TW201209404A - LOC device for genetic analysis which performs nucleic acid amplification before removing non-nucleic acid constituents in a dialysis section - Google Patents

Abstract

A lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device having an inlet for receiving the sample, a supporting substrate, a plurality of reagent reservoirs, a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the sample, and, a dialysis section downstream of the nucleic acid amplification section for prehybridization filtration of amplicon produced by the nucleic acid amplification section, the dialysis section being configured to remove cell debris from the amplicon, wherein, the nucleic acid amplification section and the dialysis section are both supported on the supporting substrate.

Description

201209404 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus employing microsystem technology (MST). In more detail, the present invention relates to the processing and analysis of microfluidics and biosynthesis for molecular diagnostics. [Prior Art] Molecular diagnostics has emerged as an area that guarantees effective early detection of disease before the onset of disease symptoms. Molecular diagnostic tests can be used to detect: • Hereditary diseases • Acquired diseases • Infectious diseases • Genetic quality of susceptible diseases that are highly susceptible to health and high turnover due to the high degree of accuracy and rapid turnover of molecular diagnostic tests Invalid health care services, improve medical outcomes, improve disease management and provide individualized patient care. Molecular diagnostics Many techniques are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobase allows the synthetic DN A short sequence (oligonucleotide) to bind (hybrid) to a particular nucleic acid sequence for nucleic acid testing. If a hybrid occurs, the complementary sequence will appear in the sample. This will, for example, make it possible to predict a person's future illness, determine the identity and pathogenicity of an infectious pathogen, or -5 - 201209404 to determine that someone will respond to a drug. Nucleic Acid-Based Molecular Diagnostic Testing Nucleic acid-based testing has four distinct steps: 1. Sample preparation 2. Nucleic acid extraction 3 (optional) nucleic acid amplification 4- Detection of many sample types available for genes Analysis, such as blood, urine, sputum and tissue samples. Since not all samples represent disease progression, this diagnostic test determines the type of sample required. These samples have a variety of different compositions, but usually only one composition is of interest. For example, high red blood cell concentrations in blood can inhibit the detection of pathogenic organisms. Therefore, purification and/or concentration steps are typically required at the beginning of the nucleic acid test. Blood is a type of sample that is more commonly used. It has three main components: white blood cells (white blood cells), red blood cells (red blood cells), and blood clotting cells (platelets). Blood coagulation cells promote coagulation and remain active outside the cell. In order to inhibit blood agglutination, the sample is mixed with a chemical such as ethylenediaminetetraacetic acid (EDTA) before purification and concentration. Red blood cells are usually removed from the sample to concentrate the target cells. In humans, red blood cells account for about 99% of blood cell material, but because they do not have a nucleus, they do not carry DNA. Furthermore, red blood cells containing certain components such as heme can interfere with downstream nucleic acid amplification processes (described below). Removal of red blood cells can be achieved by distinguishing cytolytic red blood cells in a cytolytic solution to preserve the integrity of other cellular material, and then -6 - 201209404 using centrifugation to separate intact cellular material from the sample. Thus, a concentrate of the target cells can be provided and the nucleic acid can be extracted from the concentrates. The precise procedure for extracting the nucleic acids will depend on the sample and the diagnostic assay to be performed. For example, the protocol used to extract viral RNA is significantly different from the protocol used to extract the genomic DNA DN A. However, the extraction of nucleic acids from a target cell typically involves a cell lysis step followed by nucleic acid purification. The cytolysis step of the cell disrupts the cells and cell membranes and releases the genetic material. This is usually done with a cytosolic detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cell. The nucleic acid can then be purified by an alcohol (usually ice-cooled ethanol or isopropanol) precipitation step or by a solid phase purification step, typically based on a ruthenium oxide substrate on a column, resin or paramagnetic beads. The granules are carried out in the presence of a high concentration of chaotropic salts, followed by washing, followed by dissolution with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protease that digests the protein to further purify the sample. Other methods of cytolysis also include mechanical cytolysis by ultrasonic oscillations and heating of the sample to 94 ° C to disintegrate the thermolysis of the cell membrane. The extracted material may also contain a very small amount of target DN A or RN A, especially when the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target at a low concentration to a detectable level. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). PCR has been known in the art and a detailed description of this type of reaction can be found in 201209404 E. van Pelt-Verkuil et a 1., Principles and Technical Aspects of PCR A m p 1 i f i c a t i ο n, S p r i n g e r, 2 0 0 8. PCR is a powerful technique for amplifying a target DNA sequence in a complex DNA background. If you want to amplify RNA (by PCR), you must first use an enzyme called reverse transcriptase to transcribe RNA into cDNA (complementary DNA). Then, the prepared cDNA was further amplified by PCR. PCR is an exponential process that continues as long as the conditions supporting the reaction permit. The ingredients of this reaction are:

1 · Primer pair: a single strand of short DNA complementary to the flanking region of the target sequence and about 10-30 nucleotides long. 2. DNA polymerase: a thermostable enzyme capable of synthesizing DNA 3. Deoxyribonucleoside III Phosphoric acid (dNTPs): provides nucleotides that are incorporated into newly synthesized DNA strands. 4. Buffer: The best chemical environment for DNA synthesis. PCR typically involves the addition of such reactants to small tubes containing extracted nucleic acids (~ 10-50 microliters). The tube is placed in a thermal cycler (a device that allows the reaction to react at different temperatures for varying lengths of time). The standard operating procedures for each thermal cycle involve the denaturation phase, the binding phase, and the extension phase. The extension period is sometimes referred to as the primer extension period. In addition to this three-step process, a two-step thermal process can be used, in which the combination period and extension period are combined. The denaturation period typically involves raising the reaction temperature to 90-95 °C to denature the DNA strand; during the binding phase, the temperature is lowered to about 50-60 °C to allow the primer to bind; then during the extension, the temperature is l 201209404 up to 60-72 °C for DNA polymerase activity for primer extension. This process is repeated approximately 20-40 times, with the end result of producing a copy of the target sequence between millions of two primers. There are also a number of variations relative to the PCR standard protocol, including, for example, multiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase pCR, which have been developed for use in Molecular diagnosis. Multiplex PCR uses multiple primer sets in a single PCR mix to create amplicons of different lengths that are specific for different DN A sequences. By targeting multiple genes at the same time, more information can be obtained in a single test-operation, otherwise several experiments will be required. However, the optimization of multiplex PCR is more difficult and it is necessary to select primers with similar binding temperatures and amplicon of similar length and base composition to ensure that the amplification efficiency of each amplicon is equal. Also known as junction adapter PCR is a method for nucleic acid amplification of substantially all of the DN(R) sequences in a complex DNA mixture without the need for a target-specific primer. This method involves first digesting the target DNA population with an appropriate restriction enzyme (enzyme). A double-stranded oligonucleotide linker (also known as an adaptor) having a suitable overhanging end is then ligated to the end of the target DN A fragment with a ligase enzyme. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. In this way, all DNA source fragments that are sandwiched by the linker oligonucleotide can be amplified. Direct PCR describes a system for performing PCR directly on a sample without any nucleic acid extraction or only some micronucleus -9-201209404 acid extraction. It has long been agreed that various components (e.g., heme components in blood) in unpurified biological samples inhibit the PCR reaction. Traditionally, PCR requires thorough purification of the target nucleic acid prior to preparation of the reaction mixture. However, by appropriate changes in chemical properties and sample concentrations, PCR or direct PCR can be performed with minimal DNA purification. Direct PCR modulating PCR chemistry includes increasing buffer strength, using polymerases with high activity and continuous potency, and adding additives that sequester possible polymerase inhibitors. Tandem PCR uses two independent fields of nucleic acid amplification reactions to increase the likelihood that the pair of amplicons will be amplified. One form of tandem PCR is nested PCR, which uses two pairs of PCR primers to amplify a single locus in a separate nucleic acid amplification field. The first pair of primers will hybridize to the nucleic acid sequence located in the region outside the target nucleic acid sequence. A second pair of primers (nested bed primers) for the second round of amplification will be incorporated into the first PCR product and produce a second PCR product containing the target nucleic acid which will be shorter than the first PCR product. The logic behind this strategy is that if an incorrect locus is incorrectly amplified in the first round of acid