. Referring to FIG. 85, the LED 26 is directly disposed on each surface of the LOC device 301 from each chamber. Hybridization chamber array 1 1 〇. In the hybridization chamber Opposite to column 1 10 is a photosensor 44 consisting of an array of photodiodes 1 84 for detecting fluorescent signals (see Figures 53, 54 and 64). Figures 86, 87 and 88 Other specific embodiments for exposing the probe to excitation light are described. In the LOC device 30 shown in Fig. 86, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 over the hybridization chamber array 110. The pulse excitation excites the LED 26 and the photodetector 44 detects the fluorescent emission. In the LOC device 30 shown in FIG. 87, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first aperture 712, and the second aperture 714 onto the hybridization chamber array 110. The pulse excites the excitation LED 26 and the photodetector 44 detects the fluorescent emission. Similarly, the LOC device 30 shown in FIG. 88, 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 LED 26 is pulse activated again and the fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 relies on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for the P u 1 s a r 65 0 dye. SET UVT0P3 3 5T039BL LED is a suitable excitation source for the ruthenium chelate label. Table 3: Phil ips LXK2-P R14-R00 LED Specifications

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

Parameter Symbol 单位 Unit Wavelength 340 run Transmit frequency Ve 8.82(10)14 Hz Power Pi 0.000240 (minutes) @ 20mA W Pulse forward current I 200 mA Radiation pattern Lambertian N/A

