TW201211539A - LOC device for pathogen detection and genetic analysis with chemical lysis, incubation and tandem nucleic acid amplification - Google Patents

Abstract

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

Description

201211539 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 emerged as a hot area, bringing hope to disease detection in the early stages (before symptoms may be revealed). Molecular diagnostic tests are used to detect: • hereditary diseases • acquired disorders • infectious diseases • genetic susceptibility molecular diagnostics associated with health status with high precision and fast processing time, and reduced ineffective health care Services, improving patient outcomes, improving disease management and the possibility of personalizing patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids extracted and amplified from biological samples (e. g., blood or saliva), including both deoxyribonucleic acid (DNA) and ribonucleic acid (RN A ). The complementary nature of the nucleobase allows the synthesis of a short sequence of DN A (oligonucleotide) to bind (hybrid) to a particular nucleic acid sequence and can be used in nucleic acid assays. If a heterozygous reaction occurs, it means that there is a complementary sequence in the sample. This makes it possible, for example, to predict the disease that an individual will suffer in the future, to determine the nature and pathogenicity of the infectious agent, or to determine the individual's response to the drug. Nucleic Acid-Based Molecular Diagnostic Tests Nucleic acid-based assays have four distinct steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types can be used for genetic analysis, For example, blood, urine, saliva, and tissue samples. The diagnostic test determines the type of sample required, and not all samples are representative of the disease course. These samples have various components, but usually only one of these components is the subject of interest. For example, in the blood, high concentrations of red blood cells may inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required early in the nucleic acid assay. Blood is one of the most commonly sought sample types. Blood has three main components: white blood cells, red blood cells, and platelets. Platelets help to clotting and remain active in vitro. To inhibit coagulation, the sample is mixed with a reagent (e.g., ethylenediaminetetraacetic acid (EDTA)) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In humans, the number of red blood cells accounts for about 99% of the cellular material, but since the red blood cells do not have a nucleus, the red blood cells do not carry DNA. Furthermore, red blood cells contain a variety of components, such as heme, and hemoglobin may interfere with downstream nucleic acid amplification -6 - 201211539 treatment (described below). Red blood cell removal can be achieved by differentially dissolving the red blood cells in the lysis solution, and the remaining cellular material is completely retained, and the remaining cellular material can then be separated from the sample by centrifugation. This provides concentration of the target cells to extract nucleic acids from the target cells.

The extraction method used to extract nucleic acids depends on the sample and the diagnostic assay to be performed. For example, the method of extracting viral RNA will be quite different from the method of extracting DNA from the gene. However, the extraction of nucleic acids from a target cell typically involves a lysis step followed by a nucleic acid purification following the lysis step. The lysis step breaks the cell and the nuclear membrane to release the genetic material. This step is usually accomplished using a lytic detergent (eg, sodium lauryl sulfate), which also denatures a large amount of protein present in the cell. 0 followed by an alcohol precipitation step (usually using ice-cold ethanol or Isopropanol) Purification of nucleic acids, or purification of nucleic acids by a solid phase purification step, usually in the presence of a high concentration of chaotropic salt on a silica matrix, resin or paramagnetic beads in a column The solid phase purification step, followed by washing, and subsequent elution with a low ionic strength buffer. The optional step performed prior to precipitation of the nucleic acid is the addition of a protein-degrading protease to further purify the sample. Other lysis methods include mechanical lysis by ultrasonic vibration and thermal lysis of the cell membrane by heating the sample to 94 ° C. The target DNA or RNA present in the extracted material may be extremely small. This is especially true if the target is the DNA or RNA of the pathogenic microorganism. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a low concentration of a particular target core 201211539 acid to a detectable concentration. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The PCR method is well known in the art and is based on the "Principles and Technical Aspects of PCR Amplification" by E. Van Pelt-Verkuil et al. and published by Springer Press in 2008. A comprehensive description of such reactions is provided in the book. PCR is a powerful technique for amplifying target DNA sequences from complex DNA backgrounds. If RNA is to be amplified (by PCR), it is necessary to first transcribe RNA into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the generated cDNA was amplified by PCR. PCR is an exponential method and PCR can be carried out as long as the conditions for maintaining the reaction are acceptable. The components of the reaction are as follows: 1. Primer pair ~ Short single-stranded DNA of about 10 to 30 nucleotides complementary to the region flanking the target sequence. 2. DNA polymerase ~ a thermostable enzyme for the synthesis of DNA. 3_deoxyribonucleoside triphosphates (dNTPs) ~ provide nucleotides that are incorporated into the newly synthesized DNA strand. 4. Buffer ~ provides the best chemical environment for DNA synthesis. PCR generally involves placing such reactants in a small tube containing the extracted nucleic acid (about 1 〇 to 15 μl). The tube is placed in a temperature circulator; the temperature circulator is an instrument that allows the reaction to be carried out at a range of different temperatures for different periods of time. The standard procedure for each temperature cycle involves the denaturation phase, the annealing phase, and 201211539

Extension phase. The extension phase is sometimes referred to as the primer extension phase. In addition to this three-step procedure, a two-step temperature program can be used to combine the bonding phase with the extended phase in a two-step temperature program. The denaturation stage generally involves raising the reaction temperature to 90 to 95 ° C to denature the DNA strands; in the bonding stage, lowering the temperature to about 50 to 60 ° C to bond the primers; and subsequently in the elongation phase In the middle, the temperature is raised to an optimum activity temperature of DN A polymerase at 60 to 72 ° C for the primer extension reaction. This process is repeated for about 20 to 40 cycles, and the end result is the creation of millions of copies of the target sequence between the primers. Standard PCR procedures are available in a variety of variations, such as multiplex PCR for molecular diagnostics, linker-primer PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR. Multiplex PCR uses multiple sets of primers in a single PCR reaction mixture to create different sizes of amplicons that are specific for different DNA sequences. By locking multiple genes at once, additional information can be obtained from a single test round, otherwise additional experiments may be required to obtain additional information. However, optimization of multiplex PCR is difficult and requires the selection of primers with similar binding temperatures, and the amplicons have similar lengths and base compositions to ensure equal amplification efficiency for each amplicon. Linker-initiator PCR, also known as ligation adaptor PCR, is a method that allows nucleic acid amplification of virtually all DNA sequences in a complex DNA mixture without the use of a target specificity primer. The method first involves the cleavage of the target DNA population using an appropriate restriction endonuclease-9 - 201211539 (enzyme). The double-stranded oligonucleotide linker (also known as a adaptor) with the appropriate cantilever ends is then ligated to the ends of the target DN A fragment using a ligase enzyme. Nucleic acid amplification is then performed using oligonucleotide primers that are specific for the linker sequences. Thus, all fragments of the DNA source that are pinched by the linker oligonucleosides can be amplified.

Direct PCR describes a system for performing PCR directly on a sample without any nucleic acid extraction or minimal nucleic acid extraction. It has long been believed that many components present in unpurified biological samples (e.g., blood red components in blood) inhibit the PCR reaction. Traditionally, PCR requires extensive purification of the target nucleic acid prior to preparation of the reaction mixture. However, by appropriately changing the chemical reagent and the sample concentration, it is possible to perform PCR with minimal DNA purification or directly perform PCR. Modification of PCR chemistries for direct PCR involves increasing buffer strength, using a polymerase with high activity and persistence, and additives that can be chelated with potential polymerase inhibitors. Tandem PCR utilizes two different rounds of nucleic acid amplification reactions to increase the probability of amplifying the correct amplicon. One form of tandem PCR is Nested PCR, in which two pairs of PCR primers are used to amplify a single locus in two different rounds of nucleic acid amplification reactions. The first pair of primers is hybridized to a nucleic acid sequence located at a region outside the target nucleic acid sequence. A second pair of primers (nested primers) for use in the second round of nucleic acid amplification reaction binds within the first PCR product and produces a second PCR product comprising the target nucleic acid sequence, the second PCR product -10- 201211539 The substance will be shorter than the first PCR product. The rationale behind this method is that if the wrong locus is amplified during the first round of nucleic acid amplification, the possibility of an incorrect locus is amplified when the second amplification is performed with the second primer. Extremely low to ensure specificity.

Instant PCR (or quantitative PCR) is used to measure the amount of a PCR product in real time. The starting amount of nucleic acid in the sample can be quantified by using a probe containing a fluorescent luminescent group or a fluorescent dye in the reaction in conjunction with a set of standards. This method is particularly useful for molecular diagnostics where processing options may vary with the pathogen content of 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) and then amplifies the complementary DNA by PCR. RT-PCR is widely used in the study of gene expression to determine the expression of a gene or to identify sequences of RNA transcripts including the start and end positions of transcription. Reverse transcription enzyme PCR is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. The isothermal amplification system is another form of nucleic acid amplification method which does not depend on the thermal denaturation of the target DNA during the amplification reaction, and thus does not require a sophisticated machine. Therefore, the thermostatic nucleic acid amplification method can be performed at the original location or easily operated outside the laboratory environment. A variety of thermostatic nucleic acid amplification methods have been disclosed, including strand displacement amplification, transcription-mediated amplification, nucleic acid sequence-dependent amplification, recombinase polymerase amplification, rolling circle amplification, and branched Amplification, helicase-dependent isothermal DNA amplification, and circular nucleic acid-mediated amplification. -11 - 201211539 Constant temperature nucleic acid amplification method does not rely on continuous heating to denature template DNA to produce a single-stranded molecule as a template for further amplification, instead of using alternative methods, such as the use of specific restriction endonucleases Enzyme-catalyzed cleavage to form a gap in the DNA molecule or to digest the double strand of the DNA using an enzyme at a constant temperature.

The strand displacement amplification method (SDA) relies on the ability of certain restriction enzymes to form gaps in the unmodified strand of semi-modified DNA and the 5'-3' endonuclease-deficient polymerase has the ability to prolong and replace downstream DNA. The ability of the chain. Exponential nucleic acid amplification is then achieved by combining a synonymous reaction with an antisense reaction (a strand displacement system derived from a synonymous reaction in such reactions as a template for an antisense reaction). Instead of cutting DNA in a conventional manner, nicking enzymes (e.g., N. Alwl, N. BstNBl, and Mlyl) which use a gap in one of the DNA double strands are useful for such reactions. The strand displacement amplification method has been improved by using a combination of a thermostable restriction enzyme (jval) and a thermostable outer polymerase (polymerase). This combination has been shown to increase the amplification efficiency of the reaction from 1 to 8 fold amplification to 101 (fold) amplification, and thus it may be possible to utilize this technique to amplify a particular single replication molecule. Transcription-mediated amplification (TMA) and nucleic acid sequence-dependent amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than replicating corresponding genomic DNA. This technique uses two primers and two or three enzymes, respectively, RNA polymerase, reverse transcriptase and (if reverse transcriptase does not have RNA hydrolytic enzyme activity) selective RNA hydrolase H (RNase Η). One of the primers contains a promoter sequence for use by RNA polymerase. In the first step of nucleic acid amplification, the primer is at the defined position and the target -12-201211539

Target ribosomal RNA (rRNA) is heterozygous. The reverse transcriptase creates a purine replica of the target rRNA by extending from the 3' end of the promoter primer. The RNA in the DNA double-stranded complex produced by the reverse transcriptase RNA hydrolase activity (if the reverse transcriptase has RNA hydrolase activity) or the added RNase Η degradation. Next, a second primer binds to the DNA replica. A double-stranded DNA molecule is created by synthesizing a new DNA strand from the end of this primer by reverse transcriptase. RNA polymerase recognizes the promoter sequence in the DN A template and begins transcription. Each newly synthesized RNA amplicon is re-entered into the above procedure and used as a template for a new round of replication reactions. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a particular DNA fragment is achieved by binding the opposite sequence of the oligonucleotide primer to the template DNA and extending the primer with a DNA polymerase. Denaturation of double-stranded DNA (dsDNA) templates without the use of heat. Instead, RPA uses a recombinase-introduction complex to scan double-stranded DNA and aid in strand exchange at homologous positions. The resulting structure is stabilized by interaction of the single-stranded DNA binding protein with the replaced template strand, thereby avoiding the priming of the primer due to branch migration. The disassembly of the recombinase allows the 3' end of the oligonucleotide to be contacted with a strand displacement DNA polymerase (the strand replaces a DNA polymerase such as a large fragment of Bacillus subtilis polymerase I ( )) and subsequently The primer is used to extend the reaction. Exponential nucleic acid amplification is achieved by repeating this procedure. The helicase-dependent thermostated DNA amplification (HDA) system uses a DNA helicase to generate a single-stranded model for primer hybridization -13 - 201211539

The plate was then subjected to primer extension reaction using DN A polymerase. In the first step of the HD A reaction, the helicase travels along the target DNA and breaks the hydrogen bond connecting the two DNA strands, and then the two DNA strands bind to the single-stranded binding protein. Exposing the single-stranded target region with a helicase allows the primer to bind to the single-stranded target region. The DNA polymerase then uses the free deoxyribonucleoside triphosphates (dNTPs) to extend the 3' end of each primer to generate two DNA replicas. The two replicated double stranded DNAs each enter the next HDA cycle, resulting in exponential nucleic acid amplification of the target sequence.

Other constant temperature techniques relying on DN A include the rolling circle amplification method (RC A ), in which the DNA polymerase continuously extends the primer around a circular DNA template to generate a circular shape from multiple repeats. A long DNA product consisting of a template replica. Until the end of the reaction, the polymerase produces replicas of thousands of circular templates, and the length of the copies is related to the original target DNA. This method allows rapid nucleic acid amplification for spatial resolution of the target and for the information. This method can generate up to 1 0 1 2 template replicas in 1 hour. A branching amplification method is a variant of the RCA method that utilizes a closed circular probe (C-probe) or a padlock probe and a high persistence The DNA polymerase exponentially amplifies the C-probe under constant temperature conditions. The circular nucleic acid-mediated isothermal amplification method (LAMP) provides high selectivity and employs a DNA polymerase and a set of four specially designed primers that recognize a total of six different sequences on the target D N A . LAMP is initiated starting with the inner primer containing the sequence of the syntactic and antisense strands of the target DNA. Subsequent chain replacement by lateral primer guidance 〇ΝΑ -14- 201211539

The synthesis reaction releases single-stranded DNA. The single-stranded DNA is used as a template to guide DNA synthesis reaction by the second inner primer and the second outer primer, and the second inner primer and the second outer primer are hybridized with the other end of the target to generate dry- Stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer is hybridized to the loop on the product and a displacement DN A synthesis reaction is initiated to obtain the original dry-cyclic DN A and a new stem-loop having a double backbone length. DNA. This cycle was continued at a rate that accumulated 1 〇 9 target replicas in one hour. The final product has a number of inverted repeat target sequences, and the final product is cross-linked by reverse-repetitive target sequences in the same chain to form a dry-loop of broccoli-like structures with multiple loops. DN A. After completion of the nucleic acid amplification reaction, it is necessary to analyze the amplification products to determine whether or not the expected amplicon (amplification amount of the target nucleic acid) is generated. The method of analyzing the product can range from simply judging the size of the amplicon by gel electrophoresis to identifying the nucleotide composition of the amplicon using the DN A hybrid method. Gel electrophoresis is one of the simplest methods for confirming whether a nucleic acid amplification process produces an expected enhancer. Gel electrophoresis separates DNA fragments by applying an electric field on a gel matrix. Negatively charged DNA fragments move through the matrix at different rates, which are primarily dependent on the size of the DNA fragments. After the electrophoresis is completed, the fragments in the gel can be stained to see the fragments. It is usually dyed with ethidium bromide, which emits fluorescence under ultraviolet light. The DNA size marker and the amplicon can be electrophoresed side by side on the gel, and the fragments can be judged by comparison with DNA size markers (-15-201211539 stepped DNA bands) containing DNA fragments of known size. The size. Since the oligonucleotide primers bind to a specific position on both wings of the target DNA, the size of the amplification product can be predicted, and the amplification product can be detected on the gel to a strip of known size. band. In order to confirm the identity of the amplicon or the generation of several amplicon, the DNA probe heterozygous reaction is usually performed on the amplicon.

DNA hybridization refers to the formation of double-stranded DNA by complementary base pairing. DNA hybridization reactions for positive identification of specific amplification products require the use of DNA probes of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DNA sequence, a hybrid reaction will occur under conditions of suitable temperature, pH and ion concentration. If a heterozygous reaction occurs, it indicates that there is a gene or DNA sequence of interest in the original sample.

Optical detection is the most common method for detecting heterozygous reactions. Any of the amplicons or probes are labeled to emit light by means of fluorescence luminescence or electrochemiluminescence (ECL). The difference in these methods is that the means for generating the excited state of the luminescent group are different 'but both methods can covalently label the nucleoside chain. In electrochemiluminescence (ECL), current is stimulated to cause luminescent molecules or luminescent complexes to illuminate. In the luminescence method, irradiation is performed by laser light which causes luminescence. The detection unit and the illumination source for providing excitation light (the wavelength of which is absorbed by the fluorescent molecules) are used to detect fluorescence. The detection unit includes a photo sensor for detecting a transmitted signal (for example, a photomultiplier tube or -16-

201211539 Charge Coupled Device (CCD) arrays and mechanisms used to avoid signals in the sensor output signal (eg, wavelengths: the fluorescent molecules should illuminate the light to release stokes shifted), and by this detection Single: The Stokes displacement system emits light and the absorbed excitation: or wavelength difference. ECL luminescence is detected using a luminescent wave of the ECL species employed. For example, transition metal transition metal-ligand complexes release lines, so conventional photodiodes and charge couplers can be used as photosensors. The advantage of ECL is that, if excluded, ECL light may be an increase in sensitivity that occurs within the detection system. Microarrays allow hundreds of thousands of DNA sequences to be simultaneously powerful tools with the potential to screen thousands of genetically harmful pathogens in a single test. A microarray consists of different DNA probes immobilized on a substrate. Fluorescent molecules or luminescence are used during or after the reaction: DN A (amplicon)' and subsequent target DNA (amplicons) are applied to the probe array. The temperature is controlled and the microarray is maintained for hours or days, during which time a heterozygous reaction occurs. The microarray is washed with a buffer of incubation to remove unbound and closed, using an air stream (usually nitrogen) to dry _ excitation light is incorporated into the optical filter. Listen to the Tox displacement (collecting this release of light. The difference in frequency between the long-sensing light-sensing i-ligand complexes (the visible wavelength of the photosynthetic device (CCD) is the only light in the ambient light, thus the hybrid Experiments. Micro-array disease or detection of many sensations is carried out by a number of spots, first of all, after nucleic acid amplification, indicating that the target is between the needle and the amplicon in the labeled wet environment. DNA strands. Cleaning microarrays. Hybridization -17- 201211539 should be very important with the severity of cleaning. Strict and specific combination. Excessive rigor may lead to insensitivity. Can be detected by complementary Detecting the light emitted by the amplicon to identify the hybrid anti-system. Using the microarray scanner to detect the origin of the micro-column scanner is usually a computer-controlled inverted microscope (inverted scanning fluoresce: microscpe, which is generally used Laser: Use a light sensor (for example, a photomultiplier tube to write a number. The fluorescent molecules release the fluorescence released by the detection unit to collect the Stokes displacement light as described above must be collected Separating and transmitting to the detector. The micro-array sweep yoke configuration is configured to provide common out-of-focus information at the imaging plane. This mode allows only light to be measured above and below the focus plane of the target, Thereby increasing the signal to noise ratio (signal to detector converts the measured fluorescent photons into electrical energy into digital signals. This digital signal is translated into a number of generational light intensities. Each feature of the array is composed of such pixels. The final result of the scan is the exact sequence and position of each probe on the microarray. The identification and analysis of the heterozygous target sequences can be obtained at the following URL. The fluorescent probes may lead to a high degree of inappropriate binding. , thereby reducing the conformation of the needle forming hybrid. The array of fluorescent light, the microarray vertical scanning conjugate focal fluorescing ace confocal coated fluorescent dye), and ζ CCD ) detecting the illuminating trust s displacement light, and In the unexcited excitation wavelength applicator, the conjugated pinhole is usually used to eliminate the focus portion of the line. The source line cannot enter the detector impedance ratio. The power transfer table is derived from the image of the specified pixel by one or more images of the array surface. The system is known, so it can be more information: -18- 201211539 https://www.premierbiosoft.com /tech_notes/FRET_probe. html; and https://www.invitrogen.com/site/us/en/home/References/Mo lecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance -Energy-Transfer-FRET.html.

Molecular Diagnostics for Key Care Although molecular diagnostic tests offer many advantages, such tests have a lower-than-expected development in clinical testing and a small number of practical applications remaining in laboratory medicine. The reason for this is mainly due to the complexity of the nucleic acid test and the high cost associated with the test relying on methods that do not involve nucleic acids. The universal adaptability of molecular diagnostic tests in clinical management and the development of instruments and equipment (significant Cost reduction, from the beginning (sample processing) to the end (generating results) provides fast and automated analytical assays and can be operated without human intervention. Key care technologies for clinics, hospital clinics, or even consumers at home can bring many benefits, including: • Fast results to facilitate immediate processing and improved care quality. ' • Tests can be obtained by testing very few samples. • Reduce clinical effort. • Reduce inspection effort and increase office efficiency by reducing administrative work. -19 - 201211539 Improve the cost of each patient by shortening the length of hospital stay, summarizing the consultations of newly diagnosed outpatients and reducing the handling, storage and delivery of samples. Helps make clinical management decisions such as infection control and antibiotic use. Molecular diagnostics based on on-wafer laboratory (LOC)

Molecular diagnostic systems based on microfluidics provide a tool to automate and accelerate molecular diagnostic analysis. The primary reason for the faster detection time of this system is the diagnostic method steps of a built-in tandem device that can be extremely small, automated, and often inexpensive in a microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. The on-wafer laboratory (LOC) device is a common form of microfluidic device. The LOC device has a plurality of MST structures located within a MST layer for fluid processing on a single carrier substrate (typically germanium). Fabrication of the LOC device using the ultra-large integrated circuit (VSLI) lithography technology of the semiconductor industry allows each LOC device to maintain an extremely low unit cost. However, large external piping systems and electronics are required to control the flow of fluid through the LOC unit, to add reagents, to control reaction conditions, and to perform such operations. Efficiently connecting I:OC devices to such external devices limits the use of LOC devices for molecular diagnostics in the test environment. The cost of external devices and the complexity of their operation hinders LOC molecular diagnostics as a focus for care. Practical options for the environment. In view of the above reasons, there is a need for a molecular diagnostic based on the on-wafer-20-201211539 laboratory (LOC) device that can be used for priority care. [Inventive content] The present invention will now be described in the following paragraphs: GCF020.1 This system provides - on-wafer laboratory LOC devices for pathogen detection and genetic analysis comprising: an inlet for receiving the sample; a plurality of reagent storage tanks; a lysis section for dissolving white blood cells for release The pathogen and the genetic material in the white blood cell are in fluid communication with one of the reagent storage tanks containing the agent for dissolving the disease in the lysate section; the incubation section is located in the lysate section Compatible with one of the reagent storage tanks containing reagents for the plurality of enzymes of the genetic material; a nucleic acid amplification section for amplifying the nucleic acid sequence from the genetic material: wherein the lysis is performed The segments, the incubation segments, and the nucleic acids are expanded on the carrier substrate. Preferably, the first nucleic acid amplification reaction (PCR) segment is GCF020.2. GCF020.3 preferably, the LOC device system. Various systems. a biological sample (LOC) device,

The pathogen and substance in the sample, the lysate segment is downstream of the lysate segment of the original and white blood cells, and the culture is subjected to an enzymatic catalytic reaction and the downstream of the culturing segment is all loaded with an increased segment system. The polymerase chain also comprises a hybrid segment, -21 - 201211539, the hybrid segment is located downstream of the PCR segment, and the hybrid segment has a probe array and a photosensor, the probe array being used for The target nucleic acid sequence in the sample is heterozygous and the photosensor is used to detect the heterozygous reaction of any of the probes in the array. G C F 0 2 0.4 Preferably, the photosensor is less than 1600 microns from the array of probes.

