TW201209403A - LOC device for genetic analysis which performs nucleic acid amplification after sample preparation in a dialysis section - Google Patents

Abstract

A lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a dialysis section for separating small constituents from larger constituents in the sample, a plurality of reagent reservoirs, a nucleic acid amplification section downstream of the dialysis section for amplifying nucleic acid sequences in the sample, wherein, the dialysis section and the nucleic acid amplification section are both supported on the supporting substrate.

Description

201209403 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to the processing and analysis of microfluidics and biochemicals for molecular diagnostics. [Prior Art] Molecular diagnostics have been used to provide areas for early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genetically related diseases Pathogenic Factors Due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the incidence of ineffective health care, improve patient outcomes, improve disease management, and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the 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 receive in the future, determine the type and pathogenicity of an infectious pathogen, or determine the individual's response to the drug. 201209403 Nucleic Acid-Based Molecular Diagnostic Tests Nucleic acid-based assays have four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine , sputum and tissue samples are used for genetic analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required at the beginning of a nucleic acid assay. Blood is one of the more frequently requested sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombotic cells promote agglutination and maintain activity in vitro. To inhibit coacervation, the sample is mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of cellular material, but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by differentially lysing the red blood cells in the lysis solution, leaving the remaining intact cellular material, which can then be separated from the sample by centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. The exact protocol used to extract the nucleic acid depends on the sample and the diagnostic analysis to be performed. 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 done using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The step of precipitating with alcohol (usually ice ethanol or isopropanol) in the presence of a high concentration of chaotropic salt, usually in the cerium oxide matrix, resin or paramagnetic beads in the fractionation column, prior to washing, Alternatively, the nucleic acid is purified via a solid phase purification step followed by elution 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 °C to destroy the thermal lysis of the cell membrane. Target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively 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. 201209403 PCR is a useful technique for amplifying target DNA sequences against a relatively complex DNA background. If RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. 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. A pair of short strands of DN A having about 10-30 nucleotides complementary to the E-flanking target sequence. 2. DNA polymerase-synthesis of DNA thermostable enzymes 3. Deoxyribose Nucleoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - The best chemical environment for DNA synthesis - PCR involves placing these reagents in small tubes containing extracted nucleic acids ( ~1〇-5〇μL) Place the tube in a polymer thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. Standard procedures for each thermal cycle include the denatured phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denatured phase generally involves ramping the reaction temperature to 90-95 ° C to denature the DNA strand: in the adhesive phase, the temperature is lowered to ~50-60 °C for adhesion of the primer: then in the extended phase, the temperature is raised The optimal DN A polymerase activity temperature is 60-72 ° C for extension of the primer. This method repeats the cycle for about 20-40 times, and the end result is a sequence of targets -8 - 201209403 that produce millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multiple primer set PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single experiment by targeting multiple genes at once (in other ways, several trials are required). Optimizing multiple primer set PCR is more difficult because it requires the selection of primers with approximate adhesion temperatures and amplicon with approximate length and base composition to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR, is a method for enabling nucleic acid amplification of virtually all DNA sequences in complex DNA mixtures without the need for target-specific Sexual introduction. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). Using a 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 DN A fragment. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. Thereby, all fragments of the DNA source adjacent to the linker oligonucleotide can be amplified. Direct PCR describes a system that performs PCR directly on a sample without any nucleic acid extraction (or minimal nucleic acid extraction). It has long been believed that the PCR reaction is inhibited by 201209403, a component of many components present in unpurified biological samples, such as the raw heme component in the blood. Traditionally, PCR requires enhanced purification of target nucleic acids prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentration, PCR can be performed or direct PCR can be performed with minimal DNA purification. The adjustment of PCR chemistry for direct PCR involves enhanced buffer strength, the use of high activity and processivity polymerases, and the addition of honey to potential polymerase inhibitors. Repeated sequence PCR utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. Repetitive Sequence One type of PCR is nested PCR in which two pairs of PCR primers are used to perform single locus amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. The second pair of primers (nested primers) used in the second amplification binds to the first PCR product and produces a second PCR product (short in the first PCR product) containing the target nucleic acid. The rationale used in this strategy is that if the wrong locus is amplified 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. Instantaneous PCR or quantitative PCR is 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 probe containing a fluorophore or a fluorescent dye and a set of reference standards in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DN A from RNA. The reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (c DNA) -10- 201209403, and then the 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 augment RNA viruses such as human immunodeficiency virus or hepatitis C virus. Constant temperature amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DNA during the amplification reaction, and thus does not require complicated machinery. The thermostatic nucleic acid amplification method can be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Some of the constant temperature nucleic acid amplification methods of Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification Has been described. The thermostatic nucleic acid amplification method does not rely on the continuous heat denaturation of the template DNA to produce a single-stranded molecule as a template for further amplification, but relies on an enzymatic cleavage such as a DNA molecule that specifically limits the endonuclease at normal temperature' or It is another way to separate DNA strands using enzymes. The ability of strand-substituted amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi-modified DNA, and 5,-3' exonuclease-deficient polymerases to extend and replace Downstream stocks. However, after -11 - 201209403, exponential nucleic acid amplification is achieved by a combination of sense and antisense, wherein the strand of the sense reaction is substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) which produces a nick in one of the DNA strands without cutting the DNA in a conventional manner is useful for this reaction. SDA has been improved by using a combination of thermostable restriction enzyme (/να 1 ) and thermostable exo-polymerase (polymerase). This combination appears to increase the amplification efficiency of the reaction from 1 to 8 fold amplification to 101. () amplification so that this technique can be used to amplify unique single copy molecules. Transcription-mediated amplification (TMA) and nucleic acid-dependent sequence amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than 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 RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to a target ribosomal RNA (rRNA) at a defined position. The reverse transcriptase produces a DNA copy of the target rRN A by extending 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 sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon is reintroduced into the process and serves as a template for new replication. In recombinant enzyme polymerase amplification (RPA), a constant amplification of a specific DNA fragment is achieved by binding a relative oligonucleotide primer to the template DN A and extending it by DN A polymerase to -12-201209403. Denatured double-strand templates do not require heat. Conversely, RPA utilizes recombinant fermentation: scanning dsDNA and promoting the homologous (cognate) position of the structure obtained by the single-stranded DNA-binding protein and the substituted template strand, thus preventing the primer from being displaced by the branched mobilin to replace the DNA polymerase Sub til is a large segment of Pol I (m )) close to the end, and the primer then begins to extend. This nucleic acid amplification is repeated by circulation. The helicase amplification (HDA) mimics the in vivo system using DNA helicase to generate primers for hybridization with DNA polymerase extension primers. In the HDA reaction, the helicase passes through the target DNA, destroying the two strands and binding by a single-stranded binding protein. The primer is allowed to adhere by the heliostat. The DNA polymerase uses autologous triphosphate (dNTP) to subsequently replicate the DNA of each primer. Two replicates into the next HD A cycle 'cause the target sequence to be followed by other DNA-based thermostats including RCA' in which the DNA polymerase is formed by a number of circular repeats around the circular 〇ΝΑ primer. . By terminating the reaction, the polymerase produces thousands of copies of the copy strand tethered to the original target DNA. The resulting DNA (dsDNA)-introduction mismatch comes from: stock exchange. By interaction, it is stable and released. Recombinant fermentation (such as the 31 step of Bacillus oligonucleotides to reach the index, in the first step of the single-strand template merging in the in vivo system, the hydrogen bond, the deoxygenation of the two strands with the single-strand target area The 3' end of the ribonucleotide to generate two dsDNA strands independently exponentially nucleic acid amplification by rolling-cycle amplification (templates are continuously extended. Long DNA products. Circular template, which has: a spatial solution to the target-13 - 201209403 Rapid nucleic acid amplification of resolution and signal. Up to 12 copies of the template can be generated in 1 hour. Branched amplification is a variant of RCA and utilizes a closed circular probe (C-probe) or a lock Probes and highly progressive DNA polymerases to exponentially amplify C-probes at ambient temperatures. Circular thermostat amplification (LAMP) provides high selectivity and utilizes DNA polymerase and contains four specially designed primers In the introduction group, the primer group recognizes a total of six different sequences on the target DNA. The inner primer of the sense strand containing the target DNA and the antisense strand sequence starts the LAMP. The subsequent strand-initiated DNA synthesis release sheet is triggered by the external primer. Strand DNA. This is used as the second inner and outer primer The template for DNA synthesis initiated, the second inner and outer primers hybridize to the other end of the target to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer hybridizes to the loop on the product. The initial substitution DNA synthesis produces the original stem-loop DN A and the new stem-loop DNA with twice the stem length. The cyclic reaction is continued for less than one hour to accumulate 1 〇 9 copies of the target. The final product has several Stem-loop DNA of an inverted repeat target, and a cauliflower-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 DNA hybridization to identify the nucleotide composition of the amplicon. Electrophoresis is one of the simplest ways to check whether a nucleic acid amplification step produces the desired amplicon. Colloidal electrophoresis uses an electric field applied to a colloidal matrix to separate DNA fragments. Negatively charged DNA fragments will be at different rates ( The main-14-201209403 depends on its size) moving through the matrix. After the electrophoresis is completed, the fragments in the color colloid make it visible. Fluorescent phenanthrene in the UV light is the most commonly used dye. The size marker (DNA standard fragment (DNA)) is compared to determine the size of the fragment, and the DNA size label contains a DNA fragment of a size that is run along with the amplicon. Because the oligonucleon binds to a specific position adjacent to the target DNA, Amplification yields can be predicted and detected in bands of known size on the colloid. When so, or if several amplicons are produced, DNA probe hybridization is often used. DNA hybridization means complementary bases. DNA pairing to form a double strand for positive recognition of a particular amplification product requires the use of a DNA probe of about 20 nucleotides. If the probe has a sequence complementary to the amplicon DNA sequence, hybridization will occur under favorable temperature, ion concentration conditions. If hybridization occurs, it indicates that the DNA sequence of interest appears in the original sample. Optical detection is the most common method of detecting hybridization. The label extension is a probe that emits light by fluorescing or electrochemiluminescence. These trigger the excited states of the luminescent portion in different ways, but both are also covalently labeled with acid stocks. In electrochemiluminescence (ECL), when excited, light is generated by a luminophore molecule or a complex. When the fluorescent light is emitted, the emitted excitation light emits light. A luminescent source is used to detect fluorescence, and the illuminating source provides excitation light having a wavelength of fluorescence absorption and a detection unit. The detection unit contains photosensing, and the dyeable bromination ladder has a large amplification and amplification of DNA with known nucleotide primers. The length is (target pH and gene or enhancer or method of nucleoside current thorns to generate light molecules (the -15-201209403 such as photomultiplier tubes or charge coupled device (CCD) arrays) to detect the emitted signal, And a mechanism that prevents excitation light from being included in the output of the photosensor (such as a wavelength-selective filter). In response to the excitation light, the fluorescent molecules emit Stokes-shifted light, and the emitted light is detected. Unit collection. The Tox displacement is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. The light sensor is used to detect the ECL emission, 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, thus using a conventional photodiode and a CCD as a photosensor. The advantage of ECL is that if the ambient light is excluded, the ECL emission can be the only light present in the detection system. Thus, the sensitivity of the microarray enables 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 The presence or absence of several infectious pathogens is detected in a single assay. The microarray consists of a number of different DNA probes immobilized on a substrate and spotted. First, the target DNA (amplifier) is labeled with fluorescent or luminescent molecules. (during or after nucleic acid amplification), then applying it to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment, where the probe and the amplicon occur. Hybridization. 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. This results in a highly non-specific binding. Excessive stringency may result in inability to properly bind to a reduced sensitivity. Hybridization is identified by detecting the light emission from the labeled amplicon that hybridizes with the complementary probe to detect-16-201209403. The microarray scanner detects fluorescence from the microarray, which is typically a computer controlled inverted scanning fluorescent conjugated focus microscope. A laser and photosensor (such as a photomultiplier tube or CCD) that excites the fluorescent dye is typically used to detect the emitted signal. The fluorescent molecules emit down-converted light (as described above) and the light is collected by the detection unit. The fluorescent light 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 by conjugated focal pin holes located in the image plane. Information. This allows only the focused portion of the light to be detected. Light above or below the focal plane of the object is prevented from entering the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, electrical energy. It is then converted to a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. Find out more about fluorescent probes here: http : "www.premierbiosoft.com/tech_notes/FRET_probe.html and http : "www.invitrogen.com/site/us/en/home/References/ Molecular -probes-The-Handbook/Technical-Notes-and-Product-

