TW201211538A - LOC device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification - Google Patents

Abstract

A lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a dialysis section for separating pathogens from larger constituents in the sample, a plurality of reagent reservoirs, a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the cells in the lysis section, a first nucleic acid amplification section downstream of the lysis section for amplifying first nucleic acid sequences in the genetic material, and, a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section, wherein, the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.

Description

201211538 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatments and assays for molecular diagnostics. [Prior Art] 0 Molecular diagnosis has been used to provide an area for early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the occurrence and improvement of ineffective health care Patient outcome G, improved disease management and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobases allows for the binding (hybridization) of synthetic DNA (oligonucleotide) short sequences to specific nucleic acid sequences for nucleic acid assays. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will get in the future, to determine the species and pathogen of the infectious pathogen, or to determine the individual's response to the drug. 201211538 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 outset. Blood is one of the more commonly sought sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombotic cells promote agglutination and maintain activity in vitro. To inhibit coacervation, the sample is mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of the cellular material but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by differentially lysing the red blood cells in the lysate, leaving the remaining intact cellular material, which can then be separated from the sample using centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. The exact procedure used to extract nucleic acids depends on the sample and the diagnosis to be performed -6- 201211538 analysis. 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 and subsequent nucleic acid purification. The cell lysis step ruptures the cell and nuclear membrane and releases the genetic material. This is often accomplished using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The ruthenium nucleic acid is then purified by an alcohol (usually ice ethanol or isopropanol) precipitation step, or via a solid phase purification step, prior to washing in the presence of a high concentration of chaotropic salt, usually in a fractionation column. The ruthenium oxide matrix, resin or paramagnetic beads are then eluted with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protease that digests the protein to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 t to destroy the hot lysis of the cell membrane. Target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogen 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 target DNA sequences relative to complex DNA backgrounds. If RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNa) using the enzyme called reverse transcriptase 201211538. 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 approximately 10-30 nucleotides complementary to the flanking target sequence 2 - DNA polymerase - thermostable enzyme for the synthesis of DNA 3. Deoxyribose nucleus Glycoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - the best chemical environment for DNA synthesis. PCR typically involves placing these reagents in small tubes containing extracted nucleic acids (~10). -5 〇 microliters). The tube is placed in a polymer thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocols for each thermal cycle include the denatured phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denaturation phase generally involves heating the reaction temperature to 90-95 t to denature the DNA strand; in the adhesive phase, the temperature is lowered to ~50-6 CTC for adhesion of the primer; then in the extended phase, the temperature is raised to the optimum DNA polymerization. The enzyme activity temperature is 60-72 ° C for extension of the primer. This method repeats the cycle by about 20 to 40 times, and the final result is 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 prime (linker-primed 201211538) PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcription Enzyme 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 an approximation with an approximate adhesion temperature and an amplicon of approximate length and base 0 to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR, is a method for enabling nucleic acid amplification of virtually all DNA sequences in complex DNA mixtures without the need for target-specific Sexual introduction. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). Using a zymase enzyme, a double-stranded oligonucleotide linker (also known as a zygote) with a suitable overhanging end is then ligated to the terminal of the target DNA fragment. Nucleic acid amplification is then carried out using an oligonucleotide primer 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, using appropriate changes in chemical properties and sample concentration, DNA purification can be performed to minimize 201211538 PCR or direct PCR. Modification of PCR chemistries for direct PCR involves potentiating buffer strength, using high activity and processivity polymerases and additions to potential polymerase inhibitors. Cascade PCR utilizes two independent nucleic acids Amplification to increase the probability of amplifying the correct amplicon. One type of tandem 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 containing the target nucleic acid (short than the first PCR product). The rationale used in this strategy is that if the wrong locus is amplified by mistakes during the first nucleic acid amplification, the probability of re-amplifying the wrong locus by the second pair of primers is very low, thus ensuring specificity. Instant PCR or quantitative PCR was used to measure the amount of PCR product in real time. The initial amount of nucleic acid in a sample can be determined by using a fluorophore containing a probe or fluorescent dye and a reference standard in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. 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- 201211538 Thermostatic amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DN A during the amplification reaction' thus does not require complex machinery. The constant temperature nucleic acid amplification method can thus be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Some thermostated nucleic acid amplifications of Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification The method has been described. The thermostatic nucleic acid amplification method does not rely on the continuous heat denaturation of the template DN A to produce a single-stranded molecule as a template for further amplification, but relies on the enzyme property of a DNA molecule such as a specific nuclease at a constant temperature. Cutting, or other methods of separating DNA strands with enzymes. The ability of strand-substituted amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi-modified DNA, and the ability to rely on 5'-3' exonuclease-deficient polymerases to extend and Replace the downstream stocks. An exponential nucleic acid amplification is then achieved by a coupling sense and an antisense reaction, wherein the strands from the sense reaction are substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) -11 - 201211538 which is not nicked in a normal manner but which nicks a strand of DN A is used for this reaction. SDA has been improved by the use of a combination of a thermostable restriction enzyme (J να 1 ) and a thermostable exo-polymerase (polymerase). This combination appears to increase the amplification efficiency of the reaction from 108-fold amplification to l〇C) amplification so that this technique can be used to amplify unique single-copy molecules. Transcription-mediated amplification (TMA) and nucleic acid sequence-dependent amplification (NASBA) use RN A polymerase to replicate RN A sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RN A polymerase. In the first step of nucleic acid amplification, the primer hybridizes to the target ribosome RN A (rRN A ) at a defined position. The reverse transcriptase produces a DNA copy of the target rRNA by extension from the 3' end of the promoter primer. If another RNase is present, the RNA in the obtained RNA:DNA double strand is decomposed by the RNase activity of the reverse transcriptase. Next, the second primer binds to the DNA copy. A double-stranded DNA molecule is produced by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RNA polymerase recognizes the promoter in the DNA template and initiates transcription. Each newly synthesized RN A amplicon is re-entered and used as a template for new replication. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a specific DNA fragment is achieved by binding the opposite oligonucleotide to the template DNA and extending it by the DNA polymerase. Denaturation of the double-stranded DNA (dsDNA) template does not require heat. Conversely, RP A uses a recombinant enzyme-primer mismatch to scan dsDNA and promote share exchange at the cognate position. The resulting -12-201211538 structure is stabilized by the interaction of a single strand of DNA binding protein with a 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 strand replacing the large fragment of the DNA polymerase (such as Pol I C ), and the primer then begins to extend. This step is repeated by cycling to achieve exponential nucleic acid amplification. The 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 unwinding enzyme traverses the target DNA, destroying the hydrogen bonds that bind the two strands, which are then bound by a single-stranded 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 produce 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 polymerase continuously extends the primer around the circular DNA template to produce a long DNA product consisting of a number of circular repeat copies. By terminating the reaction, the polymerase produces thousands of copies of the circular template with a copy strand tethered to the original target DNA. This results in a spatial resolution of the target and rapid nucleic acid amplification of the signal. A template of up to 1012 copies can be produced in one hour. Branch-type amplification is a variant of RCA, and a circular probe (C-probe) or a latching probe and a highly progressive DNA polymerase are used to exponentially expand C at room temperature. - Probe. Circular thermostat amplification (LAMP) provides high selectivity and utilizes DNA polymerase and a primer set containing four specially designed primers that recognize a total of six different sequences on the target DNA of the -13-201211538. The inner primer of the sense strand containing the target DNA and the antisense strand sequence initiates LAMP °. The subsequent strand-derived DNA synthesis initiated by the exogenous primer releases a single strand of DNA. This serves as a template for DNA synthesis initiated by the second internal and external primers. The second inner and outer primers are hybridized with the other end of the target to produce a stem-loop (stem_l〇〇P) 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, producing the original stem-loop DNA and the new stem-loop DNA with twice the stem length. The cycle was continuously reacted within one hour to accumulate 1 〇 9 copies of the target. The final product is a stem-loop DN A with several inverted repeat targets and a broccoli-like structure with a plurality of loops (alternatingly adhering to inverted repeat targets in the same strand). After completion of 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 DN A hybrid 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, depending primarily on their size. After the electrophoresis is completed, the fragments in the colloid can be stained to make them visible. Brominated phenanthrenequinone, which is fluorescent under UV light, 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. Since the oligonucleotide primer binds to a specific position adjacent to the target DN A from -14 - 201211538, the size of the amplified product can be predicted and detected using a band of known size on the colloid. To confirm the amplification or if several amplicons are produced, the DN A probe is often used to hybridize to the amplicon. DN A hybridization means the formation of double stranded DNA by complementary base pairing. A DN A probe of about 20 nucleotides in length is required for DN A hybridization for positive recognition of a particular amplification product. If the probe has a sequence complementary to the amplicon (target) DN A 0 sequence, hybridization will occur at favorable temperature, pH and 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 a current, light is generated by a luminophore molecule or a complex. When fluorescing, the illuminating light is emitted to emit 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 transmitted signal and a mechanism to prevent excitation light from being included in the photosensor output (such as a wavelength-select filter). Back stress luminescence, fluorescence molecular emission history Stokes-shifted light, and this emitted light is collected by the detection unit. Stokes converts the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. -15- 201211538 The light sensor is used to detect ECL emissions, and the light sensor is sensitive to the emission wavelength of the ECL type used. For example, a transition metal coordination complex emits light of a visible wavelength, and thus a conventional photodiode and C CD are used as photosensors. The advantage of ECL is that if ambient light is excluded, the ECL emission can be the only light present in the detection system, thus increasing sensitivity. Microarrays allow hundreds of thousands of DNA 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 experiment. The microarray consists of a number of different DN A probes that are attached to the substrate and are spotted. The target DNA (amplicon) is first labeled with fluorescent or luminescent molecules (during or after nucleic acid amplification) and then applied to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment where hybridization occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. Less stringent may result in highly non-specific binding. Excessive rigor may result in inability to properly combine and result in reduced sensitivity. Identification of hybridizations by detecting light emission from labeled amplicon-forming hybrids. The microarray scanner is typically a computer-controlled inverted scan using a microarray scanner to detect fluorescence from the microarray. Fluorescent conjugated focus microscopy, which typically uses a laser and photosensor that excites a fluorescent dye, such as a photomultiplier tube or CCD, to detect the emitted signal. The fluorescent molecules emit light converted by Stokes (as described above), and the light is collected by the detecting unit. -16- 201211538 The emitted fluorescence must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. A conjugate focal configuration is often used in microarray scanners to remove out-of-focus information by conjugated focal pinholes located in the image plane. This makes it possible to detect only the focused portion of the light. Light that is prevented above or below the focal plane of the object enters the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, which is then converted into a digital signal. This digital signal is converted to a 0 number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. More information on fluorescent probes can be found below: http://www. premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/ Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET. html o In-situ medical molecular diagnostics Although molecular diagnostic tests offer advantages, clinical testing in this type of testing Growth is not as good as expected and still only accounts for a small portion 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 closely related to the rapid and automated analysis that can significantly reduce costs, from initial (sample processing) to end (production), and the development of instruments that do not require extensive human intervention. -17- 201211538 For physicians' clinics, proximity or user-based hospitals, home-based in-home healthcare technologies offer the following advantages: • Rapid results are achieved to quickly promote treatment and improve care quality • Tested through very small samples Get the ability to test defects. • Reduce clinical workload. • Reduce laboratory workload and increase work efficiency by reducing management efforts. • Improve the cost per patient by reducing hospital stays, getting results from outpatient visits at the first visit, and simplifying the handling, storage and delivery of samples. • Promote clinical management decisions such as vaccination control and antibiotic use. Molecular Diagnostics Based on On-Wafer Laboratory (LOC) Based on methods for providing automated and accelerated molecular diagnostic analysis for molecular diagnostic systems for fluid technology. The shorter detection time is mainly due to the use of very small amounts of automation, built-in and low overhead cascades in the diagnostic method steps in the microfluidic device. The use of nanoliters and micro-upgrades also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are a common form of microfluidic devices. The on-wafer laboratory device has an MST structure in the MST layer to integrate fluid processing into a single support substrate (typically germanium). The use of the VLSI (Very Large Integral Circuit) technology of the semiconductor industry enables the unit cost of each device to be very low. However, controlling the flow of fluid through the LOC device, adding reagents, controlling reaction conditions, etc. requires a large volume. External piping and electronics. Connecting the LOC unit to -18-201211538 to these external devices virtually limits the use of the LOC device for molecular diagnostics to inspection. The cost of external equipment and its operational complexity are eliminated. The use of LOC-based molecular diagnosis as a practical option for in situ medical treatment. In view of the above, there is a need for a LOC device-based molecular diagnostic system for in situ care. [Invention] The present invention will be described in the following paragraphs. Various aspects of the invention. GCF001.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting a pathogen in a biological sample, the LOC device comprising: an inlet for receiving a sample; a support substrate; and causing pathogens in the sample a dialysis section in which a larger component is separated from the sample; a lysate located downstream of the dialysis section for lysing pathogens a genetic material derived therefrom; and a nucleic acid amplification portion located downstream of the lysis unit for amplifying a nucleic acid sequence in the genetic material; wherein the dialysis portion, the lysis portion, and the nucleic acid amplification portion are supported on the support substrate 〇GCF001 .2 Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GCF001.3 Preferably, the LOC device has a hybridization-19-201211538 portion downstream of the PCR portion, and the PCR portion has a sample for use in the sample A probe array for hybridization of a target nucleic acid sequence and a photosensor for detecting hybridization of any probe in the array. GCF001.4 Preferably, the dialysis portion has a first channel in fluid communication with the inlet, and is lysed with the lysis portion a second passage in fluid communication and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component 'the second passage is in fluid communication with the first passage via the orifice such that the pathogen flows into the second passage' and the larger component is retained In the first channel, GCF001.5 preferably, the first channel and the second channel group constitute a sample by capillary action. GCF00 1.6. Preferably, the second channel group is constituted by capillary action. Pathogen Inhalation of the lysis unit. GCF00 1.7 Preferably, the lysis unit has a heater for the hot lysing pathogen. GCF001.8 Preferably, the LOC device also has a plurality of reagent storage tanks, wherein one of the formula storage tanks contains A lytic reagent for a chemical lytic pathogen in the lysate. GCF001.9 Preferably, the LOC device also has a flow path from the inlet to the hybridization portion, wherein the flow path group constitutes a sample to be sucked from the inlet by capillary action Hybridization. GCF001.10 Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the combined PCR portion, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit system The group configuration uses a temperature sensor output to feedback control the PCR section.

GCF00 1.il Preferably, the PCR section has a PCR-20-201211538 microchannel for the heating cycle sample to amplify the nucleic acid sequence, and the PCR microchannel is defined for the sample and the cross-sectional area of the traverse flow is less than 1〇〇,部分 square micron part of the flow path. GCF001.12 Preferably, the LOC device also has at least one heater element for heating the extension of the nucleic acid sequence within the PCR microchannel, the elongated heater element extending parallel to the PCR microchannel. GCF001.13 Preferably, at least one portion of the PCR microchannel forms an elongated PCR chamber. 0 GCF001.14 Preferably, the PCR part has a plurality of elongated PCR chambers each formed by a separate portion of the PCR microchannel, and the PCR microchannel has a 蜿蜒 structure formed by a series of wide tortuous lines, and each wide zigzag system is formed. The channel portion of one of the elongated PCR chambers.

GCF001.15 Preferably, the LOC device also has a reagent reservoir for retaining reagents for pCR; and a surface tension valve having an orifice that forms a meniscus of the immobilization reagent such that the meniscus retains the reagent The meniscus is removed from the reagent reservoir until it contacts the fluid sample and the reagent flows out of the reagent reservoir. GCF001.16 Preferably, the LOC device also has an array of hybrid chambers containing probes. GCF001.17 Preferably, the photosensor is an array of photodiodes that are positioned with the hybridization chamber. GCF001.18 Preferably, the CMOS circuit has a digital interface for storing hybridization data from the output of the photosensor and a data interface for transmitting the hybridization data to an external device. GCF001.19 Preferably, the PCR section has an active valve for allowing liquid to flow to the hybridization chamber during the thermal cycle for -21 - 201211538 to retain liquid in the PCR section and in response to an activation signal from the CMOS circuit. GCF00 1 .20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater The liquid is boiled and the meniscus is released from the meniscus retainer to restore the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lysses pathogens in its chemical and/or hot lysate The nucleic acid sequence of the sample is analyzed by releasing the genetic material of the pathogen, amplifying any target gene sequence, and by hybridizing with the oligonucleotide probe and detecting its internal imaging array and using the reagent stored in the reagent reservoir of the LOC device. . The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, low system component volume, and simple system manufacturing steps to provide an inexpensive analytical system. The use of chemical and thermal lysis methods has been chosen to simplify analytical chemistry requirements and provide versatility for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides a solution that is easy to use, mass-processable, and inexpensive with an integrated system component. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, low-system, integrated solution that is compact, lightweight, and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the optical Q component string. The reagent storage tank, which is integrated with the LOC unit and holds the analysis of the total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF002.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting a pathogen in a biological sample, the LOC device comprising: an inlet for receiving a sample; a support substrate; a pathogen in the sample compared to the sample a large component separated dialysis section; a lysate located downstream of the dialysis section 'for lysing pathogens to release genetic material therein; a first nucleic acid amplification section located downstream of the lysis compartment' for amplification from lysis a nucleic acid sequence in the genetic material of the first part of the sample stream, and a second nucleic acid amplification portion located downstream of the lysis unit for amplifying the genetic material of the second part of the sample stream from the lysis unit a nucleic acid sequence; wherein the 'dialysis portion, the lysis unit, the first nucleic acid amplification unit, and the second nucleic acid amplification unit -23-201211538 are all supported on the support substrate. 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 PC R unit. GCF002.3 Preferably, the first PCR portion has a first set of primer pairs that are bonded to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs that are bonded to the second set of complementary nucleic acid sequences, first The set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF002.4 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 repetition, and temperature during specific phases of PCR. GCF002.5 Preferably, the LOC device also has a first hybridization portion downstream of the first PCR portion, having a first probe array hybridized with the first target nucleic acid sequence and a second hybridization portion downstream of the second PCR portion a second probe array for hybridization to a second target nucleic acid sequence, and a photosensor for detecting hybridization of any of the probes in the first array and the second array. GCF002.6 Preferably, the dialysis section has at least two passages in fluid communication with the plurality of orifices, the plurality of orifices being sized to correspond to a predetermined threshold. -24 - 201211538 GCF002.7 Preferably, at least two channels and a plurality of orifices are configured to allow the sample to flow through the channels and orifices by capillary action. GCF002.8 Preferably, the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the lysis portion, and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component, the second passage The first channel is in fluid communication via the orifice such that the pathogen flows into the second channel and the larger component remains in the first channel. 0 GCF002.9 Preferably, the LOC device also has a plurality of reagent reservoirs, wherein one of the reagent reservoirs contains a lysis reagent for the chemical lysing pathogen in the lysate. GCF002.1 0 Preferably, the dialysis section has a heater for use in thermally lysing pathogens when the sample is in the lysis chamber. GCF002.il 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, The CMOS circuit system consists of using a temperature sensor output to feedback control the first and second PCR sections. GCF002.1 2 Preferably, the first PCR portion has a PCR microchannel for a heating cycle sample, and the PCR microchannel defines a flow path GCF002.1 3 having a cross-sectional area of less than 100,000 square microns. Preferably, the PCR microchannel There is at least one elongated heater element extending parallel to the PCR microchannel. GCF002.14 Preferably, the PCR portion has a plurality of elongated PCR chambers formed by respective portions of the PCR microchannels. The PCR microchannel has a 蜿蜒 structure formed by a series of width tortuos of -25-201211538, each width The tortuous line forms the channel portion of one of the elongated PCR chambers.