GCF020.5 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to achieve a predetermined enzyme catalytic reaction temperature. GCF020.6 Preferably, one of the reagent reservoirs contains a adapter primer for binding to the nucleic acid sequence within the incubation segment. GCF020.7 Preferably, the nucleic acid amplification segment is a thermostatic nucleic acid amplification segment.

GCF02 0.8 Preferably, the reagent storage tanks each have a surface tension valve for leaving the reagent in the reagent storage tank, the surface tension valve having a meniscus anchor, the meniscus anchor system being used for pinning The meniscus of the reagent is removed until the meniscus of the reagent contacts the sample fluid to remove the meniscus to allow reagent to flow out of the reagent reservoir. GCF 020.9 Preferably, the LOC device also has a flow path from the inlet to the hybrid section, wherein the flow path is configured to direct the sample from the inlet to the hybrid section by capillary action. GCF020.1 0 Preferably, the LOC device also has a complementary metal oxide semiconductor (CMOS) circuit, a temperature sensor and a microsystem technology (-22-201211539 MST) layer, the MST layer comprising the PCR segment, wherein The CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to achieve feedback control of the PCR segment using the output of the temperature sensor.

GCF020.il preferably, the PCR segment has a PCR microchannel for thermal cycling of a sample to amplify a nucleic acid sequence, the PCR microchannel defining a portion of the sample flow path, and the PCR micro The channel has a cross-sectional cross-sectional area of less than 100,000 square microns. Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequence within the PCR microchannel, the elongate heater element extending parallel to the PCR microchannel. GCF020.1 3 Preferably, at least one segment of the PCR microchannel forms an elongated PCR chamber. Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by an individual segment of the PCR microchannel having a series of A wide curved channel formed by a meandering configuration, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. GCF020.1 5 preferably, the PCR section has an active valve that leaves the sample within the PCR section during amplification of the target nucleic acid sequences such that the CMOS circuit is configured to expand The active valve is opened after the increase to allow the capillary drive flow to continue. GCF020.1 6 preferably, the LOC device also has an array of hybrid chambers containing the probes such that the probes such as -23-201211539 in each chamber are configured to One of the target nucleic acid sequences is heterozygous. GCF020.1 7 Preferably, the photo sensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF020.1 8 preferably, the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface is used to The combined data is transmitted to an external device.

GCF020.1 9 preferably, the PCR section has an active valve that retains liquid within the PCR section during thermal cycling and allows liquid to flow to the impurities in response to an activation signal originating from the CMOS circuit Combined chamber

GCF011.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, and the meniscus anchor is constructed to define a bend for preventing the capillary driving flow of the liquid The liquid level, and the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, so that the capillary drive flow continues. The LOC device for pathogen detection and genomic analysis, which is simple to use, mass-produced, and inexpensive, receives a biological sample through a sample placement tank of the LOC device, and utilizes a reagent storage tank stored in the LOC device. The reagent dissolves white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, pretreats the genetic material of the sample in the incubation section of the LOC device, and amplifies the target gene sequence, and Hybridization with an oligonucleotide probe and sensing the hybridization of the probes by an integral imaging array of the LOC device should be performed to analyze the nucleic acid sequence of the sample. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides processing and analysis of the continuity of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system.

In the incubation section, the genetic material undergoes various pretreatments, such as nucleic acid-limited shearing and conjugation of the primers, to provide optimal or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to enhance the analysis. Verify system sensitivity, signal-to-noise ratio, and reliability. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely compact. Easy to carry system. The integrated imaging sensor increases read sensitivity and eliminates the need to use multiple optical components in an optical collection system by benefiting from large light collection angles. The reagent reservoirs integrated into the LOC device and holding all of the reagent requirements for the assay can provide a low number of system components and a simple manufacturing procedure to obtain an inexpensive analytical inspection system. -25- 201211539 GCF02 1.1 This system of the present invention provides a wafer-on-a-lab (L0C) device for pathogen detection and genetic analysis of biological samples. The LOC device comprises: an inlet for receiving the sample; a carrier substrate; a plurality of reagent storage tanks;

a lysis segment for dissolving pathogens and white blood cells in the sample to release genetic material in the pathogen and white blood cells, the lysing segment and the containing for dissolving the lysing segment The pathogen is in fluid communication with one of the reagent storage tanks of the leukocyte lysing reagent; the incubation section is located downstream of the lysis section and the cultivating section is associated with the inheritance One of the reagent storage tanks of the plurality of enzymes in which the substance is subjected to an enzymatically catalyzed reaction is in fluid communication;

a first nucleic acid amplification section downstream of the incubation section for amplifying a nucleic acid sequence within the genetic material of the first portion of the sample fluid derived from the incubation section: and a second a nucleic acid amplification segment, the second nucleic acid amplification segment being located downstream of the incubation segment for amplifying a nucleic acid sequence within the genetic material of the second portion of the sample fluid derived from the incubation segment; The lysis segment, the incubation segment, the first nucleic acid amplification segment, and the second nucleic acid amplification segment are all carried on the carrier substrate. GCF02 1.2 Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second polymerase chain reaction (PCR) segment . -26-

201211539 GCF021.3 preferably, the first PCR segment has a first set of pairs and the first set of primer pairs is used to bind a first set of complementary nucleic acid sequences, the second PCR segment has a second set of primer pairs and A second set of primers is used to bind a second set of complementary nucleic acid sequences that differ in the second set of complementary nucleic acid sequences. GCF021.4 Preferably, the first PCR segment and the second segment are constructed and operable with different amplification parameters, at least one of the following parameters: reverse transcriptase species; polymerase Species; concentration of deoxyribonucleoside triphosphate; buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. GCF021.5 Preferably, the LOC device also includes a first miscellaneous segment, a second hybrid segment and a photo sensor, the first hybrid segment being located downstream of a PCR segment and having the first hybrid segment a first probe array for hybridization with the first nucleic acid sequence, and the second hybrid segment is downstream of the second PCR segment and the second hybrid segment has a second for hybridization with the second nucleic acid sequence A probe array, and the photosensor is used for the hybridization reaction of any of the probes in the first array or the second array. GCF02 1.6 Preferably, the photosensor is less than 1 600 microns from the first probe array and the second probe array. Primer, and pair and PCR addition regions, the target is in the target detection column or -27-201211539 GCF02 1.7 preferably, the incubation segment has a heater that heats the genetic material and such The enzyme is to achieve a predetermined enzyme catalytic inverse. GCF02 1 .8 Preferably, one of the reagent storage tanks contains a primer primer' such adapter adapters for engagement with a sequence within the incubation section. GCF021.9 Preferably, the first nucleic acid amplification segment is a first nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic amplification segment. GCF02 1.10 Preferably, the reagent storage tanks each have a surface tension valve for leaving the reagent in the reagent storage tank, the surface tension having a meniscus anchor, and the meniscus anchor is used to fix the meniscus of the reagent. The meniscus of the reagent contacts the sample fluid to remove the meniscus to allow the agent to flow out of the reagent reservoir. Preferably, the LOC device also has a complementary metallization semiconductor (CMOS) circuit, a temperature sensor and a micro-system (MST) layer, the MST layer comprising the first PCR segment and the second segment, Wherein the CMOS circuit is disposed between the carrier substrate and the layer, the C Μ 电路 S circuit is configured to use the temperature sensor to achieve feedback control of the first PCR segment and the second PCR segment String GCF021.12 preferably 'this PCR segment has a PCR microchannel for the thermal cycling' that defines a flow path with a flow cross-sectional area of 100,000 square microns. GCF021.13 preferably 'the PCR microchannel has at least "heating temperature", rotating nucleic acid, constant temperature, acid expansion

For the valve to the test, the oxygen sample MST is less than the length of the sample.

-28- 201211539 A heater element that extends parallel to the PCR microchannel. Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The channel formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers.

GCF02 1.15 Preferably, the LOC device also has a reagent storage tank containing reagents for PCR; and a surface tension valve having a hole, the surface tension valve having a hole configured to hold the meniscus of the reagent, The meniscus leaves the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. Preferably, the LOC device also includes an array of first hybrid chambers for receiving the first probes such that the first probes within each hybrid chamber are configured to One of the first target nucleic acid sequences is heterozygous. GCF02 1.17 Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF021.18 Preferably, the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the hybrid data to External device. Preferably, the first PCR section has an active valve, 29 - 201211539. The active valve retains liquid in the first PCR section during thermal cycling and responds to an activation signal originating from the CMOS circuit. Liquid is allowed to flow to the first hybrid chamber array. GCF02 1.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, so that the capillary drive flow continues.

The LOC device for pathogen detection and genomic analysis, which is simple to use, mass-produced, and inexpensive, receives a biological sample through a sample placement tank of the LOC device, and utilizes a reagent storage tank stored in the LOC device. Resolving the cells of the sample in a chemical lysis chamber of the LOC device to release the genetic material of the sample, pretreating the genetic material in the incubation section of the LOC device, and amplifying the target gene sequence, And analysing the nucleic acid sequence of the sample by heterozygous with the oligonucleotide probe and sensing the hybridization reaction of the probes by the integrated imaging array of the LOC device. The lysis method is from the sample The cells extract the analytes for analysis and diagnosis and provide processing and analysis of the continuity of the targets. The lysing subunit integrated in the device provides a simple analytical test procedure, low system component count, and a simple system manufacturing process to obtain an inexpensive analytical test system. In the culture section, the genetic material undergoes various pretreatments, such as nucleic acid-restricted shearing and conjugation of the primers, to provide the most appropriate or necessary conditions for subsequent analysis stages -30-201211539, and to improve the information content of the analysis results. And improve the sensitivity, signal noise ratio and reliability of the analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the parallel amplification chambers allow different target or target groups to appropriately use different primer pairs or primer groups and also allow for the use of different optimal amplification parameters, thereby increasing Analyze the sensitivity, signal-to-noise ratio, and reliability of the test.

The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, productive, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent reservoirs integrated into the LOC device and holding all of the reagent requirements for the assay can provide a low number of system components and a simple manufacturing procedure to obtain an inexpensive analytical inspection system. GCF022.1 This system of the invention provides a on-wafer laboratory (LOC) device for pathogen detection and genetic analysis of biological samples, the LOC device comprising: an inlet for receiving the sample; a carrier substrate; 201211539 a plurality of reagent storage tanks; a lysis section for dissolving pathogens and white blood cells in the sample to release and genetic material in the pathogen and white blood cells' the lytic segment and the containing a reagent storage tank for dissolving the pathogen in the lysing section and the lysis reagent of the white blood cell;

a growing section, the growing section is downstream of the lysis section and the culturing section is in fluid communication with one of the reagent storage tanks containing a plurality of enzymes for enzymatically reacting with the genetic material; a nucleic acid amplification section, the first nucleic acid amplification section being located downstream of the incubation section for amplifying a first nucleic acid sequence in the genetic material; and a second nucleic acid amplification section, the second nucleic acid amplification An extension segment is located downstream of the first nucleic acid amplification segment for amplifying a second nucleic acid sequence derived from an amplicon derived from the first nucleic acid amplification segment; wherein the lysis segment, the culturing The segment, the first nucleic acid amplification segment, and the second nucleic acid amplification segment are all carried on the carrier substrate.

Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second polymerase chain reaction (PCR) region. segment. GCF022.3 preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences.

GCF022.4 Preferably, the first PCR segment and the second PCR-32-201211539 segment are constructed and operable with different amplification parameters, the amplification parameters being at least one of the following parameters: Transcriptase species; polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time;

The number of thermal cycle repetitions; and the temperature during a particular phase of the PCR. Preferably, the LOC device also has a hybrid segment located downstream of the second PCR segment, and the hybrid segment has a probe array and a photo sensor. A needle array is used to hybridize to a target nucleic acid sequence and the photosensor is used to detect a heterozygous reaction of any of the probes in the array. Preferably, the photosensor is less than 1600 microns from the array of probes. GCF022.7 Preferably, the incubation section has a heater that heats the genetic material and the enzymes to achieve a predetermined temperature of the enzyme catalyzed reaction. GCF022.8 Preferably, one of the reagent storage tanks contains a adaptor primer for binding to the nucleic acid sequence within the incubation section. Preferably, the first nucleic acid amplification segment is a first thermosensitive nucleic acid amplification segment, and the second nucleic acid amplification segment is a second thermostated nucleic acid amplification segment. Preferably, the reagent storage tanks each have a surface tension valve that remains in the reagent storage tank, the surface tension valve has a liquid surface anchor, and the meniscus anchor is used to fix the meniscus of the reagent. The meniscus is contacted with the sample fluid to remove the meniscus to allow the agent to flow out of the reagent reservoir. GCF022.il Preferably, the LOC device also has a CMOS, temperature sensor and micro system technology (MST) layer, the MST layer, the first PCR segment and the second PCR segment, wherein the CMOS system is disposed on Between the carrier substrate and the MST layer, the CMOS is configured to achieve feedback control of the first PCR and the second PCR segment using the output of the temperature sensor. GCF022.1 2 preferably, the first PCR segment has a PCR channel for thermal cycling of the sample, the microchannel defining a flow path having a flow cross-section of less than 100,000 square microns . GCF022.1 3 Preferably, the PCR microchannel has at least one shaped heater element extending axially from the PCR microchannel. GCF022.14 Preferably, the PCR segment has a plurality of PCR chambers, and each elongate PCR chamber is formed by the PCR microchannel segment having a series of wide curved channels The crucible configuration, and each wide curved channel is a channel section for forming one of the PCR chambers. Make the test bend, the test, the test circuit,

Road section micro-pass PCR area length long flat shape long shape

-34- 201211539 GCF022.15 Preferably, the LOC device also has a reagent storage tank containing reagents for PCR; and a surface tension valve having a hole, the surface tension valve having a hole configured to hold the reagent The meniscus is such that the meniscus leaves the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir.

GCF022.1 6 preferably, the LOC device also has an array of hybrid chambers for receiving the probes such that the probes within each hybrid chamber are configured to interact with the standard nucleic acid sequences One of them is heterozygous. G C F 0 2 2.1 7 Preferably, the photosensor is associated with an array of photodiodes arranged by the hybrid chambers. GCF022.18 Preferably, the CMOS circuit has a digital memory and a data interface, the digital system is used to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the hybrid data. To an external device. Preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid flow in response to an activation signal originating from the CMOS circuit To the first hybrid chamber array. GCF022.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, so that the capillary drive flow can continue. -35- 201211539

The LOC device for pathogen detection and genomic analysis, which is simple to use, mass-produced, and inexpensive, receives the biological sample by the sample placement tank of the LOC device, and utilizes the reagent storage tank stored in the LOC device. Resolving the cells of the sample in the chemical lysis chamber of the LOC device to release the genetic material of the sample, pretreating the genetic material in the incubation section of the LOC device, and amplifying the target gene sequence And analyzing the nucleic acid sequence of the sample by heterozygous with the oligonucleotide probe and sensing the hybridization reaction of the probes by the integrated imaging array of the LOC device. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides processing and analysis of the continuity of the targets. The lysing subunit integrated in the device provides a simple analytical test procedure, low system component count, and a simple system manufacturing process to obtain an inexpensive analytical test system.

In the incubation section, the genetic material undergoes various pretreatments, such as nucleic acid-limited shearing and conjugation of the primers, to provide optimal or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to enhance the analysis. Verify system sensitivity, signal-to-noise ratio, and reliability. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the cascaded amplification chambers allow for segmental local optimization of the early and late cycles of the amplification process, thereby increasing sensitivity, signal to noise ratio and reliability of the assay. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an easy-to-use, mass-produced, integrated solution that is inexpensive and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles.

The reagent reservoirs integrated into the LOC device and holding all of the reagent requirements for the assay can provide a low number of system components and a simple manufacturing procedure to obtain an inexpensive analytical inspection system. [Embodiment] Summary This review indicates the main components of the molecular diagnostic system incorporating the specific embodiment of the present invention. Comprehensive details of the structure and operation of the system will be set forth later in this specification. Referring to Figures 1, 2, 3, 93 and 94, the system has the following primary components: Inspection Module 1 and Inspection Module 1 1 are the dimensions of typical USB memory keys and the cost of such modules is extremely low. The inspection module 10 and the inspection module 1 1 each comprise a microfluidic device, typically in the form of a laboratory on-wafer (LOC) device 30, which has been preloaded for molecular and diagnostic purposes. Analyze the tested reagents and usually more than 1 000 probes (see Figures 1 and 93). The test module to be displayed in Figure 1 〇 -37- 201211539

Fluorescence-based detection techniques are used to identify target molecules' while assay module 1 in Figure 93 uses electrochemiluminescence-based detection techniques. The LOC device 30 has an integrated photosensor 44 for use in fluorescence detection or electrochemiluminescence detection (the detection methods will be described in more detail below). Both the inspection module 1 and the inspection module 1 1 use a standard micro USB plug 14 for power supply, data transmission and control, both of which have a printed circuit board (PCB) 5 7, and the two modules The groups each have an external supply capacitor 32 and an inductor 15. Both the inspection module 10 and the inspection module 1 1 are single-use for mass production and sales in ready-to-use sterilization packaging.

The outer casing 13 has a large placement groove 24 for receiving a biological sample and a tearable sterilizing sealing tape 22 (the adhesive tape 22 preferably has a low viscous adhesive) that covers the large placement groove 24 before use. The sealing film 408 having the film guard 410 forms part of the outer casing 13 to reduce the dehumidification effect in the inspection module while simultaneously releasing the pressure caused by small fluctuations in the air pressure. The film protector 410 protects the sealing film 40 8 from damage. The inspection module reader 12 supplies power to the inspection module 10 or the inspection module 11 via the micro USB slot 16. The inspection module reader 12 can take many different forms and the selection of such forms will be described later. The version of the reader 12 shown in Figures 1, 3 and 93 is a smart phone embodiment. A block diagram of the reader 12 is shown in FIG. The processor 42 runs application software originating from the program storage 43. The processor 42 also connects the display screen 18 and the user interface (UI) touch screen 17 and the button 19 , the cellular radio 2 1 , the wireless network connector 23 and the satellite navigation -38-

201211539 System 25. The cellular radio 21 communicates with the wireless network connector 23. The satellite navigation system 25 is used to update the accompanying location data library. Alternatively, the location data can be entered via the touch screen 17 or the button 19. The data store 27 contains genetic and diagnostic test results, patient data, analytical tests and probe data for identifying each probe and the probe. Data storage 2 7 and program 43 may be shared with a shared memory device. The application software installed on the set of readers 12 provides results analysis and is accompanied by diagnostic and diagnostic messages. For diagnostic testing, the test module 10 (or test 11) is inserted into the micro USB slot on the test module reader 12 to act as the sterilizing sealing tape 22 and to take the sample (in liquid form). Place the slot 24 large. Pressing the start button 20 begins the test by this. The sample flows into the LOC device 30, and the plate analysis test extracts and grows the sample nucleic acid (the target) and performs hybridization with the nucleic acid using a pre-synthesized hybrid reactive oligonucleotide probe. Hehe. In the case of the inspection module 10 (the inspection mode is based on a fluorescence-based detection method), the probes are excited by a light-emitting diode (LED) 26 housed in the housing 13 by a fluorescent cursor. Light is used to induce the heterozygous probe to emit fluorescence (see 1st). In the case of the inspection module 1 1 (the inspection module 1 1 uses a detection method based on learning illumination), the LOC device 30 is equipped with an ECL probe, and it is not necessary to use a light-emitting diode (LED) 26 to generate light. Role (see Figures 1 and 2). The electrode 860 and the electrode system are used for the popular manual transmission, and the inspection module of the array storage test module is used for the division of the injection software, and the sample is expanded. The electroluminescence of Figure 2 and the above-mentioned luminescence of 870-39-201211539 for excitation current (see Figure 94). The illuminating effect (fluorescent or luminescent) is detected using a photo sensor 44 integrated in a CMOS circuit of each LOC device. The measured signal is amplified and converted to a digital output 値, and the digital output 値 is analyzed by the test module reader 12. The reader then displays the result. This information can be stored locally and/or uploaded to a web server containing patient records. The inspection module 1 or the inspection module 1 1 is removed from the inspection module reader 1 2 and the inspection module is properly disposed.

Figures 1, 3, and 93 show the construction of a test module reader 12 that constitutes a mobile phone/smartphone. In other aspects, the test module reader can be used in a hospital, a private practice clinic or a laboratory. A portable personal computer/notebook 101, a dedicated reader 1〇3, an e-book reader 107 , tablet 1〇9 or desktop computer 105 (see Figure 95). The reader can be used to link a variety of additional application uses, such as patient records, bills, online databases, and multi-person environments. The reader can also be connected to a variety of local peripherals or remote peripherals such as printers and patient smart cards. Referring to Figure 96, the host system of the epidemiological database built into the host system 111 that can update the epidemiological data via the reader 12 and the network 125 using the data generated by the test module 1 ' 113 built-in genetic database, electronic health record (EHR) host system 115 built-in electronic health record, electronic medical record (EMR) host system 121 built-in electronic medical record and personal health record (piiR) host A personal health record built into system 123. On the other hand, the epidemiological database built into the host system of the epidemiological data, the genetic database built in the host system 113 of the genetic data, and the electronic data can be used via the network 125 and the -40-201211539 reader. The health record (EHR) built-in electronic health record, electronic medical record (EMR) host system 121 built-in electronic medical record and personal health record (PHR) host system 123 built-in personal health record update The digital memory in the LOC device 30 of the inspection module 10.