amplification, the probability that the locus will be amplified again by the second pair of primers will be extremely low. To ensure its specificity, real-time PCR or quantitative PCR is used to measure the instantaneous amount of PCR products. The initial amount of nucleic acid in the sample can be quantified by using a probe containing a fluorophore or a fluorescent dye and a set of reaction standards. This is particularly useful for molecular diagnostics because the therapeutic regimen in molecular diagnostics will vary depending on the pathogen load within the sample. -10- 201209404 Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. The reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used for gene expression characterization to determine gene expression or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It can also be used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. Isothermal amplification is another form of nucleic acid amplification whose amplification does not depend on the thermal denaturation of the target DN A, so no complicated mechanical means are required. Therefore, the isothermal nucleic acid amplification method can be carried out in situ or easily in an environment outside the laboratory. A variety of isothermal nucleic acid amplification methods have been described, including strand displacement amplification, transcription vector amplification, nucleic acid sequence amplification, recombinase polymerase amplification, rolling circle amplification, mesh Branch amplification, helicase-dependent isothermal DN A amplification and circular medium isothermal amplification. The isothermal nucleic acid amplification method does not rely on the continuous heat denaturation of the template DN A to produce a single strand of the molecule as a template for further amplification, but at a constant temperature by other methods such as the use of a specific restriction endonuclease to the enzyme. A gap is cut in the DN A molecule, or an enzyme is used to separate the DNA strand. The strand displacement amplification method (SD A ) relies on the ability of a specific restriction enzyme to cleave the unmodified gap in the semi-modified DNA and the 5'_3' exonuclease-deficient polymerase. The ability to replace the downstream strands. Exponential nucleic acid amplification is achieved by combining meaningful and antisense reactions, in which a meaningful reaction strand substitution (the resulting sequence) is used as a template for an antisense reaction. This reaction uses a nicking enzyme (which does not cut the DNA in a conventional manner, but instead cuts out a gap in the double-stranded DNA), such as N. -11 - 201209404

Alwl, N. BstNBl and Mlyl. SDA has been improved by the use of a combination of a thermostable restriction endonuclease (aval) and a thermostable exonuclease deficiency-polymerase polymerase. This combination shows an increase in amplification efficiency of amplification of 1 〇 8 fold response to amplification of 1 01 () times, so this technique can be used to amplify unique single copy molecules. Transcription vector amplification (TMA) and nucleic acid sequence-based amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes: RN A polymerase, reverse transcriptase and optionally RNase Η (if the reverse transcriptase does not have RNase activity). A primer contains a promoter sequence of RNA polymerase. In the first step of nucleic acid amplification, the primer is hybridized to one of the target ribosomal RNA (rRNA). The reverse transcriptase can produce a DNA copy of the target rRN A by extending from the 3' end of the promoter. In the resulting RNA: DNA, the RNA is decomposed by the RNase activity possessed by the reverse transcriptase or the added RNase. Next, the second primer is allowed to bind to the DNA copy. A new DN Α strand is synthesized from the end of the primer by reverse transcriptase to form a double stranded DNA molecule. RNA polymerase recognizes the promoter sequence within the DN A template and initiates transcription. Each newly synthesized RNA amplicon will re-enter this process and serve as a template for a new round of replication. In the recombinase polymerase amplification method (RPA), isothermal amplification of a specific DNA fragment is achieved by allowing the opposite oligonucleotide primer to bind to the template DNAJ and by the extension of the DNA polymerase. There is no need to use heat to denature the double strand DNA (dsDNA) template. Alternatively, RPA uses a recombinase-primer complex to scan dsDNA and facilitate share exchange of homologous sites. The structure of -12-201209404 is stabilized by the interaction between the single DN A binding protein and the replaced template strand. This prevents the primer from being ejected due to branch migration. The dissociation of the degrading enzyme leaves the 3' end of the oligonucleotide of the parental replacement DN A polymerase (e.g., a large fragment of Bacillus subtilis eaciZ/Ms Pol I (Bsu). Exponential nucleic acid amplification can be accomplished by cyclically repeating this process. The helicase-dependent amplification method (HDA) mimics the living system, using DN A helicase to generate a single-strand template for hybridization of the primer and then extending the primer with DN A polymerase. In the first step of the HAD reaction, the helicase moves along the target DN A, disintegrating the two hydrogen bonds, and the two separated molecules will bind to the single-stranded binding protein. Single-stranded target areas allow primer binding. The DN A polymerase then extends from the 3' end of each primer using free deoxyribonucleoside triphosphates (dNTPs) to form two DNA replicators. These two replicated dsDNA strands will each enter the next HAD cycle, causing exponential nucleic acid amplification of the target sequence. Other DN A-based isothermal techniques include rolling circle amplification (RCA), in which the DNA polymerase allows the primer to be extended along the circular DNA template to produce repeats containing a number of circular templates. Copy the strip of DN A product. At the end of the reaction, the polymerase generates a copy of thousands of circular templates that are linked to the original target DN A. This allows for spatial resolution of the target and rapid nucleic acid amplification of the signal. Up to 101 2 template copies can be produced in one hour. The reticular branch is amplified as a change in RCA using a locked circular probe (C-probe) or a padlock probe and a high continuous potency DN A polymerase under isothermal conditions exponential-13- The C-probe was amplified by 201209404. The circular medium isothermal amplification method (LAMP) is highly selective and employs DNA polymerase and a set of four primers that are specifically designed to recognize a total of six unique sequences on the target DNA. An inducible strand containing the target DNA and an intron within the antisense strand sequence can trigger LAMP. Subsequent replacement of DN A by an external leader leads to the release of single-stranded DNA. These single-stranded DN A can be used as a second intro and an external primer (which is heterozygous to the other end of the target sequence). A template for DNA synthesis that forms a stem-loop DNA structure. In the next LAMP cycle, an internal primer will hybridize to the loop of the product and initiate a replacement DN A synthesis reaction, producing the original stem-loop DNA and the new stem-loop DNA (the stem length is twice the original ). This cycle of reaction continues and less than an hour can accumulate 1 〇 9 copies of the target. The final product is a stem-loop DNA having a plurality of inverted copies of the target and a polycyclic broccoli structure formed by the incorporation of a replica of the target in the same strand. After completion of the nucleic acid amplification, the amplification product must be analyzed to determine if the desired amplicon (amplification amount of the target nucleic acid) has been produced. The method of analyzing the product is to determine the nucleotide composition of the amplicon by simply measuring the size of the amplicon by gel electrophoresis to heterozygous using DN A. Gel electrophoresis is the easiest way to check if a nucleic acid amplification reaction produces the desired amplicon. Gel electrophoresis applies an electric field to the gel matrix to separate the DN A fragments. The negatively charged DN A fragment moves at different speeds (mainly by its size) on the substrate. When the electrophoresis is over, the fragments in the gel can be stained to make them visible to the naked eye. Ethidium bromide is a commonly used dye of -14-201209404, which emits fluorescence under UV light. The size of the fragments is aligned with a DNA size standard reference (DNA ladder containing DN A fragments of known size) to determine that the reference will be placed next to the amplicon and run at the same time. Since the oligonucleotide primer binds to a specific site that encloses the target DNA, the size of the amplification product can be estimated and detected by a known size spectrum on the gel. In order to reliably identify the amplicon, or if multiple amplicons are simultaneously produced, the amplicon is usually hybridized with a DNA probe. DN A heterozygous refers to the formation of a double-stranded DN A by a pairing reaction of complementary bases. The DN A hybridization reaction used to positively identify a specific amplification product requires the use of a DNA probe of about 20 nucleotides in length. If the sequence of the probe is complementary to the amplicon (target) DNA sequence, the hybridization reaction will occur under favorable conditions of temperature, pH and ionic strength. If heterozygous occurs, the gene or DN A sequence indicating interest has been found in the original sample. Optical detection is the most common method for detecting heterozygosity. Amplicon or probe can be labeled by fluorescence or electrochemiluminescence to emit light. These methods differ in how the luminescent group is excited to form, but both can covalently label the nucleotide strand. In electrochemiluminescence (ECL), the light line is produced after the luminophore molecules or complexes are stimulated by current. In the case of fluorescence, it is irradiated with excitation light that causes a radiation reaction. The fluorescent system uses an illumination source (which provides excitation light of a wavelength that can be absorbed by the fluorescent molecules) and a detection unit to detect. The detecting unit includes a photo sensor capable of detecting a radiation signal (for example, a photomultiplier tube or a charge coupled -15-201209404 device (CCD) array), and a mechanism for preventing excitation light from being included in the output of the photo sensor. (eg wavelength-select filter). The fluorescent molecules emit Stokes-shifted light to react to the excitation light, which is collected by the detection unit. Stokes shifts the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. The ECL illumination is detected using a light sensor that is sensitive to the radiation wavelength of the ECL species being used. For example, a transition metal-ligand complex will emit light at visible wavelengths, so conventional photodiodes and CCDs can be used as photosensors. An advantage of ECL is that if the ambient light is excluded, the ECL illumination is the only light present in the detection system that enhances detection sensitivity. Microarrays allow hundreds or thousands of DN A heterozygous experiments to be performed simultaneously. Microarrays are powerful tools in molecular diagnostics that can screen thousands of diseases or detect the presence of multiple infectious pathogens in a single test. The microarray consists of a number of different DN A probes that are fixed to the substrate and are spotted. The target DNA (amplicon) is first labeled with a fluorescent or luminescent molecule (either during or after nucleic acid amplification) and then applied to the probe array. The microarray is incubated in a controlled temperature, humid environment for hours or days, during which time the probe and the amplicon will hybridize. After incubation, the microarray must be rinsed with a series of buffers to remove unbound strands. Once cleaned, air jets (usually nitrogen) are used to dry the surface of the microarray. The stringency of hybridization and cleaning is severe. Insufficient stringency can result in a high degree of non-specific binding. Excessive rigor can result in inability to properly combine, resulting in reduced sensitivity. The heterozygous line is detected by the emission of light from the labeled amplicons, which have been hybridized with complementary probes from -16 to 201209404. Fluorescence from the microarray is detected using a microarray scanner, typically a computer-controlled inverted scanning fluorescent conjugated focus microscope that typically uses a laser to excite the fluorescent dye. And using a light sensor (such as a photomultiplier tube or cc D ) to detect the transmitted signal. The fluorescent molecules emit Stokes-shifted light (as described above) which are collected by the detection unit. The emitted fluorescence must be collected, separated from the wavelength of the unabsorbed excitation light, and transmitted to the detector. Conjugate focal designs are often used in microarray scanners to remove defocus information by conjugated pinholes located on the image plane. This will only allow the light in the focus portion to be detected. Avoid light from above and below the object's focal plane into the detector, thereby increasing the ratio of signal to noise. The detected fluorescent photons are converted into electrical energy by the detector and then converted into a digital signal. This digital signal is translated into a number representing the intensity of the fluorescence of a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. The exact sequence and position of each probe on the microarray is known, so that the hybrid target sequences can be identified and analyzed simultaneously. More information on fluorescent probes can be found at: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/Molecular- Probes-The-Handbook/Technical-Notes-and-Product-Highlights/ Fluorescence-Resonance-Energy-Transfer-FRET. html -17- 201209404 Molecular Diagnostics for Point-of-care Although molecular diagnostic tests can provide these benefits, clinical laboratories The growth of this type of test is slower than expected and these tests are only a small part of the laboratory medical practice. This is primarily due to the fact that testing with nucleic acids is more complicated and more expensive than other testing methods that do not involve nucleic acids. The widespread use of molecular diagnostic tests in clinical settings is closely related to the development of equipped instruments. These instruments must be able to significantly reduce costs and provide fast and automated verification from scratch (sample processing) to tail (resulting results). And it does not require a lot of human intervention. The point-of-care technology used to serve doctors' clinics, hospital clinics, or even home-based homes has many benefits, including: • Rapid results can be achieved, resulting in immediate treatment and improved care quality. • Ability to obtain laboratory-quality test results by testing very few samples. • Reduce clinical effort. • Reduce laboratory load and improve office performance by reducing administrative work. • Improve the cost of treatment for each patient by reducing the time spent on hospitalization, the time required to reach a conclusion for first-time medical outpatient consultation, and reducing the time spent on sample handling, storage, and transportation. • Accelerate clinical governance conclusions such as infection control and antibiotic use -18- 201209404 On-wafer laboratory (LOC)-based molecular diagnostics Micro-fluid-based molecular diagnostic systems provide automated and accelerated molecular diagnostic assays Device. The faster detection time is mainly based on the active small size of the body involved, the automated operation and the diagnostic process steps of the microfluidic device as a low overhead built-in cascade. The volume of nanoliters and microliters also reduces reagent wear and cost. The on-wafer laboratory (LOC) device is in the form of a common microfluidic device. The LOC unit has an MST structure (for fluid processing) integrated into the MST layer on a single struts (usually ruthenium). The use of the VLSI (very large scale integrated) uranium engraving technology of the semiconductor industry can make the unit cost of the LOC device very low. However, large amounts of external piping and electronics are required to control the flow of fluid through the L Ο C device, to add reagents, to regulate reaction conditions, and the like. In fact, the connection of LOC devices to such external devices limits the use of LOC devices for molecular diagnostics in laboratory environments. The cost of such external devices and the complexity of their operation make LOC-based molecular diagnostics a practical solution for point-of-care devices.

Based on the above observations, people need a molecular diagnostic system based on LOC devices that can be used for fixed-point care. SUMMARY OF THE INVENTION Various aspects of the invention are described in the following numbered paragraphs. GCF028.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis of a biological sample, the LOC device -19-201209404 comprising: a receiving inlet for a sample; a support base a dialysis device having a smaller component separated from a larger component, the smaller component being less than a predetermined threshold size and the larger component being greater than the predetermined threshold size: and a nucleic acid sequence for amplifying the genetic material a nucleic acid amplification region; wherein the dialysis region and the nucleic acid amplification region are both supported on the support substrate 〇GCF028.2. Preferably, the LOC device further has a photo sensor and is located in the nucleic acid amplification region. a downstream heterozygous region having a hybrid probe array for hybridization with a target nucleic acid sequence within an amplicon produced by the nucleic acid amplification region, the probe being designed to hybridize to the target nucleic acid sequence Together, a probe-target hybrid is formed, wherein the photosensor is configured to detect the probe-target hybrid. Preferably, the larger component comprises a pathogen and cells larger than a predetermined size, and the cells and cells larger than a predetermined threshold size include target cells containing the genetic material for analysis. GCF02 8.4 Preferably, the dialysis zone is located upstream of the nucleic acid amplification zone and upstream of the hybrid zone to filter the amplicons prior to hybridization, the dialysis zone being configured to remove cell debris from the amplicon. GCF02 8.5 preferably 'the LOC device further has a first dialysis zone upstream of the nucleic acid amplification region to separate the pathogen in the sample and the cells larger than the predetermined threshold 値-20-201209404 from the smaller component, according to The cells larger than the predetermined size include target cells containing the genetic material for analysis. GCF02 8.6 Preferably, the first dialysis zone has a first passage in fluid communication with the inlet of the upstream end, a second passage in fluid communication with the waste passage at the downstream end, and a plurality of such pathogens And a smaller pore and smaller pores than the smaller ones, the second channel being in fluid communication with the first channel via the pores such that the pathogens and target cells are left in the first channel and the The smaller components then flow into the second channel. GCF028.7 Preferably, the first channel and the second channel are configured to be filled with the sample by capillary action. GCF028.8 Preferably, the second dialysis zone has a large component channel, a small component channel, and a plurality of second apertures capable of fluidly connecting the large component channel to the small component channel, the second small The pores are sized to allow the nucleic acid sequence to flow from the large component channel into the small component channel, while cell debris larger than the second pore is retained in the large component channel, and the small component channel is associated with the hybrid region Fluid communication. GCF028.9 Preferably, the nucleic acid amplification region is an isothermal nucleic acid amplification region. GCF028.1 0 Preferably, the LOC device further has a reagent reservoir for containing reagents for isothermal nucleic acid amplification; and a surface tension valve having a small aperture configured to hold the meniscus of the reagent The meniscus allows the reagent to remain in the reagent reservoir until it contacts the fluid sample to remove the meniscus, and the reagent then flows out of the reagent reservoir. -21 - 201209404 GCF02 8.1 1 Preferably, the nucleic acid amplification region is a polymerase chain reaction (P C R ) amplification region. GCF02 8.1 2 Preferably, the LOC device further has a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer having the