Ultraviolet excitation 吸收 absorbs a small amount of light in the UV spectrum. Therefore, it is advantageous to use UV excitation light. A UV LED excitation source can be used, but the broad spectrum of LED26 -110- 201211532 reduces the effectiveness of this method. To illustrate this, use LEDs. Optionally, the UV laser can be an excitation source unless it is not practical for a particular test module market. The LED driver LED driver 29 is stationary LED 26 for the desired duration. The low-power USB 2.0 certified device can be obtained with a minimum operating voltage of 4.4 volts up to 1 φ (1 mA). The standard circuit is used for this purpose. Light Diode Figure 54 shows an optical diode 184 that incorporates a LOC mounted CMOS circuit 86. Light diode 184 is part of CMOS circuit 86 without additional masking. This is a significant advantage of CMOS optical CCDs. CCD is another sensing technology. φ is integrated onto the same wafer or on a wafer using non-standard processing steps.” On-wafer detection is inexpensive and reduces the short optics of the analysis system. The path length reduces the noise from the surrounding environment with effective optical signals and suppresses the traditional optical requirements for lenses and filters. The quantum efficiency of the photodiode 184 is that the photon collides with its effective fraction, and the photon is efficiently converted into photoelectrons. For standard visible light, the quantum efficiency is in the range of 0.3 to 0.5 depending on processing parameters such as the nature of the overlay. Filtered UV High Laser Current Drive Unit Load Quasi-Power Settings 301 or steps The electrode body is superior to the process and can be adjacent to the crystal size. Compared with the collection of the required area of the firefly assembly, the minimum intensity of the fluorescent signal that can be detected is determined by the number and the detection valve of the -111 - 201211532 light diode. The detection valve 値 also determines the size of the photodiode 184 to the number of hybridization chambers 1 80 in the hybridization and detection section 52 (see Figure 52). The size and number of chambers are technical parameters 'which are limited by the scale of the LOC device (1760 micron x 5824 m in the example of LOC device 301) and incorporate other functional modules (such as pathogen dialysis and expansion) The addition 1 12) is limited by the size of the non-animal pieces that can be used afterwards. For standard 矽 processing, the photodiode 1 84 detects at least 5 photons. However, to confirm reliable detection, the minimum 値 can be set to 10 sub-. Thus, with a quantum efficiency range of 〇·3 to 0.5 (as discussed above), the fluorescence emission of the self-equal probe is a minimum of 17 photons, and a suitable error bound for reliable detection of 30 photons is included. Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, auto-fluorescence, and residual excitation photon flux that are not fully attenuated introduces background noise and shifts the output signal. Use one or more calibration signals to divide the background from each output signal. The 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 label not reacting with the probe. A high calibration source represents a positive result from the probe-targeted composition. In the specific embodiment described herein, the low school light source is provided by the calibration chamber 382 in the hybridization chamber array 110: it does not contain any probes; and the cavity is 70 light to measure the migration target Quasi-112-201211532 contains a probe that does not have a fluorescent indicator: or a probe that includes an indicator and a configuration such that quenching is always expected to occur. 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 hybridization chambers in the LOC device. The output signal from the other hybridization chamber minus the calibration signal substantially removes the background and leaves the signal generated by the fluorescent emission (if any φ is generated). Signals generated by ambient light in the area of the array of chambers are also removed. It will be appreciated that the negative control set probes described above with reference to Figures 118 through 121 can be used to calibrate the chamber. However, as shown in Figures 〇1 and 1-2, which are enlarged views of the inserts DG and DH shown in LOC variant X728 of Figure 94, another option is to isolate the calibration chamber 382 from the ampersons. . When hybridization is prevented by fluid isolation, background noise and compensation can be cleared by a chamber that isolates the fluid or by a probe containing a missing indicator or any "standard" probe that has both an indicator and a quenching agent. To judge. The calibration chamber 3 82 provides a high calibration source to generate a high signal for the corresponding photodiode. The high signal corresponds to all probes in the hybridized chamber. The indicator, with no quencher or only the indicator spotting probe, will consistently provide a signal that approximates the hybridization chamber signal, and the majority of the probes have been hybridized within the hybridization chamber. It will be appreciated that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 throughout the array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by the relatively closest calibration chamber 382-113-201211532, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has a 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 hybridization chambers 180. In this configuration, the hybridization chamber 180 is calibrated by the adjacent hybridization chambers 382. Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, Figure 117 shows a differential imager circuit 78 8 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. Differential imager circuit 78 8 samples the signal from pixel 790 and "virtual" pixel 792. In one embodiment, the "virtual" pixel 792 is shielded from light illumination, so its output signal provides a dark reference pixel. Alternatively, the "virtual" pixel 792 can be exposed to the excitation light and the remainder of the array. In one embodiment, the "virtual" pixel 792 is light permeable and the signal from the ambient light in the region of the array of cells can also be subtracted. The signal from pixel 790 is weak (e.g., close to the dark signal), and it is difficult to distinguish between background and very weak signals without reference to the dark signal level. During use, "read_row" 794 and "read_ro.w_d" 795 are started and the M4 797 and MD4 80 1 transistors are turned on. Switches 807 and 809 are turned off such that the outputs from pixel 79 and "virtual" pixel 792 are each stored on pixel capacitor 803 and virtual pixel capacitor 085. After the pixel signal is stored, switches 807 and 809 are disabled. The "read_C〇1" switch 81 1 and the dummy "read_c〇l" switch 813 are then turned off, and the converted capacitor amplifier 815 is amplified at the output of the differential signal 817. -114- 201211532 Inhibition and Enable of Photodiode The photodiode 184 must be suppressed during the LED 26 excitation and the photodiode 184 must be enabled during the fluorescence period. 65 is a circuit diagram of a single photodiode 184 and FIG. 66 is a timing diagram of an optical diode control signal. The circuit has a photodiode 184 and six MOS transistors, Mshunt 3 94, Mtx 3 96, Mreset 3 9 8, Msf 400, Mread 402, and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 3 94, and Mreset 398 are turned on by pulling Mshunt gate 384 and resetting φ gate 38 8 high. During this period, the photons are excited to generate carriers in the photodiode 184. These carriers must be removed when the amount of carrier produced is sufficient to saturate the photodiode 184. During this cycle, Mshunt 3 94 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or due to excitation-generated carrier diffusion in the substrate, while Mreset 398 resets to accumulate at the node' Any carrier of NS' 4 06. After excitation, the capture cycle begins at t4. In this loop, the response from the emission of the fluorophore is captured and integrated into the circuit on node φ lNS' 406. This is achieved by dragging the tx gate 3 86 high, which turns on the transistor Mtx 3 96 and any accumulated carriers on the transfer photodiode 184 to the node 'NS' 406. The capture cycle can be as long as a fluorescent emission. The outputs from all of the photodiodes 184 in the hybridization chamber array 110 are simultaneously captured. There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. This delay is due to the need to read the respective photodiodes 184 in the hybridization chamber array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, while the -115-201211532 final 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-following 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. Inhibition Methods Figures 114, 115 and 116 show three possible configurations 778, 780, 782 for Mshunl transistors 394. Mshunt transistor 3 94 has a very high turn-off ratio when the maximum 値1^1 = 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. 1 15, the Mshunt Gate 3 84 can be configured to surround the photodiode 1 84. The 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 effective 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 384. In FIG. 114, the Mshunt gate 384 is attached to one side of the photodiode 184. This is the simplest to manufacture and the smallest impact on the active area 185 of the photodiode. However, any carrier remaining at the far end of the photodiode 184 takes a long time to diffuse through the Mshunt gate 3 84. In Figure 115, the Mshunt gate 3 84 surrounds the photodiode 1 84. This further reduces the average path length of the carrier in the photodiode 184 to the Mshunt gate 384. However, extending the Mshunt gate around the photodiode 184 -116 - 201211532 pole 384 causes the optical diode active area 185 to be substantially reduced. Configuration 78 2 in Figure 116 positions Mshunt Gate 384 in active area 185. This provides the shortest average path length to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active area 185 is greatest. It also creates a wide leak path. 2- Enabled method φ a- The flip-flop photodiode drives the parallel transistor in a fixed delay. 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. d. Drive the shunt transistor as a 2c but with a programmable delay. 68 is a schematic cross-sectional view showing the photodiode 184 and the flip-flop photodiode 187 buried in the CMOS circuit 86 φ through the hybridization chamber 180. The small area in the corner of the photodiode 184 is replaced by the flip-flop photodiode 187. 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 2 44 is extinguished and the photodiode 184 is activated after a brief delay of At 3 00 (see Fig. 2). This delay allows the fluorescent photodiode 184 to detect fluorescent emissions from the FRET probe 186 without the excitation light 244. This enables detection and improvement of the signal-to-noise ratio. -117- 201211532 Under each hybridization chamber 180, both photodiode 184 and flip-flop photodiode 187 are located in CMOS circuit 86. The photodiode array is combined with an electronic component to form a photosensor 44 (see Figure 64). The photodiode 184 is a pn plane made during the fabrication of the CMOS structure without the need for additional masking or steps. During MST fabrication, a dielectric layer (not shown) over the photodiode 184 is arbitrarily etched using standard MST photolithography techniques to cause more of the phosphor to illuminate the active region 185 of the photodiode 184. Photoreceptor 184 has a field of view such that fluorescent signals from probe-target hybrids within hybridization chamber 180 are incident on the surface of the sensor. The converted fluorescent light becomes a photocurrent that can be measured using the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to triggering the photodiode 187. These choices can be used in combination with 2a and above. Fluorescent Delay Detection The following derivation shows the fluorescence delay detection for life cycle fluorophores for the above LED/fluorescent combination. Fluorescence is derived as a function of time after an ideal pulse excitation of the fixed intensity Ie between ^ and ~ as shown in Figure 60. Let Κ1] (0 at time t equal to the intensity of the excited state, then between and after excitation 'the number of excited states per unit volume per unit time is described by the following equation: d[Sl] | [S1](Q IBec dt U Tf ~J7e where C is the molar concentration of the fluorophore, ε is the molar extinction coefficient, 〃^ is -118- extremely thin external target 2b long-term strong micro-excitation 201211532 frequency, and h = 6·62606896(10)·34 Js is the Planck constant. This differential equation has the general formula: ^- + p(x)y = q(x) αχ There is a solution: y(^) = jp(x)dq {x, dx + k • (2) Now use this to solve equation (1), • [simj^ii.+ke-^ ... ο) hye then at time n, [Sl](h) = 〇, And the formula (3) h