Returning to Figures 1, 2, 93 and 94, the reader 12 uses the battery power of the mobile phone configuration. The mobile phone reader contains all the inspection and diagnostic information that has been pre-uploaded. It can also be loaded or updated via some wireless interface or contact interface to communicate with peripheral devices, computers or online servers. The micro USB slot 16 is for connecting to a computer or to a mains power supply for charging the battery. Figure 62 shows a specific embodiment of the test module 10 for testing a positive or negative result of only a particular target, such as for testing whether an individual is infected, for example, H1N1 Type A. flu virus. A module 47 that is solely powered by USB and is an indicator built for a particular purpose is sufficient for this task. No other readers or application software is required. Only the USB powered and indicator 45 on the module 47 as an indicator signals to indicate a positive or negative result. This structural configuration is ideal for a large number of inspections. Additional tools provided with the system may include test tubes containing reagents (for the pretreatment of certain samples) with a spatula and lancet for collecting samples. Figure 62 shows a specific embodiment of an inspection module including a -41 - 201211539 spring-loaded lancet 390 and a lancet release button 3 92 for ease of use. Satellite phones can be used in remote areas. Inspection module electronic component

Figures 2 and 94 are block diagrams of the electronic components of the test module 1 and the test module 1 1 , respectively. 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' power conditioner 31, a random access memory (RAM) 38, and a program and data. Flash marks billion body 40. These components are the entire inspection module 1 or inspection module 1 1 (including light sensor 44, temperature sensor 170, liquid sensor 174, various heaters 152, 154, 182, 234 along with associated driver 37). And 39 and recorders 35 and 41) provide control and memory functions. Only the light-emitting diode (LED) 26 (as in the example of the test module 10), the external power supply capacitor 32 and the micro-U S B plug 14 are located outside the LOC device 30. The LOC device 30 includes a plurality of bond pads for connection to such external components. The random access memory (RAM) 380 and the program and data flashing device 40 have the application software and diagnostic and verification information for more than 1000 probes (eg, encrypted flash/security storage) ). In the case of the inspection module 1 1 constructed for ECL detection, no light-emitting diode (LED) 26 is required (see Figures 93 and 94). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 is equipped with electrochemiluminescent probes, and each of the hybrid chambers has a pair of ECL excitation electrodes 860 and 870. -42- 201211539 A variety of inspection modules 1 are manufactured in a variety of inspection types for off-the-shelf use. The difference between these types of tests depends on the on-board analytical tests performed by the reagents and probes. Specific examples of some infectious diseases that are rapidly identified by this system include: Influenza-type influenza virus, Influenza-type influenza virus, Influenza-type influenza virus, Insect salmon anemia virus (Isavirus), Tohogotovirus

• Pneumonia-respiratory cell fusion virus (RSV), adenovirus, interstitial pneumonia virus, Streptococcus pneumoniae, Staphylococcus aureus • Tuberculosis-Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium tuberculosis, Carbachol branch Mycobacterium and M. vaccae • Plasmodium falciparum, Toxoplasma gondii and other protozoan parasites • Salmonella enteric serovar typhi • Salmonella • Human immunodeficiency virus (HIV) • Dengue-yellow virus • Type A, B, C, D, and E hepatitis • In-hospital infections such as Clostridium difficile, vancomycin-resistant enterococci, and xylene penicillin-resistant Staphylococcus aureus • Herpes simplex virus (HSV) -43- 201211539 Cytomegalovirus (CMV) Epstein Barr's virus (EBV) Encephalitis - Japanese encephalitis virus, Chandipura virus Pertussis I. Hemp virus

Meningitis-Streptococcus pneumoniae and meningococcus, anthrax, and anthrax, can be identified by some of the hereditary diseases identified by this system: • Cystic fibrosis • Hemophilia • Sickle anemia • Tessa's disease (Tay-Sachs disease, also known as familial black dementia)

• Hemochromatosis • cerebral arteriopathy • Crohn's disease • Polycystic kidney disease • Congenital heart disease • Rett syndrome A few cancer options that can be identified by this diagnostic system Includes: • Ovarian Tumor-44- 201211539

Colorectal cancer multiple endocrine neoplasia retinoblastoma turcot syndrome The above options are not exhaustive, and the diagnostic system can be used to detect diseases far superior to the above types by using nucleic acid and proteosome analysis techniques. With physical condition. Detail construction of the system components LOC device L Ο C device 30 is the core of the diagnostic system. The L Ο C-loaded microfluidic platform relies rapidly on molecular diagnostic diagnostic assays for nucleic acids. The main step is to prepare samples 'extracting nucleic acids, amplifying nucleic acids, and detecting: LOC devices have different uses, and will be described in detail later. As mentioned above, the inspection module 1 and the inspection module can be configured differently to detect different targets. Similarly, the LOC device 30 is one of a plurality of different implementations of the LOC device 30 tailored to the target of interest for fluorescence detection of the target nucleic acid sequence in the whole blood sample. L0C device 3 0 1. For the purpose of explanation, the structure and operation of the 301 will now be described in detail with reference to Figs. 4 to 26 and 27 to 57. Fig. 4 is a diagram showing the convenience of the construction of the LOC device 301, using four tests for use with the LOC device 301 for performing the various devices. The structure of these uses has a number of examples. The cause of this is to achieve LOC mapping. The corresponding component symbols for the functional sections of the method phase -45- 201211539 are labeled with the method stages shown in Figure 4. The method stages associated with each of the major steps in the diagnostic assay of nucleic acid molecules are also indicated as: sample insertion and preparation stage 288, extraction stage 290, incubation stage 291, amplification stage 292, and detection stage 2 94. The various reservoirs, chambers, valves, and other components of the LOC device 301 will be described in more detail later.

Figure 5 is a perspective view of the LOC device 301. The LOC device 301 is fabricated using mass production techniques of CMOS and MST (Microsystem Technology). The layered structure of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) in FIG. The LOC device 301 has a germanium substrate 84 for carrying a CMOS + MST wafer 48 that includes a complementary metal oxide semiconductor (CMOS) circuit 86 and a microsystem technology (MST) layer 87 with overlying A cap layer 46 on the MST layer 87. For the purposes of this patent specification, the term "microsystem technology (MST) layer" refers to an assembly of structures and layers that process samples using various reagents. Accordingly, the structures and components are constructed to define a plurality of flow paths having characteristic dimensions that support the liquid during the processing period (the liquid has similar physical properties to the sample) for capillary drive flow. In view of this, the MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and components that are sized to achieve capillary drive flow and that can handle very small volumes. The particular embodiment described in the cold description shows that the MST layer is carried on the CMOS circuit 86 but does not include a plurality of features of the cover layer 46 and active components. However, those skilled in the art will appreciate that the MST layer does not have to have an underlying CMOS or does not have an upper cap layer to process the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 microns X 5 82 microns. Of course, LOC devices made for different applications may have different sizes.

Figure 6 shows the features of the MST layer 87 that overlap the features of the cap layer. The illustrations AA to AD and the illustrations AG to AH shown in Fig. 6 are enlarged and displayed in the figures 13, 13, 35, 56, 55 and 58, respectively, and the illustrations AA to AD and the illustration AG-AH are detailed. The following is provided for a comprehensive understanding of the various structures in the LOC device 301. Figures 7 through 10 show the features of the cover layer 46 separately, while Figure 11 shows the structure of the CMOS + MST device 48 separately. Layered Structures Figures 12 and 22 are schematic diagrams showing the layered structure of the CMOS + MST device 48, the cap layer 46, and the fluid interaction between the two. The drawings are not to scale and are intended to be illustrative. Figure 12 is a cross-sectional view through the sample inlet 68, and Figure 22 is a cross-sectional view through the reservoir 54. Preferably, as shown in Fig. 12, the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86 and that operates the active components located above the MST layer 87. The passivation layer 88 seals and protects the CMO S layer 8.6 from the fluid flowing through the MST layer 87. • 47- 201211539

The fluid flows through the capping channels 94 and the microsystem technology (MST) channels 90 located in the cap layer 46 and the MST channel layer 100, respectively (see Figures 7 and 16). Cell transport occurs in the larger channels 94 fabricated in the cap layer 46 while biochemical processing is performed in the smaller MST channels 90. The cell transport channel is sized to transport cells in the sample to predetermined locations within the MST channels 90. Cells carrying sizes greater than 20 microns (e.g., certain white blood cells) require channel sizes greater than 20 microns, and thus the cross-sectional cross-sectional area is greater than 400 square microns. The MST channel (especially the MST channel located at the location within the LOC device where no cells are transported) can be significantly smaller.

It will be understood that the cover channel 94 and the MST channel 90 system, and in particular the MST channel 90, may also be referred to as a heated microchannel or a dialysis M S T channel depending on the particular function of the channel. The MST channel 90 is formed by patterning with photoresist and etching through the MST channel layer 100 deposited on the passivation layer 88. The MST channels 90 are covered by a top layer 66 and the top layer 66 forms the top of the CMOS + MST device 48 (refer to the orientation shown in the figures). Although the cover channel layer 8 and the reservoir layer 78 are sometimes shown as different film layers, the cover channel layer 80 and the reservoir layer 78 are formed from a single piece of material. Of course, the piece of material may not be a whole piece. Both sides of the sheet of material are etched to form a capping channel layer 8 (and the capping channels 94 are saturated with the layer) and a reservoir layer 78 (the reservoirs 54, 56 are etched in this layer, 5S, 60 and 62). Alternatively, the storage tanks and the cover channels may be formed by micro-molding. Etching Techniques and Micro-Molding Techniques -48-201211539 Both are used to fabricate channels having flow cross-sectional areas of up to about 20,000 square microns and as small as about 8 square microns.

At different locations in the LOC device, the flow cross-sectional area of the channels may have an appropriate range of choice. When the channel contains a large number of samples or the composition of the sample contained in the channel is large, it is suitable to use a cross-sectional area of up to 20,000 square micrometers (for example, a channel having a width of 200 micrometers in a thick layer of 100 micrometers). When the channel contains a small amount of liquid or a mixture containing no large cells, it is preferred to use a very small flow cross-sectional area. The lower sealing layer 64 seals the cap channel 94 and the upper sealing layer 82 encloses the reservoirs 54, 56, 58, 60 and 62. The five storage tanks 54, 56, 58, 60, and 62 are pre-loaded with reagents for analysis and inspection. In the specific embodiments described herein, the storage tanks are prefilled with the following reagents, but may be readily substituted with other reagents, such as: • Storage tank 54: anticoagulant and optionally included Red blood cell lysis buffer • Storage tank 56: Lysis reagent • Storage tank 58: Restriction enzyme, ligase and linker (for linker-primer PCR, see Figure 61, this is an excerpt from T. Stanchan Et al., "Human Molecular Genetics 2" published by Garland Science in New York and London in 1999). • Storage tank 60: amplification mixture (deoxyribonucleoside triphosphates (dNTPs), primers, buffer), and -49- 201211539 • Storage tank 62: DNA polymerase. The cap layer 46 and the CMOS + M ST layer 48 are in fluid communication by corresponding openings in the lower sealing film 64 and the top layer 66. These openings are referred to as upper suction holes 96 and lower suction holes 92 depending on whether fluid flows from the MST passages 90 to the cover channels 94 or vice versa.

Operation of the LOC device

The following is a step-by-step description of the operation of the LOC device 301 by analyzing pathogenic DNA in a blood sample. Of course, other types of biological or abiotic fluids can also be analyzed using a suitable (or appropriate combination) of reagents, testing procedures, LOC device variants, and detection systems. Returning to Figure 4, the analysis of the biological sample involves five major steps, including the implantation and preparation of the sample 288, the nucleic acid extraction step 290, the nucleic acid incubation step 291, the nucleic acid amplification step 2 92, and the detection and Analysis step 294. The sample placement and preparation step 28 8 involves mixing the blood with the anticoagulant 1 16 and then using the pathogenic dialysis section 70 to separate the pathogen from the white blood cells and red blood cells. Preferably, as shown in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action draws the blood sample along the cover channel 94 to the reservoir 54. When the blood sample fluid causes the surface tension valve 118 to open, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants avoid the formation of agglomerates that may block fluid flow. -50- 201211539 Preferably, as shown in Fig. 22, the anticoagulant 116 is aspirated from the reservoir 54 by capillary action and the anticoagulant 116 is introduced into the MST channel 90 via the lower suction hole 92. The lower suction aperture 92 has a capillary action priming feature (CIF) 102 to shape the geometry of the meniscus such that the meniscus does not park at the edge of the lower suction aperture 92. When the anticoagulant 1 16 is aspirated from the reservoir 54, the vent 122 in the upper seal layer 82 allows air to enter in place of the anticoagulant 116.

The MST channel 90 shown in Fig. 22 is part of a surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and positions the meniscus 120 at the meniscus anchor 98 of the upper suction port 96. Prior to use, the meniscus 120 remains settled at the upper suction aperture 96 such that the anticoagulant does not flow into the cover passage 94. When the blood flows through the capping passage 94 to the upper suction hole 96, the meniscus 120 is removed and the anticoagulant is drawn into the blood fluid. Figures 15 to 21 show an illustration of AE' which is part of the illustration AA shown in Fig. 13 of the illustration AE. As shown in Figures 15, 16 and 17, the surface tension valve 118 has three separate MST channels 90' which extend between the respective upper and lower suction holes 92, 96. Surface tension The number of MST channels 90 in the valve can be varied to vary the flow rate of reagents flowing into the sample mixture. When the sample mixture is mixed with the reagents by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample fluid. Thus, the surface tension valve of each reservoir is constructed to meet the desired reagent concentration. Blood flows into the pathogen dialysis section 70 (see Figures 4 and 15). In the -51 - 201211539 pathogen dialysis section 70, an array of a plurality of apertures 164 having a predetermined threshold 値 size is used from the sample. The target cells are concentrated in the medium. Cells smaller than the critical enthalpy can pass through the wells while larger cells cannot pass through the wells. The unwanted cells may be any of the larger cells that are blocked by the array of holes 1 64 or smaller cells that pass through the holes, and direct the unwanted cells to turn to the waste unit 76, and at the same time The target cells continue to be analyzed for analysis.

In the pathogenic dialysis section/sputum described in this case, the pathogens derived from the whole blood sample are concentrated for microbial DNA analysis. The array of holes is formed by a plurality of holes 1 64 having a diameter of 3 microns, and the holes 1 64 allow the feed fluid in the cover channel 94 to be fluidly coupled to the target channel 74. The 3 micron diameter holes 164 are coupled to the dialysis uptake holes 168 of the target channel 74 by a series of dialysis MST channels 2〇4 (best shown in Figures 15 and 21). The pathogen is small enough to pass through the 3 micron diameter holes 164 and the pathogen is injected into the target channel 74 via the dialysis MST channels 204. Cells larger than 3 microns (e.g., red blood cells and white blood cells) remain in the waste channel 72 in the cap layer 46, which leads to the waste storage tank 7 6 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate specific pathogens or other target cells, such as white blood cells for human DNA analysis. A more detailed description of the variation of the dialysis section and the dialysis method will be provided later. Referring again to Figures 6 and 7, the fluid is drawn through the target channel 74 to the surface tension valve 1 of the lysis reagent reservoir 56. 28. Table - 52 - 201211539 Face 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 by the sample fluid, 'assuming that the physical properties of the fluids are substantially equal', the flow rate from all seven MST channels 90 will be greater than the source from the anticoagulant storage tank 54 (storage tank 54) The surface tension valve 118 has a flow rate of three MST passages 90). Therefore, the proportion of lytic reagent in the sample mixture is greater than the proportion of anticoagulant.

The lysis reagent is mixed with the target cells in the target channel 74 in the chemical lysis section 130 by diffusion. The boiling start valve 126 stops the flow of the fluid for a period of time sufficient for diffusion and lysis to release the genetic material within the target cells (see Figures 6 and 7). The structure and operation of the boiling start valve will be described in more detail below with reference to Figures 31 and 32. The applicant has also issued other active valve types that can be used in the present invention to replace the boiling start valve (active valves are relative to passive valves such as surface tension valve 1). These alternative valve designs are also described later. When the boiling start valve 126 is opened, the dissolved cells flow into the mixing section 133 to perform the limiting shear reaction and the linker engagement reaction before amplification. Referring to Fig. 13, when the fluid releases the meniscus at the surface tension valve 132 at the starting point of the mixing section 131, the restriction enzyme, the linker and the ligase are released from the reservoir 58. The mixture flows through the entire length of the mixing section 133 for diffusion mixing. The lower suction opening 134 at the end of the mixing section 133 is the entrance to the incubation chamber of the cultivation section 114 -53 - 201211539 133 (see Figure 13). The incubation chamber inlet channel 133 feeds the mixture into a crucible configuration comprised of a plurality of heated microchannels 210. The crucible configuration of the microchannels 210 can be provided to limit shear reactions and linkers. The incubation chamber for holding the sample during the ligation reaction (see Figures 13 and 14).

Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in Figure AB of Figure 6. Figures 23 to 29 each show the case where the layers of the CMOS + MST device 48 and the cap layer 46 are sequentially increased. The inset AB shows the end of the incubation section 114 and the beginning of the amplification section 112. Preferably, as shown in Figures 14 and 23, the fluid is injected into the microchannels 210 of the incubation section 114 until the fluid reaches the boiling start valve 106 where the fluid stops flowing while simultaneously Spread. As described above, the microchannel 210 in the upstream of the boiling start valve 106 becomes an incubation chamber containing a sample, a restriction enzyme, a ligase, and a linker. The heaters 154 are then activated and maintained at a constant temperature for a specific period of time for the limiting shear reaction and the linker ligation reaction. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is an optional step and that only some types of nucleic acid amplification assays require this incubation step 291. In addition, in some cases, a heating step may be required at the end of the incubation phase to raise the temperature above the incubation temperature. Increasing the temperature before the fluid enters the amplification section 112 removes the restriction enzymes and the binding enzyme. The inactivation of restriction enzymes and ligases is particularly relevant when a constant temperature nucleic acid amplification method will be performed. After incubation, the boiling start valve 106 is activated (turned on) and the -54-201211539 fluid continues to flow into the amplification section 112. Referring to Figures 31 and 32, a plurality of microchannels 158 form one or more amplification chambers which are injected into the crucible configuration of the heated microchannels 158 until the mixture reaches the boiling start valve 1 0 8. Preferably, as shown in the cross-sectional view of FIG. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and then the polymerase is released from the storage tank 62 to flow into the incubation section 1 1 4 and an intermediate MST channel 2 1 2 of the amplification section 1 1 2 .

Figures 35 through 51 show the layers of the LOC device 301 in the illustration AC of Figure 6. Figures 35 through 51 each show the case where the layers of the CMOS + MST device 48 and the cap layer 46 are sequentially incremented. The illustration Ac is located at the end of the amplification segment 112 and at the beginning of the hybrid and detection segment 52. The incubated sample, amplification mixture, and polymerase flow through the microchannels 1 58 to the boiling start valve 1 〇 8. After sufficient time has elapsed for diffusion mixing, the heaters 154 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 DN A . After the nucleic acid amplification process, the boiling start valve 〇8 is turned on, and the fluid continues to flow into the hybrid and detection section 52. The operation of these boiling start valves will be further described later. As shown in Fig. 52, the hybrid and detection section 52 has a hybrid chamber array 110. Figures 52, 53, 54 and 56 show details of the hybrid chamber array 1 1 and individual hybrid chambers 180. A diffusion barrier 1 75 is located at the entrance of the hybrid chamber 180, which prevents the target nucleic acid, probe strand and - from occurring between the hybrid chambers 180 during hybridization. 55 - 201211539 The diffusion of hybrid probes to avoid false hybrid detection results. The diffusion barriers 175 provide a length of flow path 'the length of the flow path is long enough for the probe to hybridize with the nucleic acid molecule and to prevent diffusion of the target sequence and probe during the time the signal is detected One chamber is left out and the other chamber is contaminated to avoid erroneous results.

Another mechanism for preventing erroneous readings is to have the same probe in a plurality of such hybrid chambers. A plurality of photodiodes 184 correspond to the hybrid chambers 180 containing the same probes, and the CMOS circuit derives a single result from the photodiodes 184. Abnormal results in the derivation of a single result can be omitted from calculation or differential weighting.

The thermal energy required for the hybridization reaction is provided by a CMOS controlled heater 182 (the heaters 182 are described in further detail below). After activation of the heaters, a hybrid reaction occurs between the complementary target sequence and the probe sequence. The LED driver 29 in the CMOS circuit 86 sends a signal to a light emitting diode (LED) 26 located in the test module 10 to cause the LED to illuminate. These probes only illuminate when a heterozygous reaction has taken place, thus eliminating the cleaning and drying steps typically used to remove unbound probe strands. The hybrid reaction forces the dry-loop structure of the fluorescent resonance energy transfer (FRET) probe 186 to open, allowing the fluorescent luminescent group to emit fluorescent energy in response to the excitation light of the LED, which will be done later More detailed description. Fluorescence is detected by photodiode 184 located in CMOS circuit 86 below each of the hybrid chambers 180 (see below for a description of the hybrid chamber). The photodiodes 184 and associated electronic components for all of the hybrid chambers collectively form the photosensor 44 (see Figure 59). In other embodiments -56-201211539, the photosensor may be a charge coupled device array (CCD array). The signal measured from the photodiode 184 is amplified and converted to a digital output 値, and the digital output 値 is analyzed by the test module reader 12. Further details of this detection method are described later. Additional details of the LOC device modularization of the design

The LOC device 301 has a number of functional sections including the reagent storage tanks 54, 56, 58, 60 and 62, the dialysis section 70, the lysis section 130, the incubation section 114, and the amplification section 112, various types Valves, humidifiers and humidity sensors. In other embodiments of the LOC device, such functional segments may be omitted and additional functional segments may be added, or such functionality may be used to achieve different uses than those described above. For example, the incubation section 1 14 can serve as the first amplification section 1 1 2 of the tandem amplification assay assay system, and the chemical lysis reagent storage tank 56 is used for the addition of primers, dNTPs, and buffers. The first amplification mixture, and reagent storage tank 58, is used to add reverse transcriptase and/or polymerase. If chemical lysis of the sample is desired, the chemical lysis reagent may also be added to the reservoir along with the amplification mixture, or by heating the sample for a predetermined period of time in the incubation section. Perform hot lysis. In some embodiments, if chemical lysis is desired and a mixture of the primer, dNTP, and buffer is desired to be separated from the chemical lysis reagent, an additional storage tank can be incorporated immediately upstream of the storage tank 58 A mixture of the primer, dNTP and buffer is contained. -57- 201211539

In some cases, it may be desirable to omit a certain step' such as incubation step 291. In this case, the LOC device may be specially manufactured to omit the reagent storage tank 58 and the incubation section 112, or may simply not load the reagent into the storage tank, or may have such an active valve if it has an active valve The active valve is not activated without dispensing the reagents into the sample fluid, and at this point the incubation section is simply turned into a channel to deliver the sample from the lysis section 130 to the amplification section 112. The heaters can be operated independently, and when the reaction is carried out depending on thermal energy (e.g., the thermal lysis reaction depends on thermal energy), the heaters can be programmed to prevent the heaters from starting during this step to ensure The hot lysis reaction does not occur in a LOC device that does not require a hot lysis reaction. The dialysis section 70 can be located at the starting point of the fluid system within the microfluidic device as shown in Figure 4, or the dialysis section 70 can be located anywhere else within the microfluidic device, for example, in some cases It may be beneficial to perform dialysis after the amplification phase 292 to remove cell debris prior to the hybridization and detection step 294. Alternatively, two or more dialysis sections can be inserted at any location throughout the LOC device. Similarly, the LOC device can incorporate a plurality of additional amplification segments 1 1 2 to ensure amplification of multiple targets in parallel or in series, followed by specific nucleic acid probes in the hybrid chamber array 1 1 0 The needle detects the targets. For analysis of a sample that does not require dialysis (e. g., a whole blood sample), the dialysis section 70 can simply be omitted from the placement and preparation section 28 of the sample designed for the LOC device. In some cases, the dialysis section 70 need not be omitted from the LOC device even if the analysis does not require dialysis. If the presence of the dialysis section does not create a geometrical impediment to the analytical test, the LOC device with the dialysis section 70 in the insertion and preparation section of the sample -58-201211539 can still be used without damaging the necessary functions. . Furthermore, the detection section 2 94 can comprise a plurality of protein body chamber arrays that are identical to the hybrid chamber array, but are loaded into the protein body chamber arrays to be designed for use with A probe that binds or hybridizes to a sample target protein present in an unamplified sample, rather than a nucleic acid probe designed to hybridize to a target nucleic acid sequence.

It will be appreciated that the LOC device manufactured for use in this diagnostic system selects different combinations of multiple functional segments depending on the particular LOC application. Most of the LOC devices in these LOC devices have most of the functional segments, and are suitably composed of multiple functional segments by extensive options from the functional segments used within existing LOC devices. The combination can be designed with additional LOC devices for new uses. Only a few LOC devices are shown in this specification, and some LOC devices are only 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 in the present specification are not exhaustively listed and are derived from a wide range of options from the functional segments used in existing LOC devices. Many additional LOC designs can be made with the appropriate combination of multiple functional sections. Sample Types A variety of LOC device variants that accept and analyze nucleic acid components or protein components in various sample types of liquids, including but not limited to blood and blood products, saliva, cerebrospinal fluid, urine, semen-59 - 201211539, amniotic fluid, cord blood, breast milk, sweat, pleural exudate, tears, pericardial fluid, ascites, environmental water samples and dips. The LOC device can also be used to analyze amplicons obtained from amplification of a large number of nucleic acids; in this case, all reagent storage tanks will be emptied or configured so as not to release components in the storage tank, and such dialysis The segment, lysis segment, incubation segment, and amplification segment will only be used to deliver the sample from the sample inlet 68 to the hybrid chambers 180 for nucleic acid detection as described above.

