TW201211240A - LOC device for pathogen detection with dialysis, thermal lysis, nucleic acid amplification and prehybridization filtering - Google Patents

Abstract

A lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a first dialysis section for separating pathogens from larger constituents in the sample, a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section having a lysis chamber and a heater for lysing the pathogens while the sample is in the lysis chamber, a nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material, and, a second dialysis section downstream of the nucleic acid amplification section for prehybridization filtration of amplicon produced by the nucleic acid amplification section, the second dialysis section being configured to remove cell debris from the amplicon, wherein, the first dialysis section, the lysis section, the nucleic acid amplification section and the second dialysis section are all supported on the supporting substrate.

Description

201211240 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatment and analysis for molecular diagnostics. [Prior Art] Molecular diagnostics have been used to provide an early indication of disease detection before symptoms develop. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the occurrence and improvement of ineffective health care Patient outcome, improved disease management, and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobases allows for the binding (hybridization) of synthetic DNA (oligonucleotide) short sequences to specific nucleic acid sequences for nucleic acid assays. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will get in the future, to determine the species and pathogen of an infectious pathogen, or to determine the individual's response to the drug. 201211240 Nucleic Acid-Based Molecular Diagnostic Tests Nucleic acid-based assays have four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3 • Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine , sputum and tissue samples are used for genetic analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required at the outset. Blood is one of the more frequently requested sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombotic cells promote agglutination and maintain activity in vitro. To inhibit coacervation, the sample is mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of the cellular material but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by differentially lysing the red blood cells in the lysate, leaving the remaining intact cellular material, which can then be separated from the sample using centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. The exact procedure used to extract nucleic acids depends on the sample and the diagnosis to be performed -6 - 201211240 Analysis. For example, the protocol used to extract viral RN A is quite different from the protocol used to extract genomic DN A. However, self-targeting cell extraction of nucleic acids typically involves a cell lysis step and subsequent nucleic acid purification. The cell lysis step ruptures the cell and nuclear membrane and releases the genetic material. This is often accomplished using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The nucleic acid is then purified in a precipitation step with alcohol (usually ice ethanol or isopropanol) or via a solid phase purification step prior to washing in the presence of a high concentration of chaotropic salt, usually in a fractionation column. The substrate, resin or paramagnetic beads are extensively eluted with a low ionic strength buffer. Any step before the nucleic acid sink is to add a protein-cleaving protease to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to disrupt thermal lysis of the cell membrane. The target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogen source. Nucleic acid amplification provides the ability to selectively amplify (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 a comprehensive comprehensible description of such reactions is provided in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 20〇8. PCR is a useful technique for amplifying a standard DNA sequence with respect to a relatively complex DNA background. If RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using the 201211240 enzyme called reverse transcriptase. Subsequently, the obtained cDNA was amplified by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is:

1. Primer pair - a short single strand of DN A having approximately 10-30 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis DNA thermostable enzyme 3. Deoxyribose nucleus Glycoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands - Buffer - The best chemical environment for DNA synthesis PCR generally involves placing these reagents in small tubes containing extracted nucleic acids (~10 -5 0 microliters). The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocols for each thermal cycle include the 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 phases are combined. The denaturation phase generally involves heating the reaction temperature to QO-pSY to denature the DNA strand; in the adhesive phase, the temperature is lowered to ~50-60 ° C for adhesion of the primer: then in the extended phase, the temperature is raised to the optimum The DNA polymerase activity temperature is 60-72 ° C for extension of the primer. This method repeats the cycle approximately 20-40 times, with the end result being a target sequence between millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multi-primer PCR, linker-primed 201211240 PCR, direct PCR, tandem IJ (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 sub-targeting multiple genes (in other ways, several trials are required). It is more difficult to optimize multi-primer PCR because it requires the selection of primers with approximate adhesion temperature and amplicon with approximate length and base composition to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR, is a method for enabling nucleic acid amplification of virtually all DNA sequences in complex DNA mixtures without the need for target-specific Sexual introduction. This method first digests the target DNA population with a suitable restriction endonuclease (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 PCR reactions are inhibited by many components present in unpurified biological samples, such as protohemoglobin components in blood. Traditionally, PCR requires enhanced purification of target nucleic acids prior to preparation of the reaction mixture. However, using appropriate changes in chemical properties and sample concentration, DNA purification can be minimized for -9 - 201211240 PCR or direct PCR. Modification of PCR chemistries for direct PCR involves potentiating buffer strength, using high activity and processivity polymerases and additions to potential polymerase inhibitors. Repetitive PCR utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. One type of repeat PCR is nested PCR in which two pairs of PCR primers are used to perform single-nucleotide amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. The second pair of primers (nested primers) used in the second amplification binds to the first PCR product and produces a second PCR product containing the target nucleic acid (shorter than the first: PCR product). The rationale used in this strategy is: If the wrong locus is amplified due to a mistake during the first nucleic acid amplification, the probability of re-amplifying the wrong locus by the second pair of primers is very low, thus ensuring specificity. Instant PCR or quantitative PCR was used to measure the amount of PCR product in real time. The initial amount of nucleic acid in a sample can be determined by using a fluorophore containing a probe or fluorescent dye and a reference standard in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. A reverse transcriptase is an enzyme that reverse transcribes RNA into a complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. -10- 201211240 Thermostat amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DN A during the amplification reaction, thus eliminating the need for complex machinery. The constant temperature nucleic acid amplification method can thus be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent I sotherma 1 DNA Amp 1 ificati ο η and Loop-Mediated Isothermal Amplification Some of the constant temperature nucleic acid amplification methods have been described. The constant temperature nucleic acid amplification method does not rely on the continuous heat denaturation of the template DNA to produce a single strand of the molecule as a template for further amplification, but relies on an enzymatic cleavage such as a DNA molecule that specifically limits the endonuclease at a 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 the ability to rely on 5'-3' exonuclease-deficient polymerases to extend and Replace the downstream stocks. An exponential nucleic acid amplification is then achieved by a coupling sense and an antisense reaction, wherein the strands from the sense reaction are substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) -11 - 201211240 which is used to cleave DNA on one of the strands of DNA without using an ordinary method to cut DNA is useful for this reaction. SDA has been improved by the use of a combination of a thermostable restriction enzyme (Χναΐ) and a thermostable exo-polymerase (polymerase). This combination appears to increase the amplification efficiency of the reaction from 1 〇 8 fold amplification to 10 115 fold 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 RN A sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to the target ribosome RN A (rRN A ) at a defined position. The reverse transcriptase produces a DNA copy of the target rRNA by extension from the 3' end of the promoter primer. If another RNase is present, the RNA in the obtained RNA:DNA double strand is decomposed by the RNase activity of the reverse transcriptase. Next, the second primer binds to the DNA copy. A double-stranded DN A molecule is produced by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RN A polymerase recognizes the promoter in the DN A template and initiates transcription. Each newly synthesized RN A amplicon is re-entered and used as a template for new replication. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a specific DNA fragment is achieved by binding the opposite oligonucleotide to the template DNA and extending it by the DNA polymerase. Denaturation of the double-stranded DNA (dsDNA) template does not require heat. Conversely, RP A uses recombinant enzyme-primer mismatches to scan dsDNA and promote share exchange at cognate locations. The resulting -12-201211240 structure is stabilized by the interaction of a single DN A binding protein with a substituted template strand, thus preventing the primer from being released due to branch migration. The recombinant enzyme decomposes off the V-terminus of the oligonucleotide which is adjacent to the strand replacing the large fragment of the DNA polymerase (such as whi/b P〇l I μ), and the primer then begins to extend. This step is repeated by cycling to achieve exponential nucleic acid amplification. The helicase amplification (HDA) mimics the in vivo system, using DN Α helicase in an in vivo system to generate a single-strand template for primer hybridization and then extending the primer with a DNA polymerase. In the first step of the HDA reaction, the helicase passes through the target DNA, destroying the hydrogen bonds that bind the two strands, which are then bound by a single-stranded binding protein. The single-strand target region exposed by the helicase allows the primer to adhere. DN A polymerase uses free deoxyribonucleoside triphosphate (dNTP) to subsequently extend the 3' end of each primer to create two DNA replicas. The two replicated dsDNA strands independently enter the next HD A cycle, resulting in exponential nucleic acid amplification of the target sequence. Other DNA-based thermostating techniques include rolling cycle amplification (RCA) in which a DNA polymerase continuously extends a primer around a circular DNA template to produce a long DNA product consisting of a number of circular repeat copies. By terminating the reaction, the polymerase produces thousands of copies of the circular template with a copy strand tethered to the original target DNA. This results in a spatial resolution of the target and rapid nucleic acid amplification of the signal. A maximum of 101 2 copies of the template can be produced in one hour. Branched amplification is a variant of RCA and utilizes a closed circular probe (C-probe) or a snap-on probe and a highly progressive DN A polymerase for exponential amplification at ambient temperature C-probe. Circular thermostat amplification (LAMP) provides high selectivity and utilizes DNA polymerase and a primer set containing four specially designed primers, and the primer set recognizes a total of six different sequences on the target DNA of the -13-201211240. The inner primer, which contains the sense strand of the target DNA and the antisense strand sequence, initiates the LAMP. The subsequent strand-derived DNA synthesis initiated by the external primer releases a single strand of DNA. This serves as a template for the synthesis of DN A initiated by the second internal and external primers. The second inner and outer primers hybridize to the other end of the target to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer hybridizes to the loop on the product and initiates the replacement DNA synthesis, producing the original stem-loop DNA and the new stem-loop DNA with twice the stem length. The cycle reaction was continued for one hour to accumulate a 109 copy target. The final product is a stem-loop DNA with several inverted repeat targets and a broccoli-like structure with a plurality of loops (formed alternately with inverted repeat targets in the same strand). After completion of the nucleic acid amplification, the amplified product must be analyzed to determine whether the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product is to simply measure the size of the amplicon by colloidal electrophoresis and use DNA hybridization to identify the nucleotide composition of the amplicon. Colloidal electrophoresis is one of the simplest ways to check the nucleic acid amplification step to produce the desired amplicon. Colloidal electrophoresis utilizes an electric field applied to a colloidal matrix to separate DNA fragments. Negatively charged DNA fragments will move through the matrix at different rates, depending primarily on their size. After the electrophoresis is completed, the fragments in the colloid can be stained to make them visible. Brominated phenanthrenequinone, which is fluorescent under UV light, is the most commonly used dye. The size of the fragment is determined by comparison with a DNA size marker (DNA ladder) containing a DN A fragment of known size which is run along with the amplicon. Since the oligonucleotide primer binds to a specific position adjacent to the target DNA from -14-201211240, the size of the amplified product can be predicted and detected using a band of known size on the colloid. In order to confirm the amplification or if several amplicon are produced, a DNA probe is often used to hybridize with the amplicon. DNA hybridization means the formation of double-stranded DNA by complementary base pairing. DNA hybridization for positive recognition of a particular amplification product requires the use of a DN A probe of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DN A sequence, hybridization will occur at favorable temperature, pH and ion concentration conditions. If hybridization occurs, the gene or DN A sequence of interest is present in the original sample. Optical detection is the most common method of detecting hybridization. The amplicon or probe is labeled to emit light via fluorescing or electrochemiluminescence. These methods have different ways of inducing the excited state of the luminescent moiety, but both are equally capable of covalent labeling of the nucleoside stock. In electrochemiluminescence (ECL), when excited by a current, light is generated by a luminophore molecule or a complex. When the fluorescent light is emitted, the emitted light is emitted to emit light. The illuminating source is used to detect fluorescence, and the illuminating source provides excitation light having a wavelength absorbed by the fluorescent molecules and a detecting unit. The detection unit includes a photosensor, such as a photomultiplier tube or a charge coupled device (CCD) array, to detect the transmitted signal and a mechanism to prevent excitation light from being included in the photosensor output (such as a wavelength-select filter). Back stress luminescence, fluorescence molecular emission history Stokes-shifted light, and this emitted light is collected by the detection unit. Stokes converts the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. -15- 201211240 The light sensor is used to detect ECL emissions, and the light sensor is sensitive to the emission wavelength of the ECL type used. For example, a transition metal coordination complex emits light of a visible wavelength, and thus a conventional photodiode and a CCD are used as photosensors. The advantage of ECL is that if ambient light is excluded, the ECL emission can be the only light present in the detection system, thus increasing sensitivity. Microarrays allow hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are useful molecular diagnostic tools that can screen thousands of genetic diseases or detect the presence of several infectious pathogens in a single experiment. The microarray consists of a number of different DN A probes that are attached to the substrate and are spotted. The target DNA (amplicon) is first labeled with fluorescent or luminescent molecules (during or after nucleic acid amplification) and then applied to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment where hybridization occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. Less stringent may result in highly non-specific binding. Excessive rigor may result in inability to properly combine and result in reduced sensitivity. Identification of hybridizations by detecting light emission from labeled amplicon-forming hybrids. The microarray scanner is typically a computer-controlled inverted scan using a microarray scanner to detect fluorescence from the microarray. Fluorescent conjugated focus microscopy, which typically uses a laser and photosensor that excites a fluorescent dye, such as a photomultiplier tube or CCD, to detect the emitted signal. The fluorescent molecules emit light converted by Stokes (as described above), and the light is collected by the detecting unit. -16- 201211240 The emitted fluorescence must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. A conjugate focal configuration is often used in microarray scanners to remove out-of-focus information by conjugated focal pinholes located in the image plane. This makes it possible to detect only the focused portion of the light. Light that is prevented above or below the focal plane of the object enters the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, which is then converted into a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. More information on fluorescent probes can be found below: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/ Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET.html In-situ Care Molecular Diagnostics Despite the advantages of molecular diagnostic tests, the growth of this type of test in clinical tests Not as expected and still only account for a small portion of the implementation of laboratory medicine. This is mainly due to the complexity and cost associated with nucleic acid testing compared to experiments based on non-amino acid methods. The broad applicability of molecular diagnostic tests to clinical treatment is associated with rapid and automated analysis that significantly reduces costs, from initial (sample processing) to final (results), and the development of instruments that do not require extensive human intervention. It is closely related. For physicians' clinics, proximity or user-based hospitals, home-based healthcare technologies offer the following advantages: • 彳 $ insults! 1 , resulting in rapid promotion of treatment and improved care quality • through trials The ability to test defects with a small sample. • Reduce clinical workload. • Reduce laboratory workload and increase work efficiency by reducing management efforts. • Improve the cost per patient by reducing hospital stays, getting results from first visits, and simplifying the handling, storage, and delivery of samples 〇 • Promoting clinical management decisions such as vaccination control and antibiotic use. Molecular Diagnostics Based on On-Wafer Laboratory (LOC) Based on methods for providing automated and accelerated molecular diagnostic analysis for molecular diagnostic systems for fluid technology. The shorter detection time is mainly due to the use of very small amounts of automation, built-in and low overhead cascades in the diagnostic method steps in the microfluidic device. The use of nanoliters and micro-upgrades also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are a common form of microfluidic devices. The on-wafer laboratory device has an MST structure in the MST layer to integrate fluid processing into a single support substrate (typically germanium). The use of the VLSI (Ultra Large Integrated Circuit) technology of the semiconductor industry makes the unit cost of each LOC device very low. However, controlling the flow of fluid through the LOC device, adding reagents, controlling the reaction -18-201211240 conditions, etc. requires a large volume of external piping and electronics. Connecting the LOC device to these external devices actually limits the use of the LOC device for molecular diagnostics to inspection processing. The cost of external equipment and its operational complexity preclude the use of LOC-based molecular diagnostics as a practical option for in situ care. In view of the above, there is a need for a molecular diagnostic system based on LOC devices for in situ care. SUMMARY OF THE INVENTION Various aspects of the present invention will be described in the following paragraphs. GCF007.1 This aspect of the invention provides a wafer-on-lab (LOC) device for detecting a pathogen in a biological sample, the LOC device comprising: an inlet for receiving a sample; a support substrate; a pathogen and a larger component in the sample The first dialysis section is separated; the lysis part located downstream of the dialysis section is used for lysing the pathogen to release the genetic material therein, and the lysis part has a lysis chamber and a heater to dissolve the sample in the lysis chamber. a nucleic acid amplification unit located downstream of the lysis unit for amplifying a nucleic acid sequence in the genetic material; and a second dialysis unit located downstream of the nucleic acid amplification unit for pre-hybridization filtration generated by the nucleic acid amplification unit The second dialysis section is configured to remove cell debris from the amplicon; wherein, the first dialysis section, the lysis unit, the nucleic acid amplification section, and the second dialysis section are supported by -19-201211240 On the support substrate. GCF007.2 Preferably, the lysis unit has a heater for the hot lytic pathogen. GCF007.3 Preferably, the LOC device also has a hybridization portion downstream of the second dialysis section, having a probe array for hybridization with a target nucleic acid sequence in the sample; and a photosensor for detecting Hybridization of any probe in the array. GCF007.4 Preferably, the first dialysis portion has a first passage in fluid communication with the inlet, a second passage in fluid communication with the lysis portion, and a plurality of first orifices, the first orifice being larger than the pathogen and less than The larger component, the second channel is in fluid communication with the first channel via the first orifice such that the pathogen flows into the second channel and the larger component remains in the first channel. GCF007.5 Preferably, the first channel and the second channel are configured to buffer the sample by capillary action. GCF007.6 Preferably, the second dialysis portion has a large component passage, a small component passage, and a plurality of second orifices fluidly connecting the large component passage to the small component passage, the second orifice being sized to allow The nucleic acid sequence flows from the large component channel to the small component channel leaving cell fragments larger than the second port in the bulk channel, the small component channel being in fluid communication with the hybrid. GCF007.7 Preferably, the nucleic acid amplification unit is a constant temperature nucleic acid amplification unit 〇GCF007.8. Preferably, the LOC device also has a reagent storage tank for holding a reagent for constant temperature nucleic acid amplification; and -20-201211240 surface tension A valve having an orifice configured to hold 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 and the reagent exits the reagent reservoir. GCF007.9 Preferably, the nucleic acid amplification unit is a polymerase chain reaction (PCR) amplification unit. GCF007.1 Preferably, the LOC device also has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer of the combined PCR portion, wherein the CMOS circuit is located between the support substrate and the MST layer, and the CMOS circuit system is configured The temperature sensor output is used to feedback control the PCR section. GCF007.il Preferably, the PCR portion has a PCR microchannel for heating a circulating sample to amplify a nucleic acid sequence, the PCR microchannel defining a portion of the sample flow path and having a cross-sectional area of less than 100, 〇〇〇 square micron across the flow path . GCF007.1 2 Preferably, the LOC device also has at least one elongated heater element for heating the nucleic acid sequence in the elongated PCR microchannel, the elongated heater element extending parallel to the PCR microchannel. GCF007.1 3 Preferably, at least one portion of the PCR microchannel forms an elongated PCR chamber. GCF007.14 Preferably, the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel, and the PCR microchannel has a crucible structure formed by a series of wide tortuous curves, each of which is curved to form a The channel portion of the elongated PCR chamber. GCF007.1 5 Preferably, the LOC device also has a reagent storage tank for holding the reagent used for the PCR; and a surface tension valve having an orifice 21 - 201211240 configured to fix the meniscus of the reagent, so that the meniscus The reagent is retained in the reagent reservoir until the liquid sample is contacted to remove the meniscus and the reagent flows out of the reagent reservoir. GCF007.1 6 Preferably, the LOC device also has an array of hybrid chambers containing probes such that the probes in the hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. GCF007.1 7 Preferably, the photosensor is an array of photodiodes that are registered with the hybridization chamber. GCF007.1 8 Preferably, the CMOS circuit has a digital memory for storing hybridization data from the photosensor output and a data interface for transmitting the hybridization data to an external device. GCF007.1 9 Preferably, the PCR section has an active valve for retaining liquid in the PCR section and in response to an activation signal from the CMOS circuit to allow liquid to flow to the hybridization chamber during thermal cycling » GCF007.20 is preferred The active valve is a boiling pilot valve having a meniscus holder and a heater. The meniscus holder is configured to fix the meniscus and stop the liquid flow driven by the capillary action, and the heater causes the liquid to boil and self-bend. The level holder releases the meniscus and restores the capillary action drive flow. The easy-to-use, mass-produced and inexpensive pathogen detection LOC device receives biological samples through its sample container, utilizes its dialysis section to separate any pathogens contained in the sample, and lyses pathogens in its hot lysate to release pathogens. The genetic material, amplifies any target gene sequence, and analyzes the nucleic acid sequence of the sample by hybridizing to the oligonucleotide probe and detecting its internal imaging array and utilizing reagents stored in a reagent reservoir of the LOC device. The dialysis function takes additional information from the sample and increases the sensitivity, the -22-201211240-to-noise ratio and the dynamic range of the analysis system. The dialysis unit and device are of a unitary structure that provides low system component quantities and simple manufacturing steps to provide an inexpensive analytical system. The lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides subsequent target treatment and analysis. The lysing unit is integral with the device' which provides a simple analytical step, a low amount of system components, and a simple manufacturing step' to provide an inexpensive analytical system. The thermal lysis method simplifies the analytical chemistry requirements and provides processing power for a wide range of samples. Amplification of the target gene sequence increases the sensitivity of the analytical system and the signal-to-noise ratio. Pre-hybridization filters remove hot lysed fragments from sample cells, improving the efficiency of the hybridization method and subsequent analytical sensitivity, signal-to-noise ratio, and reliability. Pre-hybridization filtration also allows the hot lysis method to be utilized in a wide range of sample types due to its advantages. The probe hybridization provides target analysis via hybridization. The integrated probe hybrid provides an easy-to-use, mass-processable and inexpensive solution with a low level of system component integration. The integrated image sensor eliminates the need for expensive external imaging systems and provides an inexpensive, low-system component integration solution that is small, lightweight, and easy to carry. Due to the large light collection angle, the integrated image sensor increases read sensitivity and eliminates the need for optical components in the string of optical components. A reagent storage tank that is integral with the LOC unit and maintains the analysis of total reagent requirements, provides low system component quantities and simple manufacturing steps, resulting in an inexpensive analytical system from -23 to 201211240. [Embodiment] This general specification indicates the main components of the molecular diagnosis including the specific embodiment of the present invention. Details of the system structure and operation are discussed in the following description. Referring to Figures 1, 2, 3, 109 and 1 10, the system has the following most components: The test modules 10 and 11 are of the size of a conventional USB flash drive and are easily manufactured. The test modules 1 and 1 each contain a microfluidic device in the form of a conventional crystallographic laboratory (LOC) device 30 and preloaded with reagents, and generally more than 1000 probes for molecular diagnostic analysis (see Figure 1). And 109 the test module 1 shown in FIG. 1 uses a fluorescence-based detection technique to identify the target molecule, and the test module 11 in FIG. 09 uses an electroluminescence-based detection technique. The LOC device 30 has An integrated photosensor 44 for fluorescent or electrochemographic detection (described in detail below). Both test sets 10 and 11 use standard micro-joints 14 for power, data, and control, all with printing A circuit board (PCB) 57, and a capacitor 3 2 and an inductor 15 each having an external power. The test modules 1 and 11 are both for manufacturing single use and are distributed in aseptic packaging for use. There is a large container 24 for receiving a biological sample and a removable sterile sealing strip 22, which preferably has a low-viscosity adhesive 'for large containers before use. A membrane seal 40 with a membrane guard 410 forms part The external system is integrated and important on the film.) -USB test mode for large portions of the cover casing 13 in addition to reduction assay -24-201211240 'module in the moisture resistance, is provided by a small change in pressure relief role. The membrane guard 410 protects the membrane seal 408 from damage. The test module reader 12 is powered to the test module 10 or 11 via a micro-U S B埠1 6. The test module reader I2 can be in many different forms, and its selection is described later. The reader I2 version shown in Figures 1, 3 and 109 is a specific embodiment of a smart phone. A block diagram of the reader 12 is shown in FIG. The processor 42 executes application software from the program storage 43. The processor 42 is also interfaced with a display screen 18 and a user interface (UI) touch screen 17 and button 19, a cellular radio 21, a wireless network connection 23, and a satellite navigation system 25. The cellular radio 21 and the wireless network connection 23 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location data. Alternatively, the location data can be input by the touch screen 1 7 or the button 1 9 person. The data store 27 holds genetic and diagnostic information, test results, patient information, analysis and probe data for identifying each probe, and its array position. The data storage 27 and the program storage 43 can be shared by a common memory device. The application software installed in the Test Module Reader 1 2 provides results analysis and additional test and diagnostic information. To perform a diagnostic test, the test module 10 (or test module 1 1) is inserted into the micro-USB port 16 on the test module reader 12. The sterile sealing strip 22 is turned up and the biological sample (in liquid form) is loaded into the large sample container 24. The start button 20 is pressed to initiate the test by applying the software. The sample is passed to LOC unit 30 where it is analyzed for extraction, culture, amplification and hybridization of the pre-synthesized hybrid-reactive oligonucleotide probe to the sample nucleic acid (target). In the case of the test module 1 ( (which uses fluorescence-based detection), probes 25-201211240 are fluorescently labeled and LEDs 26 placed in the shell 13 provide the necessary excitation light to induce self-hybridization Fluorescent emission of the needle (see Figures 1 and 2). In a test module (which uses electrochemiluminescence (ECL) based detection), the LOC device 30 carries an ECL probe (as described above) and the LED 26 is not necessary to generate a photoluminescence. Conversely, electrodes 860 and 807 provide excitation current (see Figure 110). The emission (fluorescence or photoluminescence) is detected using a photo sensor 44 integrated with a CMOS circuit on each LOC device. The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the results. Data can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 11 is removed from the test module reader 12 and processed as appropriate. 1, 3 and 109 show a test module reader 12 configured as a mobile/smartphone 28. In other forms, the test module reader is a laptop/notebook 1 〇1, a dedicated reader 103, an e-book reader 107, a tablet 109 or a table used in a hospital, private clinic or laboratory. Computer 105 (see Figure 111). 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 peripherals, such as printers and patient smart cards. Referring to Figure 12, through the reader 12 and the network 125, the data generated by the test module 10 can be used to update the epidemiological database maintained by the host system for epidemiological data 1 1 1 for genetic data. The genetic database maintained by the host system of 113, the electronic health record maintained by the host system for electronic health record (EHR) 115 -26- 201211240, and the host system for electronic medical record (EMR) 121 Maintained electronic medical records, as well as personal health records maintained by the host system for personal health records (PHR) 123. Conversely, epidemiological data held by the host system for epidemiological data 1 1 1 , genetic data held by the host system for genetic data 113, for electronic use via the network 125 and the reader 12 An electronic health record maintained by the host system of the Health Record (EHR) 115, an electronic medical record maintained by the host system for the Electronic Medical Record (EMR) 121, and a host for personal health record (PHR) 123 The personal health record maintained by the system can be used to update the digital memory in test module 1 〇 LOC 30. Referring again to Figures 1, 2, 109 and 110, in a mobile phone configuration, reader 12 uses battery power. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be loaded or uploaded via some network or contact interface to enable communication with peripheral devices, computers or online servers. Set the Micro-USB port 16 to connect the computer or main power supply to recharge the battery. Figure 70 shows a specific embodiment of the test module 10 for testing that only requires the presence or absence of a particular target, such as whether the test individual is infected with, for example, influenza A virus H1N1. It is only suitable as a built-in module 47 for USB power/indicator only. No other readers or application software is required. The indicator 45 on the module 47 of the USB power/indicator only shows a positive or negative result. This configuration is ideal for a large number of screenings. Additional items supplied to the system may include test tubes containing reagents for pre-treating a particular sample, and a tongue depressor and lancet containing sample collection. -27- 201211240 For convenience, the test module of the embodiment shown in Fig. 7A includes a spring-loaded retractable lancet 3 90 and a lancet release button 392. Satellite phones can be used in remote areas. Test Module Electronics Figure 2 and 1 1 0 are block diagrams of the electronic components in test modules 10 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a clock 33, a power conditioner 31, a RAM 38, and a program and data flash memory 40. These are provided for test module 10 or 11 integral and associated driver 37 including light sensor 44, temperature sensor 170, liquid sensor I 74 and various heaters 152, 154, 182, 234 And 39 and the control and memory of the registers 35 and 41. Only LED 26 (in the case of test module 10), external power supply capacitor 32 and micro USB connector 14 are external to LOC device 30. The LOC device 30 includes an adhesive pad for attachment to these external components. RAM 38 and program and data flash memory 40 have application software and diagnostic and experimental information for more than 1 000 probes (flash/security storage, such as via encryption). In the case of the test module 1 1 configured for ECL detection, there is no LED 26 (see Figures 109 and 110). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 is loaded with an electrochemiluminescent probe and a hybrid chamber, each having an ECL excitation electrode pair 8 60 and 87 〇. Many types of test modules are manufactured in a number of test formats that are ready for ready-to-use users. The test format differs in the reagents and probes of the onboard assay. -28- 201211240 Some examples of infectious diseases that are rapidly identified by this system include: • Influenza-influenza virus A, B, C, infectious salmon anemia virus, toco soil virus • pneumonia-respiratory fusion virus ( RSV), adenovirus, interstitial pneumonia virus, pneumococci, staphylococcus aureus • tuberculosis - mycobacterium tuberculosis, mycobacterium bovis, mycobacteria, mycobacteria, and mycobacterium vulgaris • malignant Plasmodium, Toxoplasma and other parasitic protozoa • Typhoid-cold bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue-yellow virus • Hepatitis (A to E) • Iatrogenic infection - for example, Clostridium botulinum, vancomycin-resistant enterococci and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) • Encephalitis- Japanese encephalitis virus, Zhangdipula virus • Pertussis-pertussis • Hemorrhoids - paramyxovirus • Meningitis - Streptococcus pneumoniae and meningococcus • Anthracnose - Bacillus anthracis Some examples of hereditary diseases identified by this system include: -29- 201211240 • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black idiots • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic kidney disease • Congenital heart disease • A few options for disease identified by the diagnostic system: • 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 proteomic analysis. Detailed structure of system components LOC device LOC device 30 is the center of the diagnostic system. It uses microfluidic platforms to rapidly perform four important steps in nucleic acid-based molecular diagnostic analysis, namely sample preparation, nucleic acid extraction, nucleic acid amplification and detection. The LOC device also has a use for the -30-201211240 generation and will be detailed below. As discussed above, test modules 10 and 1 1 can take many different configurations to detect different targets. Similarly, the LOC device 30 has many different embodiments for targeting the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a pathogen of a whole blood sample. For purposes of illustration, the structure and operation of LOC device 301 is described in detail with reference to Figures 4 through 26 and 27 through 57. 4 is a schematic view of 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 LOC device 301 that implements the processing stage. The processing stages associated with the major steps of each nucleic acid-based molecular diagnostic assay also represent: sample input and preparation 28, extraction 290, culture 291, amplification 292, and detection 294. The various reservoirs, chambers, valves, and other components of the LOC device 301 are described in more detail below. FIG. 5 is a perspective view of the LOC device 301. It is manufactured using high volume CMOS and MST (microsystem technology) manufacturing techniques. The layered construction of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) of Figure 12. The LOC device 301 has a germanium substrate 84 supporting a COMS + MST wafer 48, including a CMOS circuit 86 and an MST layer 87, covering the MST layer 87 with a cover 46. For the purposes of this patent specification, the term "MST layer" relates to a collection of structures and layers of a sample treated with different reagents. Accordingly, these structures and components are configured to define a flow path having a characteristic size that supports a capillary action driven liquid stream having physical properties similar to the physical properties of the sample during processing. Accordingly, MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce fluids that are driven by capillary action -31 - 201211240 and process structures and components that are very small in size and size. The particular embodiment described in this specification shows that the MST layer is a structure and active component supported on CMOS circuitry 86, but excludes the features of cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 microns X 5824 microns. Of course, LOC devices fabricated for different applications may have different dimensions. Figure 6 shows the features of the MST layer 87 overlaid with the cover features. The inserts AA to AD, AG and AH shown in Fig. 6 are individually enlarged in Figs. 13, 14, 35, 56, 55 and 63, and a sufficient understanding of the respective structures in the LOC device 301 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 of the layered configuration of the CMOS + MST device 48, the cover 46, and the fluid interaction therebetween. The drawings are not drawn to scale for the purpose of illustration. Figure 12 is a cross-sectional view through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12, CMOS + MST device 48 has a germanium substrate 84 that supports CMOS circuitry 86 that operates the active components within MST layer 87 described above. Passivation layer 88 seals and protects CMOS layer 86 from fluid flow through MST layer 87.