GCF002.1 5 Preferably, the LOC device also has a reagent storage tank for retaining reagents for PCR; and a surface tension valve having a plurality of orifices forming a meniscus of the fixed reagent so that the meniscus is reagent Retained in the reagent reservoir until the meniscus is removed by contact with the fluid sample and the reagent flows out of the reagent reservoir. GCF002.1 6 Preferably, the LOC device also has a first array of hybridization chambers containing a first probe. GCF002.1 7 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF002.1 8 Preferably, the CMOS circuit has a digital interface for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. GCF002.1 9 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. GCF002.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus and stop the capillary flow driven liquid flow 'heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. The easy-to-use, mass-produced, and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lyses in its chemical and/or hot lysate- 26- 201211538 Pathogens to release pathogen genetic material, amplify any target gene sequence, and analyze by interfering with an oligonucleotide probe and detecting its internal imaging array and using reagents stored in the reagent reservoir of the LOC device The nucleic acid sequence of the sample. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, low system component volume, and simple system manufacturing steps to provide an inexpensive analytical system. The use of chemical and thermal lysis methods has been chosen to simplify analytical chemistry requirements and provide processing capabilities for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. In addition, parallel amplification chambers allow different target or target groups to optimally use different primer pairs or different primer pair sets, and use different optimal amplification parameters to enhance subsequent analytical sensitivity and signal- Pair-noise-ratio and reliability. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective solution for the integration of low-system components in large quantities, -27-201211538, which is a compact, lightweight and easy-to-carry system. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integrated with the LOC unit and holds the analysis of the total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF003.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting a pathogen in a biological sample, the LOC device comprising: an inlet for receiving a sample; a support substrate; making the pathogen in the sample larger than the sample a dialysis section in which the components are separated; a lysis unit located downstream of the dialysis section for lysing the pathogen to release the genetic material therein, and a first nucleic acid amplification section located downstream of the lysis unit for amplifying the genetic material a first nucleic acid sequence, and a second nucleic acid amplification portion located downstream of the first nucleic acid amplification portion for amplifying a second nucleic acid sequence in the amplicon obtained from the first nucleic acid amplification portion: wherein the dialysis portion dissolves The cell, the first nucleic acid amplification unit, and the second nucleic acid amplification unit are both supported on the support substrate. GCF003.2 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 PCR unit. GCF003.3 Preferably, the first PCR portion has a first set of primer pairs bonded to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primers bonded to the complementary nucleic acid sequence of the second set -28-201211538 Yes, the first set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF003.4 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; 0 buffer solution; thermal cycle time; thermal cycle repetition, and temperature during specific phases of PCR. GCF003.5 Preferably, the LOC device also has a hybridization portion downstream of the second PCR portion, having a second probe array for hybridization with the target nucleic acid sequence, and a light perception for hybridization of any of the probes in the array Detector. GCF003.6 Preferably, the dialysis portion has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis portion, and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component, the second The passage is in fluid communication with the first passage via the orifice such that the pathogen flows into the second passage and the larger component remains in the first passage. GCF003.7 Preferably, the first channel and the second channel group constitute a sample by capillary action. GCF003.8 Preferably, the second channel group constitutes the inhalation of the pathogen into the lysis portion by capillary action. GCF003.9 Preferably, the lysis unit has a -29-201211538 heater for hot lytic pathogens. GCF003.1 0 Preferably, the LOC device also has a plurality of reagent reservoirs, wherein one of the reagent reservoirs contains a lysis reagent for the chemical lysing pathogen in the lysate. GCF003.il 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, CMOS The circuit system consists of using the temperature sensor output to feedback control the first and second PCR sections. GCF003.1 2 Preferably, the first PCR portion has a PCR microchannel for the heat cycle sample, and the PCR microchannel defines a flow path having a cross-sectional area of less than 100,000 square microns across the flow. GCF003.1 3 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel.

GCF003.1 4 Preferably, the first PC R portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, and the PCR microchannel has a crucible structure formed by a series of wide tortuosities, each width The tortuous line forms the channel portion of one of the elongated PCR chambers. GCF003.15 Preferably, the LOC device also has a reagent storage tank for retaining reagents for PCR; and a surface tension valve having an orifice forming a meniscus constituting the fixed reagent so that the meniscus retains the reagent The meniscus is removed in the reagent reservoir until contact with the fluid sample and the reagent flows out of the reagent reservoir. GCF003.1 6 Preferably, the LOC device also has a hybrid array of -30-201211538 containing probes. GCF003.1 7 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF003.1 8 Preferably, the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. GCF003.1 9 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 second PCR portion during thermal cycling. GCF003.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, dissolves in its chemical and/or hot lysate The pathogen is used to release the genetic material of the pathogen, amplify any target gene sequence, and analyze the sample by hybridizing with the oligonucleotide probe and detecting its internal imaging array and using reagents stored in the reagent reservoir of the LOC device. Nucleic acid sequence. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. -31 - 201211538 The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, a low system component amount, and a simple system manufacturing step, resulting in an inexpensive analytical system. The use of chemical and thermal lysis methods has been chosen to simplify analytical chemistry requirements and provide processing capabilities for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. In addition, parallel amplification chambers allow different target or target groups to optimally use different primer pairs or different primer pair sets, and use different optimal amplification parameters to enhance subsequent analytical sensitivity and signal- Pair-noise-ratio and reliability. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, integrated solution with low system component quantities that are small, lightweight and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integral with the LOC unit and retains the need for analysis of total reagents, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF0 (Ml of the present invention is directed to a wafer-on-lab (LOC) device for detecting pathogens in a biological sample, LOC device package -32-201211538 comprising: an inlet for receiving a sample; a support substrate; a pathogen in the sample a dialysis section separated from a larger component of the sample; a lysis section located downstream of the dialysis section for lysing the pathogen to release the genetic material therein, the lysis unit having a lysis chamber and a heater for the sample to be in the lysis For lysing pathogens in the chamber; and 0 for the nucleic acid amplification portion located downstream of the lysate for amplifying the nucleic acid sequence in the genetic material; wherein the dialysis section, the lysis part, and the nucleic acid amplification section are all supported Preferably, the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion on the support substrate. GCF004.3 Preferably, the LOC device has a hybridization portion downstream of the PCR portion, which is used in the sample A probe array 杂交 hybridized to a target nucleic acid sequence and a photosensor for detecting hybridization of any probe in the array. GCF004.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet, and lysis a second passage in fluid communication and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component, the second passage being in fluid communication with the first passage via the orifice such that the pathogen flows into the second passage, and the larger component Preferably, the first channel and the second channel group constitute a sample by capillary action. GCF004.6 Preferably, the second channel group is constituted by capillary action. -33- 201211538 The pathogen is inhaled into the lysis unit. GCF004.7 Preferably, the lysis unit has a heater for the hot lytic pathogen. GCF004.8 Preferably, the LOC device is also provided for retention for constant temperature. a reagent storage tank for a reagent for nucleic acid amplification; and a surface tension valve having an orifice forming a meniscus constituting the immobilization reagent such that the meniscus retains the reagent in the reagent reservoir until the fluid sample is contacted to remove the meniscus The reagent flows out of the reagent reservoir. GCF004.9 Preferably, the LOC device also has a flow path from the inlet to the hybridization section, wherein the flow path is configured to suck the sample from the inlet to the hybridization section by capillary action GCF004.1 0 Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer combined with the PCR portion, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit system is used. The temperature sensor output is used to feedback control the PCR portion. GCF004.il Preferably, the PCR portion has a PCR microchannel for the heating cycle sample to amplify the nucleic acid sequence, and the PCR microchannel defines the sample and the cross-sectional area of the traverse flow is less than A portion of the flow path of 1 00,000 square microns. GCF004.1 2 Preferably, the LOC device also has at least one heater element for heating the extension of the nucleic acid sequence within the PCR microchannel, the elongated heater element being parallel to the PCR microchannel And extended. GCF004.1 3 Preferably, at least one of the PCR microchannels forms an elongated P c R chamber. GCF004.1 4 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the pcR micro-pass-34-201211538 channel, and the PCR microchannel has a crucible structure formed by a series of wide tortuosity, Each wide tortuous line forms a channel portion of one of the elongated PCR chambers. GCF004.1 5 Preferably, the LOC device also has a reagent reservoir for holding reagents for PCR; and a 'surface tension valve having an orifice that forms a meniscus of the immobilization reagent such that the meniscus will reagent Retain in the reagent reservoir until the fluid sample is contacted to remove the meniscus and the reagent flows out of the reagent reservoir. GCF004.1 6 Preferably, the LOC device also has an array of hybrid chambers containing probes. GCF004.17 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF004.1 8 Preferably, the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. 〇 GCF004.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section and in response to an activation signal from the CMOS circuit to allow liquid to flow to the hybridization chamber during thermal cycling. GCF004.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to isolate any pathogens contained in the sample, and dissolves in its hot lysate. The pathogen extracts the genetic material of the pathogen, amplifies any target gene sequence, and analyzes the nucleic acid of the sample by hybridizing with the oligonucleotide probe and detecting its internal imaging array and using the reagent stored in the reagent reservoir of the LOC device. sequence. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, low system component volume, and simple system manufacturing steps to provide an inexpensive analytical system. The hot lysis method simplifies the analytical chemistry requirements and provides processing power for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides a solution that is easy to use, mass-processable, and inexpensive with an integrated system component. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, low-system, integrated solution that is compact, lightweight, and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. An analytical system that integrates with the LOC unit and retains the reagent storage tank for the analysis of total reagent requirements, providing low system component quantities and simple manufacturing steps, which in turn leads to an inexpensive -36-201211538. GCF005.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting pathogens in a biological sample. The LOC device comprises: an inlet for receiving a sample; a support substrate; a larger pathogen in the sample and the sample. a dialysis section in which the components are separated; 0 a lysis part located downstream of the dialysis section for lysing the pathogen to release the genetic material therein, and the lysis part has a lysis chamber and a heater for use when the sample is located in the lysis chamber a lytic pathogen; a first nucleic acid amplification portion located downstream of the lysate portion for amplifying a nucleic acid sequence in the genetic material of the first portion of the sample stream from the lysate; and 'located downstream of the lysis portion a nucleic acid amplification unit' for amplifying a nucleic acid sequence in the genetic material of the second part of the sample stream from the lysis unit; wherein the dialysis unit, the lysis unit, the first nucleic acid amplification unit, and the second nucleic acid amplification unit The crucibles are supported on the support substrate. Preferably, the first nucleic acid amplification unit is a first polymerase chain reaction (PCR) portion and the second nucleic acid amplification unit is a second PCR unit 〇GCF005.3. Preferably, the first PCR portion has a first set of primer pairs that bind to the first set of complementary nucleic acid sequences, and a second set of primer pairs that bind to the second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different from the second set of complementary nucleic acid sequences . Preferably, the first PCR portion and the second PCR portion organization -37-201211538 are operated with different amplification parameters, and the amplification parameters are 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 specific phases of PCR. GCF005.5 Preferably, the LOC device also has a first hybridization portion downstream of the first PCR portion, having a first probe array hybridized with the first target nucleic acid sequence and a second hybridization portion downstream of the second PCR portion a second probe array for hybridization to a second target nucleic acid sequence, and a photosensor for detecting hybridization of any of the probes in the first array and the second array. GCF005.6 Preferably, the dialysis section has at least two passages that are in fluid communication with the plurality of orifices, the plurality of orifices being sized to correspond to a predetermined threshold. GCFO 〇 5.7 Preferably, at least two channels and a plurality of orifices are configured to cause the sample to flow through the channels and orifices by capillary action. GCFO 〇 5.8 Preferably, the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the lysis portion, and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component, the second passage The first channel is in fluid communication via the orifice such that the pathogen flows into the second channel and the larger component remains in the first channel. GCF005.9 Preferably, the lysis unit has a -38-201211538 heater for the hot lytic pathogen. GCF005.10 Preferably, the LOC device also has a reagent storage tank for retaining reagents for constant temperature nucleic acid amplification; and a surface tension valve having a plurality of orifices constituting a meniscus of the immobilization reagent, such that the meniscus The reagent is retained in the reagent reservoir until the meniscus is removed by contact with the fluid sample and the reagent flows out of the reagent reservoir. GCF005.il 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. The CMOS circuit system consists of using a temperature sensor output to feedback control the first and second PCRs. GCF005.1 2 Preferably, the first PCR portion has a PCR microchannel for a heating cycle sample, and the PCR microchannel defines a flow path having a cross-sectional area of less than 100,000 square microns across the flow. GCF005.13 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GCF005.14 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, and the PCR microchannel has a 蜿蜒 structure formed by a series of wide tortuous lines, and each of the wide tortuous lines forms an elongation The channel section of one of the PCR chambers.

GCF005.1 5 Preferably, the L0C device also has a reagent storage tank for retaining reagents for PCR; and a surface tension valve having an orifice forming a meniscus constituting the fixed reagent so that the meniscus is reagent Retain in the reagent reservoir until the fluid sample is removed -39-201211538 to remove the meniscus and the reagents flow out of the reagent reservoir. GCF005.1 6 Preferably, the LOC device also has a first array of hybridization chambers containing a first probe. GCF005.1 7 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF005.1 8 Preferably, the CMOS circuit has a digital memory for storing the hybridization data from the photosensor output and a data interface for transmitting the hybridization data to the external device. GCF005.1 9 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. GCF005.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lyses pathogens in its hot lysate to release pathogens. The genetic material, amplifies any target gene sequence, and analyzes the nucleic acid sequence of the sample by hybridizing to the oligonucleotide probe and detecting its internal imaging array and utilizing reagents stored in a reagent reservoir of the LOC device. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and the device are of a unitary structure, which provides a low system component quantity and a simple manufacturing step, thereby obtaining an inexpensive analysis system of -40 - 201211538. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integral with the device' which provides a simple analytical step, a low system component volume, and a simple system manufacturing step to provide an inexpensive analytical system. The hot lysis method simplifies the analytical chemistry requirements and provides processing power for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. In addition, 'parallel amplification chambers enable different target or target groups to optimally use different primer pairs or different primer pair groups, and use different optimal amplification parameters to enhance subsequent analytical sensitivity and signal- Pair-noise-ratio and reliability. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and provides a cost-effective, low-system component integration solution that is small, lightweight, and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integrated with the LOC unit and holds the analysis of the total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF006.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting pathogens in a biological sample, LOC device package -41 - 201211538 comprising: an inlet for receiving a sample; a support substrate; a pathogen in the sample a dialysis section separated from the larger component of the sample; a lysis section located downstream of the dialysis section for lysing the pathogen to release the genetic material therein, the lysate having a lysis chamber and a heater for the sample to be in the lysis a lysing pathogen in the chamber; a first nucleic acid amplification portion located downstream of the lysate portion for amplifying the first nucleic acid sequence in the genetic material; and a second nucleic acid amplification downstream of the first nucleic acid amplification portion a second nucleic acid sequence for amplifying the amplicon obtained from the first nucleic acid amplification portion; wherein the dialysis portion, the lysis portion, the first nucleic acid amplification portion, and the second nucleic acid amplification portion are both supported Support on the substrate. GCF006.2 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 PCR unit. GCF006.3 Preferably, the first PCR portion has a first set of primer pairs that are bonded to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs that are bonded to the second set of complementary nucleic acid sequences, first The set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF006.4 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; -42- 201211538 Buffer solution; Thermal cycle time; Thermal cycle repeat 'and temperature during specific phases of PCR. GCF006.5 Preferably, the LOC device also has a hybridization portion downstream of the second PCR portion, having a probe array for hybridizing to the target nucleic acid sequence' and light sensing for detecting hybridization of any probe in the array Device. 0 GCF006.6 Preferably, the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the lysis portion, and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component, the second The channel is in fluid communication with the first channel via the orifice such that the pathogen flows into the second channel' while the larger component remains in the first channel. GCF006.7 Preferably, the first channel and the second channel group constitute a sample by capillary action. GCF006.8 Preferably, the lysis unit has a helium heater for the hot lytic pathogen. GCF006.9 Preferably, the first nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. GCF006.10 Preferably, the LOC device also has a reagent storage tank for retaining reagents for constant temperature nucleic acid amplification; and a 'surface tension valve having an orifice forming a meniscus constituting the immobilization reagent, such that the meniscus Retaining the reagent in the reagent reservoir until the fluid sample is contacted to remove the meniscus and the reagent flowing out of the reagent reservoir is & ° GCF006.il preferably, the LOC device also has a CM0S circuit, a temperature-43-201211538 degree 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 system is configured to use the temperature sensor output to feedback control the first and second PCR section. GCF006.1 2 Preferably, the first PCR portion has a PCR microchannel for the heating cycle sample, and the PCR microchannel defines a flow path having a cross-sectional area of less than 100,000 square microns across the flow. GCF006.1 3 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GCF006.14 Preferably, the PCR portion has a plurality of elongated PCR chambers formed by respective portions of the PCR microchannels. The PCR microchannels have a crucible structure formed by a series of wide tortuous lines, and each of the wide tortuous lines forms an elongation. The channel section of one of the PCR chambers. GCF006.1 5 Preferably, the L0C device also has a reagent storage tank for retaining reagents for PCR; and a surface tension valve having a plurality of orifices forming a meniscus of the fixed reagent so that the meniscus will reagent Retained in the reagent reservoir until the meniscus is removed by contact with the fluid sample and the reagent flows out of the reagent reservoir. GCF006.1 6 Preferably, the LOC device also has a hybrid chamber array containing probes. GCF006.1 7 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF006.1 8 Preferably, the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data -44 - 201211538 to an external device. GCF006.1 9 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 second P CR portion during thermal cycling. GCF006.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus retainer to restore the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lyses pathogens in its hot lysate to release pathogens. The genetic material, amplifies any target gene sequence, and analyzes the nucleic acid sequence of the sample by hybridizing to the oligonucleotide probe and detecting its internal imaging array and utilizing reagents stored in a reagent reservoir of the LOC device. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, low system component volume, and simple system manufacturing steps to provide an inexpensive analytical system. The hot lysis method simplifies the analytical chemistry requirements and provides processing power for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the letter-to-noise ratio. In addition, parallel amplification chambers allow different target or target groups to optimally use different primer pairs or different primer pair sets, and use different optimal amplification parameters to enhance subsequent analytical sensitivity and signal- Pair-noise-ratio and reliability. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, low-system, integrated solution that is compact, lightweight, and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integrated with the LOC unit and holds the analysis of the total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF008.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting pathogens in a biological sample. The LOC device comprises: an inlet for receiving a sample; a support substrate; a larger pathogen in the sample and the sample a dialysis section in which components are separated; a plurality of reagent storage tanks; a lysis unit located downstream of the dialysis section for lysing pathogens to release genetic material therein, a lysate and cells containing cells for lysis in the lysis unit -46 - 201211538 One of the reagent storage tanks of the lysis reagent is in fluid communication; and a nucleic acid amplification section located downstream of the lysis unit for amplifying the nucleic acid sequence in the genetic material; wherein, the dialysis section, the lysis section And the nucleic acid amplification unit is supported on the support substrate 〇GCF00 8.2. Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. 0 GCF008.3 Preferably, the LOC device has a hybridization portion downstream of the PCR portion, having a probe array for hybridization with a target nucleic acid sequence in the sample, and a photosensor for detecting hybridization of any probe in the array . GCF008.4 Preferably, the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the lysis portion, and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component, the second passage The first channel is in fluid communication via the orifice such that the pathogen flows into the second channel and the larger component remains in the first channel. 〇 GCF008.5 Preferably, at least two channels and a plurality of orifices are configured to cause the sample to flow through the channels and orifices by capillary action. GCF008.6 Preferably, the first channel and the second channel group constitute a sample by capillary action. G C F 0 0 8.7 Preferably, the nucleic acid amplification unit is a thermostatic nucleic acid amplification unit. GCF008.8 Preferably, the reagent reservoirs each have a surface tension valve for holding the reagent therein, and the surface tension valve has a meniscus holder 'meniscus holder for fixing the meniscus of the reagent until the sample flow Contacting and removing the meniscus allows the reagent to flow out of the reagent reservoir. -47- 201211538 GCF008.9 Preferably, the LOC device also has a flow path from the inlet to the hybridization section, wherein the flow path is configured to draw the sample from the inlet to the hybridization section by capillary action. GCF008.1 0 Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the combined PCR section, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit system is formed. The temperature sensor output is used to feedback control the PCR section. GCF008.il Preferably, the PCR portion has a PCR microchannel for the heating cycle sample to amplify the nucleic acid sequence, and the PCR microchannel defines a portion for the sample and the cross-sectional area of the traverse flow is less than 1 〇〇, 〇〇〇 square micrometer Flow path. GCF008.1 2 Preferably, the LOC device also has at least one heater element for heating the extension of the nucleic acid sequence within the PCR microchannel, the elongated heater element extending parallel to the PCR microchannel. GCF008.1 3 Preferably, at least one portion of the PCR microchannel forms an elongated PCR chamber. GCF008.1 4 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, and the PCR microchannel has a crucible structure formed by a series of wide tortuosity, and each of the wide tortuous lines is formed. The channel portion of one of the elongated PCR chambers. GCF008.1 5 Preferably, the LOC device also has a reagent storage tank for retaining reagents for PCR; and a surface tension valve having a plurality of orifices forming a meniscus of the fixed reagent so that the meniscus will reagent Retained in the reagent reservoir until the meniscus is removed by contact with the fluid sample and the reagent flows out of the reagent reservoir. -48- 201211538 GCF008.1 6 Preferably, the LOC device also has an array of hybrid chambers containing probes. GCF008.1 7 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF008.1 8 Preferably, the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. 0 GCF008.1 9 Preferably, the PCR section has an active valve for retaining liquid in the PCR section and in response to an activation signal from the CMOS circuit to allow liquid to flow to the hybridization chamber during thermal cycling. GCF008.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. An easy-to-use, mass-produced and inexpensive pathogen detection LOC device that receives a biological sample through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lyses pathogens in its hot lysate for release The genetic material of the pathogen, amplifying any target gene sequence, and analyzing the nucleic acid sequence of the sample by hybridizing with the oligonucleotide probe and detecting its internal imaging array and using reagents stored in a reagent reservoir of the LOC device. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and the device are of a unitary structure' which provides a low system component volume and simple manufacturing steps, resulting in an inexpensive analytical system. -49- 201211538 The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integral with the device' which provides a simple analytical step, a low system component volume, and a simple system manufacturing step to provide an inexpensive analytical system. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, integrated solution with low system component quantities that are small, lightweight and easy to carry. The integrated image sensor increases the read sensitivity due to the large light collection angle, which eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integrated with the LOC unit and holds the analysis of the total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF009.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting a pathogen in a biological sample, the LOC device comprising: an inlet for receiving a sample; a support substrate; making the pathogen in the sample larger than the sample a dialysis unit for separating components; a plurality of reagent storage tanks; -50- 201211538 a lysis unit located downstream of the dialysis section for lysing pathogens to release genetic material therein, and the lysate is contained for lysis in lysis One of the reagent storage tanks of the lysis reagent of the cells in the chamber is in fluid communication, and is located in the first nucleic acid amplification portion downstream of the lysis unit for amplifying the first portion of the sample stream obtained from the lysis unit a first nucleic acid sequence in the genetic material; and a second nucleic acid amplification portion located downstream of the lysis portion for amplifying a second nucleic acid sequence in the genetic material in the second portion of the sample stream obtained from the lysing portion The dialysis unit, the lysis unit, the first nucleic acid amplification unit, and the second nucleic acid amplification unit are all supported on the support substrate. GCF009.2 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 PCR unit. GCF009.3 Preferably, the first PCR portion has a first set of primer pairs that are bonded to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs that are bonded to the second set of complementary nucleic acid sequences, A set of complementary nucleic acid sequences differs from a second set of complementary nucleic acid sequences. GCF009.4 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; deoxyribose nuclear tone Di-ortho acid concentration, buffer solution; thermal cycle time; -51 - 201211538 thermal cycle repetition; and, temperature during a specific phase of PCR. GCF009.5 Preferably, the LOC device also has a first hybridization portion of the first PCR portion, having a first needle array hybridized with the first target nucleic acid sequence and a second hybrid portion downstream of the second PCR portion, having A second probe array hybridized with a second target nucleic acid sequence, and a photosensor for detecting hybridization of any of the probes in the second array. GCF009.6 Preferably, the dialysis portion has a first passage fluidly connected to the inlet, a second passage in fluid communication with the lysis portion, and a plurality of orifices larger than the pathogen and smaller than the larger component, the second passage being through the orifice One channel is in fluid communication such that the pathogen flows into the second channel and the more component remains in the first channel. GCF009.7 Preferably, the first channel and the second channel structure are capillary-charged to sample. G C F 0 0 9.8 Preferably, the second channel group constitutes the inhalation of the pathogen into the lysis portion by capillary. GCF009.9 Preferably, the first nucleic acid amplification unit is a second constant temperature amplification unit and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. GCF009.1 0 Preferably, the reagent storage tanks each have a surface tension valve for being tested therein, and the surface tension valve has a meniscus fixed meniscus holder for fixing the meniscus of the reagent until it is connected to the sample stream. In addition to the meniscus, the reagent flows out of the reagent reservoir. GCF009.il Preferably, the LOC device also has a CMOS circuit level sensor and a microsystem technology for combining the first and second PCR sections (