For some sample types, a pretreatment step is required. For example, the semen may need to be liquefied before the sample is placed in the LOC device, and the mucus may need to be pretreated with an enzyme to reduce the viscosity. Sample entry

Participating in Figures 1 and 12, the sample is added to the large placement slot 24 of the inspection module 1 . The large placement slot 24 is a frustoconical cone and the sample is fed into the inlet 68 of the LOC device 301 by capillary action. The sample is here flowed into a 64 micron wide x 60 micron deep cap channel 94 and the sample is attracted to the anticoagulant reservoir 54 by capillary action in the cap layer channel 94. Reagent Storage Tanks Analytical inspection systems that use microfluidic devices (e.g., LOC device 301) require a small volume of reagents, thus allowing the reagent storage tanks to contain all of the reagents required for biochemical processing, each reagent reservoir having a small volume. This volume is less than about 1, 〇〇〇, 〇〇〇, 〇〇〇 cubic microns, and in most cases -60-201211539 this volume is less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns, and in these patterns In the example of the LOC device 301 shown, this volume is less than 20,000,000 cubic microns. Dialysis section

Referring to Figures 15 through 21, 33 and 34, the pathogenic dialysis section 70 is designed to concentrate pathogenic target cells from the sample. As previously described, there are a plurality of holes in the top layer 66 which are in the form of holes 1 64 having a diameter of 3 microns which filter out target cells from a large number of samples. As the sample flows through the 3 micron diameter holes 164, microbial pathogens pass through the holes into a series of dialysis MST channels 204, and the microbial pathogens flow back up to the target via a 16 micron dialysis uptake 168. In channel 74 (see Figures 33 and 34). The remainder of the sample (e.g., red blood cells, etc.) remains in the capping channel 94. Downstream of the pathogenic dialysis section 70, the cover channel 94 is turned into a waste channel 72 to the waste storage tank 76. For biological sample types that produce a significant amount of waste, a bubble-like insert or other porous element 49 is disposed in the outer casing 13 of the test module 1 to be in fluid communication with the waste storage tank 76 (see Figure 1). . The operation of the pathogen dialysis section 70 relies entirely on the capillary action of the fluid sample. The 3 micron diameter holes 164 located at the upstream end of the pathogenic dialysis section 70 have a capillary action priming feature (CIF) 166 (see Figure 33) to attract the fluid downwardly into the dialysis MST channel below. . The first upper suction hole 198 of the target passage 74 also has a capillary action priming feature (CIF) 202 (see Fig. 15) to prevent the fluid from being easily settled on the dialysis surface -61 - 201211539 The suction hole is 1 6 8 .

The small component dialysis section 682 to be shown in Fig. 78 may have a structure similar to the pathogenic dialysis section 70. The small component dialysis section separates the small target cells from the sample by shaping the size of the pores (and, if necessary, shaping the shape of the pores) such that the pores are adapted to allow small target cells or molecules to pass through or Molecules, such that the small target cells or molecules enter the target channel and continue for further analysis. Larger sized cells or molecules are sent to waste storage tank 766. Thus, the LOC device 30 (see Figures 1 and 93) is not limited to isolation of pathogens having a size of less than 3 microns, but can also be used to separate cells or molecules of any desired size. Lysis segment

Returning to Figures 7, 11, and 13, the genetic material in the sample is released from the cells by chemical lysis. As described above, the lysis reagent from the lysis reagent reservoir 56 is mixed with the sample fluid in the target channel 74 downstream of the surface tension valve 128 of the lysis reagent reservoir 56. However, some diagnostic assays are more suitable for use with the hot lysis method, or even for the target cells using the chemical lysis method and the hot lysis method. The LOC device 301 provides the heated microchannels 210 of the incubation section 112 for thermal lysis. The sample fluid is injected into the incubation section 1 14 and stops at the boiling start valve 106. Incubate the microchannels 210 to heat the sample to reach the temperature at which the cell membrane ruptures. In some hot lysis applications, the enzymatic reaction in the chemical lysis section 130 is not necessary, and the hot lysis reaction completely replaces the enzymatic reaction in the chemical lysis section -62-201211539 130. Boiling Start Valve As discussed above, LOC unit 301 has three boiling start valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of a boiling start valve 108 located at the end of the heated microchannels 158 of the amplification section 112, respectively.

The sample fluid 1 1 9 is attracted by capillary action along the heated microchannels 158 until the sample fluid reaches the boiling start valve 108. The leading meniscus 120 of the sample fluid settles at the meniscus anchor 98 at the valve inlet 146. The geometry of the meniscus anchor 98 stops the leading meniscus to stop the capillary flow. As shown in Figures 31 and 32, the meniscus anchor 98 is provided by an opening provided from the MS T-channel 90 to the upper suction opening of the cover channel 94. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 1 5 2 is located at the perimeter edge of the valve inlet 1 46. The ring heater 152 is controlled by C Μ Ο S via the heater contact point 1 5 3 of the boiling start valve. To open the valve, CMOS circuit 86 delivers an electrical pulse to heater contact 153 of the valve. The annular heater 152 heats the liquid sample 1 1 9 until the liquid sample boils. The boiling action causes the meniscus 1 20 to disengage from the valve inlet 1 46 and begin to wet the cap channel 94. Once the cover channel 94 begins to wet, capillary flow is again performed. Fluid sample! i 9 is injected into the cap channel 94 and flows through the lower suction port 150 of the valve to the valve outlet 148 ′ at the valve outlet 148, the capillary drive fluid exiting the channel 1 along the augmentation-63-201211539 segment. Continue to flow into the hybrid and detection section 5 2 . A plurality of liquid sensors 1 74 are disposed at the front and rear of the valve for judgment. It will be appreciated that once the boiling start valves are opened, the boiling start valves can no longer be closed. However, when the LOC device 301 and the inspection module 10 are single-use devices, there is no need to close the valves again. Incubation section and nucleic acid amplification section 6, 6, 13, 14, 23, 24, 25, 35 to 45, 50 and 51

The figure shows the incubation section 114 and the amplification section 112. The incubation section 114 has a single heated incubation microchannel 210 that is positioned in the MST channel layer 100 and etched from the lower suction aperture 134 to the boiling start valve 106.蜿蜒 pattern (see Figures 13 and 14). Controlling the temperature of the incubation section 112 can enable an enzymatic reaction with higher efficiency. Similarly, the amplification section U 2 has heated amplifying microchannels 158 from the boiling start valve 106 to the boiling start valve 108 in a 蜿蜒 configuration (see Figures 6 and 14). . The valves terminate the flow of the fluid to allow the target cells to remain in the heated incubation microchannel 210 or amplification microchannel 158 and to simultaneously mix, incubate and nucleic acid amplification. The pattern of the microchannels is also advantageous to some extent to the mixing of the target cells and reagents. In the incubation section 1 14 and the amplification section 112, the heaters 154 are controlled by the CMOS circuit 86 to heat the sample cells and reagents using pulse width modulation (PWM). Each of the curved channels of the heated incubation microchannel 2 1 0 and the amplification microchannel 158 has three independent operations - 64 - 201211539

a heater 154 extending between respective heater contact points 156 of the heaters (see Figure 14), the heater contact points 156 providing input heat flux density Dimensional control. Preferably, as shown in Figure 5i, the heaters 154 are carried on the top layer 66 and are embedded in the lower sealing layer 64. The heater material is titanium aluminum alloy (TiAl), but a variety of other conductive materials are also suitable. The elongate heaters 154 are in parallel with the length of each of the channel segments (each of the channel segments forming the wide curved channel of the crucible shape). In the amplification section 112, each wide curved flow path can be operated as a separate PCR chamber by individual heater control. This analytical assay system using a microfluidic device (e.g., LOC device 301) requires a small volume of amplicons, thus allowing amplification reactions to be performed in the amplification section 112 with a low volume of amplification mixture. This volume is less than about 400 nanoliters, in most cases less than 1 70 nanoliters, typically less than 70 nanoliters and in the case of LOC device 301 this volume is between 2 nanoliters to 30 millimeters. Between microliters. Increased heating rate and higher diffusion mixing The small cross section of each channel section increases the heating rate of the augmented fluid mixture. All fluids are at a relatively short distance from the heater 154. It can be seen that reducing the cross-section of the channel (i.e., the cross section of the amplifying microchannel 158) to less than 100,000 square microns results in a heating rate that is higher than that provided by a larger gauge device. The lithography manufacturing technique allows the fabrication of a -65-201211539 microchannel 158 with a cross-sectional cross-sectional area of a flow path of less than 1 6,000 square microns, which provides a substantial cross-sectional area of the flow path below 1 6,000 square microns. High heating rate. Feature sizes of up to 1 micron can be easily achieved using lithography. If very few amplicons are required (as is the case with LO C device 301), the cross-sectional area can be reduced to less than 2,500 square microns. In order to achieve the diagnostic analysis test using 1 000 to 2000 probes on the LOC device and completing the "put sample, output result" requirement within one minute, the flow between 400 square microns and 1 square micrometer The cross sectional area can meet this requirement.

The heater elements within the amplification microchannel 158 heat the nucleic acid sequences at a rate above 80 Kelvin per second (K), and in most cases are above 1 〇〇K per second. The nucleic acid sequences are heated at a rate. In general, the heater element heats the nucleic acid sequences at a rate of 1 〇〇〇K or more per second, and in many cases, the heater element heats the nucleic acid sequences at a rate of more than 10 000 K per second. . Generally, according to the requirements of the analysis and inspection system, the heater element is at a rate of more than 100,000 K per second, a rate of 1,000,000 K or more per second, a rate of ιο, οοο, οοο K or more per second, 20,000,000 K per second. The nucleic acid sequences are heated at a rate of above 40,000,000 κ per second, at a rate of more than 80,000,000 K per second, and at a rate of more than 1600,000,000 K per second. The passage of the small cross-sectional area is also beneficial for the diffusion mixing of any reagent with the sample fluid. The diffusion of one liquid into the other prior to the diffusion mixing is maximized by the diffusion near the interface between the two liquids. The concentration will decrease with distance from the interface. The use of microchannels with a relatively small cross-sectional area of flow direction ensures two fluids -66- 201211539

Flow next to the interface for faster diffusion mixing. It can be seen that the mixing rate achieved by narrowing the cross-section of the channel to less than 100,000 square microns is higher than the mixing rate provided by equipment using larger specifications. The lithography manufacturing technique allows the fabrication of microchannels having cross-sectional cross-sectional areas of flow paths below 16,000 square microns, which provide a significantly higher mixing rate than the cross-sectional area of 16,000 square microns. If a very small volume is required (as is the case with LOC unit 301), the cross-sectional area can be reduced to less than 2500 square microns. In order to achieve a diagnostic analysis test using 1000 to 2000 probes on the LOC device and completing the "implantation sample, output result" requirement within one minute 'between 400 square micrometers to 1 square micrometer The cross-sectional area of the flow crosses this requirement. A short thermal cycle time keeps the sample mixture close to the heaters and uses a very small fluid volume to allow for rapid thermal cycling during the nucleic acid amplification process. For target sequences up to 150 base pairs (bp) in length, each thermal cycle (i.e., denaturation, adhesion, and primer extension) is completed in 30 seconds. In most diagnostic assays, individual thermal cycling times are within 11 seconds, and most thermal cycling time is within 4 seconds. The LOC device 30 for performing a portion of the most commonly used diagnostic assays has a thermal cycle time of between about 0.45 seconds and 1.5 seconds for a target sequence of up to 150 base pairs in length. Thermal cycling at this rate allows the assay module to complete the nucleic acid amplification procedure for much less than 1 minute, and typically less than 2 20 seconds. For most analytical assays, the amplified segment can produce sufficient amplicons within 80 seconds from the sample -67-201211539 fluid entering the sample inlet. For most analytical tests, sufficient amplicons are produced within 30 seconds. The amplicon is fed to the hybrid and detection section 52 via a boiling start valve 108 after a predetermined number of amplification cycles have been completed. Hybrid chamber

Figures 52, 53, 54, 56 and 57 show the hybrid chambers 180 in the hybrid chamber array 11A. The hybrid and detection section 52 has an array 110 of 24x45 composed of a hybrid chamber 180, and each hybrid chamber has a hybrid sensitive FRET probe 186, a heater element 182, and an integrated photodiode 184. » The photodiode 184 is incorporated for detection of fluorescence produced by a hybrid reaction of a target nucleic acid sequence or protein with the FRET probes 186. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. Any material between the FRET probe 186 and the photodiode 184 must allow release of light. Therefore, the partition wall section 97 between the probes 1 8 6 and the photodiodes 1 84 is also permeable to the emitted light. In the LOC device 301, the partition wall section 97 is a thin layer of hafnium oxide (about 0.5 microns). Incorporating a photodiode 184 directly beneath each of the hybrid chambers 180 allows the probe-target hybrid to be minimally small while still producing a detectable fluorescent signal (see Figure 54). A small amount of probe-target hybrids allow the use of small volumes of hybrid chambers. The amount of probe required to achieve detectable amount of probe-target hybrids prior to hybridization is less than about -68-201211539

2 70 pg (equivalent to 900,000 cubic microns), in most cases less than about 60 pg (equivalent to 200,000 cubic microns), usually less than about 12 pg (equivalent to 40,00 cubic microns) and In the case of the LOC device 301 shown in the drawings, it is less than about 2.7 pg (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybrid chambers allows for a higher chamber density on the LOC device and thus more probes. In LOC device 301, the hybrid section has more than 1000 chambers in an area of 1500 microns by 1500 microns (i.e., the area of each chamber is less than 2250 square microns). Smaller volumes also reduce reaction time, making hybridization and detection faster. An additional advantage of requiring a small amount of probe in each chamber is that only a minimal amount of probe solution needs to be dispensed into each chamber during manufacture of the LOC device. A specific embodiment of the LOC device of the present invention is formed using a volume of a probe solution of 1 〇 12 liters or less. After nucleic acid amplification, the boiling start valve 108 is activated and the amplicon flows along the flow path 176 and flows into each of the hybrid chambers 1 8 〇 (see Figures 5 2 and 5 6). The end liquid sensor 178 indicates when the amplicon is injected into the hybrid chambers 180 and when the heaters can be activated 182° after sufficient hybridization time, the light emitting diodes (LEDs) ) 26 is activated. The openings in each of the hybrid chambers 180 provide an optical window 130 for exposing the FRET probes to excitation light (see Figures 52, 54 and 56). The light emitting diode (LED) 26 continues to illuminate for a sufficient amount of time to induce the probes to emit high intensity fluorescent signals. During the period of -69 - 201211539, the photodiodes 1 84 were short-circuited. After a pre-progemmed delay time of 300 (see Figure 2), the photodiode 1 84 operates and detects fluorescence illumination without excitation light. Incident light (see Fig. 54) on the active region 185 of the photodiode 184 is converted to photocurrent, which can then be measured using a CMOS circuit 86.

The hybrid chambers 180 are each equipped with a probe for detecting a single target nucleic acid sequence. If desired, probes can be placed in each of the hybrid chambers 180 to detect more than one or more different targets. Alternatively, the same probe can be loaded into multiple hybrid chambers or all hybrid chambers to repeatedly detect the same target nucleic acid. Repeated loading of the probes throughout the hybrid chamber array 110 in this manner results in increased confidence in the results obtained and, if desired, adjacent to the hybrid chambers The multiple results measured by the photodiodes are combined to provide a single result. Those skilled in the art will appreciate that depending on the specifications of the analytical test, the hybrid chamber array 110 may have from one to more than one different probe. Hybrid Chambers Using Electrochemiluminescence Detection Figures 86, 98, 123 and 124 show such hybrid chambers 180 for use in the LOC device ECL variant (LOC device variant L 729 ). In this particular embodiment of the LOC device, an array of 10 x 45 hybrid chambers 180 (each heterozygous chamber having a hybrid sensitive ECL probe 237) is registered by a plurality of integrated The corresponding array of light diodes in the CMOS, 70-201211539 1 84, is configured. Each photodiode 184 is incorporated in a manner similar to the LOC device constructed for the fluorescence detection method for detecting electrochemiluminescence produced by the hybridization reaction of the target nucleic acid sequence or protein with the ECL probe 23 Function (ECL). Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. Similarly, the release light can pass through a transparent partition section 97 between the probes 186 and the photodiode 184 «

The photodiode 184 in close proximity to each of the hybrid chambers 180 allows the amount of probe-target hybrid to be minimized while still producing a detectable ECL signal (see Figure 8 6). A small amount of probe-target hybrid allows the use of a small volume of hybrid chamber. The detectable amount of probe-target hybrid is less than about 270 pg (equivalent to a chamber volume of 900,000 cubic microns) prior to hybridization, and in most cases less than about 60 Picograms (equivalent to 200,000 cubic microns), typically less than about 12 pg (equivalent to 40,000 cubic microns) and less than about 2.7 pg in the case of the LOC device shown in the figures (equivalent to 9,000 cubic meters) Micron chamber volume). Of course, reducing the size of the hybrid chambers allows for a higher chamber density on the LOC device and thus more probes. In the LOC device shown, the hybrid segment has more than 100 cells in an area of 1500 microns by I5 〇〇 microns (i.e., the area of each chamber is less than 2250 square microns). Smaller volumes also reduce reaction time, making hybridization and detection faster. An additional advantage of requiring a small amount of probe in each chamber is that only a minimal amount of probe solution needs to be dispensed into each chamber during manufacture of the LOC device. In the case of the LOC 201211539 device shown in these figures, the required amount of probe can be made using 1 (Γ 12 liters or less than 12 liters of probe solution volume point). After nucleic acid amplification, boiling start Valve 108 is activated and flows along flow path 176 and into each of the hybrid chambers 180 (see Figures 52 and 124). Endpoint liquid sensor 1 78 indicates when the amplification is to be performed Sub-injecting the hybrid chambers 180 to facilitate activation of the heaters 182°

After sufficient hybridization time, the photodiode 1 8 4 is activated to prepare for the collection of ECL signals. The ECL excitation driver 39 (see Fig. 94) then activates the ECL electrodes 860 and electrodes 870 for a predetermined length of time. The photodiode 184 is continuously activated for a short period of time after the ECL excitation current is stopped to maximize the signal noise ratio. For example, if the photodiode 184 is continuously activated for five times of the luminescence luminescence decay period, the signal will decay to 1% of the initial »» the incident light on the photodiode 184 is converted into a photocurrent. The photocurrent can then be measured using CMOS circuit 86. Humidifier and Humidity Sensor The illustration AG of Figure 6 indicates the location of the humidifier 1 96. The humidifier prevents evaporation of the reagents and probes during operation of the LOC device 301. Preferably, as shown in the enlarged view of Figure 5, the water storage tank 108 is in fluid communication with the three evaporators 90. During the manufacturing process, the biological storage water is injected into the water storage tank 1 8 8 and the water storage tank 1 8 8 is sealed. Preferably, as shown in Figures 5 and 60, water is drawn into the three lower suction holes 194 of the -72-201211539 by capillary action and flows along the respective water supply channels 192 to the evaporators 190. A set of upper suction holes 193. The meniscus is positioned at each of the upper suction holes 193 to retain water. The evaporators have an annular heater 191 that surrounds the upper suction holes 193. The ring heaters 191 are connected to the CMOS circuit 86 by a conductive post 376 that leads to the top metal layer 195 (see Figure 37). When activated, the annular heater 191 heats the water, causing moisture to evaporate and humidifying the device environment.

The position of the humidity sensor 232 is also shown in Figure 6. However, as best shown in the enlarged view of the inset AH in Fig. 58, the humidity sensor has a capacitive comb structure. The lithographically etched first electrode 296 and the lithographically etched second electrode 298 face each other such that the teeth of the first electrode 296 and the second electrode 298 are interleaved. The opposing electrodes form a capacitor having a capacitance and the capacitor can be monitored by CMOS circuitry 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, causing the 'capacitance to increase.' Humidity sensor 232 abuts the hybrid chamber array 110, and humidity measurement at the hybrid chamber array 110 is extremely important to retard evaporation of the solution (containing the exposed probe). Feedback Sensors Temperature sensors and liquid sensors are included in each of the L Ο C devices 310 to provide feedback and diagnostics during device operation. Referring to Figure 35, nine temperature sensors 170 are distributed throughout the amplification section 112. Similarly, the incubation section 112 also has nine temperature sensors 170. Each of these sensors uses a 2 X 2 bipolar junction transistor (BJTs) to monitor the temperature of the fluid -73 - 201211539 and provide feedback to the CMOS circuit 86. The CMOS circuit 86 uses this feedback to precisely control the thermal cycling during the nucleic acid amplification procedure and to precisely control any heating during thermal lysis and incubation. In the hybrid chamber 180, the CMOS circuit 86 uses the hybrid heaters 182 as temperature sensors (see the resistance system temperature dependence of the hybrid heaters 182 in Fig. 56, and the CMOS circuit 86 utilizes The resistance is temperature dependent, which pushes out the heater readings of each of the chambers of the hybrid chambers 180.

The LOC device 301 also has a plurality of MST channel liquid sensors 174 and a capping channel liquid sensor 208. Figure 35 shows a list of MST channel liquid sensors 174 at the end of one of every other meandering channel of the heated microchannel 158. Preferably, as shown in Figure 37, the MST channel liquid sensors 174 are a pair of electrodes formed by exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid breaks the circuit between the electrodes to indicate that liquid is present at the location of the sensor. Figure 25 shows an enlarged perspective view of the capping channel liquid sensor 208. A plurality of pairs of opposing titanium aluminum (TiAl) electrodes 218 and 220 are disposed on the top layer 66. A gap 222 is interposed between the electrodes 218 and 220 to keep the circuit open when there is no liquid. The presence of liquid breaks the circuit and the C Μ S circuit 86 uses this feedback signal to monitor the fluid. Gravity-independent test module 1 〇 system position independent. It is not necessary to operate it. • 74- 201211539 The test module is fixed on a flat and stable surface. The capillary drive fluid flows without external piping to the auxiliary equipment, thus allowing the modules to be easily carried and easily inserted into a portable portable reader (e.g., a mobile phone). A gravity-independent mode of operation indicates that the test module is also acceleration-independent in all practical situations. The inspection modules are shock and shock resistant, and the inspection modules will operate on a moving vehicle or while holding a mobile phone.

A dialysis section of a dialysis variant having a flow channel for preventing bubble collapse is described below as a LOC device embodiment of LOC device variant VIII 518, and the LOC device variant VIII 518 is shown at 64, 65, 66 and 67 in the picture. This LOC device has a dialysis section that can inject a fluid sample without leaving air bubbles trapped in the channel. The LOC device variant VIII 518 also has a layer of additional material, referred to as the interface layer 594. The interface layer 5 94 is between the capping channel layer 80 and the MST channel layer 100 of the CMOS + MST device 48. The interfacial layer 5 94 allows for a more complex fluid interconnect structure between the reagent reservoirs and the MST layer 87 without increasing the size of the tantalum substrate 84. Referring to Fig. 65, the bypass channel 600 is designed to introduce a delay in the flow of the fluid sample from the interface waste channel 604 to the interface target channel 602. This time delay allows the fluid sample to flow through the dialysis MST channel 204 to the dialysis uptake 168 that holds the meniscus. The dialysis of the sample fluid from all of the dialysis MST channels 204 is utilized by a capillary action priming feature (CIF) 202 located at the suction port from the bypass channel 600 to the interface target channel 602 -75-201211539. The interface target channel 602 is injected into the upstream end of the upper suction hole 168.

Without the bypass channel 600, the sample fluid will still be injected from the upstream end of the interface target channel 62, but finally the forward advancing meniscus arrives and passes through the upper suction hole belonging to the unfilled MST channel. , causing the air to fall at this point. This trapped air reduces the sample flow rate through the leukocyte dialysis section 3 28 . Nucleic Acid Amplification Variants Parallel Polymerase Chain Reactions Several variants of LOC devices have multiple expansion sections that operate in parallel. For example, the LOC device variant VII 492 shown in Fig. 63 has a plurality of parallel amplification sections 1 1 2 2 . 1 to 1 1 2.4, and the parallel amplification sections 1 1 2.1 to 1 1 2.4 allow simultaneous execution Multiple nucleic acid amplification analysis tests.