Iff€CTf t /r k = --~~f-eh'Tt ...(4) hve Substituting (4) into (3): mm=

Iescr^ Iescr^ hve hve

At time ί 2,: [幻](,2) = 〜(5) hve hve 于ϊ 2 The excited state is exponentially decayed and described by equation (6): [51](〇= [51](i2)e -(^)/r^ (6) Substituting (5) into (6): [S\](t) = ^^-[1 - eHll't,)lT/ ]e{,~hVli ...(7) hve The fluorescence intensity is obtained by the following equation: 119- 201211532

Where is the fluorescence frequency, η is the quantum yield, and 1 is the optical path length. Then we can see from (7): (9) (1)=_ e~(h~t\V^{ -,·<·-<ι)Ιτί dt hve 』 Substituting (9) into (8):

If{t) = Ie£c^^-[l-eHh"'),Tf ]e-(,-,2)/r/ Because r/ v« Therefore, we can write the following approximation, which Describe the attenuation of the fluorescence intensity after a sufficiently long excitation pulse (ΗΐΗΐ>gt; Tf): When -(11) In the previous section, we summarize the case for Tf, when 2 is /〆/) = dagger. From the above equation, we can derive the following formula: rif{t) = ne£c^e'{t',l)lTf (12) where '(0 Αν. is per unit area per unit The number of fluorescent photons of time and hv· is the number of excitation photons per unit area per unit time -120- «/(0 201211532 Therefore, 〇0 nf{t) = [nf(t)dt ...(13) ' 3 where ' is the number of fluorescent photons per unit area and ~ is the time point at which the photodiode is turned on. Substituting (12) into (13): 〇〇nf=jfieec^e~{t"l)lTfdt ...(14) h At present, the number of fluorescent photons φ per unit area per unit time reaches the photodiode, which is obtained by: 0 = & (, ¥〇...(15) where & is the light of the optical system Collecting efficiency. Substituting (12) into (15) we find that η\(ί) = φϋηεε€ΐηβ-(,-'^ (16) Similarly, the number of fluorescent photons per unit of fluorescent area~ reaching the photodiode Will be as follows: co ns =\nsm Φ h and substituting (16) and integrating: ns = φ^εοίητ fe(h'hVx, therefore, ns = φ^εοίητ^'^ ...(17) ί3 ideal値When the photon is generated in the photodiode 184 due to fluorescence photons, the electron rate is equal to When the illuminator is generated at the electron rate in the photodiode 184, the excitation photon flux decays more rapidly than the fluorescence photon flux. The output electron ratio of the sensor per unit of fluorescence area of the fluorescence is: έ&gt ;(〇= ^η;(〇-121 - 201211532 where ¢5 / is the quantum efficiency of the sensor at the fluorescent wavelength. Substituting (17) we get: ef{t) = ...(18) The output electron rate of the sensor due to the per-unit fluorescence area of the excited photon is _ = -(19) where & is the quantum efficiency of the sensor at the excitation wavelength, and % is the p-cut relative to the excited LED The time constant of the characteristic. After the time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and prolongs its decay time, but we assume a negligible effect on If(t), so we adopt a conservative approach. At present, as mentioned earlier, the ideal ~ of ̄ is: ef{h) = et{h) Therefore, from (18) and (19) we get: = Φβηββ~(,3-, ι)/τ&gt And after the reorganization we get: ln(a:/7-) -i ab - ...(20)

Tf Tc From the above two paragraphs, we get the following two working equations: ns =Φ^τί^ΙΧι ...(21) -122- 201211532 ψΜ] Δί= 11 ...(22) τί ^ where F _ ^ and Δί ~ ' We also understand that, in fact, ί2 - heart" Γ /. The ideal time for fluorescence detection and the number of fluorescent photons detected using the Philips LXK2-PR1 4-R00 LED and Pulsar 650 dye are determined as follows. The ideal detection time of φ is determined by equation (22): Recall the concentration of the amplicon, and assume that all the amplicons are hybridized, and then the concentration of the fluorescent fluorophore is: c = 2.8 9 (10 guang 6 mol/L) The height of the chamber is the optical path length 1 = 8 (10) · 6πι. The fluorescent area has been regarded as equivalent to the photodiode area, but the actual light-emitting area is substantially larger than the photodiode area; Assume that == 5 ^ = 10 A_' is the light collection efficiency of the optical system. The characteristics of the photodiode, & is the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength The ratio is extremely conservative 値 with a typical LED attenuation life cycle of 0.5 nanoseconds and use

Pulsar650 specification can be determined ~ : F = [1.48(10)6][2.89(10)-6][8(10)'6](1) = 3.42(10)-5 1η([3.42(10Γ5]( 10)(0.5)) _ 1 1 Γ(10)-6 a 0.5(1 〇)-9 =4.34(10)-9 s The number of detected photons is determined using equation (21). First of all 'per single The number of excitation photons emitted by the -123- 201211532 bit time is determined by the detection illumination geometry.

The Philips LXK2-PR14-R〇〇LED has a Lambertian radiation pattern, so: n) = ril0 cos(0) ---(23) where % is the solid angle per unit angle of the LED in the direction of the forward axis The number of photons emitted per unit time 'and ^ is the direction in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: /i, =|ϋ;</Ω η =fw;oc〇s(0)i/n Ω (24) Now ΔΩ = 2π[ 1 - cos(0 + △ 0)] — 2π[1 — cos(0)] ΔΩ = 2n[cos{B) - cos{9 + Δ^)] : 4;rsin(0)cos Δ0 ^ sinlj + 4 ^cos(^)sin^

(ΑΘ;, J dQ = 2π$ΐη(θ) (1θ substitution (24): l ; i, = cos(0)sin(0)i/0 rearrange, we get */0