The fluid flows through both the capping channel 94 and the MST channel layer 100 and the MST channel 90 (see, for example, Figures 7 and 16). When biochemical treatment is performed on the smaller MST-32-201211240 channel 90, cell delivery occurs in the larger channel 94 made in the cover 46. The cell delivery channels are sized to deliver cells in the sample to predetermined locations in the MST channel 90. Cells that deliver a size greater than 20 microns (e.g., certain white blood cells) require channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. In particular, the MST channel at a location in the LOC that does not require delivery of the cells can be significantly smaller. It will be understood that the cover channel 94 and the MST channel 90 are common references and that the particular MST channel 90 can also be due to its particular function ( For example) heated microchannels or dialyzed MST channels. The MST channel 90 is formed by etching through a 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 80 and the sump layer 78 are formed from a single piece of material. Of course, the piece of material can also be non-singular. A sheet of material is etched from both sides to form a cap channel layer 80 and a sump layer 78, a cap channel 94 is etched in the cap channel layer 80, and sumpes 54, 56, 58, 60 and 62 are etched in the sump layer 78. Alternatively, the sump and cover channels are formed by micro-forming. Both etching and microforming techniques are used to fabricate channels having traverse fluids of up to 2 Å, 〇〇〇 square microns and up to 8 pm microns. Appropriate choices for the cross-sectional area of the channel across the fluid are available at different locations in the LOC device. A large number of samples or samples having a large component are accommodated in the channel 'up to 20, and the cross-sectional area of the square micron (e.g., a 200 micron wide channel in a layer of 100 micrometers thick) is suitable. Among them, a small amount of -33-201211240 liquid or a mixture free of large cells is contained in the channel'. Preferably, it is a very small cross-sectional area across the fluid. The lower seal 64 surrounds the lid passage 94 and the upper seal layer 82 surrounds the sump 54, 56, 580, 60 and 6 2 °. The five sump 54, 56, 58, 60 and 62 are preloaded with reagents for specific analysis. In the embodiments described herein, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant, optionally including red blood cell lysis buffer • Storage tank 56: lysis reagent • Slot 58: Restriction enzymes, ligases, and junctions (for junction initiation PCR (see Figure 69, excerpted from T. Stachan et al., Human)