The downstream is located in the orifice of the test and the large port of the nucleic acid agent, touch and temperature, MST-52-201211538) layer, wherein the CMOS circuit is located between the support substrate and the MST layer, CMOS circuit system The group configuration uses the temperature sensor output to feedback control the first and second PCR sections. GCF.009.1 2 Preferably, the first PCR portion has a PCR microchannel for a heating cycle sample, and the PCR microchannel defines a flow path having a cross-sectional area of less than 100,000 square microns across the flow. GCF009.1 3 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. GCF009.1 4 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, and the PCR microchannel has a crucible structure formed by a series of wide tortuous lines, and each of the wide tortuous lines is formed. The channel portion of one of the elongated PCR chambers. GCF009.1 5 Preferably, the LOC device also has a reagent storage tank for holding reagents for PCR; and a surface tension valve having an orifice 组 which constitutes a meniscus of the fixed reagent, so that the meniscus will The reagent remains in the reagent reservoir until the meniscus is removed by contact with the fluid sample and the reagent flows out of the reagent reservoir. GCF009.16 Preferably, the LOC device also has a first array of hybridization chambers containing a first probe. GCF009.1 7 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF009.1 8 Preferably, the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. -53- 201211538 GCF009.1 9 Preferably, the first PCR portion has an active for retaining liquid in the first PCR portion and in response to an activation signal from the CMOS circuit during the thermal cycle to allow liquid to flow to the first hybridization chamber array. valve. GCF009.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lyses pathogens in its hot lysate to release pathogens. The genetic material, amplifies any target gene sequence, and analyzes the nucleic acid sequence of the sample by hybridizing to the oligonucleotide probe and detecting its internal imaging array and utilizing reagents stored in a reagent reservoir of the LOC device. The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, low system component volume, and simple system manufacturing steps to provide an inexpensive analytical system. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. In addition, parallel amplification chambers allow different target or target groups to optimally use different primer pairs or different primer pairs, and -54-201211538 uses different optimal amplification parameters to enhance subsequent Analyze sensitivity, signal-to-noise-ratio and reliability. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, low-system component solution solution that is compact, lightweight, and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integrated with the LOC unit and holds the analysis of the total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. GCF01 0.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting pathogens in a biological sample, the LOC device package Q comprising: an inlet for receiving a sample; a support substrate; a pathogen in the sample compared to the sample a large component separated dialysis section; a plurality of reagent storage tanks; a lysis unit located downstream of the dialysis section for lysing pathogens to release genetic material therein, the lysate and the lysis unit for lysis in the lysis unit One of the reagent sump of the lysis reagent of the cell is in fluid communication, and is located at a first nucleic acid amplification portion downstream of the lysis portion for amplifying the first nucleic acid sequence in the genus -55-201211538; a second nucleic acid amplification unit downstream of the first nucleic acid amplification unit for amplifying a second nucleic acid sequence in the amplicon obtained from the first nucleic acid amplification unit; wherein the dialysis unit, the lysis unit, and the first nucleic acid amplification unit The addition portion and the second nucleic acid amplification portion are both supported on the support substrate. 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 PCR unit. GCF010.3 Preferably, the first PCR portion has a first set of primer pairs that are bonded to the first set of complementary nucleic acid sequences, and the second PCR portion has a second set of primer pairs that are bonded to the second set of complementary nucleic acid sequences, first The set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF010.4 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 repetition; and, temperature during a particular phase of the PCR. GCF010.5 Preferably, the LOC device also has a hybridization portion downstream of the second PCR portion, having a probe array for hybridization with the target nucleic acid sequence and a photosensor for detecting hybridization of any of the probes in the array. GCF010.6 Preferably, the dialysis section has a first passage of -56-201211538 in fluid communication with the inlet, a second passage in fluid communication with the lysis unit, and a plurality of orifices, the orifice being larger than the pathogen and smaller than the larger component The second passage is in fluid communication with the first passage via the orifice such that the pathogen flows into the second passage and the larger component remains in the first passage. GCF010.7 Preferably, the first channel and the first channel group constitute a sample by capillary action. GCF010.8 Preferably, the second channel group constitutes the inhalation of the pathogen into the lysis portion by capillary action 0. GCF010.9 Preferably, the first nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit and the second nucleic acid amplification unit is a second constant temperature nucleic acid amplification unit. GCF010.10 Preferably, the reagent storage tanks each have a surface tension valve for holding the reagent therein, the surface tension valve has a meniscus holder, and the meniscus holder is used to fix the meniscus of the reagent until the sample flow Contacting and removing the meniscus allows the reagent to flow out of the reagent reservoir. GCF010.il Preferably, the LOC device also has a CMOS circuit, a temperature sensitivity 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. The CMOS circuit system consists of using a temperature sensor output to feedback control the first and second PCR sections. GCF010.12 Preferably, the first PCR portion has a PCR microchannel for a heat cycle sample, and the PCR microchannel defines a flow path having a cross-sectional area of less than 100,000 square microns across the flow. GCF0 1 0.1 3 Preferably, the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. -57- 201211538 GCFO 1 0.14 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel. The PCR microchannel has a 蜿蜒 structure formed by a series of wide tortuosities, each width The tortuous line forms the channel portion of one of the elongated PCR chambers. GCF 010.15 Preferably, the LOC device also has a reagent storage tank for retaining reagents for PCR; and a surface tension valve having an orifice forming a meniscus constituting the fixed reagent so that the meniscus retains the reagent The meniscus is removed in the reagent reservoir until contact with the fluid sample and the reagent flows out of the reagent reservoir. GCF 010.16 Preferably, the LOC device also has an array of hybrid chambers containing probes. GCF010.17 Preferably, the photosensor is an array of photodiodes that are positioned in engagement with the hybridization chamber. GCF 010.18 Preferably, the CMOS circuit has a digital interface for storing hybridization data from the output of the photosensor and a data interface for transmitting the hybridization data to an external device. GCF 010.19 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 second PCR portion during thermal cycling. GCF010.20 Preferably, the active valve is a boiling pilot valve having a meniscus holder and a heater, and the meniscus holder is configured to fix the meniscus to stop the liquid flow driven by the capillary action, the heater system The liquid is boiled and the meniscus is released from the meniscus holder to restore the capillary action drive flow. Easy-to-use, mass-produced and inexpensive pathogen detection LOC device-58- 201211538 Receive biological samples through its sample container, use its dialysis section to separate any pathogens contained in the sample, and lyse pathogens in its hot lysate The nucleic acid sequence of the sample is analyzed by releasing the genetic material of the pathogen, amplifying any target gene sequence, and by hybridizing with the oligonucleotide probe and detecting its internal imaging array and using the reagent stored in the reagent reservoir of the LOC device. . The dialysis function takes additional information from the sample and increases the sensitivity, signal-to-noise ratio, and the dynamic range of the analysis system. The dialysis unit and device are of a squat structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integrated with the device, which provides a simple analysis step, low system component volume, and simple system manufacturing steps to provide an inexpensive analytical system. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. In addition, parallel amplification chambers allow different target or target target sets to optimally use different primer pairs or different primer pair sets, and use different optimal amplification parameters to enhance subsequent analytical sensitivity and signal - Pair - Noise - Ratio and reliability. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and offers a cost-effective, low-system, integrated solution that is compact, lightweight, and easy to carry. Due to the large light collection -59-201211538 Angle's integrated image sensor increases readout sensitivity and eliminates the need for optical components in the string of optical components. The reagent storage tank, which is integral with the L 0 C unit and retains the need for analysis of total reagents, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system. [Embodiment] This general specification indicates the main components of the molecular diagnostic system including the specific embodiment of the present invention. The details of the system structure and operation are discussed in the following description. Referring to Figures I, 2, 3, 133 and 134, the system has the following most important components: The test modules 10 and 11 are of the size of a conventional USB flash drive and can be manufactured inexpensively. The test modules 10 and 11 each contain a microfluidic device, which is typically in the form of a lab-on-lab (LOC) device 30 and preloaded with reagents, and typically has more than 1,000 probes for molecular diagnostic analysis (see Figure 1 and 133). The test module 1 shown in Figure 1 uses a fluorescence-based detection technique to identify target molecules, while the test module 11 in Figure 13 uses an electrochemiluminescence-based detection technique. The LOC device 30 has an integrated photosensor 44 (described in detail below) for fluorescence or electrochemiluminescence detection. The test modules 1 and 1 1 each use a standard micro-u s B connector 14 for power, data, and control, each having a printed circuit board (PCB) 57, and a capacitor 32 and an inductor 15 each having an external power supply. The test modules 10 and 11 are for a single use of a large number of -60-201211538 and are distributed in aseptic packaging for use. The outer casing 13 has a large container 24 for receiving biological samples and a removable sterile sealing strip 22 which preferably has a low viscosity adhesive to cover the large container prior to use. The membrane seal 408 having the membrane shield 410 forms part of the outer casing 13 to reduce the moisture resistance in the test module while providing a pressure relief effect by small pressure changes. The membrane guard 410 protects the membrane seal 408 from damage. The test module reader 12 supplies power to the test module group 10 or 11 via the micro-USB port 16. The test module reader 12 can be in many different forms, and its selection is described below. The reader 12 version shown in Figures 1, 3 and 133 is a specific embodiment of a smart phone. A block diagram of the reader 12 is shown in FIG. The processor 42 executes the application software from the program storage 43. The processor 42 also displays the screen 18 and the user interface (UI) touch screen 17 and buttons 19, the cellular radio 21, the wireless network connection 23, and the satellite navigation. System 25 is interfaced. The cellular radio 21 and the wireless network connection 23 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location data.替代 Alternatively, you can enter the location data with the touch screen 1 7 or button 1 9 people. The data store 27 holds genetic and diagnostic information, test results, patient information, analysis and probe data for identifying each probe, and its array position. The data storage 27 and the program storage 43 can be shared by a common memory device. The application software installed in the test module reader 12 provides results analysis and additional test and diagnostic information. To perform a diagnostic test, test module 10 (or test module 1 1) is inserted into micro-USB port 16 on test module reader 12. The sterile sealing strip 22 is turned up and the biological sample (in liquid form) is loaded into the large sample container 24-61 - 201211538. The start button 20 is pressed to initiate the test by applying the software. The sample is passed to LOC unit 30 where it is analyzed for extraction, culture, amplification and hybridization of the pre-synthesized hybrid-reactive oligonucleotide probe 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 electrochemiluminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to generate photoluminescence. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 134). The emission (fluorescence or photoluminescence) is detected using a photo sensor 44 integrated with a CMOS circuit on each LOC device. The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the results. Data can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 1 1 is removed from the test module reader 1 2 and processed as appropriate. Figures 1, 3 and 1; 33 show the group of test module readers 12 constituting a mobile phone/smart phone 28. In other forms, the test module reader is a laptop/notebook for use in a hospital, private clinic, or laboratory, a dedicated reader 103, an e-book reader 107, a tablet computer, or a table. Computer 105 (see Figure 135). The reader can interface with a number of additional applications such as patient records, accounts, online databases and multi-user environments. It can also interface with some local or remote peripherals, such as printers and patient smart cards. -62- 201211538 Referring to Figure 1 3, through the reader 1 2 and the network 1 2 5, the data generated by the test module 10 can be used to update the epidemiology of the host system for epidemiological data 111. The database, the genetic database maintained by the host system for genetic data 1 1 3, the electronic health record maintained by the host system for electronic health record (EHR) 115, and used for electronic medical records (EMR) The electronic medical record maintained by the host system of 121, and the personal health record maintained by the host system for personal health record (PHR) 123. Conversely, the epidemiological data held by the host system for epidemiological data 1 1 1 via the network 1 2 5 and the reader 1 2 1 , the genetic data held by the host system for the genetic data 113, Electronic health records maintained by the host system of the Electronic Health Record (EHR) II5, electronic medical records maintained by the host system for electronic medical records (EMR) 121, and for personal health records (PHR) The personal health record maintained by the host system of 123 can be used to update the digital memory in the test module 1〇LOC 30. G Referring again to Figures 1, 2, 133 and 134, in the mobile phone configuration, the reader 12 uses battery power. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be loaded or uploaded via some network or contact interface to enable communication with peripheral devices, computers or online servers. Set the Micro-USB port 16 to connect the computer or main power supply to recharge the battery. Figure 7A shows a specific embodiment of the test module 10 for testing that only requires the presence or absence of a particular target, such as whether the test individual is infected with, for example, influenza A virus η 1 N 1 . It is only suitable as a built-in module 47 for U S B power/indicators. No other readers are required or should be used -63- 201211538. The indicator 45 on the module 47 of the USB power/indicator only shows a positive or negative result. This configuration is well suited 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 3 90 and a lancet release button 3 92. Satellite phones can be used in remote areas. Test Module Electronics Figures 2 and 13 4 are block diagrams of the electronic components of test modules 10 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a clock 33, a power conditioner 31, a RAM 38, and a program and data flash. These are provided for the test module 10 or 11 including the photo sensor 44, the temperature sensor 170, the liquid sensor 1 74, and the various heaters 152, 154, 182, 234, and the associated driver 37. And 3 9 and the registers of the registers 3 5 and 4 1 and the body of the billion. Only LED 26 (in the case of test module 10), external power supply capacitor 32 and micro-USB connector 14 are external to LOC device 30. The LOC device 30 includes an adhesive pad for attachment to these external components. RAM 38 and program and data flash memory 40 have application software and diagnostic and experimental information for more than one probe (flash/security storage, e.g., via encryption). In the case of the test module 11 constructed for ECL detection, there is no LED 26 (see Figures 133 and 134). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 is loaded with an electrochemiluminescent probe and a hetero-64-201211538 chamber, each having an ECL excitation electrode pair 860 and 870. Many types of test modules 10 are manufactured in a number of test formats that are ready for ready-to-use users. The test format differs in the reagents and probes of the onboard assay. Some examples of infectious diseases that are rapidly identified by this system include: • Influenza-influenza virus A, B, C, infectious salmon anemia virus, tocovirus Q • pneumonia-respiratory fusion virus (RSV), Adenovirus, 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- typhoid bacillus • Ebola virus • Human immunodeficiency virus (HIV) 〇 • Dengue fever – Yellow fever virus • Hepatitis (A to E) • Iatrogenic infections – for example Clostridium pneumoniae, vancomycin-resistant enterococci and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Audemars Piguet-Barr virus (EBV). Encephalitis-Japan Encephalitis virus, Zhangdipula virus • Pertussis-pertussis-65- 201211538 • Measles-paramyxovirus • Meningitis - Streptococcus pneumoniae and meningitis double ball m • Anthrax - Bacillus anthracis identified by this system Some examples of hereditary diseases include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black Mongolian idiots • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic kidney disease • Congenital Sexual Heart Disease • A small selection of cancers identified by the diagnostic system for the disease. • Ovarian cancer • Colon cancer • Multiple endocrine tumors • Retinoblastoma • Turcot syndrome The above list is not An exhaustive 'and diagnostic system can be organized to detect many different diseases and symptoms using nucleic acid and proteomic analysis. Detailed structure of system components -66' 201211538 LOC device LO C device 30 is the center of the diagnostic system. It uses a microfluidic platform to rapidly perform the four main steps of nucleic acid-based molecular diagnostic analysis, namely sample preparation, nucleic acid extraction, nucleic acid amplification and detection. LOC devices also have alternative uses' and will be described in more detail below. As discussed above, the test modules (7) and ^ can take many different configurations to detect different targets. As such, the LOC device 30 has a number of different embodiments for targeting the target of interest. The 〇C device 30 is in the form of 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 is described in detail with reference to Figures 4 through 26 and 27 through 57. 4 is a schematic diagram showing the structure of the LOC device 301. For the sake of convenience, the processing stages shown in Fig. 4 are represented by the component symbols corresponding to the functional portions of the LOC device 301 that implements the processing stage. The processing stages associated with the major steps of each nucleic acid-based molecular diagnostic assay also represent: sample input and preparation 28, extraction 290, culture 291, amplification 292, and detection 294. Various tanks, chambers, valves, and other components of the LOC unit 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 construction of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) of Figure 12. The LOC device 301 has a germanium substrate 84 supporting a COM S + 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 organized to define a flow path having a characteristic size of -67 - 201211538 that supports a capillary action driven liquid stream 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 1760 microns X 58 24 microns. Of course, LOC devices made for different applications can have different sizes. Figure 6 shows the features of the MST layer 87 overlaid with the cover features. The illustrations 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 independently shows the structure of the CMOS + MST device 48, FIGS. 7 through 10 independently show the features of the cover 46. Layered Structure Figures 12 and 22 are schematic views of the layered configuration of the CMOS + MST device 48, the cover 46, and the fluid interaction therebetween. The drawings are not drawn to scale for the purpose of illustration. Figure 12 is a cross-sectional view through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12, CMOS + MST device 48 has a germanium substrate 84 that supports a CMOS circuit 86 that operates the active components in the -68-201211538 MST layer 87 described above. Passivation layer 88 seals and protects CMOS layer 86 from fluid flow through MST layer 87. The fluid flows through both the cap layer 46 and the MST channel layer 100 and the MST channel 90 (see, for example, Figures 7 and 16). When biochemical treatment is performed on the smaller MST channel 90, cell delivery occurs in the larger channel 94 made in the cover 46. The cell delivery channels are sized to deliver cells in the sample to predetermined locations in the MST channel 90. Cells that deliver a size greater than 0 20 microns (e.g., certain white blood cells) require channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. In particular, the MS T channel at a location in the LOC that does not require delivery of the cells can be significantly smaller. It will be understood that the cover channel 94 and the MST channel 90 are common references and that the particular MST channel 90 can also be For example, heated microchannels or dialyzed MST channels. The MST channel 90 is formed by etching through an MST channel layer 1 deposited on the passivation layer 88 and patterned with a photoresist. 〇 The MST channel 90 is surrounded by a top layer 66 that forms the top of the CMOS + MST device 48 (relative to the orientation shown in the figure). Although sometimes shown as a separate layer, the cover channel layer 80 and the sump layer 78 are formed from a single piece of material. Of course, the piece of material can also be non-singular. The sheets of material are etched from both sides to form the cover channel layer 80 and the sump layer 78, the cover channels 94 are etched in the cover channel layer 80, and the sumpes 54, 56, 58, 60 and 62 are etched in the sump layer 78. Alternatively, the sump and cover channel are formed by micro-forming. Both worming and microforming techniques are used to produce channels having a cross-flow of up to 20, 〇〇〇 square microns and as small as 8 square microns. -69- 201211538 There are appropriate options for the cross-section of the passage through the fluid at different locations in the LOC unit. A large number of samples or samples having a large component are accommodated in the channel, and a cross-sectional area of up to 20,000 square micrometers (e.g., a 200 micrometer wide channel in a layer of 100 micrometers thick) is suitable. A small amount of liquid or a mixture free of large cells is contained in the channel, preferably a very small cross-sectional area across the fluid. The lower seal 64 surrounds the lid passage 94 and the upper seal layer 82 surrounds the sump 54, 56, 580, 60 and 6 2 °. The five sump 54, 56, 58, 60 and 62 are preloaded with reagents for specific analysis. In the embodiments described herein, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant, the selectivity of which includes red blood cell lysis buffer • storage tank 5 6 : lysis reagent • Storage tank 5 8: Restriction enzymes, ligases and junctions (for junction initiation PCR (see Figure 69, excerpt from t. Stachan et al., Human Molecular Genetics 2, Garland Science, NY and London, 1 999) • Storage tank 60: amplification mixture (deoxyribonucleoside triphosphate (TP), primer, buffer), and • storage tank 62: DNA polymerase. Cover 40 and CMOS + MST layer 48 are in fluid communication via respective openings in lower seal 64 and top layer 66. The upper conduit 96 and the lower conduit 92 are representative of whether the fluid flows from the MST passage 90 to the cover passage 94 or vice versa. -70-201211538 L O C Device Operation The operation of the LOC device 301 is described step by step with reference to the analysis of pathogenic DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using a suitable kit or combination of reagents, test protocols, LOC variants, and detection systems. Referring to Figure 4, the analysis of a biological sample involves five main steps, including: sample input and preparation 28 8. 〇 nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294 ° sample input and preparation steps 2 8 8 The blood and anticoagulant 1 16 are mixed and then the pathogen is separated from the white blood cells and red blood cells by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, blood samples enter the device via sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood stream opens its surface tension valve 118, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants prevent the formation of blood clots that can block 〇 flow. As best shown in Figure 22, the anticoagulant 116 is withdrawn from the reservoir 54 by capillary action and enters the MS T channel 90 via the lower conduit 92. The lower duct 92 has a capillary action initiation feature (CIF) 102 to form a meniscus geometry that is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent 122 in the upper seal 82 allows air to replace the anticoagulant 1 16 . The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 is filled with a surface tension valve 118 and fixed to the bend of the upper conduit 96 - 71 - 201211538 The liquid level 120 is at the meniscus holder 98. Prior to use, the meniscus 120 remains fixed to the upper conduit 96 such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the lid channel 94 to the upper tube 96, the meniscus 120 is removed and the anticoagulant is drawn into the fluid. Figures 15 through 21 show an inset AE which is a portion of the inset AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 118 has three separate MST passages 90 extending between the 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. When the sample mixture and reagents are mixed by diffusion, the flow rate away from the sump determines the concentration of the reagent in the sample stream. Thus, the surface tension valve of each sump is configured to meet the desired reagent concentration. Blood is passed to the pathogen dialysis section 70 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of orifices 1 64 sized according to a predetermined valve. Cells smaller than the valve stomata pass through the orifice, while large cells cannot pass through the orifice. As the target cells continue to be part of the analysis, the undesired cells are reintroduced into the waste unit 76. Undesired cells are large cells that are blocked by an array of orifices 1 64 or small cells that pass through the orifice. In the pathogen dialysis section 70 described herein, the pathogen from the whole blood sample is concentrated for microbial DNA analysis. The array of orifices is formed by fluidly communicating the input in the lid channel 94 to a plurality of 3 micron diameter orifices 164 of the target channel 74. The 3 micron diameter orifice 164 and the dialysis suction orifice 168 for the target channel 74 are connected by a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The pathogen is small enough to pass through the dialysis M S T-72-201211538 channel 204 through the 3 micron diameter orifice 164 and to fill the target channel 74. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 of the lid 46, which leads to the waste reservoir 76 (see Figure 7). Other orifice shapes, sizes, and aspect ratios can be used to isolate specific pathogens or other target cells, such as white blood cells for human DN A analysis. More detailed details of the dialysis section and dialysis variants are provided later. Referring again to Figures 6 and 7, fluid is drawn through target channel 74 to surface tension valve 128 in helium lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending between the lysis reagent reservoir 56 and the target channel 74. When the meniscus is removed from the sample stream, the flow rate of all seven MST channels 90 will be greater than the flow rate of the anticoagulant reservoir 54, wherein the surface tension valve 118 has three MST channels 90 (assuming the physical properties of the fluid are approximately equal) . Thus the proportion of lytic reagent in the sample mixture is greater than the ratio of anticoagulant. The lysis reagent and the target cells are mixed by diffusion in the target channel 〇 74 in the chemical lysis unit 130. Boiling the pilot valve 126 stops the flow until diffusion and lysis are performed for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). Referring to Figures 31 and 32, the construction and operation of the boiling pilot valve will be described in detail below. Other active valve types (as opposed to passive valves, such as surface tension valve 118) have also been developed by the applicant, which can be used in place of the boiling pilot valve. These alternative valve designs are also described below. When the boiling pilot valve 1 2 6 is turned on, the lysed cells flow into the mixing portion 131 to pre-amplify restriction digestion and linker ligation. -73- 201211538 Referring to Figure 13, when the fluid removes the meniscus on the surface tension valve 132 at the beginning of the mixing portion 131, the restriction enzyme, linker and ligase are released from the reservoir 58. For diffusion mixing, the mixture flows through the length of the mixing section 1 31. At the end of the mixing portion 131 is a lower duct 134 leading to the incubator inlet passage 133 of the culture portion II4 (see Fig. I3). The incubator inlet channel 133 feeds the mixture into the heated microchannel 2 1 蜿蜒 structure, which provides a culture chamber for retaining the sample during restriction enzyme cleavage and junction ligation (see Figures 13 and 14) .

Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 within the inset AB of Figure 6. The figures show successive additions to form layers of the CMOS + MST layer 48 and cover 46 structures. The inset AB shows the end of the culture portion 1 14 and the start of the amplification portion 1 12 . As best shown in Figures 14 and 23, the fluid fills the microchannel 210 of the culture portion 14 until it reaches the boiling pilot valve 106, where the fluid stops when diffusion occurs. As discussed above, the microchannel 210 upstream of the boiling pilot valve 106 becomes a culture chamber containing a sample, restriction enzymes, zymase, and linker. The heater 154 is then activated and maintained at a steady temperature to cause the restriction enzyme shear and junction bonding to occur for a specific period of time. Those skilled in the art will appreciate that this culturing step 29 1 (see Figure 4) is arbitrary and is only required for some types of nucleic acid amplification assays. 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. The temperature is increased to inactivate the limiting enzyme and ligase before entering the amplification unit 1 1 2 . Limiting the inactivation of enzymes and ligases has a specific effect when amplified with isothermal acid. After the incubation, the boiling pilot valve 106 is activated (opened) and the fluid is re-introduced -74-201211538 into the amplification section 112. Referring to Figures 31 and 32, the mixture is filled with a structure of microchannels 158 until it reaches the boiling pilot valve ,8, which forms one or more amplification chambers. As best shown in the cross-sectional view of Fig. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and the polymerase is then released from the storage tank 62 to enter the junction culture section and the amplification section (114 and 112, respectively). The middle MST channel 212. Figures 35 through 51 show the layers of the LOC device 301 in the inset AC of Figure 6. 0 The figures show successive layers of layers forming the CMOS + MST device 48 and cover 46 structures. The inset A C shows the end of the amplification unit 1 1 2 and the start of the hybridization and detection unit 52. The cultured sample, amplification mixture, and polymerase flow through the microchannel 1 5 8 to the boiling pilot valve 1 〇 8. After diffusion mixing for a sufficient time, the heater 154 in the microchannel 158 is activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification procedure, the boiling pilot valve 108 is opened and the fluid re-enters the hybridization and detection portion 52. The operation of the boiling pilot valve is described in more detail below. The hybridization and detection unit 52, as shown in Figure 52, has an array 110 of hybridization chambers. Figures 52, 53, 54 and 56 show the hybridization chamber array 11 and the individual hybrid chambers 180 in detail. The entrance to hybridization chamber 180 is a diffusion barrier 175 that prevents the target nucleic acid, probe strands, and hybridization probes from diffusing between hybridization chambers 180 during hybridization to prevent erroneous hybridization assay results. The flow path of the diffusion barrier 175 is long enough to prevent the target sequence and probe from diffusing out of one chamber and contaminating another during the time the probe and nucleic acid hybridize and detect the signal, thus avoiding erroneous results. -75- 201211538 Another mechanism to prevent erroneous reading is to have the same probe in some hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 184 corresponding to the hybridization chamber 18 containing the same probe. The results of the exceptions in the derived single results can be ignored or given different weights. The thermal energy required for hybridization is provided by a CMOS controlled heater 182 (described in more detail below). Hybridization occurs between the complementary standard probe sequences after the heater is activated. The LED driver 29 in the CMOS circuit 86 transmits a message to cause the LEDs 26 located in the test module 10 to illuminate. These probes only fluoresce when hybridization occurs, thereby eliminating the cleaning and drying steps that are often required to remove unbound strands. The hybrid forced F R E T probe 186 stem and loop structure opens 'which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as detailed below. Fluorescence is detected by photodiode 184 located in CMOS circuit 86 under each hybridization chamber 180 (see the description of the hybridization chamber below). Photodiodes 184 and associated electronics for all hybrid chambers together form 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 180 is amplified and converted to a digital output that can be analyzed by the test module reader 丨2. Further details of the detection method are described below. Other Detailed Description of the LOC Device The modular design LOC device 301 has a number of functional components including reagent reservoirs 5 4, 5 6 , 58, 60 and 62, dialysis section 70 lysate 130, culture section 114 and amplification Department 1 1 2, valve type, humidifier and humidity sensor. In other LOC devices of the example -76-201211538, the functional portions may be omitted, but another functional portion or a functional portion different from the use of the above-described device may be added. For example, the culture portion 1 14 can be used as the first amplification portion 112 of the tandem amplification analysis system, and the lysis reagent reservoir 56 can be used to add the first amplification mixture of the primer, the dNTP, and the buffer, and the reagent storage tank is used. 58 to add reverse transcriptase and / or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 5 6 or, alternatively, the sample may be heated for a predetermined period of time to cause thermal lysis in the culture section. . In some embodiments, if chemical lysis is desired and the chemical lysis reagent is mixed therewith, additional reservoirs can be combined upstream of the sump 58 for mixing the primers, dNTPs, and buffer. In some cases, steps such as incubation step 291 are omitted. In this case, the LOC device can be specially manufactured to avoid the reagent storage tank 58 and the culture portion 114 or the storage tank can carry only the reagent, or when there is an active valve, it is not activated to dispense the reagent into the sample flow, and the culture portion It is simply a channel for transferring the sample from the lysis unit 130 to the amplification unit 112. The heaters operate independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in L〇C devices that do not require hot lysis. The dialysis section 7 can be located at the beginning of the fluid system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, prior to the amplification phase 292, prior to the 'hybridization and detection step 294, dialysis is performed to remove cell debris. Alternatively, two or more dialysis sections can be combined at any location on the L0C 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 hybridization chamber arrays -77 - 201211538 11 . 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 for the LOC device to omit the dialysis section 70. If the presence of the dialysis section does not cause geometrical obstruction, the sample input and preparation section can still have the LOC of the dialysis section 7 without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to a protein present in the non-amplified sample, rather than being designed to A nucleic acid probe that hybridizes to a target nucleic acid sequence. It will be appreciated that the LOC devices manufactured for use with this diagnostic system are different from the combinations of functional components selected for the particular LOC application. The vast majority of functional units are common to many LOC devices and the design of additional LOC devices for new applications has functional components that are appropriately combined in the configuration of the large functional units 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 other LOC designs are associated with a combination of suitable functional components. Sample Type LOC Variants Accept and Analyze Nucleic Acids or Protein Contents of Various Sample Types in Liquid Form 'Liquid Forms include, but are not limited to, 'Blood and Blood-78-201211538 Liquid Products, Saliva, Cerebrospinal Fluid, Urine, Semen Amniotic fluid, cord blood, breast milk, sweat, pleural effusion, tears, pericardial fluid, peritoneal fluid, environmental water samples and dips. Amplicon derived from meganucleic acid amplification can also be analyzed using a LOC device; in this case, all reagent storage tanks will be empty or the constitutive composition will not release its contents, and only dialysis, lysis, The culture and amplification section delivers the sample from the sample inlet 68 to the hybridization chamber for nucleic acid detection 180, as described above. 0 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 10. 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 Tanks A microfluidic device, such as LOC unit 301, is used to analyze the system with a small amount of reagent that allows the reagent reservoir to contain all of the necessary reagents for biochemical treatment, and each reagent reservoir is in a small volume. This volume is indeed less than 1, 〇〇〇, 〇〇〇, 〇〇〇 cubic micron, in most cases less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns, and the LOC device 301 shown in the figures In the case of less than 20,000,00 cubic microns. -79- 201211538 Dialysis Department Referring to Figures 15 to 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate the pathogen target cells from the sample. As previously described, the top layer 66 has a plurality of orifices 316 of a diameter of 3 microns that filter the target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice 1 64, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via a 16 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. Foam illustrations or other porous elements 49 in the outer casing 13 of the test module 10 are in fluid communication with the waste reservoir 76 for the type of biological sample that produces a substantial amount of waste (see Figure 1) ° pathogen dialysis The Department 70 series operates with the capillary action of the fluid sample. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Figure 33) so that fluid is drawn down into the dialysis MST channel 204 below. The first extraction aperture 198 for the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus over the dialysis extraction aperture 168. The small component dialysis section 682, which is schematically shown in Fig. 88, may have a structure similar to the pathogen dialysis section 70. The small component dialysis section separates any small standard cells or molecules from the sample by sizing (and shaping, if necessary) to accommodate orifices that allow 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 the waste reservoir 766. Since -80-201211538, the LOC device 30 (see Figures 1 and 133) is not limited to isolation of pathogens having a size of less than 3 μηη, but can be used to separate cells or molecular lysates of any desired size. Referring again to Figure 7, 1 1 and 1 3, by chemical lysis, the genetic material in the sample is released from the cell. As described above, the lysate G reagent from the lysis tank 56 is mixed with the sample flowing in the target passage 74 downstream of the surface tension valve 182 of the lysis tank 5. However, some diagnostic assays preferably use hot lysis treatment, or even a combination of chemical and thermal lysis of target cells. The LOC device 301 accommodates the heated microchannel 2 1 0 of the culture portion 1 1 4 . The sample stream is flooded with the culture portion 1 14 and stopped at the boiling pilot valve 106. The culture microchannel 210 heats the sample to a temperature at which the cell membrane ruptures. In some hot lysis applications, the enzyme reaction is not required in the chemical lysis unit 130, and the hot lysis completely replaces the enzyme reaction in the chemical lysis unit 130. Boiling Pilot Valve As discussed above, LOC unit 301 has three boiling pilot valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling pilot valve 108 on the heated microchannel 158 side of the amplifying portion 112. By capillary action, the sample stream 119 is attracted along the heated microchannel 158 until it reaches the boiling pilot valve 1〇8. The meniscus 120 at the leading edge of the sample flow is fixed to the meniscus holder of the valve inlet 146. The meniscus holder 98 is slightly delayed. -81 - 201211538 How to stop the meniscus from moving forward to prevent capillary flow. As shown in Figures 31 and 32, the meniscus holder 98 is a conduit on the orifice provided by the upper opening of the pipe from the MST passage 90 to the cover passage 94. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 152 is located around the valve inlet 146. The loop heater 152 is CMOS controlled by boiling the valve heater contact 153. To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater contact 153. The ring heater 152 is resistively heated until the liquid sample 119 is boiled. Boiling removes meniscus 1 20 from valve inlet 1 46 and begins to wet cover passage 94. Once the wet cover channel 94 is started, the capillary action is restored. The fluid sample 119 is filled with the passage 94 and flows through the sub-valve conduit 150 to the valve outlet 148, wherein the capillary driven liquid flow advances along the expansion 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 appreciated that once the boiling pilot valve is opened, it is not possible to close again. However, since the LOC device 301 and the test module 1 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 114 and the amplification unit 112. The culture portion II4 has a single, heated culture microchannel 210, which is etched to form a ruthenium pattern in the MST channel layer 100 from the lower conduit opening 134 to the boiling pilot valve 106 (see Figure and 14). . Controlling the temperature of the culture portion 1 14 enables a more efficient enzyme reaction. In the same manner as the -82-201211538, the amplifying portion 112 has a heating microchannel 158 (see Figs. 6 and 14) which is heated from the boiling pilot valve 106 to the boiling pilot valve 108. The valves stop flow to retain the target cells in the heated culture or amplification microchannels 210 or 158 during mixing, culture, and nucleic acid amplification. The microchannel 蜿蜒 pattern also promotes (to some extent) the target cells to mix with the reagents. In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated via a heater 154 controlled by a CMOS circuit 86 whose pulse width modulation (PWM) is used. 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 FIG. Μ)), It provides two-dimensional control of the input heat flux density. As best shown in Figure 51, the heater 154 is supported on the top layer 66 and buried in the lower seal 64. The heater material is TiAl, but many other conductive metals are also suitable. The elongated heater 154 is parallel to the longitudinal length of each of the channel portions forming the meandering meandering. In the amplification unit 112, each of the wide meanders can be operated as an independent PCR chamber via individual heater control. The use of a microfluidic device, such as LOC device 301, requires a small volume of amplicons required for the analysis system to allow amplification of the small volume of amplification mixture in amplification portion 112. This volume is less than 400 nanoliters, in most cases less than 170 nanoliters, typically less than 70 nanoliters, and in the case of LOC device 301, this volume is between 2 nanoliters and 30 nanoliters. . Increased heating rate and better diffusion mixing -83 - 201211538 The small cross-sectional area of each channel section 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 1 〇〇, 〇〇〇 square micron' and has a significantly higher heating rate than the "large scale" device. The lithography manufacturing technique allows the amplifying microchannels 158 to have a cross-section that provides a higher heating rate across substantially less than 1 6,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small amount of amplicons are required (as is the case in LOC unit 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1,000 to 2,000 probes on a LOC device and "sample in, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and 1 square micron. between. The heater element in the amplification microchannel 1 58 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 κ 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 0000 K per second. Typically, based on the requirements of the analytical system, the heater elements are greater than 100 per second, 〇〇〇K, greater than 1,000,000 K per second, greater than 10,000,000 K per second, greater than 20.000. 000 K per second, greater than 40,000,000 K per second, per second. The nucleic acid sequence is heated at a rate greater than 80.000. 000 K and greater than 1 60,000,000 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 phenomenon occurs when the density is -84- 201211538 degrees and decreases with distance from the interface. The microfluids ‘crossing the direction of the fluid with a relatively small cross-sectional area' are used to keep the two fluids flowing through the interface for rapid diffusion mixing. Reducing the channel cross-section to less than 100, 〇〇〇 square microns results in a significantly higher diffusion rate than "large-scale" devices. The lithography manufacturing technique allows the microchannels to have cross sections that traverse less than 16,000 square microns and substantially provide a higher mixing rate. If only a very small amount of amplicons are required (as is the case in LOC unit 301), the cross section can be reduced to less than 2,500 square microns. Acupuncture for diagnostic analysis of 1,000 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 short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid, resulting in rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e., denaturation 黏, 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 between 0.45 seconds and 1.5 seconds. This rate of thermal cycling allows the test module to complete the nucleic acid amplification procedure in less than 10 minutes; often within 2,200 seconds. For most analyses, the amplification section produces sufficient amplicons from the sample fluid entering the sample inlet within 80 seconds. For most of the analysis, sufficient amplicons were generated within 30 seconds. Upon completion of the predetermined number of amplification cycles, the -85-201211538 amplicon is fed to the hybridization and detection section 52 via the boiling pilot valve 1〇8. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chambers 180 in the hybrid chamber array 11〇. Hybridization and detection portion 52 has a 24 X 45 array 110 of hybridization chambers 180 each having a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. Photodiode 1 84 is incorporated to detect camp light from a target nucleic acid sequence or protein that hybridizes to FRET probe 186. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. For the emitted light, any material between the FRET probe 186 and the photodiode 184 must be transparent. Therefore, the wall portion 7 7 between the probe 186 and the photodiode 1 8 4 must also be optically transparent to the emitted light. In LOC device 301, wall portion 97 is a thin layer of hafnium oxide (about 0.5 microns). Direct intrusion of photodiode 184 beneath each hybridization chamber 180 allows the use of a very small volume of probe-target hybridization while still producing a detectable fluorescent signal (see Figure 54). A small volume of hybridization chamber can be used because of the small amount. Prior to hybridization, the amount of probe required for detectable probe-target hybridization is greater than 270 pg (?4〇§1^111) (corresponding to 900,000 cubic microns), which is less than in most cases. 60 picograms (corresponding to 200,000 cubic micrometers) 'normally less than 12 picograms (corresponding to 4 inches, 000 cubic micrometers), and less than 2.7 picograms in the case of L Ο C device 301 shown in the drawing (corresponds to a chamber with a volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes on the LOC device. In the LOC device 301, the -86-201211538 hybrid has more than one chamber (i.e., less than 2,250 square micrometers per chamber) in an area of 1,500 micrometers by 1,500 micrometers. 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 dispensed into each chamber. A specific embodiment of the LOC device according to the present invention may be configured with a probe solution of 1 nanoliter or less. After nucleic acid amplification, the boiling pilot valve 108 is activated and the amplicon flows along the zero flow path 176 and into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates when the hybridization chamber 180 is flooded with the amplicon and the heater 182 can be activated. After sufficient hybridization time, LED 26 is activated (see Figure 2). The opening in each hybrid chamber 180 is provided with an optical window 136 to expose the FRET probe 186 to the excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a sufficiently long period of time to induce a high intensity fluorescent signal from the probe. The photodiode 184 is shorted during excitation. After a preprogrammed delay of 300 (see Figure 2), the photodiode 1 84 is enabled and the fluorescent emission is detected in the absence of 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 a CMOS circuit 86. Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. If desired, each hybridization chamber 180 can carry probes that detect more than 1,000 different targets. Alternatively, many or all of the hybridization chambers may carry the same probe that repeatedly detects the same target nucleic acid. Copying the probe in this manner in the hybrid chamber array 11 使得 increases the confidence of the results obtained, and if desired, the results can be combined by the photodiodes of adjacent hybridization chambers to obtain a single junction -87-201211538 fruit. Those skilled in the art will appreciate that there may be from 1 to over 1, 00 different probes on the hybrid chamber array 1 10, depending on the analytical details. Humidifier and Humidity Detector The inset AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of evaporation of reagents and probes during operation of the LOC device. As best shown in the enlarged view of Fig. 55, the water sump 188 is fluidly coupled to three evaporators 1 90. The water storage tank 18 8 is filled with molecular bio-grade water and is sealed during manufacture. As best shown in Figures 55 and 67, by capillary action, water is drawn to the three lower conduits 194 and along the individual water supply passages 192 to the three upper conduits 193 of the evaporator 190. The meniscus is fixed to each of the upper pipes 193 to retain water. The evaporator has a ring heater 191 that surrounds the upper pipe 193. With a thermally conductive post 376, a ring heater 191 is coupled to the CMOS circuit 86 to the top metal layer 195 (see Figure 37). At startup, the ring heater 197 heats the water causing the water to evaporate and wet the surrounding equipment. The position of the humidity sensor 232 is also shown in FIG. However, preferably, as shown in the enlarged view of the illustration 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 constant of the air gap between the electrodes increases, causing the capacitance to increase. The humidity sensor 2 3 2 abuts the hybridization chamber array 1 1 〇 (mostly the humidity measurement location) to slow the evaporation of the solution containing the exposed probe. -88- 201211538 Feedback Sensors Temperature and liquid sensors are incorporated into the LOC unit 3 0 1 to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors 170 are assigned to all of the amplification unit 1 1 2 . Similarly, the culture unit 1 14 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to the CM〇S circuit Ο 86 . The CMOS circuit 86 utilizes this to accurately control thermal cycling during nucleic acid amplification as well as thermal lysis and any heating during incubation. In hybridization chamber 180, CMOS circuit 86 uses hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of the hybrid heater 182 is temperature dependent and the CMOS circuit 86 utilizes this to drive the temperature reading of each of the hybrid chambers 180. The LOC device 301 also has a number of MST channel liquid sensors 174 and a cover channel liquid sensor 208. Figure 35 shows the line of the MST channel liquid sensor 174 at one end of each of the spaced turns in the heated microchannel 158. Preferably, as shown in FIG. 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the current between the electrodes to indicate their presence at the sensor. Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. The opposite electrode pairs 218 and 220 are deposited on the top layer 66. A gap 222 is provided between the electrodes 218 and 22 0 to keep the circuit open in the absence of liquid. The circuit is closed when the liquid is present and the CMOS circuit 86 utilizes this feedback to monitor the flow. -89 - 201211538 The GRAVITATIONAL INDEPENDENCE test module 10 is self-directed. It does not need to be fastened to a smooth surface to operate. The fluid flow driven by capillary action and the lack of an external conduit to the auxiliary device make the module truly portable and can be easily inserted into a similar portable handheld reader, such as a mobile phone. The gravity autonomous operation represents that the test module is also acceleration independent of all practical ranges. It is shock and vibration resistant and can be operated on a moving carrier or on a mobile phone that is carried. Dialysis Variants A dialysis section having channels to avoid trapped bubbles. The following is a specific embodiment of the LOC device of LOC variant VIII 5 18 shown with reference to Figures 72, 73, 74 and 75. This LOC device has a dialysis section that is trapped in a fluid sample and trapped in a channel without bubbles. LOC Variant VIII 518 also has an additional layer of material, with reference 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. Interfacial layer 5 94 causes a more complex fluid interconnection between the reagent sump and MST layer 87 without increasing the size of ruthenium substrate 84. Referring to Figure 73, the bypass channel 600 is designed to introduce a time delay into the fluid sample from the interface waste channel 604 to the interface target channel 6〇2. This time delay causes the fluid sample to flow through the dialysis MST channel 204 to the dialysis draw 168 of the fixed meniscus. The capillary action initiation feature (CIF) of the bypass channel 600 to the interface target channel 602 at the upper conduit 202' is the point upstream of all dialysis draws 168 of the dialysis MS T channel 2〇4. -90- 201211538 Surface target channel 602. Without the bypass channel 600, the interface target channel 602 still begins to charge from the upstream end, but eventually, the traveling meniscus arrives and passes through the untwisted MST channel above the pipe, leading to the point capture air. The trapped air reduces the sample flow rate through the white blood cell dialysis section 328.