Highlights/Fluorescence-Resonance-Energy-Transfer- -17- 201209403 FRET. Html In-situ Nursing Molecular Diagnostics Despite the advantages offered by molecular diagnostic tests, this type of test in clinical tests has grown less than expected and still accounts for only 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, provide initial (sample processing) to final (results), and the development of instruments that do not require extensive human manipulation. For physicians' clinics, proximity or user-based hospitals, home-based in-home healthcare technologies offer the following advantages: • Quick results are obtained to quickly promote treatment and improve care quality • Tested by testing a very small number of samples ability. • Reduce clinical workload. • Reduce laboratory workload and increase work efficiency by reducing management efforts. • Improve the cost per patient by reducing hospital stays, getting results from first visits, and simplifying the handling, storage, and delivery of samples. • Promoting clinical management decisions, such as infection control and antibiotic use. Laboratory (LOC)-based molecular diagnostics-18- 201209403 Microfluidic-based molecular diagnostic systems provide devices that automate and accelerate molecular diagnostic analysis. The shorter detection time is primarily due to the fact that the required sample volume is less active, automated, and low-cost built-in cascaded diagnostic method steps within the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are in the form of common microfluidic devices. The LOC device has an MST structure within the MST layer for integrating fluid processing onto a single support substrate (typically helium). The unit cost of each LOC device is very low by using the VLSI (Ultra Large Integrated Circuit) lithography technology of the semiconductor industry. However, controlling the flow of fluid through the LOC unit, adding reagents, controlling reaction conditions, and the like requires large external piping and electronics. Connecting the LOC devices to these external devices greatly limits the molecular diagnostic use of the LOC devices in the laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as a practical option in a local healthcare setting. In view of the above, there is a need for a molecular diagnostic system based on LOC devices for in situ care. SUMMARY OF THE INVENTION Various aspects of the invention are now described in the following paragraphs. GCF032. 1 The aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis of a biological sample. The LOC device comprises: an inlet for receiving the sample; a support substrate; a dialysis portion for the sample The group is separated from the larger component; -19- 201209403 a plurality of reagent storage tanks; a nucleic acid amplification section downstream of the dialysis section for amplifying the nucleic acid sequence in the sample; wherein the dialysis section and the nucleic acid amplification section Both are supported on the support substrate. GCF03 2. Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) unit. GCF03 2. Preferably, the LOC device also has a photodetector and a hybridization portion downstream of the PCR portion, the hybrid portion having an array of probes for hybridizing to the target nucleic acid sequence in the sample to form a probe-label A target hybrid, wherein the photosensor is configured to detect the probe-target hybrid. GCF03 2. Preferably, the dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the PCR portion, and a plurality of holes larger than the plurality of components and smaller than the large components. The two channels are in fluid communication with the first channel via the apertures such that when the larger components remain in the first channel, the plurality of components flow into the second channel. GCF03 2. Preferably, the first channel, the second channel, and the plurality of apertures are configured to buffer the sample by capillary action. GCF03 2. Preferably, the second channel is configured to attract the sample to the nucleic acid amplification portion by capillary action. GCF03 2. Preferably, the nucleic acid amplification unit is a thermostatic nucleic acid amplification unit 〇 GCF0 3 2. Preferably, the reagent storage tanks each have a surface tension valve for retaining the reagent therein, the surface tension valve having a meniscus holder -20-201209403 for fixing the meniscus of the reagent until The sample stream contacts the meniscus to allow the reagents to flow out of the reagent reservoir. GCF032. Preferably, the LOC device also has a flow path from the hybridization portion, wherein the flow path is configured to aspirate the sample from the inlet to the hybridization portion. GCF03 2. Preferably, the LOC device also has a CMOS, temperature sensor and micro system technology (MST) layer, the MST layer of the PCR portion, wherein the CMOS circuit is disposed between the support substrate MST layers, the CMOS The circuitry is configured to utilize temperature sensing to feedback control of the PCR section. GCF032. Il preferably, the PCR portion has a PCR microchannel that, during use, thermally cycles to amplify a nucleic acid sequence that defines a portion of the sample flow path and has a cross-section that is less than 100,000 square microns across the flow. GCF032. Preferably, the LOC device also has at least one elongated heater element for heating the nucleic acid in the extended PCR microchannel, the extended heater element extending in parallel to the PCR microchannel. GCF032. Preferably, at least one of the PCR microchannels is an extended PCR chamber. GCF032. Preferably, the PCR portion has a plurality of extended PCR chambers, each of which has a respective portion of the PCR microchannel, the microchannel having a meandering structure formed by a series of wide curved tracks. Each line forms a channel portion of one of the extended PCR chambers. GCF032. Preferably, the LOC device also has a function for receiving the removal port to be included in the hair circuit and the measurement and transmission, and the PCR area is formed in a length of the sequence length. • 21 · 201209403 reagent for PCR a reagent reservoir; and a surface tension valve having a bore configured to secure a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the liquid sample removes the Meniscus. GCF032. Preferably, the hybridization portion has an array of hybridization chambers for receiving the probes. GCF032. Preferably, the photosensor is an array of photodiodes arranged in alignment with the hybridization chamber. GCF03 2. Preferably, the CMOS circuit has a digital memory for storing the hybrid data output from the photo sensor and a data interface for transmitting the hybrid data to the external device. GCF032. Preferably, the PCR portion has an active valve for retaining liquid in the PCR portion during thermal cycling and allowing fluid to respond to activation signals from the CMOS circuit into the hybridization chamber. GCF032. Preferably, the active valve has a boiling initiation valve configured to secure a meniscus retainer that blocks the meniscus of the liquid drive flow, and to boil the liquid from the meniscus holder The heater that lifts the meniscus to retract the capillary drive flow. This LOC device design uses a better method than simple filtering, which reduces clogging and has the advantage of being able to separate the desired sample components from the unwanted sample components on a size basis. Such LOC devices have the advantages provided by amplification of a particular sequence, including: sensitivity provided by amplification; wide dynamic range; and high specificity for the target DNA sequence. -22-201209403 [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview This general specification indicates the main components of the molecular diagnostic system including the specific embodiments of the present invention. The details of the construction and operation of this system are explained below. Referring to Figures 1, 2, 3, 96 and 97, the system has the following heaviest components: Test modules 10 and 11 are typical USB flash drives and are inexpensive to manufacture. The test modules 10 and 1 each comprise a microfluidic device in the form of a lab-on-lab (LOC) device 30, and pre-reagents and typically more than one or more of these are used for the molecular diagnostic assay needle (see Figures 1 and 96)»When the test module 1 in Fig. 96 makes electrochemiluminescence-based detection technology, the module 1 shown in Fig. 1 uses a fluorescence-based detection technique to identify Targeting The LOC device 30 has an integrated light sensor 44 for fluorescence or electrochemiluminescence detection (below). Both test modules 10 and 1 1 are provided with a standard micro USB plug 14 for power, data and control, a printed circuit board (PCB) 57, and each having an externally supplied capacitor 彳 and inductor 15. Both test modules 10 and 11 are for a single use for large-scale use and are distributed in sterile packaging for use. The outer casing 13 has a large container 24 for receiving a biological sample and a sterile sealing tape 22 which preferably has a low viscosity adhesive to cover the large container. The membrane seal 408 having the membrane guard 4 10 is formed in a very general book, and the test sample is used. The details are all reduced to 32. Remove the front part -23- 201209403 Shell 1 3 to reduce the moisture resistance of the test module, and the pressure is released by the small pressure. The membrane guard 410 protects the membrane seal 40 8 from the test module 1 or the test module 1 2 via the micro-USB 埠1. The test module reader 12 can be many, and its selection is described later. The 12 version shown in Figures 1, 3 and 96 is a specific embodiment of a smart phone. The reader 12 is shown in Figure 3. The processor 42 executes from the program storage 43 software processor 42 and also interfaces with the display screen 18 and the user interface (control screen 17 and button 19, cellular radio 21, wireless network 23, and satellite navigation system 25. Honeycomb The radio 21 network connection 23 is used for communication. The satellite navigation system 25 is used for the data update epidemiological database. Alternatively, the location data can be manually input by touching the P or the button 19. The data storage 27 transmits and diagnoses the information. , test results, patient information, used to identify each analysis and probe data and their array location. Data storage 27 and memory 43 can be shared in a common memory device. Test module reads f installed application software provides results analysis In addition to the test and diagnosis to perform a diagnostic test, the test module 10 (or the test strip is inserted into the micro-USB port 16 on the test module reader 12). The sealing band 22 is turned up and the biological sample is presented. The liquid form is loaded into the large container 24. The start button 20 is pressed to be tested by the application software. The sample flows into the LOC device 30 and is extracted, cultured, amplified and analyzed by onboard analysis (0) assay. The synthesized hybrid-reactive acid probe hybridizes with the sample nucleic acid (target). The damage is detected in the test module 1. The application UI of the same type reader block diagram is connected to the touch screen and wireless to position the screen. The information stored in the program is stored in the I. 12. An) sterilized to the sample to start the test of n-board oligonuclear -24-24 - 201209403 (using fluorescence-based detection), the probe is The LEDs 26 that are fluorescently labeled and placed in the shell 13 provide the necessary excitation light to induce fluorescence emission from the hybridized probe (see Figures 1 and 2). In test module 1 1 (which uses electrochemiluminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to produce illumination. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 97). A light sensor 44 integrated with a CMOS circuit on each LOC device is used to detect the emission (fluorescence or photoluminescence). The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the result. Data can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 1 1 is removed from the test module reader 1 2 and processed as appropriate. 1, 3 and 96 show a test module reader 12 configured as a mobile/smartphone 28. In other forms, the test module reader is a laptop/notebook 101, a dedicated reader 1〇3, an e-book reader 107, a tablet 109 used in a hospital, a private clinic, or a laboratory. Or a desktop computer 1〇5 (see Figure 98). The reader can be interfaced with additional applications such as patient records, accounting, online databases and multi-user environments. It can also interface with some local or remote peripheral devices, such as printers and patient smart cards. Referring to FIG. 99, the data generated by the test module 1 through the reader 12 and the network 125 can be used to update the epidemiological database contained in the host system for the epidemiological data 111 for genetic data. 1 1 3 Host-25- 201209403 The genetic database contained in the system, the electronic health record contained in the host system for Electronic Health Record (EHR) 115, for electronic medical records (EMR) 121 The electronic medical record contained in the host system and the personal health record contained in the host system for Personal Health Record (PHR) 123. Conversely, the epidemiological data contained in the host system for epidemiological data 111 via the network 1 2 5 and the reader 1 2 ', the genetic data contained in the host system for the genetic material 113, Electronic health records contained in the host system for Electronic Health Record (EHR) 115, electronic medical records contained in the host system for Electronic Medical Record (EMR) 121, and for personal health records (PHR) 123 The personal health record contained in the host system can be used to update the digital memory in the LOC 30 of the test module 10. Referring again to Figures 1, 2, 96 and 97, in the mobile phone configuration, read The device 12 uses battery power. The mobile phone reader contains all preloaded test and diagnostic information. Data can also be loaded or updated via some wireless or contact interface to enable communication with peripheral devices, computers or online servers. A micro-USB port 16 is provided to connect a computer or a primary power supply to recharge the battery. Figure 70 shows a specific embodiment of the test module 10 for testing that only needs to know the presence or absence of a particular target, such as an experiment. Whether the individual is infected with, for example, influenza A virus Η 1 N 1 . It is only suitable as a built-in module 47 for USB power/indicator only. No other readers or application software is required. For example, the indicator 45 on the module 4 of the S B power/indicator does not produce positive or negative results. This configuration is ideal for a large number of inspections. Additional items supplied to the system by beta -26-201209403 may include test tubes containing reagents for pre-treatment of specific samples, and tongue depressors and lancets containing sample collection. For convenience, the test module of the embodiment shown in Fig. 70 includes a spring-loaded retractable lancet 309 and a lancet release button 392. Satellite phones can be used in remote areas. Test Module Electronics Figures 2 and 9 are each a block diagram of the electronic components of test modules 1 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a timer 33, a power conditioner 31, a RAM 38, and a program and data flash memory 40. These are provided for the test module 10 including the photo sensor 44, the temperature sensor 170, the liquid sensor 174 and the various heaters 1 5 2, 1 5 4, 182, 234. Or 11 and associated drivers 37 and 39 and the control and memory of registers 35 and 41. Only the LED 26 (in the example of the test module 10), the external power supply capacitor 32, and the micro USB plug 14 are external to the LOC device 30. The on-wafer laboratory device 30 includes an adhesive pad for joining to these external components. The RAM 38 and the program and data flash memory 40 have application software and diagnostic and detection information for one probe (flash/security storage, such as via encryption). In the example of the test module 1 1 configured with ECL detection, there is no LEd 26 (see Figures 96 and 97). The data is encrypted by the LOC device 30 to preserve storage and communicate with external devices. The L0C device 30 is fabricated in a number of test forms with electrochemiluminescence probes and the hybridization chamber load 'each having many types of ECL excitation electrode pairs 860 and 870 -27-201209403 test module 10' ready for ready use . These test formats differ in the onboard analysis of reagents and probes. Some examples of infectious diseases that are quickly identified by this system include: • Influenza-influenza virus A, B, C, infectious salmon anemia virus, tocovirus • pneumonia-respiratory fusion virus (RSV), gland Virus, interstitial pneumonia virus, pneumococci, Staphylococcus aureus • tuberculosis - Mycobacterium tuberculosis ' Mycobacterium bovis 'Mycobacterium tuberculosis, Mycobacterium vaccae and Mycobacterium vaccae • Plasmodium falciparum, Toxoplasma and other parasitic protozoa • Typhoid- typhoid • Ebola • Human immunodeficiency virus (HIV) • Dengue fever • Flavivirus • Hepatitis (A to E) • Iatrogenic infections – such as refractory spores Bacteria, vancomycin-resistant enterococci and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) • Encephalitis - Japanese encephalitis virus, chapter Deppa virus • Pertussis-Bertus bacillus -28- 201209403 • Ma Yang-paramyxovirus • Meningitis - Streptococcus pneumoniae and meningococcus • Anthracnose - Bacillus anthracis Some examples of sexually transmitted diseases include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black scrophular idiots • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic renal rickets • Congenital Cardiac Disease • The few options for the diagnosis of cancer by Leier's disease include: • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinoblastoma • Turcot syndrome The above list is not exhaustive. And the diagnostic system can be configured to detect many different diseases and symptoms using nucleic acid and protein analysis. Detailed structure of the system components -29- 201209403 LOC device L 0 C device 30 is the center of the diagnostic system. It uses microfluidics to rapidly perform four important steps in nucleic acid-based molecular diagnostic analysis: sample preparation, nucleic acid extraction, nucleic acid amplification, and detection. The LOC device has an alternative use and will be detailed below. As discussed above, trials 1 and 1 can take many different configurations to detect different targets. Again, the LOC device 30 has a number of different embodiments for targeting the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a body of a whole blood sample. For the purposes of this description, the structure and operation of the LOC device 301 are described in detail with reference to Figures 4 and 27 through 57. 4 is a schematic diagram showing the structure of the LOC device 301. For convenience, the processing stages shown in Fig. 4 are represented by the component symbols corresponding to the functional portions of the apparatus 301 that implements the processing stage. The processing stages associated with each of the major steps in the diagnostic analysis of nucleic acids are also indicated: Input and Preparation 288, Extraction 290, Culture 291, and Expansion 292 to determine 2 94. The various reservoirs, chambers, valves, and components of the LOC unit 30 1 will be described more closely below. Figure 5 is a perspective view of the LOC device 301. It is manufactured using high CMOS and M ST (microsystem technology) manufacturing techniques. The layered structure of LOC 3 〇1 is a partial cross-sectional view of Fig. 12 (not to be described in detail. LOC device 301 has a board 84 supporting COMS + MST wafer 48, including CMOS circuit 86 and MST layer 87, to cover 46 MST layer 87. For the purposes of this patent specification, the term "MS T layer platform, also has a specific sample of the pathogens. The basic sample of the LOC and the sample of the volume of the inspection group" 30- 201209403 A collection of structures and layers of samples treated with different reagents. Thus, these structures and components are configured to define a flow path having a characteristic size that supports a capillary action driven liquid flow having physical properties similar to the physical properties of the sample during processing. Accordingly, MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and assemblies that are sized for liquid flow driven by capillary action and that are processed to a very small volume. The particular embodiment described in this specification shows that the MST layer is a structural and active component supported on CMOS circuitry 86, but excluding the features of cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 microns X 5824 microns. Of course, LOC devices made for different applications can have different sizes. Figure 6 shows the features of the MST layer 87 overlaid with the cover features. The 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 will be 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 showing the layered configuration of the CMOS + MST device 48, the cover 46, and the fluid interaction therebetween. The figures are not drawn to scale for illustrative purposes. Figures -31 - 201209403 12 are schematic cross-sectional views through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12', the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86»passivation layer 88 that operates the active components in the MST layer 87 described above to seal and protect the CMOS layer 86 from Fluid flows through the MST layer 87. Fluid flows through both the cover channel 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 100 (see, for example, Figures 7 and 16). While the biochemical treatment is being performed on the smaller MST channel 90, cell transport occurs in the larger channel 94 made in the cover 46. The cell transport channel is sized to carry the cells in the sample to a predetermined point in the MST channel 90. Transporting cells larger than 20 microns in size (e.g., certain white blood cells) requires channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. The MST channel, particularly at locations in the LOC where transport of cells is not required, can be significantly small. It will be understood that the cover channel 94 and the MST channel 90 are of the same reference and that the particular MST channel 90 can also be referred to as, for example, a heated microchannel or a dialysis MST channel depending on its particular function. The MST channel 90 is formed by etching through an MST channel layer 1 deposited on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is surrounded by a top layer 66 that forms the top of the CMOS + MST device 48 (relative to the orientation shown in the figure). Although sometimes shown as a separate layer, the cover channel layer and the reservoir layer 78 are formed from a single piece of material. Of course, the sheet of material may also be non-uniform. The sheet of material is etched from both sides to form a lid channel layer and a reservoir -32 - 201209403 layer 78, which is etched in the lid channel layer 80, and uranium engraved tanks 54, 56, 58, 60 and 62 are stored in the reservoir layer 78. Further, the sump and the cover passage are formed by a micro-forming process. Both etching and microforming techniques are used to fabricate channels having a cross-sectional area across the fluid that is as large as 20,000 square microns and as small as 8 square microns. There are a series of suitable choices for one of the cross-sectional areas of the channel across the fluid at different locations in the LOC device. A large number of samples or samples having a large component are accommodated in the channel, and a cross-sectional area of more than 20,00 square micrometers (e.g., a 200 micrometer wide channel in a layer of 100 micrometers thick) is suitable. A small amount of liquid or a mixture free of large cells is contained in the channel, preferably across a very small cross-sectional area of the fluid. A lower seal 64 surrounds the cover passage 94 and the upper seal layer 82 surrounds the sump 54, 56, 58, 60 and 62. The five reservoirs 54, 56, 58, 60 and 62 are preloaded to analyze specific reagents. In the particular embodiment described, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant with a choice of red blood cell lysis buffer • Storage tank 5 6 : Dissolution Reagents • Storage tank 5 8 : Restriction of enzymes, ligases and junctions (for junction-priming PCR (see Figure 69, from τ· Stachan et al.) , Human Molecular Genetics 2, Garland Science, NY and London, 1 999 excerpt) • Storage tank 60: amplification mixture (deoxynucleotide triphosphate (ciNTPs -33 - 201209403), primer, buffer) and • storage tank 62: The DNA polymerase cover 46 and the CMOS + MST layer 48 are in fluid communication via respective openings in the lower seal 64 and the top layer 66. The openings represent the upper conduit 96 and the lower conduit 92 depending on whether fluid flows from the MST passage 90 to the cover passage 94 or vice versa. LOC Device Operation The operation of the LOC device 301 is described step by step with reference to the analysis of pathogen DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using appropriate reagents, assay protocols, L Ο C variants, and detection systems. Referring to Figure 4, the analysis of biological samples involves five major steps, including • sample input and preparation 288, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294. The sample input and preparation step 288 involves mixing the blood with the anticoagulant 1 16 and then separating the pathogen from the white blood cells and red blood cells by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, the blood sample enters the device via the sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood flow opens its surface tension valve 1 18, the anticoagulant is released from the sump 54 (see Figures 15 and 22). The anticoagulant prevents the formation of blood clots that can block flow. As best shown in Figure 22, the anticoagulant 116 is withdrawn from the sump 54 by capillary action and enters the MST channel 90 via the lower conduit 92. The lower duct 92 has a capillary initiation configuration feature (CIF) 102 to form a meniscus geometry of -34 - 201209403 such that it is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent 122 in the upper seal 82 allows air to replace the anticoagulant. The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and is secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. The meniscus 120 remains fixed to the upper conduit 96 prior to use such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the lid channel 94 to the upper conduit 96, the meniscus 110 is removed and the anticoagulant is drawn into the fluid. Figures 15 through 21 show an inset AE which is part of the inset AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 18 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. The flow rate of the sample mixture in the sample stream is determined by the flow rate of the sample mixture as it is mixed by diffusion and the reagents. Thus, the surface tension valve for each of the sump is configured to meet the desired reagent concentration. The blood passes through the pathogen dialysis section 70 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of wells 1 64 sized according to a predetermined valve. Cells smaller than the valve pass through the holes, and large cells cannot pass through the holes. While the target cells continue to be part of the analysis, the unwanted cells are reintroduced into the waste unit 76. The undesired cells are large cells that are blocked by the array of such holes 1 64, or small cells that pass through the holes -35-201209403. In the pathogen dialysis section 70 described herein, the whole blood pathogen is concentrated for microbial DNA analysis. The array of apertures is formed by communicating the input flow in the cover channel 94 to the aperture 1 64 of the target channel 74. The 3 micron diameter hole 1 64 and the dialysis suction port 168 of the target channel 74 are connected by a series of through channels 206 (best shown in Figures 15 and 21). The pathogen is filled with the target channel 74 through the dialysis MST channel 204 through the 3 micron diameter aperture. Cells larger than 3 microns, such as red blood cells, remain in the waste channel 72 of the lid 46, which passes through the waste reservoir 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate specific target cells such as white blood cells for human DNA analysis. More detailed details for the dialysis department and dialysis variants. Referring again to Figures 6 and 7, the fluid is drawn through the target 3 to the surface tension valve 128 in the lysis reagent reservoir 56. The force valve 128 has seven MST channels 90 extending between the lysis test and the target channel 74. When the meniscus flows from the sample, the flow rate from all seven of the MST channels 90 will be greater than the flow rate from the agent sump 54, wherein the surface tension valve 118 has three 1 channel 90 (assuming the physical properties of the fluid are The ratio of the lysing