Molecular Genetics 2, Garland Science, NY and London, 1999)) • Storage tank 60: amplification mixture (deoxyribonucleoside triphosphate (dNTP), primer, buffer), and • sump 62: DNA polymerase. Cover 46 and CMOS+MST layer 48 are in fluid communication via respective openings in lower seal 64 and top layer 66. The upper conduit 96 and the lower conduit 92 are representative of whether the fluid flows from the MST passage 90 to the cover passage 94 or vice versa. LOC Device Operation The operation of LOC device 301 is described step by step with reference to the analytical pathogenic DN A in the blood sample. Of course, other organisms -34- 201211240 or non-biological fluid species are also analyzed using a suitable kit or combination of reagents, test protocols, LOC variants, and detection systems. Referring to Figure 4, the analysis of a biological sample involves five major steps, including: sample input and preparation 288, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294 〇 sample input and preparation steps 2 8 8 mixed blood The pathogen is separated from the white blood cells and red blood cells by the anticoagulant 1 16 and then by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood stream opens its surface tension valve 118, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants prevent the formation of blood clots that can block flow. As best shown in Figure 22, the anticoagulant 116 is withdrawn from the reservoir 54 by capillary action and enters the MST channel 90 via the lower conduit 92. The lower duct 92 has a capillary action initiation feature (CIF) 102 to form a meniscus geometry that is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent 122 in the upper seal 82 allows air to replace the anticoagulant 1 16 . The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 is filled with a surface tension valve 118 and secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. Prior to use, the meniscus 120 remains fixed to the upper conduit 96 such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the lid channel 94 to the upper tube 96, the meniscus 120 is removed and the anticoagulant is drawn into the fluid. -35- 201211240 Figures 15 through 21 show the insert AE which is part of the insert AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 118 has three separate MST passages 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent entering the sample mixture. When the sample mixture and reagents are mixed by diffusion, the flow rate away from the sump determines the concentration of the reagent in the sample stream. Therefore, the surface tension valve of each sump is configured to meet the desired reagent concentration. Blood is passed to the pathogen dialysis section 70 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of orifices 1 64 sized according to a predetermined valve. Cells smaller than the valve stomata pass through the orifice, while large cells cannot pass through the orifice. As the target cells continue to be part of the analysis, the undesired cells are reintroduced into the waste unit 76. Undesired cells are large cells that are blocked by an array of orifices 164 or small cells that pass through the orifice. In the pathogen dialysis section 70 described herein, the pathogen from the whole blood sample is concentrated for microbial DNA analysis. The array of orifices is formed by fluidly communicating the input in the lid passage 94 to a plurality of 3 micron diameter orifices 1 64 of the target passage 74. The 3 micron diameter orifice 1 64 and the dialysis extraction orifice 168 for the target channel 74 are connected by a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The pathogen is small enough to pass through the dialysis MS T channel 204 through the 3 micron diameter orifice 164 and to fill the target channel 74. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 of the lid 46, which leads to the waste reservoir 76 (see Figure 7). Other orifice shapes, sizes, and aspect ratios can be used to isolate specific pathogens -36 - 201211240 or other target cells, such as white blood cells for human DNA analysis. More detailed details of the dialysis section and dialysis variants are provided later. Referring again to Figures 6 and 7, fluid is drawn through target channel 74 to surface tension valve 128 in lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending between the lysis reagent reservoir 56 and the target channel 74. When the meniscus is removed from the sample stream, the flow rate of all seven MS T channels 90 will be greater than the flow rate of the anticoagulant reservoir 54, which has three MS T channels 90 (assuming physical properties of the fluid) To be roughly equal). Thus the proportion of lytic reagent in the sample mixture is greater than the ratio of anticoagulant. The lysis reagent and the target cells are mixed by diffusion in the target channel 74 in the chemical lysis unit 130. Boiling the pilot valve 126 stops the flow until diffusion and lysis are performed for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). Referring to Figures 31 and 32, the construction and operation of the boiling pilot valve will be described in detail below. Other active valve types (as opposed to passive valves, such as surface tension valve 1 18) have also been developed by the applicant, which can be used in place of the boiling pilot valve. These alternative valve designs are also described below. When the boiling pilot valve 1 2 6 is turned on, the lysed cells flow into the mixing portion 13 1 to pre-amplify restriction digestion and linker ligation. Referring to Figure 13, when the fluid removes the meniscus on the surface tension valve 132 at the beginning of the mixing portion 131, the restriction enzyme, linker and ligase are released from the reservoir 58. For diffusion mixing, the mixture flows through the length of the mixing portion 131. At the end of the mixing portion 131 is a lower duct 134 (see Fig. 13) leading to the incubator inlet passage -37 - 201211240 133 of the culture portion 114. The incubator inlet channel 133 feeds the mixture into the heated microchannel 2 10 '蜿蜒 structure' which provides a culture chamber for retaining the sample during restriction enzyme cleavage and junction ligation (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 within the insert AB of Figure 6. The figures show successive layers forming a layer of CMOS + MST layer 48 and cover 46 structure. The insert AB shows the end of the culture portion 1 14 and the start of the amplification portion 1 12 . As best shown in Figures 14 and 23, the fluid fills the microchannel 2 1 0 of the culture section 1 1 4 until it reaches the boiling pilot valve 106, where the fluid stops when diffusion occurs. As discussed above, the microchannel 210 upstream of the boiling pilot valve 106 becomes a culture chamber containing samples, restriction enzymes, binding enzymes, and linkers. The heater 1 54 is then activated and maintained at a steady temperature to cause the restriction enzyme shear and junction bonding to occur for a specific period of time. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is arbitrary and is only required for some types of nucleic acid amplification assays. Again, in some instances, it may be desirable to have a heating step at the end of the incubation period to increase the temperature above the culture temperature. The temperature is increased to inactivate the limiting enzyme and the ligase before entering the amplification section 112. Limiting the inactivation of enzymes and ligases has a specific effect when amplified with isothermal acid. After the incubation, the boiling pilot valve 106 is activated (opened) and the fluid is again introduced into the amplifying portion 1 1 2 . Referring to Figures 31 and 3 2 ', the mixture is filled with the microchannel 1 5 8 structure until it reaches the boiling pilot valve 108, which forms one or more amplification chambers. As best shown in the cross-sectional view of Figure 30, the amplification mixture (dNTP 'primer, buffer) is released from the storage tank 60 and the polymerase is then released from the -38-201211240 storage tank 62 into the junction culture section and the amplification section (respectively Intermediate MST channel 212 of 11 4 and 112). Figures 35 through 51 show the layers of the LOC device 301 in the insert AC of Figure 6. The figures show successive layers of layers forming the CMOS + MST device 48 and cover 46 structures. The insert AC shows the end of the amplification section 112 and the initiation of the hybridization and detection section 52. The cultured sample, amplification mixture, and polymerase flow through microchannel 158 to boiling pilot valve 108. After diffusion mixing for a sufficient time, the heaters 15 in the microchannels 158 are activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification procedure, the boiling pilot valve 108 is opened and the fluid re-enters the hybridization and detection portion 52. The operation of the boiling pilot valve is described in more detail below. As shown in Fig. 52, the hybridization and detection section 52 has an array 1 10 of hybridization chambers. Figures 5, 5, 3, 5 and 5 show the hybridization chamber array 1 1 〇 and individual hybrid chambers 180 in detail. The entrance to hybridization chamber 180 is a diffusion barrier 175 that prevents the target nucleic acid, probe strands, and hybridization probes from diffusing between hybridization chambers 108 during hybridization to prevent erroneous hybridization assay results. The flow path of the diffusion barrier 175 is long enough to prevent the target sequence and probe from diffusing out of one chamber and contaminating the other during the time the probe and nucleic acid hybridize and detect the signal, thus avoiding erroneous results. Another mechanism to prevent erroneous reading is to have the same probe in some hybridization chambers. The CMOS circuit 86 derives a single-result from the photodiode 184 corresponding to the hybridization chamber 180 containing the same probe. The exported orders—the results of the exceptions in the results can be ignored or given different weights. -39- 201211240 The thermal energy required for hybridization is provided by a CMOS controlled heater 1 82 (described in more detail below). Hybridization occurs between the complementary target probe sequences after the heater is activated. The LED driver 29 in the CMOS circuit 86 transmits a message to cause the LEDs 26 located in the test module 1 to emit light. These probes only fluoresce when the hybridization occurs, thereby eliminating the cleaning and drying steps that are often required to remove unbound strands. The stem and loop structure of the hybrid forced FRET probe 186 is opened, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as detailed below, by the photodiode in the CMOS circuit 86 located below each hybrid chamber 180. Detected by body 184 (see the description of the hybridization chamber below). Photodiodes 184 and associated electronics for all hybrid chambers together form photosensor 44 (see Figure 64). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected by the photodiode 184 is amplified and converted to a digital output that can be analyzed by the test module reader 12. Further details of the detection method are described below. Other Detailed Description of the LOC Device The modular design LOC device 301 has a number of functional components including reagent reservoirs 54, 56, 58, 60 and 62, dialysis section 70, lysis section 130, culture section 114, and amplification section 1 1 2 , valve type, humidifier and humidity sensor. In the LOC device of other specific embodiments, these functional portions may be omitted, but another functional portion or a functional portion different from the use of the above-described device may be added. For example, the culture portion 1 14 can be used as the first amplification portion 112 of the repetitive sequence amplification analysis system, and the lysis reagent reservoir 56 can be used to add the first amplification mixture of the primer, -40-201211240 dNTP, and buffer, and 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 5 6 or alternatively 'heat lysing may occur in the culture section by heating the sample for a predetermined period of time. In some embodiments, if chemical lysis is required and the chemical lysis reagent is mixed with the mixture, additional sump can be combined upstream of the sump 58 for mixing the primer, dNTP, and buffer. In the above, the steps such as the cultivation step 291 are omitted. In this case, the LOC device can be specially manufactured to avoid the reagent storage tank 58 and the culture portion H4 or the storage tank can carry only the reagent, or when there is an active valve, it is not activated to dispense the reagent into the sample flow, and the culture portion It is simply a channel for transferring the sample from the lysis unit 130 to the amplification unit 11 2 . The heaters operate independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the fluid system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after the amplification phase 292, hybridization Prior to detection step 294, dialysis is performed to remove cell debris. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplifications 1 1 2 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using specific nucleic acid probes in hybridization array U0. To analyze, for example, a sample of whole blood in which dialysis is not required, the dialysis portion 70 is simply omitted from the sample input and preparation portion 288 of the LOC design. In some cases, even if the analysis does not require dialysis, it is not necessary to omit the dialysis section 70 from the LOC device. If the presence of the dialysis section does not cause geometrical obstruction, the sample input and preparation section can still have the LOC of the dialysis section 70 without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to a protein present in the non-amplified sample, rather than being designed to A nucleic acid probe that hybridizes to a target nucleic acid sequence. It will be appreciated that the LOC devices manufactured for use with this diagnostic system are different from the combinations of functional components selected for the particular LOC application. The vast majority of functional units are common to many LOC devices and the design of additional LOC devices for new applications has functional components that are appropriately combined in the configuration of the large functional units used in existing LOC devices. Only a few LOC devices are shown in this description, and some others are shown to illustrate the design flexibility of the LOC devices manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices shown herein are not exhaustive' and that many other LOC designs are associated with a combination of suitable functional components. Sample type LOC variants accept and analyze a variety of nucleic acid or protein contents in a 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 -42-201211240 can be configured to not release its contents and use only dialysis The lysis, culture and amplification sections are used to transfer the sample from the sample inlet 68 to the hybridization chamber for nucleic acid detection 180, as described above. For some sample types, a pre-treatment step is required, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and possibly pre-treat the mucus with enzyme to reduce stickiness. Sample Input Referring to Figures 1 and 12, a sample is added to the large container 24 of the test module 10. The large container 24 is a truncated cone that is fed into the inlet 68 of the LOC unit 301 by capillary action. Here, it flows into the 64 μm wide 60 μm deep cover channel 94 and is also attracted to the anti-coagulant storage tank 54 by capillary action. Reagent Tanks A microfluidic device, such as LOC unit 301, is used to analyze the system with a small amount of reagent that allows the reagent reservoir to contain all of the necessary reagents for biochemical treatment, and each reagent reservoir is in a small volume. This volume is indeed less than 1,000,000,000 cubic microns, in most cases less than 3,000,000,000,000 cubic microns, typically less than 70, 〇〇〇, 〇〇〇 cubic microns, and in the case of the LOC device 301 shown in the figures. The medium system is less than 20,000,000 cubic microns. Dialysis Section Referring to Figures 15 through 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate the pathogen target cells from the sample. As previously described, -43-201211240 in the top layer 66 is a plurality of orifices of orifices 164 having a diameter of 3 microns, filtering target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice 1 64, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via a 16 μιη dialysis extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) is retained in the cover channel 94. Downstream of the pathogen dialysis section 70, the cover channel 94 becomes a waste channel 72 to the waste reservoir 76. Foam inserts or other porous elements 49 in the outer casing 13 of the test module 10 are configured to be in fluid communication with the waste reservoir 76 (see Figure 1) for a biological sample type that produces a substantial amount of waste. The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Figure 33) such that fluid is drawn down into the underlying dialysis MST channel 204. The first extraction aperture 198 for the target channel 74 also has a CIF 2〇2 (see Figure 15) to prevent fluid from easily securing the meniscus above the dialysis extraction aperture 168. The small component dialysis section 682 shown in Fig. 79 may have a structure similar to that of the pathogen dialysis section 70. The small component dialysis section separates any small target cells or molecules from the sample by size (and shaping, if necessary) suitable for the orifices that allow the small target cells or molecules to pass to the target channel and continue to be further analyzed. Large size cells or molecules are removed to the waste reservoir 7 66. Therefore, the LOC device 30 (see FIGS. 1 and 109) is not limited to the isolation of pathogens having a size of less than 3 μηι, and can be used to separate cells or molecules of any desired size. 44-201211240 Lysis Department Referring again to Figures 7, 1 1 and 1 3, by chemical lysis, the genetic material in the sample is released from the cell. As described above, the lysing reagent from the lysis tank 56 is mixed with the sample flowing in the target channel 74 downstream of the surface tension valve 128 of the lysis tank 56. However, some diagnostic assays preferably use hot lysis treatment, or even a combination of chemical and thermal lysis of target cells. The LOC device 301 accommodates the heated microchannel 2 1 0 of the culture portion 1 1 4 . The sample stream is swelled to the culture portion 114 and stopped at the boiling pilot valve 1〇6. 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 Pilot Valve As discussed above, the LOC device 301 has three boiling pilot valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling pilot valve 108 on the heated microchannel 158 side of the amplifying portion 112. By capillary action, the sample stream 1 1 9 is attracted along the heated microchannel 1 58 until it reaches the boiling pilot valve 1〇8. The meniscus 120 at the leading edge of the sample stream is secured to the meniscus holder 98 of the valve inlet 146. The meniscus holder 98 causes the meniscus to stop moving forward to prevent capillary flow. As shown in Figures 31 and 3, the meniscus holder 98 is a conduit on the orifice provided by the upper opening of the pipe from the MST passage 90 to the cover passage 94. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 1 52 is located around the valve inlet 146. Ring-45-201211240 shaped heater 152 is CMOS controlled by boiling the valve heater contact 153. To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater contact 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 wet cover channel 94 is started, the capillary action is restored. The fluid sample 119 is filled with the passage 94 and flows through the sub-valve conduit 150 to the valve outlet 148, wherein the capillary-driven liquid flow advances along the expansion outlet passage 160 into the hybridization and detection portion 52. The liquid sensor 174 is placed before and after the valve for diagnosis. It will be appreciated that once the boiling pilot valve is opened, it is 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' which is etched to form a serpentine pattern in the MST channel layer 100 from the lower conduit opening 134 to the boiling pilot valve 106 (see Figures 13 and 14). Controlling the temperature of the culture portion 1 14 enables a more efficient enzyme reaction. Similarly, the amplifying portion 1 12 has a heating microchannel 158 (see Figs. 6 and 14) which is heated from the boiling pilot valve 1〇6 to the boiling pilot valve 108. The valves stop flow to retain the target cells in the heated culture or amplification microchannels 210 or 158 during mixing, culture, and nucleic acid amplification. Micro-46- 201211240 The channel pattern also promotes (to some extent) the mixing of target cells and reagents. In the culture unit 1 1 4 and the amplification unit 1 1 2, the sample cells and reagents are adjusted by using pulse width. The heater 154 controlled by the CMOS circuit 86 of the variable (PWM) is heated. Each of the meandering structures of the heated culture microchannel 210 and the amplification microchannel 158 has three independently operable heaters 154 (extending between the individual heater contacts 1 5 6 (see Figure 1 4). )), which provides two-dimensional control of the input heat flux density. As best shown in FIG. 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 unit 1 1 2, 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 301, requires a small volume of amplicons required by the analysis system to allow for the amplification of a small volume of amplification mixture in the amplification portion 1 1 2 . This volume is less than 400 nanoliters, in most cases less than 170 nanoliters, typically less than 7 nanoliters, and in the case of LOC device 301, this volume is between 2 nanoliters and 30 nanoliters. between. Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a relatively short distance from the heater 1 54. The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 100,000 square microns, while the "high scale" equipment has a significantly higher heating rate. The lithography fabrication technique - 47 - 201211240 allows the amplification microchannel 158 to have a cross section that provides a higher heating rate across substantially less than 16,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small amount of amplicons are required (as is the case in LOC unit 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1,000 to 2,000 probes on a LOC device and "sample in, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and 1 square micron. between. The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate greater than 80 absolute temperatures (K) per second, in most cases at a rate greater than 100 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than 1000 K per second, and in many cases, the heater element heats the nucleic acid sequence at a rate greater than 1 0000 K per second. Typically, based on the requirements of the analysis system, the heater elements are greater than 〇〇, 〇〇〇 κ, greater than 1,000,000 K per second, greater than 10 per second, 〇〇〇, 〇〇〇 κ, greater than 20.000 per second, per second. . 000 κ, greater than 40,000,000 K per second, greater than 80.000. 000 K per second, and a nucleic acid sequence at a rate greater than 160,000,000 K per second. A small cross-sectional area channel is also beneficial for the diffusive mixing of any reagent with the sample fluid. Before the diffusion mixing is completed, near the interface between the two liquids, the diffusion of one liquid to the other is most pronounced. The density of the phenomenon decreases with distance from the interface. The microfluids ‘crossing the direction of the fluid with a relatively small cross-sectional area' are used to keep the two fluids flowing through the interface for rapid diffusion mixing. Reducing the channel cross-section to less than 100,0 〇〇 square micron' results in a significantly higher diffusion rate than "large-scale" devices. The lithography manufacturing technique allows the micro-48-201211240 channel to have a cross-section that provides a higher mixing rate across less than 1600 square microns. If only a very small amount of amplicons are required (eg LOC device 3 〇1) In the case of ), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis performed on a LOC device with 1 to 2,000 probes and a "sample into 'answer" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and 1 Between square microns. The short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid, resulting in rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e., denaturation, adhesion, and primer extension) was completed in 30 seconds for a target sequence of up to 150 base pairs (bp) long. In most diagnostic analyses, individual thermal cycle times are less than 11 seconds and most are less than 4 seconds. For a target sequence of up to 1 50 base pairs (bp) long, the thermal cycle time of the LOC device 30 for some of the most common diagnostic assays is between 0.45 seconds and 1.5 seconds. This speed of thermal cycling allows the test module to complete the nucleic acid amplification procedure in less than a 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 analyses, sufficient amplicons are generated within 30 seconds. When the predetermined number of amplification cycles is completed, 'the amplicon is fed into the hybridization and detection section via the boiling priming valve 108. 2 2 Hybridization chambers. Figures 52, 53, 54, 56 and 57 show hybridization in the hybridization chamber array 11〇- 49- 201211240 Room 180. The hybridization and detection unit 52 has a 24 χ 45 array 1 10 of hybridization chambers 180 each having a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. Photodiode 184 is incorporated to detect fluorescence from a target nucleic acid sequence or protein that hybridizes to FRET probe 186. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. For the emitted light, any material between the FRET probe 186 and the photodiode 184 must be transparent. Therefore, the wall portion 97 between the probe 186 and the photodiode 184 must also be optically transparent to the emitted light. In LOC device 301, wall portion 97 is a thin layer of hafnium oxide (about 0.5 microns). Direct intrusion of photodiode 184 beneath each hybridization chamber 180 allows the use of a very small volume of probe-target hybridization while still producing a detectable fluorescent signal (see Figure 54). A small volume of hybridization chamber can be used because of the small amount. Prior to hybridization, the amount of probe required for detectable probe-target hybridization is greater than 270 pg (卩丨<:〇8"111) (corresponding to 900,000 cubic microns), for most In the case less than 60 picograms (corresponding to 200,000 cubic micrometers), typically less than 12 picograms (corresponding to 4 inches, 〇〇〇 cubic micrometers), and in the case of the LOC device 301 shown in the figures, less than 2.7 pico Grams (corresponding to a chamber with a volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes on the LOC device. In the LOC device 301, at 1,500 micron. Within the uoo micron area, the hybrid has more than one chamber (ie, less than 2,250 square microns per chamber). The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of the needle is that during the manufacture of the LOC device, only a very small amount of probe solution needs to be dispensed into each chamber. According to the specific embodiment of the LOC device of the invention according to -50 to 201211240, 1 nanoliter or less can be configured. Probe solution After nucleic acid amplification, the boiling priming valve 108 is activated and the amplicon flows along the flow path 176 and flows into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates that the hybridization chamber 180 is charged. The amplicon and the starter heater are 182. After sufficient hybridization time, the LED 26 is activated (see Figure 2). The opening in each hybridization chamber 180 is provided with an optical window 136 to expose the FRET probe 186 to Excitation radiation (see Figures 52, 54 and 56). LED 26 emits light for a sufficient period of time to induce a high intensity fluorescent signal from the probe. During excitation, photodiode 184 is shorted. Preprogrammed delay After 3 00 (see Fig. 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 184 (see Fig. 54) is converted into usable. The photocurrent measured by the CMOS circuit 86. Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. If desired, each hybridization chamber 180 can carry a detection of more than one, different targets. Probe. Alternatively, many or all of the hybridization chambers may be loaded with repeated detection of the same standard The same probe of the nucleic acid. The probe is replicated in this manner in the array of hybridization chambers in such a way that the confidence of the results obtained is increased, and if desired, all results can be combined by photodiodes of adjacent hybridization chambers. A single result is obtained. Those skilled in the art will appreciate that there may be from 1 to over 1,000 different probes on the hybrid chamber array 110 depending on the analysis details. Humidifier and Humidity Sensor - 51 - 201211240 The insert AG indicates the position of the humidifier 196. The humidifier is free of evaporation of reagents and probes during operation of the LOC device 310. As best shown in the enlarged view of Fig. 55, the water sump 188 is fluidly coupled to the three evaporators 190. The water storage tank 188 is filled with molecular biological grade water and is sealed during manufacture. As best shown in Figures 55 and 67, by capillary action, water is drawn to the three lower conduits 194 and along the individual water supply passages 192 to the three upper conduits 193 of the evaporator 190. The meniscus is fixed to each of the upper ducts 193 to retain water. The evaporator has a ring heater 191 which surrounds the upper pipe 193. With a thermally conductive post 376, a ring heater 191 is coupled to the CMOS circuit 86 to the top metal layer 195 (see Figure 37). At start-up, the annular heater 191 heats the water causing the water to evaporate and wet the surrounding equipment. The position of the humidity sensor 23 2 is also shown in FIG. However, preferably, as shown in the enlarged view of the insert AH shown in Fig. 63, the humidity sensor has a capacitive comb structure. The lithographically etched first electrode 296 and the lithographically etched second electrode 298 are opposed to each other such that their teeth are interleaved. The opposing electrodes form a capacitor having a capacitance that can be monitored by CMOS circuitry 86. As the humidity increases, the dielectric constant of the air gap between the electrodes increases, causing the capacitance to increase. Humidity sensor 23 2 abuts the hybridization chamber array 1 1 〇 (the most important humidity measurement location) to slow the evaporation of the solution containing the exposed probe. Feedback Sensor The temperature and liquid sensor system is integrated into the LOC unit 3 0 1 to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors - 52 - 201211240 170 are assigned to all of the amplification section 112. Similarly, the culture unit 114 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control thermal cycling during nucleic acid amplification as well as thermal lysis and any heating during incubation. In the hybridization chamber 180, the CMOS circuit 86 uses the hybridization heater 182 as a temperature sensor (see FIG. 56). The resistance of the hybrid heater 182 is temperature dependent, and the CMOS circuit 86 utilizes this to drive the temperature of each hybrid chamber 180. take. 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 M S T channel liquid sensor 147 at one of the intervals of each of the heated microchannels 158. Preferably, as shown in FIG. 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the current between the electrodes to indicate their presence at the sensor. Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. Opposite TiAl electrode pairs 218 and 220 are deposited on top layer 66. A gap 222 is provided between electrodes 218 and 220 to keep the circuit open in the absence of liquid. The circuit is closed when liquid is present and CMOS circuit 86 utilizes this feedback to monitor flow. GRAVITATIONAL INDEPENDENCE test module 10. Directional autonomy. It does not need to be fastened to a smooth surface to operate. The fluid flow driven by capillary action and the lack of access to the auxiliary equipment make the module truly portable and easily plugged into a similar portable handheld reader, such as a mobile phone. The gravity autonomous operation represents that the test module is also acceleration independent of all practical ranges. It is resistant to shock and vibration and can be operated on a moving vehicle or on a mobile phone. The dialysis variant has a passage to avoid trapped bubbles. The following is shown in Figures 72, 73, 74 and 75. A specific embodiment of the LOC device of LOC variant VIII 5 18 . This LOC device has a dialysis section that is trapped in a fluid sample and trapped in a channel without bubbles. LOC Variant VIII 518 also has an additional layer of material, reference interface layer 5 94. Interface layer 5 94 is disposed between cover channel layer 80 and MST channel layer 100 of CMOS + MST device 48. Interfacial layer 594 causes a more complex fluid interconnection between the reagent sump and MST layer 87 without increasing the size of ruthenium substrate 84. Referring to Figure 73, the bypass channel 600 is designed to introduce a time delay into the fluid sample from the interface waste channel 604 to the interface target channel 602. This time delay causes the fluid sample to flow through the dialysis MST channel 204 to the dialysis draw 168 of the fixed meniscus. The capillary action initiation feature (C IF) 2 02 of the bypass channel 600 to the interface target channel 602 at the upper conduit, the point upstream of all dialysis draws 168 from the dialysis MST channel 204, the sample fluid fill interface Target channel 602. Without the bypass channel 600, the interface target channel 602 still begins to charge from the upstream end, but eventually, the traveling meniscus arrives and passes through the untwisted MST channel above the pipe, leading to the point capture air. The trapped air -54- 201211240 gas reduced the sample flow rate through the white blood cell dialysis section 3 2 8 . The pre-hybridization filter variant of the LOC device, LOC variant XII 758, uses a small component dialysis section 682 (see Figures 93 to 100) located at the outlet of the amplification section 112. The small component dialysis section 68 2 provides a pre-hybridization filtration purification stage 293 (see Figure 93). Pre-hybridization filters remove cell debris that remains in the sample stream after cell lysis. Hybridization efficiency can be affected by cell debris, so it is advantageous to reduce the concentration of cell debris prior to hybridization. Referring to Figures 98, 99 and 100, the small component dialysis section 682 has three adjacent channels in the bottom channel layer 100; two small component channels 762 are adjacent to both sides of the large component channel 760. A series of apertures along the large tapered passage 764 of the large component passage 760 provide fluid connection to the small component passages 726. In most practical applications, the apertures are 1 to 8 microns wide and 1 to 8 microns high. When the sample flows down to the large component channel 760, particles that are small enough to pass through the inverted tapered opening (eg, amplicons) flow through the small component channel 716, while larger particles (eg, cell debris). It is left in the large component channel that ends up in the blind terminal 766. The smaller particles continue to flow along the small component channels to the opposite side of the hybridization chamber array 110, and the two smaller particle streams all travel along the enthalpy path through the array to the respective blind terminals 7 6 8 (see Figure 1). 〇). Prior to detection, the panel amplicon was filled with all independent hybridization chambers 18 〇.