PCR region, wherein the CMOS circuit is located on the support substrate and the MST Between the layers, the CMOS circuit is configured to use the temperature sensor output to feedback control the PCR region. GCF02 8.13 Preferably, the PCR region has a PCR microchannel that is thermally cycled during use to amplify the nucleic acid sequences, the PCR microchannel defining a portion of the flow path of the sample And its cross-sectional area perpendicular to the flow is less than 100, 〇〇〇 square micron. GCF02 8.1 4 Preferably, the LOC device further has at least one elongated heating element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heating element extending parallel to the PCR microchannel. GCF028.1 5 Preferably, at least one of the PCR microchannels forms an elongated PCR chamber. GCF028.1 6 Preferably, the PCR region has a plurality of elongated PCR chambers respectively formed by respective blocks of the PCR microchannels, the PCR microchannels having a 蜿蜒 configuration formed by a series of wide meandering flows, each The wide meander stream is a channel block that forms the elongate PCR chamber. GCF02 8.1 7 Preferably, the LOC device further has a reagent reservoir for containing the reagent for PCR; and a surface tension valve having a small hole configured to fix the meniscus of the reagent, thus in the sample with the fluid The bend -22-201209404 level will leave the reagent in the reagent reservoir before contacting to remove the meniscus. GCF028.1 8 Preferably, the LOC device further has a cultivating zone downstream of the first dialysis zone and upstream of the PCR zone, the culturing zone and a reagent reservoir containing an enzyme capable of performing an enzymatic reaction with the genetic material Connected in fluid. GCF02 8.1 9 Preferably, the photo sensor is a photodiode array, and the positions of the photodiodes are registered with the hybrid chamber. GCF028.20 Preferably, the PCR zone has an active valve to allow liquid to remain in the PCR zone during thermal cycling and to react to the activation signal from the CMOS circuitry to allow liquid to flow to the hybrid chamber. This LOC device has the advantage of separating the desired sample components from the sample components based on the size of the components, and the method used to reduce clogging is superior to the simple filtration method. This LOC device has the advantages provided by sequence-specific amplification methods, including sensitivity provided by amplification, wide dynamic range, and high specificity for the target DNA sequence. GCF03 1.1 This aspect of the invention provides a on-wafer laboratory (LOC) device for pathogen detection and genetic analysis of biological samples, the LOC device comprising: - receiving a sample containing a sample of genetic material; a support substrate; a plurality of reagent reservoirs; a culture zone downstream of the inlet, the culture zone being in fluid communication with a reagent reservoir containing an enzyme capable of reacting with the genetic material; and -23-201209404 a nucleic acid amplification region downstream of the incubation region to amplify the nucleic acid sequence in the genetic material; and a dialysis region downstream of the nucleic acid amplification region to filter the amplicon produced by the nucleic acid amplification region before hybridization The dialysis zone is configured to remove cell debris from the amplicon; wherein the incubation zone, the nucleic acid amplification zone, and the dialysis zone are supported on the support substrate. GCF03 1.2 Preferably, the incubation zone has a heater to heat the genetic material and enzyme to a predetermined enzyme reaction temperature. Preferably, the LOC device further has a photo sensor and a hybrid region located downstream of the nucleic acid amplification region, the hybrid region having an array of hybrid chambers, each hybrid chamber containing a different probe The probe is designed to hybridize to the target nucleic acid sequence to form a probe-target hybrid, wherein the photosensor & is configured to detect The probe-target hybrid was measured. GCF03 1.4 Preferably, each of the hybrid chambers has a volume of less than 900,000 cubic microns. GCF031.5 Preferably, each of the hybrid chambers has a volume of less than 200,000 cubic microns. GCF031.6 Preferably, the dialysis zone has a large component channel, a small component channel, and a plurality of small holes capable of fluidly connecting the large component channel to the small component channel, the small holes being sized to allow nucleic acid A sequence flows from the large component channel to the small component channel, and cell debris larger than the small holes is retained in the large component channel, the small component channel being in fluid communication with the hybrid region at -24-201209404. GCF031.7 Preferably, the nucleic acid amplification region is an isothermal nucleic acid amplification region. GCF031.8 Preferably, the LOC device further has a reagent reservoir for containing reagents for isothermal nucleic acid amplification: and a surface tension valve having a small aperture configured to hold the meniscus of the reagent, The meniscus thus leaves the reagent in the reagent reservoir prior to contact with the fluid sample to remove the meniscus. GCF031.9 Preferably, the nucleic acid amplification region is a polymerase chain reaction (PCR) amplification region. Preferably, the LOC device further has a CMOS circuit, a temperature sensor, and a microsystem technology (MST) layer having the PCR region, wherein the CMOS circuit is located on the support substrate and Between the MST layers, the CMOS circuit is configured to use the temperature sensor output to feedback control the PCR region. GCF031.il preferably, the PCR region has a PCR microchannel that is thermally cycled to amplify the nucleic acid sequences during use, the PCR microchannel defining a portion of the flow path of the sample and The cross-sectional area perpendicular to the flow is less than 1 〇〇, 〇〇〇 square micron. GCF031.12 Preferably, the LOC device further has at least one elongated heating element for heating the nucleic acid sequence within the elongated PCR microchannel, the elongated heating element extending parallel to the PCR microchannel. GCF031.13 Preferably, at least one of the PCR microchannels forms an elongated PCR chamber. -25- 201209404 GCF031.14 Preferably, the PCR region has a plurality of elongated PCR chambers respectively formed by respective blocks of the PCR microchannel, the PCR microchannel having a structure formed by a series of wide meandering streams Type, each wide meander stream is a channel block that can form the elongated PCR chamber. GCF03 1.15 Preferably, the LOC device further has a reagent reservoir for containing reagents for PCR; and a surface tension valve having a small hole configured to fix a meniscus of the reagent, the meniscus The reagent can be left in the reagent reservoir until the meniscus is removed by contact with the fluid sample, and then the reagent will flow out of the reagent reservoir. Preferably, the GCF 031.16 probe of each of the hybrid chambers having a hybrid chamber array containing probes is designed to hybridize to one of the sequences of the target nucleic acid sequences. GCF03 1 .17 7 Preferably, the photosensor is an array of photodiodes, which are located in the hybrid chamber of which they are recorded. G C F 0 3 1 . 1 8 Preferably, the C Μ Ο S circuit has a digital memory for storing the hash data from the photosensor output, and a data interface for transmitting the hybrid data to an external device. GCF031.19 Preferably, the PCR zone has an active valve to allow liquid to remain in the PCR zone during thermal cycling and to react to the activation signal from the CMOS circuitry to allow liquid to flow to the hybrid chamber. GCF03 1.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and the meniscus anchor is configured to fix a meniscus to block the capillary drive flow of the liquid and a heating The heater is added to the liquid -26-201209404 to boil to remove the meniscus from the meniscus anchor, so that the capillary drive flow resumes. This LOC device has the advantage of separating the desired sample components from the sample components based on the size of the components, and the method used to reduce clogging is superior to the simple filtration method. An advantage of this LOC design is that it can enrich the effective target concentration of the sample portion for further processing by the LOC device. This LOC device also has the advantages provided by sequence-specific amplification methods, including the sensitivity provided by amplification, wide dynamic range, and high specificity for the target DNA sequence. This LOC device also has the advantage that the sample can be grown under controlled conditions. GCF03 3.1 This aspect of the invention provides a wafer-on-lab (LOC) device for genetic analysis of a biological sample, the LOC device comprising: a receiving inlet for receiving a sample; a supporting substrate; a plurality of reagents a nucleic acid amplification region downstream of the incubation region to amplify the nucleic acid sequence in the sample; and a dialysis region downstream of the nucleic acid amplification region to filter the amplification region of the nucleic acid amplification region before hybridization The dialysis region is configured to remove cell debris from the amplicon; wherein the nucleic acid amplification region and the dialysis region are both supported on the support substrate. GCF03 3.2 Preferably, the LOC device further has a photo sensor and a hybrid region located downstream of the dialysis zone, the hybrid region having a hybrid probe array capable of interacting with a target nucleic acid in the sample The sequences are heterozygous to form a probe-target hybrid, wherein the photosensor is configured to detect the probe-target hybrid. GCF0 3 3.3 square micron volume. Preferably, each of the hybrid chambers has a volume of less than 900,000 angstroms GCF 033.4 square microns. Preferably, each of the hybrid chambers has a volume of less than 200,000 GCF 03 3.5 microns. Preferably, each of the hybrid chambers has less than 40,000 cubic GCF 033.6. Preferably, the dialysis zone has a large component channel, a small component channel, and a plurality of channels capable of fluidly connecting the large component channel to the small component channel. An aperture, the size of the aperture being such that a nucleic acid sequence flows from the large component channel to the small component channel, and cell debris larger than the small hole is retained in the large component channel, the small component