...(26) The output power of the LED is 0.515 watts and ve= 6.52 (10) 14 Hz, therefore: -124- ...(27) 201211532 =_0.515_ ~[6.63(10)·34][6.52 (10)14] = 1.19(10)18 Photon 渺 Bring this 入 into (26) We get: ηιο = 1.19(10)18 π = 3.79(10)17 Photon 渺 渺 Refer to Figure 61, Optical Center 252 and The lens 254 of the LED 26 is as shown in the schematic. 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 furnace [16 ( 10)~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. The photosensor at the edge of the array receives only 2% of the photons for the Lambertian excitation source intensity distribution at the hybridization event. 0 The number of excitation photons emitted per unit time: ne = ή, Ω ... (28) = [ 3.79(10)π][3.21(10)-5] =1.22(10)13 Photon 渺 Now refer to equation (29): -125- 201211532 ns = ^0«β^/β'Δ//Γ/ ” ,=(0.5)[1.22(10/3 ][3.42(10) _i][l(l O)"4]〆靡广'清'4 = 208 photons per sensor so 'use Philips LXK2- With the PR14-ROO LED and the Pulsar 650 fluorophore, we can easily detect any hybridization events that cause the number of excited photons. The SET LED illumination geometry is shown in Figure 62. At Id = 20 mA, the LED has minimal optics. The power output Pl = 240 microwatts, the wavelength center is at λβ = 3 40 nm (the absorption wavelength of the ruthenium chelate). The LED is driven linearly at ID = 200 mA to increase the output power to ρι = 2,4 mW. By placing the optical center 252 of the LED at a distance of 17.5 mm from the hybridization chamber array 110, we concentrated the output flux to a dot size with a maximum diameter of 2 mm. 2 m in the hybrid array plane The photon flux in the diameter point is obtained by Equation 2 》 hve 2.4(10)-3 _ [6.63(10)~34][8.82(10)14] = 4.10(10)15 Photon 渺 Using Equation 2 8 , we get: he = ή, Ω = 4.10(10)15»]; π[1(1〇)·3]2 = 3.34(10)11 Photon proud now 'recall equation 22 and use the previously listed Tb Chelate-126- 201211532 sex, ^ ln[(6.94(10)-5)(10)(0.5)] Δ^=-i" 1 1(10)-3 ~ 0.5(10)-9 = 3.98 (10) 9 seconds now from the equation 21: ns = (0.5)[3.34(10)n][6.94(10)-5][l(10)'3]e'398('0)',/, (,())'3 = 11,600 photons per sensor. The photon theoretical number emitted by the SET LED and the ruthenium chelate system from the hybridization event can be easily detected and far exceeds 30 photons. The lower limit is required for the reliable detection of the above-mentioned light sensor. The maximum separation between the probe and the photodiode is detected on the wafer to avoid confocal The need for detection by a microscope (see background of the invention). Deviating from traditional detection techniques is an important factor in saving time and cost associated with the system. Conventional detection requires imaging optics that must use lenses and curved mirrors. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. Placing the photodiode in a very close proximity to the probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode is 1 micron, the collection angle of the emitted light is as high as 173°. This angle is calculated by considering the light emitted from the probe closest to the center of the hybrid chamber surface of the photodiode, which has a planar active surface region parallel to the surface of the chamber. The emission cone system in which light can be absorbed by the photodiode is defined as sensing at its apex and around its plane. -127 - 201211532 The corner of the device has a transmitting probe. For a 16 micron χ 16 micron sensor, the apex angle of the cone is 1 70°; in the case where the photodiode is expanded such that its area conforms to the hybrid chamber area of 2 9 μm X 1 9.7 5 μm, The angle is 1 7 3 °. A separation of 1 micron or less between the surface of the chamber and the active surface of the photodiode is easily achieved. The application of a non-imaging optical method requires that the photodiode 184 be in close proximity to the hybridization chamber to collect sufficient photons of the fluorescent radiation. The maximum spacing between the photodiode and the probe is determined as shown in Figure 54 below. Using the ruthenium chelate fluorophore and the SET UVT0P3 3 5T039BL LED, we calculated 1,1,600 photons from individual hybridization chambers 1 80 to 16 micron X 16 micron photodiodes 184. In carrying out this calculation, we assume that the light collection region of the hybridization chamber 180 has the same bottom area as the photodiode effective 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 & = (λ5. More precisely we can write & = [(the bottom area of the light collection area of the hybrid chamber) / (photodiode area)] [Ω / 4π], Where Ω = solid angle is the opposite of the photodiode at the base of the hybridization chamber at the representative point. For the correct square pyramid geometry: Q = 4arCSin(a2/(4d〇2 + a2)), where d〇= The distance between the chamber and the photodiode, and α is the size of the photodiode. Each hybridization chamber releases 23,200 photons, and the selected photodiode has a detection low limit of 17 photons. The minimum optical efficiency required is: Team: 1 7/23200 = 7.33 x 1 0' 201211532 The bottom area of the light collection area of the hybridization chamber 180 is 29 microns x 19.75 microns. The solution of dQ will be obtained in the hybridization chamber and The maximum limiting distance between photodiodes 184 is d 〇 = 249 μm. In this limitation, the collecting cone angle as defined above is only 0.8. It should be noted that this analysis ignores the negligible effect of refraction. Variants The LOC device 301, described and illustrated in detail above, is only one of many possible LOC device designs. Some possible combinations will now be described with a flow chart (from sample input to detection) and/or display of LOC device variants using different combinations of the various functional units described above. The process is appropriately divided into sample inputs and Preparation stage 28 8 , extraction stage 290 , culture stage 291 , amplification stage 292 , pre-hybridization stage 293 , and detection stage 2 94. For clarity and conciseness, only a brief description or summary of all LOC variants is shown. The detailed configuration is not shown. 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. The appropriate location.