The LOC device variant XI 746 shown in Fig. 82 also has a plurality of parallel amplification sections 112.1 to 112.4, but additionally has a plurality of parallel incubation sections 1 1 4.1~1 Μ . 4 , so that the sample can be performed Different treatments are performed before amplification. Other LOC device variations (e.g., LOC device variant XIV 641 to be shown in Figure 69) indicate that the plurality of parallel amplified segments can be "X", which is limited only by the size of the LOC device. The larger the LOC device, the more multiple amplification reaction segments can be accommodated in parallel. The separate amplification segments can be configured to perform different cycles and/or temperatures for a particular target size or a particular amplification mixture composition -76-201211539

. The LOC device can perform a multiplex nucleic acid amplification method or a single nucleic acid amplification method in each segment by a plurality of amplification segments operating in parallel. In multi-nucleic acid amplification, more than one target sequence is amplified using one or more primers. A parallel nucleic acid amplification system having "m" chambers can perform an amplification reaction equivalent to η weight, where n = n(l)+n(2)+n(3)+·.· + n(m And n(i) indicates the primer number to be used for the chamber "i" in the different primer pairs used for the multiplex amplification reaction, and it is necessary to keep in mind the signal noise in the parallel amplification system. The ratio (SNR) is higher than the signal-to-noise ratio of the η-wei amplification reaction performed in a single-chamber system. In the special case of n(i) =1, the amplification reaction in chamber [i] is just a single amplification reaction.

Figures 70, 71, 72, 73 and 74 of the drawings illustrate the LOC devices operating in series with the amplification sections 112.1 and 112.2 in the apparatus. The first amplification section 112.1 comprises a reagent storage tank 60.1 of the amplification mixture and two reagent storage tanks 62.1 of the polymerase. Each of the amplification sections applied after the initial section also contains two reagent storage tanks, a storage tank of the amplification mixture 6〇.2 and a storage tank 62.2 of the polymerase. Multiple amplification segments in series allow for tandem PCR analysis to verify that the first amplification segment 112.1 is used to perform a pre-amplification reaction to enhance subsequent nucleic acid expansion in segment 112.2 Increase the sensitivity of the reaction. Multiple amplification segments in tandem can also be used to perform nested polymerase chain reaction (PCR). -77- 201211539

In tandem PCR for pre-amplification, the first amplification segment 112.1 amplifies the nucleic acid sequence within the sample containing the target sequence. This amplification reaction does not require specificity for the target sequence (e.g., amplification reaction of the whole genome), but the amplification reaction does increase the concentration of the target sequence. After the pre-amplification reaction is carried out, the sample is mixed with the reagents derived from the storage tank 60.2 and the storage tank 62.2 and then the sample mixture is flowed into the second amplification section 112.2. The reagents stored in reservoir 60.2 comprise specific probes that only amplify the target sequences in the preamplified sample mixture. It should be noted that similar methods of replacing PCR with a thermostatic technique, such as the first amplification phase or the second amplification phase, can also be employed to achieve the advantages of pre-amplification.

Nested PCR is a special form of tandem PCR that has the added advantage of high target specificity. In the nested PCR method, the nucleic acid amplification step in the first amplification section 112.1 is to use a primer to amplify a sequence having a length greater than the final target sequence, and the primers are formed in the storage tank 60.1. A portion of the amplified amplification reagent is stored, and the primers are complementary to the region at the outside of the target sequence. The reaction in the first amplification segment 1 12.1 produces an amplicon consisting of the target sequence plus a flanking section. This amplified mixture is mixed with a reagent derived from storage tank 60.2 and a polymerase derived from storage tank 62.2. The reagents stored in storage tank 60.2 comprise primers that complement the position at both ends of the target sequence (i.e., the sequence of sub-regions derived from the amplicon of the first amplification stage). When performing a nucleic acid amplification reaction in the second amplification section 112.2, since the concentration of the amplicon derived from the first amplification stage is much larger than the concentration of the original sample molecule, at a sequence position unrelated to the target The probability of an increase in the -78-201211539 increase is greatly reduced. The sensitivity and specificity of the nested PCR method can also be achieved when one or both of these PCR amplification stages are replaced by sequence-specific thermostatic amplification techniques.

The advantage of separately storing the polymerase and separately adding the polymerase to the sample mixture is that different polymerases can be selected for the pre-amplification step and the final nucleic acid amplification step. For example, this approach allows for a low error rate (eg, 'readable') polymerase for the preamplification step to prevent the creation of a faulty target sequence or an incorrect target sequence while allowing for the final amplification step. Use a higher speed or temperature resistant polymerase. Direct Polymerase Chain Reactions Traditionally, PCR requires large-scale purification of target DNA prior to preparation of the reaction mixture. However, by appropriately changing the chemical reagent and the sample concentration, it is possible to perform nucleic acid amplification or directly perform amplification by minimizing D N A purification. When the nucleic acid amplification method is a PCR method, this method is called direct PCR. In a LOC device that performs nucleic acid amplification at a controlled constant temperature, the method is a direct isothermal amplification method. The use of direct nucleic acid amplification techniques in LOC devices, particularly in terms of simplifying the required fluid design, has considerable advantages. Adjustments to amplification chemistries for direct PCR or direct constant temperature amplification include increasing buffer strength, using polymerases with high activity and high persistence, and additives that can be chelated with polymerase inhibitors. It is also important to dilute the inhibitor present in the sample. To take advantage of direct nucleic acid amplification techniques, the LOC device design incorporates two additional features. The first feature is a reagent storage tank (eg, 8 - 79 - 201211539

The storage tank 58) is suitably sized to supply a sufficient amount of amplification reaction mixture or diluent such that the final concentration of sample components that may interfere with the amplification chemical is low enough to allow for successful nucleic acid amplification. The expected dilution of non-cellular sample components is between 5 and 20 times. Different LOC structures (e.g., pathogenic dialysis section 70 in Figure 4) can be used at appropriate times to ensure that the concentration of the target nucleic acid sequence is maintained at a sufficiently high concentration for amplification and detection. In this particular embodiment (further depicted in Figure 6), a dialysis section is employed upstream of the sample extraction section 290, which effectively concentrates the pathogens small enough to enter the amplification section 292 and rejects larger cells The larger cells are brought into the waste storage tank 76. In another embodiment, the dialysis section selectively rejects proteins and salts in the plasma while leaving the cells of interest.

The second LOC structural feature (characteristic for supporting direct nucleic acid amplification) is a channel aspect ratio design to adjust the mixing ratio between the sample and the components of the amplification mixture. For example, to ensure that the inhibitor of the sample is preferably diluted 5 to 20 times by a single mixing step, the length and cross section of the sample channel and the reagent channel are designed such that the mixing action is initiated. The flow impedance of the upstream sample channel is 4 to 19 times greater than the flow impedance of the channel through which the reagent mixture flows. The flow resistance in the control microchannel can be easily achieved by controlling the geometry of the design. For a constant cross section, the flow impedance of the microchannel increases linearly with channel length. The focus of the hybrid design is that the flow impedance in the microchannel is more dependent on the smallest cross-sectional dimension. For example, when the aspect ratios of microchannels are inconsistent, the flow impedance of a microchannel with a rectangular cross section is inversely proportional to the cube of the smallest vertical • 80 - 201211539 size. Reverse transcriptase-polymerase chain reaction (RT-PCR)

When the sample nucleic acid species RNA to be analyzed or extracted, such as RNA or message RNA derived from RNA virus, is first subjected to PCR amplification, the RNA is first reverse transcribed into complementary DNA (cDNA). The reverse transcription reaction (single-step rt-PCR) can be performed in the same chamber as the PCR, or the reverse transcription reaction can be performed as an independent initial reaction (two-step RT-PCR). In the variants of the LOC devices described in the present application, the reverse transcriptase can be added to the reagent storage tank 62 together with the polymerase, and the heaters 154 can be programmed to perform the reverse transcription step followed by the nucleic acid. A single-step RT-PCR can be performed simply by cycling through the amplification step. The reverse transcription step is carried out by using the incubation section 114 and storing and dispensing the buffer, primer, dNTP and reverse transcriptase using the reagent storage tank 58, and then performing the amplification in the amplification section 1 1 2 by a general method. Amplification can also easily achieve two-step RT-PCR. Thermostatic Nucleic Acid Amplification Method For some applications, the constant temperature nucleic acid amplification method is a preferred nucleic acid amplification method, thereby eliminating the need to repeat the cycle of the reaction components, undergoing various temperature cycles, and instead making the amplification The section is maintained at a constant temperature (typically about 37 ° C to 41 ° C). A variety of thermostatic nucleic acid amplification methods have been disclosed, including strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), and recombinant enzyme polymerization -81 - 201211539 Addition (RPA), helicase-dependent isothermal DNA amplification (HDA), rolling circle amplification (RCA), ramification amplification (RAM), and circular nucleic acid-mediated amplification (LAMP) And any one of these methods or other isothermal amplification methods may be employed in a particular embodiment of the LOC device described herein.

To perform the thermostatic nucleic acid amplification, the reagent storage tank 60 and the reagent storage tank 62 adjacent to the amplification section are loaded with a reagent 'supplied for performing the constant temperature method to replace the PCR amplification mixture and the polymerase. For example, to perform strand displacement amplification (SDA), reagent storage tank 60 contains amplification buffer, primers, and dNTPs, and reagent storage tank 62 contains appropriate nickase and exo-DNA polymerase (exo) -DNA polymerase). For performing recombinase polymerase amplification (RPA), reagent storage tank 60 contains amplification buffer, primers, dNTPs, and recombinase, and reagent storage tank 62 contains a strand displacement DNA polymerase (e.g., 5^). Similarly, to perform helicase-dependent isothermal DNA amplification (HDA), reagent storage tank 60 contains amplification buffer, primers, and dNTPs, and reagent storage tank 62 contains suitable DNA polymerase and substitution heating for solution. Open the double-stranded DNA strand helicase. Those skilled in the art will appreciate that such necessary reagents can be dispensed between the two reagent reservoirs in any manner suitable for nucleic acid amplification. Since nucleic acid sequence-dependent amplification (NASBA) or transcription-mediated amplification (TMA) does not require transcription of RNA into cDNA, NASBA or TMA is suitable for amplification of RNA viruses (eg HIV or Viral nucleic acid of hepatitis C virus). In this example, the reagent reservoir 60 is filled with amplification buffer, primer and dNTP, and the reagent storage tank -82-

RN A polymerase, reverse transcriptase and randomly selected in 201211539 62

I enzyme H ( RNase Η ). For some forms of thermophilic nucleic acid amplification, it may be necessary to have an initial change to separate the double-stranded DNA template prior to the temperature of the Wiver nucleic acid amplification method. Since the temperature of the amplification section 12 can be carefully controlled by the heater 154 in the extension 158 (see Figure 14), this step can be easily achieved in the specific embodiment of the LOC described in the present application. . The thermostatic nucleic acid amplification method is more tolerant of the potential in the sample. As described above, the thermostatic nucleic acid amplification method is generally suitable when it is desired to perform a direct nucleic acid amplification reaction. Therefore, thermostatic nucleic acid amplification is applicable to both LOC device variant XLIII 673, LOC XLIV 674 and LOC device variant XLVII 677, which are shown in the 79, 80 and 81 isothermal amplification methods, respectively. The pre-amplification dialysis step 70, 686 or 682 and/or the pre-hybrid pre-dialysis step 682 as shown in Figures 79 and 81 may be used in combination with the dialysis step 682, respectively. Helping to partially concentrate the target cells in the sample or to enter the heterozygous chamber array 1 1 〇 to remove unwanted cells. Those skilled in the art will appreciate that the above-described precipitation step and pre-hybridization dialysis step can be used. random combination. The thermostatic nucleic acid amplification method can also be performed in multiple amplification sections in parallel (such as the amplification section to be shown in the 63th and 68th teeth), and the constant temperature nucleic acid amplification method (for example, 'circular nucleic acid-mediated amplification art

RNA hydrolysis is carried out in a constant cycle to increase the preparation of the mixing device in the microchannel, and the sample is sometimes subjected to variations such as a variant. Directly or one of the nucleic acids in the > 80 plot is extended to the sample residue. Pre-amplification 69 69 Figure 槪 More or some t (LAMP • 83- 201211539 )) is compatible with the initial reverse transcription step for amplification of RNA. Light diode

Figure 54 shows the photodiode 184 integrated in the CMOS circuit 86 of the LOC device 301. Light diode 184 is formed as part of CMOS circuit 86 without the need for additional masking or steps. This is the advantage of the CM0S* diode over C C D (C C D is an alternative sensing technique that uses a non-standard processing step to integrate the CCD on the same wafer or on an adjacent wafer). The on-wafer detection method is low cost and can reduce the size of the analytical inspection system. The shorter optical path length reduces noise from ambient environments for efficient collection of fluorescent signals and eliminates the need for conventional optical components consisting of lenses and filters.

The quantum efficiency of the photodiode 184 is caused by photons impinging on the active region 185 of the photodiode 184 to be efficiently converted into photoelectrons. For standard 矽 process, the quantum efficiency for visible light ranges from 0.3 to 0.5 depending on process parameters (for example, the number of cover layers and the absorption properties). The detection threshold of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection threshold also determines the size of the photodiode 184 and thereby determines the number of hybrid chambers 180 in the hybrid and detection section 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (in the case of LOC device 301, which is 1 760 microns x 5 8 2 4 microns) and incorporates other The actual footprint available after the functional module (eg, pathogen dialysis section 70 and amplification section 1 1 2). -84- 201211539 For standard 矽 process, photodiode 1 84 detects a minimum of 5 photons. However, to ensure reliable detection, the minimum 値 can be set to 10 photons. Therefore, as described above with a quantum efficiency of 0.3 to 0.5, the fluorescent light emitted from the probes must have a minimum of 17 photons, but 30 can be included in the appropriate error range for reliable detection. Photon. Electrochemiluminescence as an alternative detection method

Electrochemiluminescence (ECL) involves the formation of a variety of species at the surface of the electrode and subsequent exposure of the species to an electron transfer reaction to form an excited state that illuminates. The difference between electrochemiluminescence and general chemiluminescence is that electrochemiluminescence relies on species that are excited by oxidation or reduction of the luminescent group or co-reactant at the electrode. In the present context, a coreactant is an additional reagent added to an ECL solution that enhances ECL luminescence efficiency. In general chemiluminescence, the excited species are produced by a mixture of various suitable reagents. The luminescent atom or complex is conventionally referred to as a luminescent group. In short, the ECL method relies on the formation of an illuminating luminescent group in an excited state, and will emit photons when an luminescent layer of an excited state is generated. When utilizing any such method, another method may be employed to cancel the excited state resulting in a desired luminescence (i.e., matting). A specific example of an inspection module using the ECL method instead of the fluorescence detection method

I don't need to use an excitation LED. Fabricating electrodes in the hybrid chambers to provide

I is used to generate an electrical pulse of the ECL, and the photosensor 44 is used to detect the photons. The duration and voltage of the electrical pulse are controlled; in some specific embodiments, the control current is used instead of the control voltage. Cold light emitting group and extinction group

The aforementioned ruthenium complex [Ru(bpy)3]2 + used as a fluorescent reporter in the probe can also be used in the ECL reaction in the hybrid chambers as a luminescent light-emitting group, and using TPrA (Tri-n-propylamine, (CH3CH2-CH2)3N) as a co-reactant. The advantage of the co-reactant ECL is that it does not consume luminescence luminescent groups after the emission of photons, and such reagents can be reused for this method. Furthermore, the [Ru(bpy)3]2 + /TPrA ECL system provides good signal intensity in aqueous solution under physiologically acidic pH conditions. A co-reactant system N-butyldoethanolamine and 2-(dibutylamino) ethanol which can be used in combination with ruthenium complexes and produce results equal to or better than TPrA. Ethanol ) 〇

Figure 84 illustrates the reactions that occur during ECL processing in which [RU(bpy)3]2+ is the luminescent light-emitting group 864 and TPrA is the co-reactant 866. In the [Ru(bpy)3]2 + /TPrA ECL system, the oxidation reaction of [Ru(bpy)3]2 + and TPrA occurs at the anode 860 followed by chemical electroluminescence. 8 62. These reactions are as follows: -86- (1)201211539

Ru(bpy)32+ -e' Ru(bpy)33+ TPrA -e -> [TPrA*]+ TPrA* + H+

Ru(bpy)33+ +TPrA·^^ Ru(bpy)3'2+ + product Ru(bpy)3,2+ -> Ru(bpy)32+ +hu

The wavelength of the release light 862 is about 620 nm with respect to the Ag/AgCl reference electrode, and the anode potential is about 1.1 volts (V). For the [Ru(bpy)3]2 + /TPrA ECL system, any of the aforementioned black hole type matting group (BHQ2) or Iowa black extinction group RQ may be a suitable extinction group. In a specific embodiment described in the present invention, the matting group is a functional group attached to the probe initially, but in other embodiments the extinction group may also be freely present in the solution. Independent molecule. Hybrid probe for ECL detection

(2) (3) (4) Figures 101 and 102 show heterozygous sensitive ECL probes 23 7 . Such probes are commonly referred to as molecular beacons and are dry-loop probes that are produced from single-stranded nucleic acids and that emit cold light when the probes are hybridized to complementary nucleic acids. Figure 101 shows a single ECL probe 237 prior to hybridization with the target nucleic acid sequence 238. The probe has a ring portion 420, a stem 242, a luminescent light-emitting group 864 at the 5' end, and a matting group 248 at the end. The loop portion 240 is composed of a sequence complementary to the target nucleic acid sequence 238. Complementary sequences located on both sides of the probe sequence are fused together to form the backbone 242 87 -87 - 201211539 When a complementary target sequence is lacking, the probe remains closed as shown in Figure 101. The stem 242 keeps the pair of the luminescent group and the extinction group close to each other, so that significant resonance energy transfer can occur between the luminescent group and the extinction group, and the luminescence group is substantially eliminated. Luminescence ability after chemical excitation.

Figure 102 shows the ECL probe 23 7 in an open or hybrid configuration. When hybridized with the target nucleic acid sequence 23 8 , the dry-loop structure disintegrates 'the luminescent light emitting group 864 and the extinction group 248 are spatially separated, thereby restoring the luminescent ability of the luminescent light emitting group 8 64 . Optically measuring the ECL release light 862 means that the probe has been hybridized.

Since the backbone helical structure of the probe is designed such that the backbone helical structure is more stable than the probe-target helical structure that is not complementary to a single nucleic acid, the probes have very high specificity and complementarity The target is heterozygous. Since the double-stranded DNA system is relatively rigid, it is unlikely that the double-stranded DNA is stereostructured to coexist with the probe-target helix structure and the backbone helix structure. The ECL probe with the primer is followed by a dry-loop probe with a primer and a linear probe (or 蝎 probe) with a primer, which is a substitute for the molecular beacon, and the probe can be used for the LOC In the device for immediate and quantitative nucleic acid amplification. The instant amplification method can be performed directly in the hybrid chambers of the LOC device. The benefit of using a probe with a primer is that the probe molecule is physically linked to the primer, so that only a single heterozygous event occurs during the nucleic acid amplification period, without the need for a heterozygous reaction of -88-201211539 The hybrid reaction with the probe is carried out separately. This ensures that the reaction is carried out efficiently and immediately, and that the probe with the primer provides a stronger signal, shorter reaction time and better discrimination than the separate use of the primer and the probe. The probes (along with the polymerase and amplification mixture) can be placed into the hybrid chambers 180 during manufacture without the need for an additional amplification section on the LOC device. Alternatively, the amplification segments can be retained but not used or used for other reactions.

Linear ECL probes with primers Figures 103 and 104 show a linear ECL probe 693 with primers during the initial round of the nucleic acid amplification reaction and a linear ECL probe during the subsequent round of nucleic acid amplification reactions, respectively. Hybrid structure. Referring to Fig. 103, the linear probe 693 with the primer has a double-stranded main cadre 242. One of the double strands comprises the probe sequence 696 with the primer, the probe sequence 696 is identical to a region on the target nucleic acid sequence 696, and the 5' end of the probe sequence 696 is labeled with luminescence The group 864 and the Y-terminus of the probe sequence 696 are ligated to the oligonucleotide primer 700 via amplification blocker 694. The matting group 248 is marked at the V-terminus of the other chain of the stem 242. Upon completion of the nucleic acid amplification reaction of the initial round, the probe can be looped and hybridized to the extended nucleic acid strand using the now complementary sequence 698. During the nucleic acid amplification reaction of the initial round, the oligonucleotide primer 700 is adhered to the target DNA 23 8 (see FIG. 103) and then the primer 700 is extended to form the probe sequence and the extension. The DNA strand of both products is increased. The amplification blocker 694 prevents polymerase reading -89- 201211539

The probe area 696 is taken and copied. When the subsequent denaturation reaction is carried out, the extended oligonucleotide primer 700 is dissociated from the hybrid system of the template, and the double-stranded stem 242 of the linear probe with the primer is also dissociated to release the extinction base. Mission 248. Once the temperature is lowered for the binding step and the extension step, the primer-attached probe sequence 696 of the primer-attached linear ECL probe is rolled up and the amplified complementary sequence on the extended nucleic acid strand 698 is heterozygous and the measured release light indicates the presence of the target DNA. Unextended linear probes with primers retain their own double-stranded backbone and remain in a matte state. Since this detection method relies on a single molecular process, this detection method is particularly suitable for fast detection systems. Dry-ring ECL probe with primer

Figures 105A through 105F show the operation of the dry-loop ECL probe 705 with the primer attached. Referring to Figure 〇5, the dry-loop ECL probe 705 with primer has a stem 242 consisting of complementary double stranded DNA and a loop 240 containing the probe sequence. The luminescence luminescent group 864 is labeled on the 5th end of one of the backbone chains 708. The other backbone strand 710 is ligated to the 3, terminally labeled matting group 248, and the backbone strand 710 carries both the amplification blocker 6 94 and the oligonucleotide primer 700. During the initial denaturation phase (see Figure 105B), the nucleic acid strands of the target nucleic acid 238 are separated as the backbone 242 of the stem-loop ECL probe 705 with the primer attached. When the temperature is cooled to carry out the bonding stage (see Fig. 105C), the oligonucleotide primer 700 on the stem-loop ECL probe 705 with the primer and the nucleic acid sequence of the -90-201211539 target are arbitrarily Hehe. Complementing the complementary sequence 7〇6 complementary to the target nucleic acid sequence 23 8 during the elongation phase (see Figure 5D)

I

A DNA strand is formed which contains both the probe sequence 705 and the amplification product. Amplification blocker 094 prevents the polymerase from reading and replicating the probe region 7〇5. The probe is then ligated after the denaturation step (see 105E), the probe sequence of the loop portion 240 of the dry-loop probe attached to the primer (see Figure 105F) and the complementary sequence 706 on the extended strand. Bonding. This structure allows the luminescent group 864 to be relatively far from the extinction group 248 to significantly enhance the luminescence.