reagent in the sample mixture is substantially equal to the anticoagulation ratio. The lysis reagent and the target cell are used in the chemical lysing portion of the sample by using 3 micrometers for the MST to be small to the foot 164 and the ball and the white channel are led to the original body or the surface of the channel 74 is later introduced. Anticoagulant circle MST when the agent storage tank is removed. Therefore, the target of the blood agent -36- 201209403 is mixed by diffusion in channel 74. The boiling initiation valve 126 stops the flow until diffusion and lysis occur for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling initiation valve are described in detail below with reference to Figs. 31 and 32. Other active valve types (as opposed to passive valves such as the surface tension valve 118) have also been developed by the applicant, which can be used in place of the boiling initiation valve. These alternative valve designs are also described below. When the boiling initiation valve 126 is opened, the lysed cells flow into the mixing portion 131 for pre-amplification restriction digestion and linker ligation. Referring to Fig. 13, when the fluid is removed from the meniscus on the surface tension valve 132 starting from the mixing portion 131, the restriction enzyme, the linker and the ligase are released from the reservoir 58. The mixture flows through the length of the mixing portion 131 for diffusion mixing. At the end of the mixing portion 131 is a lower duct 134 (see Fig. 13) leading to the incubator inlet passage 133 of the cultivating portion 114. The feeder inlet channel 133 feeds the mixture into the crucible configuration of the heated microchannel 210, which provides a culture chamber that retains the sample during restriction enzyme digestion and junction assembly (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the inset AB of Figure 6. The various figures show the continuous attachment of the layers forming the CM0S + MST layer 48 and the structure of the cover 46. The inset AB shows the end of the culture portion 114 and the start of the amplification portion 112. As best shown in Figures 14 and 23, the fluid breaks into the microchannels 210 of the culture portion 114 until the boiling initiation valve 106 is reached, wherein the fluid stops while diffusion occurs. As discussed above, the boiling initiation valve 106 on the -37-201209403 swims the microchannel 210 into a culture chamber containing the sample, restriction enzymes, and junction junctions. The heater 154 is then activated and maintains stability for the occurrence of restriction enzyme digestion and coupling for a specific period of time. Those skilled in the art will understand that this incubation step 29 1 (see Figure 4) is only required for some nucleic acid amplification. Increase the type of analysis. Furthermore, in some cases, it may be desirable to have a heating step at the end of the incubation period to increase the temperature above the culture temperature. This increase before entering the amplification section 1 1 2 causes the restriction enzyme and ligase to be inactivated. When thermostated nucleic acids are used, there is a specific association between restriction enzymes and ligase inactivation. After the cultivation, the boiling initiation valve 106 is activated (turned on) and flows back to the amplifying portion 112. Referring to Figures 31 and 32, the mixture is heated to the structure of the microchannel 1 58 until the boiling initiation valve is reached, the microchannels forming one or more amplification chambers. As best shown in cross-sectional view of 30, the amplification mixture (dNTPs, primers, buffers are released from storage tank 60 and polymerase is then released from storage tank 62 into the junction and the amplification portion (each 1 1 4 and 1 1 2 ) The intermediate MST channels are shown in Figures 35 through 51 as layers of the LOC device in the inset AC of Figure 6. Each of the figures shows a layer that is continuously stacked to form a CMOS + MST device 48 46. The end of the AC line amplification portion 112 is illustrated. Hybridization and initiation of the moiety 52. The cultured sample, amplification mixture and polymerase microchannel 158 are passed to the boiling initiation valve 1〇8. After the diffusion mixing is sufficient, the heater 154 in the microchannel 158 is activated. Heating cycle or amplification. The amplification mixture undergoes a predetermined number of thermal cycles or preset enzyme expansion and temperature stabilization. Select an example to charge the temperature amplification fluid to the liquid) culture 2 12 30 1 and cover detection The flow time is constantly increased from -38 to 201209403 to amplify sufficient target DNA. The operation of the nucleic acid amplification procedure to initiate valve 108 opening and fluid re-entry into the hybridization and detection section is described in more detail below. As shown in Fig. 52, the hybridization and detection section 52 has a matrix 110. Figures 52, 53, 54, and 56 show hybridization and individual hybridization chambers 180 in detail. The inlet 175 of the hybridization chamber 180 prevents diffusion between the target nucleic acid, the probe strand, and the hybridization chamber 180 during hybridization to prevent the erroneous hybridization detection barrier 175 from having a flow path length long enough for the probe and During the time of detecting the signal, the target sequence and the probe are prevented from escaping and contaminating another chamber, thus avoiding erroneous results. Another mechanism to prevent erroneous reading is in some of the same probes that hybridize. The CMOS circuit 86 derives a single result from the photodiode 184 containing the hybridization chamber 180. The result of the anomaly in the result can be ignored or the thermal energy required to supply the hybridization with different specific gravity is controlled by CMOS heating (more) Detailed description is given below). After the heater is activated, it is complementary between the target probe sequences. The CMOS circuit 86 driver 29 transmits a message to the test module 1 . These probes only fluoresce when hybridization occurs to avoid washing and drying steps to remove unbound strands. The stem-and-loop structure of the hybrid strong probe 186 is opened, which allows the fluorescent energy of the LED to excite the light, as detailed below. After the light in the CMOS circuit 86 under the hybridization chamber 180, the light is boiled 52. The boiling chamber is introduced into the chamber. The chamber array 1 1 0 is the diffusion barrier. The hybrid probe is used to test the results. The nucleic acid hybridization has the same probe in one chamber expansion chamber. Deriving the hybridization of the single-shot device 182, the LED LED 26 emits light, and usually needs to be used to detect the FRET light group to emit light. The detection is performed by the respective polar bodies 1 - 8 - 39 - 201209403 (see the hybrid cavity below). The photodiode 184 and associated electronics for all of the hybridization chambers together form the photosensor 44 (see Figure 64). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected by the photodiode 1 84 is amplified and converted to a digital output that can be analyzed by the test module reader 12. Further details of this 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 54, 56, 58, 60 and 62, a dialysis section 70, a lysis section 130, a culture section 114, and an amplification section 1 12, Valve type, humidifier and humidity sensor. In other embodiments of the LOC device, such functional portions may be omitted and additional functional portions or functional portions for alternative uses of the above-described devices may be added. For example, the culture portion 1 14 can be used as the first amplification portion 1 1 2 of the repeated sequence amplification analysis system, and the chemical lysis reagent storage tank 56 can be used to add the first amplification mixture of the primer, the dNTP, and the buffer. A reagent reservoir 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 56, or alternatively, the sample may be heated for a predetermined period of time to cause thermal lysis in the culture. In some embodiments, if chemical lysis is required and the chemical lysis reagent is mixed with the mixture, additional storage tanks may be combined upstream of the sump for mixing the primers, dNTPs, and buffers. In the case, the steps such as the cultivation step 291 are omitted. In the case of -40-201209403, the LOC device can be specially manufactured to prevent the reagent storage tank 58 and the culture portion 114 or the storage tank from carrying more than the reagent, or if there is an active valve, it is not activated to dispense the reagent to the sample flow. The medium and the culture unit are simply passages for transferring the sample from the lysis unit 130 to the amplification unit 112. The heater operates independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the flow system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after the amplification phase 292, prior to the hybridization and detection step 2 94, dialysis is performed to remove the cell debris system. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplifications 1 1 2 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using specific nucleic acid probes in the hybridization chamber array 1 1 . To analyze, for example, a sample of whole blood in which dialysis is not required, the dialysis portion 70 is simply omitted from the sample input and preparation portion 2 88 of the LOC design. In some cases, even if the analysis does not require dialysis, it is not necessary to omit the dialysis section 70 from the LOC device. If the presence of the dialysis section does not cause geometrical obstruction, the sample input and preparation portion can still have the LOC of the dialysis section 70 without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybridization chamber array but carries a probe that is designed to conjugate or hybridize to a sample target protein present in the non-amplified sample, rather than a design A nucleic acid probe for hybridization to a target nucleic acid sequence. It will be appreciated that the LOC device 201209403, which is manufactured for use in this diagnostic system, differs in the combination of functional components selected for the particular LOC application. Most of the functional components are common to many LOC devices, and the design of additional LOC devices for new applications has a combination of appropriate functional components in the bulk of the functional options used in existing LOC devices. Only a few LOC devices are shown in this description, and some others are shown to illustrate the design flexibility of the LOC devices manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices shown herein are not exhaustive, and that many additional LOC designs are related to the combination of appropriate functional components. Sample Type LOC Variants can accept and analyze a variety of nucleic acid or protein contents in liquid form, including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord Blood, breast milk, sweat, pleural effusion, tears, pericardial fluid, peritoneal fluid, environmental water samples and beverage samples. Amplicon derived from meganucleic acid amplification can also be analyzed using a LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, and only use dialysis, lysis The culture and amplification section delivers the sample from the sample inlet 6 8 to the hybridization chamber for nucleic acid detection 180, as described above. For some sample types, a pre-treatment step is required, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and possibly pre-treat the mucus with enzyme to reduce stickiness. Sample Input Refer to Figures 1 and 12 to add the sample to the large container 24-42 - 201209403 of the test module. 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 μηι wide 60 μπη deep cover channel 94 and is also attracted to the anticoagulant storage tank 54 by capillary action. Reagent Tanks The use of microfluidic devices, such as the small amount of reagents required by the analysis system of the LOC device 301', 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 1,0 0 0,0 0 0,0 0 0 micron, in most cases less than 300,000,000 cubic microns, generally less than 70,000,000 cubic microns, and shown in the figure In the case of LOC device 301, it is less than 20,000,000 cubic microns. Dialysis section 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, a plurality of apertures in the top layer 66 are apertures 164 having a diameter of 3 microns, filtering the target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice 164, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via a 16 [mu] dialysis extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) is retained in the cover channel 94. Downstream of the pathogen dialysis section 70, the cover channel 94 becomes a waste channel 72 to the waste reservoir 76. The foam (f0am) insert-43-201209403 or other porous elements in the outer casing 13 of the test module 10 are configured to be fluid with the waste reservoir 76 for a type of biological sample that produces a relatively large amount of waste. Connected (see Figure 1). The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. An orifice 164 having a diameter of 3 microns at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Fig. 33) such that the fluid is pulled downward into the dialysis MST channel 2〇4 below. The first suction port 198 for the standard channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus above the dialysis suction port 168. The small component dialysis section 682 shown in Fig. 7 4 may have a structure similar to that of the pathogen dialysis section 70. The small component dialysis section separates the sample from any small target cells or molecules by sizing (and shaping, if necessary) to allow small target cells or molecules to pass to the target channel and continue to further analyze the well. Large size cells or molecules are removed to waste reservoir 766. Thus, LOC device 30 (see Figures 1 and 96) is not limited to isolation of pathogens smaller than 3 μηη, but can be used to isolate cells or molecules of any desired size. Lysis Department Referring again to Figures 7, 11, and 13, the chemical species in the sample are released from the cells by chemical lysis. As described above, the lysis reagent from the lysis tank 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 128 of the lysis tank 56. However, some diagnostic assays are preferably suitable for hot lysis treatment, or even a combination of chemical and thermal lysis of target cells. The LOC device 301 houses the heated microchannels 210-2012-201209403 of the culture portion 114. The sample stream is flooded with the culture portion 114 and stopped at the boiling initiation valve 106. The culture microchannel 210 heats the sample to the temperature at which the cell membrane ruptures. In some hot lysis applications, the enzyme reaction is not required in the chemical lysis unit 130, and the hot lysis completely replaces the enzyme reaction in the chemical lysis unit 130. Boiling Initiating Valve As discussed above, LOC unit 301 has three boiling inducing valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling initiation valve 108 at the end of the heated microchannel 158 of the amplification section Π2. By capillary action, the sample stream 1 1 9 is drawn along the heated microchannel 158 until it reaches the boiling initiation valve 108. The meniscus 120 at the leading edge of the sample stream is secured to the meniscus holder 98 of the valve inlet 146. The meniscus holder 9 8 geometry stops the meniscus and prevents capillary flow. As shown in Figures 31 and 32, the meniscus holder 98 is an orifice provided by the opening of the pipe from the MST passage 90 to the cover passage 94. The surface tension of the meniscus 使 2〇 keeps the valve closed. Ring heater 1 52 is located around valve inlet 1 46. The ring heater 1 5 2 is controlled by C Μ Ο S via the boiling induced valve heater contact 1 5 3 . To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater contact 1 53. The ring heater 1 52 is resistively heated until the liquid sample 1 19 is boiled. Boiling removes meniscus 120 from valve inlet 146 and begins to wet cover passage 94. Once the lid passage 94 is wetted, the capillary action is restored. The fluid sample 119 is filled with the passage 94 and flows through the lower valve conduit 15 and -45-201209403 to the valve outlet 1 48, wherein the capillary-driven liquid flow advances along the expansion outlet passage 160 to the hybridization and detection portion 52. . The liquid sensor 174 is placed before and after the valve for diagnosis. It will be understood that once the boiling trigger valve is opened, it is impossible to close it again. However, since the LOC device 301 and the test module 10 are single-purpose devices, it is not necessary to close the valve. Culture section and nucleic acid amplification section The culture section 114 and the amplification section 112 are shown in Figs. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51. The culture portion 114 has a single, heated culture microchannel 210 that is etched to form a ruthenium pattern in the MST channel layer 100 from the lower conduit opening 134 to the boiling initiation valve 〇6 (see Figures 13 and 14). ). Controlling the temperature of the culture portion 114 enables a more efficient enzyme reaction. Similarly, the amplifying portion 112 has an amplifying microchannel 158 (see Figs. 6 and 14) which is heated from the boiling inducing valve 106 to the boiling initiation valve 108. Upon mixing, culture, and nucleic acid amplification, the valves stop flow to retain the target cells in the heated culture or amplification microchannels 210 or 158. The microchannel 蜿蜒 pattern also promotes (to some extent) the target cells to be mixed with the reagents. In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated via a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each of the turns of the heated culture microchannel 210 and the amplification microchannel 158 has three independently operable heaters 1 54 extending between the individual heater contacts 156 (see Figure 14). 46- 201209403), which provides two-dimensional control of the input heat flux density. As best shown in Figure 51, heater 154 is supported on top layer 66 and buried in lower seal 64. The heater material is TiAl, but many other conductive metals are also suitable. The elongated heater 154 is parallel to the longitudinal length of each of the channel portions forming the meandering meandering. In the amplification section 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 the LOC device 30, requires a small volume of amplicons required for amplification in the amplification portion 112 to use a small volume of amplification mixture. This volume is less than 400 nanoliters, in most cases less than 170 nanoliters, less than 70 nanoliters, and in the case of LOC device 310, this volume is between 2 nanoliters and 30 nanoliters. between. Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a relatively short distance from the heater 154. The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 100,00 square microns, while the "high scale" device has a significantly higher heating rate. The lithography manufacturing technique allows the amplifying microchannel 158 to have a cross-section that spans a flow path that provides a relatively high heating rate of less than 16,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small number of amplicons (as is the case in LOC device 301) are required, 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 a "sample into" answer within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and -47- 201209403 1 square micron. The heater element in the amplification microchannel 1 58 heats the nucleic acid sequence at a rate of greater than 80 absolute temperatures (K) per second, which in most cases is greater than 1 〇〇 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than 1 000 K per second, and in many cases, the heater element heats the nucleic acid sequence at a rate greater than 10, 〇〇〇 K per second. Typically, based on the requirements of the analytical system, the heater elements are greater than 100,000 K per second, greater than 1,000,000 K per second, greater than 10,000,000 K per second, greater than 20,000,000 K per second, and greater than 40,000,000 K per second. The nucleic acid sequence is heated at a rate greater than 80,000,000 K per second 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 density of the phenomenon decreases with distance from the interface. A microchannel with a relatively small cross-sectional area across the flow direction is used while keeping the two fluids flowing through the interface for rapid diffusion mixing. Reducing the cross-section of the channel to less than 1 〇〇, 〇〇〇 square micron, results in a significantly higher diffusion rate than the "large scale" devices. The lithography manufacturing technique allows the microchannels to have a cross-section that spans a flow path that provides a higher mixing rate of less than 16,000 square microns. 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 entry, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 40 0 square microns and 1 square. Micro-48- 201209403 • Between meters. A short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid' to cause rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e., variability, adhesion, and primer extension) is completed in 30 seconds for a sequence of up to 15 〇 base pairs (bp) long. In most diagnostic analyses, the individual thermal cycle times are less than 11 seconds and most are less than 4 seconds. For a target sequence of up to 150 base pairs (bp) long, the thermal cycle time of the LOC device 30 for some of the most common diagnostic analyses is zero. 45 seconds to K5 seconds. This rate of thermal cycling allows the test module to perform nucleic acid amplification procedures in less than one minute; often within 220 seconds. For most analyses, the amplification section produces sufficient amplicons from the sample fluid entering the sample inlet within 80 seconds. For most of the analysis, sufficient amplicon is generated in 30 seconds. Upon completion of the predetermined number of amplification cycles, the amplicon is fed to the hybridization and detection section 52 via the boiling initiation valve 108. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chambers 180 in the hybridization chamber array 11A. 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 184 is incorporated to detect fluorescence from a target nucleic acid sequence or protein that hybridizes to FRET probe 186 -49-201209403. 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 97 between the probe 186 and the photodiode 1 8 4 must also be optically transparent to the emitted light. In the LOC device 301, the wall portion 97 is a thin layer of cerium oxide (about 〇. 5 microns). Direct intrusion of photodiode 184 under each hybridization chamber 180 allows the use of a very small volume of probe-target hybrid, yet still produces 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 to detect the probe-target hybrid is greater than 2 70 picograms (corresponding to 900,000 cubic micrometers), in most cases less than 60 pico G (corresponding to 200,000 cubic microns), typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and less than 2. in the case of the LOC device 301 shown in the figures. 7 picograms (corresponding to a chamber 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 hybrid has more than one chamber (i.e., less than 2,250 square microns per chamber) in an area of 1,500 microns by 1,500 microns. The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of the small number of probes required for each chamber is that during the manufacture of the L0C 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 using a probe solution of 1 nanoliter or less. After nucleic acid amplification, the boiling initiation valve 108 is activated and the amplicon flows along the -50-201209403 flow path 176 and into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates the point at which the hybridization chamber 180 is filled with the amplicon and the heater 182 can be activated. After sufficient hybridization time, the LED 26 (see Figure 2) is activated. » The opening in each of the hybrid chambers 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 illumination continues for a sufficiently long period of time to induce a high intensity fluorescent signal from the probe. The photodiode 184 is shorted during the excitation. After a preprogrammed delay of 3 〇〇 (see Figure 2), the photodiode 184 is enabled and the fluorescent emission is detected under no excitation light. The incident light on the active region 1 85 of the photodiode 1 84 (see FIG. 54) is converted into a photocurrent that can be measured using the CMOS circuit 86. Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. needle. If desired, each hybridization chamber 180 can carry probes that detect more than 1,000 different targets. Alternatively, many or all of the hybridization chambers may carry the same probe that repeatedly detects the same target nucleic acid. Copying the probes in this manner in the hybrid chamber array 1 1 使得 increases the confidence of the results obtained, and if desired, all results can be combined by photodiodes of adjacent hybridization chambers to obtain a single result. Those skilled in the art will appreciate that there may be from 1 to over 1,000 different probes on the hybridization chamber array 110, depending on the analysis details. Humidifier and Humidity Detector The inset AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of -51 - 201209403 LOC device 3 0 1 evaporation of reagents and probes during operation. As best seen in the enlarged view of Fig. 55, the water sump 188 is fluidly coupled to the evaporator 190. The water storage tank 188 is filled with molecular biological grade water and sealed during manufacture. As best shown in Figures 55 and 67, by the effect, water is drawn to the three lower conduits 1 94 and along the individual water supply passages 192 to the three upper conduits 193 of the evaporator 190. The meniscus is placed on each of the upper tubes 193 to hold the water. The evaporator has a ring-shaped addition 191 which surrounds the upper pipe 193. With a thermally conductive post 3 76, a ring-shaped 191 is connected to the CMOS circuit 86 to the top metal layer 195 (see |). At startup, the ring heater 191 heats the water causing the water to evaporate around the device. The position of the humidity sensor 232 is also shown in FIG. However, as shown in the enlarged view of the inset AH shown in Fig. 63, the humidity sensing has a capacitive comb structure. The lithographically etched first electrode 296 and the lithographically etched second electrode 298 are opposite each other to make their teeth. The opposing electrodes form a capacitor with a capacitance that can be monitored by CMOS electricity g. As the humidity increases, the dielectric of the air gap between the electrodes increases, causing the capacitance to increase. Humidity sensor 23 2 is adjacent to the hybridization chamber 1 1 〇 (the most important humidity measurement location) to slow the evaporation of the solution containing the exposed needle. The feedback sensor temperature and liquid sensor system is integrated into the LOC device 301 to provide feedback and diagnostics during operation. Referring to Figure 3 5, the nine temperatures are displayed in three by the hair-in-the-surface heat exchanger I 37 and the wet optimal instrument and the interpolated ^ 86 constant chamber array of the sense of installation -52-201209403 170 is distributed to the entire amplification unit 112. Similarly, the culture portion 114 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control the thermal cycling during the nucleic acid amplification process and any heating during thermal lysis and incubation in the hybridization chamber 180, which uses the hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of hybrid heater 182 is temperature dependent and CMOS circuit 86 utilizes this to obtain a temperature reading of each hybridization chamber 180. The LOC device 301 also has a number of MST channel liquid sensors 174 and a cover channel liquid sensor 208. Figure 35 shows the line of the MST channel liquid sensor 174 at one of the ends of each of the heated microchannels 158. Preferably, as shown in Figure 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the circuit between the electrodes to indicate where they are located in the sensor. Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. Pairs of TiAl electrode pairs 218 and 220 are deposited on top layer 66. A gap 222 is provided between the electrodes 218 and 220 to keep the circuit open in the absence of liquid. The circuit is closed when the liquid is present and the CMOS circuit 86 uses this feedback to monitor the flow. The GRAVITATIONAL INDEPENDENCE test module 10 is self-directed. It does not need to be fastened to a smooth surface -53- 201209403 to operate. The fluid flow driven by capillary action and the lack of access to the external tubing make the module a portable and easily portable handheld reader such as a mobile phone. The gravity self test module is also acceleration independent of all practical ranges. Vibration-resistant and mobile-carrying vehicles or carrying actions