Nucleic Acid Amplification Variants ◎ Parallel PCR Many variants of LOC devices have multiple amplifications operating in parallel. For example, the LOC variant VII 492 shown in Fig. 71 has parallel amplification sections 112.1 to 112.4 which allow simultaneous multiplex amplification analysis. The LOC variant XI 746 shown in Fig. 116 also has a parallel amplification section 112.1. Up to 112.4, but with additional parallel cultures 114.1 through 114.4, so that the samples can be processed differently before amplification. Other LOC variants, such as LOC XIV 641 shown in Figure 78, show that the number of parallel amplifications 〇 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. The respective amplification sections can be grouped to perform at different cycle times and/or temperatures for a particular target size or a particular amplification mixing component. When a plurality of amplification sections are performed in parallel, the LOC apparatus can operate a multiplex nucleic acid amplification process or a single-uniform 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 n-fold amplification, where n = n ( 1 ) + η ( 2 ) + ... + η ( i ) + ... + η ( m ) And n(i) is -91 - 201211538 for a different number of primer pairs for multiplex amplification performed in chamber "i", to know the SNR (signal-to-noise ratio) in a parallel amplification system Greater than the SNR of the η reamplification performed in a single chamber system. In the special case of n(i) =1, the amplification in chamber "i" becomes only a single amplification.

Tandem PCR (in which) Figures 79, 82, 85, 86, and 87 outline the LOC devices in which the amplification units 1 12. 1 and 12.2 are operated in series. The first amplification section 1 12.1 comprises two reagent reservoirs, 60.1 for amplification mixing and 62.1 for polymerase. Each of the amplification sections added after the start portion also includes two reagent storage tanks, and the storage tank 60.2 is used for amplification mixing and storage tank 62.2 for the polymerization enzyme. The amplification in series allows for tandem PCR analysis whereby the first amplification portion 112.1 is used for pre-amplification to increase the sensitivity of subsequent nucleic acid amplification performed by portion 112.2. The amplification unit in series can also be used for nested PCR reactions. In tandem PCR for pre-amplification, the first amplification portion 112.1 is used to amplify a nucleic acid sequence in a sample comprising a target sequence. This amplification does not require specificity for the target sequence (e. g., whole genome amplification), but still increases the target sequence concentration. After pre-amplification, the sample is mixed with reagents from reservoirs 60.2 and 62.2. The sample then enters the second amplification section 1 1 2.2. The reagents stored in sump 60·2 include specific primers to amplify only the target sequences in the pre-amplified sample mix. It should be noted that a similar method in which P C R is replaced by a thermostatic technique in the first or second amplification stage can also be employed to achieve advantageous pre-amplification. Nested PCR is a special type of tandem continuous PCR with the added benefit of high target -92 - 201211538 specificity. In the nested PCR, in the nucleic acid amplification step of the first amplification unit 12.1, by using the primer to form an amplification mixing reagent partially stored in the storage tank 6 0.1, the amplification is greater than the final target sequence. A sequence complementary to the outer region of the target sequence. The amplicon obtained by the reaction in the first amplification section 1 1 2 · 1 is the target sequence plus the adjacent portion. This amplified mixture was mixed with reagents from storage tank 60.2 and polymerase from 62J. The reagents stored in sump 6 0.2 include a sub-portion complementary to the position of each end of the target sequence, i.e., the amplicon obtained from the first amplification step. When nucleic acid amplification is performed in the second portion 12.2, the chance of amplification of the sequence position unrelated to the target is greatly reduced, and the concentration of the amplicon obtained from the first amplification stage is much larger than the concentration of the original sample molecule. The sensitivity and specificity of nested PCR can also be achieved when one or two PCR amplification stages are replaced by sequence-specific constant temperature amplification techniques. The advantage of separately storing the polymerases and adding them separately to the sample mix is that different polymerases can be selected for the pre-amplification and final nucleic acid amplification steps. For example, this allows for the selection of low error rate (eg, calibration) for the pre-amplification step to avoid the generation of target nucleic acids with false or pseudo-target sequences, while still allowing for higher speed or thermal tolerance. The more highly potent polymerase is finally amplified.

Direct PCR Traditionally, PCR requires extensive purification of target DNA prior to preparation of the reaction mixture. However, by appropriately changing the chemical and sample concentrations, nucleic acid amplification can be performed using a minimum amount of DN A purification, or direct amplification can be performed. When nucleic acid amplification is performed with -93 - 201211538 PCR, this method is called direct PCR. When nucleic acid amplification is carried out at a normal temperature controlled in a LOC apparatus, this method is direct constant temperature amplification. Direct nucleic acid amplification techniques have considerable advantages when used in LOC devices, especially with regard to the simplification of the desired fluid design. Amplification chemical adjustments for direct PCR or direct isothermal amplification include increasing buffer strength, using highly active and highly progressive polymerases, and additions to potential polymerase inhibitors. It is also important to dilute the inhibitor in the sample. To exploit direct nucleic acid amplification techniques, LOC devices are designed to incorporate two additional features. The first feature is a reagent reservoir (eg, sump 58 in Figure 8) that is appropriately sized to supply a sufficient amount of amplification reaction mix or diluent such that the final concentration of sample components that may affect the amplification chemistry is sufficient Low to successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LOC structure, such as the pathogen dialysis section 70 of Figure 4, is used. In this particular embodiment (further illustrated in Figure 6), the concentration of the pathogen sufficient to enter the amplification portion 292 is effectively concentrated upstream of the sample extraction portion 290 and the larger cells are discharged to the waste container 76. Dialysis department. In another embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of 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, design the length of the sample and reagent channels to -94-201211538 cross section 'to make the mixing start position The upstream sample channel constitutes a flow group that is 4 to 19 times more resistant to flow than the flow of the reagent mixture. The flow group resistance in the microchannel is easily controlled by controlling the design. The flow group resistance for 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 depends more on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannels of square cross-section is inversely proportional to the cube of the smallest vertical dimension. ❹ Reverse transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RN A, such as from RNA virus or messenger RNA, RNA must be reversed as complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction (one-step RT-PCR) can be carried out in the same chamber as the PCR, or it can be a separate initial reaction (two-step RT-PCR). In the LOC variant described herein, the reverse transcription step and the subsequent step of performing the nucleic acid amplification step by adding the reverse transcriptase and the polymerase to the reagent storage tank 62 and the stylized heater 154 can be simply implemented. Step RT-PCR. The buffer, the primer, the dNTP, and the reverse transcriptase are stored and distributed by the reagent storage tank 58, and the culture unit 14 is used for the reverse transcription step, and then amplified in the amplification unit 112 in a normal manner. The two-step RT-PCR is simply done. Constant temperature nucleic acid amplification For some applications, the preferred nucleic acid amplification method is constant temperature nucleic acid amplification, so that it is not necessary to repeatedly circulate the reaction components at various temperature cycles, but the -95-201211538 amplification portion is maintained at normal temperature, usually About 37 ° C to 4 plant C. Some methods for thermostatic nucleic acid amplification have been described, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASB A ), recombinant enzyme polymerase amplification (RPA), and 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 type can be specifically used for LOC herein. In a specific embodiment of the device. To perform a constant temperature nucleic acid amplification, reagent reservoirs 6 and 62 adjacent to the amplification section will carry appropriate reagents for a particular constant temperature method rather than carrying PCR amplification and polymerase. For example, for S D A, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reagent reservoir 62 contains appropriate endonucleases and exo-DNA polymerases. For RPA, reagent reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins, and reagent reservoir 62 contains strand-substituted DNA polymerase, such as. Similarly, for HDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reservoir 62 contains the appropriate DNA polymerase and helicase (rather than 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 the amplification of viral nucleic acids from RNA viruses, such as HIV or hepatitis C virus, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDNA. In this example, the reagent reservoir 60 is filled with amplification buffer, primers, and dNTPs, and the reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase H. For some thermostatic nucleic acid amplification types, an initial denaturation cycle must be employed to separate the two-strand DN A template before maintaining the temperature of the thermostated nucleic acid amplification at -96 - 201211538 for continued reaction. This denaturation cycle can be easily accomplished in all of the specific embodiments of the LOC device described herein because the temperature of the mixing in the amplification portion 112 can be tightly controlled by the heaters 15 in the amplification microchannel 158. I4) ° Constant temperature nucleic acid amplification is more tolerant to potential inhibitors in the sample and is therefore generally suitable for direct nucleic acid amplification from a desired sample. Therefore, constant nucleic acid amplification is particularly useful for LOC variant XLni 673, LOC variant XLIV 674 and LOC variant XLVII 677 shown in Figures 89, 90 and 91, respectively. Direct thermostatic amplification can also be combined with one or more of the preamplification dialysis steps 70, 686 or 682 as shown in Figures 89 and 91 and/or the pre-hybridization dialysis step 682 as shown in Figure 90, To facilitate partial concentration of the target cells in the sample prior to nucleic acid amplification, respectively, or to remove unwanted cell debris before the sample enters the hybridization chamber array 1 1 . Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be used.恒温 It is also possible to carry out constant temperature nucleic acid amplification in parallel amplification sections 'such as those described in Figures 7 1 '76 and 78'. Multiplex and some thermostatic 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 be combined with a CMOS controlled flow rate sensor 740' as illustrated in Figure 132 and LOC Variant X 728 ( See Figures 99 to 115). These sensors are used to determine the flow rate in the two steps -97- 201211538. In the first step, the temperature of the xenon heater element 814 is determined by applying a low current and measuring the voltage to determine the resistance of the xenon heater element 814, and thus using the resistance between the heater element and the temperature 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 蜿蜒 heater element such that the temperature of element 814 increases and some of the heat is lost by the flowing liquid. At the same time as the higher voltage is applied, the new resistance of element 814 is determined by again measuring the voltage across element 814 and the increased temperature is again calculated by CMOS circuit 86. The flow rate of the liquid is determined using the new temperature of the crucible heater element 8 1 4 and the known sample liquid temperature 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 23 6 prior to hybridization to the target nucleic acid sequence 2 3 8 . The probe has a ring 240, a stem 2 42 , a fluorophore 246 at the 5' end, and a quencher 248 at the 3' end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are bonded together to form stem 242. In the absence of a complementary target sequence' as shown in Figure 58 the probe remains closed. The stem 242 maintains the fluorophore-quenching agent relatively close to each other such that the resonance energy of the amount of -98*201211538 can be transmitted between each other, and substantially eliminates the fluorescing fluorophore when irradiated with the excitation light 244. ability. Figure 59 shows FRET probe 236 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 23 8 , the stem-and-loop structure is disrupted, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to fluoresce. Optically detecting the ground fluorescence emission 250 as an indicator that the probe has been hybridized. The probe hybridizes with the complementary target with extremely high specificity, since the stem helix of the probe is designed to be a probe with a single non-complementary nucleotide. - The target spiral is stable. Due to the relatively strong double-stranded DNA, it is impossible for the probe-target helix and the stem helix to coexist. Primer-Linked Probe Primers - Linked Stem-and-Ring Probes and Primer-Linked Linear Probes, also known as Spider Probes, are a substitute for molecular beacons and can be used for LOC loading Instant and 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 that only a single hybridization is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an immediate and efficient response and produces a stronger signal, shorter reaction times, and better discrimination when using separate primers and probes. During manufacture, the probe (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 18 without the need for a separate amplification portion on the LOC device. Alternatively, the amplification portion is not used or used for other reactions. -99 - 201211538 Primer-Linked Linear Probes Figures 92 and 93 show the primer-linked linear probe 692 during the first round of nucleic acid amplification and the hybridization configuration during subsequent nucleic acid amplification, respectively. Referring to Figure 92, the primer-coupled probe 692 has a double stem section 242. One of the probe sequences 696, which binds to the primer, is homologous to the region on the target nucleic acid 696 and is labeled with the fluorophore 24 6 at its 5' end, and its 3' end is linked via the amplification blocker 694. To the oligonucleotide primer 700. The 3' end of the other strand of stem 2 42 is labeled with quenching portion 248. After completion of the first round of nucleic acid amplification, using the currently complementary sequence 698, the probe can surround and hybridize to the extended strand. During the first round of nucleic acid amplification, the oligonucleotide primer 7 is adhered to the target DNA 238 (Fig. 92) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the reading of polymerase through and copying probe region 696. At the time of subsequent denaturation, the hybridization of the extended oligonucleotide primer 700/template and the primer-linked linear probe of the double stem 242 were separated, thereby releasing the quencher 24 8 . Once the temperature for the adhesion and extension steps is lowered, the primer-joined probe sequence of the primer-joined probe sequence is 296-curved and hybridized to the amplified complementary sequence 698 on the extended strand' and the detected fluorescence is indicated 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 94A through 94F show the operation of the primer-coupled stem-and-loop probe 704 -100-201211538. Referring to Figure 94A, the primer-ligated stem-and-loop probe 704 has a stem 242 of complementary double stranded DNA and a loop 240 of the combined probe sequence. The 5' end of one of the stem strands 70 8 is marked with a fluorophore 246. The other strand 710' is labeled with a 3'-end quencher 248 and the other strand 710 carries both an amplification blocker 694 and an oligonucleotide primer 700. In the initial denaturing phase (see Figure 94B), the strands of the target nucleic acid 238 and the introduced stem 242 are separated from the stem-and-loop probe 704. When the temperature is cooled for the adhesive phase (see Figure 94C), the oligonucleotide primer 7 of the primer-linked stem-and-loop probe 704 hybridizes to the target nucleic acid sequence 23 8 . During extension (see Figure 94D), the complement 70 6 of the target nucleic acid sequence 238 is synthesized to form a DN A strand containing both the probe sequence 704 and the amplified product. Amplification blocker 694 prevents the polymerase from reading through and copying probe region 704. After denaturation, the probe sequence of the loop-coupled stem-and-loop probe loop segment 240 (see Figure 94F) is adhered to the complementary sequence on the extended strand, when the probe is attached. This configuration causes the fluorophore 246 to be at a great distance from the quencher 248, resulting in a significant increase in fluorescence emission. ❹ Control Probes The hybridization chamber array 1 1 〇 includes some hybridization chambers 180 with positive and negative control probes for analytical quality control. Figures 128 and 129 illustrate negative control probes without fluorophore 796, and Figures 130 and 131 depict positive control probes without quencher 79 8 . 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. -101 - 201211538 Referring to Figures 128 and 129, the negative control probe 796 does not have a 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 129) or the probe maintains its stem-and-loop configuration (see Figure 128), the response to the excitation light 244 can be ignored. Alternatively, the negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing loop 240, a probe sequence will be obtained that will not hybridize to any of the nucleic acid sequences in the sample of interest, the stem 242 of the probe molecule will rehybridize with itself, and the fluorophore and quencher will Stay in close proximity and will not emit visible fluorescence. This negative control signal corresponds to a low order emission from hybridization chamber 180 where the probe is not hybridized but the quencher does not quench all of the emission from the reporter. Conversely, a positive control probe 798 without quenching is constructed, as shown in Figures 130 and 131. Back to the stress luminescence 244, whether or not the positive control probe 798 hybridizes to the target nucleic acid sequence 23, the no substance causes the fluorescent emission 250 from the fluorophore 246 to quench. Figure 52 shows the possible distribution of positive and negative control probes (378 and 380, respectively) in the hybridization chamber array 110. Control probes 378 and 380 are placed in hybridization chamber 180 and positioned across the line of hybridization chamber array 1 1 0. However, the configuration of the control probes within the array is arbitrary (as in the case of hybrid chamber arrays, the configuration of the fluorophores requires a fluorophore with a long fluorescence lifetime to allow the excitation light to have sufficient time to decay to a higher level. The intensity of the fluorescent emission of the photosensor 44 is as low as -102-201211538 degrees, thereby increasing the sufficient signal-to-noise ratio. And the longer fluorescent lifetime represents a larger integrated fluorescent photon. The fluorescent lifetime of the fluorophore 246 (see Figure 59) is greater than nanoseconds, often greater than 200 nanoseconds, more often greater than 300 nanoseconds, and in most cases greater than 400 nanoseconds. Or lanthanide metal-based metal-coordination complexes with long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high heat, chemical and photochemical stability, all of which are characterized by It is a favorable characteristic related to the requirements of the fluorescence detection system. The metal-coordination complex which is particularly thoroughly studied based on the transition metal ion ruthenium (Ru(II)) is the reference (2, 2'-linked Pyridine) ruthenium (II) ([Ru ( bpy ) 3] 2+ ), which has a lifetime of about 1 μ8. The compound is commercially available from Biosearch Technologies under the trade name Pulsar 650. Table 1: Photophysical properties of Pulsar 050 (钌 chelate) Parameter symbol 値 Unit absorption wavelength ^abs 460 nm One emission wavelength λ έ πι 650 nm Absorption coefficient Ε 14800 M4 cm·1 fluorescence lifetime Tf 1.0 lis a quantum yield Η 1 (deoxygenated) N/A monoterpene metal-coordination complex, ruthenium chelate, has been successfully shown as a FRET probe system Fluorescent reporter in medium with a long life of 1 600". -103- 201211538 Table 2: Photophysical properties of ruthenium chelate parameters Symbol 値 Unit absorption wavelength ^abs 330-350 nm Emission wavelength λβτη 548 nm Absorption coefficient Ε 13800 (hbs, and ligand-dependent, up to 30000 @ λβ=340ηιη) Mlcml fluorescence lifetime Tf 1600 (hybridization probe) μΞ quantum yield Η 1 (coordination dependent) Ν/Α LOC device 301 The fluorescent detection system used does not utilize filtering to remove unwanted background fluorescence. If quencher 248 has no natural emission to increase the signal-to-noise ratio, it is therefore advantageous. Without natural emission, quenching Agent 248 does not contribute Fluorescence to the background. High quenching efficiency is also important, which results in no fluorescence before hybridization. The black hole quencher (BHQ) from Biosearch Technologies, Inc., Novato, Calif., does not have natural emission and has high quenching. Extinction efficiency, as well as suitable quenching agents for the system. The maximum absorption enthalpy of BHQ-1 occurs at 543 nm and the quenching range is 480-5800 nm, making it a suitable quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 579 nm and the quenching range is 560-670 nm, making it a suitable quencher for P s ar 650. Iowa Black F quenchers (Iowa Black FQ and RQ) from Integrated DNA Technologies, Coralville, Iowa, are suitable alternative quenchers with little or no background emission. The Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorption enthalpy at 531 nm and is therefore a suitable quencher for Tb-chelating fluorophores. Iowa Black RQ has a maximum absorption enthalpy at 656 nm and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 65 0 . -104- 201211538 In the specific embodiments described herein, the quencher 24 8 is a functional portion that is initially attached to the probe, but in other embodiments, the quencher may be free in solution. Molecule. Excitation Sources In the specific embodiment based on the fluorescence detection described herein, LEDs are selected to replace laser diodes, high power lamps, or laser excitation sources because of low power consumption, low cost, and small size. Referring to Fig. 95, LED 26 is directly placed on hybrid array array UO on the outer surface of LOC device 301. Opposite to the hybridization chamber array 110 is a photosensor 44 consisting of an array of photodiodes 184 from each chamber for detecting fluorescent signals (see Figures 53, 54 and 64). Other embodiments for exposing the probe to excitation light are outlined at 97 and 98. In the LOC device 30 shown in Fig. 96, the excitation light 2 44 generated by the excitation LED 26 is directed by the lens 254 over the hybrid cell array Ο 1 10 . The LED 26 is pulsed and the fluorescent emitter is detected by the photo sensor 44. In the LOC device 30 shown in FIG. 97, the excitation light 24 generated by the excitation LED 26 is composed of a lens 254, a first aperture 712, and The second aperture 71 4 is directed over the hybridization chamber array 110. The LED 26 is pulsed and the fluorescent emission is detected by the photo sensor 44. Similarly, the LOC device 30 shown in FIG. 98, 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 pulsed again and the fluorescent emission is detected by the light sensor -105 - 201211538 detector 44. The excitation wavelength of LED 26 is dependent on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for the Pulsar 6 50 dye. The SET UVT0P335T039BL LED is the appropriate excitation source for the bismuth melamine.