I ECL Control Probes Hybrid chamber arrays 110 contain a number of hybrid chambers 180 containing positive control ECL probes and negative control ECL probes for quality control of analytical assays. Figures 1 06 and 1 07 illustrate the negative control ECL probe 78 6 without the luminescent group, and the 108 and 109 diagrams are not

A summary of the positive control ECL probe 78 7 containing the extinction group. The positive control ECL probe and the negative control ECL probe have a dry-loop structure similar to the ECL probe described above. However, regardless of whether the probes are heterozygous or not

I open structure or remain closed, positive control ECL probe 787 will always

I issues an ECL signal 862 (see Figure 102), and the negative control ECL probe 78 6 never issues an ECL signal 8 62. Referring to Figures 106 and 107, the negative control ECL probe 786 has no luminescent light-emitting groups (and may or may not have an extinction group 248). ^ Thus, regardless of whether the target nucleic acid sequence 23 8 is as shown in Figure 07 General with -91 - 201211539

The probe is heterozygous, or the probe maintains its own backbone 242 and loop 240 structure as shown in Fig. 106, and the ECL signal is negligible. Alternatively, the negative control ECL probe can be designed such that the negative control ECL probe remains matte forever. For example, by having an artificial probe (loop) sequence 240 that will not hybridize to any nucleic acid sequence within the sample under investigation, the backbone 242 of the probe molecule will re-hybridize with the probe molecule itself, The luminescent group and the matting group will remain close and no visible ECL signal will be detected. This negative control signal can correspond to any low luminescence that may occur when incomplete extinction. Conversely, as shown in Figures 108 and 109, the positive control ECL probe 78 7 is constructed to contain no matting groups. Whether or not the positive control ECL probe 78 7 is hybridized to the target nucleic acid sequence 2 3 8 does not eliminate the ECL release light 862 of the luminescent light-emitting group 864.

Figures 99 and 100 show another possible method for constructing a positive control chamber. In this case, the calibration chambers 382 are sealed from the amplicon (or any fluid containing the target molecules), and the ECL luminescent luminescent group solution can be injected into the calibration chambers 382. A positive signal is permanently detected at the electrode. Similarly, the E C L signal can never be detected because the control chamber lacks an inlet to prevent any target from contacting the probes, and thus the control chamber can be a negative control chamber. Figure 5 2 shows the possible distribution of positive control probes and negative control probes throughout the array of heterozygous chambers (3 7 8 and 380, respectively). For the ECL method, the positive control ECL probe 786 and the negative control -92-201211539 ECL probe 78 7 replaced the control fluorescent probes 3 7 8 and 380, respectively. The control probes are placed in the hybrid chambers 180 arranged along the diagonal line extending across the array of hybrid chambers 110. However, the arrangement of the control probes in the array is arbitrarily configured (depending on the configuration of the hybrid chamber array 110 at the time). Calibration chamber for ECL detection

The photodiode 184 responds to ambient ambient light present at the sensor array and electrical inconsistencies from light rays at other locations in the array that introduce background noise and offset into the output signal. This background 每个 in each output signal is removed by a plurality of calibration chambers 382 in the hybrid chamber array 110, wherein the calibration chambers 382 are free of any probes or contain non-ECL luminescent bases. a probe of a group, or a probe containing a luminescent light-emitting group and an extinction group, but the groups are configured to expect the extinction to occur all the time.

I. The number and configuration of the calibration chambers 382 in the entire array of hybrid chambers can be arbitrarily determined. However, if the photodiodes 184 are calibrated by the relatively closest calibration chamber 382, the calibration is more accurate. Referring to Fig. 124, the hybrid chamber array 11A has a calibration chamber 382 for use by all eight hybrid chambers. That is, the calibration chamber 382 is located in the center of the 3 χ 3 square array comprised of the hybrid chambers 180. In such a configuration, the hybrid chambers ISO are calibrated by the immediately adjacent calibration chamber 382. Fig. 83 shows a differential imaging circuit 788 for subtracting the application of electrical pulses from the ECL signals surrounding the hybrid chambers 180 to create a photodiode corresponding to the calibration chamber 3 82. Signals sent by 184 -93- 201211539. The differential imaging circuit 788 samples signals originating from pixel 790 and "virtual" pixel 792. Signals caused by ambient light in the array of cells are also subtracted. The signal originating from pixel 790 is small (i.e., close to a dark signal)' and it is difficult to distinguish between the background 値 and the minimum signal without reference to the dark signal.

During use, "read column line (read_row)" 794 and "virtual pixel read column line (read_row_d)" 795 are activated, and M4 transistor 797 and MD4 transistor 801 are turned on. Switch 807 and switch 8 09 are turned off so that the output 源自 from pixel 790 and "virtual" pixel 792 are stored on pixel capacitor 803 and virtual pixel capacitor 805, respectively. After the pixels are stored, the switch 807 and the switch 809 are stopped. Subsequently, the read read line (read_C〇l) switch 81 1 and the virtual pixel "read line" switch 8 1 3 are turned off, and the switched capacitor amplifiers 815 at the output are amplified. Differential signal 817

ECL Intensity and Signal Efficiency The standard efficiency measure in an ECL reaction is the number of photons obtained for each "Faraday" electron (ie, each electron participating in the electrochemical reaction). The ECL efficiency is expressed as ψΕα : \ΐάτ Φεα = TNAr (5) JJo where I is the intensity (photons/second), the i-line current (amperes), the F-system Faraday constant, and the NA-based Yafotian constant. -94- 201211539 ECL reaction efficiency of co-reactants

The ion-eliminating electroluminescent reaction (annihilation ECL) in an oxygenated aprotic solution (eg, a nitrogen-treated acetonitrile solution) is sufficiently pure to allow for measurement efficiency, and the consensus is about 5%. . However, this co-reactant system has been generally considered to measure helium directly beyond meaningful efficiency. Instead, the release light intensity can be measured in the same manner by scaling up in relation to a readily prepared standard solution (e.g., Ru(bpy)32+). Such documents (see, for example, K. Leland and MJ Powell, 1990, J. Electrochem. Soc., 137, p. 3127, and R. pyati and M. Richter, 2007, published in Annu. Rep. Prog Chem. C No. 103, pp. 12-78, states that the peak enthalpy of the efficiency of the Ru(bpy)32+ and TPrA co-reactant is equivalent to acetonitrile in the absence of a promoter (eg, a surfactant). Performing 5% of the peak enthalpy seen in the ion-eliminating ECL reaction (for example, 2% efficiency, see I. Rubinstein and AJ Bard, 1981, J. Am. Chem. Soc. 103, 512~516 Page paper). ECL potential The voltage at the working electrode of the Ru(bpy)32 + /TPrA ECL system is about +1.1 volts (generally referred to as the control Ag/AgCl reference electrode). Such a high voltage will shorten the electrode life, but this is not a problem for a single-use device such as the LOC device used in the diagnostic system of the present invention -95-201211539.

The desired voltage between the anode and the cathode depends on the combination of the solution components and the electrode material. Choosing the correct voltage may require a compromise between the highest signal strength, the stability of the reagents and electrodes, and the occurrence of undesirable side reactions such as the electrolytic reaction of water in the chamber. In experiments using buffered Ru (bp y) 32 + /co-reactant aqueous solutions and platinum electrodes, the ECL luminescence reached a maximum at 2.1 volts to 2.2 volts (depending on the selected co-reactant). When the voltage is lower than 1.9 volts and higher than 2.6 volts, the emitted light intensity drops to less than 75% of the peak ,, and when the voltage is lower than 1.7 volts and higher than 2.8 volts, the emitted light intensity drops to less than 50% of this peak. Therefore, for ECL operation in such systems, the preferred anode-cathode voltage difference is between 1.7 volts and 2.8 volts, and the preferred system is between K9 volts and 2.6 volts. The voltage difference in this range allows the maximum light intensity to be released as a function of voltage while preventing significant gas formation at the electrodes.

ECL luminescence wavelength The wavelength of the release light 8 62 derived from the ECL reaction has a strong peak (measured under air or vacuum) at about 620 nm, and the release light spans a relatively broad wavelength range. Significant release of light occurs at wavelengths from about 5 50 nm to 700 nm. In addition, the peak light emission wavelength can have a variation of about 10% due to changes in the chemical environment surrounding the active species. The LOC device embodiment described herein (which does not contain a specific wavelength filter) has two advantages in capturing signals having such a wide and variable spectrum with -96-201211539. The first advantage is sensitivity: any wavelength filter will reduce the light transmittance, even if the light falls within the passband of the filter, so the efficiency of the device is improved by the absence of a filter. Advantages: Flexibility: There is no need to adjust the passband of the filter after a change in the secondary reagent, and the signal is less dependent on small differences in the non-target components of the implanted sample.

Solution volume involved in the ECL reaction

The I ECL reaction relies on the availability (a v a i 1 a b 1 i. t y ) of the luminescent light-emitting groups (and co-reactants) in the solution. However, as shown in Fig. 8 6 , the excited species 8 68 are generated only near the electrodes 860 and the electrodes 870. The boundary layer depth in the module of the present invention refers to the depth at which the solution layer 872 of the excited species 868 can be generated near the electrode 860. 1 This depth is simplified because solution kinetics may drive the available thick

I degree increases or _ low: • Increases availability: diffusion and electrophoresis effects will allow more

The solution of I is exchanged. • Reduced availability: Reagents may be adsorbed on these electrodes and become unavailable for ECL reactions. For a 0.5 micron boundary layer depth 値, the following observations were obtained: observed in an experiment using a luminescent luminescent group 864 bonded to a magnetic bead having a diameter of up to 4.5 microns to attract the luminescent light-emitting group 864 onto the anode 860 ECL reaction. -97- 201211539 For the interdigitated electrode array, the Ru(bpy)32 + /TPrA luminescence 862 is a function of electrode spacing and the Ru(bpy)32 + /TPrA ECL luminescence is found at 0.8 microelectrode spacing The effect of 8 62 is large. Co-reactant 866 may be required in aqueous solution 872 when the electrode spacing is about 2 microns. This represents the number of diffusions of the germinated species 868, suggesting that these species in the ground state may undergo similar scale exchanges. Steady State and Pulse Operation During the pulsed activity of the electrodes 860 and 870, the intensity of the illuminating effect 862 (see Figure 102) is typically higher than the intensity of the illuminating effect 862 during steady state activity. Therefore, the CMOS circuit 86 (see Fig. 91) modulates the pulse width of the enable signal for transmitting the electrodes 860 and . Recycling of reagents and life cycle of species The ruthenium (Ru) mismatch in the Ru(bpy)32 + /TPrA ECL system is not consumed, so the 862 degree does not decrease when the continuous reaction cycle is performed. If the total reaction cycle time is about 1 millisecond, the life cycle of the rate limit is about 0.2 milliseconds. ECL meters to the most use micron diffusion

ECL is the strongest step by the 870 system

Electrophoretic effects and other constraints Due to the complexity of the solutions in the hybrid chamber, many phenomena occur when the voltage is turned on. Electrophoresis of macromolecules, ohmic transmission ECL and -98 -

201211539 The capacitive effects from small ion migration occur simultaneously. Since the DNA system is highly electronegative and is attracted to the anode 860 by the anode 860, the use of oligonucleotides (probes and amplicons) may make probe-target hybrids more difficult to detect. get on. The time is usually very short (about a few milliseconds). Due to the anode 860

The spacing between I 8 70 is small, so even if a gentle voltage (special) is applied, the electrophoretic effect is still strong. In some embodiments of the LOC device, electrophoresis can enhance luminescence as 862, and in other embodiments electrophoresis can reduce the effect. This problem can be solved by increasing or decreasing the electrode spacing to increase or decrease the power. The anode located above the photodiode 1 84 is an interdigitated configuration of the cathode 8 70 which is an example of minimizing this spacing. Even in the carbon electrode 860 and the carbon electrode 870, the ECL reaction can be produced without such a common electrode configuration. Ohmic Heating (Direct Current) Referring to the ECL reaction chamber 874 shown in Fig. 87, the current required for the ECL voltage of the f volt is determined as follows. The direct current (DC) current through the chamber is electrically separated from the interface between the electrodes 860 and 870 and the bulk solution by a resistance Rs of the solution, the Rs resistance being derived from the electrical path of the bulk solution. Geometric shape. For solutions with ionic strength (related to conditions within the device), the chamber resistance is primarily determined by the interfacial resistance at the electrode electrodes 870, and Rs is negligible. Leading the electrophoresis to this movement and the cathode to about 1 volt into the ECL. The luminescent effect is 860 with the extreme reactants, about 2.2 determines: .V and rate and guide a loc 8 60 and 99 - 201211539 can be used for the LOC device The electrode geometry in the scale measures the macroscopic current through the same solution to estimate the effect of the interface resistance. The current density at the platinum electrode when the current passes through the same solution is measured in a macroscopic manner. Consistent with the worst-case conditions (high current) that may be encountered, the total ionic strength and ECL reactant concentration in the test solution is higher than the total ionic strength and ECL reactant concentration used in the LOC device. The anode area is smaller than the cathode area and the anode is surrounded by a cathode having an equal area and having a toroidal geometry. For an anode having a circular shape of 2 mm in diameter, the measured current was 1 · 1 milliamperes (mA) and a current density of 305 amps per square meter was obtained. In the heating model, the electrode area exhibits a square ring geometry as shown in Fig. 87. The anode is a ring having a width of 1 μm and a thickness of 1 μm. This surface area is 196 square microns and thus a current of 1 = 69 nanoamperes (nA) is calculated. This model of heating (power = V2/R) is established for the worst case (worst case where all heat is used to raise the temperature of the water in the chamber). If the heat removed by the LOC device body is not considered, this heating causes the chamber contents to be heated at a rate of 5.8 ° C / sec at a voltage difference of 2.2 volts. Increasing the temperature of the chambers by about 20 °C can cause most of the hybrid probes to denature. For highly specific probes for mutation detection, it is preferred to limit heating to only 4 ° C or less. By such a degree of temperature stability, the single-base mismatch sensitivity heterozygous -100-201211539 reaction with a suitably designed sequence becomes feasible. This method allows for the detection of mutations and allelic differences in the degree of single nucleotide polymorphism. Therefore, the direct current is applied to the 1* electrode 860 and the electrode 870 for 0.69 seconds to limit the heating to 4 °C. 1

The current of about 69 nanoamperes that is compatible with the chamber is well above the amount of Faraday current that the ECL species of the micromolar concentration can provide. Thus, the small duty cycle (l〇w-duty-cycle) of the electrodes 8 60 and 870 further reduces the heating (to 1 ° C or lower) while maintaining sufficient ECL luminescence 862 without Problems associated with depletion of reagents will be introduced. In other embodiments, the current is reduced to 0.1 nanoamperes (this current eliminates the need for pulsed activation of the electrodes). Even with currents as low as 0.1 nanoamperes, the ECL luminescence 8 62 is still limited by the luminescent light-emitting groups. The geometry of the chamber and the electrode The optical coupling between the ECL luminescence I and the photosensor maximizes the ECL luminescence. The instant chemical precursor is generated within a few nanometers of the working electrode. See Figure 86 again. The excited species 868) typically occur within a few microns or less of the location. Since the corresponding photodiode 1 84 of the photo sensor 44 can observe the volume of the working electrode (anode 8 60). Therefore, the electrodes 8 60 and the electrodes 8 搂 are directly adjacent to the active surface region 185 of the corresponding photodiode 184 in the photo sensor 44. In addition, the shape of the anode 860 is shaped to increase the length of the circumference of the anode side visible to the photometric body 184. Such

I -101 - S; 201211539 The purpose of the configuration is to maximize the volume of the excited species 868 detected by the lower photodiode 184. Figure 85 shows three specific embodiments of the anode 8 60. The comb-like anode 87 8 has the advantage that the parallel fingers 880 can be interdigitated with the fingers of the cathode 870. The interdigitated structure is shown in Figure 92 and a partial view of the LOC layout shown in Figures 98 and 100. The interdigitated structure provides a uniform dielectric void 8 76 (see Figure 86) that is relatively narrow (1 micron to 2 microns) and is fabricated for lithographic fabrication methods. The comb structure is relatively simple. As described above, since the excited species 868 will diffuse between the anode and the cathode, the relatively narrow dielectric voids 8 76 between the electrodes 860 and the electrodes 870 are exempt from certain solutions 8 The need to use a co-reactant in 72. Eliminating the need for a co-reactant eliminates the chemical shocks that the co-reactant can have on the chemicals of the various analytical tests and provides a wider range of available analytical test methods. Referring again to Fig. 85, certain embodiments of the anode 8 60 have a tortuous structure 8 82. To achieve a high perimeter length while maintaining acceptable manufacturing tolerances, the structure is suitable for fabrication into a plurality of wide rectangular curved channels 8 84 . The anode may have a more complex structure 886 if desired or desired. For example, the anode may have a serrated section 888, a branched structure 8 90, or a combination of both. A partial view of the LOC device design including the branched structure 890 is shown in Figures 1 23 and 24. This more complex structure (e.g., structure 886) provides a longer side perimeter length, and since the step of patterning -102-201211539 closely spaced relative cathodes is more difficult, this complexity is best suited for solutions using co-reactants. Chemical reagents. Electrode Thickness Typically, an ECL cell contains a working electrode that looks flat. In addition, the conventional technique for fabricating a metal layer is easy to make a flat structure having a metal thickness of about 1 micrometer. As previously described and briefly shown in Figures 85, 8 8 and 8 9, increasing the side circumference enhances the use of the ECL release light with the photodiode 184. Further enhancing the collection of the emitted light by the photodiode 184

I see the second option for the collection efficiency of Figure 102: Figure) to increase the thickness of the anode. This scheme is shown in Figure 86. The portion of the reference electrode adjacent to the wall surface of the working electrode is a region where the photodiode is operatively coupled. Therefore, for the finger of the working electrode 860, the collection efficiency of the release light 862 can be improved by increasing the thickness of the electrode. "Furthermore, since the high current carrying capacity is not required, the width of the working electrode 860 can be reduced. It is practical that the thickness of the electrode 860 and the electrode 8 70 cannot be increased indefinitely. The tip and pitch dimensions of the electrode may be about 1 micron, and the liquid step makes the width of the gap unsuitable for the depth of the gap. The optimum actual thickness of the equal electrode is between 0.25 micrometers and 2 micrometers.

Look at the long coupling that has been described before microfabrication as 862 (the maximum width of the pole 860 and the volume 184 is therefore the same. This is the equivalent of the larger, the -103-201211539 the electrode 860 and the electrode 870 The spacing between the two is important for the signal quality of the LOC device, especially in the particular embodiment where the electrodes are interdigitated. When the anode 8 60 is in a branched structure (eg, 8th) In the specific embodiment of the structure shown in Figures 5 and 89, the spacing between the adjacent elements is also quite important. Both the ECL luminous efficiency and the collection efficiency of the emitted light need to be maximized. It tends to prefer an electrode spacing of 1 micron or less. When performing an ECL reaction in the absence of a co-reactant, it is particularly preferred to use a small pitch, which may be equivalent to the fact that the wavelength of light 8 62 is released. The nature is limited. Therefore, in many embodiments in which light is not required to pass through the position between the electrodes 8 60 and the electrode 870 (see Figure 102), the goal is usually to maximize the electrode spacing However, in the specific embodiment where the release of light 862 must pass between the electrodes 860 and the electrode 870, it is necessary not only to take into account the ECL illumination method, but also to consider the wave properties of the light. From [Ru(bpy 3] 2 + ECL reaction release light 8 62 wavelength is about 620 nm, and therefore its wavelength in water is 460 nm (0.46 μm). The photodiode 184 and the ECL excited species 868 series In a particular embodiment where the electrode structure is on a different side and the electrode structure is a metal, the release light 862 must pass through a gap between the elements of the metal structure. If the gap corresponds to the wavelength of the emitted light. The diffracting effect generally reduces the intensity of the propagating light reaching the photodiode 184. However, when the illuminating light 862 is irradiated onto the gap at a large angle, the dissipative mode coupling phenomenon can be controlled to enhance the collected The strength of the signal. Available at -104-

In 201211539, two measurement methods are adopted in the LOC ft to improve the coupling efficiency between the light-emitting diodes 8 6 2 . The first method is that the spacing between the metal elements is not low in the wavelength of light in water, i.e., about 0.4 microns. The optimum range of pitch spacing is between 0.4 microns and 2 microns when combined with other observations of the small spacing between the electrodes. The second method is to minimize the gap between the electrodes to the optical distance. In the L0C device of the present invention, this distance indicates that the total thickness of the film layer between the electrodes 860 and 870 and the light is 1 micron or less. In a specific embodiment in which a plurality of film layers are present between the photodiodes, the thickness of the layers is configured to have a quarter-wavelength or three-quarters of a series of further excellent electrode models having a reflection inhibiting release light 862. A cross-sectional view of the electrodes 860 in the hybrid chamber. On the side circumference of the anode 860

The volume occupied by the stimulated species is sometimes referred to as the trapping region 894 with the participating volume above the anode 860, and the optical coupling is negligible. Therefore, the containment region 894 is referred to below on pages 8, 8 and 8 Figure 9 presents a technique for the electrode structure to provide the strength that can be used for the underlying light two-volume ECL release light 862. Figure 87 is a ring-shaped geometric structure in which the electrode 184 and the electrode body 184 of the body of the electrode body 184 which is not mutually exclusive with the release or the like The film layer point of the film wavelength. And the electrode 8 70 is surrounded by the 892. Since the polar body 184 is negligible. Judging the geometry of a particular body 184 -105-201211539 The anode 8 60 is surrounded by the photodiode 184. The anode 860 is located in the photodiode. Figure 89 shows a more complicated The structural configuration 'on the anode 860 has a series of fingers 880 to increase the length of the space. For all of the above structures, the model is calculated as follows for the volume of the solution Vecl 892, emitter: Nem - N|um . Xp / TECL - ^ECL^lNa · "tp / ^ECL where the luminescent group The number of participants N|um = the lifetime of VeClI ECL, the concentration of CL-based luminescent group, and the & The number of photons emitted by the isotropic direction is NPH. 1 Series: Nph〇t— φε〇ί Nem where to· is the ECL efficiency and is defined as the average number of photons emitted by a single luminescence. The electrode signal S derived from the photodiode is S ~ Nph〇t · φοψς, where φ. Optical coupling efficiency (the photodiode 1 84 ® and \ is the quantum efficiency of the photodiode. Therefore the signal s = vECLcLNA ^ EMq TECL for the electrode structure of Figures 87 and 88, t system: 丨 long edge. Within the perimeter of the 84th. i The configuration shows the side edge of the k-pole. The effective total number of Nem (6) 〕 LNA, τ Ε α_ is the τρ is the ECL (8) of the pulse week (7) The number of photons): (9)

-106-

201211539 Lu 0 = (2 5 % of the light directed to the photodiode 1 8 4 (10% unreflected photons) ie 鈇 for the structure of Figures 8 and 8 8

I for the electrode structure configuration of Fig. 89 '50% The direction of the photodiode 184 is emitted, but the equation of the absorption effect is unchanged, so: ??? = (50% of the photons directed to the photodiode) x (10% unreflected photons) That is, for the structure of Fig. 89, nine =: 5% The participating volume 892 depends on the electrode structure, and a detailed description is provided. The input parameters used for the calculation are shown below: F) χ = 2.5% of the photonic system is written to the angle and is in the corresponding paragraph -107- 201211539 Table 5, the number of input parameter parameters 値 indicates the concentration of the luminescent group 2.89 μΜ Previously calculated probe concentration ECL recirculation period (life) rEa_ 1 millisecond luminescence luminescent group of multiple reaction steps of Shoukou 卩 boundary layer depth D 0.5 micron effective volume of solution involved in ECL reaction (including diffusion With electrophoresis) The current application time Γρ 0.69 sec is selected to limit ohmic heating to 4 Τ: (as mentioned above) X-axis size of the chamber 28 μm chamber Υ axis size 28 μm chamber height Ζ 8 μm photodiode X-axis size 16 micron photodiode x-axis size 16 micron electrode thickness (ie, exposed edge height) 1 micron electrode layer minimum width and void 1 micron process critical dimension electrode interfacial current density 350 amps per square meter Volume resistivity for ohmic heating solution 0.5 ohm x metric for voltage application of ohmic heating (working electrode - counter electrode) 2_2 volt

Annular Geometry Around the Peripheral of the Light Dipole Referring to Figure 87, the anode 8 60 is an annulus surrounding the edge of the photodiode 184. In this configuration, the participating volume 8 92 is VECX=4x [(the film layer on the side of the electrode wall) + (a quarter of the cylinder above the electrode wall)] calculated = -108 - 201211539 Photon generated by the 0.5 micron boundary layer: 3·1χ105 The electron count in the photodiode 184: 2.3xl03 This signal can be obtained by the photodiode of the L〇C device photosensor 44 below. Easy to measure. Additional fingers to increase edge length