Nucleic Acid Amplification Variants Direct PCR 201211240 Traditionally, P C R required extensive purification of target DNA prior to preparation of the reaction mixture. However, by appropriately changing the chemical and sample concentrations, nucleic acid amplification can be performed using a minimum amount of DNA purification, or direct amplification can be performed. When nucleic acid amplification is performed by PCR, this method is called direct PCR. When nucleic acid amplification is carried out at a normal temperature controlled in a LOC apparatus, this method is direct constant temperature amplification. Direct nucleic acid amplification techniques have considerable advantages when used in LOC devices, especially with regard to the simplification of the desired fluid design. Amplification chemical adjustments for direct PCR or direct isothermal amplification include increasing buffer strength, using highly active and highly progressive polymerases, and additions to potential polymerase inhibitors. It is also important to dilute the inhibitor in the sample. To exploit direct nucleic acid amplification techniques, LOC devices are designed to incorporate two additional features. The first feature is a reagent reservoir (eg, sump 58 in Figure 8) that is appropriately sized to supply a sufficient amount of amplification reaction mix or diluent such that the final concentration of sample components that may affect the amplification chemistry is sufficient Low to successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LOC structure is used, such as the pathogen dialysis section 70 of Fig. 4. In this particular embodiment (further illustrated in Figure 6), the concentration of the pathogen sufficient to enter the amplification portion 292 is effectively concentrated upstream of the sample extraction portion 290 and the larger cells are discharged to the waste container 76. Dialysis department. In another embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of channel-56-201211240 to adjust the mixing ratio between the sample and the amplified mixed components, for example, to ensure correlation with the sample via a single mixing step. The dilution of the inhibitor is preferably in the range of 5 to 20 times, and the length and cross section of the sample and the reagent channel are designed such that the sample channel formed upstream of the mixing start position is resistant to the flow of the flow channel of the reagent mixture. The group resistance was 4 times -1 to 9 times higher. The flow group resistance in the microchannel is easily controlled by controlling the design. The flow group resistance for a constant cross-sectional area 'microchannel' increases linearly with channel length. It is important for the hybrid design that the flow group resistance in the microchannel depends more on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannels of square cross-section is inversely proportional to the cube of the smallest vertical dimension. Reverse Transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RNA, such as from RNA virus or messenger RNA, RNA must be reversed as complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction (one-step RT-PCR) can be carried out in the same chamber as the PCR, or it can be a separate initial reaction (two-step RT-PCR). In the LOC variant described herein, a reverse transcription step can be performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the stylized heater 154 to successively perform the nucleic acid amplification step, and simply perform a step. RT-PCR. The buffer, the primer 'dNTP and the reverse transcriptase are stored and distributed by the reagent storage tank 58, and the culture unit 14 is used for the reverse transcription step, and then amplified in the amplification unit 112 in an ordinary manner. The two-step RT-PCR is simply done. -57- 201211240 Constant Temperature Nucleic Acid Amplification For some applications, the preferred nucleic acid amplification method is constant temperature nucleic acid amplification, so that it is not necessary to repeatedly circulate the reaction components at various temperature cycles, but the amplification portion is maintained at normal temperature, usually About 37 ° C to 41 ° C. Some methods for thermostatic nucleic acid amplification have been described, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), uncoupling thermostated DNA Amplification (HDA), rolling cycle amplification (RCA), branched amplification (RAM), and circular thermostat amplification (LAMP), and any or other isostatic amplification methods of this type may be particularly useful for LOC devices herein. In a specific embodiment. To perform a constant temperature nucleic acid amplification, reagent reservoirs 60 and 62 adjacent to the amplification section will carry appropriate reagents for a particular constant temperature method rather than carrying PCR amplification and polymerase. For example, for SDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reagent reservoir 62 contains appropriate endonucleases and exo-DNA polymerases. For RPA, reagent reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins, and reagent reservoir 62 contains strand-substituted DNA polymerase, such as. Similarly, for HDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reservoir 62 contains the appropriate DN A polymerase and helicase (rather than heat) to unwind the double strands of DN A. Those skilled in the art will appreciate that the necessary reagents can be dispensed into two reagent reservoirs in any manner suitable for nucleic acid amplification. For the amplification of viral nucleic acids from RNA viruses, such as HIV or hepatitis C virus, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDNA. In this example, the reagent reservoir 60 is filled with an amplification buffer, a -58-201211240 and dNTP, and a reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase. For some thermostatic nucleic acid amplification types, an initial denaturation cycle must be employed to separate the two-strand DN Α template before maintaining the temperature of the thermostated nucleic acid amplification for the reaction to continue. Since the temperature of the mixing in the amplifying portion U2 can be tightly controlled by the heater 154 in the amplifying microchannel 158, this denaturation cycle can be easily accomplished in all of the specific embodiments of the LOC device described herein (see Figure 14). ). 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 shown in Figures 80, 81 and 82, respectively. Direct thermostatic amplification can also be combined with one or more preamplification dialysis steps 70, 686 or 682 as shown in Figures 80 and 82 and/or a pre-hybridization dialysis step 682 as shown in Figure 81, Partial concentration of the target cells in the sample is facilitated prior to nucleic acid amplification, respectively, or unwanted cell debris is removed before the sample enters the hybridization chamber array 110. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be used. Thermostatic nucleic acid amplification can also be performed in parallel amplification sections, such as those described in Figures 71, 76, and 77. Multiplex and some constant temperature nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA. 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 by -59-201211240 and are stem-and-loop probes produced from single-stranded nucleic acids and fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 23 prior to hybridization to the target nucleic acid sequence 23 8 . The probe has a ring 240, a stem 242, a fluorophore 246 at the 5' end, and a quencher 248 at the 3' end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequences are glued together to form stems 24 2 . In the absence of a complementary target sequence, as shown in Figure 58, the probe remains closed. The stem 2U maintains the fluorophore-quenching agents relatively close to each other such that a large amount of resonant energy can be transmitted between each other and substantially eliminates the ability of the fluorophore to emit fluorophores when illuminated by the excitation light 244. Figure 59 shows FRET probe 236 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 23 8 , the stem-and-loop structure is disrupted, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore to fluoresce. Optical detection of the fluorescence emission of 250 to serve as a probe for hybridization. The probe hybridizes to the complementary target with extremely high specificity, because the probe's stem helix is designed to be more unique than a single non-complementary nucleotide. The needle-target helix is stable. Due to the relatively strong double-stranded DNA, it is impossible for the probe-target helix and the stem helix to coexist. Primer-Linked Probe Primers - Linked Stem-and-Ring Probes and Primer-Linked Linear Probes, Also known as Mule Probes, are Alternatives to Molecular Beacons and Can Be Used for Instant and Quantitative LOC Devices Nucleic acid amplification. Timely amplification can be directly implemented in the hybridization chamber of LOC-60-201211240. The advantage of using a primer-ligated probe is that the probe element is physically linked to the primer' so only a single hybridization is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an immediate and efficient response and produces a stronger signal, shorter reaction times, and better discrimination when using separate primers and probes. During manufacture, the probe (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 180 without the need for a separate amplification portion on the LOC device. Alternatively, the amplification portion is not used or used for other reactions. Primer-Linked Linear Probes Figures 83 and 84 show the arrangement of the primer-ligated linear probe 694 during the first round of nucleic acid amplification and the hybridization during subsequent nucleic acid amplification, respectively. Referring to Figure 83, the primer-coupled probe 692 has a double stem section 242. One of the probe sequences 696, which binds to the primer, is homologous to the region on the target nucleic acid 696 and is labeled with its 5' end by fluorophore 246, and its 3' end is coupled via amplification blocker 694 to Oligonucleotide primer 700 » marks the other 3' end of stem 242 with quenching moiety 248. After completion of the first round of nucleic acid amplification, the currently complementary sequence 698' probe can be used to wrap around and hybridize to the extended strand. During the first round of nucleic acid amplification, oligonucleotide primer 700 is attached to target DNA 238 (Fig. 83) and then extended to form a DN A strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the reading of polymerase through and copying probe region 696. Upon subsequent denaturation, the hybridized extended oligonucleotide primer 700/template and the primer-linked linear probe of the double stem 242 are separated, thereby releasing the quencher 248. ~ But for the temperature-61 - 201211240 degree reduction of the adhesion and extension steps, the primer-linked probe sequence 696 of the primer-joined linear probe is crimped and hybridized with the amplified complementary sequence 698 on the extended strand' and detected Fluorescence indicates the presence of the target DN A. The unextended primer-linked linear probe retains its double stem and the fluorescence remains quenched. This test method is particularly suitable for rapid detection systems because it relies on a single molecular process. Primer-Linked Stem-and-Ring Probes Figures 85A through 8F show the operation of the primer-coupled stem-and-loop probes 7〇4. Referring to Figure 85A, the primer-ligated stem-and-loop probe 704 has a stem 242 of complementary double stranded DNA and a loop 240 of the combined probe sequence. The 5' end of one of the stems 708 is marked with a fluorophore 246. The other strand 710 is labeled with a 3'-end quencher 248 and the other strand 710 carries both an amplification blocker 694 and an oligonucleotide primer 700. In the initial denaturing phase (see Figure 85B), the stem of the target nucleic acid 23 8 and the stem 242 to which the primer is ligated are separated from the stem-and-loop probe 704. When the temperature is cooled for the adhesive phase (see Figure 85C), the oligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 hybridizes to the target nucleic acid sequence 23 8 . During extension (see Figure 85D), the complementary 706 of the target nucleic acid sequence 2 38 is synthesized to form a DN A strand containing both the probe sequence 704 and the amplified product. Amplification blocker 694 prevents the polymerase from reading through and copying probe region 704. After denaturation, the probe sequence of the loop-and-loop probe loop segment 240 (see Figure 85F) is adhered to the complementary sequence 706 on the extended strand when the probe is subsequently attached. This configuration leaves the fluorophore 246 far from the quencher 248, resulting in a significant increase in fluorescence emission. - 62 - 201211240 Control Probes The hybridization chamber array 110 includes a number of hybridization chambers 180 having positive and negative control probes for analysis of quality control. Figures 105 and 106 illustrate negative control probes without fluorophore 796, and Figures 107 and 108 depict positive control probes without quencher 798. The positive and negative control probes have a stem-and-loop structure as described above for the FRET probe. However, whether the probe hybridizes to an open configuration or remains closed, the fluorescent signal 250 will always be emitted from the positive control probe 798 and the negative control probe 796 will never emit the fluorescent signal 250. Referring to Figures 105 and 106, the negative control probe 796 does not have a fluorophore (and may or may not have a quencher 248). Thus, regardless of whether the target nucleic acid sequence 298 hybridizes to the probe (see Figure 1-6) or the probe maintains its stem-and-loop configuration (see Figure 105), the response to excitation light 244 can be ignored. Alternatively, the negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing loop 240, a probe sequence will be obtained that will not hybridize to any of the nucleic acid sequences in the sample in question, and stem 242 of the probe molecule will rehybridize with itself' and the fluorophore and quencher will Keep it close and will not emit visible, fluorescent light. This negative control signal corresponds to the low order emission from the hybridization chamber 180. The probe is not hybridized in the hybridization chamber 180 but the quencher does not quench all of the emission from the reporter. Conversely, a non-quenching positive control probe 7 9 8 was constructed as shown in Figures 1 〇 7 and 108. Back stress luminescence 2 44, regardless of whether the positive control probe 798 hybridizes to the target nucleic acid sequence 23 8 , no material quenches the fluorescent emission 250 from the fluorophore 246. Figure 5 2 shows the possible distribution of positive and negative control probes in the hybrid chamber array 1 1 ( (points -63 - 201211240 and 3 7 8 and 380). Control probes 3 78 and 380 are placed in the hybrid chamber 180 and positioned across the line of the hybrid chamber array 1 1 。. However, the configuration of the control probes within the array is arbitrary (as is the configuration of the hybridization chamber array 110). The fluorophore design requires a fluorophore with a long fluorescence lifetime to allow the excitation light to have sufficient time to decay to a lower intensity than the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal. For the noise ratio. Moreover, the longer fluorescent lifetime represents a larger integrated fluorescence count. The fluorescence lifetime of fluorophore 2 46 (see Figure 59) is greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and in most cases greater than 400 nanoseconds. Metal-coordination complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal, chemical, and photochemical stability. Both are advantageous features related to the needs of fluorescent detection systems. A particularly well-studied metal-coordination complex based on a transition metal ion ruthenium (Ru(II)) is ginseng (2,2'-bipyridyl) ruthenium (II) (Ru (bpy) 3] 2+), the lifetime of which is about 1 μ 8. This complex is available from Biosearch Technologies under the trade name Pulsar 650. -64- 201211240 Table 1: Photophysical properties of Pulsar 650 (钌 chelate) Symbol 値 unit absorption wavelength ^-abs 460 nm emission wavelength λέπι 650 nm absorption coefficient Ε 14800 ntW1 fluorescence lifetime Xf 1.0 μδ quantum yield Η 1 (deoxidized) Ν/Α lanthanide metal-coordination complex, The ruthenium chelate has been successfully shown as a fluorescent reporter in the FRET probe system and has a long lifetime of 1 600 μ5. Table 2: Photophysical properties of the ruthenium chelate. Symbol 値 Unit absorption wavelength ^abs 330- 350 nm emission wavelength ^em 548 nm absorption coefficient Ε 13800 (, bs, and ligand-dependent, up to 30000 @ λβ = 340nm) M'1 cm'1 fluorescence lifetime tf 1600 (hybridization probe) μδ quantum Yield Η 1 (coordination dependent) Ν/Α LOC device 3 0 1 The fluorescence detection system used is not Filtering is used to remove unwanted background fluorescence. If quencher 248 has no natural emission to increase the signal-to-noise ratio, then there is an advantage. Without natural emission, quencher 248 does not contribute to background fluorescence. High quenching efficiency is also important, which makes no fluorescence before the occurrence of the father. Biosearch -65- 201211240 from Novato, California

The Black Hole Quencher (BHQ) from Technologies, Inc. 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 560-670 nm making it a suitable quencher for Pulsar 650. Iowa Black FQ and RQ, available from Integrated DNA Technologies of Coralville, Iowa, are suitable alternative quenchers with little or no background emission. The Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorption enthalpy at 531 nm and is therefore a suitable quencher for Tb-chelating fluorophores. Iowa Black RQ has a maximum absorption enthalpy at 65 6 nm and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 650. In the specific examples described herein, quencher 248 is the initial It is attached to the functional portion of the probe, but in other embodiments, the quencher 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 to replace the laser diode 'high power lamp or laser excitation source because of low power consumption, low cost, and small size. Referring to Figure 86, LEDs 26 are directly placed on hybrid array 1 10 on the exterior surface of LOC device 301. Opposite to the hybrid chamber array Π 0 is a photo sensor 44 consisting of an array of photodiodes 184 from each chamber for detecting fluorescent signals (see Figures 53, 54 and -66-201211240 6 4). ° Figures 87, 88 and 89 illustrate other specific embodiments for exposing the probe to excitation light. In the LOC device 30 shown in Fig. 87, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 onto the hybridization chamber array 110. The LED 26 is pulsed and the fluorescent emitter is detected by the photo sensor 44. In the LOC device 30 shown in FIG. 88, the excitation light 244 generated by the excitation LED 26 is emitted by the lens 254, the first aperture 712, and the first The two-beam 7 14 is directed over the hybrid array 1 1 〇. The LED 26 is pulsed and the fluorescent emission is detected by the photo sensor 44. Similarly, the LOC device 30 shown in FIG. 89, the excitation light 24 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 onto the hybridization chamber array 110. The LED 26 is pulsed again and the fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 is dependent on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for the Pulsar 650 dye. SET UVT0P3 3 5T039BL LED is the appropriate excitation source for the ruthenium chelate label. . Table 3 : Philips LXK2-PR14-R00 LED Specifications

Parameter Symbol 单位 Unit Wavelength λεχ 460 nm Transmit frequency Vem 6.52 (10) 14 Hz Output power Pi 0.515 (min) @ ΙΑ W Emission pattern Lambertian data sheet N/A -67- 201211240 Table 4: SET UVT0P334T039BL LED specifications