Table 3: Philips LXK2-PR14-R00 LED gauge) Grid parameter symbol 値 cell wavelength λεχ 460 nm Transmit frequency Vem 6.52(10)14 Hz Output power Pi 0.515(min) @ 1A W Transmit mode Lambertian data map N/A Table 4 : SET UVT0P334T039BL LED Specifications

Parameter symbol 値cell wavelength λε 340 nm emission frequency Ve 8.82(10)14 Hz power Pi 0.000240(min) @ 20mA W pulse forward current I 200 mA emission mode Lambertian N/A ultraviolet excitation pupil 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 LED 26 reduces the effectiveness of this method. For this, a filtered UV LED can be used. Incidentally, the UV laser can be an excitation source unless it is not practical for a particular test module market due to the relatively high cost of the laser. LED Driver -106- 201211538 LED driver 29 drives the LED 26 at a fixed current for the desired duration. The low-power USB 2.0 certified device can be obtained with a minimum operating voltage of 4.4 volts at up to 1 unit load (1 mA). Standard power conditioning circuits are used for this purpose. Light Diode Figure 54 shows an optical diode 184 that is combined with the 0 CMOS circuit 86 of the LOC device 301. The dimming diode 184 is part of the CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCDs, another sensible technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard processing steps. On-wafer inspection is inexpensive and reduces the size of the array system. The shorter optical path length reduces noise from the surrounding environment to efficiently collect fluorescent signals and reduce the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the fraction of photons that collide with the active region 185, and the photon is efficiently converted into photoelectrons. For standard enthalpy treatment, the quantum efficiency of visible light is in the range of 0.3 to 0.5 depending on processing parameters such as the number of cover layers and absorption characteristics. The detection valve of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and the number of hybrid chambers 180 in the hybridization and detection section 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (1,760 μm x 5,824 μm in the example of LOC device 301) and are subject to other functional modules (such as pathogen dialysis). 70 -107- 201211538 and the expansion part 1 1 2 ) The size of the non-animal parts that can be used afterwards is limited to the standard sand treatment, and the photodiode 184 is detected at a minimum of 5, in order to confirm the reliable detection 'minimum 値 can be set to 1 0 of these quantum efficiencies range from 〇.3 to 0.5 (as discussed above) 'self-light emission is a minimum of 17 photons' and 30 photons contain a suitable margin for error. The non-uniform electrical characteristics of the photodiode 184 and the residual excitation photon flux of the auto-fluorescence full attenuation introduce background noise into the signal. The calibration signal is generated from each of the output signals by one or more calibration signals using a calibration source that will calibrate one or more of the calibration photodiodes 184 in the array. A low calibration source is used to determine the negative result of the target probe reaction. A high calibration source represents a positive result from the probe-target. In the specific embodiment described herein, the calibration chamber 382 in the low calibration hybridization chamber array 10 is provided, which: does not contain any probes; contains probes that do not have a fluorescent reporter; or contains reporters The probes and groups constitute a quenching agent that is always expected. The output signal from such a calibration chamber 3 82 is very close to the noise and bias in the output signals from all of the hybrid chambers in the medium. The output signal generated by the chamber is subtracted from the calibration signal, and the signal generated by the fluorescent emission is substantially removed (if any signal photon is generated. However, the photon detected by the probe is not finished to the output number. Removed. The light source exposed to the respective target is not hybridized with the background of the source and the other in the raw quenched LOC device. -108- 201211538 Signals generated by ambient light in the area of the self-room array are also removed. It will be appreciated that the negative control group probes described above with reference to Figures 128 through 131 can be used in the calibration chamber. However, as shown in Figures 111 and 112, which is an enlarged view of the inset DG and DH of the LOC variant X 728 shown in Figure 104, another option is to fluidly isolate the calibration chamber 382 from the amplicons. When hybridization is prevented by fluid isolation, background noise and bias can be emptied from the chamber that is fluidly isolated or by any probe containing a lack of reporter or indeed having both a reporter and a quencher. Needle to judge" The calibration chamber 382 can provide a high calibration source to generate a high signal to the corresponding photodiode. The high signal corresponds to all probes in the hybridized chamber. The use of a reporter with no quencher or only a reporter spotting probe will consistently provide a signal that a large number of probes in the hybridization chamber have hybridized within the hybridization chamber. It is also understood that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 for the entire array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by a relatively close calibration chamber 382, the calibration 较 is more accurate. Referring to Figure 56, hybridization chamber array 1 10 has one calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybrid chambers 180. In this configuration, the hybridization chamber 180 is calibrated by the immediately adjacent calibration chamber 382. Due to the excitation of the self-fluorescent signal from the surrounding hybridization chamber 180, Figure 127 shows a differential imager circuit 878 for subtracting the signal from the photodiode 18 4 of the corresponding calibration chamber 382. The differential imager circuit 7 8 8 samples the signal from the pixel 790 and the "virtual" pixel 792. In one embodiment, the "virtual" pixel 792 is masked to prevent light illumination, so its output signal provides a dark reference. Or 109· 201211538, the "virtual" pixel 792 can be exposed to the excitation light with the rest of the array. In a particular embodiment where "virtual" pixel 792 is light absorbing, the signal produced by ambient light in the area of the array is also 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 because there is no reference to the dark level. During use, "read-column" 794 and "read_column_(1" 795 are enabled and M4 797 and MD4 801 transistors are turned on. Off switches 807 and 809 are turned off such that outputs from pixel 790 and "virtual" pixel 792 are turned off. Stored separately on pixel capacitor 803 and virtual pixel capacitor 805. After the pixel signal is stored, switches 807 and 809 are disabled. The "read-line" switch 81 1 and the virtual "read_row" switch 813 are then turned off. And the switched capacitor amplifier 815 amplifies the differential signal 817. The suppression and enabling of the photodiode must suppress the photodiode 184 during the excitation of the LED 26 and must enable the photodiode 184 during the fluorescent period. The circuit diagram of the single photodiode 184 and the timing diagram of the photodiode control signal are shown in Fig. 66. The circuit has a photodiode 184 and six MOS transistors, Mshunt 394, Mtx 396, Mreset 3 98, Msf 4 00, Mread 4 02 and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 394, and Mreset 3 98 are turned on by pulling Mshunt gate 3 84 and reset gate 3 88 high. During this period, the photon is excited. Yuguang Dipole 1 The carrier is generated in 84. When the amount of the generated carrier is sufficient to saturate the photodiode 1 84, the carriers must be removed. During this cycle, due to leakage of the transistor or excitation due to excitation in the substrate The carrier -110- 201211538 diffusion, Mshunt 3 94 directly removes the carriers generated in the photodiode 181 2 3 4, and the Mreset 398 resets any carrier accumulated in the node 'NS' 406. After the excitation The capture cycle begins at t4. In this cycle, the response from the emission of the fluorophore is captured and integrated into the circuit on node 'NS' 406. This is achieved by dragging the tx gate 3 86 high. The transistor Mtx 396 and any accumulated carrier on the transfer photodiode 184 to the node 'NS' 406. The capture cycle can be as long as the fluorescent emission. From the 0 of the hybrid cell array 1 1 0 has the photodiode 184 The output is 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 cell array 110 after the capture cycle ( See Figure 52). The first light to be read The polar body 184 will have the shortest delay before the read cycle, and the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the read gate 3 93 is turned on by the drag. Transistor Mread 402. The source-slave transistor Msf 400 is used to buffer and read the voltage of the 'NS' node point 406. Any other method of enabling or suppressing the photodiode is discussed below: -111 - 1 . Suppression method 2

Figures 124, 125 and 126 show possible configurations 778, 780, 782 for Mshunt transistor 394. Mshunt transistor 394 has a very high turn-off ratio when the maximum 値 |FCS| = 5 V 3 is enabled during excitation. As shown in Fig. 124, the Mshunt gate 3 84 system is formed on the edge of the photodiode 184. Optionally, as shown in FIG. 125, the Mshunt gate 3 84 can be grouped to form a loop 201211538 light-emitting diode 184. A third option is to fabricate the Mshunt gate 3 84 within the photodiode 184, as shown in FIG. According to the third option, the photodiode active region 185 is less. The 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. 124, the Mshunt gate 3 84 is attached to one side of the photodiode 184. This is the simplest fabrication and the smallest impact on the active region of the photodiode 185. However, any carrier remaining at the far end of the photodiode 184 takes a long time to diffuse through the Mshunt gate 3 84. In FIG. 125, the Mshunt gate 386 surrounds the photodiode 184. This further reduces the average path length of the carriers in the photodiode 184 to the Mshunt gate 3 84. However, extending the Mshunt gate 3 84 around the photodiode 184 causes the photodiode active region 185 to be substantially reduced. The fabric 782 in FIG. 126 positions the Mshunt gate 3 84 in the active region 185. This provides the shortest average path to the Mshunt gate 3 8 4 and thus the shortest transition time. However, the impact on the active zone 185 is greatest. It also creates a wide leak path. 2. Enable method a. The flip-flop photodiode drives the shunt transistor with a fixed delay 〇 b. The flip-flop photodiode drives the shunt transistor with a programmable delay. 〇. The parallel drive transistor is driven by the LED drive pulse with a fixed delay 〇 d. The parallel transistor is driven as a programmable delay. -112- 201211538 Fig. 68 is a schematic view showing the light diode 184 and the flip-flop photodiode 187 buried in the CMOS circuit 86 through the hybridization chamber 180. The small area in the corner of the photodiode 184 is replaced by the flip-flop light diode 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 244 has extinguished and activates the photodiode 184 (see Fig. 2) after a brief delay of At3 00. This delay allows the fluorescent Q photodiode 184 to detect fluorescent emissions from the FRET probe 1 86 without the excitation light 244. This enables detection and enhancement of signal to noise ratio. Under each of the hybrid chambers 180, both the photodiode 184 and the flip-flop photodiode 18 7 are located in the CMOS circuit 86. The photodiode array is combined with appropriate electronic components to form a photosensor 44 (see Figure 64). The photodiode 184 is a pn junction made during the fabrication of the CMOS structure without the need for additional masking or steps. During MST fabrication, the dielectric layer (not shown) over the photodiode 184 is arbitrarily thinned using standard MST photolithography techniques to cause more erbium to illuminate the active region 185 of the photodiode 184. The photodiode 184 has a field of view such that a fluorescent signal from the probe-target hybridization within the hybridization chamber 180 is incident on the surface of the sensor. The converted fluorescence becomes the photocurrent that can then be measured using the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to the trigger photodiode 187. These choices can be used in combination with 2a and 2b above. Fluorescence Delay Detection -113- 201211538 The following derivation is based on the above-mentioned LED/fluorescent combination using long-life fluorescence luminescence detection. After an ideal pulse excitation of the fixed intensity Ie between time ~ and 〇 shown in Figure 60, the fluorescence intensity is derived as a function of time. Let [^1] (〖) be equal to the intensity of the excited state at time t, then the number of excited states per unit volume per unit time during and after the excitation is described by the following differential equation: ...(1) d[Sl] | [^1 ](〇dt tF hve where C is the molar concentration of the fluorophore, ε is the molar quenching coefficient' is the excitation frequency, and h = 6.62606896(1 0)-34 Js is the Planck constant. This differential equation has General formula: dy_ dx + p(x)y^q(x) There is a solution: y(x) q(x)dx + k \p{x)dx ...(2) Now use this to solve the equation [s\ m=i^iL+ke-^ hve then at time h, ...(3) [51 ] ( tx ) =〇, and from (3 ): k =—

Lsct hve

Substituting (4) into (3) -114- 201211538 hve hve at time h,: ^-(/2^1)^/ (5) hve hve at i 2 〇, the excited state is exponentially decayed and is expressed by equation (6) Description: _) = _2Κ(ί-,2)~ ...(6) Substituting (5) into (6): [51](〇= I-^J-[\ - e(h~h),^ y(,- hVT^ ...(7)

Ave The intensity of the fluorescence is obtained by the following equation: where ν / is the fluorescence frequency, η is the quantum yield, and 1 is the optical path length, then from (7)

c/[51](/) _ Ie€C ^ ^-(^2)/^ dt hve ...(9) Substituting (9) into (8):

If (〇 = ε£εαΙη^-[l-e^t{),Xl ...(10) ^ e because ^! — 〇〇,

r/ K Therefore, we can write the following approximation, which describes the attenuation of the fluorescence intensity after a sufficiently long excitation pulse ():

Ifit) = Iesc^-e~(,~h)lTlfor t>t2 ...(11) -115- 201211538 In the previous section, we summarized the situation of i2-il >>Tf

If(t)=Ie£C^ and for t>h If(t)=Ie£clr]'e-(t-h)/r, in . From the above equation, we can derive the following expression: nf(t) = neGclne-〇-,l),T/ ...(12) «>(0 =