Referring to Figure 89, parallel fingers 880 are added over the entire anode 860. The figure shows that only the horizontal edges contribute to the participating volume 892 to avoid double counting the vertical edges. At this time, the participating volume is as follows: VE<x;i8x2)><[(membrane layer on the side of the electrode wall)+ (a quarter of the cylinder above the electrode wall)] 8 9 The calculation result of the structure of the figure: photons generated by the boundary layer of 0.5 μm: the electron count in the UxlO6 photodiode 184: 8.0 x 103 This photodiode 1 84 can easily detect this signal. Full coverage The structure shown in Figures 90 and 91 is the limit of the maximum coupling surface area. In practice, the coupling between the surface area of the electrode and the active surface of the photodiode 184 reaches 90% or better to achieve near-optimal results, even if the photodiode active surface region 185 and the electrode A 50% coupling of the surface area also provides most of the benefits of a fully covered structure. Full coverage can be achieved in two specific embodiments: the first type -109 - 201211539 is as shown in Fig. 90, by using a transparent anode 860 in a plane parallel to the surface of the photodiode 184 and The area of the anode 860 is comparable to the area of the photodiode, and the anode is disposed in close proximity to the photodiode 184 such that the release light 862 passes through the anode and illuminates the photodiode. In a second embodiment, which is explicitly shown in FIG. 91, the anode 860 is also disposed in parallel with the surface area of the photodiode and is configured to match the area of the photodiode, but the solution 872 is interposed in the anode. Within the gap between the 860 and the photodiode 184. For the signal model establishment of the fully covered structure, it is assumed that the anode is completely covering the film layer above the photodiode 184, and half of the photons are directed toward the photodiode 184 (the absorption rate is still 10%). Photons generated by the 0.5 micron boundary layer: 7·7χ105 The electron count in the photodiode: 1.2χ104 In addition to the above modules, the signal and analysis can be improved by using a surfactant and fixing the probe to the anode. . Maximum spacing between the ECL probe and the photodiode The on-wafer detection of the hybridization reaction eliminates the need for detection by a conjugated focus microscope (see paragraph of the prior art). Such detection from the conventional detection technology is an important factor in saving time and cost in the system of the present invention. Traditional detection methods require imaging optics that require many lenses or curved mirrors. By employing non-imaging optics, the diagnostic system eliminates the need for complex and bulky strings of optical components. The advantage of placing the photodiode in close proximity to the probes is extremely high efficiency: when the -110-201211539

When the material thickness between the probe and the photodiode is 1 micron, the collection angle of the released light is up to 1 74. . The angle is calculated from the light emitted by the probe at the center of mass of the hybrid chamber closest to the surface of the photodiode, wherein the photodiode has a flat active surface region parallel to the surface of the chamber . The light in the cone of the illuminating angle can be absorbed by the photodiode' and the conical system of the illuminating angle is defined as having a luminescence at the sensor corner at the perimeter of the flat chamber surface and at the apex of the cone needle. For a 16 micron χ 16 micron sensor, the apex angle of the cone is 170°; the limit is extended when the photodiode is enlarged to make the area of the photodiode comparable to a 28 micron x 2 6.5 micron hybrid chamber. Next, the apex angle is 174°. It is easily achieved that the separation distance between the surface of the chamber and the active surface of the photodiode is 1 micron or less. Systems employing non-imaging optics must have photodiodes 1 84 very close to the hybrid chamber to collect sufficient fluorescent photons. The maximum spacing between the photodiode and the probe can be determined as follows. Using the ruthenium chelate luminescent group and the electrode configuration of Fig. 89, assuming that the quantum efficiency of the sensor is 30%, we calculate 16 microns: < 16 micron sensor absorbs 27,000 from each Do not mix the photons of the chamber to produce 8000 electrons. When performing this calculation, we assume that the collection region of the hybrid chamber 180 has a base area equal to the photodiode active region 185 and that one quarter of the total number of photons has reached a photon conversion angle The sensor, and conservatively estimated that the photon ratio of the sensor-dielectric interface that did not leave the scattering due to scattering was 10%. That is, the light collection efficiency of the optical system is Α =0 025. -111 - 201211539 More precisely, we can write a formula = [(base area of the collection area of the hybrid chamber) / (photodetector area)] [Ω / 4π] [1 〇% absorption rate] Where Ω = the solid angle at which the photodetector is located at a representative point on the substrate of the hybrid chamber. For the geometry of a regular pyramid: where 4 = the distance between the chamber and the photodiode, and a is the size of the photodiode. Each hybrid chamber releases l.lxl6 photons. The selected photodiode has a detection threshold of 17 photons; and when < 値 is 1 〇 larger than the size of the sensor (ie, substantially perpendicular incidence), it is not at the surface of the sensor The proportion of reflected photons can be increased from 10% to 90%. Therefore, the minimum optical efficiency required is: φ0 = 17 / (1.lxlO6 χ 0.9) = 1.72X10'5 The base area of the light collecting region of the hybrid chamber 180 is 29 μm X 19.75 μm. Solving the name, we will get the maximum limit distance between the bottom of the hybrid chamber and the photodetector < = 1600 microns. Under this constraint, the above-defined light collecting cone angle is only 0.8 degrees. It should be noted that this analysis ignores this negligible refraction effect. LOC Device Variations The LOC device 301, described and illustrated above, is only one of many possible LOC device designs. It will now be described and/or illustrated in a summary flow chart (from sample entry to detection) using multiple combinations of different combinations -112- 201211539

The LOC device variations of the various functional segments are described to illustrate one of the possible combinations. The flow diagrams are suitably divided into sample placement and preparation stages 288, extraction stage 290, incubation stage 291, expansion section 292, pre-hybridization stage 293, and detection stage 294. For all LOC devices that are briefly described above or only in the form of abbreviations, for the sake of clarity and conciseness, the drawings of the complete layout are not shown. Also for clarity, the smaller functional units (eg, liquid sensors and temperature sensors) are not shown, but it will be appreciated that such smaller functional units have been incorporated into each of the following LOCs. Appropriate position in the design of the device"

LOC Device Variant XXIX The LOC device variant XXIX 659 shown in Figure 75 is a LOC device for pathogen detection and genetic analysis. The placement and preparation section 288 of the sample is simplified and therefore does not include any dialysis steps. The removal of the dialysis step improves the detection sensitivity of the pathogens that may adversely affect the dialysis function. A whole blood sample is applied to the sample inlet 6 8 ' and the anticoagulant i derived from the reservoir 54 is added by the surface tension valve 1 18 . The extraction nozzle section 290 is used in the chemical lysis section 130 to dissolve the pathogens and white blood cells using a lysis reagent derived from the reservoir 56. The incubation stage 291 includes incubating the sample with restriction enzymes, ligase and linker-derived from storage tank 58 and culturing in the incubation section 114, and the sample enters the amplification stage 292 after incubation. In the amplification stage 292, the amplification mixture derived from the storage tank 60 and the polymerase derived from the storage tank 62 are added to the sample, and the sample is amplified in the amplification section 112-113-201211539 this. The detection phase 294 occurs within the array of heterozygous chambers 1 1 .

LOC Device Variant XXX The LOC Device Variant XXX 660 shown in Figure 76 is a LOC device for pathogen detection and gene analysis. The placement and preparation of the sample is simplified and therefore does not include any dialysis steps. Removal of the dialysis step can increase the sensitivity of detection of such pathogens that may adversely affect dialysis function. A whole blood sample is applied to the sample inlet 68, and an anticoagulant derived from the storage tank 54 is added by the surface tension valve 118. The extraction stage 290 is used in the chemical lysis section 130 to dissolve the pathogens and white blood cells using a lysis reagent derived from the reservoir 56. The sample is then incubated with the restriction enzyme, ligase and linker derived from the storage tank 58 in the incubation section 112, and then expanded in parallel amplification sections 112.1, 112.2...112. The sample is added and the sample is detected in an individual hybrid chamber array 1 1 0.1, 1 1 0.2 ... 1 1 0 . X.

LOC Device Variant XXXI The LOC device variant XXXI 661 shown in Figure 77 is a LOC device for pathogen detection and genetic analysis. The placement and preparation of the sample is simplified and therefore does not include any dialysis steps. Removal of the dialysis step can increase the sensitivity of detection of such pathogens that may adversely affect dialysis function. A whole blood sample is applied to the sample inlet 68, and an anticoagulant derived from the storage tank 54 is added by the surface tension valve 118. The extraction stage 290 is used in the chemical lysis section 130 to dissolve the pathogens and white blood cells using a lysis reagent derived from reservoir 114-201211539 reservoir 56. Following incubation of the sample with restriction enzymes, ligases, and linkers in the incubation section 114, the samples are amplified in tandem amplification sections 112.1 and 12.2, and in a single hybrid chamber array. The sample was detected in 1 1 0. A microfluidic device having a dialysis device, a LOC device, and an interconnecting cap layer having a dialysis device 784, a LOC device 785, and an interconnect cap layer 51

The microfluidic device 78 3 provides greater modularity and improved sensitivity. In response to the LOC device design incorporating all of the functions (e.g., LOC device variant 729 of Figure 97), the dialysis function is separated from the LOC device to allow for the selection of different specialized dialysis devices 784 for different targets. These tailored dialysis devices 784 can be combined with the LOC device 785 and the interconnect cap layer 51 to form a complete analytical inspection system. Furthermore, different LOC devices 785 optimized for different analytical testing methods can be issued, and the different LOC devices 78 5 can cooperate with different dialysis devices to provide a powerful and flexible system bursting method. For some applications, the LOC device can also be configured to have no dialysis device or incorporate multiple LOC devices 78 5 . Each of the surface micromachined wafers comprised of the microfluidic device 783 can be fabricated using the best and most cost effective manufacturing method. For example, the dialysis device 784 does not require a CMOS circuit, so the dialysis device 784 can be fabricated using less expensive materials and fewer process steps. Moreover, the larger and optimized dialysis device 784 provides improved sensitivity, signal to noise ratio, and dynamic range for the analytical inspection system. -115- 201211539 As shown in Fig. 110, the fluid device 783 prepares the sample 288), and then extracts (290), incubates (291), and amplifies using the same amplification chamber (1 12.1 to 12.12). (292) and detecting) the pathogenic DNA. The assembly employs multiple amplification chambers to increase the sensitivity of the assay and improve the signal to noise ratio. The LOC device performs an ECL reaction by means of a hybrid chamber array 1 10.1 to Π0.12 to detect a needle-target hybrid. The system modularly provides different dialysis 784 for detecting other targets in a liquid sample, such as white blood red blood cells, pathogens or molecules such as free proteins or DNA, and the description describes pathogenic DNA detection, but is familiar with It is understood by the art that the microfluidic device 783 is not limited to detecting only one such target, and that the microfluidic device 78 3 having the dialysis device 784, the LOC device 78 5 with the cap layer 51 is shown. The interconnect cap layer 51 is comprised of a reservoir layer 78, a capping channel layer 80, and an interfacial layer 594. The interface layer is between the capping channel layer 80 and the channel layer 100 of the CMOS + MST device 48. The interfacial layer 594 allows for a more complex fluid interconnect structure between the MST layers 87 for storage of such reagents and does not squash the size of the substrate 84. Fig. 112 overlays the top channels and the interface channels, such as the storage tanks, to illustrate the use of the interface layer to achieve more sophisticated piping engineering. Referring to Figures 1 1 2 and 1 1 3, the sample (e. g., blood) is introduced into the sample inlet 68, and capillary action is applied to the anticoagulant surface tension valve 1 18 along the cap channel 94. Preferably, the anticoagulant surface tension valve 118 has a location in the interface layer 5 94 as shown in Figure 1 1 3 (the 294 is detected in the probe device ball, although the object is present. The trough 594 MST trough and the two-116-201211539 interface ramps 59ό and 598 which are added to the attraction, the storage tank side interface channel 596 connects the storage tank outlet to the lower suction holes 92. Together, and the sample side interface channel 5 98 connects the upper suction holes 96 with the cover layer channel 94. The anticoagulant from the storage tank 54 flows through the MST channel via the interface channel 596 on the storage tank side. 90 until the meniscus is settled in the upper suction holes 96

At the office. The sample fluid flowing along the cover channel 94 wets the interface side 598 of the sample side to remove the meniscus such that when the sample fluid continues to flow to the pathological dialysis section 70, the anticoagulant is combined with the blood sample. Referring to Figures 1 1 2 and 1 1 3, the pathogenic dialysis section 70 in this embodiment includes an interface target channel 602 and an interface waste channel 604, and is fluidly connected by a plurality of pores having a predetermined critical dimension. The interface target channel 062 and the interface waste channel 604 are coupled to the interface. The pores located upstream of the poles of the dialysis section 70 are different from the pores located downstream of the dialysis section 70; the downstream pores select pores having a diameter of less than 8.0 microns to allow the target to pass through the pores Go to the interface target channel 602. In a particular embodiment of the invention, the holes are selected to have a pore size 164 of 3.0 microns to allow passage of the pathogen to the interface target channel 602. The pathogenic dialysis section 70 is constructed such that the sample flows through the channels and the pores under capillary action. Referring to Figures 1 1 2 and 1 1 3, the blood sample flows through the capping channel 94 to the upstream end of the interface spent cell channel 604. The interface waste cell channel 604 is open toward the 3.0 micron diameter apertures 64 and the holes 164 are routed to the dialysis MST channel 204. Each of the dialysis MST channels 204 is routed from the 3.0 mm diameter holes 164 to the respective -117-201211539 dialysis uptake holes 168. The dialysis upper suction holes 168 are open toward the passage 602. However, the upper suction holes are constructed to continue with the liquid level without allowing the capillary to drive the flow. The pathogenic dialysis section 70 includes a bypass channel 600 channel 600 system for accommodating the fluid channel structures without trapping. The 600 located at the extreme upstream end of the pathogenic dialysis section 70 has a capillary priming feature (C IF) 202 to facilitate capillary flow from the bypass channel 600 into the interface target channel 602 (see Figure K 113). The bypass channel also has a wide curved flow path to extend the waste cell channel 604 to the interface target channel 602. The longer flow path delays the sample fluid such that after the meniscus is formed at the most M ST channel 204, the sample flow target channel 602. The sample fluid begins at the upstream end and the sample fluid moves downstream along the interface target channel 602. The sample fluid is released from the lysate at each dialysis uptake 168. All channels are filled with sample fluid. Some dialysis MST channels cannot be filled when there is no such bypass channel 600 or when there is no dialysis upper suction port 168 for the liquid level. Likewise, air bubbles may be present at the interface target channel 602. In either case, it is possible to substantially stop the flow of the section. The interface waste channel 604 is injected into the waste channel 72, and the target channel 602 is injected into the target channel 74 (see Figure 11). The five transmissive interfaces are targeted by the capping channel 72 and the capping channel 74, and the bypass is driven by the air-bypass channel to drive the fluid; 1 12 and the length of the dialysis body injection boundary upstream of the boundary flow path, And when moving, the face. When the bottom of the sample passes through the set bend 204, an air flow may be formed through the pass and the interface 2 and the 1 1 3 split section are connected in series -118-201211539 to increase the efficiency of the dialysis procedure. Located at the exit of the fifth dialysis section, the sample fluid containing the target is drawn from the dialysis device 784 by capillary action into the LOC device 785 along the target channel 74 in the cap channel layer. The waste channel 72 leads to the waste storage tank 76 (see Figure 1 1 1).

The L〇C device 785 can also be used as a stand-alone microfluidic device having a replacement cap layer suitable for a single device, and in this configuration, an anticoagulant can be optionally used. The tank 55 is used to supply an anticoagulant. An example of such a stand-alone LOC device 785 is for whole blood analysis. Referring to Figures 1 14 and 151, the target flows into the LOC device 78 5 along the cover channel 74. The target passes through an optional anticoagulant surface tension valve 117 (this surface tension valve 117 is used to add the contents of the optional anticoagulant reservoir 55) and the target continues Traveling until the target reaches the lysis surface tension valve 128. In the configuration described herein, the optional reservoir and surface tension valve do not affect the flow of the sample or the operation of the LOC device, even when not in use. If the anticoagulant surface tension valve 118 is provided, the lysis reagent surface tension valve 128 has an interface channel 606 on the lysis reagent storage tank side and an interface channel 608 on the lysis sample side (see Fig. 1 15). The lysis reagent flows from the storage tank 56 through the capping passage 94 to the interface passages 60, 6 on the side of the lysis reagent storage tank. The reagent flows into the lower suction holes 92, flows through the MST passage 90 to the upper suction holes 96, and the reagents are at the upper suction holes 96.

I Hold the meniscus (see Figure 1 1 5). Sample fluid from target channel 74 is injected into interface channel 608 on the lysis sample side. The sample fluid removes the meniscus at the upper suction port 96 from -119 to 201211539, and the lysing reagent merges with the sample as the sample fluid flows into the chemical lysis cell segment 130.

In the chemical lysis section 130, a lysis reagent is diffused and mixed throughout the sample fluid to dissolve the target cells and release the genetic material within the target cells. The sample fluid stops at the mixing section outlet valve 206. The mixing section inlet valve is a boiling start valve 206. A liquid sensor 174 located upstream of the valve provides feedback that the sample flow system generally reaches the suction port 151 of the valve. If the CMOS circuit 86 is programmed to write a delay time to ensure that the target cells are completely dissolved, the liquid sensor feedback will begin to perform the delay time. After any delay time has elapsed, the boiling start valve 206 is activated and the downstream liquid sensor 1 74 indicates that the fluid has begun to flow again along the MST passage 90.

The lysed sample fluid continues to flow to the surface tension valve 132 of the restriction enzyme, ligase, and linker. The operation of the surface tension valve 132 is the same as that of the anticoagulant surface tension valve 118 described previously. Referring to FIG. 115, when the sample fluid reaches the settled meniscus at the surface tension valve 132, the restriction enzyme, ligase, and linker-primer are released from the reservoir 58 and The sample fluids meet. The sample then flows through the MST channel 90 to the heated microchannel of the incubation section 146. Referring to Figures 1 15 and 116, the incubation section 112 is constructed of a single microchannel 210 and is heated by a plurality of heaters 154. Referring to Figure 116, the sample flow system Stop at the incubation chamber outlet valve 2〇7 for a sufficient period of time. The chamber exit valve 207 is a boiling valve - 120 - 201211539, which is similar to the mixing section outlet valve 206. The liquid sensor 1 74 at the beginning of the incubation section, in conjunction with the flow rate sensor 74 (see, Figure 12) and the CMOS circuit 86, begins performing a incubation time delay. After sufficient incubation, the incubation chamber outlet valve 207 is activated and fluid continues to flow along the MST incubation outlet passage 630 to the polymeric enzyme surface tension valve 140 (see Figure 117). The polymerase originating from reservoir 62 and the sample as the sample fluid travels through the amplification injection channel 632

Fluid is combined. Returning to Figures 116 and 117, the amplification injection channel 632 directs the sample fluid through the twelve amplification mixture surface tension valves 138. Each amplification

The amplification mixtures in the mixture storage tanks 60.1 to 60.12 (see Figure 117) are passed through respective cover channels 94 to position the meniscus at the surface tension valve 138 of the expansion mixture. The sample fluid sequentially opens each of the surface tension valves, and the amplification mixture from the individual amplification mixture storage tanks 60.1~60.12 enters the 12 amplification chambers 112.1~112.12 together with the sample fluid. By. The LOC device has a CMOS circuit that allows operative control of the amplification section by a temperature sensor and a heater. Referring to Figure 118, each of the twelve amplification chambers I12.1 to 112.12 has one of the amplification outlet valves 108, respectively. The amplifying outlet valve 108 is a boil-start valve like the culturing section outlet valve 207 = the sample cartridge is stopped at each of the amplifying outlet valves 108. After amplification, the amplification outlet valve 108 is opened to cause the amplicon to flow into the hybrid chamber array 1 1 0 · 1.~1 1 〇. 1 2, the hybrid chamber array 1 1 1. 1~1 1 〇.1 2 -121 - 201211539 A probe constructed to form a probe-conjugate with the target nucleic acid sequence, in which case the target nucleic acid sequence is ill. The sample flows along the flow path 176 through each of the non-110.1~110.12 and through the respective diffusion barrier/into the individual hybrid chamber 180 (see Figures 98 and 119). In Figures 99 and 119, when the sample fluid reaches the final sensor 1 78, the hybrid heaters 182 are activated and held for a delay time to facilitate the generation of probe-target hybrids. The flow rate 740 (see Fig. 120) is included in the pathogen incubation section 1 14 to determine the delay time. After a suitable delay time for hybridization, the excitation current applied to the ECL electrode 860 and the electrode 870 (see Figure 122) causes photons of the probe-target hybrid line and utilizes the underlying CMOS The light perception in circuit 86 detects the photons. The photosensor is comprised of an array of a plurality of photodiodes 1 84, and the photodiodes 184 are configured to be adjacent to each of the chambers. Figures 121 and 122 show the calibration chambers 382. As described elsewhere in this document, the calibration chambers are used to calibrate the polar body 1 84 to adjust system noise and background artifacts. In addition, the positive-illuminated probe 787 and the negative ECL control probe 786 are placed in some of the chambers 180 for analysis of the quality of the test. The Figure 1 shows the ECL electrodes with interdigitated cross-sections. The change of the school's 3 82. The humidifier 196 and the humidity sensor 23 2 are used to control the liquid of the 靶 target heterogeneous DNA, the 175 〇V point liquid; the continuation of a sensor to determine the reaction 1 19 and the emission photodetector 44 The composition of the hybrid cavity case is explained by the optical ECL pair hybrid cavity system. The first chamber LOC-122-201211539 is placed in 785 (especially in the hybrid chamber array 110) for evaporation and condensation. Figure 1 18 shows the main components of the humidifier 96, the water storage tank 188 and the evaporator 190.

Referring to Figure 117, the evaporative indicator 189 indicates whether the package of the device has been damaged during poultry storage and thereby indicates whether the integrity and reliability of the microfluidic device has decreased. During manufacture, a small drop of liquid is dropped onto liquid sensor i 74 at the center of the evaporation indicator 189. If the sealed package has broken during storage, the droplet will evaporate. The presence or absence of the liquid can be detected by the liquid sensor 1 74 to indicate the seal integrity on the microfluidic device. Conclusion The devices, systems, and methods described in this case facilitate molecular diagnostic testing at low cost, high speed, and focused care. The above-described system and the components of the system are purely illustrative, and those skilled in the art will be able to readily comprehend many variations and modifications without departing from the spirit and scope of the broad inventive concept. BRIEF DESCRIPTION OF THE DRAWINGS A number of preferred embodiments of the present invention, which are described with reference to the drawings, are for illustrative purposes only, and the drawings are as follows: Figure 1 shows a test constructed for fluorescence detection. Module and inspection module reader. Figure 2 is a summary view of the sub-components of the electric-123-201211539 sub-assembly in the inspection module constructed for fluorescence detection. Figure 3 is the 电子 of the electronic components in the inspection module reader. To map the map. Figure 4 is a schematic representation of the construction of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of the LOC device having features and structures derived from all of the overlapping film layers. Figure 7 is a plan view of the LOC device showing the cover structure separately. Figure 8 is a top perspective view of the cover layer having internal passages and storage slots, the internal passages and storage slots being shown in dashed lines. Figure 9 is a top exploded perspective view of the cover layer having internal passages and storage tanks. The internal passages and storage tanks are shown in dashed lines. The first drawing is a bottom perspective view of the cover, the bottom perspective showing the structural configuration of the top channels. The πth diagram is a plan view of the LOC device, which shows the structure of the CMOS + MST device separately. Fig. 1 is a schematic cross-sectional view of the LOC device at the entrance of the sample. Fig. 13 is an enlarged view of the inset AA shown in Fig. 6. Fig. 14 is an enlarged view of the illustration AB shown in Fig. 6. Fig. 15 is an enlarged view of the illustration AE shown in Fig. 13. The figure is a partial perspective view showing the layered structure of the LOC device in the illustration AE. -124- 201211539 Figure 17 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. First, Fig. 18 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 19 is a partial perspective view showing the layered structure of the LOC device in the illustration AE.