Nucleic Acid Amplification Variants Direct PCR Traditionally, PC R requires target DNA prior to preparation of the reaction mixture. However, when appropriately changing the chemical and sample concentration, the minimum amount of DNA purification is performed for nucleic acid amplification, or when PCR is performed for nucleic acid amplification, the method is referred to as performing a nucleic acid amplification at a controlled normal temperature in a straight 3 LOC device. When directly heated at a constant temperature. When used in LOC devices, especially in the simplification of bulk design, direct nucleic acid amplification technology has a large number of PCR or direct constant amplification amplification chemical adjustment package strength, using high activity and high progressive polymerase And an additive that chelate with the inhibitor. Dilute the presence of inhibition in the sample. In order to utilize direct nucleic acid amplification techniques, features of the LOC device are designed. A first feature is a reagent reservoir (eg, Figure 58) that is appropriately sized to supply a sufficient amount of amplification diluent such that the insertion of an auxiliary device that may interfere with the sample component of the amplification chemistry is representative of a similar primary operation A large amount of purification is required for impact resistance and telephone operation, and amplification can be utilized. When 妾PCR is used, this method is an advantage over the required flow. Adding buffers directly to potential polymerases is also important. Into two tanks in the 8 tanks, the reaction mixture or the final concentration is -54-201209403 low enough to successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LOC structure, 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 storage tank 76. Dialysis department. In another specific embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of the channel to adjust the mixing ratio between the sample and the amplified mixture. For example, to ensure that the dilution of the inhibitor associated with the sample via a single mixing step is in the range of preferably 5 to 20 times, the length and cross section of the sample and reagent channels are designed such that the sample channel upstream of the mixing start position The composition of the flow group is 4 to 1 times higher than that of the channel through which the reagent mixture flows. The flow group resistance in the microchannel is easily controlled by controlling the design. For a constant cross-sectional area, the flow resistance of the microchannel increases linearly with the length of the channel. It is important for the hybrid design that the flow group resistance in the microchannel is more dependent on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannels of the square cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse Transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RNA, such as from RNA virus or messenger RNA, RNA anti-55-201209403 must be transcribed into complementary DNA (cDNA) prior to PCR amplification. . The reverse transcription reaction (one-step RT-PCR) can be carried out in the same chamber as the PCR, or it can be a separate initiation reaction (two-step RT-PCR). In the LOC variant described herein, a reverse transcription step and a subsequent step of performing a nucleic acid amplification step can be performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the stylized heater 154, and simply performing a step. RT-PCR. The reagent storage tank 58 is used to store and dispense buffers, primers, dNTPs, and reverse transcriptase, and the culture portion 1 1 4 is used for the reverse transcription step, followed by amplification in the amplification unit 丨丨2 in a conventional manner. 'Two-step RT-PCR can also be done simply. Thermostatic Nucleic Acid Amplification For some applications, the preferred nucleic acid amplification method is constant temperature nucleic acid amplification. Therefore, it is not necessary to repeatedly circulate the reaction components at various temperature cycles, but the amplification portion is maintained at normal temperature, usually about 37 ° C. To 41〇C. __ Some thermostatic nucleic acid amplification methods, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), unwinding Constant temperature DNA amplification (HDA), rolling cycle amplification (RCA), branched amplification (RAM) and circular, 丨 ̄ ̄ temperature amplification (LAMP), and any other or other isothermal amplification methods available for this article In a specific embodiment of the LOC 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 mixes and polymerases. For example, for SDA, the reagent reservoir 6A contains amplification buffer, primers, and dNTPs, and the reagent reservoir 62 contains an endonuclease and an exo-DNA polymerase suitable for -56-201209403. For RPA, reagent reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins, and reagent reservoir 62 contains a stock-substituted DNA polymerase, such as. Similarly, the HDA' reagent reservoir 60 contains amplification buffer, primer and dNTP' and reagent reservoir 62 contains appropriate DN A polymerase and helicase (without heat) to unwind the double stranded DNA. Those skilled in the art will appreciate that the necessary reagents can be dispensed into two reagent reservoirs in any manner suitable for nucleic acid amplification. For amplification of viral viruses, such as HIV or hepatitis C virus nucleic acids, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDNA. In this example, the reagent reservoir 60 is filled with amplification buffer, primer, and dNTP, and the reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase. For some types of thermostatic nucleic acid amplification, an initial denaturation cycle must be employed to separate the double-stranded DNA template before maintaining the temperature of the thermostated nucleic acid amplification for continued reaction. Since the temperature of the mixing in the amplification section 112 can be tightly controlled by the heater 154 in the amplification microchannel 158, this denaturation cycle can be easily accomplished in all of the specific embodiments of the LOC apparatus described herein (see Figure 1 4). Thermostatic nucleic acid amplification is more tolerant to potential inhibitors in the sample and is therefore generally suitable for direct nucleic acid amplification from a desired sample. Thus, constant temperature nucleic acid amplification is particularly useful for LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677, respectively, shown in Figures 75, 76 and 77. Direct thermostatic amplification can also be combined with one or more preamplification dialysis steps 70, 686 or 682 as shown in Figures 75 and 77 and/or a pre-hybridization dialysis step as shown in Figure 76. The combination of 682 facilitates partial concentration of the target cells in the sample prior to nucleic acid amplification, respectively, or removal of unwanted cellular debris prior to entry of the sample into 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. Thermostatic nucleic acid amplification can also be performed in parallel amplification sections, such as those described in Figures 71, 72, and 73. Multiplex and some constant temperature nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA. Additional details of the fluorescence detection system. Figures 58 and 59 show hybridization-reactive FRET probes 23 6 . These are often referred to as molecular beacons and are stem-and-loop probes produced from single-stranded nucleic acids and fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization to the target nucleic acid sequence 238. The probe has a ring 240, a stem 242, a fluorophore 246 at the 5' end, and a quencher 248 at the 31 end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are joined together to form stem 242. In the absence of a complementary target sequence, as shown in Figure 58, the probe remains closed. The stems 242 maintain the fluorophore-quenching agents in close proximity to each other such that a large amount of resonant energy can be transmitted between each other and substantially eliminate the ability of the fluorophore to emit fluorophores when illuminated by the excitation light 244. Figure 59 shows a FRET probe 236 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 2 3 8 , the stem-and-loop structure is destroyed, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to emit -58-201209403 light. The optical detection of the ground fluorescence emission is 2 5 〇 as a probe impurity indicator. The probe hybridizes to the complementary target with extremely high specificity, because the stem of the probe is designed to be a probe with a single non-complementary nucleotide - the target is stable due to the relatively strong 'stereo-probe-label The target helix and the snail may coexist. Primer-Linked Probes-Linked Stem-and-Ring Probes and Probe-Linked Linear Probes, also known as scorpion-type probes, are alternatives to molecular beacons and can be used for both immediate and quantitative nucleic acid amplification of devices. . Timely amplification can be performed directly into the hybridization chamber of the device. The advantage of using a primer-coupled probe is that the element is physically linked to the primer, so that only one event is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an effective response and produces a stronger, shorter reaction time with better identification when using separate primers and probes. The needle (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 180 during manufacture to require a separate amplification portion on the LOC device. Alternatively, the amplification section is used or used for other reactions. Primer-Linked Linear Probes Figures 78 and 79 show the introduction of the linear probe 692 during the first round of nucleic acid amplification and the hybridization during subsequent nucleic acid amplification, as shown in Figure 78, the primer-linked linear probe 692. A spiral with a double stem section. The needle is not rotated, and the LOC LOC probe is mixed with the instant signal, and the probe is not uncoupled. Ref. -59- 242 201209403. One of the primer-binding probe sequences 696' is homologous to the region on the target nucleic acid 696 and is labeled 5' to its fluorophore 246, and its 3' end is coupled via amplification blocker 694 to Oligonucleotide primer 700. The other end of the stem 242 is labeled with a quencher portion 248. After completion of the first round of nucleic acid amplification, using the currently complementary sequence 698, the probe can wrap around and hybridize to the extended strand. During the first round of nucleic acid amplification, oligonucleotide primer 700 is attached to target DN A 238 (Fig. 78) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the polymerase from reading through and copying probe region 696. Upon subsequent denaturation, the hybridized extended oligonucleotide primer 700/template and the primer-coupled linear probe of the double stem 242 are separated, thereby releasing the quencher 248. Once the temperature for the adhesion and extension steps is reduced, the primer-linked probe sequence 696 of the primer-joined linear probe is crimped and hybridized to the amplified complementary sequence 698 on the extended strand, and the detected fluorescent light indicates the target. DNA exists. 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-molecule process. Primer-Linked Stem-and-Ring Probes Figures 80A through 80F show the operation of the primer-coupled stem-and-loop probes 7〇4. Referring to Fig. 80A, the primer-ligated stem-and-loop probe 704 has a stem 242 which complements the double stranded DNA and a loop 24〇 of the combined probe sequence. The 5' end of one of the stem strands 708 is marked with a fluorophore 246. Another strand 710' is labeled with 3'-end quencher 248 and the other strand 710 carries both amplification blockers 694-60-201209403 and oligonucleotide primer 700. In the initial denaturing phase (see the strand of the target nucleic acid 23 8 and the stem 242 linked by the primer separate stem -704. When the temperature is cooled for the adhesive phase (see Figure 80C), the stem-and-loop probe 704 is attached. The oligonucleotide primer 70 is hybridized to sequence 238. During extension (see Figure 80D), the complement 706 of sequence 23 is synthesized to form a DNA strand containing both probe sequence 704 and both. Amplification blocker 694 The polymerase is prevented from passing and copying the probe region 704. After denaturation, the probe sequence 706 on the extended strand is adhered to the probe (Fig. 80F) of the loop segment 240 of the stem-and-loop probe followed by adhesion. This fluorophore 246 is far removed from the quencher 248, resulting in enhanced fluorescence. Control Probes The hybridization chamber array 1 1 includes some hybridization chambers 180 having analytical negative control probes. Figures 92 and 93 负 the negative control probe 796 of the fluorophore, and the positive control probe 798 depicted in Figures 94 and 95. The positive and negative control probes have a stem-and-loop structure as described above. However, regardless of whether the probe hybridization is on and off, the fluorescent signal negative control probe 796 will never be emitted from the positive control probe 798. Referring to Figures 92 and 93, negative control probe 796 is not fluorescent with or without quencher 248). Thus, regardless of whether the target 23 8 hybridizes to the probe (see Figure 93) or the probe retains its stem. 3 8 0B ), and the - loop probe, the primer L-target nucleic acid: the target nucleic acid. Production: When reading the probe, the sequence (see configuration makes the shot significant and correct without the quenching agent FRET probe configuration or No. 25 0: group (and nucleic acid sequence and - ring) Configuration 61 - 201209403 (see Figure 92), the response to excitation light 244 can be ignored. Alternatively, negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing ring 240 it will not The probe sequence that hybridizes 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 remain in close proximity and will not emit visible fluorescein This negative control signal corresponds to the low order emission from the hybridization chamber 180 where the probe is not hybridized but the quencher does not quench all of the emission from the indicator. Conversely, the construction is not quenched. The positive control probe 798 of the extinguishing agent, as shown in Figures 94 and 95, responds to the excitation light 244, Whether or not the control probe 798 is hybridized to the target nucleic acid sequence 2 3 8 , the no substance quenches the fluorescent emission from the fluorophore 246. Figure 52 shows the positive and negative in the hybrid chamber array 1 1 0 A feasible distribution of control probes (3 78 and 3 80 respectively). Control probes 3 78 and 380 are placed in hybridization chamber 180 and positioned across the line of hybridization chamber array 110. However, within the array The configuration of the control probes is arbitrary (as is the configuration of the hybrid chamber array 1 1 0.) Some examples of protein detection variant LOC devices use homogeneous protein detection assays to detect specific proteins in crude cell lysates Many homogeneous protein detection assays have been developed for these specific embodiments of LOC devices. Typically these assays utilize antibodies or aptamers to capture target proteins. In one type of assay, aptamers that bind to a particular protein 142-62- 201209403 141 uses two different fluorophores or luminophores 143 as the precursor and acceptor in the fluorescence resonance energy transfer (FRET) or electrotransfer (ERET) reaction. Both the host 143 and the acceptor 144 are connected to the same Target to target At protein 142, the conformational changes result in the formation of a conformation and shape in the absence of the target aptamer 141 (see Figure 1 〇〇A); when bound to the target, a larger separation from the receptor (see Figure 1 〇〇B) The precursor is a luminophore and the effect of binding to the target is 862 (see Figure 1 00B).