Is the number of fluorescent photons per unit area per unit time and

Je_ is the number of excited photons per unit area per unit time. Therefore, (0 = J if)dt (13) where \ is the number of fluorescent photons per unit area and ί3 is the time point at which the photodiode is turned on. Substituting (12) into (13): 〇〇hf = jnesc^eHt't2)lT/dt (14) At present, the number of fluorescent photons reaching the photodiode per unit area per unit time, & , obtained by: ";(0 = n>(0^〇-.(15) where nine is the light collection efficiency of the optical system. Substituting (1 2 ) into (1 5 ) we find «; = ^0n>/7e'(,_,i)/r/ (16) Similarly, the number of fluorescent photons per unit of fluorescent area\to the photodiode will be as follows: -116- 201211538

CO

Hs=jms(t)dt ll and substituting '(16) and integrating: »s =Φ〇ηβεαΙητίβ'{, ί~, ι)'Τ/ Therefore, ns =φ0ήΐεοΙητ/β~ί'"Τι ... (17) The ideal enthalpy of i3 is when the electron spectroscopy generated in the photodiode 184 due to the fluorescence photon is equal to the electron enthalpy generated by the excitation photon in the photodiode 184 because of the excitation photon flux attenuation ratio. The fluorescence photon flux decays more quickly. The output electron rate of the sensor due to the fluorescence area per unit of fluorescence is: έ>(7)=(d)) where the quantum efficiency of the sensor at the wavelength of the fluorescence is used. Substituting (1 7 ) we get: ef(t) = (l)f(l)0neec^e~{,~h)lTf (18) ^ Similarly, due to the per-unit fluorescence area of the excited photons The output electron enthalpy rate is: where is the quantum efficiency of the sensor at the excitation wavelength, and ^ is the time constant relative to the "off" characteristic of the excited LED. After time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends its decay time, but we assume that this pair has a negligible effect on If (t), so we take a conservative approach. At present, as mentioned earlier, the ideal for ί3 is: • έ > (ί3) = · έ: (ί3) -117- 201211538 Therefore 'by (1 8 ) and ( 〗 9 ) we get: φ / φ0ηβεϋΙηβΗ ,,~'2),Τί = and after reforming we get: Ιη(εοΙη^^-) ~-'2=-l~~p~ ...(20)

Tf Te From the above two paragraphs, we get the following two expressions: ns =ΦύΚ¥τί^Ιτ,...(21)

In Δί = __Α_2 ...(22) τί ^ where corpus = £c/;7 and Δί = ί3 -i2 'We also know that, in fact, q » r〆 ideal time for fluorescence detection and using Philips LXK2 -PR 1 The number of fluorescent photons detected by the 4-ROO LED and the Pulsar 650 dye is determined as follows. The ideal detection time is determined using equation (2 2 ): Recall the concentration of the amplicon, and assuming that all amplicons are hybridized, the fluorescence concentration of the fluorescing is: c = 2.89 (10) _ 6 mol / L. The height of the chamber is 1 = 8 (1 〇Γ 6 m). The fluorescent area is considered to be equivalent to the photodiode area, but the actual fluorescent area is substantially larger than the photodiode area; therefore, can it be assumed? 20.5 is the light collection efficiency of the optical system. The characteristics of the photodiode, t = 10 is the extremely conservative ratio of the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength. Typical LED attenuation lifetime ~ = 0.5 nanoseconds and using Pulsar 650 gauge -118- 201211538 grid, can be determined & : F = [1.48(10)6 ][2.89(10)-6 ] [8(10)~6 ](1) =3.42(10)5 1η([3.42(10)-5](10)(0.5)) 1(10)-6_ 0.5(10)-9 = 4.34(10)*9 s Detected The number of photons is determined using equation (2 1 ). First, the number of excitation photons emitted per unit time is determined by examining the illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian emission mode, so: n, = rilQ cos(0) ...(23) where) ϊ, the angle to the direction of the forward axis of the LED is 每 per unit solid angle per The number of photons emitted per unit time, and the heart is % in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: n,=J}i; dn Ω = fnwcos(6>)i/n Ω -(24) Now, △Ω = 2π[1 _ cos(0 + Δ0)] _ 2; τ [1 — cos(0)] ΔΩ = 2; r[cos(0) - cos(0 + Δ0)] (ΑθΛ . —sin 1 2 J 1 2 J + 4^cos(^ )sin2 = 4;rsin(0)cos άζΐ = 2π%\η{θ)άθ -119- 201211538 Substitute (24): π 2 ^=| 2ml0 cos(0)sin(e)d0 o =砵0 Rearrange , we get: Λ ΊΟ ...(26) The output power of the LED is 515·515 watts and ve = 6.52(10)14 Hz, so 0.515 [6.63(1〇Γ34][6.52(10)14] = 1.19( 10) 18 photon 値 bring this 入 into (26) we get: ...1.19(10)18 Ί0 π = 3.79(10)17 photon luminosity Refer to Figure 61, optical center 252 and LED 26 lens 254 system The luminescence diode is 16 μm X 16 μm, and for the photodiode in the middle of the array, the solid angle ( Ω ) of the light cone emitted from the LED 26 to the photodiode 184 is approximately: Ω = sensor area /r2 [16(10)-6] [16(10)-6] shows 2.825(10)-3] = 3.21(10)_5 Sphericality will understand the central photodiode of photodiode array 44! 8 4 is Use for these calculations The sensor at the edge of the array is only connected to -120-201211538 at the hybridization event to reduce the photon distribution by 2% for the Lambertian excitation source intensity distribution. The number of excitation photons emitted per unit time: he = η, Ω ... (28 ) =[3.79(10)17][3.21(10)-5] = 1.22(10)13 Photon/sec Now refer to equation (29): ns =Φ〇ΚΡτίβ~^ΙΤ/ η, = (0.5)[ 1·22(10)13][3·42(10)_5][1(10 宽 l〆3400)—9/100)·" =208 Photon/Sensor Therefore, use Philips LXK2-PR14-R00 With LEDs and Pulsar 650 fluorophores, we can easily detect any hybridization events that cause these numbers of photons to be excited. The SET LED illumination geometry is shown in Figure 62. At ID = 20 mA, the LED has a minimum optical power output of p1 = 240 μW and a wavelength center at λβ = 340 nm (absorption wavelength of the ruthenium chelate). Drive the LED at Id = 2 〇〇 mA, linearly increasing 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 11, we approximate the output flux to a dot size having a maximum diameter of 2 mm in the 2 mm diameter dot of the hybridization array plane. The photon flux is obtained from Equation 27. 2.4(10)'3 _[6.63(10)-34][8.82(10)14] = 4.10(10)15 Photons/sec -121 - 201211538

Using Equation 28, we get: K=n, Q 4.10(10)15 [16(10)6]2 vs. 1(1〇)·3]2 3.34(10)" Photon 渺 Now, back to the equation 22 and using the previously listed Tb chelate properties,

Af_ln[(6.94(10)-5)(10)(0.5)] 1(10)-3 a 0.5(10)-9 = 3.98(10)-9 seconds Now theorem 2 1 : ns = (0.5) [3.34(10)n][6.94(10)-5][l(l〇)-3]e-398(1〇r,/'(,0)'3 = 11,600 photon/sensor by hybrid event The photon theory number emitted using the SET LED and the ruthenium chelate system can be easily detected and well beyond the lower limit of 30 photons, which is reliable for use with the photosensors indicated above. On-wafer detection to detect the maximum spacing between the desired probe and the photodiode avoids the need to detect with a conjugate focal microscope (see prior art). This departure from traditional detection techniques is important in system-related time and cost savings. The traditional detection requires the use of lens and curved mirror imaging optics. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. Place the photodiode very close to the probe with polar collection The advantage of efficiency. When the material thickness between the probe and the photodiode is 1 micron, the emission angle of the emitted light is as high as -122-201211538 173°. This angle is obtained by Calculated from the light emitted by the probe closest to the center of the surface of the hybridization chamber of the photodiode, the photodiode has a planar active surface region parallel to the surface of the chamber. The emission pyramid in which light can be absorbed by the photodiode The system is defined as having a transmitting probe at its apex and at the sensor corners around its plane. For a 16 micron X16 micron sensor, the apex angle of the cone is 170°; the photodiode is expanded In a limit such that the area conforms to the area of the 29 micron X 19.75 micron hybridization chamber, the apex angle is 173° 〇. The separation between the chamber surface and the active surface of the photodiode is 1 micron or less is easy to achieve. Applying 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 emission. The maximum spacing between the photodiode and the probe is determined by reference to Figure 54 below. For the fluorophore and SET UVT0P335T039BL LEDs, we calculated 11600 photons from individual hybridization chambers 180 to 16 micron x 16 micron photodiodes 184. In performing this calculation, we assume that the hybrid chamber Ο 180 The region has the same bottom area as the photodiode active region 185, and half of the total number of hybrid photons reaches the photodiode 184. That is, the light collection efficiency of the optical system is nine = 0.5. More precisely, we can write out = [( The bottom area of the light collection area of the hybridization chamber) / (photodiode area)] [Ω / 4π], where Ω = solid angle is opposite to the photodiode of the representative point on the substrate of the hybridization chamber. (right) square pyramid geometry: QMarcsinU^Hdc^ + a2)), where d〇 = the distance between the chamber and the photodiode, and α is the size of the photodiode. -123- 201211538 23 200 photons were released from each hybrid chamber. The selected lower limit of the selected photodiode is 17 photons. Therefore, the minimum optical efficiency required is: ^0= 1 7/23200 = 7.33xl0 The bottom area of the light collection region of the '4 hybridization chamber 180 is 29 microns Χ 19·75 microns. Solving do will result in a maximum limit distance between the hybridization chamber and the photodiode 184 of d 〇 = 249 μm. In this limitation, the collection cone angle as defined above is only 〇. . It should be noted that this analysis ignores the negligible effect of refraction. LOC Variants The LOC device 301, described and illustrated in detail above, is only one of many possible LOC device designs. Some possible combinations will now be described in the context of a flow chart (from sample input to detection) and/or display of LOC device variants using different combinations of the various functionalities described above. The flow chart is appropriately divided into a sample input and preparation stage 288, an extraction stage 290, a culture stage 291, an amplification stage 292, a pre-hybridization stage 293, and a detection stage 294. For the sake of clarity and conciseness, only a brief description or summary of all LOC variants is shown and no detail configuration is shown. Also for clarity, smaller functional units, such as liquid sensors and temperature sensors, are not shown, but it should be understood that such functional units have been incorporated into the proper location of each of the following LOC device designs.

LOC Variant XI-124-201211538 Figures 116-123 show LOC variant XI 746. The LOC device extracts 290, cultures 291, expands 292, and detects 294 pathogen DNA. Sample input and preparation stage 28 8 and extraction 290 are the same as in LOC unit 301 (see Figure 4). In the culture stage 291, four parallel culture sections 114.1 to 114.4 were used. Referring to Figures 118, 119, and 121' shared restriction enzymes, binders, and junction reservoirs 58 are added to the sample stream through the surface tension valve 132 to the sampler feed channel 748. The co-cultivator feed channel 74 8 Q is filled with the culture portions 114.1 to 114·4 via the first, second, third, and fourth incubator inlets 750, 752, 754, and 756, respectively. Referring to Fig. 122, the culture portions 114.1 to 114.4 flow to the four parallel amplification portions 1 1 2.1 to 1 1 2 · 4, respectively. After sufficient incubation time, the boiling pilot valve at the outlet of each incubator is opened 207. Each of the amplification sections 112.1 to 112.4 has individual amplification mixing devices 60.1 to 60.4 and polymerase devices 62.1 to 62.4 (see Figs. 116 to 119). The polymerase is added immediately prior to nucleic acid amplification to optimize the amplification process. Referring to Fig. 123, each of the amplification sections 112.1 to 112.4 has a boiling pilot valve 108 at its individual outlet. After the thermal cycle, the boiling pilot valve 108 is opened such that the amplicon from each of the amplification sections 1 12.1-1 12.4 flows into the individual hybridization chamber arrays 110.1 to 110.4. The hybrid chamber arrays 110.1 through 110.4 are surrounded by strip of titanium nitride to provide an LED wafer support surface 634 for exciting the LEDs 26 (see Figure 3). The excitation LED is sealed to the LED wafer support surface 634. As shown in Figures 104 and 108, the gas pressure in the hybridization chamber 180 is equal to the gas pressure through the venting holes 122 and the venting passages 636 in the MST layer 87 (see Figure 1A). As shown in FIG. 121, the pathogen dialysis section 70 has bypass passages 600-125-201211538 to prevent air recruitment. The LOC variant XI 746 also has a flow sensor 740 and a liquid sensor 1 74 (see FIG. 1 18) for use in heater elements in various functional sections (eg, hybrid arrays, culture and amplification sections, etc.). Time-limited operation.

LOC Variant XIII Figure 77 shows the operation of the LOC variant XIII 640 for pathogen detection. This uses the pathogen dialysis unit 70, the chemical lysis unit 130, and the thermal lysis unit 638. This LOC variant XIII 640 does not have restriction enzymes, binders and linker sump sump (see, for example, sump 5 8 in Figure 4). The single flow stream (relative to the parallel flow stream) nucleic acid amplification function (see amplification mixing reservoir 60, polymerase reservoir 62, and amplification portion 112) is maintained. The boiling pilot valve 108 (other types of active valves may also be used) is located at the outlet of the chemical lysis unit 130 and the hot lysis unit 638, and at the amplification unit 112. Surface tension valves 1 18, 1 2 8 , 1 3 8 and 1 40 are located at the outlets of reagent reservoirs 5 4 , 56 , 60 and 62 .

LOC Variant XIV Figure 78 is a schematic representation of the LOC variant XIV 641 for pathogen detection. The LOC device uses both the single pathogen dialysis unit 70 and the chemical lysis unit 130 and the thermal lysis unit 63 8 . The LOC variant XIV 641 has parallel nucleic acid amplification functions (parallel-operated amplification units 丨12·1 '112·2...112·Χ), and individual hybridization chamber arrays 110.1, 110.2, ...110·χ and wafer glazing A sensor 44 (each hybrid cell array has a separate array of photodiodes). Each nucleic acid amplification stream has separate amplification mixing tanks (60.1, 60_2...60.Χ) and polymerase tanks (62.1, 62.2...62.X) independent of -126-201211538. The LOC variant XIV 641 provides the benefit of parallel nucleic acid amplification to pathogen detection. The boiling priming valve 108 is used at the outlet of the lysis unit 130 and the augmenting portion 1 12.X, and the surface tension valves 118, 128, 13 3 8.1 - 1 3 8.X and 140.1-140.X are respectively located in the reagent storage tank 54. , 56, 60.1-60.X and 62.1-62.X exports.

LOC Variant XV 〇 Figure 79 shows the LOC variant XV 642. The LOC variant XV 642 uses both the pathogen dialysis unit 70 (for pathogen detection) and the chemical lysis unit 130 and the hot lysis unit 63 8 . The LOC variant XV 642 has a tandem nucleic acid amplification function in which two amplification sections 12.1 and 12.2 are arranged in series. Adjacent to each of the amplification sections 1 12.1 and 1 12·2, the upstream of the amplification mixing tanks 60.1 and 60.2 and the polymerase storage tanks 62.1 and 62.2. The amplicon derived from the tandem amplification is used for the hybridization chamber array 1 1 0 detected by the photo sensor 44. The boiling pilot valve 108 is used at the outlet of the lysis sections 1 30 and 638 and the amplification section 112.1-112.2. The surface tension valves 118, 128, 138.1-138.2, and 140.1-140.2 are located at the outlets of reagent reservoirs 54, 56, 60.1-60.2, and 62.1-62.2, respectively.

LOC Variant XVI Figure 80 is a LOC variant XVI 643 for pathogen detection. It has a pathogen dialysis unit 70, a hot lysate 638, and a nucleic acid amplification function (amplification unit 112 and reagent storage tanks 60 and 62). And hybridization on the wafer (photosensor 44) (hybridization chamber array 1 1 〇). -127- 201211538

LOC Variant XVII Figure 81 is a schematic representation of the LOC variant XVII 644 for pathogen detection. The LOC device uses a single pathogen dialysis unit 70 and a thermal lysis unit 63 8 . The LOC variant XVII has a parallel nucleic acid amplification function (parallelly operated amplification sections 112.1, 112.2...112.X), and separate hybridization chamber arrays 110.1, 110.2 ... 1 10.X and on-wafer photosensor 44 (Each hybrid cell array has an independent array of photodiodes). Each nucleic acid amplification stream has separate amplification mixing tanks (60.1, 60.2 ... 60.X) and separate polymerase storage tanks (62_1, 62.2 ... 62.X). The LOC variant XVII 644 provides the benefit of parallel nucleic acid amplification for pathogen detection. The outlet of the lysis unit 130 and the amplification unit 1 12.1-1 12.X use the boiling pilot valve 108, and the surface tension valves 118, 128, 138.1-138.X and 40.1-140.X are respectively located in the reagent storage tank 54. , 56, 60.1 -60.X and 62.1-62.X exports.

LOC Variant XVIII Figure 82 shows the LOC variant XVIII 645. This variant uses the pathogen dialysis section 70 (for pathogen detection) and the hot lysis section 63 8 . The LOC variant XVIII 645 has a tandem nucleic acid amplification function in which two amplification sections 112, 1 and 1 12.2 are arranged in series. Adjacent to each of the amplification sections 1 12.1 and 12.2, the upstream of the amplification mixing tanks 60.1 and 60.2 and the polymerase tanks 62.1 and 62·2. The amplicon from the tandem amplification is used for the hybridization chamber array 1 1 0 detected by the photo sensor 44. The boiling pilot valve 108 is used at the outlet of the hot lysis section 63 8 and the amplification section 112.1-112.2. The surface tension valves 118, 138.1_ 1 3 8.2 and 1 4 0.1 -1 4 0.2 are respectively located at the outlets of the reagent storage tanks 5 4 , 6 0 _ 1 - 6 0.2 and -128 - 201211538 62·1·62·2.

The LOC variant XX is not shown in the LOC variant XX 647 lineage configuration in Figure 83 for pathogen detection' which has a pathogen dialysis section 70, a chemical lysis section 130, and a single amplification section 112.