I. The figure shows the layered structure of the LOC device in the illustration AE.

Partial perspective. Figure 21 is a partial perspective view showing the layered structure of the LOC device in the inset AE. Fig. 22 is a schematic cross-sectional view showing the lysis reagent storage tank shown in Fig. 21. Figure 23 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 24 is a partial perspective view showing the layered structure of the LOC device in the inset AB.

I Fig. 25 is a partial perspective view showing the layered structure of the LOC device in the illustration AI. Figure 26 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 27 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 28 is a partial perspective view showing the layered structure of the LOC device in the inset AB. -125- 201211539 Figure 29 is a partial perspective view showing the layered structure of the LOC device in the illustration AB. Figure 30 is a schematic cross-sectional view of the amplification mixture storage tank and the polymerase storage tank. Figure 31 is a separate illustration of the characteristics of a boiling start valve. Figure 32 is a cross-sectional view of the boiling start valve taken along the line 3 3 - 3 3 shown in Figure 31. Fig. 33 is an enlarged view of the illustration AF shown in Fig. 15. Figure 34 is a schematic cross-sectional view of the upstream end of the dialysis section taken along line 35-35 of Figure 33. Fig. 35 is an enlarged view of the illustration AC shown in Fig. 6. Figure 36 shows a further enlarged view of the inside of the illustration AC of the amplified section. Figure 37 is a further enlarged view of the inside of the illustration AC of the enlarged section. Figure 38 is a further enlarged view showing the inside of the illustration AC of the amplified section. Figure 39 is a further enlarged view of the illustration AK shown in Figure 38. Figure 40 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 41 is a further enlarged view showing the inside of the illustration AC of the enlarged section. Fig. 42 is a further enlarged view showing the inside of the illustration AC of the amplification chamber -126 - 201211539 Fig. 43 is a further enlarged view of the inside of the illustration AL shown in Fig. 42. Figure 44 is a further enlarged view showing the inside of the illustration AC of the amplified section. Fig. 45 is a further enlarged view of the inside of the illustration AM shown in Fig. 44.

Figure 46 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 47 is a further enlarged view of the illustration AN shown in Fig. 46. Figure 48 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 50 is a further enlarged view showing the inside of the illustration AC of the amplification section. Figure 51 is a schematic cross-sectional view of the amplified section. Figure 52 is an enlarged plan view of the hybrid section. Figure 53 is a further enlarged cross-sectional view showing two hybrid chambers separately. Figure 54 is a cross-sectional view of a single hybrid chamber. Fig. 55 is an enlarged view of the humidifier shown in the illustration AG shown in Fig. 6. Fig. 56 is an enlarged view of the illustration ad shown in Fig. 52. Figure 57 is an exploded perspective view of the L〇c device in the illustration AD. • 127- 201211539 Figure 5 8 is an enlarged plan view of the humidity sensor shown in Figure AH of Figure 6. Figure 5 is a schematic diagram showing a portion of the photodiode array of the photosensor. Fig. 60 is an enlarged view of the evaporator shown in the illustration AP of Fig. 55. Figure 61 is a diagram of the PCR of the linker primer. Figure 62 is a schematic representation of a test module equipped with a lancet. Fig. 63 is a diagrammatic view showing the configuration of the LOC device variation VII. Fig. 64 is a plan view showing the configuration of the LOC device variation VIII derived from all the features and structures of the mutually overlapping film layers. Fig. 65 is an enlarged view of the inset CA shown in Fig. 64. Fig. 66 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 64. . Fig. 67 is an enlarged view of the illustration CE shown in Fig. 65. Figure 68 is a schematic representation of the structure of the LOC device variant VIII. Figure 69 is a schematic diagram of the structure of the LOC device variant XIV. Figure 70 is a schematic diagram showing the structure of the variable XV of the LOC device. Figure 71 is a schematic view of the structure of the LOC device variant XVIII. Figure 72 is a schematic diagram of the structure of the LOC device variant XXII - 128 - 201211539 Figure 73 is the structure of the LOC device variant XXV BRIEF DESCRIPTION OF THE DRAWINGS Fig. 74 is a schematic diagram of the structure of the LOC device variant χχνΙΠ. Fig. 76 is a schematic diagram of the structure of the LOC device variant XXIX. Fig. 76 is a schematic diagram of the structure of the LOC device variant XXX.

Fig. 77 is a schematic view showing the structure of the LOC device variation type XXXI. Fig. 78 is a schematic view showing the structure of the LOC device variation type XLI. Figure 79 is a schematic view of the structure of the LOC device variant XLIII. Figure 80 is a schematic view of the structure of the LOC device variant XLIV. Figure 81 is a schematic diagram of the structure of the LOC device variant XLVII. The figure is a schematic diagram of the structure of the LOC device variant XI. Figure 83 is a circuit diagram of the differential imager. Figure 84 shows the reactions that occurred during electrochemiluminescence (ECL) processing. Figure 85 shows three different anode configurations. Figure 86 is a fragmentary cross-sectional view of the anode and cathode of the hybrid chamber. -129- 201211539 Figure 87 is a diagram showing an anode having a ring-shaped geometry around the perimeter edge of the photodiode. Fig. 88 is a view showing an anode which is located inside the peripheral edge of the photodiode and has a ring-shaped geometry. Figure 89 is a diagram showing an anode having a set of fingers for increasing the lateral edge length of the anode. Figure 90 illustrates the use of a transparent anode to maximize surface area for coupling and ECL signal detection. Figure 91 illustrates the use of an anode fixed to the top of the hybrid chamber to maximize surface area for coupling and ECL signal detection. Fig. 92 is a view showing an anode which is interdigitated with the cathode. Figure 93 shows the test module and test module reader that are constructed to be used with ECL detection. Figure 94 is a summary overview of the electronic components in the test module that can be constructed for use with ECL detection. Figure 95 shows the inspection module and the alternative inspection module reader. Figure 96 shows an inspection module and an alternative inspection module reader with a host system that houses various databases. Fig. 97 is a plan view showing a variation L of the LOC device, which shows all the features overlapping each other and shows the position of the illustration GA-GL. Fig. 98 is an enlarged view of the inset GD shown in Fig. 97. Fig. 99 is an enlarged view of the illustration GG shown in Fig. 97. Fig. 100 is an enlarged view of the illustration GH shown in Fig. 97. The first 〇 1 diagram is an electrochemiluminescence resonance energy transfer in a closed structure. -130- 201211539 Diagram of the shift probe. Figure 102 is an illustration of an electrochemiluminescence resonance energy transfer probe in an open and heterozygous structure. Fig. 03 is a diagram showing the initial round _ of the amplification reaction with an illuminating linear probe of the primer. Figure 1 04 is an illustration of a light-emitting linear probe with primers for subsequent amplification cycles.

Figures 105A-105F illustrate thermal cycling of a luminescent dry-loop probe with primers attached thereto. Figure 1 06 shows a negative control cold light probe in a dry loop configuration. Figure 107 is a schematic illustration of the negative control group luminescence probe in Figure 106 of the open configuration. Figure 1 08 shows a negative control cold light probe in a dry loop configuration. Figure 109 is a schematic illustration of the negative control group luminescence probe in Figure 106 of the open configuration. Figure 110 is a graphical representation of a microfluidic device having a dialysis device 'LOC device, interconnect capping layer, and electrochemiluminescence (ECL) detection. Figure 111 is a perspective view of a microfluidic device having a dialysis device, a LOC device, and an interconnecting cap layer. Figure 112 shows the features of the dialysis device of the microfluidic device and shows the location of the inset JA. Fig. 113 is an enlarged view of the illustration JA shown in Fig. 112. -131 - 201211539 Figure 114 shows these features of the LOC device of the microfluidic device and shows the position of the inset JB-JJ. Fig. 115 is an enlarged view of the illustration JB shown in Fig. 114. Fig. 116 is an enlarged view of the illustration JC shown in Fig. 114. Fig. 117 is an enlarged view of the illustration JD shown in Fig. 114. Fig. 118 is an enlarged view of the illustration JE shown in Fig. 114. Fig. 119 is an enlarged view of the illustration JF shown in Fig. 114. Fig. 120 is an enlarged view of the illustration JG shown in Fig. 114. Fig. 121 is an enlarged view of the inset JH shown in Fig. 114. Figure 122 is an enlarged view of the illustration shown in Figure 114. Figure 123 is an enlarged view of the hybrid chamber of the LOC device variant L. Figure 124 is an enlarged view of the hybrid chamber array of the LOC device variant L showing the distribution of multiple calibration chambers. [Main component symbol description] 1 0 : Inspection module 1 1 : Inspection module 1 2 : Inspection module reader 13 : Housing 14 : Micro USB plug 15 : Inductor 16 : Micro USB slot 1 7 : Touch Screen -132- 201211539 18 : Display Screen 19 : Button 20 : Start Button 2 1 : Honeycomb Radio 22 : Tornable Sealing Tape 23 : Wireless Network Connector 24 : Large Placement Slot 25 :: Satellite Navigation System

26: Light-emitting diode (LED) 27: Data storage 28: Mobile phone/smart phone 2 9 : LED driver 30: On-wafer laboratory (LOC) device 3 1 : Power conditioner 32: Capacitor 33: Clock 1 34: Controller

I 3 5: Recorder 36: l|SB Device Driver 3 7: Driver 38: Random Access Memory (RAM) 39: Driver 40 = Program and Data Flash Memory 41: Recorder - 133 - 201211539 42 : Processor 43: Program Memory 44: Light Sensor/Photo Sensor Array 45: Indicator 4 6: Cover Layer 47: Powered by USB only and as an indication 48: CMOS + MST Wafer 49: Foam Insert /Porous element 51: Interconnecting cap layer 5 2 : Hybrid and detecting section 5 4 : Storage tank 5 6 : Storage tank 5 7 : Printed circuit board 5 8 : Storage tank 60 : Storage tank 60. 1 : Storage Tank 60.2: Storage tank 60.3: Storage tank 60.4: Storage tank 60.5: Storage tank 60.6: Storage tank 6 〇. 7: Storage tank 60.8: Storage tank 60.9: Module of storage tank

-134- 201211539 60.10 : Storage tank 60.1 1 : Storage tank 60.12 : Storage tank 60.X : Storage tank 62 : Storage tank 62.1 : Storage tank 62.2 : Storage tank 62.3 : Storage tank

62.4: Storage tank 62.X: Storage tank 64: Lower sealing layer 66: Top layer 68: Sample inlet 70: Dialysis section/dialysis step 72: Waste liquid channel 74: Target channel 76: Waste unit/waste storage tank 78: Storage tank layer 80: Cover channel layer 8 2: Upper sealing layer 84: 矽 substrate (CMOS) circuit 86: Complementary metal oxide semiconductor 87: Microsystem technology (MST) layer 8 8 : Passivation layer -135 - 201211539 90 : Microsystem Technology (MST) Channel 92: Lower Suction Hole 94: Cover Channel 96: Upper Suction Hole 9 7 : Partition Wall Section 9 8 : Meniscus Anchor

100: Microsystem Technology (MST) channel layer 1 〇1: Portable personal computer/notebook computer 102: Capillary action priming feature (CIF) 103: Dedicated reader 105: Desktop computer 106: Boiling start valve 107 : e-book reader 108: boiling start valve / amplification outlet valve 1 0 9 : tablet

1 1 〇: Hybrid chamber array 1 1 〇. 1 : Hybrid chamber array 1 1 〇. 2 : Hybrid chamber array 1 1 〇. 3 : Hybrid chamber array 1 1 〇. 4 : Hybrid Chamber array 1 1 〇. 5 : Hybrid chamber array 1 1 〇. 6 : Hybrid chamber array 1 1 〇. 7 : Hybrid chamber array 1 1 〇. 8 : Hybrid chamber array - 136- 201211539 110.9: Hybrid chamber array 1 1 0.1 0 : Hybrid chamber array 1 1 0.1 1 : Hybrid chamber array 1 10.12: Hybrid chamber array 1 10.X: Hybrid chamber array 1 η:: Epidemiological data host system 1 1 2: amplification section

1 1 2.1: Amplified segment 1 12.2: Amplified segment 1 12.3: Amplified segment 1 12.4: Amplified segment 1 1 2.5: Amplified segment 1 1 2.6: Amplified segment 1 12.7: Amplification Section 112.8: Amplification section 1 1 2.9 : Amplification section 1 1 2.1 0 : Amplification section 1 1 2.1 1 : Amplification section 1 1 2 . 1 2 : Amplification section 112.X: expansion Increased section 1 1 3 : host system of genetic data 1 1 4 : incubation section 1 1 4.1 : incubation section 1 1 4.2 : cultivation section -137- 201211539 1 1 4.3 : cultivation section 1 1 4.4 : cultivation area Paragraph 1 15: Host system for electronic health record (EHR) 1 1 6 : Anticoagulant 117: Anticoagulant surface tension valve 1 1 8 : Surface tension valve 1 1 9 : Sample fluid 1 2 0 : Meniscus 121: Electronic medical record (EMR) host system 1 2 2 : vent 123: personal medical record (PHR) host system 125: network 126: boiling start valve 1 2 8 : surface tension valve 1 3 0 : ( Chemical) lysis section 1 3 1 : mixing section 132 : surface tension valve 1 3 3 : incubation chamber inlet channel 1 3 4 : lower suction hole 1 36 : optical window 1 3 8 : amplification mixture surface tension valve 1 3 8 . 1 : Amplification mixture surface tension valve 138.2 : expansion Mixture surface tension valve 1 3 8 . 3 : Amplification mixture surface tension valve 201211539 13δ.4: Amplification mixture surface tension valve 1 38 .X : Amplification mixture surface tension valve 140: Polymerase surface tension valve 14〇.1 : Polymerase Surface Tension Valve 140.2: Polymerase Surface Tension Valve

140.3: polymerase surface tension valve Μ 0.4: polymerase surface tension valve 14 〇 · Χ : polymerase surface tension valve 146 : valve inlet 1 4 8 : valve outlet 150 : lower suction hole 1 5 1 : valve upper suction hole 1 5 2 : Heater 1 53 : Heater contact point of boiling start valve 1 5 4 : Heater 1 5 6 : Heater contact point 158 : Microchannel 160 : Amplification section leaves channel 164 : Hole 165 : Hole 166 : capillary action priming feature (CIF) 168: dialysis upper suction hole 170: temperature sensor 174: liquid sensor - 139 - 201211539 175 : diffusion barrier 176 : flow path

1 7 8 : End point liquid sensor 1 80 : Hybrid chamber 1 8 2 : Heater 1 84 : Light diode 18 5: Active area 186: FRET probe 1 8 8 : Water storage tank 189 : Evaporation indicator 190: evaporator 1 9 1 : ring heater 192: water supply passage 193: upper suction hole 1 9 4 : lower suction hole

195: exposed area of the top metal layer 1 9 6 : humidifier 197: cap layer channel liquid sensor 198: first upper suction hole 202: capillary action priming feature (CIF) 204: dialysis MST channel 2 06 : mixing zone Segment outlet valve/boiling start valve 207: incubation chamber outlet valve 2 0 8 : mixing section outlet valve -140- 201211539 2 1 0 : heated microchannel 212: intermediate MST channel 2 1 8 : titanium electrode 220: Titanium-aluminum electrode 222: gap 232: humidity sensor

2 3 4 : Heater 23 7 : ECL probe 23 8 : Target nucleic acid sequence / target DNA 240 : Ring 242 : backbone 248 : extinction group 28 8 : sample insertion and preparation stage 290 : extraction stage ( Extraction section) 291: incubation stage/cultivation step 292: amplification stage/amplification step/amplification section 293: pre-hybridization stage 294: detection stage/detection section 296: first electrode 29 8 : Two electrodes 3 00 : Preprogrammed delay time 301 : On-wafer laboratory (LOC) device 3 2 8 : Dialysis section 3 7 6 : Conductive column -141 - 201211539 3 78 : Control probe 3 8 0 : Control probe 3 82 : Calibration chamber 3 90 : Retractable lancet 3 9 2 : lancet release button 408 : sealing film 410 : film guard

492: LOC Device Variant VII

518: LOC device variant VIII 594: interface layer 5 96 : interface channel 5 98 : interface channel 600 : bypass channel 602 : interface target channel 604 : interface waste (cell) channel

6 06 : Interface channel 608 on the side of the lysis reagent storage tank: interface channel 610 on the lysis sample side: interface conduit 6 1 2 : mixing section outlet lower suction hole 614: valve interface channel 616: valve interface groove 630: MST Nutrient outlet channel 63 2 : Amplification injection channel 63 8 : Hot lysis chamber -142- 201211539

641 : LOC device variant XIV

659 : LOC device variant XXIX

660 : LOC device variant XXX

661 : LOC device variant XXXI 673: LOC device variant XLIII 674: LOC device variant XLIV 677: LOC device variant XLVII

6 82 : dialysis section / dialysis section / pre-hybridization dialysis step 684: further sample analysis 6 8 ό :, pre-amplification dialysis step 693: linear ECL probe 694 with primer: amplification blocker 696: Probe sequence with primer 698: amplified complementary sequence 700: oligonucleotide primer 705: stem-looped ECL probe/probe region 706: complementary sequence 708: backbone strand 710: backbone Chain 7 1 2 : first optical 稜鏡 729 : LOC device variant. 74 〇: 感 rate sensor 746 : L0C device variant XI 766 : waste storage tank - 143 - 201211539 7 6 8 : waste storage tank 7 8 3: microfluidic device 7 84 : dialysis device 7 8 5 : LOC device 7 86 : negative control ECL probe 7 8 7 : positive control ECL probe 7 8 8 : differential imaging circuit 7 9 0 : pixel

7 9 2 : Virtual pixel 794 : Read column line 795 : Virtual pixel read column line 797 : M4 transistor 8 0 1 : M D 4 transistor 8 03 : Pixel capacitor 8 05 : Virtual pixel capacitor

8 07 : switch 8 09 : switch 8 1 1 : read line switch 8 1 3 : virtual pixel read line switch 815 : capacitor amplifier 8 1 7 : differential signal 8 6 0 : electrode / anode 8 62 : ECL signal/release light 8 64 : luminescence luminescent group -144- 201211539 8 6 6 : co-reactant 86 8 : ECL excited species 8 70 : electrode 8 7 2 : solution 874 : ECL reaction chamber 8 7 6 : dielectric Air gap 8 78: comb-shaped anode

8 8 0 :: Finger 8 8 2 ; 蜿蜒 structure 8 8 4 : Curved channel 886: More complex structure 8 8 8 : Serrated structure 8 90 : Branched structure 892 : Participation volume 894 : Containment region

Claims (1)

201211539 VII. Patent Application Range: 1 · A wafer-on-lab (LOC) device for pathogen detection and genetic analysis of biological samples, the LOC device comprising: an inlet for receiving the sample; a carrier substrate; a plurality of reagents a lysis zone for dissolving pathogens and white blood cells in the sample to release genetic material in the pathogens and white blood cells, and the lysing section is used with the lysate segments Dissolving one of a reagent storage tank of the pathogen in the lysing section and the lysing reagent of the white blood cell; the culturing section, the culturing section is located downstream of the lysing section, and the cultivating section is associated with the And one of the reagent storage tanks containing a plurality of enzymes for performing an enzymatic reaction with the genetic material; the first nucleic acid amplification section, the first nucleic acid amplification section being located downstream of the incubation section for use Amplifying the first nucleic acid sequence in the genetic material; and a second nucleic acid amplification segment, the second nucleic acid amplification segment being located downstream of the first nucleic acid amplification segment for amplification derived from the first One core a second nucleic acid sequence within the amplicon of the acid amplification segment; wherein the lysis segment, the incubation segment, the first nucleic acid amplification segment, and the second nucleic acid amplification segment are all carried On the carrier substrate. 2. The LOC device of claim 1, wherein the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second polymerase Chain reaction (PCR) segment. 3. The LOC device of claim 2, wherein the first-146-201211539 PCR segment has a first set of primer pairs, and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and The second pCR segment has a second set of primer pairs, and the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. 4. The LOC device of claim 3, wherein the first PCR segment and the second PCR segment are constructed to operate on different amplification parameters, the amplification parameters being at least One: reverse transcriptase species; polymerase 1 enzyme species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of thermal cycle repeats; and temperature during a particular phase of PCR. 5. The LOC device of claim 4, further comprising a hybrid segment located downstream of the second PCR segment, the hybrid segment having a probe array and light A sensor, the probe array is for hybridization with a target nucleic acid sequence, and the photosensor is for detecting a heterozygous reaction of any of the probes in the array. 6. The LOC device of claim 3, wherein the photosensor is less than 1600 microns from the array of probes. 7. The LOC device of claim 1, wherein the incubation section has a heater for heating the genetic material and the enzyme -147-201211539 to a predetermined enzyme catalyzed reaction temperature. 8. The LOC device of claim 1, wherein one of the reagent reservoirs contains a adapter primer for engagement with a nucleic acid sequence in the incubation segment. 9. The LOC device of claim 1, wherein the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment 10. The LOC device of claim 1, wherein the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a bay surface anchor, A meniscus anchor is used to hold the meniscus of the reagent until the meniscus contacts the sample stream to remove the skim surface to allow the reagent to flow out of the reagent reservoir. 11. The LOC device of claim 4, further comprising a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer, the MST layer combining the first PCR segment and the second PCR region a segment, wherein the CMOS circuit is disposed between the carrier substrate and the MST layer, and the CMOS circuit is configured to use an output of the temperature sensor to achieve the first PCR segment and the second PCR segment Feedback control. 12. The LOC device of claim 11, wherein the first PCR segment has a PCR microchannel for thermal cycling of the sample, the PCR microchannel being defined to have less than 100000 Flow path of a square micron flow transverse cross-sectional area. 13. The LOC device of claim 12, wherein the -148-201211539 PCR microchannel has at least one elongated heater element 'the elongated heater element system It extends in parallel with the PCR microchannel. 14. The LOC device of claim 13, wherein the PCR segment has a plurality of elongated PCR chambers, and each of the elongated PCR chambers is formed by an individual segment of the PCR microchannel, the PCR The microchannel has a crucible configuration formed by a series of wide curved channels, and each of the wide curved channels is used to form a channel section of one of the elongate PCR chambers. 15. The LOC device of claim 14, wherein the LOC device further comprises: a reagent storage tank for containing a PCR reagent; and a surface tension valve having a hole, the surface tension valve having a hole configured to hold the The meniscus of the reagent is such that the meniscus retains the reagent in the reagent reservoir until the meniscus contacts the sample stream to remove the meniscus and the reagent exits the reagent reservoir. 16. The LOC device of claim 14, wherein the LOC device further comprises an array of hybrid chambers for receiving the probes such that the probes within each hybrid chamber are constructed to One of the target nucleic acid sequences is heterozygous. 17. The LOC device of claim 16, wherein the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. 18. The LOC device of claim 16, wherein the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the photosensor output and the data interface is used. The hybrid data is transmitted to an external device. 19. The LOC device of claim 16, wherein the first PCR segment has an active valve that retains liquid within the first PCR segment during thermal cycling and responds to the source The liquid is allowed to flow to the first hybrid chamber array from the activation signal of the CMOS circuit. 20. The LOC device of claim 19, wherein the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the block The capillary of the liquid drives the meniscus of the flow, the heater being used to boil the liquid to disengage the meniscus from the meniscus anchor such that the capillary drive flow continues. -150-