channel The hybrid zone is in fluid communication. GCF03 3.7 extension. Preferably, the nucleic acid amplification region is an isothermal nucleic acid extension GCF03 3.8. Preferably, the LOC device further has a reagent reservoir for containing reagents for isothermal nucleic acid amplification; and a surface tension valve having a The orifice is configured to hold the meniscus of the reagent so that the meniscus leaves the reagent in the reagent reservoir prior to contact with the fluid sample to remove the meniscus. GCF033.9 Preferably, the nucleic acid amplification region is a polymerase chain reaction (P C R ) amplification region. GCF033.10 Preferably, the LOC device further has a CMOS circuit, a temperature sensor of -28-201209404, and a microsystem technology (MST) layer having the PCR region, wherein the CMOS circuit is located on the support base Between the material and the MST layer, the CMOS circuit is configured to use the temperature sensor output to feedback control the PCR region. GCF03 3.il Preferably, the PCR region has a PCR microchannel that is thermally cycled during use to amplify the nucleic acid sequences, the PCR microchannel defining a portion of the flow path of the sample Its cross-sectional area perpendicular to the flow is less than 100,000 square microns. GCF03 3.1 2 Preferably, the LOC device further has at least one elongated heating element for heating the nucleic acid sequence within the elongated PCR microchannel, the elongated heating element extending parallel to the PCR microchannel. GCF03 3.1 3 Preferably, at least one of the PCR microchannels forms an elongated PCR chamber. GCF03 3.1 4 Preferably, the PCR region has a plurality of elongated PCR chambers respectively formed by respective blocks of the PCR microchannel, the PCR microchannel having a 蜿蜒 configuration formed by a series of wide meandering flows, each width The meander stream is a channel block that forms the elongated PCR chamber. GCF03 3.1 5 Preferably, the LOC device further has a reagent reservoir for containing the reagent for PCR; and a surface tension valve having a small hole configured to fix the reagent meniscus, thus in the fluid sample The meniscus leaves the reagent in the reagent reservoir prior to contact to remove the meniscus. GCF03 3.1 6 Preferably, the hybrid region is an array of hybrid chambers containing probes, and the probes within each hybrid chamber are designed to hybridize to one of the sequences of the target nucleic acid sequence -29-201209404. GCF033.1 7 Preferably, the photosensor is an array of photodiodes whose positions are registered with the hybrid chamber. GCF03 3.1 8 Preferably, the CMOS circuit has a digital memory for storing the hash data from the photosensor output, and a data interface for transferring the hybrid data to an external device. GCF 033.1 9 Preferably, the PCR zone has an active valve to allow liquid to remain in the PCR zone during thermal cycling and to react to the activation signal from the CMOS circuitry to allow liquid to flow to the hybrid chamber. GCF03 3.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and the meniscus anchor is configured to fix a meniscus to block the capillary drive flow of the liquid, and a heating The heater is used to heat the liquid to boiling to release the meniscus from the meniscus anchor, causing the capillary drive flow to resume. The L0C device has the advantage of separating the required sample components from the undesired sample components based on the size of the components, and the method used can reduce clogging superior to the simple filtration method. An advantage of the design of the L0C device is that it can enrich the effective target concentration of the sample portion for further processing by the LOC device. The design of the L0C device also has the advantage of removing unwanted components from the treated mixture that may interfere with subsequent target detection. [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Describing -30-201209404 This description defines a main component of a molecular diagnostic system embodying many specific examples of the present invention. A full detailed description of the overall structure and operation of the system is shown later in this patent specification. Referring to Figures 1, 2, 3, 112 and 113, the system has the following top-level components: Test modules 10 and 11 are typically USB memory (memory key) sizes and are very inexpensive to manufacture. Test modules 10 and 1 each contain a microfluidic device 'typically in the form of a wafer-on-lab (LOC) device 30 that has been pre-loaded with reagents for molecular diagnostic assays and typically more than 1000 probes (see Figure 1 and Figure 1 1 2). Test Module 1 As shown in Figure 1, the ground-based detection technique is used to identify the target molecules, while the test module 1 of Figure 1 1 2 is based on the detection of electrochemiluminescence. Testing technology. The LOC device 30 has an integrated photosensor 44 (described in detail below) for fluorescence or electrochemiluminescence detection. Both test modules 1 and 1 1 use a standard Micro-USB plug 14 to transfer power, data, and control, both of which have a printed circuit board (PCB) 57, and both have external power supplies. The capacitor 32 and the inductor 15 are supplied. The test modules 1 and 1 are for single use and can be mass produced and delivered in sterile packaging. The outer casing 13 has a macroreceptacle 24 for receiving a biological sample and a detachable sterile sealing tape 22, preferably having a low viscosity adhesive to cover the large sump prior to use. A film seal 408 having a film cover 410 forms part of the outer casing 13 to reduce dewetting in the test module while providing a pressure relief to prevent small fluctuations in air pressure. The film cover -31 - 201209404 410 protects the film seal 408 from damage. The test module reader 1 2 supplies the test module 10 or 11 power through the micro-U S B埠1 6 . The test module reader 2 can take a variety of different forms and will be detailed later. The reader 1 2 version shown in Figures 1, 3, and 1 2 is an example of a smart phone. The block diagram of the reader 12 is shown in Figure 3. The processor 42 can execute application software from the program storage 43. The processor 42 also interfaces the display screen 18 with a user interface (ui) touch screen port and buttons 19, a cellular radio station 21, a wireless network connection 23, and a satellite navigation system 25. The cellular radio station 2 1 and the wireless network connection 2 3 are used for communication. The satellite navigation system 25 can update the epidemiological database with location information. Alternatively, the location data can be manually input through the touch screen 17 or the button 19. The data storage device 27 contains genetic and diagnostic information, test results, patient information, assays, and probe data for confirming the position of each probe and its array. The data store 27 and the program store 43 can share the same device. The application software installed in the test module reader 12 provides results analysis, as well as other test and diagnostic information.

For the diagnostic test, the test module 10 (or the test module 1 1 ) is inserted into the micro-U S B埠16 of the test module reader 12. The sterile sealing tape 22 is torn open and a biological sample (in liquid form) is loaded into the sample sump 24. Press the start button 20 to start the test through the application software. The sample will flow into the LOC device 30 and the plate assay will begin to extract, incubate, amplify, and hybridize with the sample nucleic acid (target) using a hybrid-reactive oligonucleotide probe prior to synthesis. In the case of the test module 1 〇 (which uses fluorescence-based detection), the probe is fluorescently marked and mounted in the housing 13 - LED -32 - 201209404 2 6 to provide triggering The probes required to emit fluorescence are 1 and 2). In the test module 1 1 (which is detected using an electrochemical 孽), the LOC device 30 is equipped with an ECL probe ( ) and does not require the LED 26 to generate luminescent radiation. Alternative poles 8 60 and 870 provide excitation current (see Figure 113). Light or luminescence) is detected using a light sensor 44 integrated in each LOC device J. The detection signal will be amplified and output, which can be analyzed by the test module reader 12. However, no results were shown. This information can be stored locally and/or uploaded to the inclusion server. Test module 1 1 or 1 1 is removed from the test module and properly discarded. Figures 1, 3, and 1 show the making of a mobile phone/smart test module reader 1 2 . In other forms, the laptop/note-only reader 103, the e-book reader 107, and the tablet-type computer 105 (see Figure 114) used in the test model hospital, private clinic or laboratory are read. The device can be connected with the wide image as a patient record, a bill, an online database, and a multi-user. It can also be associated with a variety of regional or remote peripheral devices. 