Parameter symbol 値 unit wavelength λβ 340 nm emission frequency Ve 8.82 (10) 14 Hz power Pi 0.000240 (min) @20mA W pulse forward current I 200 mA emission pattern Lambertian N/A ultraviolet excitation pupil absorbs a small amount of light in the UV spectrum . Therefore, it is advantageous to use UV excitation light. A UV LED excitation source can be used, but the broad spectrum of LED 26 reduces the effectiveness of this method. For this, a filtered UV LED can be used. Incidentally, the UV laser can be an excitation source unless it is not practical for a particular test module market due to the relatively high cost of the laser. L E D Driver The LED driver 29 drives the LED 26 at a fixed current for a desired duration. The low-power USB 2.0 certified device can be obtained with a minimum operating voltage of 4.4 volts at up to 1 unit load (1 mA). Standard power conditioning circuits are used for this purpose. Photodiode Figure 54 shows photodiode 184 that is integrated into CMOS circuit 86 of LOC device 301. Light diode 184 is part of CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCD2, another sensible technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard -68-201211240 processing steps. On-wafer inspection is inexpensive and reduces the size of the array system. The shorter optical path length reduces noise from the surrounding environment to efficiently collect fluorescent signals and reduce the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the fraction of photons colliding with its active region 185, and the photon system is efficiently converted into photoelectrons. For standard enthalpy treatment, the quantum efficiency of visible light is in the range of 0.3 to 0.5 depending on processing parameters such as the number of cover layers and absorption characteristics. The detection valve of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 1 84 and the number of hybrid chambers 180 in the hybridization and detection portion 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (1 760 μm x 5824 μm in the example of LOC device 301) and are subject to other functional modules (such as pathogen dialysis). The size of the portion 7 〇 and the amplifying portion 1 1 2 ) can be limited by the size of the animal piece that can be used. For standard 矽 processing, photodiode 184 detects a minimum of 5 photons. However, in order to confirm the reliable detection, the minimum 値 can be set to 1 光 photons. Therefore, the quantum efficiency range is from 0.3 to 0.5 (as discussed above), the fluorescence emission from the probe is a minimum of 17 photons, and the 30 photons contain a suitable margin for the error of reliable detection. The non-uniform electrical characteristics of the photodiode 184, the auto-fluorescence, and the residual excitation photon flux that is not fully attenuated introduce background noise and shift to the output -69 - 201211240 signal. The calibration signal is generated from each of the output signals by one or more calibration signals using a calibration source that will calibrate one or more of the calibration photodiodes 184 in the array. A low calibration source is used to determine the negative result of the target probe reaction. The high calibration source represents the positive result from the probe/target. In the specific embodiment described herein, the calibration chamber 382 in the low calibration hybridization chamber array 110 is provided: it does not contain any probes: contains probes that do not have a fluorescent reporter; or contains probes with reporters The needle is configured to always expect a quenching agent. The output signal from such a calibration chamber 3 82 is very close to the noise and bias in the output signals from all of the hybrid chambers in the medium. The output signal generated by the chamber is subtracted from the calibration signal, and the signal generated by the fluorescent emission is substantially removed (if any signal generated by the ambient light in the area of the array is generated, it is also referred to as H. The above negative control sets of Figures 105 through 108 are in the calibration chamber. However, as shown in Figures 91 and 92, which are enlarged views of the inserts DG and DH showing LOC variant X 728, another calibration chamber 3 82 and amplification Sub-fluidic isolation. When hybridizing from a fluid, background noise and bias can be judged by a chamber that is fluidly isolated, with a probe that lacks a reporter or that does have a reporter and a quencher, a "standard" probe. The chamber 382 can provide a high calibration source to generate a high signal in the diode. The high signal corresponds to removal of all probe numbers in the hybridized chamber. The light source exposed to the respective target is not quenched by the source. The LOC device has other hybrid backgrounds and words). turn. The probe can be used with the option of Figure 90 to block or either pass any of the packets of the package. It is reported that the -70-201211240 agent and no quencher or only the reporter spotting probe will consistently provide a signal that a large number of probes in the hybridization chamber have hybridized in the hybridization chamber. It is also understood that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 for the entire array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by a relatively close calibration chamber 382, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has one calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybrid chambers 180. In this configuration, the hybridization chamber ISO is calibrated by the immediately adjacent calibration chamber 3M. Due to the excitation of the self-fluorescent signal from the surrounding hybridization chamber 180, FIG. 104 shows a differential imager circuit 78 8 for subtracting the signal from the photodiode 18 4 of the corresponding calibration chamber 382. The differential imager circuit 788 samples the signals from the pixels 790 and the "virtual" pixels 792. In one embodiment, the "virtual" pixel 7 92 is shielded from light illumination, so its output signal provides a dark reference. Alternatively, the "virtual" pixel 792 can be exposed to the laser with the remainder of the array. In a particular embodiment where the "virtual" pixel 792 is light responsive, the signal generated by ambient light in the area of the array is also subtracted. The signal from pixel 790 is weak (eg, close to the dark signal), and it is difficult to distinguish between the background and the very weak signal because there is no reference to the dark signal level. During the use, the "read_column" 794 and "start" are activated. Read_column_<1" 795 and turn on the M4 797 and MD4 801 transistors. Turning off switches 807 and 809 causes the outputs from pixel 790 and "virtual" pixel 792 to be stored on pixel capacitor 803 and virtual pixel capacitor 805, respectively. After the pixel signal is stored -71 - 201211240, switches 807 and 809 are disabled. The "read_row" switch 81 1 and the virtual "read_row" switch 8 1 3 are then turned off, and the switched capacitor amplifier 815 at the output amplifies the differential signal 817. The suppression and enabling of the photodiode must suppress the photodiode 184 during the excitation of the LED 26 and the photodiode 184 must be enabled during the fluorescence period. FIG. 65 is a circuit diagram of the single photodiode 184 and FIG. 66 is the photodiode control. Timing diagram of the signal. The circuit has a photodiode 184 and six MOS transistors, Mshunt 394, Mtx 396, Mreset 3 98, Msf 400, Mread 402, and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 394, and Mreset 3 98 are turned on by pulling Mshunt gate 3 84 and resetting gate 3 88 high. During this period, the excitons generate carriers in the photodiode 184. When the amount of carrier generated is sufficient to saturate the photodiode 184, the carriers must be removed. During this cycle, Mshunt 394 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or due to excitation-generated carrier diffusion in the substrate, while Mreset 398 resets to accumulate at node 'NS ' 406 any carrier. After the activation, the capture cycle begins at t4. In this loop, the response from the emission of the fluorophore is captured and integrated into the circuitry on node 'NS' 406. This is achieved by dragging the tx gate 3 86 high, which turns on the transistor Mtx 396 and any accumulated carrier on the transfer photodiode 184 to the node 'NS' 406. The capture cycle can be as long as a fluorescent emission. The output from all of the photodiodes 18 4 in the hybrid cell array 1 1 同时 is simultaneously captured. There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. -72- 201211240 This delay is due to the need to read the respective photodiodes 184 in the hybridization chamber array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, while the 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 read gate 3 93 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 a photodiode is discussed below: 1. Inhibition method

Figures 101, 102 and 103 show possible configurations 778, 780, 782 for Mshunt transistor 394. Mshunt transistor 3 94 has a very high turn-off ratio when the maximum 値 |k| = 5 V is enabled during excitation. As shown in FIG. 101, the Mshunt gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in FIG. 102, the Mshunt gate 3 84 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 active region 185 is less. These three configurations 778, 7S0, and 782 reduce the average path length from all locations in the photodiode 184 to the Mshunt gate 384. In FIG. 101, an 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 region of the photodiode 185. However, any carrier that is stuck to the far end of the photodiode 1 84 takes a long time to diffuse through the Mshunt gate 3 84. -73- 201211240 In Figure 102, the Mshunt gate 3 84 surrounds the photodiode 184. This further reduces the average path length of the carriers in the photodiode 184 to the Mshunt gate 384. However, extending the Mshunt gate 3 84 around the photodiode 184 causes the photodiode active region 185 to be substantially reduced. Configuration 782 in FIG. 103 positions Mshunt gate 3 84 in active region 185. This provides the shortest average path to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active zone 185 is greatest. It also creates a wide leak path. 2. Enabling method a. The flip-flop photodiode drives the shunt transistor with a fixed delay. b. The flip-flop photodiode drives the shunt transistor with a programmable delay. c. The parallel drive transistor is driven by the LED drive pulse with a fixed delay. d. Drive the parallel transistor as a 2 c but with a programmable delay. Fig. 68 is a schematic view showing the photodiode 184 and the flip-flop photodiode 187 buried in the CMOS circuit 86 through the hybridization chamber 180. The small area in the corner of the photodiode 184 is replaced by the flip-flop light diode 187. The trigger photodiode 187 having a small area is sufficient because the intensity of the excitation light is higher than that of the fluorescent emission. The flip-flop photodiode 187 is sensitive to the excitation light 2 44 . The flip-flop photodiode 87 shows that the excitation light 2 44 is extinguished and the photodiode 184 is activated after a brief delay of At 3 00 (see Fig. 2). This delay allows the fluorescent photodiode 184 to detect the fluorescent emission from the FRET probe 1 86 when there is no excitation light. This enables detection and enhancement of signal to noise ratio. Both the photodiode 184 and the flip-flop photodiode -74-201211240 187 are located in the CMOS circuit 86 under each hybrid cell 180. The photodiode array is combined with appropriate electronic components to form a photosensor 44 (see Figure 64). The photodiode 184 is a pn junction made during the fabrication of the CMOS structure without the need for additional masking or steps. During MST fabrication, the dielectric layer (not shown) over the photodiode 184 is arbitrarily thinned using standard MST photolithography techniques to cause more of the phosphor to illuminate the active region 185 of the photodiode 184. The photodiode 184 has a field of view such that a fluorescent signal from the probe-target hybridization within the hybridization chamber 180 is incident on the surface of the sensor. The converted fluorescent light becomes a photocurrent that can be measured using the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to the trigger photodiode 187. These choices can be used in combination with 2a and 2b above. Fluorescence Delay Detection The following derivation is based on the fluorescence delay detection of long-life fluorophores for the combination of the above LED/fluorescent clusters. After excitation by the ideal pulse of the fixed intensity Ie between the time shown in Fig. 60 and (2), the fluorescence intensity is derived as a function of time. Let [] ( ί ) be equal to the intensity of the excited state at time t, then During and after excitation, the number of excited states per unit volume per unit time is described by the following differential equation: Back (〇+腿=么...(1) dt rF hve where C is the molar concentration of the fluorophore and ε is the molar quenching The coefficient, Ve is the excitation -75-201211240 frequency, and h = 6.62606896 (10) · 34 Js is the Planck constant. This differential equation has the general formula = ^- + p(x)y = q(x) αχ solution:

|p(x)dx q^dx + k eJpW<& Now use this to solve equations (1), ... (3) hve then at time h, [SI] (h) = 〇, and from (3 (4) (3) = one... hve Substituting (4) into [51] (〇人Cry hve hve at time ί 2,: I £CT r I SQX f _it * \iτ , [51] (i2) = -~f----~Le(h ύ ! ... ( 5) hve hve at / 2 G, the excited state is exponentially decayed and described by equation (6): [51] (〇 = [ 51](i2)e'(,',2)/r/ ... (6) Substituting (5) into (6): -76- 201211240 [51](〇= le^-f [1 - ]e -(,-^)/r/ ... (7) The intensity of the fluorescence is obtained by the following equation:

If{t) = -^^-hvfnl ... ( 8 ) where v7 is the optical frequency of the egg ' η is the quantum yield, and i is the optical path length 〇 then from (7 ): c-(hh)lu- y£<th)lu ( 9 ) dt hve Substituting (9) into (8):

If {t) = Ιε€ϋΙη^[1 -e~{,1-ll)/^]e{,"lVtf ... ( 1 0 ) ^ e because ^~-->〇〇, I At ) -> I εοΙη—β~(ι~,ι)ΙΤ/

Tf Ve therefore 'we can write the following approximation, which describes the attenuation of the camping light intensity after a sufficiently long excitation pulse (i2-il>>Tf): for t>t2 ΙΜ) = ΙεεοΙη^-βΗί~ , 2), Τί ... (11) In the previous section, we summarized the situation for h-il >>Tf, and for tth 丨(7):/household. ^ e From the above equation, we can derive the following formula: nf{t) = ηεεοΙηβ~{,'(ι)'τ, ... ( 12) where -77- 201211240 \(0=_^7 is The number of photons per unit area per unit time and & is the number of excitation photons per unit area per unit time. rlVe Therefore, 00 rif{t) = \nf{t)dt ... ( 13 ) where & The number of fluorescent photons per unit area and ί3 is the time point at which the photodiode is turned on. Substituting (1 2 ) into (1 3 ): 〇〇hf = jyie8cl”e...dt ... ( 14) h Currently, the number of fluorescent photons per unit area per unit time reaches the photodiode, & (0, It is obtained by: ns(t) = nf(t)<f>0 (15) where is the light collection efficiency of the optical system. Substituting (1 2 ) into (.1 5 ) we find ris (t) = φ^ηβεοΙηβΛι~, ι)ΙΤί ... (16) Similarly, the number of fluorescent photons reaching the photodiode per unit of fluorescent area 将 will be as follows: 圮=P; (o must be substituted ( 16) and integral: h ns = φ (; η, εα Ι η τ ί β (, ι 'Η) ΙΤ / Therefore, ns = φ ^ ήΐ εοΙ · ητfe ~ ^ IXf ... ( 17) ί3 ideal 値 is due to fluorescent The photon generated in the photodiode 184 has an electron ratio equal to that produced by the excitation photon in the photodiode 184 because the excitation photon flux decays more rapidly than the fluorescence photon flux. The output electron rate of the fluorescence per unit of fluorescence area is: ·έ>(,) = Na) where seven is the quantum efficiency of the sensor at the fluorescent wavelength. Substitute (1 7) we get: έ }(ί) = φ/φ0ηΙ!α:Ιηβ~ΙΙ~,2)ΙΤ/ ... (18) Similarly, the output electron ratio per unit of fluorescence area of the excited photons is: €(0 W...(19) What is the quantum efficiency of the sensor at the excitation wavelength, and τβ is the time constant relative to the "cut" characteristic of the excited LED. After time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends it. Decay time, but we assume that this pair of I f ( t ) is negligible, so we take a conservative approach. Currently, as mentioned earlier, the ideal ί of ί3 is: ef(t3) = ee (t3) Therefore, from (18) and (19) we get ··Φ/Φ〇η€εοΙηβ'{,ι~,ι)ΙΤί = and after reforming we get: \η{εοΙη Φ/Φο,IT ..(20)

Te -79- 201211240 From the above two paragraphs, we get the following two expressions: ns ~Φ^ιΐΡτfe ^,Xi ...(21) ln(V plex, Δ/ = —^__tlA ( 22 ) r/ re where F = £c//7 and Δ/ = ί3-ί2, we also know, in fact, ί2-ί, »,. Ideal time for fluorescent detection and use? 11丨1丨?3 1^尺2-? The number of fluorescent photons detected by the 1114-1100 LED and the Pulsar 650 dye is determined as follows. The ideal detection time is determined using the formula (22): recalling the concentration of the amplicon, and assuming that all the amplicons are hybridized, the fluorescent light is emitted. The concentration of the light group is: c = 2.89(10) '6mol/L. The height of the chamber is the optical path length l = 8 (l〇V6m. ) The fluorescent area has been regarded as equivalent to the photodiode area, but the actual firefly The light region is substantially larger than the light diode region: therefore, it can be assumed that the light collection efficiency of the optical system is greater than 0.5. The characteristics of the photodiode, ^ = 10 Φβ is the quantum efficiency of the photodiode at the fluorescence wavelength. The ratio of the quantum efficiency of the excitation wavelength of the photodiode is extremely conservative. With a typical LED attenuation lifetime of 0.5 nanoseconds and using the Pulsar 650 specification Can be determined ~: F = [1.48(10)6 ][2.89(10)·6 ][8(10广6 ](1) =3.42 ( 10 ) '5 A. ln([3.42(10)-5] (10)(0.5)) Heart 1 1 1(10)·6 ~ 0.5(10)-9 = 4.34(10) -80- 201211240 The number of detected photons is determined using equation (2 1 ). First, The number of excitation photons emitted per unit time is determined by examining the illumination geometry