The second type of analysis uses two bodies 141 that must be joined independently of the epitope or target protein 142 region (see Figures 101A, 101B, 102A and 145 or aptamer 141 with different fluorophores or luminescent groups that can be used as fluorescence Resonance Energy Transfer (FRET) Energy Transfer (ERET) reactions in which the donor and acceptor groups 143 and 144 form part of a pair of short complementary oligonucleotides 147 via a long elastic antibody or aptamer ( ). Once antibody 145 or aptamer 141 is linked to the complementary oligonucleotide 147 to each other and hybridize to each other ( ) such that the precursor and the receptors 1 43 and 1 44 can be used as FRET for the target protein detection signal in order to determine no, or very A few background antibodies 145 or aptamer 141 oligonucleotides 147 and 144 are indicated, which are chemiluminescent resonance energy L maps 100A and 100B] aptamer 141, and when the formation is changed in intervals. The receptor is located at the proximal end. The new configuration results in a donor when the acceptor is a quencher and I add a light emission of 250 or to a different and non-overlapping antibody 145 or two suitable 102B). These antibodies 143 and 144 are labeled, or electrochemiluminescent resonators. The fluorophore or luminescent linker 14 9 is bonded to see Figure 101A and 102A! Target protein 142, which is adjacent to Figures 101B and 102B, resulting in a valid 250 or ERET 862. No. Because the ligation to the two hybridizes to each other without binding to the protein-63-201209403 11 42 'The sequence and length of the complementary oligonucleotide 147 need to be carefully selected so that the dissociation constant (kd) of the double strand is quite high (~5 μΜ). Therefore, 'when the free antibodies or aptamers labeled with these oligonucleotides are mixed at a nanomolar concentration, they happen to be lower than the molar concentration of their kd, the probability of double-strand formation and the FRET 25 0 or ERET 8 62 produced. The signal is negligible. However, when both antibody 145 or aptamer 141 are conjugated to the standard IE protein 142, the regional concentration of oligonucleotide 147 will be much higher than its kd' causing almost complete hybridization and producing detectable FRET 250 or ERET 862 signal. The choice of fluorophores or luminophores is an important consideration when designing a homogeneous protein assay. Crude cell lysates are typically turbid and may contain substances that autofluorescent. In this case, it is desirable to use molecules with long-acting fluorescence or electrochemiluminescence as well as donor-acceptor pairs 143 and 144 that are optimized to emit maximum FRET 250 or ERET 862. One such donor-receptor pair is a ruthenium chelate and Cy5, when compared to other host-receptor pairs, 'this type of receptor-receptor has previously been shown to significantly enhance the signal in such systems- The background ratio is such that the signal is read by attenuating background fluorescence, electrochemiluminescence, or scattered light attenuation. The ruthenium chelate and the AlexaFluor 647 or ruthenium complex and the Fluorescein FRET or ERET pair also work well. The sensitivity and specificity of this method is similar to that of enzyme-linked immunosorbent assay (ELIS As), but does not require manipulation of the sample. In some embodiments of the LOC device, one of the antibodies 145 or one of the aptamers 141 is ligated to the proteomic analysis chamber 124 (see, for example, Figures 103 and 104), and the protein lysate is lysed during lysis Chemistry -64 - 201209403 The lysate 130 binds to another antibody 145 or aptamer 141 to facilitate binding to the first antibody 145 or 141 prior to entry into the proteomic analysis chamber 124. When only one bond or miscellaneous is required in the proteomic analysis chamber, this increases the speed of the connection and the detectable signal produced. The fluorophore design requires a fluorophore with a long fluorescence lifetime to allow the excitation light to be sufficiently attenuated to the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal to the noise. ratio. Moreover, a longer lifetime represents a larger integrated fluorescence count. Fluorescence lifetime of fluorophore 246 (see Figure 59) is greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and greater than 4000 nanoseconds in large cases. Metal-coordination-based long-life (from hundreds of nanoseconds to milliseconds) based on transition metals or lanthanide metals, appropriate quantum yields, thermal, chemical, and photochemical stability, all of which are characterized by thermal, chemical, and photochemical stability. A beneficial feature related to the needs of the fluorescent system. The metal-coordination complex based on the transition metal ion ruthenium (Ru(II)) is a ruthenium (2,2'-bipyridyl) ruthenium ([Ru(bpy)3]2 +), the lifetime of his is about 1μ3. This complex is available from Biosearch Technologies under the trade name Pulsar 650. Fluorescent, versatile and high-detection in the accommodating time of the aptamer (II purchased from -65- 201209403