LOC Variant XI Figures 106 through 113 show the LOC variant XI 746. This LOC device extracts 290, cultures 291, expands 292, and detects 294 pathogen DNA. The sample input and preparation phase 288, as well as the extraction phase 290, are identical to those of the LOC device -129-201211532 301 (see Figure 4). In the cultivation stage 291, four parallel culture sections 114.1 to 114.4 were used. Referring to Figures 1-8, 1〇9 and 111, the shared restriction enzyme, binding enzyme and linker reservoir 58 adds the enzyme to the sample stream via the surface tension valve 132 to the co-incubator feed channel 748. The same incubator feed channel 748 is filled with all the culture portions 114.1 to 114.4 through the first, second, third and fourth incubator inlets 750, 752, 754 and 756, respectively. Referring to Figure 1 1 2 'cultivation portion 1 1 4.1 Up to 1 1 4.4 flows to the four parallel amplification sections 1 1 2 2 . 1 to 1 1 2 · 4, respectively. After sufficient incubation time, the boiling initiation valve 2 07 at the outlet of the respective culture section is opened. Each of the amplification sections 1 12.1 to 112.4 has a respective amplification mix reservoir 60.1 to 60.4 and polymerase reservoirs 62.1 to 62.4 (see Figs. 106 to 109). A polymerase is added to the horse prior to nucleic acid amplification to optimize the amplification procedure. Referring to Fig. 113, each of the amplification sections 112.1 to 112.4 has a boiling initiation valve 108 at each of its outlets. After the thermal cycle, the boiling initiation valve 108 is opened such that the amplicon from each of the amplification sections 1 1 2.1 -1 1 2.4 flows into the respective hybridization chamber arrays 110.1 to 110.4. The hybrid chamber arrays 110.1 through 110.4 are surrounded by a piece of titanium nitride to protect the LED wafer support surface 63 4 (see Figure 3) for exciting the LEDs 26. The excitation LED is sealed to the LED wafer support surface 634. The air pressure in the hybridization chamber 180 as shown in Figs. 94 and 98 is equal to the atmosphere passing through the vent hole 122 and the vent passage 63 6 in the MST layer 87 (see Fig. 90). As shown in Figure 111, the pathogen dialysis section 70 has a bypass passage 600 to avoid gas trapping. LOC Variant XI 746 also has flow rate sensing -130-201211532 740 and liquid sensor 174 (see Figure 108) for timing operation of heater elements in various functional sections (eg hybrid array, culture and amplification) Ministry, etc.). Conclusion The devices, systems, and methods described herein promote molecular diagnostic testing as a low cost, rapid, and focused care test. The system and its components described above are fully described, and those skilled in the art will be able to readily recognize many variations and modifications without departing from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which: FIG. 1 shows a test module and a test module reader configured for fluorescence detection. . Figure 2 is a schematic representation of the electronic components in a test module configured for fluorescence detection. Figure 3 is a schematic diagram of the electronic components in the test module reader. Figure 4 is a schematic view of the structure of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of an LOC device having features and structures from all of the layers superimposed on each other. Figure 7 is a plan -131 - 201211532 diagram of a LOC device having a lid structure that is independently shown. Figure 8 is a top perspective view of the lid with the inner channel and reservoir shown in phantom. Figure 9 is an exploded top perspective view of the inner channel and reservoir cover shown in phantom. Figure 10 is a bottom perspective view showing the cover of the top channel configuration. Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device independently. Figure 12 is a schematic cross-sectional view of the LOC device at the sample inlet. Figure 13 is an enlarged view of the insert AA shown in Figure 6. Figure 14 is an enlarged plan view of the insert AB shown in Figure 6. Figure 15 is an enlarged view of the insert AE shown in Figure 13. Figure 16 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AE. Figure 17 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AE. Figure 18 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AE. Figure 19 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AE. Figure 20 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AE. Figure 21 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AE. -132- 201211532 Figure 22 is a schematic cross-sectional view showing the lysis reagent reservoir of Figure 21. Figure 23 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AB. Figure 24 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AB. Figure 25 is a partial perspective view showing the layered structure of the LOC device inside the insert AI. Figure 26 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AB. Figure 27 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AB. Figure 28 is a partial perspective view illustrating the layered configuration of the LOC device inside the insert AB. Figure 29 is a partial perspective view showing the layered structure of the LOC device inside the insert AB. Figure 30 is a schematic cross-sectional view of the amplified hybrid reservoir and polymerase reservoir. Figure 31 shows the characteristics of a separate boiling initiation valve. Figure 32 is a schematic cross-sectional view showing the boiling initiation valve of the line 32-32 of Figure 31. Figure 33 is an enlarged view of the insert AF shown in Figure 15. Figure 3 is a schematic cross-sectional view showing the upstream end of the dialysis zone of the line 3 4 - 3 4 of Figure 3. -133- 201211532 Figure 35 is an enlarged view of the insert AC shown in Figure 6. Fig. 36 is a further enlarged view showing the inside of the insert AC of the amplifying portion. Fig. 37 is a further enlarged view showing the inside of the insert AC of the amplifying portion. Fig. 38 is a further enlarged view showing the inside of the insert AC of the amplifying portion. Figure 39 is a further enlarged view of the interior of the insert AK shown in Figure 38. Figure 40 is a further enlarged view showing the inside of the insert AC of the amplification chamber. Fig. 41 is a further enlarged view showing the inside of the insert AC of the amplifying portion. Figure 42 is a further enlarged view showing the inside of the insert AC of the amplification chamber. Figure 43 is a further enlarged view of the inside of the insert AL shown in Figure 42. Fig. 44 is a further enlarged view showing the inside of the insert AC of the amplifying portion. Figure 45 is a further enlarged view of the interior of the insert AM shown in Figure 44. Figure 46 is a further enlarged view showing the inside of the insert AC of the amplification chamber. Figure 47 is a further enlarged view of the insert AN shown in Figure 46 - 134 - 201211532. Figure 48 is a further enlarged view showing the inside of the insert AC of the amplification chamber. Figure 49 is a further enlarged view showing the inside of the insert AC of the amplification chamber. Fig. 50 is a further enlarged view showing the inside of the insert AC of the amplifying portion. Figure 51 is a schematic cross-sectional view of the amplifying portion. Figure 52 is an enlarged plan view of the hybridization section. Figure 53 is a further enlarged plan view of two separate hybridization chambers. Figure 54 is a schematic cross-sectional view of a single hybridization chamber. Figure 55 is an enlarged view of the humidifier illustrated in the insert AG of Figure 6. Figure 56 is an enlarged view of the insert AD shown in Figure 52. Figure 57 is an exploded perspective view of the LOC device in the insert AD. Figure 58 is a diagram of a FRET probe in a closed configuration. Figure 59 is a diagram of a FRET probe in an open and hybrid configuration. Figure 60 is a graph of excitation light density as a function of time. Figure 61 is a diagram of the excitation illumination geometry of the array of intersecting chambers. Figure 262 is a graphical representation of the illumination geometry of the sensor electronics L E D . Figure 63 is an enlarged plan view of the humidity sensor of the insert AH shown in Figure 6. Fig. 64 is a view showing a summary of the photodiode array of a portion of the photosensor - 135 - 201211532. Figure 65 is a circuit diagram of a single photodiode. Figure 66 is a timing diagram of the photodiode control signal. Figure 67 is an enlarged view of the evaporator of the insert AP shown in Figure 55. Figure 68 is a schematic cross-sectional view showing the detection of the photodiode and the triggering photodiode through the hybridization chamber. Figure 69 is a diagram of the linker-to-head PCR. Figure 7A is a schematic view showing a test module having a lancet. Figure 71 is a graphical representation of the structure of LOC Variant VII. Figure 72 is a plan view of a LOC variant VIII having features and structures from all layers overlapping each other. Figure 73 is an enlarged view of the insert CA shown in Figure 72. Figure 74 is a partial perspective view showing the layered structure of the LOC variant shown in the insert CA of Figure 72. Figure 75 is an enlarged view of the insert CE shown in Figure 73. Figure 76 is a graphical representation of the structure of the LOC variant VIII. Figure 77 is a diagram showing the structure of the LOC variant XIV. Figure 78 is a diagram showing the structure of the LOC variant XLI. Figure 79 is a diagram showing the structure of the LOC variant XLIII. Figure 80 is a diagram showing the structure of the LOC variant XLIV. Figure 81 is a diagram showing the structure of the LOC variant XLVII. Figure 82 is a diagram of a primer-linked linear fluorescent probe during initial amplification. -136- 201211532 Figure 83 is a diagram of a linear fluorescent probe coupled to a primer during a subsequent amplification cycle. Figures 84A through 84F illustrate the thermal cycling of the primer-coupled fluorescent stem-and-loop probe. Figure 85 is a schematic illustration of the excitation chamber array and the excitation LED of the photodiode. Figure 86 is a schematic illustration of the excitation LED and optical lens that direct light into the hybrid chamber array of the LOC device. Figure 87 is a schematic illustration of an excitation LED, an optical lens, and an optical raft for directing light into the hybrid chamber array of the LOC device. Figure 88 is a schematic illustration of the excitation LED, optical lens and mirror arrangement for directing light into the hybrid chamber array of the LOC device. Figure 89 is a graphical representation of the LOC variant X. Figure 90 is a perspective view of the LOC variant X. Figure 91 is a plan view showing the LOC variant X of a separate CMOS + MST device structure. Figure 92 is a perspective view of the bottom surface of the lid of the reagent reservoir shown in phantom. Figure 93 is a plan view showing only the features of the separate cover. Figure 94 is a plan view showing all the features overlapping each other and showing the positions of the inserts DA to DK. Figure 95 is an enlarged view of the insert DA shown in Figure 94. Figure 96 is an enlarged view of the insert DB shown in Figure 94. Figure 97 is an enlarged view of the insert DC shown in Figure 94. -137- 201211532 Figure 98 is an enlarged view of the insert DD shown in Figure 94. Figure 99 is an enlarged view of the insert DE shown in Figure 94. Figure 1A is an enlarged view of the insert DF shown in Figure 94. The figure is an enlarged view of the insert DG shown in FIG. Figure 102 is an enlarged view of the insert DH shown in Figure 94. Figure 1A is an enlarged view of the insert DJ shown in Figure 94. Figure 104 is an enlarged view of the insert DK shown in Figure 94.