1C Smart Card is now referenced. Group 10 generated picker 12 and network 125 to update the epidemiological data host? The etiology database, the electro-luminescence line in the electronic health record (EHR) host system 115 in the gene data host system 113 (see [Luminescence (ECL), as discussed above, is based on electrical radiation (Firefly II) After the CMOS circuit and the digital device is converted into a digital position, the reader will record the network of the reader 12 on the network reader 12, and the test set reader can be a computer 1 〇 1, a camphor 109 or a table pan. Other application environments, such as printers or patient data can be read through the flow gene database in the system 1 1 1 , sub-health records, -33- 201209404 electronic medical record (EMR) host system 121 electronic medical records and individuals Health record (PHR) personal health record in the host system 123. Conversely, the epidemiological database in the epidemiological data host system 1 1 1 , the gene database in the gene data host system 113, and the electronic health record ( EHR) Electronic Health Record in Host System 115, Electronic Medical Record in Electronic Medical Record (EMR) Host System 121, and Personal Health Record (PHR) Personal Health Record in Host System 123 may also be transmitted through the network 125 The reader 12 updates the digital memory in the LOC device 30 of the test module 10. Referring now back to Figures 1, 2, 1 12 and 1 13, the reader 12 uses battery power in the mobile phone configuration. The mobile phone reader contains all pre-loaded test and diagnostic information. The data can also be loaded or updated via a variety of wireless or contact interfaces to communicate with peripheral devices, computers or online servers. Micro-USB埠16 is available. Connect to the computer or maintain the power supply while the battery is charging. Figure 7 shows the test module 1 具体 specific example of the test only needs to show the result of a positive or negative reaction to a specific target, such as testing Whether someone is infected with, for example, the H1N1 influenza A virus. A USB-only power/indicator module 47 built for a specific purpose is suitable. No other reader or application software is required. On the USB power/indicator module 47 The indicator 45 will display the signal of the positive or negative reaction result. This configuration is quite suitable for a large number of screenings. Other items provided by this system also include a test tube containing a pretreatment reagent for a specific sample, The tongue depressor and the lancet for sample collection. The specific example of the test module shown in Figure 7 conveniently contains the collapsible type that is held by the spring -34- 201209404 lancet 390 and lancet release button 392 Satellite phones can be used in remote areas. Test Module Electronics Devices 2 and 1 1 3 are block diagrams of the electronic components of test modules 10 and 11. The CMOS circuits integrated in LOC device 30 have a USB. A device driver 36, a controller 34, a USB-compatible LED driver 29, a clock 33, a power conditioner 3 1 , a RAM 38, and a program and data flash memory 40 are provided. These devices provide control and memory for the entire test module 1 or 11 including a photo sensor 44, a temperature sensor 170, a liquid sensor 1 74, and different heaters 152, 154, 182. 234, and the accompanying drivers 37 and 39 and the registers 35 and 41. Only the LED 26 (in the example of the test module 10), the external power supply capacitor 32, and the micro-USB plug 14 are external to the LOC device 30. The LOC device 30 includes bond-pads that are coupled to such external components. RAM 38 and program and data flashing billions of body 40 with application software and diagnostic and test information for more than 1 000 probes (flash / secure storage, such as through encryption). For the example of test module 1 using ECL detection, the module does not have LED 26 (see Figures 1 12 and 1 13). 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 an electrochemiluminescent probe and a hybrid chamber having a pair of ECL excitation electrodes 860 and 870. Many test module types 10 are manufactured in different test formats and are easy to use immediately. The difference between these test formats is based on plate assays using reagents and probes. -35- 201209404 Some examples of infectious diseases that can be quickly identified by this system include: • Influenza: influenza A, B, C, infectious salmon virus Isavirus, Thogotovirus • Pneumonia: Respiratory tract fusion virus (RSV), adenovirus, interstitial pneumonia virus, Streptococcus pneumoniae, Staphylococcus aureus • Tuberculosis: Mycobacterium tuberculosis (co 6α ci eh iw) 6 ec: w / ois ), Bovine M. tuberculosis (6 ov ζ· ί ), Mycobacterium tuberculosis (α/Wcflnwm), Mycobacterium tuberculosis (caneii/) and Mycobacterium vaccae (microti) • Plasmodium falciparum Protozoa (corpse/a/c/parwm), Toxoplasma gondii () and other protozoan parasites • Typhoid: Salmonella typhimurium s er ον ar typhi) • Ebola virus • Human immunodeficiency virus ( Human immunodeficiency virus (HIV)) • Dengue fever: F 1 avivirus • Hepatitis (types A to E) • Hospital-acquired infections: such as Clostridium difficile Vancomycin-resistant enterococcus (5; and methicillin-resistant Staphylococcus aureus • Herpes simplex virus (HSV) -36- 201209404 • Cytomegalovirus (CMV) • Istamaba virus ( Epstein-Barr virus (EBV)) • Encephalitis: Japanese encephalitis virus, Chandipura virus • Pertussis: Haemophilus pertussis (5orcfeie//a) • Measles: Paramyxovirus • Meningitis: pneumonia Diplococcus and meningitidis • Anthracnose: Bacillus anthracis (^aci/ZMsaniAz-aci·?) Some examples of hereditary diseases that can be identified by this system include: • Cystic fibrosis • Hemophilia Disease • Sickle cell anemia • Black scorpion idiot • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic kidney disease • Congenital heart disease • A small group of Reiter's syndrome can be identified with this diagnostic system Cancers include: • Ovarian cancer • Colon cancer -37- 201209404 • Multiple endocrine neoplasms • Retinoblastoma • List of Perk's syndrome Not exhaustive and the diagnostic system can be configured to detect many different types of diseases and conditions using nucleic acid and proteomic analysis. Detailed Configuration of System Components LOC Device The LOC device 30 is the center of this diagnostic system. By using a microfluidic platform, it is capable of rapidly performing four major steps in nucleic acid-based molecular diagnostic assays, namely sample preparation, nucleic acid extraction, nucleic acid amplification and detection. There are other uses for this LO C device, which will be explained in more detail later. As discussed above, test modules 10 and 1 1 can take many different configurations to detect different targets. Similarly, the L O C device 30 also has a number of specific examples for the design of the target. A LOC device 30 is in the form of a LOC device 310 that uses fluorescence to detect a target nucleic acid sequence of a pathogen in a whole blood sample. For purposes of illustration, the structure and operation of the LOC device 301 will now be described in detail with reference to Figures 4 through 26 and Figures 27 through 57. Fig. 4 is a schematic representation of the overall structure of the LOC device 301. For convenience, the process stage shown in Figure 4 is represented by the reference number of the functional area of the LOC device 301 performing the process stage. The process stages associated with each of the major steps in nucleic acid-based molecular diagnostic assays have also been identified as: sample input and preparation 2 8 8 , extraction 290, incubation 291, amplification 292, and detection 294 . The LOC device -38 - 201209404 3 0 1 different reservoirs, chambers, valves and other components will be described in detail later. 〇 Figure 5 is a perspective view of the LOC device 301. It is manufactured using high volume CMOS and MST (microsystem technology) manufacturing technology. The layered structure of the LOC device 301 is shown in a schematic (not to scale) partial cross-sectional view of Fig. 12. The LOC device 301 has a germanium substrate 84 supporting a CMOS + MST wafer 48, a CMOS circuit 86 and an MST layer 87, and a cap 46 overlying the MST layer 87. For the purposes of this patent specification, the term "MST layer" refers to a collection of structures and layers of film that will be treated with different reagents. Accordingly, the structures and members are configured to define flow paths having characteristic dimensions that support capillary drive flow of liquid having similar physical properties to the samples during sample processing. In this regard, the MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing techniques can also produce such structures and components that can be sized to produce capillary drive flow and that can handle very small volumes. The specific embodiment described in this patent specification shows the MST layer and the structure and active components supported on the CMOS circuit 86 with the MST layer, but the features of the top cover 46 are eliminated. However, readers familiar with this technology should be aware that the MST layer can handle samples without the need for a substrate CMOS or an actual overlying cap. The overall size of the LOC device shown in the following figures is 1760 μπιχ 5 8 24 μηι. Of course, LOC devices of different applications may have different sizes -39 - 201209404 Figure 6 shows the feature components of the MST layer 87 on which the cap features are superimposed. Figure 6 shows the illustrations AA to AD, AG, and AH magnified in Figures 13, 14, 4, 5, 5, 5, and 63, respectively, and is described in detail below to give a thorough understanding of the LOC. Each structure within the device 3〇1. Figures 7 through 1 show the features of the top cover 46 independently and the 11th view shows the structure of the CMOS + MST device 48 independently. Layered Structures Figures 12 and 22 are sketches showing the layered structure of the CMOS + MST device 48, the top cover 46, and the fluid interaction therebetween. For the sake of explanation, these drawings are not shown in the actual scale. Fig. 1 is a schematic cross-sectional view through the sample inlet 68 and Fig. 22 is a schematic cross-sectional view through the reservoir 54. As best shown in Fig. 12, the CMOS + MST device 48 has a germanium substrate 84 that supports a CMOS circuit 86 that can manipulate the active components within the upper MST layer 87. A passivation layer 88 seals and protects the CMOS layer 8.6 from contact with the flow of liquid through the MST layer 718. Fluid flows through the top cover channel 94 of the cap layer 46 and the MST channel 90 located at the MST channel layer 100 (see, for example, Figures 7 and 16). Cell transport occurs in a larger channel 94 built into the top cover 46, while biochemical treatment takes place in a smaller M S T channel 90. The size of the cell transport channel is capable of transporting cells within the sample to a predetermined location within the MST channel 90. The transport of cells larger than 20 microns (e. g., specific white blood cells) requires that the channel diameter be greater than 20 microns, so that the channel will have a cross-sectional area perpendicular to the flow that is greater than 400 square microns. The MS channel is significantly smaller when it is not in the LOC to transport the fine -40-201209404 cell position. It should be understood that the cap channel 94 and the MST channel 90 are common terms, and the particular MS T channel 90 can be referred to as a heated microchannel or a dialysis MS T channel, depending on its particular function. The MST channel 90 is formed by etching a MS T channel layer 100 deposited over the passivation layer 88 and patterning with a photoresist. The MST channel 90 is surrounded by a top wall layer 66 that forms the top end of the CMOS + MST device 48 (as viewed from the orientation of the drawing). Although sometimes represented by individual layers, The top channel layer 8 and the reservoir layer 78 are formed on a single piece of material. Of course, this piece of material may not be a single whole. The sheet