〇 L0C variant XXI Figure 84 is a schematic representation of the l〇c variant XXI 648 for pathogen detection. The L0C device uses a single-pathogen dialysis unit 7 and a chemical lysis unit 13A. l〇C variant XXI 648 has parallel nucleic acid amplification functions (parallel-operated amplification sections 112·1, 112·2...112.X), and separate hybridization chamber arrays 110.1, 110.2...110. A sensor 44 (each hybrid cell array has a separate array of photodiodes). Each nucleic acid amplification stream has separate amplification mixing tanks (60.1, 60.2...60·Χ) and separate polymerase storage tanks (62.1 Q, 62.2 ... 62.Χ). The LOC variant XXI 648 provides the benefit of parallel nucleic acid amplification for pathogen detection. The boiling priming valve 108 is used at the outlet of the lysis unit 130 and the amplifying portion 112.1-112.X, and the surface tension valves 118, 128, 1 3 8.1 - 1 3 8. Χ and 140.1-140. Exports of 54, 56, 60.1 - 60.X and 62.1-62.X. LOC Variant XXII Figure 85 shows the LOC variant XXII 649. The LOC variant XXII 649 uses a pathogen dialysis unit 70 (for pathogen detection) and a chemical lysis unit 130. -129- 201211538 LOC Variant XXII 649 has a tandem nucleic acid amplification function in which two amplification portions 112.1 and 11 2 are arranged in series. Adjacent to each of the amplification sections 11 2, 1 and 1 12.2, the upstream of the amplification mixing tanks 60.1 and 60.2 and the polymerase storage tanks 62.1 and 62.2. The amplicon derived from the tandem amplification is used for the hybridization chamber array 1 1 以 detected by the photosensor 44. A boiling pilot valve 108 is used at the outlet of the chemical lysis unit 130 and the amplification unit 112.1-112.2. Surface tension valves 118, 128, 13 3 8.1 - 1 3 8.2 and 140.1 - 140.2 are located at the outlets of reagent reservoirs 54, 56, 60.1 -60.2 and 62.1 -62.2, respectively. Conclusion The devices, systems, and methods described herein facilitate molecular diagnostic testing at low cost and high speed and in situ care. The above-described system and its components are merely illustrative, and many variations and modifications will be readily apparent to those skilled in the art without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will be described with reference to the accompanying drawings, wherein: FIG. 1 shows a test module and a test module configured for fluorescence detection. Figure 2 is a schematic view of the electronic components in the test module for fluorescence detection; Figure 3 is a schematic diagram of the electronic components in the test module reader; -130- 201211538 Figure 4 is a schematic view showing the structure of the LOC device; Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device having all the layer structures and features stacked on each other, and Figure 7 is a stand-alone display Figure 8 is a plan view of the top surface of the LOC device with the inner channel and the sump shown in phantom. Figure 9 is a perspective view of the exploded top surface of the inner channel and the sump shown in phantom; A bottom view of the cover of the upper channel assembly is shown; Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device; Figure 12 is a schematic view of the sample inlet of the LOC device; Figure 13 is a view of Figure 6 Figure A is an enlarged view; Figure 14 is Figure 6. An enlarged view of the illustrated illustration AB; Ο Figure 15 is an enlarged view of the illustration AE shown in Figure 13; Figure 16 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE, Figure 17 is an illustration of the illustration A partial perspective view of the laminated structure of the LOC device in the AE, FIG. 18 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE; FIG. 19 is a view illustrating the laminated structure of the LOC device in the illustration AE Partial perspective view, -131 - 201211538 Figure 2A is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE, and Figure 21 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE, Figure 22 is a schematic view of the lysing reagent storage tank shown in Figure 21; Figure 23 is a partial perspective view illustrating the lamination structure of the LOC device in the illustration AB, and Figure 24 is a diagram illustrating the LOC device in the illustration AB a partial perspective view of the laminate structure, FIG. 25 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AI, and FIG. 26 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AB. 27 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AB; A partial perspective view of the laminated structure of the LOC device in the illustration AB; FIG. 29 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AB; FIG. 30 is a view of the amplification mixing tank and the polymerase storage tank. Figure 31 shows the characteristics of a separate boiling pilot valve; Figure 32 is a schematic view of the boiling pilot valve taken along line 3 3 - 3 3 shown in Figure 31; Figure 33 is shown in Figure 15. An enlarged view of the illustration AF; -132- 201211538 Figure 34 is a schematic view of the upstream end of the dialysis section taken along line 3 5-3 5 shown in Figure 33; Figure 35 is an illustration AC shown in Figure 6. Figure 36 is a further enlarged view showing the amplification portion in the illustration AC; Figure 37 is a further enlarged view showing the amplification portion in the illustration AC; Figure 38 is a further enlarged view showing the amplification portion in the illustration AC; 39 is a further enlarged view of the illustration AK shown in FIG. 38; FIG. 40 is a further enlarged view showing the amplification chamber in the illustration AC; FIG. 41 is a further enlarged view showing the amplification portion in the illustration AC; A further enlarged view of the amplification chamber is shown in the illustration AC; FIG. 43 is the illustration in the illustration AL shown in FIG. Figure 14 is a further enlarged view showing the amplification section in the illustration AC; Figure 4 is a further enlarged view of the illustration AM shown in Figure 44; Figure 46 is a further enlargement of the amplification chamber shown in the illustration AC Figure 47 is a further enlarged view of the illustration AN shown in Figure 46; Figure 48 is an enlarged view of the enlargement chamber in the illustration AC; Figure 49 is a further illustration of the amplification chamber shown in the illustration AC Figure 5 is a further enlarged view showing the amplification section in the illustration AC; Figure 51 is a schematic view of the amplification section; Figure 52 is an enlarged plan view of the hybridization section; Figure 53 is an enlarged view of two independent hybridization chambers. Figure 54 is a schematic view of a single hybridization chamber; Figure 55 is an enlarged view of the humidifier illustrated in the illustration AG shown in Figure 6 - 133 - 201211538 Figure 56 is shown in Figure 52 Figure 57 is an exploded perspective view of the LOC device in the inset AD; Figure 58 is a FRET probe in a closed configuration; Figure 59 is a FRET probe in an open and hybrid configuration. Figure 60 is the plot of excitation light versus time; Figure 61 is the excitation illumination geometry of the hybrid chamber array (excitati〇n ill Fig. 62 is a diagram of the LED illumination geometry of the sensor electronics; 'Fig. 63 is an enlarged plan view of the humidity sensor shown in the illustration AH of Fig. 6; Fig. 64 is a partial light sensing 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 shown in the illustration AP of Figure 55. Plan view » Figure 68 is a schematic diagram of the photodiode and the photodiode of the trigger through the hybridization chamber; Figure 69 is a diagram of the junction-primed PCR; Figure 7 is a test module with a lancet Figure 71 is a graphical representation of the structure of the LOC variant VII; Figure 72 is a plan view of the LOC variant VIII having all of the layer structures and features superposed on each other; Figure 73 is an illustration CA shown in Figure 72 Figure 74 is a partial perspective view showing the layered structure of -134 - 201211538 of the LOC variant VIII in the inset CA shown in Figure 72; Figure 75 is an enlarged view of the inset CE shown in Figure 73; Figure 76 is a graphical representation of the structure of LOC variant VIII; Figure 77 is the structure of LOC variant XIII Figure 78 is a graphical representation of the structure of the LOC variant XIV; Figure 79 is a graphical representation of the structure of the LOC variant XV; Figure 80 is a graphical representation of the structure of the LOC variant XVI; Figure 81 is a LOC variant XVII Figure 82 is a graphical representation of the structure of LOC variant XVIII; Figure 83 is a graphical representation of the structure of LOC variant XX; Figure 84 is a graphical representation of the structure of LOC variant XXI; Figure 85 is a LOC variant Figure 86 is a graphical representation of the structure of the LOC variant XXV; Figure 87 is a graphical representation of the structure of the LOC variant XXVIII; Figure 88 is a graphical representation of the structure of the LOC variant XLI; 89 is a graphical representation of the structure of the LOC variant XLIII; Figure 90 is a graphical representation of the structure of the LOC variant XLIV; Figure 91 is a graphical representation of the structure of the LOC variant XLVII; Figure 92 is the primer-joined during the initial amplification Figure of linear fluorescent probe; Figure 93 is a diagram of the primer-linked linear fluorescent probe during the subsequent amplification cycle; Figures 94A to 94F graphically illustrate the primer-linked fluorescent stem-and-loop probe Thermal cycle; -135- 201211538 Figure 95 is related to the hybrid chamber array and BRIEF DESCRIPTION OF THE DRAWINGS FIG. 96 is a schematic illustration of an excitation LED and an optical lens for directing light onto a hybrid chamber array of a LOC device; FIG. 97 is a hybridization chamber for directing light to a LOC device. An illustration of the excitation LEDs, optical lenses, and apertures on the array; Figure 98 is a schematic illustration of the excitation LEDs, optical lenses, and mirror configurations for directing light onto the hybrid chamber array of the LOC device; Figure 99 is LOC Schematic representation of the structure of variant X; Figure 100 is a perspective view of LOC variant X;

Figure 101 is a plan view showing the LOC variant X of the structure of the independent CMOS + MST device; Figure 102 is a perspective view of the bottom side of the cover (the reagent reservoir is shown in dashed lines) * Figure 103 is a plan view showing only the features of the separate cover; 104 is a plan view showing all the features superimposed on each other, and shows the positions of the illustrations DA to DK; FIG. 105 is an enlarged view of the illustration DA shown in FIG. 104; FIG. 106 is an enlarged view of the illustration DB shown in FIG. Figure 107 is an enlarged view of the illustration DC shown in Figure 104; Figure 108 is an enlarged view of the illustration DD shown in Figure 1-4; Figure 109 is an enlarged view of the illustration DE shown in Figure 1-4; Figure 110 is an enlarged view of the illustration DF shown in Figure 1-4; Figure 111 is an enlarged view of the illustration DG shown in Figure 1-4; -136-201211538 Figure 112 is an illustration DH shown in Figure 104 Figure II3 is an enlarged view of the illustration DJ shown in Figure 1-4; Figure 114 is an enlarged view of the illustration DK shown in Figure 104; Figure 115 is an enlarged view of the illustration DL shown in Figure 104; Figure 1 is a schematic representation of the structure of LOC variant XI; Figure 117 is a perspective view of LOC variant XI; Figure 118 is a diagram showing LOC variant XI overlaying each other Flat view of all the features of the square, and the display position of the EA to EC of illustration; Figure 119 is a plan view showing only the cover member LOC XI of the features of the independent variable >

Figure 120 is a plan view showing a LOC variant XI of a stand-alone CMOS + MST device structure, Figure 121 is an enlarged view of the inset EA shown in Figure 118; Figure 122 is an enlarged view of the inset EB shown in Figure 118; An enlarged view of the illustration EC shown in Fig. 18; Ο Fig. 124 shows an embodiment of a parallel transistor for an optical diode; Fig. 125 shows an embodiment of a parallel transistor for an optical diode; 26 shows an embodiment of a parallel transistor for an optical diode; FIG. 127 is a circuit diagram of the differential imager; and FIG. 128 schematically depicts a negative control fluorescent probe having a stem-and-ring structure; A negative control fluorescent probe of Figure 128 is shown in an open configuration; Figure 130 depicts a positive control fluorescent probe in the form of a stem-and-loop structure; Figure 131 depicts a positive view of the open structure 130. Control Fluorescence -137- 201211538 Needle; Figure 1 3 2 Briefly describe the flow sensor controlled by c MO S; Figure 1 33 shows the test module and test module assembled by the ECL test Reader; Figure 134 is a schematic diagram of the electronic components in the test module used with ECL detection. FIG 135 is not significant alternative test module and test module reader; display module and the host system tests alternative test module reader and a variety of storage in database 136, and FIG. [Main component symbol description] 1 〇: Test module 1 1 : Test module 1 2 : Test module reader 1 3 : Case 14 : Micro-USB connector 1 5 : Sensor 16: Micro-USB 埠 17: Touch Control screen 1 8 : Display screen 1 9 : Button 2 0 : Start button 2 1 : Honeycomb radio -138 - 201211538 22 : Aseptic sealing tape 23 : Wireless network connection 24 : Large container 2 5 : Satellite navigation system 26 : Light-emitting diode 27: data storage 2 8 : telephone 29 : LED driver 30 : LOC device 3 1 : power conditioner 32 : capacitor 33 : clock 3 4 : controller 35 : register 36 : USB device driver 3 7 : Drive 3 8 : Random Access Memory 3 9 : Driver 40 = Flash Memory 41 : Register 42 : Processor 43 : Program Memory 44 : Light Sensor Indicator - 139 - 201211538 46 : Cover 47 Module 48: CMOS+MST device 49: porous element 52: detection portion 54: sump 56, 56.1, 56.2, 56.3: sump 5 7: printed circuit board 58, 58.1, 58.2: sump 60, 60.1-60.12, 60. X: sump 62, 62.1, 62.2, 62.3, 62.4, 62.X: sump 6 4 : lower seal 66: top layer 6 8 : sample inlet 70: Analysis section 72: waste channel 74: target channel 76: waste reservoir 78: sump layer 80: cover channel layer 8 2: upper sealing layer 8 4: 矽 substrate 86: CMOS circuit 87: MST layer - 140 - 201211538 8 8 : Passivation layer 90 : MST channel 92 : Lower pipe 94 : Cover channel 96 : Upper pipe 9 7 : Wall 98 : Meniscus holder 〇 1 00 : MST channel layer 101 : Laptop / notebook 102 : Capillary action Starting feature 103: dedicated reader 105: desktop computer 106: boiling priming valve 107: e-book reader 108: boiling priming valve 〇 109: tablet 110, 110.1-110.12, 110.X: hybrid chamber array 1 1 1 : Epidemiological data 112, 112.1-112.12, 112.X: Amplification part 1 1 3 : Genetic data 1 1 4 · 1 -1 1 4 · 4 : Culture part 1 1 5 : Electronic health record 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve -141 - 201211538 1 1 9 : Liquid sample 1 2 0 : Meniscus 1 2 1 : Electronic medical record 1 2 2 : Vent 1 2 3 : Personal health record 125: Network 126: boiling pilot valve 128, 128.2, 128.3: surface tension valve 1 3 0, 1 3 0.1 - 1 3 0 · 3 : lysis unit 1 3 1 : mixing unit 132, 132.1, 132.3: table Tension valve 1 3 3 : incubator inlet channel 134: lower pipe 1 36 : optical window 138, 138.1, 138.2, 138.X: surface tension valve 140, 140.1, 140.2, 140.X: surface tension valve 1 4 6 : valve Inlet 1 4 8 : Valve outlet 1 5 0 : Down valve 1 5 2 : Ring heater 1 5 3 : Valve heater contact 1 5 4 : Heater 1 5 6 : Heater contact 158 : Microchannel 142 - 201211538 1 60 : outlet channel 1 6 4 : orifice 166 : capillary action initiation feature 168 : dialysis extraction aperture 170 : temperature sensor 174 : liquid sensor 175 : diffusion barrier 176 : flow path 1 7 8 : liquid sensation Detector 180: hybridization chamber 1 8 2 : heater 1 84 : photodiode 1 8 5 : active region 1 8 6 : probe 1 87 : photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : Annular heater 192: water supply passage 193: upper pipe 194: lower pipe 1 9 5: top metal layer 196: humidifier 1 9 8 : extraction hole - 143 201211538 202: capillary action starting feature 204: MST channel 206: Boiling pilot valve 207: Boiling pilot valve 208: Liquid sensor 210: Microchannel 2 1 2 : MST channel 2 1 8 : Electrode 220: Electrode 222: Gap 23 2 : Humidity sensor 2 3 4 : Heater 23 6 : FRET probe 23 8 : Target nucleic acid sequence 240 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 2 4 8 : Quencher 250 : Fluorescent signal 2 5 2 : Optical center 2 5 4: Lens 2 8 8 : sample input and preparation 2 9 0 : extraction stage -144 - 201211538 291 : culture stage 292 : amplification stage 293 : pre-hybridization filtration purification stage 294 : detection stage 296 : first electrode 298 : Two electrodes 3 0 0 · Delay 〇 301 : LOC device 3 2 8 : White blood cell dialysis unit 3 76 : Thermal conduction column 3 7 8 : Positive control probe 3 8 0 : Negative control probe 3 8 2 : Calibration chamber 3 8 4 : Wenji 3 8 6 : Noisy Ο 3 8 8 : Gate 3 9 0 : Retractable lancet 3 92 : Lancet release button 393: Smell 3 9 4 : Μ Ο S transistor 3 96 : MOS Transistor 398: MOS transistor 400: MOS transistor 402: MOS transistor-145-201211538 404: MOS transistor 406: node 408: film seal 4 1 0: film guard 492: LOC variant 518: LOC change Body 594: Interfacial layer 600: Bypass channel 602: Interface target channel 604: Waste channel 63 6 : Ventilation channel 63 8 : Thermal lysis part 640 : LOC Variant 641 : LOC Variant 642 : L OC Variant 643 : LOC Variant 644 : LOC Variant 645 : LOC Variant 647 : LOC Variant 648 : LOC Variant 649 : LOC Variant 6 8 2 : Dialysate 6 8 6 : Dialysis Step 692 : Primer-Join Linear probe-146-201211538 694: amplification blocker 6 9 6 : probe region 698: complementary sequence 700: oligonucleotide primer 704: stem-and-loop probe 706: complementary sequence 708: stem 7 1 0 ··s 7 1 2 : first aperture 7 1 4 : second aperture 7 1 6 : first mirror 71 8 : second mirror 728 ·· LOC variant 740 : flow rate sensor 746 : LOC variant 748 : feed channel 750 : inlet 7 5 2 · inlet 754 : inlet 756 : inlet 75 8 : LOC variant 7 6 0 : large component channel 7 6 2 : small component channel 764 : opening -147 - 201211538 7 6 6 : Blind terminal 7 6 8 : Blind terminal 7 7 8 : Fabric 7 8 0 : Fabric 7 8 2 : Fabric 7 8 8 : Differential imager circuit 7 9 0 : Pixel 792 : Virtual pixel 7 9 4 : Read_Column 7 9 5 : Read_Column 7 9 6 : Negative control probe 7 9 7 : (Chip) 7 9 8 : Positive control probe 8 0 1 : (Crystal) 8 0 3 : Pixel capacitor 805: Virtual pixel capacitor 8 0 7 : Switch 8 0 9 : On 811: switch 813: switch 814: Heating elements 815: capacitor amplifier 817: Differential Signal 8 60: ECL excitation electrode 8 70: ECL excitation electrode -148-

Claims (1)

201211538 VII. Application for Patent Park: 1. A wafer-on-lab (LOC) device for detecting pathogens in a biological sample, the LOC device comprising: an inlet for receiving a sample; a support substrate; and a pathogen in the sample a plurality of separate dialysis sections; a plurality of reagent storage tanks; a lysate located downstream of the dialysis section for lysing pathogens to release genetic material therein, the lysate being contained in the lysis unit One of the reagent sump of the cell lysing lysis reagent is in fluid communication; a first nucleic acid amplification portion located downstream of the lysis portion for amplifying the first nucleic acid sequence in the genetic material; and being located at the first nucleic acid a second nucleic acid amplification unit downstream of the amplification unit, configured to amplify a second nucleic acid sequence in the amplicon from the first nucleic acid amplification unit; wherein the dialysis portion, the lysis unit, and the first nucleic acid amplification The portion and the second nucleic acid amplification portion are both supported on the support substrate. 2. The LOC device of claim 1, wherein the first nucleic acid amplification portion is a first polymerase chain reaction (PCR) portion and the second nucleic acid amplification portion is a second PCR portion. 3. The LOC device of claim 2, wherein the first PCR portion has a first set of primer pairs for annealing with the first set of complementary nucleic acid sequences, and the second PCR portion has a second and second PCR portions A second set of primer pairs to which a complementary nucleic acid sequence is affixed, the first set of complementary nucleic acid sequences being different from the second set of -149-201211538 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, and the amplification impurities are at least one of the following: a reverse transcriptase type ; polymerase type; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; thermal cycle repetition; and temperature during a specific phase of PCR. 5. The LOC device of claim 4, further comprising a hybridization portion downstream of the second PCR portion, the hybrid portion having a probe array for hybridization with the standard nucleic acid sequence and for detecting the array Any probe hybrid & light sensor. 6. The LOC device of claim 3, wherein the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the lysis portion, and a plurality of orifices, the orifice being larger than The pathogen is smaller than the larger components, the second channel is in fluid communication with the first channel via the orifice such that the pathogen flows into the second channel, and the larger component remains in the first channel. 7. As claimed in the patent scope The LOC device of the sixth item, wherein the first channel and the second channel group constitute a sample by capillary action. 8. The LOC device according to claim 7 of the patent scope, wherein the second channel is -150-201211538 The line constitutes the inhalation of the pathogen into the lysis portion by capillary action. 9. The LOC device of claim 1, wherein 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. 10. The LOC device of claim 1, wherein the reagent storage tanks each have a surface tension valve for holding the reagent therein, the surface tension valve having a meniscus holder, and the meniscus holder for fixing the reagent The meniscus is kneaded until it contacts the sample stream to remove the meniscus so that the reagent flows out of the reagent reservoir. 11. The LOC device of claim 4, further comprising a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer merging the first and second PCR sections, wherein the CMOS circuit is located at the support Between the substrate and the MST layer, the C Μ 电路 S circuit group constitutes a feedback control of the first and second PCR sections using a temperature sensor output. 12. The LOC device of claim 11, wherein the first ❹ PCR portion has a PCR microchannel for a heating cycle sample. The PCR microchannel defines a cross-sectional area of the traverse flow of less than 1 〇〇, 〇〇〇 Square micron flow path. 13. The LOC device of claim 12, wherein the PCR microchannel has at least one elongated heater element extending parallel to the PCR microchannel. 14. The LOC device of claim 13, wherein the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, the PCR microchannel having a series of wide tortuous turns In the 蜒 structure, each of the wide tortuous lines forms a channel portion of one of the elongated PCR chambers. -151 - 201211538 15. The LOC device of claim 14, further comprising a reagent reservoir for retaining reagents for PCR; and a surface tension valve having a meniscus that constitutes a fixed reagent; Port L allows the meniscus to retain reagents in the reagent reservoir until the fluid sample is contacted to remove the meniscus and the reagents flow out of the reagent reservoir. 16. The LOC device of claim 15 further comprising an array of hybridization chambers for housing the probe. 17. The LOC device of claim 16, wherein the photosensor is an array of light diodes positioned in registration with the hybridization chamber. 18. The LOC device of claim 16, wherein the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. 19. The LOC device of claim 16, wherein the first PCR portion has a means for retaining liquid in the first PC R portion during the thermal cycle and in response to an activation signal from the CMOS circuit to allow liquid to flow to the second Active valve of the PCR section. 20. The LOC device of claim 19, wherein the active valve is a boiling pilot valve having a meniscus holder and a heater, the meniscus holder being configured to fix the meniscus and to stop the capillary The action-driven liquid flow' is used to boil the liquid and release the meniscus from the meniscus holder to restore the capillary action drive flow. -152-