Table 1: Photophysical properties of Pulsar 65 0 (钌 chelate) Parameter symbol 値 Unit absorption wavelength λ abs 460 nm Emission wavelength λ em 650 nm Absorption coefficient E 1 4800 M'1 cm'1 Camp light lifetime Tf 1.0 Quantum yield H ^ 1 (deoxygenated) N/A lanthanide metal-coordination complex, ruthenium chelate, has been successfully shown as a fluorescent indicator in the FRET probe system with 1 600 μ5 Long life. Table 2: Photophysical properties of ruthenium chelate

Parameter symbol 値 unit absorption wavelength λ abs 330-350 nm emission wavelength λ em 548 nm absorption coefficient E 1 3 800 Qabs, and ligand-dependent, up to 30000 @ X c = 340 nm) fluorescence lifetime tf 1600 (hybridization Probes) μδ Quantum Yield H 1 (Coordination Dependent) N/A LOC Device 3 0 1 The fluorescence detection system used does not use filters to remove unwanted background fluorescence. It is therefore advantageous if the quencher 248 has no natural emission to increase the signal-to-noise ratio. Without natural emission, the quencher 24 8 does not contribute to background fluorescence. High quenching efficiency is also important, which makes -66 - 201209403 no egg light before the occurrence of the father. The black hole quencher (BHq), available from Biosearch Technologies, Inc. of Novato, Calif., does not have natural emissions and has high quenching efficiency, as well as suitable quenchers for use in systems. The maximum absorption enthalpy of BHQ-1 occurs at 534 nm and the quenching range is 480-580 nm, making it a suitable quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 579 nm and the quenching range is 5 60-670 nm making it a suitable quencher for Pulsar 650. Iowa Black Quench (Iowa Black FQ and RQ) from Integrated DNA Technologies of Coralville, Iowa, is an alternative quenching agent with little or no background emission. Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorption enthalpy at 53 1 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 . In the specific embodiments described herein, quenching agent 248 is a functional portion that is initially attached to the probe, but in other embodiments, the quenching agent can be a separate molecule that is free of solution. Excitation Sources In the specific embodiment based on the fluorescence detection described herein, LEDs are selected as excitation sources instead of laser diodes, high power lamps, or lasers because of low power consumption, low cost, and small size. Referring to Fig. 81, LEDs 26 are disposed directly on the outer surface of the LOC device 301 on the array of hybridization chambers 1 1 from each chamber. On the opposite side of the hybridization chamber array 1 1 is a light-sensing -67-201209403 detector 44 consisting of an array of photodiodes 1 84 for detecting fluorescent signals (see Figures 53, 54 and 64). . Figures 82, 83 and 84 illustrate other specific embodiments for exposing the probe to excitation light. In the LOC device 30 shown in FIG. 82, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 onto the hybridization chamber array 110. The pulse excitation excites the LED 26 and the photodetector 44 detects the fluorescent emission. In the LOC device 30 shown in Fig. 83, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first aperture 712 and the second aperture 714 onto the hybridization chamber array 110. The pulse excites the excitation LED 26 and the photodetector 44 detects the fluorescent emission. Similarly, the LOC device 30 shown in FIG. 84, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 onto the hybridization chamber array 110. The LED 26 is pulse activated again and the fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 relies on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for Pulsar 650 dyes. SET UVT0P3 3 5T039BL LED is the appropriate excitation source for the ruthenium chelate label. -68- 201209403

Table 3: Philips LXK2-PR14-R00 LED Specifications Parameter 値 Unit wavelength λ e X 460 nm Transmit frequency ^ em 6.52 ( 1 0) 14 Hz Output power Pl 0.5 1 5 (minutes) @ 1 AW Radiation pattern Lambertian Data sheet N /A

Table 4: SET UVT0P3 3 4T039BL LED Specifications Parameter 値 Unit wavelength λ e 340 nm Transmit frequency Ve 8.82(10)14 Hz Power Pi 0.000240 (minutes) @ 20mA W Pulse forward current I 200 m A Radiation pattern Lambertian N/A The 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. To illustrate this, a filtered UV LED is used. Optionally, a UV laser can be an excitation source unless a relatively high laser cost is not practical for a particular test module market. LED Driver The LED driver 29 drives the LED 26 at a fixed current for the desired duration. The low-power USB 2.0 certified device is available with a minimum operating voltage of 4·4 volts for up to 1 unit load (1 〇〇 mA). Standard power conditioning circuits are used for this purpose. -69- 201209403 Photodiode Figure 54 shows an optical diode 184 that is integrated into a CMOS circuit 86 of the L〇c device 3〇1. Light diode 184 is formed as part of CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCDs, which are another sensing technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard processing steps. The on-wafer detection system is inexpensive and reduces the size of the analysis system. The shorter optical path length reduces noise from the surrounding environment to efficiently collect the fluorescent signal' and suppresses the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is photon collision with its active region 185. The fractional 'photon system is effectively converted into photoelectrons. For standard enthalpy treatment, the quantum efficiency is in the range of 0.3 to 0.5 depending on processing parameters such as the number of capping layers and the absorptive properties for visible light. The detection valve of the photodiode 1 8 4 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 hybridization 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 (in the example of LOC device 301, which is 1:760 microns X 5 8 24 microns) and incorporates other functional modules ( The size of the non-animal pieces that can be used after the pathogen dialysis section 70 and the amplification section 1 1 2) are limited. For standard 矽 processing, photodiode 1 84 detects at least 5 photons. However, to confirm reliable detection, the minimum 値 can be set to ten photons. -70- 201209403 Thus with a quantum efficiency range of 0.3 to 0.5 (as discussed above), the fluorescence emission from the probes is a minimum of 17 photons, and the appropriate error bounds for reliable detection of 30 photons are included. Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, autofluorescence, and residual excitation photon flux that are not fully attenuated introduces background noise and shifts to the output signal. Use one or more calibration signals to remove the background from each output signal. The calibration signal is generated by exposing one or more of the calibration photodiodes 1 84 in the array to respective calibration sources. A low calibration source is used to determine the negative result of the target 尙 not reacting with the probe. A high calibration source represents a positive result from the probe-target complex. In the specific embodiments described herein, the low calibration source is provided by a calibration chamber 382 in the hybridization chamber array 110, which does not contain any probes; includes a probe that does not have a fluorescent indicator; or contains an indication The probe and configuration of the agent is such that the quenching quenching agent is always expected to occur. The output signal from such a calibration chamber 382 is very close to the noise and bias in the output signal from all of the hybridization chambers in the LOC device. The output signal from the other hybridization chamber minus the calibration signal substantially removes the background and leaves the signal generated by the fluorescent emission (if any signal is generated). Signals generated by ambient light in the area of the array of chambers are also removed. -71 - 201209403 It will be appreciated that the above negative control group probes with reference to Figures 92 through 95 can be used in the calibration chamber. However, as shown in Figures 86 and 87, which is an enlarged view of the inset DG and DH of the LOC variant X728 shown in Figure 85, another option is to isolate the calibration chamber 382 from the amplicon fluid. When hybridization is prevented by fluid isolation, background noise and bias can be cleared by chambers that isolate the fluid or by any probe that contains a missing indicator or any "standard" probe that has both an indicator and a quencher. Judge. The calibration chamber 3 82 provides a high calibration source to generate a high signal for the corresponding photodiode. The high signal corresponds to all probes in the hybridized chamber. The indicator, with no quencher or only the indicator spotting probe, will consistently provide a signal that approximates the hybridization chamber signal, and the majority of the probes have been hybridized within the hybridization chamber. It will be appreciated that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 throughout the array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by the relatively closest calibration chamber 382, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has a calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybridization chambers 180. In this configuration, the hybridization chamber 180 is calibrated by the adjacent hybridization chambers 382. Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, Figure 91 shows a differential imager circuit 788 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. Differential imager circuit 78 8 samples the signal from pixel 790 and "virtual" pixel 792. In one embodiment -72-201209403, the "virtual" pixel 792 is shielded from light illumination, so its output signal provides a dark reference pixel. Alternatively, the "virtual" pixel 792 can be exposed to the excitation light and the remainder of the array. In one embodiment, the "virtual" pixel 792 is light absorbing, and the signal generated by the ambient light in the region of the array of cells can also be subtracted. The signal from pixel 790 is weak (e.g., close to the dark signal), and it is difficult to distinguish between background and very weak signals without reference to the dark signal level. During use, start "read_row" 794 and "read_row_d" 795 and turn on the M4 797 and MD4 80 1 transistors. The switches 807 and 809 are turned off such that the outputs from pixel 790 and "virtual" pixel 792 are each stored on pixel capacitor 803 and virtual pixel capacitor 805. After the pixel signal is stored, switches 8 07 and 809 are disabled. The "read_col" switch 81 1 and the dummy "read_c〇l" switch 813 are then turned off, and the converted capacitor amplifier 815 is amplified at the output of the differential signal 817. The suppression and enabling of the photodiode must be such that the photodiode 184 must be suppressed during the excitation of the LED 26 and the photodiode 184 must be enabled during the luminescence. 65 is a circuit diagram of a single photodiode 184 and FIG. 66 is a timing diagram of an optical diode control signal. The circuit has a photodiode 184 and six MOS transistors, MshurU 394, M, x 396, Mreset 3 98, Msf 400, Mread 402 and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 3 94, and Mreset 398 are turned on by pulling Mshunt gate 384 and reset gate 388 high. During this period, the photons are excited to generate carriers in the photodiode 184. When the amount of carrier produced at -73-201209403 is sufficient to saturate the photodiode 184, these carriers must be removed. During this cycle, Mshunt 3 94 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or due to excitation-generated carrier diffusion in the substrate, while Mreset 3 98 resets the accumulation at the node. Any carrier of 'NS' 406. After excitation, the capture cycle begins at t4. In this loop, the response from the emission of the fluorophore is captured and integrated into the circuit on node 'NS' 608. This is achieved by dragging the tx gate 386 high, which turns on any accumulated carrier on the transistor Mtx 3 96 and the transfer photodiode 184 to node 'NS' 406. The capture cycle can be as long as a fluorescent emission. The outputs from all of the photodiodes 184 in the hybrid chamber array 110 are simultaneously captured. There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. This delay is due to the need to read the respective photodiodes 184 in the hybridization chamber array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, and the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Mread 402 is turned on by dragging the gate 393 high. The source-slaffer transistor Msf 400 is used to buffer and read the voltage of the 'NS' node 406. Any other method of enabling or suppressing the photodiode is discussed below: 1 Suppression Method Figures 88, 89 and 90 show three possible configurations 778, 7 80 ' 7 82 for Mshunt transistor 394. The maximum 値-74- 201209403 |h|= 5 V is enabled during the excitation period, and the Mshunt transistor 394 has a very high shutdown ratio. As shown in FIG. 88, the Mshunt Gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in FIG. 89, the Mshunt gate 384 can be configured to surround the photodiode 184. A third option is to group the Mshunt gate 384 within the photodiode 184, as shown in FIG. According to the third option, the photodiode effective area 185 is less. These three configurations 7 7 8 , 7 8 0 and 7 8 2 reduce the average path length from all positions in the photodiode 1 8 4 to the Mshunt gate 3 84. In Fig. 88, the Mshunt gate 3 84 is attached to one side of the photodiode 184. This is the simplest configuration to make and the smallest impact on the active area of the photodiode 1 85. However, any carrier remaining at the distal end of the photodiode 184 requires a longer time to diffuse through the Mshunt gate 3 84. In FIG. 89, the Mshunt gate 384 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 384 around the photodiode 184 causes the photodiode active area 185 to be substantially reduced. The configuration 782 in Figure 90 positions the Mshunt gate 384 in the active area 185. This provides the shortest average path length to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active area 185 is greatest. It also creates a wide leak path. 2. Enabling method a. The flip-flop photodiode drives the parallel transistor with a fixed delay. b. The flip-flop photodiode drives the shunt transistor with a programmable delay. -75- 201209403 c. Driving the parallel transistor with a fixed delay by the LED drive pulse d. Drive the parallel transistor as a 2c but with a programmable delay. 68 shows a schematic cross-sectional view of the photodiode 184 and the flip-flop photodiode 187 embedded in the CMOS cell through the hybridization chamber 180. The flip-flop photodiode 187 replaces the product in the corner of the photodiode 184. . Since the intensity of the excitation light is higher than that of the fluorescent emission, the facet trigger photodiode 187 is sufficient. The trigger photodiode 187 excitation light 244 is sensitive. The flip-flop photodiode 187 shows that the excitation light has extinguished and activates the photodiode 184 (2) after a brief delay of At 300. This delay allows the fluorescent photodiode 1 84 to detect the fluorescent emissions from the FRET probe 186 when there is no excitation 244. This enables and enhances the signal-to-noise ratio. Under each of the hybridization chambers 180, both the photodiode 184 and the flip-flop light 187 are located in the CMOS circuit 86. Photodiode Array The electronic components are merged to form a photosensor 44 (see Figure 64). The light body 184 is a mask or step other than the pn junction made during the fabrication of the CMOS structure. During the MST fabrication, the dielectric layer (not shown) of the photodiode 184 is thinned by standard MST photolithography techniques to allow more of the active area 185 of the fluorescent illuminating diode 184 to have a field of view. The fluorescent signal from the right target hybrid within the hybridization chamber 180 is incident on the surface of the sensor. The conversion flicker is 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 contact 86 map. The small area of the pair of 244 is shown in the illuminating detection of the two poles and the appropriate two poles need to be any other. Light Please - Light into the hair -76- 201209403 Light diode 187. These choices can be used in combination with 2a and 2b above. Fluorescent Delay Detection The following derivation shows the use of long-life fluorophore fluorescence delay detection for the above LED/fluorescent combination. After an ideal pulse excitation of the fixed intensity Ie between times ίι and Ο shown in Figure 60, the fluorescence intensity is derived as a function of time. Let [S1] (when the time t is equal to the intensity of the excited state, then during and after the excitation, the number of excited states per unit volume per unit time is described by the following differential equation: dt tf hve where C is the fluorophore Concentration, ε is the molar extinction coefficient, 1; 6 is the excitation frequency, and h = 6.62606896 (10) -34 JS is the Planck constant 〇 This differential equation has the general formula: ~- + p(x)y = q (x)

OX has a solution:

\e^P{x)dx q{x)dxJrk ΛΧ) = ——.. Now use this to solve the equation [sim=!^iL+ke-^ hve -77- 201209403 and then at time [S\] ( n ) = 0, and A:: k ηεοτ L〇''lzi hve of equation (3) substituting (4) into (3) [just\εοτ{ IeecTf ^_(,_,i)/r/ hve hve at time T2, just 2)

eSCTf Ie€CT f a-^h)/rf hv„ h\ at / 2 G, the excited state is exponentially decayed and described by equation (6) [51] (〇= [51](/2)e-(, -,j)/r^ (6) Substituting (5) into (6): ...(7) [51](〇= ^^[1- g-(hWl )/r/ ]e_( iW2)/r/ hv„ The fluorescence intensity is obtained by the following equation: j {〇= _d^mhv ^ ... ( 8 ) αχ where ν/ is the fluorescence frequency, η is the quantum yield, and 1 is the optical path The length is then known from (7): ^1](0 丨1call-"-'2)/1·, (9) dt " hve ... Substituting (9) into (8): IAt) = /efc/ 7—[1 -e'(,2',,)/r/>'(,',ί)/Γ/ ... ( 10) -78- 201209403 because If(t) ieSclv^J-e' {,~,l)lTf

Τί K Therefore 'we can write the following approximation, which describes the attenuation of the fluorescence intensity after a sufficiently long excitation pulse (>> Tf): for l^t2 If{t)^Iesc^^-eH ,~'l)lTf ...(11) In the previous section, we summarize the situation for >> Tf, and for t > tl Ι^ΐπΐη'ε-(four)~. From the above equation, we can derive the following formula: «/(0 = ηΒεοΙηβ~{'~, ι)ΙΤ/ ... ( 12) where = is the number of fluorescent photons per unit area per unit time and Κ =4~ is the number of excitation photons per unit area per unit time. Therefore, 〇〇 nfit)=\fif{t)dt ... ( 13 ) , 3 where seven is the number of fluorescent photons per unit area and 3 is the time point at which the photodiode is turned on. Substituting (12) into (13): «/ = ^ηεεοΙηβΛ,~,ι)ΙΤ/ dt (14) At present, the number of fluorescent photons per unit area per unit time reaches the photodiode, & , obtained by: ^(〇= «>(〇^0 ... ( 15 ) -79- 201209403 where 彳 is the light collection efficiency of the optical system. Substituting (1 2 ) into (1 5 ) Found = Φοη, εαΙηβ'0''1'11^ (16) Fluorescent photons Similarly, the number of photodiodes per unit of fluorescent area t will be as follows: 〇〇圮(7)汾 and substituted into (16) Integral: ^3 ns = φΰηΒεοΙητ fe<h~h)'Tf Therefore, ns = φ^ιΒεοΙητfe~^'11 ... ( 17) The electron rate in body 184 is much faster. Sub-rate: ideal for ί3 When the electron rate generated by the photon generated by the photon is equal to that generated by the excitation photon in the photodiode 184, since the excitation photon flux decays more than the fluorescence photon flux is attenuated due to the fluorescence per unit area of the fluorescence. The sensor output is electrically β/{ί) = φίη!{ί) where 0 / is the quantum efficiency of the sensor at the fluorescent wavelength. Substituting (1 7 ) we get: έ}(〇= φ/φ0ηΒεαΙηβΗ,-,ι)Ι^ (18) Sensor output is the same, because the electron emission rate per unit of fluorescent area of the excited photon : ee(0 = ΦΑε ...(19) -80- 201209403 where is the quantum efficiency of the sensor at the excitation wavelength for a long time, and τβ is the time constant with respect to the F-cut characteristic of the excited LED. At time t2 Then the 'attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends its decay time, but we assume a negligible effect on I f ( t ), so we take a conservative approach. Currently, as mentioned earlier, The ideal of /3 is as follows: ·έ>(6)=研3) Therefore, from (1 8 ) and (1 9 ) we get: and after reforming we get: ln(£c/7—) h-t2= —i~~- ( 20) T/ From the above two paragraphs, we get the following two working equations: ns =Φ〇ΚΡτ/β~6"τί ...(2 1 ) = ... ( 22) where corpse = Fc/?7 and Δ,= ί3-, we also understand, in fact,. The ideal time for fluorescence detection and the number of fluorescent photons detected using the Philips LXK2-PR1 4-R00 LED and Pulsar 650 dye is determined as follows: 201209403 The ideal detection time is determined using equation (22): Recall the amplicon Concentration, and assuming that all amplicons hybridize, then the fluorescence concentration of the fluorescing is: c = 2.89 (10) _ 6 mol / L. The height of the chamber is the optical path length 1 = 8 (10) '6 m. The fluorescent region has been considered to be equivalent to the photodiode region, but the actual fluorescent region is substantially larger than the photodiode region: therefore, it is assumed that Lu is a large factor. =0.5 is the light collection efficiency of the optical system. The characteristics of the photodiode, ^ = 10 fc is the extremely conservative ratio of the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength. With a typical LED decay life cycle & 0.5 nanoseconds and use

Pulsar650 specification, can be determined △ /: F = [1.48 (10) 6] [2.89 (10) -6] [8 (10) '6] (1) = 3.42 (10) '5 A. ln ([3.42 ( 10)-s](10)(0.5))

Ar =-— 1 1(10)-6 _ 0.5(10)-9 =4.34(10)-9 s The number of photons detected is determined using equation (21). First, the number of excitation photons emitted per unit time is determined by the detection illumination geometry.

Philips LXK2-PR14-R00 LED has Lambertian radiation mode, therefore: h) = nl0 cos(0) ... (23) where A; is the unit with the angle of the forward axis of the LED is 0 -82 - 201209403 The number of photons emitted per unit time, and ~ is the β in the direction of the forward axis. • The total number of photons emitted by the LED per unit time is:

Ω ,0 cos (9) ί/Ω Now, ΑΩ = 2π[1 — cos(0 + Α0)] - 2π[1 — cos(0)] ΔΩ = 2n[cos{0) - cos(9 + Δ ^)] 4;rsin(0)cos[·^ |sin Αθ_ Cl j 47rcos(0)sin2

LG = 27rsin(^)ii?0 Substitute (24) Rearrange, we get:

...(26) The output power of the LED is 0.515 watts and ve = 6.52 (10) 14 Hz, therefore: hve -83- ...(27 ) 201209403 =_0515_ ~ [6.63(10)-34][6.52( 10)14] =1.19(10)18 photons/sec. Bring this 値 into (26) we get: ... 1.19(10)18 η'0=~ΊΓ~ =3.79(10)17 photons/second /Sphericality Referring to Figure 61, the optical center 252 and the lens 254 of the LED 26 are as shown in the schematic. The photodiode is 16 microns χ 16 microns, 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] 2.825(10)-3]2 =3.2 1 (1 〇Γ5 Sphericality will understand that the central photodiode 184 of the photodiode array 44 is used for these calculations The photo sensor located at the edge of the array receives only 2% of the photons for the Lambertian excitation source intensity distribution during the hybridization event. The number of excitation photons emitted per unit time: hc = η, ςΐ ... (28 ) =[3.79(10),7][3.21(10)·5] =1 .22 ( 1 0 ) 13 Photons/sec -84- 201209403 Now refer to equation (29): ns =</>^ eFTfe'A,lt/ ns = (0.5)11.22(10)^^3.42(10)-^^(10)-6^-43400)^^0^ =208 photons per sensor So, use Philips LXK2 -PR14-R00 LED and Pulsar 65 0 fluorophore, we can easily detect any hybridization event that causes the number of excited photons. The SET LED illumination geometry is shown in Figure 62. At Id = 20 mA, the LED has a minimum optical power output of Pl = 240 microwatts and a wavelength center of λε = 3 40 nm (the absorption wavelength of the ruthenium chelate). The drive LED linearly increases the output power to ρι = 2.4 mW at ID = 205 mA. By placing the optical center 25 2 of the LED at a distance of 17.5 mm from the hybridization chamber array 110, we concentrated the output flux to a dot size having a maximum diameter of 2 mm. The photon flux in the 2 mm diameter dot of the hybrid array plane is obtained from Equation 27. η,'·

Pi hve 2.4(10)- [6.63(10)-34] [8.82(10)14] 4· 10 ( 10 ) 15 Photons/sec Using Equation 2 8, we get: ne = -85- 201209403 4.10(10 ) 15 [16(10)6]2 vs. 1(10).3]2 photons/sec 3.34(10) Now, call equation 22 again and use the previously listed Tb chelate properties, ln[(6.94(10) )-5)(10)(0.5)] 1(10)-3 — 0.5(10)-9 =3.98 ( 10 ) ·9 seconds Now theorem 2 1 : ns = (0.5)[3.34(10)&quot ;][6.94(10)-5][1(10)-3>'398(,0),/1(,0)'3 =1 1,600 photons per sensor are used by hybrid events using SET LEDs and 铽The photon theory number emitted by the chelate system can be easily detected and is well below the lower limit of 30 photons, which is the reliable for the above-mentioned photosensors. Detection required. The maximum spacing between the probe and the photodiode is detected on the wafer to avoid detection by a confocal microscope (see the background of the present invention). Deviating from traditional detection techniques is an important factor in saving time and cost associated with the system. Conventional detection requires imaging optics that must use lenses and curved mirrors. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. Placing the photodiode in a very close proximity to the probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode -86-201209403 is 1 micron, the emission angle of the emitted light is 173°. This angle is calculated by considering the light emitted from the probe closest to the center of the hybridization surface of the photodiode, which has a planar active surface region of the parallel chamber surface. The light may be defined therein by an emission pyramid of the photodiode having a radiation probe at its apex and at a corner of the periphery of its plane. The apex angle of this cone was 1 70° for a sensing of 16 μm x 16 μm; it was 173 ° in the case where the photodiode was expanded to face a hybrid chamber area of 29 μm x 9.75 μm. A separation of micrometers or less between the surface of the chamber and the active surface of the photodiode is easily achieved. Applying a non-imaging optical method requires the photodiode 184 to be very close to the chamber to collect enough photons of the fluorescent radiation. The maximum spacing between the photodiode and the probe is determined as shown in Figure 54 below. Using the ruthenium chelate fluorophore and SET UVT0P3 3 5 T039BL, we calculated 11600 photons from individual hybridization chambers 180 to 16 micron X 16 low-light diodes 184. In carrying out this calculation, I set the light collection region of the hybridization chamber 180 to have the same bottom area as the active region of the photodiode, and half of the total number of hybrid photons reaches the light II 84. That is, the light collection efficiency of the optical system is A = 0.5. More precise, we can write out = [(the bottom area of the light collection area of the hybrid chamber) / (photodiode area) Π Ω / 4π] 'the Ω which is opposed by the photodiode at the representative point of the hybrid = solid angle. For the true square pyramid geometry: Ω = 4 ar cs in ( a2 / ( 4d. 2 + a2 )), where d 〇 = in the chamber chamber in the cavity absorption sensor, the apex angle is 1 hybrid The distance between the chamber of the LED meter and the 185 pole body is in the positive chamber -87- 201209403 and the photodiode, and α is the size of the photodiode. Each hybridization chamber releases 23,200 photons, and the selected photodiode has a detection low limit of 17 photons. Therefore, the minimum optical efficiency required is φ0 = 1 7/23200 = 7.3 3 X 1 Ο ' 4 The bottom area of the light collection region of the hybridization chamber 180 is 29 microns x 19.75 microns. Solving dQ will result in a maximum limiting distance between the hybridization chamber and the photodiode 1 84 of dQ = 249 microns. In this limitation, the collecting cone angle as defined above is only 〇 8 °. It should be noted that this analysis ignores the negligible effects of refraction.