material is etched from both sides to form a cap channel layer 80 etched with a cap channel 94 and a reservoir layer 78 etched with reservoirs 54, 56' 58, 60 and 62. Alternatively, the reservoir and cap channel can be formed by microforming. Both etching and microforming techniques can be used to create channels that are perpendicular to the cross-section of the flow up to 20,000 square microns or as small as 8 square microns. In LOC devices, the size of the cross-sectional area perpendicular to the flow of the channels at different locations is widely available. When the channel contains a large number of samples or when the sample contains a large structural component, the cross-sectional area of the channel can be as high as 20, 〇〇〇 square micron (for example, 200 micron wide in a 100 micron thick film layer) aisle). When the channel contains only a small amount of liquid or the mixture contains no large cells, the cross-sectional area of the channel perpendicular to the flow is preferably very small. The lower seal 64 encloses the top cover channel 94 and the upper sealing layer 82 encloses the reservoir 54. -41 - 201209404, 56, 58, 60 and 62. The assay-specific reagents have been previously installed in the five reservoirs 54, 56, 58, 60 and 62. In the specific examples described herein, the following reagents have been pre-loaded in the reservoirs, but are easily replaced with other reagents: • Reservoir 54: Anticoagulant 'optionally containing red blood cell lysis buffer• Reservoir 5 6 : lysing reagent • reservoir 5 8 : restriction endonuclease, ligase and linker sequence (for linker-to-head PCR (see Figure 70, selected from t. Stachan et al, Human) Molecular Genetics 2, Garland Science, NY and London, 1999)) • Reservoir 60: amplification mix (dNTPs, primers, buffer) and • reservoir 62: DNA polymerase. The top cover 46 and the CMOS + MST layer 48 are in fluid communication through the corresponding openings in the lower seal 64 and the top wall layer 66. These openings are referred to as uptakes 96 or downtakes 92 from the MST channel 90 to the cap channel 94 or to the reverse flow. LOC Device Operation The operation of the LOC device 310 will be described in a stepwise manner with reference to the analysis of pathogenic DNA in a blood sample. Of course, other types of biological or abiotic fluids can also be analyzed using a suitable set of reagents, test procedures, LO C variants and detection systems, or a combination thereof. Go back to the reference -42- 201209404 Figure 4, there are five main steps involved in the analysis of biological samples 'including sample placement and preparation 288, nucleic acid extraction 290, nucleic acid incubation 291, nucleic acid amplification 292 and detection and analysis 294. Sample placement and preparation step 288 involves mixing the blood with anticoagulant 116 and then separating the pathogen from the white blood cells and red blood cells in the pathogen dialysis zone 70. As best shown in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action pulls the blood sample along the top cover channel 94 to the reservoir 54. The anticoagulant is released from the reservoir 54 when the sample blood flow opens the surface tension valve (8 (see Figures 15 and 22). The anticoagulant prevents clot formation and the clot blocks the flow. As best shown in Fig. 22, the anticoagulant 116 is drawn from the reservoir 54 by capillary action and into the MST channel 90 via the downcomer 92. The downcomer 92 has a capillary initiation feature (CIF) 102 to shape the geometry of the meniscus so that it does not rest on the rim of the downcomer 92. The venting opening 122 in the upper seal 82 can replace the anticoagulant with air as the anti-coagulant 116 is pulled out of the reservoir 54. The MST channel 90 shown in Fig. 22 is part of the surface tension valve 118. The anticoagulant 116 will fill the surface tension valve 118 and secure a meniscus 120 to the meniscus anchor 98 of the upper conduit 96. Prior to use, the meniscus 120 remains attached to the upper conduit 96 so that the anticoagulant does not flow into the canopy passage 94. As the blood flows through the canopy passage 94 to the upper conduit 90, the meniscus 120 is removed and the anticoagulant is introduced into the flow. .

Figures 15 through 21 show an illustration AE, which is part of the illustration AA-43-201209404 shown in Figure 13. As shown in Figures 15, 16 and 17, there are three separate MST channels 90 extending between the respective lower guides. The MST channel 90 in the surface tension valve changes the flow rate of the reagent into the sample mixture. When the agents are mixed together by diffusion, the reagents flow out and store the concentration of the reagents in the sample stream. Therefore, the force valve will be configured to meet the desired reagent concentration and then the blood will flow into the pathogen dialysis zone 70 (the male target cell is concentrated from the sample by using a predetermined large /] array. Less than this threshold Large cells are unable to pass through the pores. Cells in the array of useless pores 1 64 or through the fine waste unit 76 of the pores and the target cells continue to remain. In the pathogen dialysis zone 70, the body is concentrated for analysis by the microorganism DN A. A small hole 3 micron hole 1 64 is formed and can fluidly connect the cap channel to the target channel 74. The diameter of 3 micrometers of the target channel 74 The dialysis upper pilot hole 168 is connected by a 204 (preferably in Figures 15 and 21 is sufficient to pass through the small hole 1 64 having a diameter of 3 μm and passing through the expansive target channel 74. For cells larger than 3 μm, The waste passage 72 remaining in the top cover 46, the passage 76 (see Fig. 7). | Surface tension valve Π The number of the tube 92 and the upper conduit 96 may vary to be used as the sample mixture and the test reservoir. The flow rate will be the surface of each reservoir. 4 and 1 5)) The pores of the pores of the pores of the cells are passed through the pores, which can be stuck in the small cells and will be redirected in the assay to become samples from the whole blood. The pathogen array consists of a number of input streams within a diameter of 94 with a small orifice 164 and a series of dialysis MST channels. Pathogens will be small enough to analyze MST channel 204 as red blood cells and white blood cells will lead to waste storage -44 - 201209404 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. More details of this dialysis area and dialysis change will be provided later. 63⁄4S5 The first test and storage reagents will be re-dissolved up to 7 times. The valve is pulled by the force flow. The surface of the sample is 4-7. The flow valve force sheet has seven MST channels 90 extending between the lysate reservoir 56 and the target channel 74. When the meniscus is collapsed by the sample stream, the flow velocity from all seven MST channels 90 will be faster than the flow rate from the anticoagulant reservoir 54 (the surface tension valve 118 has three MST channels 90) ( It is assumed that the physical properties of the streams are approximately the same). Therefore, the proportion of the lysing reagent in the sample mixture will be higher than the ratio of the anticoagulant. The cytolytic reagent and target cells are mixed by diffusion in the target channel 74 in the chemical cytolytic zone 130. Boiling-initiated valve 126 can stop the flow - enough time for diffusion and cytolysis to release hereditary material from the target cells (see Figures 6 and 7) . The structure and operation of the boiling-starting valve will be described in more detail below with reference to Figures 31 and 32. The applicant has also developed other active valve types (relative to passive valves such as surface tension valves 1 18) that can be used in place of the boiling-start valve. These additional valve designs are also described later. When the boiling-start valve 126 is opened, the lysed cells will flow into the mixing zone 133 for pre-amplification restriction endonuclease digestion and linker ligation. Referring now to Figure 13, when the flow disrupts the meniscus of the surface-45-201209404 surface tension valve 133 at the beginning of the mixing zone 131, the restriction enzyme, linker and ligase will be from the reservoir 5. Released in 8. The mixture flows along the entire length of the mixing zone 133 for diffusion mixing. The end of the mixing zone 131 is the lower conduit 134' which leads to the cultivation zone inlet passage 133 of the cultivation zone 1 > 4 (see Figure 13). The incubation zone inlet channel 133 feeds the mixture to a heated microchannel 210' of the crucible configuration which provides a chamber for holding the sample for restriction digestion and linker ligation (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the film layers inside the inset AB of the LOC device 301 of Figure 6. Each of the figures shows a sequential addition process of the layers to form the structure of the CMOS + MST layer 48 and the cap 46. The illustration AB is the end point of the incubation zone 1 14 and the starting point of the amplification zone 1 12 . As best shown in Figures 14 and 23, the flow will continue to flow into the microchannel 2 1 0 of the incubation zone until it reaches the boiling-start valve 106 where it will stop and diffuse. As discussed above, the microchannel 2 10 upstream of the boiling-start valve 106 becomes a chamber containing a sample, restriction enzyme, ligase, and linker. The heater 1 54 is then activated and maintains a constant temperature for a limited period of time for restriction enzyme digestion and linker ligation. Workers familiar with this technique should be aware that this incubation step 291 (see Figure 4) is optional and will only be required for certain nucleic acid amplification assay types. Again, in some instances, a heating step may be required at the end of the incubation period to raise the temperature above the incubation temperature. The temperature increase causes the restriction endonuclease and ligase to be deactivated before entering the amplification zone 112. The inactivation of the restriction enzymes and ligases is particularly important when isothermal nucleic acid amplification is employed. -46- 201209404 After incubation, the boiling-start valve 106 will be activated (opened) and the flow re-entered the amplification zone 112. Referring to Figures 31 and 32, the mixture will continuously flow into the heated microchannels 158 of the crucible configuration (which will constitute one or more amplification chambers) until the liquid stream reaches the boiling-start valve 108. As best shown in