Figure 1-5 is an enlarged view of the insert DL shown in Figure 94. Figure 106 is a graphical representation of the structure of the LOC variant XI. Figure 107 is a perspective view of the LOC variant XI. Figure 108 is a plan view of a LOC variant XI showing all of the features overlapping each other and showing the position of the inserts EA to EC. Figures 1-9 are plan views of LOC variant XI showing only separate cover features. Figure 110 is a plan view showing LOC variant XI of a separate CMOS + MST device structure. Figure 111 is an enlarged view of the insert EA shown in Figure 108. Figure 112 is an enlarged view of the insert EB shown in Figure 108. Figure II3 is an enlarged view of the insert EC shown in Figure 1-8. Figure 114 shows a specific embodiment of a parallel transistor for an optical diode. Figure 115 shows a specific embodiment of a parallel transistor for a photodiode. Figure 116 shows a specific embodiment of a parallel transistor for a photodiode - 138 - 201211532. Figure 117 is a circuit diagram of the differential imager. Figure 118 schematically illustrates the negative control fluorometer in the stem-and-loop configuration. Figure 1 1 shows a negative control fluorescent probe of Figure 1 18 in an open configuration. Figure 120 schematically illustrates the positive control fluorescent probe in the stem-and-loop configuration. Figure 12 is a schematic illustration of the positive control fluorescent probe of Figure 120 in an open configuration. Figure 1 22 illustrates a CMOS controlled flow rate sensor. Figure 1 23 shows the test module and test module reader that are configured for use with ECL detection. Figure 1 24 is a pictorial representation of the electronic components in a test module that is configured for use with ECL detection. Figure 1 25 shows the test module and the alternative test module reader. Figure 1 26 shows the test module and test module reader and the host system that houses the various databases. [Main component symbol description] I 〇: Test module II: Test module 1 2: Test module reader 1 3 : Case-139- 201211532 14 : Micro-USB connector 15: Sensor 1 6 : Micro- USB埠17: Touch screen 18: Display screen 19: Button 2 0: Start button 2 1 : Honeycomb radio 22: Aseptic sealing tape 2 3: Wireless network connection 24: Large container 2 5: Satellite navigation system 26: Illumination Diode 2 7 : Data storage 2 8 : Telephone 29 : LED driver 30 : LOC device 3 1 : Power conditioner 32 : Capacitor 3 3 : Timer 3 4 : Controller 35 : Register 36 : Micro USB device 1 . 1 or 2.0 3 7 : Drive -140 - 201211532 3 8 : Random Access Memory 39 : ECL Excitation Driver 40 : Program and Data Cache 41 : ECL Excitation Register 42 : Processor 43 : Program Memory 44 : Photosensor 45: Indicator 46: Cover 47: Module 48: CMOS + MST Device 49: Porous Element 52: Hybridization and Detection 54: Anticoagulant Reservoir 56, 56.1, 56.2, 56.3: Reservoir 5 7 : printed circuit board 58, 58.1, 58.2: reservoir 60, 60.1-60.12, 60.X: reservoir 62, 62.1, 62.2, 62.3, 62 .4, 62.X: reservoir 64: lower seal 6 6 : top layer 6 8 : sample inlet 7 〇: dialysis section 72 : waste channel - 141 - 201211532 74 : target channel 7 6 : waste reservoir 7 8 : Reservoir layer 80: cover channel layer 8 2 : upper seal layer 84 : 矽 substrate 86 : CMOS circuit 87 : MST layer 8 8 : passivation layer 90 : MST channel 92 : lower pipe 94 : cover channel 96 : upper pipe 97 : wall Part 98: Meniscus Holder 100: MST Channel Layer 101: Laptop/Notebook 102: Capillary Action Start Feature, 103: Dedicated Reader 105: Desktop Computer 106: Boiling Initiating Valve 107: E-book reader 108: Boiling trigger valve 109: Tablet PC-142- 201211532 110, 110.1-110.12, 110.X: Hybridization chamber array 1 1 1 : Epidemiological data 112, 112. Bu 112.12, 112.X: Amplification part 1 1 3 : genetic data 114, 114.1-114.4: culture part 1 1 5 : electronic health record 1 1 6 : anticoagulant