The Philips LXK2-PR14-R00 LED has a Lambertian emission pattern, so: n, = nl0 cos(9) ... ( 23 ) where % is the angle from the direction of the axis of the LED to 每 per unit solid angle per unit time The number of photons emitted, and the heart is % in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: ή, = |η) ί/Ω Ω =f ril0 cos(0)d〇. π ... ( 24) Now, △Ω = 2;r[ l - cos(0 + A0)] _ 2;r[l — cos(0)] + 4;rcos(0)sin2 ΔΩ = 2^-[cos(0) - cos(^ + ΑΘ)] a rm ίΔ ^Ί · (Α0λ =4^sin(^)coslsm|^ ί/Ω = 2nsm{9)d9 Substituting (24): π Ί ή, = 12mil0 cos{e)sin(9)d6 o =砵0 Rearrange ,we got:

...(26) -81 - 201211240 The output power of the LED is 0.515 watts and ve = 6·52 (10) 14 Hz, therefore: ή,: fl- ...(27) __0.515_ ~ [6.63(1 0)-34][6.52(10)u] =1 · 19 ( 10 ) 18 photons/sec brings this 入 into (26) we get: ... 1.19(10)18 =3.79 ( 10) 17 photons / Second/Sphericality Referring to Figure 61, the optical center 252 and the lens 254 of the LED 26 are shown schematically. The photodiode is 16 micrometers x 16 micrometers, and for the photodiode in the middle of the array, the solid angle (Ω) of the light cone emitted from the LED 26 to the photodiode 184 is approximately: Ω = sensor area / r2 [16 (10)^16(10)4] 2.825(10)-3]2 = 3.21 (10 Width 5 Sphere) It will be understood that the central photodiode 184 of the photodiode array 44 is used for these calculations. The sensor receives only 2% lower photons for the Lambertian excitation source intensity distribution during the hybridization event. The number of excitation photons emitted per unit time: he = η, Ω. ... ( 28 ) = [3.79(10) 17][3.21(1〇Γ5] = 1.22(10) 13 Photons/sec Now refer to equation (29): -82- 201211240 / « J-7 —Δ/1X f ns =(j>^neFTfe ! ns = (0.5)^.22(10)^1^.42(10)-^^(10)-6^-43400^^00^ =208 Photon/Sensor Therefore, use Philips LXK2-PR1 4-R00 LED With the Pulsar 650 fluorophore, we can easily detect any hybridization events that cause these numbers of photons to be excited. The SET LED illumination geometry is shown in Figure 62. The LED has a minimum optical power output when ID = 20 mA. = 24 〇 microwatt, wavelength center at λβ = 3 4 〇 nanometer (absorption wavelength of ruthenium chelate). Drive LED at ID = 200 mA, linearly increase output power to Pi = 2.4 mW. By placing the optical center 252 of the LED away from the hybrid chamber array 1 10 At a distance of 17.5 mm, we approximate the output flux to a photon flux with a maximum diameter of 2 mm and a photon flux in the 2 mm diameter point of the hybrid array plane. Equation 1 is obtained from Equation 27. 1 hve 2.4(10)- 3 ~[6.63(10)-34][8.82(10)14] =4.10 ( 10 ) 15 Photons/sec Using Equation 2 8, we get: ne - w'/Ω 4.10(10)15 [16(10 ) 6] 2 41(10)-3] 2 3.34(10)11 Photons/sec Now, return to Equation 22 and use the previously listed Tb chelate characteristics -83- 201211240 A. 1η[(6.94(10) —5)(10)(0.5)] = 11 1(10)·3 ~ 0.5(10)-9 =3.98 ( 10) "9 seconds now self-equal 2 1 : ns = (0.5) [3.34(10) π][6.94(10)-5][1(10)-3]β'398(10)'9/,(,〇Γ5 =1 1,600 photon/sensor chelating with SET LED and 铽 by hybridization event The photon theory number emitted by the object system can be easily detected and far exceeds the lower limit of 30 photons, which is used for the photosensor indicated by the above. Detecting the maximum separation on the wafer to avoid hybridization between the detection probe and the desired light diode to true confocal microscopy (see prior art) it is necessary to detect. This departure from traditional detection techniques is an important factor in the time and cost savings associated with the system. Traditional inspection requires the use of imaging optics that 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 close proximity to the probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode is 1 micron, the collection angle of the emitted light is as high as 173°. This angle is calculated by considering the light emitted from the probe closest to the center of the surface of the hybridization chamber of the photodiode. The photodiode has a planar active surface region parallel to the surface of the chamber. The cone of the cone in which the light can be absorbed by the photodiode is defined as having a firing probe at its apex and at the sensor corners around its plane. For a 16 micron XI 6 micron sensor, the apex angle of the cone is 170°: a limitation in the area of the hybrid chamber where the photodiode is expanded such that its area conforms to the 29-84-201211240 micron X 1 9.7 5 micron The apex angle is 173. . A separation of 1 micron or less between the surface of the chamber and the active surface of the photodiode is easily achieved. The application of a non-imaging optical method requires that the photodiode 184 be in close proximity to the hybridization chamber to collect sufficient photons of the fluorescent emission. The maximum spacing between the photodiode and the probe is determined as shown in Figure 54 below. Using the ruthenium chelate fluorophore and the SET UVTOP3 3 5T039BL LED, we calculated 16,000 photons from individual hybridization chambers 180 to 16 micron x 16 micron photodiodes 184. In carrying out this calculation, we assume that the light collection region of the hybridization chamber 180 has the same bottom area as the photodiode active region 185, and that half of the total number of hybrid photons reaches the photodiode 184. That is, the light collection efficiency of the optical system is ???=0.5. More precisely, we can write nine = [(the bottom area of the light collection area of the hybridization chamber) / (photodiode area)] [Ω / 4π], where Ω = solid angle is opposite to the substrate of the hybridization chamber The light diode of the representative point. For the right square pyramid geometry: Q = 4arcsin(a2/(4d〇2 + a2)), where d〇 = the distance between the chamber and the photodiode, and the size of the photodiode. Each hybrid cell releases 23,200 photons, and the selected photodiode has a lower detection limit of 17 photons. Therefore, the minimum optical efficiency required is: 焱=1 7/23 200 = 7.3 3 X 1 0_4 Hybridization chamber The bottom area of the light collection region of 180 is 29 microns x 19.75 microns. Solving, the maximum distance between the hybridization chamber and the photodiode 84 is -85- 201211240 and the limit distance is 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 effect of refraction. LOC Variants The LOC device 301 described and illustrated above is only one of many possible LOC device designs. Some possible combinations will now be described in the context of a flow chart (from sample input to detection) and/or display of LOC device variants using different combinations of the various functionalities described above. The flow chart is appropriately divided into a sample input and preparation stage 2 8 8 , an extraction stage 290, a culture stage 291, an amplification stage 292, a pre-hybridization stage 293, and a detection stage 294. For the sake of clarity and conciseness, only a brief description or summary of all LOC variants is shown and no detail configuration is shown. Also for clarity, smaller functional units, such as liquid sensors and temperature sensors, are not shown, but it should be understood that such functional units have been incorporated into the proper location of each of the following LOC device designs.

LOC Variant XII Figures 93 through 100 show LOC variant XII 758. The LOC device extracts 290, cultures 291, expands 292, and detects 294 pathogen DNA, and uses a pre-hybrid purification step 293 to increase hybridization efficiency. A sample, such as whole blood, is added to the sample inlet 68 (see Figure 95), and capillary action is applied to the surface tension valve Π8 (the anticoagulant from the sump 54 is added to the surface tension valve Π8). The sample continues to travel in the lid channel 94 to the pathogen dialysis -86 - 201211240 section 70. The dialysis section 70 has a bypass passage 600 to eliminate trapped air bubbles (see FIG. 9.5). After the dialysis section 70 performs dialysis, the red blood cells and white blood cells are directed to the waste reservoir 76 to continue the pathogen flow in the sample. To surface tension valve 128 (the lysis reagent from sump 56 is added to surface tension valve 128). The sample fills the chemical lysis chamber 130, and the chemical lysis chamber 130 is held by the boiling pilot valve 206 until the lysis reagent diffuses through the sample to release most, but not all, of the pathogen DNA. When the boiling pilot valve 206 is opened, the sample flows to the surface tension valve 132 (the restriction enzyme, ligase, and linker primer from the reservoir 58 are added to the surface tension valve 132). The sample is incubated with the culture section 1 1 4, and the sample is heated when the pathogen DN A is restricted by restriction enzyme cleavage and junction bonding (see Figure 95). After restriction enzyme shearing and junction bonding, the boiling pilot valve 207 opens to allow the sample to flow into the amplification section 112. When the sample flows into the amplification section 1 12, the amplification mix from the sump 60 and the polymerase from the sump 62 are passed through the surface tension valve 140 via the surface tension valve 138. The pathogen DNA is amplified by thermal cycling before the boiling pilot valve 108 is opened to allow the amplicon to flow to the small component dialysis section 682 where the large component has been removed. (See Fig. 95) As best shown in Figures 98 and 99, the small component dialysis section 6 82 has a large component channel between the two small component channels 762 formed in the bottom channel layer 1〇〇. 760 (see Figure 94). The large component channel 760 is coupled to the small component channel 762 by a small aperture in a series of inverted tapered openings 764 (which are smaller at the ends of the large component channels). In most practical applications, the small holes are 1 to -87 - 201211240 8 microns wide and 1 to 8 microns high. When the amplicon flows into the large component channel 760, the small component (less than the inverted tapered opening 764) begins to diffuse into the small component channel 762. As the flow proceeds to the downstream end of the small component dialysis section 682, the concentration of the small component in the large component channel 760 decreases. An additional advantage of microfabricated apertures is that the number of apertures per unit length along the channel is very high' for more efficient separation. For effective separation of components according to the size of interest, the spacing between adjacent apertures is between 1 micrometer and 1 micrometer: in the specific embodiment shown in Figure 99, the spacing between adjacent apertures It is 8 microns. Figure 100 shows the downstream end of the small component dialysis section 68 2 . The large component channel 760 lane becomes the wide tortuous end point of the blind terminal 766 as a waste reservoir. The two small component channels 762 are advanced to the opposite side of the hybridization chamber array 110, both of which pass through the tantalum path of the array to the respective turns terminal 768. The small component amplicon is flooded with all of the individual hybridization chambers 180 prior to initiation of hybridization heaters and subsequent probe-target hybridization assays (e.g., as described above). The small component dialysis section 682 removes cellular debris that may still remain in the sample stream after cell lysis. Cell debris may affect the efficiency of hybridization.