LOC Variant XLI The LOC variant XLI 671 shown in Figure 74 is used to analyze the sample, of which the lesser, substantially soluble component is of interest. The sample is added to the sample entry 68. The solid or powdered sample is combined with a suitable liquid for the capillary fluid to the sample inlet 68 of the surface tension valve 118. The reagent in the sump 54 is mixed with the sample via the surface tension valve 118 and the fluid then flows into the sub- dialysis section 682. Sample components below the lower limit of a particular size, such as salts, metabolites, DNA, and proteins, are retained in the sample. Large components such as cells, pathogens, and debris are transferred to waste storage tank 766. The purified sample is then passed to other functional units (as described in the aforementioned LOC apparatus) for further processing such as culture, nucleic acid amplification and hybridization. 684-88-201209403 Conclusions The devices, systems, and methods described herein facilitate molecular diagnostic assays. Become a low-cost, fast and a focused care test. The system and its components described above are fully described, and those skilled in the art will be able to readily recognize many variations and modifications without departing from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which: FIG. 1 shows a test module and a test module reader configured for fluorescence detection. . Figure 2 is a schematic representation of the electronic components in a test module configured for fluorescence detection. Figure 3 is a schematic diagram of the electronic components in the test module reader. Figure 4 is a schematic view of the structure of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of an LOC device having features and structures from all of the layers superimposed on each other. Figure 7 is a plan view of a LOC device having a lid structure that is independently shown. Figure 8 is a top view through the -89-201209403 with a lid of the inner channel and the sump shown in phantom. Figure 9 is an exploded top perspective view of the cover with the inner passage and the sump shown in phantom. Figure 10 is a bottom perspective view showing the cover of the top channel configuration. Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device independently. Figure 12 is a schematic cross-sectional view of the LOC device at the sample inlet. Figure 13 is an enlarged view of the inset AA shown in Figure 6. Figure 14 is an enlarged view of the inset AB shown in Figure 6. Figure 15 is an enlarged view of the inset AE shown in Figure 13. Figure 16 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 17 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 18 is a partial perspective view illustrating the layered configuration of the LOC mount inside the inset AE. Figure 19 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 20 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 21 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 22 is a schematic cross-sectional view showing the lysing reagent reservoir of Figure 21 -90 - 201209403. Figure 23 is a partial perspective view showing the layered configuration of the LOC device inside the inset AB. Figure 24 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 25 is a partial perspective view illustrating the layered configuration of the LOC device inside the illustration AI. Figure 26 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 27 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 28 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 29 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Η 30 is a schematic cross-sectional view of the amplified mixed storage tank and the polymerase storage tank. Figure 31 shows the characteristics of a separate boiling initiation valve. Figure 32 is a schematic cross-sectional view showing the boiling initiation valve of the line 32_32 of Figure 31. Figure 33 is an enlarged view of the inset AF shown in Figure 15. Figure 34 is a cross-sectional view showing the boiling initiation valve of the line 34_34 of Figure 33. Figure 35 is an enlarged view of the inset AC shown in Figure 6. Fig. 36 is a further enlarged view showing the inside of the illustration AC of the amplification section - 91 - 201209403. Fig. 37 is a further enlarged view showing the inside of the illustration AC of the amplification section. Fig. 38 is a further enlarged view showing the inside of the illustration AC of the amplification section. 39 is a further enlarged view of the inside of the illustration AK shown in FIG. 38. FIG. 40 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 41 is a further enlarged view showing the inside of the illustration AC of the amplification section. Fig. 42 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 43 is a further enlarged view of the inside of the illustration AL shown in Figure 42. Figure 44 is a further enlarged view of the inside of the AC of the display portion. Figure 45 is a further enlarged view of the inside of the illustration AM shown in Figure 44. 46 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 47 is a further enlarged view of the inside of the illustration AN shown in Figure 46. Figure 48 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. -92- 201209403 Figure 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 50 is a diagram showing the enlargement of the inside of the AC of the display portion of the display portion. Fig. 51 is a schematic cross-sectional view of the amplification portion. Figure 52 is an enlarged plan view of the hybridization section. Figure 53 is a further enlarged plan view of two separate hybridization chambers. Figure 54 is a schematic cross-sectional view of a single hybridization chamber. Figure 55 is an enlarged view of the humidifier illustrated in the inset AG of Figure 6. Figure 56 is an enlarged view of the inset AD shown in Figure 52. Figure 57 is an exploded perspective view of the LOC device in the inset AD. Figure 58 is a diagram of a FRET probe in a closed configuration. Figure 59 is a diagram of a FRET probe in an open and hybrid configuration. Figure 60 is a graph of excitation light density as a function of time. Figure 61 is a graph of excitation illumination geometry of a hybrid chamber array. Figure 62 is a graphical representation of the sensor electronics electronics LED illumination geometry. Figure 63 is an enlarged plan view showing the humidity sensor of the inset AH of Figure 6. Figure 64 is a schematic diagram showing an array of photodiodes of a portion of the photosensors. Figure 65 is a circuit diagram of a single photodiode. Figure 66 is a timing diagram of the photodiode control signal. -93- 201209403 Figure 67 is an enlarged view of the evaporator of the illustration AP shown in Figure 55. Figure 68 is a schematic cross-sectional view showing the detection of the photodiode and the triggering photodiode through the hybridization chamber. Figure 69 is a diagram of linker-priming PCR. Figure 70 is a schematic view showing a test module having a lancet. Figure 71 is a graphical representation of the structure of LOC Variant VII. Figure 72 is a graphical representation of the structure of LOC variant VIII. Figure 73 is a graphical representation of the structure of the LOC variant XIV. Figure 74 is a graphical representation of the structure of the LOC variant XLI. Figure 75 is a graphical representation of the structure of the LOC variant XLIII. Figure 76 is a graphical representation of the structure of the LOC variant XLIV. Figure 77 is a graphical representation of the structure of the LOC variant XLVII. Figure 7 is a diagram of the primer-linked linear fluorescent probe during the initial amplification. Figure 79 is a diagram of a linear fluorescent probe coupled to a primer during a subsequent amplification cycle. Figures 80A through 80F illustrate the thermal cycling of the primer-linked fluorescent stem-and-loop probe. Figure 81 is a schematic illustration of the excitation LED for the hybrid chamber array and photodiode. Figure 82 is a schematic illustration of the excitation LED and optical lens that direct light into the hybridization chamber array of the LOC device. Figure 83 is a schematic illustration of an excitation LED, an optical lens, and an optical raft for directing light into the hybrid chamber array of the LOC device. -94- 201209403 Figure 84 is a schematic illustration of the excitation LEDs, optical lenses, and mirror arrays used to direct light into the hybrid chamber array of the LOC device. Figure 85 is a plan view showing all the features overlapping each other and showing the positions of the insets da to DK. Figure 86 is an enlarged view of the inset DG shown in Figure 85. Figure 87 is an enlarged view of the inset DH shown in Figure 85. Figure 88 shows a specific embodiment of a parallel transistor for an optical diode. Figure 89 shows a specific embodiment of a parallel transistor for an optical diode. Fig. 90 shows a specific embodiment of a parallel transistor for an optical diode. Figure 91 is a circuit diagram of the differential imager. Figure 92 schematically illustrates the negative control fluorescent probe in the stem-and-loop configuration. Figure 93 illustrates the negative control fluorescent probe of Figure 92 in an open configuration. Figure 94 is a schematic illustration of a positive control fluorescent probe in a stem-and-loop configuration. Figure 95 illustrates a positive control fluorescent probe of Figure 94 in an open configuration. Figure 96 shows the test module and test module reader configured for use with ECL detection. Figure 97 is a graphical representation of the -95-201209403 electronic component in a test module that is configured for use with ECL detection. Figure 98 shows the test module and an alternative test module reader. Figure 99 shows the test module and test module reader and the host system that houses the various databases. Figures 100A and 100B are diagrams illustrating the attachment of an aptamer to a protein to make a detectable signal. Figures 101A and 101B are diagrams illustrating the attachment of two aptamers to a protein to make a detectable signal. Figures 102A and 102B are diagrams illustrating the attachment of two aptamers to a protein to make a detectable signal. Figure 103 is a plan view showing LOC deformation L of all the features overlapping each other and the positions at which the insets GA to GL are displayed. Figure 104 is an enlarged view of the inset GD shown in Figure 103. [Main component symbol description] 1 0 : Test module 1 1 : Test module 1 2 : Test module reader 1 3 : Case 14 : Micro-USB plug 1 5 : Sensor 16: Micro-USB埠1 7 : Touch Screen 1 8 : Display Screen - 96 - 201209403 1 9 : Button 2 0 : Start Button 21 : Honeycomb Radio 22 : Aseptic Sealing Band 23 : Wireless Network Connection 24 : Large Container 25 : Satellite Navigation System 26 : Illuminated Diode 2 7 : Data storage 2 8 : Telephone 29 : LED driver 30 : LOC device 3 1 : Power conditioner 32 : Capacitor 3 3 : Timer 34 : Controller 35 : Register 36 : Micro USB device 1 1 or 2.0 3 7 : Driver 3 8 : Random Access Memory 39 : ECL Excitation Driver 40 : Program and Data Cache 41 : ECL Excitation Register 42 : Processor - 97 - 201209403 43 : Program Memory 44 : Light sensor 45: indicator 46: cover 4 7 : module 48 : CMOS + MST device 49 : porous element 5 2 : hybridization and detection portion 54 : anticoagulant storage tank 56, 56.1, 56.2, 56.3: storage tank 5 7: printed circuit board 58, 58.1, 58.2: storage tank 60, 60.1-60.12, 60.X: storage tank 62, 62.1, 62.2, 62.3, 62.4, 62.X: storage 64: lower seal 66: top layer 6 8 : sample inlet 70: dialysis section 72: waste channel 74: target channel 76: waste storage tank 78: sump layer 80: cover channel layer 8 2: upper sealing layer - 98 - 201209403 84 : 矽 substrate 86 : CMOS circuit 87 : MST layer 8 8 : passivation layer 90 : MST channel 92 : lower pipe 94 : cover channel 96 : upper pipe 97 : wall portion 98 : meniscus holder I 00 : MST channel layer 101 : Laptop/Notebook 102: Capillary Action Starting Feature 103: Dedicated Reader 105: Desktop Computer 106: Boiling Initiating Valve 107: E-Book Reader 108: Boiling Initiating Valve 109: Tablet PC 110, 110.1 -110.12, 110.X: Hybridization chamber array II 1 : Epidemiological data 112, 112.1-112.12, 112.X: Amplification section I 1 3 : Genetic data II 4: Culture department -99- 201209403 1 1 5 : Electronics Health record 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Liquid sample 1 2 0 : Meniscus 1 2 1 : Electronic medical record 1 2 2 : Vent 123 : Personal health record 1 2 5 : Network 126 : boiling initiation 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 portion 132 , 132.1, 1 32.3 : Surface tension valve 1 3 3 : Incubator inlet channel 134 : Lower tube 1 36 : Optical window 141 : Aptamer 1 4 2 : Target protein 1 43 : Host 144 : Receptor 145 : Antibody 1 4 6 : valve inlet 1 4 7 : complementary oligonucleotide -100- 201209403 148 : 149 : 150 : 152 : 153 : 154 : 156 : 158 : 160 : 164 : 166 : 168 : 170 : 174 : 175 : 176 : 178 : 180 : 182 : 184 : 185 : 186 : 187 : 188 : Valve outlet coupling sub valve down pipe ring heater valve heater contact heater heater contact microchannel outlet channel orifice capillary action starting characteristic dialysis inhalation Hole temperature sensor liquid sensor diffusion barrier flow path liquid sensor hybrid chamber heater light diode active area probe light diode water storage tank -101 - 201209403 190 : evaporator 1 9 1 : ring heater 192 : Water supply channel 193: Upper pipe 194: Lower pipe 1 9 5: Top metal layer 196: Humidifier 1 9 8 : Suction hole 202: Capillary action Starting feature 204: MST Channel 208: Liquid sensor 210: Micro Channel 2 1 2 : MST Channel 2 18 : Electrode 220: electrode 222: gap 2 3 2 : humidity sensor 2 3 4 : heater 236 : FRET probe 23 8 : target nucleic acid sequence 240 : ring 242 : stem 244 : excitation light 246 : fluorophore 201209403 2 4 8: quencher 25 0 : fluorescent signal 2 5 2 : optical center 2 5 4 : lens 28 8 : sample input and preparation 290 : extraction stage 291 : culture stage 292 : amplification stage 294 : detection stage 2 9 6 : First electrode 298: second electrode 3 00: programmable delay 30 1 : L 0 C device 3 7 6 : heat conducting column 3 7 8 : positive control probe 3 8 0 : negative control probe 3 82 : calibration chamber 3 8 4 : Gate 3 8 6 : Smell 3 8 8 : Gate 3 90 : Retractable lancet 3 92 : Lancet release button 3 9 3 : No. 3 94 : MOS transistor 201209403 3 9 6 : Μ OS transistor 398: MOS transistor 400: MOS transistor 402: MOS transistor 404: MOS transistor 406: node 40 8 : film seal 4 1 0 : membrane guard 647 : AlexaFluor 67 1: LOC variant 673 : LOC Variant 674 : LOC Variant 677 : LOC Variant 6 8 2 : Dialysate 684 : Nucleic Acid Amplification and Hybridization 6 8 6 : Dialysis Step 692 : Primer-Linked Linear Probe 694 : Amplification Blocker 6 9 6 . Probe region 698: Complementary sequence 7〇〇: Oligonucleotide primer 704: Stem-and-loop probe 706: Complementary sequence 708: Stem-104 - 201209403 710: 712: 714: 716: 718 : 766 : 778 : 780 : 7 82 : 788 : 790 : 792 : 794 : 795 : 796 : 797 : 79 8 ·· 801 : 803 : 805 : 8 07 ·· 809 : 8 11: 813 : The first light edge Mirror second aperture first mirror second mirror scrap storage tank configuration configuration configuration differential imager circuit pixel virtual pixel reading_column read_column negative control probe (transistor) positive control probe (transistor Pixel capacitor virtual pixel capacitor switch switch switch -105 201209403 8 1 5 : Capacitor amplifier 8 1 7 : differential signal 860 : ECL excitation electrode 87 0 : ECL excitation electrode

Claims (1)

201209403 VII. Patent application scope: 1. A wafer-on-lab (LOC) device for genetic analysis of biological samples, the LOC device comprising: an inlet for receiving the sample; a supporting substrate; a dialysis portion for Separating the group of the sample from the larger component; a plurality of reagent storage tanks; a nucleic acid amplification section downstream of the dialysis section for amplifying the nucleic acid sequence in the sample; wherein the dialysis section and the nucleic acid amplification Both are supported on the support substrate. 2. The LOC device of claim 1, wherein the nucleic acid amplification portion is a polymerase chain reaction (PCR) portion. 3. The LOC device of claim 2, further comprising a photo sensor and a hybridization portion downstream of the PCR portion, the hybrid portion having an array of probes for hybridizing to the target nucleic acid sequence in the sample to form A probe-target hybrid, wherein the photosensor is configured to detect the probe-target hybrid. 4. 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 PCR portion, and a plurality of greater than the plurality of components and less than the The plurality of apertures 'the second channel is in fluid communication with the first channel via the apertures such that the plurality of components flow into the second channel and the larger components remain in the first channel. -107-201209403 5 - The LOC device of claim 4, wherein the track, the second channel, and the plurality of holes are configured to be filled with the sample by hair. 6. The LOC device of claim 5, wherein the tract is configured to pull the sample to the nucleic acid by capillary action. 7. The LOC device of claim 1 is wherein the addition is A constant temperature nucleic acid amplification unit. 8. The LOC device of claim 1, wherein the sump each has a surface tension valve for retaining a reagent therein, the force valve having a meniscus holder for fixing the meniscus of the reagent and the Contact of the sample stream removes the meniscus to allow the reagent to flow out of the reservoir. 9. The LOC device of claim 6, further comprising a flow path from the mouth to the hybrid, wherein the flow path draws the sample from the inlet to the hybrid by capillary action. 10. The L Ο C device of claim 4, the CMOS circuit, the temperature sensor, and the microsystem technology (MST) MST layer comprise the PCR portion, wherein the CMOS circuit is disposed on the substrate and the MST layer The CMOS circuit is configured to control the PCR portion with feedback from the sensor output. 1 1 . The LOC device of claim 10, wherein the portion has a PCR microchannel, wherein during use the sample is a thermally amplified nucleic acid sequence, the PCR microchannel defining a portion of the sample flow having across the flow The cross-sectional area is less than 100,00 () square micron. The first pass is the second pass. The nucleic acid is extended to the surface of the reagent until the reagent is inserted from the substrate to include a layer, the support utilizes the PCR cycle to follow the path and -108-201209403 12. The L0C device as part of the patent application section 11 Also included is at least one elongated heater element to heat the nucleic acid sequence in the elongated channel, the elongated heater element extending parallel to the PCR microchannel. 13. The LOC device of claim 12, wherein at least one of the microchannels forms an elongated PCR chamber. 14. The LOC device of claim 13 wherein the chamber has a plurality of extended PCR chambers, each of which is formed by a respective portion of a microchannel having a series of wide curved structures. Each of the wide curved tracks forms a channel portion of the extended PCR. 15. The LOC device of claim 14 of the patent application, further comprising a reagent reservoir for the reagent for PCR; and a surface tension valve having a meniscus configured to immobilize the reagent such that the meniscus retains the reagent The meniscus is removed in the reagent reservoir until contact with the sample. 16. The LOC device of claim 15 wherein the portion has a hybridization chamber array for receiving the probes. 17. The LOC device of claim 16, wherein the detector is in an array of photodiodes aligned with the hybridization chambers. 18. The LOC device of claim 16 has a CMOS circuit for The digital memory from the output of the photo sensor is stored and used to transmit the hybrid data to the external device interface. The PCR PCR micro-extensions to the PCR, the PCR, the PCR channel forming chamber, the hole containing the surface, the fluid, the hybrid light, and the information of the data. 109· 201209403 19. If the patent application is the 16th item The LOC device 'where the PCR portion has an active valve that is used in the PCR portion to retain liquid and respond to activation signals from the CMOS circuit during thermal cycling to allow flow to the hybridization chambers. 2. The LOC device of claim 19, wherein the active valve has a boiling initiation valve configured to fix a meniscus of a meniscus that prevents capillary flow of the liquid, and to boil The liquid is lifted from the meniscus holder to fix the meniscus to restore the capillary drive flow. -110-