1 1 8 : Surface tension valve 1 1 9 : Liquid sample 1 2 0 : Meniscus 1 2 1 : Electronic medical record 122 : Vent 123: Personal health record 125 : Network 126 : Boiling trigger valve 128, 128.2, 128.3: Surface tension valve 130, 130.1-130.3: lysis unit 1 3 1 : mixing portion 132, 132.1, 132.3: surface tension valve 1 3 3 : incubator inlet passage 134: lower tube 136: optical window 1 4 6 : valve Inlet 1 4 8 : Valve outlet - 143 - 201211532 150 : Under valve 152 : Ring heater 1 5 3 : Valve heater contact 1 5 4 : Heater 1 5 6 : Heater contact 158 : Micro channel 1 60 : outlet channel 1 6 4 : orifice 166 : capillary action initiation feature 1 6 8 : dialysis suction port 170 : temperature sensor 174 : liquid sensor 175 : diffusion barrier 176 : flow path 178 : liquid sensor 1 8 0 : hybridization chamber 1 8 2 : heater 184 : photodiode 1 8 5 : effective area 1 8 6 : probe 1 87 : photodiode 1 8 8 : water reservoir 190 : evaporator 1 9 1 : Ring heater-144- 201211532 192: Water supply channel 193: Upper pipe 194: Lower pipe 1 9 5: Top metal layer 196: Humidifier 1 9 8 : Suction hole 202: Capillary action start Sign 204 : MST channel 208 : liquid sensor 210 : micro channel 2 1 2 : MST channel 2 1 8 : electrode 2 2 0 : electrode 222 : gap 232 : humidity sensor 2 3 4 : heater 23 6 : FRET Probe 23 8 : Target nucleic acid sequence 2 40 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 2 4 8 : Quencher 250 : Fluorescence signal -145 201211532 2 5 2 : Optical center 2 5 4 : Lens 2 8 8 : Sample input and preparation 290 : Extraction stage 2 9 1 : Culture stage 292 : Amplification stage 293 : Pre-hybridization stage 294 : Detection stage 296 : First electrode 298 : Second electrode 3 00 : Programmable Delay 301: LOC device 328: white blood cell dialysis unit 3 7 6 : heat transfer column 3 7 8 : positive control probe 3 8 0 : negative control probe 3 82 : calibration chamber 3 8 4 : smell pole 3 8 6 : trouble 3 8 8 : Interpolar 3 9 0 : Retractable lancet 3 9 2 : Lancet release button 3 9 3 : Noisy 3 9 4 : Μ Ο S transistor 201211532 396: MOS transistor 398: MOS Crystal 400: MOS transistor 402: MOS transistor 404: MOS transistor 4 0 6 : node 408: film seal

4 1 0 : Membrane guard

492 : LOC Variant VII

5 1 8 : LOC variant VIII 5 94 : interface layer 600 : bypass channel 602 : interface target channel 604 : interface waste channel 63 4 : LED wafer support surface 63 6 : ventilation channel

641 : LOC XIV 67 3 : LOC Variant 674 : LOC Variant 677 : LOC Variant 682 : Dialysate 6 8 6 : Dialysis Step 692 : Primer-Linked Linear Probe 694 : Amplification Blocker - 147 201211532 6 9 6 : probe region 698 : complementary sequence 700 : oligonucleotide primer 704 : stem-and-loop probe 706 : complementary sequence 708 : stem strand 710 : strand 7 1 2 : first pupil 7 1 4 : Second aperture 7 1 6 : first mirror 71 8 : second mirror 728 : LOC variant X 740 : flow rate sensor 746 : LOC variant XI 748 : incubator feed channel 7 5 0 : incubator inlet 7 5 2 : incubator inlet 7 5 4 : incubator inlet 7 5 6 : incubator inlet 7 6 6 : waste reservoir 7 6 8 : 肓 terminal 7 7 8 : configuration 7 8 0 : configuration 7 8 2 : Configuration 201211532 788: Differential Imager Circuit 7 9 0: Pixel 792: Virtual Pixel 7 94: Read - Column 795: Virtual Read_Column 796: Negative Control Probe 7 97 : (Crystal)

7 9 8 : Positive control probe 8 0 1 : (Chip) 8 0 3 : Pixel capacitor 8 05 : Virtual pixel capacitor 8 0 7 : Switch 8 0 9 : Switch 8 1 1 : Switch 8 1 3 : Switch 8 1 4: heating element 8 1 5 : capacitor amplifier 8 1 7 : differential signal 860 : ECL excitation electrode 8 70 : ECL excitation electrode

Claims (1)