LOC Variant XIX The LOC variant XIX shown in Fig. 78 has a sample pathogen dialysis unit 70 and a hot lysate portion 638' before the nucleic acid amplification (amplification unit 112), but in the amplification 292 and the detection phase A pre-hybridization purification step 293 is added between 294. The pre-hybridization, small component dialysis section 682 removes cellular debris from the sample stream to lyse the cells. Most nucleic acid amplification protocols are sufficiently resistant to cellular debris in the sample. However, hybridization may be affected by cell debris, thus allowing -88-201211240 to be dialyzed with a pre-hybridization via a small component dialysis section 682 to substantially reduce the fragment concentration in the amplicons prior to filling the hybridization chamber array 110. Conclusion The devices, systems, and methods described herein facilitate molecular diagnostic testing at low cost and high speed and in situ care. The above-described system and its components are merely illustrative, and many variations and modifications will be readily apparent to those skilled in the art without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will be described with reference to the accompanying drawings, wherein: FIG. 1 shows a test module configured for fluorescence detection and a test module read. Figure 2 is a schematic view of the electronic components in the test module configured for fluorescence detection; Figure 3 is a schematic view of the electronic components in the test module reader; Figure 4 is a diagram showing LOC BRIEF DESCRIPTION OF THE DRAWINGS FIG. 5 is a perspective view of a LOC device; FIG. 6 is a plan view of a LOC device having all layer structures and features stacked on each other, and FIG. 7 is a LOC device having a cover structure independently shown. Figure 8 is a perspective view of the top surface of the inner channel and the sump shown in phantom -89 - 201211240 Figure 9 is an exploded top plan view of the inner channel and the sump shown in phantom; Figure 10 shows the upper channel configuration Figure 11 is a plan view showing the LOC device of the CMOS+ MST device structure; Figure 12 is a schematic view of the sample inlet of the LOC device; Figure 13 is an enlarged view of the insert AA shown in Figure 6. Figure 14 is the picture in Figure 6. Figure 15 is an enlarged view of the insert AE shown in Figure 13; Figure 16 is a partial perspective view of the laminated structure of the LOC device in the insert AE; Figure 17 is an illustration A partial perspective view of the laminated structure of the LOC device in the insert AE; FIG. 18 is a partial perspective view of the laminated structure of the LOC device in the insert AE, and FIG. 19 is a LOC in the insert AE. Partial perspective view of the laminated structure of the device; Figure 20 is a partial perspective view of the laminated structure of the LOC device in the insert AE; Figure 21 is a view showing the laminated structure of the LOC device in the insert AE Figure 22 is a schematic view of the lysing reagent sump shown in Figure 21; Figure 23 is a perspective view of a portion of the laminated structure of the LOC device in the insert AB - 90-201211240; 24 is a partial perspective view illustrating the laminated structure of the LOC device in the insert AB, and FIG. 25 is a partial perspective view illustrating the laminated structure of the LOC device in the insert AI: FIG. 26 is a view illustrating the insert AB Partial perspective view of the laminated structure of the LOC device; Figure 27 is a laminate structure illustrating the LOC device in the insert AB Figure 28 is a partial perspective view showing the laminated structure of the LOC device in the insert AB; Figure 29 is a partial perspective view showing the laminated structure of the LOC device in the insert AB; 0 is a schematic diagram of the amplification mixing tank and the polymerase storage tank; FIG. 31 shows the characteristics of the independent boiling pilot valve; FIG. 32 is a diagram of the boiling pilot valve taken along line 33-3 of FIG. Figure 3 3 is an enlarged view of the insert AF shown in Figure 15; Figure 3 is a schematic view of the upstream end of the dialysis section taken along line 3 5 - 3 5 shown in Figure 3 Figure 35 is an enlarged view of the insert AC shown in Figure 6; Figure 36 is a further enlarged view showing the amplification portion in the insert AC; Figure 37 is a further enlarged view showing the amplification portion in the insert AC; 3 8 is a further enlarged view showing the amplification portion in the insert AC; 91 - 201211240 FIG. 39 is a further enlarged view of the insert AK shown in FIG. 38; FIG. 40 is a further showing the amplification chamber in the insert AC. Figure 41 is a further enlarged view showing the amplification portion in the insert AC; Figure 42 is an amplification chamber showing the insert AC 4 is a further enlarged view of the insert AL shown in FIG. 42; FIG. 44 is a further enlarged view showing the amplification portion in the insert AC; FIG. 45 is an insert shown in FIG. Further enlarged view of the AM; Fig. 46 is a further enlarged view showing the amplification chamber in the insert AC; Fig. 47 is a further enlarged view of the insert AN shown in Fig. 46; Fig. 48 is an enlarged view of the insert AC Further enlarged view of the expansion chamber; Fig. 49 is a further enlarged view showing the amplification chamber in the insert AC; Fig. 50 is a further enlarged view showing the amplification portion in the insert AC: Fig. 5 is a diagram of the amplification portion Figure 5 is an enlarged plan view of the hybridization section; Figure 53 is a further enlarged view of two independent hybridization chambers; Figure 54 is a schematic view of a single hybridization chamber; Figure 5 is an insertion shown in Figure 6. An enlarged view of the humidifier illustrated in the article AG is an enlarged view of the insert AD shown in Fig. 52; Fig. 57 is an exploded perspective view of the LOC device in the insert AD; Fig. 58 is a FRET probe in a closed configuration. Figure of the needle; Figure 59 is a diagram of the FRET probe in an open and hybrid configuration; Figure 60 is an excitation light pair Figure 61 is a diagram of the excitation illumination geometry of the hybrid chamber array (excitation -92 - 201211240 illumination geometry); Figure 6 is a diagram of the sensor electronic technology LE D illumination geometry; Figure 63 is the insertion of Figure 6 An enlarged plan view of the humidity sensor shown in the object AH; Fig. 64 is a schematic diagram showing an array of photodiodes of a portion of the photosensor; Fig. 65 is a circuit diagram of a single photodiode; Fig. 66 is a photodiode Time chart of the control signal; Fig. 67 is an enlarged plane I c»,| · Fig. of the evaporator shown in the insert AP of Fig. 55, Fig. 68 is a photodiode and a photodiode through the hybridization chamber and the detection photodiode Figure 69 is a diagram of the junction-primed PCR; Figure 70 is a schematic diagram showing a test module with a blood needle; Figure 71 is a graphical representation of the structure of the LOC variant VII; 72 is a plan view of the LOC variant VIII having all of the layer structures and features superposed on each other; Fig. 73 is an enlarged view of the insert CA shown in Fig. 72; Fig. 74 is a view showing the insert CA shown in Fig. 72. Partial perspective view of the layered structure of LOC variant VIII: Figure 75 is shown in Figure 73 Figure 76 is a schematic representation of the structure of the LOC variant XIV; Figure 77 is a schematic representation of the structure of the LOC variant XIX; Figure 78 is a schematic illustration of the structure of the LOC variant XIX; The structure of the LOC variant XLI is described; 93 201211240 Figure 80 is a schematic diagram of the structure of the LOC variant XLIII; Figure 81 is a schematic diagram of the structure of the LOC variant XLIV; Figure 82 is the LOC variant XLVII The structure is illustrated; Figure 8 is a diagram of the primer-linked linear fluorescent probe during the initial amplification, and Figure 84 is a diagram of the primer-linked linear fluorescent probe during the subsequent amplification cycle; Figure 85A Graphical description of the thermal cycling of the primer-linked fluorescent stem-and-loop probe to 85F: Figure 86 is a schematic diagram of the LEDs associated with the hybrid chamber array and the light diode excitation; Figure 87 is for A brief description of the excitation LEDs and optical lenses on the array of hybridization chambers of the LOC device; Figure 88 is a schematic illustration of the excitation LEDs, optical lenses, and apertures used to direct light to the array of hybridization chambers of the LOC device; Figure 89 is an excitation LED for conducting light onto an array of hybrid chambers of a LOC device. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 90 is a plan view showing all the features overlapping each other and showing the positions of the inserts DA to DK; FIG. 91 is an enlarged view of the insert DG shown in FIG. Figure 92 is an enlarged view of the insert DH shown in Figure 9A; Figure 93 is a graphical representation of the structure of the LOC variant XII; Figure 94 is a perspective view of the LOC variant XII; Figure 95 is a display with overlays on each other All features and a plan view of the LOC variant XII showing the position of the insert -94-201211240 FA to FC; Figure 96 is a plan view of the LOC variant XII featuring only the features of the cover shown separately » Figure 97 shows the CMOS + MST standalone display A plan view of the LOC variant XII of the structure of the apparatus; Fig. 98 is an enlarged view of the insert FA shown in Fig. 95; Fig. 99 is an enlarged view of the insert FB shown in Fig. 95; Fig. 100 is a view of Fig. 95. FIG. 101 shows a specific embodiment of a parallel transistor for a light supply diode; FIG. 1 02 shows a specific embodiment of a parallel transistor for a light supply diode; FIG. 1 03 shows a light supply diode A specific embodiment of a parallel transistor; FIG. 104 is a differential imager Circuit diagram; Figure 105 schematically illustrates the negative control fluorescent probe in the stem-and-loop configuration; Figure 106 schematically illustrates the negative control fluorescent probe of Figure 105 in an open configuration; Figure 107 is a schematic representation of the present invention. The stem-and-loop configuration is positively controlling the fluorescent probe; Figure 108 is a schematic illustration of the positive control fluorescent probe of Figure 107 in an open configuration; Figure 109 shows the test module configured for use with ECL detection and Test module reader; Figure 1 1 shows the schematic diagram of the electronic components in the test module used with ECL detection; Figure 1 shows the test module and the alternative test module reader; and 95-201211240 Figure 1 1 2 shows the test module and the alternative test module reader and the host system that stores the various databases. [Main component symbol description] 1 〇: Test module 1 1 : Test module 1 2 : Test module reader 1 3 : Case 14 : Micro-USB connector 15 : Sensor 1 6 : Micro-USB 埠 17 : Touch Control screen 1 8 : Display screen 19 : Button 20 : Start button 2 1 : Honeycomb radio 2 2 : Aseptic sealing tape 2 3 : Wireless network connection 24 : Large container 2 5 : Satellite navigation system 26 : Light-emitting diode 2 7 : Data storage 2 8 : Telephone 29 : LED driver - 96 - 201211240 30 : LOC device 3 1 : Power conditioner 32 : Capacitor 33 : Clock 3 4 : Controller 35 : Register 36 : USB device driver 3 7 : drive 3 8 : random access memory 3 9 : drive 40 : flash memory 41 : register 42 : processor 43 : program memory 44 : light sensor 4 5 : indicator 46 : cover 47 : Module 48: CMOS + MST device 49: porous element 52: detection portion 54: sump 56, 56.1, 56.2, 56.3: sump 5 7: printed circuit board - 97 201211240 5 8, 5 8 · 1, 5 8 · 2 : Storage tank 60, 60.1-60.12, 60.X: storage tank 62, 62.1, 62.2 '62.3, 62.4' 62.X: storage tank 64: lower seal 6 6: top layer 6 8 : sample into 70: dialysis section 72: waste channel 74: target channel 76: waste reservoir 78: sump layer 80: cover channel layer 8 2: upper sealing layer 8 4: 矽 substrate 86: CMOS circuit 87: MST layer 8 8 : passivation Layer 90: MST channel 92: Lower pipe 94: Cover channel 96: Upper pipe 9 7: Wall 9 8: Meniscus holder 1 00 : MST channel layer - 98- 201211240 101 : Laptop/notebook 102: Capillary action initiation feature 103: Dedicated reader 105.: Desktop computer 106: Boiling pilot valve 107: E-book reader 108: Boiling pilot valve 109: Tablet computer 110, 110.1-110.12, 110.X: Hybridization Room array 111: Epidemiological data 112, 112.1-112.12, 112.X: Amplification part 1 1 3 : Genetic data 1 1 4 : Culture part 1 1 5 : Electronic health record 1 1 6 : Anticoagulant 118 : 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 valve 128, 128.2 , 128.3: Surface tension valve -99 - 201211240 130. > 1 3 i〇. 1 - 130.3 : 13 1: : Mixing unit 132, • 132. 1 ' 1 32.3 13 3 : incubator inlet 134: : lower pipe 13 6: optical window 13 8' 1 3 8. 1 ' 1 3 8.2 140, 140. 1 ' 1 40.2 146 : valve inlet □ 148 · · valve outlet □ 150: under the valve Pipe 152: Ring heater 153: Valve heater contact 154: Heater 156: Heater contact 15 8: Microchannel 160: Out □ Channel 164: Hole □ 166: Capillary action Start 168: Dialysis tapping hole 170: Temperature sensor 174: Liquid sensor 175: Diffusion barrier 176: Flow path feature lysis: Surface tension valve, 138.X: Surface tension valve, 140.X: Surface tension valve - 100 - 201211240 178: Liquid Sensor 1 80: hybridization chamber 1 8 2 : heater 1 84 : photodiode 1 85 : active region 1 8 6 : probe 187 : photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : Ring heater 192: water supply channel 1 93: upper pipe 194: lower pipe 1 9 5: top metal layer 196: humidifier 1 9 8 : extraction hole 202: capillary action starting feature 204: MST channel 206: boiling priming Valve 207: boiling lead Valve 208: liquid sensor 210: microchannel 2 1 2 : MST channel 2 1 8 : electrode - 101 - 201211240 220 : electrode 2 2 2 : gap 2 3 2 : humidity sensor 2 3 4 : heater 23 6 : FRET probe 2 3 8 : Target nucleic acid sequence 240 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 2 4 8 : Quencher 2 5 0 : Fluorescence signal 2 5 2 : Optical center 2 5 4 : lens 2 8 8 : sample input and preparation 2 90 : extraction stage 291 : culture stage 292 : amplification stage 293 : pre-hybridization filtration purification stage 294 : detection stage 296 : first electrode 2 9 8 : first electrode 3 00: Delay 301: LOC device-102 201211240 3 2 8: White blood cell dialysis unit 3 76: Thermal column 378: Positive control probe 3 80: Negative control probe 3 82: Calibration chamber 3 8 4: Gate 3 8 6 : Gate 3 8 8 : Gate 3 90 : Retractable lancet 3 9 2 : Lancet release button 3 9 3 : Mast 394: MOS transistor 3 9 6 : Μ Ο S transistor 3 98 : MOS Transistor 400: MOS transistor 4 0 2 : Μ Ο S transistor 404: MOS transistor 4 0 6 : node 408: film seal 4 1 〇: film guard 5 1 8 : LOC variant 594 : interface layer 600 : Bypass Channel 602: Boundary Target channel 201211240 604: waste channel 6 3 8 : hot lysate 646 : LOC variant 673 : LOC variant 674 : LOC variant 677 : LOC variant 6 8 2 : dialysis section 6 8 6 : dialysis step 692 : Primer-ligated linear probe 694: amplification blocker 6 9 6 : probe region 6 9 8 : complementary sequence 7〇〇: oligonucleotide primer 704: stem-and-loop probe 706: complementary sequence 708 : stem 7 1 0 : strand ' 7 1 2 : first aperture 7 1 4 : second aperture 7 1 6 : first mirror 7 1 8 : second mirror 75 8 : LOC variant 760 : large Component Channel 762: Small Component Channel - 104 201211240 764: Opening 766: Blind Terminal 768: Blind Terminal 778: Configuration 780: Configuration 782: Configuration 78 8: Differential Imager Circuit 790: Pixel 792: Virtual Pixel 794: Read_ Column 795: Read_Column 796: Negative Control Probe 7 9 7 : (Crystal) 798: Positive Control Probe 801: (Crystal) 803: Pixel Capacitor 805: Virtual Pixel Capacitor 8 〇 7: Switch 8 0 9 : Switch 8 1 1 : Switch 8 1 3 : Switch 8 1 5 : Capacitor amplifier 8 1 7 : Differential signal 8 60 : ECL excitation electrode 201211240 8 70 : EC L excitation electrode

Claims (1)

201211240 VII. Patent Application Range: 1 A wafer-on-lab (LOC) device for detecting pathogens in a biological sample. The LOC device comprises: an inlet for receiving a sample; a support substrate; separating the pathogen in the sample from the larger component a first dialysis portion; a lysis portion located downstream of the dialysis portion for lysing the pathogen to release the genetic material therein, the lysis unit having a lysis chamber and a heater to dissolve the sample in the lysis chamber a nucleic acid amplification unit located downstream of the lysis unit for amplifying the nucleic acid sequence in the genetic material; and a second dialysis unit located downstream of the nucleic acid amplification unit for pre-hybridization filtration generated by the nucleic acid amplification unit The second dialysis portion is configured to remove cell debris from the amplicon; wherein the first dialysis portion, the lysis portion, the nucleic acid amplification portion, and the second dialysis portion are supported on the support substrate on. 2. The LOC device of claim 1, wherein the lysis unit has a heater for the hot lytic pathogen. 3. The LOC device of claim 2, further comprising a hybridization portion downstream of the second dialysis section, having a probe array for hybridizing to a target nucleic acid sequence in the sample; and a photosensor Used to detect hybridization of any probe in the array. 4. The LOC device of claim 3, wherein the first dialysis portion has a first passage in fluid communication with the inlet, a fluid connection with the lysis portion, a second passage through which the 107-201211240 is connected, and a plurality of first holes The first orifice is generally smaller and smaller than the larger component, and the second passage is in fluid communication with the first passage through the first orifice such that the pathogen flows into the second passage and the larger one remains in the first passage. 5. The LOC device of claim 4, wherein the first and second channels are configured to impersonate the sample by capillary action. [6] The LOC device of claim 1, wherein the first portion has a large component channel, a small component channel, and a plurality of second ports of the fluid coupling channel to the small component channel, the second port is permeable to allow nucleic acid The sequence flows from the large component channel to the small component channel leaving the cell fragments of the two orifices in the large component channel, and the small component is in fluid communication with the hybridization portion. 7 The LOC device of claim 1, wherein the core portion is a thermostatic nucleic acid amplification unit. 8 a LOC device according to claim 7 of the patent scope, further comprising a reagent storage tank for holding a reagent for thermostatic nucleic acid amplification; and a surface tension valve having a meniscus configured to fix the reagent such that the meniscus will The reagent remains in the reagent reservoir until it contacts the liquid to remove the meniscus and the reagent flows out of the reagent reservoir. 9 The LOC device of claim 1, wherein the core is a polymerase chain reaction (PCR) amplification unit. 10, as claimed in claim 9 of the LOC device, which enters a CMOS circuit, a temperature sensor, and a micro-system MST) layer of the combined PCR portion, wherein the CMOS circuit is located on the support substrate and the MST layer in the pathogen of the pathogen. The second dialysis large component is larger than the first system and the acid amplification step contains, the surface of the pore sample acid amplification step contains the technology (between -108-201211240 and the CMOS circuit is configured to use the temperature sensor output) The feedback control PC R. 11 The LOC device of claim 10, wherein the PCR portion has a heating microcirculation sample to amplify a PCR microchannel of the nucleic acid sequence, and the PCR microchannel defines a partial sample flow path and has less than 100,000 square The cross-sectional area of the micrometer across the flow path. The LOC device of claim 11, wherein the PCR portion further comprises at least one elongated heater element for heating the nucleic acid sequence in the elongated PCR microchannel, elongated The heater element extends parallel to the PCR microchannel. The LOC device of claim 12, wherein at least one portion of the PCR microchannel forms an elongation A PCR chamber. The LOC device of claim 13, wherein the PCR portion has a plurality of elongated PCR chambers each formed by a respective portion of the PCR microchannel. The PCR microchannel has a series of wide tortuous turns. a 蜒 structure, each of which is curved to form a channel portion of an elongated PCR chamber. 15 The LOC device of claim 14 further comprising a reagent sump for holding a reagent used for PCR; and a surface tension valve An orifice having a meniscus configured to immobilize the reagent such that the meniscus retains the reagent in the reagent reservoir until the liquid sample is contacted to remove the meniscus and the reagent flows out of the reagent reservoir. 16 as claimed in claim 15 The LOC device further comprises an array of hybridization chambers containing probes such that the probes in each hybridization chamber are configured to hybridize to one of the target nucleic acid sequences. -109- 201211240 1 7 Patent Application No. 1 The LOC device of the 6th item, wherein the photo sensor is an array of photodiodes aligned with the registration of the hybridization chamber. 18, as in the LOC device of claim 16, wherein C The MOS circuit has a digital memory for storing the hybridization data from the photosensor output and a data interface for transmitting the hybridization data to the external device. 19 The LOC device of claim 16 of the patent application, wherein the PCR portion has An active valve for retaining liquid in the PCR section and in response to an activation signal from the CMOS circuit to allow liquid to flow to the hybridization chamber during thermal cycling, as in the LOC device of claim 19, wherein the active valve is Boiling priming valve with meniscus holder and heater 'menis surface 13 aligner configured to fix the meniscus and stop the capillary action to drive the 'liquid flow' heater to boil the liquid and fix it from the meniscus The device releases the meniscus to drive the flow. -110-