201211532 VII. Patent Application Range 1. A wafer-on-lab (LOC) device for genetic analysis of biological samples, the LOC device comprising: an inlet for accepting a sample comprising genetic material containing DNA and RNA; a plurality of reagent reservoirs; a first culture portion, the first culture portion being in fluid communication with one of the reagent reservoirs, the reagent reservoir comprising an enzyme for reacting with the genetic material enzyme; a second culture portion of the culture portion, the second culture portion being in fluid communication with one of the reagent reservoirs, the reagent reservoir comprising an enzyme for reacting with the genetic material enzyme; downstream of the first culture portion a first nucleic acid amplification unit for amplifying at least some of the genetic material; and a second nucleic acid amplification portion downstream of the second culture portion and parallel to the first nucleic acid amplification portion for amplifying at least some of the genetic a substance; wherein the first culture part, the second culture part, the first nucleic acid amplification part, and the second nucleic acid amplification part are all carried on the support substrate. 2. The LOC device of claim 1, wherein the first nucleic acid amplification portion is a first polymerase chain reaction (PC R) portion configured to amplify DN A in the genetic material, and the The second nucleic acid amplification unit is a second PCR unit configured to amplify rnA in the genetic material. 3 - The LOC device of claim 2, wherein the -150-201211532 PCR portion has a first set of primer pairs for adhering to the first set of complementary nucleic acid sequences in DN A, and the second PCR And a second set of primer pairs for adhering to a second set of complementary nucleic acid sequences in RN A, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences. 4. The LOC device of claim 3, wherein the first PCR portion and the second PCR portion are configured to operate with different amplification parameters, the amplification parameter being at least one of: | reverse transcriptase type ; polymerase type; deoxyriboribozyme triphosphate concentration; buffer solution; thermal cycle time; number of thermal cycle repetitions; and temperature during specific phase of PCR. 5. The LOC device of claim 4, further comprising a φ photosensor; a first hybridization portion downstream of the first PCR portion; a second hybridization portion downstream of the second PCR portion; the first hybridization portion a first array of probes for hybridizing to a first target nucleic acid sequence to form a probe-target hybrid and having a second hybridization portion for hybridizing to a second target nucleic acid sequence to form a probe A second array of probes of the target hybrid, wherein the light sensor is configured to detect the probe-target hybrid. 6. The device of claim 1, wherein the first nucleic acid amplification portion is a first constant temperature nucleic acid amplification portion configured to amplify DNA in the genetic material, and the second nucleic acid The amplification unit is a second thermostated nucleic acid-151 - 201211532 amplification unit configured to amplify the RNA in the genetic material. 7. The L〇C device of claim 6 is the first thermophilic nucleic acid. The amplification portion has a first set of primer pairs for adhering to the first set of complementary nucleic acid sequences in the DNA, and the second thermolabic nucleic acid amplification portion has a second set of complementary nucleic acid sequences for adhering to the RNA A set of primer pairs, the first set of complementary nucleic acid sequences differing from the second set of complementary nucleic acid sequences. 8. The L0C device of claim 7 wherein the first thermolab nucleic acid amplification portion and the second thermolab nucleic acid amplification portion are configured to operate with different amplification parameters, the amplification parameter being at least One: reverse transcriptase type; polymerase type; deoxyribonuclease triphosphate concentration; buffer solution; and temperature during nucleic acid amplification. 9. The LOC device of claim 8, further comprising a photo sensor: a first hybridization portion downstream of the first thermolabic nucleic acid amplification portion; and a second hybridization portion downstream of the second thermolabic nucleic acid amplification portion The first hybridization portion has a first array 'for hybridization with a first target nucleic acid sequence to form a probe-target hybrid, and the second hybridization portion has a second nucleic acid sequence for use with the second target A second array of probes that hybridize to form a probe-target hybrid, wherein the photosensor is configured to detect the probe-target hybrid. 10. The LOC device of claim 9, wherein the first-152-201211532 hybridization unit has a first hybridization chamber array for containing the first probes such that the first plurality of hybridization chambers a probe configured to hybridize to one of the first target nucleic acid sequences, and the second hybridization portion has a second hybridization chamber array to include the second probes such that each of the hybridization chambers The second probe is configured to hybridize to one of the second target nucleic acid sequences. 11. The LOC device of claim 10, wherein the photosensor is provided with an array of photodiodes that are aligned with the array of first hybridization chambers and the array of second hybridization chambers. 12. The LOC device of claim 11, wherein the first thermolab nucleic acid amplification portion has a nucleic acid amplification microchannel for maintaining a reaction temperature of the sample, the nucleic acid amplification microchannel defining a cross The cross-sectional area of the flow is a flow path of less than 100,000 square microns. 13. The LOC device of claim 12, wherein the nucleic acid amplification microchannel has a cross-sectional area across the flow of less than 1 6,000 square microns. 14. The LOC device of claim 1, wherein the reagent reservoirs each have a surface tension valve for retaining a reagent therein, the surface tension valve having a meniscus holder for immobilizing the reagent The meniscus is removed from contact with the sample stream to remove the meniscus. 15. The LOC device of claim 4, further comprising a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer, the MST layer including the first and second PCR sections, wherein the CMOS circuit system is configured Between the support substrate and the MST layer, the CMOS circuit is configured to control the first and second PCR-153-201211532 portions with feedback using a temperature sensor output. 16. The LOC device of claim 15, wherein the first PCR portion has a PCR microchannel, wherein during use, the sample is thermally cycled by the PCR microchannel defining a cross-sectional area across the flow. A flow path of less than 100,000 square microns. 17. The LOC device of claim 16, wherein the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. 18. The LOC device of claim 7, wherein the first PCR portion has a plurality of extended PCR chambers, each of the chambers being formed by an individual portion of the PCR microchannel, the PCR microchannel having A series of wide meandering flow formations, each of which forms a channel portion of one of the elongated PCR chambers. 19. The LOC device of claim 18, wherein the first PCR portion has an active valve to allow fluid to remain in the first PCR portion during thermal cycling and to react to activation signals from the CMOS circuit to allow fluid Entering the first hybridization chamber array. 20. The LOC device of claim 19, wherein the active valve has a boiling initiation valve configured to secure a meniscus holder that blocks a meniscus of the liquid drive flow of the liquid, and to boil the The liquid is a heater that releases the meniscus from the meniscus holder to restore the capillary drive flow. -154-