the schematic cross-sectional view of Figure 30, amplification mixtures (dNTPs, primers, buffers) are released from reservoir 60 and then polymerase is released from reservoir 62 into the junction incubation zone and expanded. The intermediate MST channel 212 of the zones (1 14 and 1 12 respectively). Figs. 35 to 51 show the film layers inside the illustration AC of the LOC device 301 of Fig. 6. Each of the figures shows a sequential addition process of the layers to form the structure of the CMOS + MST device 48 and the top cover 46. The illustration AC is the endpoint of the amplification zone 112 and the beginning of the hybrid and detection zone 52. The incubated sample, amplification mixture, and polymerase will flow through the microchannel 158 to the boiling-start valve 108. After diffusion mixing for a sufficient period of time, the heater 1 54 in the microchannel 1 58 is activated to initiate thermal cycling or isothermal amplification. The amplification mixture is subjected to a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification process, the boiling-start valve 108 will open and the flow will re-flow into the hybrid and detection zone 52. The operation of the boiling-starting valve will be described in more detail later. As shown in Table 52, the hybrid and detection zone 52 has a hybrid chamber array 110. Figures 52, 53, 54 and 56 show a hybrid chamber array 11 and a detailed individual hybrid chamber 180. The entrance of the hybrid chamber 180 is a diffusion barrier 175, which prevents the target nucleic acid, the probe strand and the hybrid probe from diffusing in the hybrid chamber during the hybridization period to avoid erroneous hybrid detection. result. The -47-201209404 diffusion barrier 1 7 5 is equal in length to the flow path, and is long enough to prevent the target sequence and probe from diffusing out of one chamber and contaminating another chamber during probe and nucleic acid hybridization and signal detection. In this way, you can avoid erroneous results. Another mechanism to prevent erroneous reading is to use the same probe in several hybrid chambers. The CMOS circuit 86 will obtain a single result from the photodiode 1 8 4 corresponding to the hybrid chamber containing the same probe. Unreasonable results in the derivation of this single result are eliminated or weighted differently. The thermal energy required for hybridization is provided by a CMOS-controlled heater 182 (described in detail below). Hybridization occurs between complementary target-probe sequences after the heater is activated. The LED driver 29 in the CMOS circuit 86 signals the LEDs 26 located in the test module 10 to illuminate. These probes fluoresce only when the hybrid occurs, thus eliminating the need to remove the cleaning and drying steps typically required for unbound strands. As will be explained in more detail later, hybridization forces the stem-loop structure of the FRET probe 186 to open, and the open structure allows the fluorophore to react to the LED excitation light to emit fluorescent energy. Fluorescence can be detected by photodiode 1 84 lining the CMOS circuit 86 at the bottom of each of the hybrid chambers 180 (see description of the hybrid chamber below). The light diodes 184 of all of the hybrid chambers and associated electronic components collectively form a light sensor 44 (see Figure 65). In other embodiments, the photosensor can be an array of charge coupled devices (CCD arrays). The detection signal from photodiode 184 can be amplified and converted to a digital output, which can be analyzed by test module reader 12. More details of this detection method will be explained later. More details of the LOC device - 48 - 201209404 Modularization of the design The LOC device 301 has a number of functional areas including reagent reservoirs 54, 56, 58, 60 and 62, dialysis zone 70, cytolysis zone 130, incubation zone 114 And the amplification zone 112, various valve types, humidifiers and humidity sensors. In other specific examples of the LOC device, such functional areas may be omitted, other functional areas may be added or the other functional areas may be used for other purposes as described above. For example, the incubation zone 114 can be used as the first amplification zone 12 of the tandem amplification assay system, and the chemical lysate reagent reservoir 56 can be used to add primers, dNTPs, and first amplification of the buffer. The mixture, reagent reservoir 58 can be used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, the chemical lysing reagent may be added to the reservoir 56 along with the amplification mixture; alternatively, the sample may be heated in the incubation zone for a predetermined period of time. Perform thermocytolysis. In some embodiments, if chemical cytolysis is required and the mixture of primers, dNTPs, and buffers is separated from the chemical lysing reagent, then a reservoir of the mixture of the primers, dNTPs, and buffer can be used. Additional reservoirs are inserted upstream. In some cases it may be necessary to omit a step, such as incubation step 291. In this case, a LOC device may be specially fabricated to omit the reagent reservoir 58 and the incubation zone 1 14; or simply do not load the reagent in the reservoir; or if there is an active valve, the active valve may not be activated. The reagent is dispensed into the sample stream, thereby simply changing the incubation zone into a channel for transporting the sample from the cytolytic zone 1 30 to the amplification zone Π 2 . The heater operates independently, and the heater can be activated when the reaction is dependent on heat (eg, hot cytolysis); however, in a LOC device that does not require thermolysis, the heater can be programmed to ensure this step -49- 201209404 The heater will not be activated during the time and the thermolysis will not occur. The permeable may be located at the beginning of the microfluidic system within the microfluidic device as shown in Figure 4 or may be located anywhere within the microfluidic device. By way of example, in some instances the dialysis system is performed after the amplification period 292 to remove the cell debris prior to the detection of step 294, which would alternatively enter the two dialysis zones anywhere in the entire LOC device. Similarly, more amplification regions 1 1 2 can also be inserted, such as allowing multiple targets to be simultaneously or simultaneously in the amplification region prior to detection by the specific nucleic acid probe hybrid chamber assay 1 Continuous amplification analysis of a sample that does not require dialysis, such as a whole blood sample, can be simply omitted from the sample placement and preparation zone 28 8 . In this example, the dialysis zone 70 is not loaded from the LOC even if dialysis is not required for analysis. If the presence of the dialysis zone does not impede the assay, the LOC can be used even if it is contained in the sample placement and preparation zone without loss of the desired functionality. Further, the detection zone 294 may contain a probe that is the same as the proteomic chamber assay and the hybrid chamber assay, but is designed to be conjugated or hybridized with the unamplified sample-like target protein, rather than being designed and sequenced. Hybrid nucleic acid probe. It should be understood that the LOC device used in this diagnostic system is a combination of different functional zones selected for a particular application. It is common to install most of the functional areas. Another design with novel applications is to thoroughly edit the appropriate combination of functional areas from the functional areas used in existing LOC devices. The analysis area 7 0 shows that, for example, heterozygous and advantageous. Or more than one. In order to set the LOC in some implementations, except for the geometric resolution zone, the LOC of the test nucleic acid is set to be selected and the LOC is selected. -50-201209404 Only a few LOC devices are shown in this description. Multiple devices are shown schematically to demonstrate the design flexibility of the LOC device used in this system. Those skilled in the art will readily appreciate that the LOC devices shown in these descriptions are not fully enumerated and many other LOC designs are related to how to edit the appropriate combination of functional areas. 'Sample type LOC variants accept and analyze nucleic acid or protein contents of sample types in a variety of liquid forms, including but not limited to blood and blood products, saliva, cerebrospinal fluid, urine, semen, Amniotic fluid, cord blood, milk, sweat, pleural effusion, tears, pericardial fluid, ascites, environmental water samples and beverage samples. Amplicon obtained from macronucleic acid amplification can be analyzed using the LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, the dialysis, cell The solubilizing, incubation and amplification zone is only used to transport the sample from the sample inlet 68 to the hybrid chamber 180 for nucleic acid detection, as described above. Some sample types may require a pretreatment step. For example, the semen may need to be liquefied prior to placing the semen and mucus into the LOC device. The mucus may need to be pretreated with an enzyme to reduce viscosity. Sample Placement Referring to Figures 1 and 12, the sample is applied to a large sump 24 of the test module 1 。. The large sump 24 is a truncated cone and the sample can be fed into the inlet 68 of the LOC unit 301 via capillary action. Here, the sample will flow into the 64 μηι wide -51 - 201209404 X 60 μηη deep top cover channel 94 where the sample is also pulled through the capillary action to the anticoagulant reservoir 54. Reagent reservoirs The volume of reagents required for assay systems using microfluidic devices (e.g., LOC device 301) is small, such that each reagent reservoir contains all of the reagents required for biochemical treatment, even if it has a small volume. This volume can easily be less than 1,000,000,000 cubic microns, in most cases less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns and the LOC device 301 shown in the figures is less than 20,000,000 cubic microns. . Dialysis Zone Referring to Figures 15 through 21, 33 and 34, the pathogen dialysis zone 70 is designed to concentrate pathogenic target cells from the sample. As previously described, a plurality of apertures in the form of a 3 micron diameter hole 164 in the top wall layer 66 filter the target cells from the sample body. As the sample flows through a 3 micron diameter orifice 1 64, microbial pathogens enter the series of dialysis MST channels 204 through the holes and flow back to the target channel 74 via the 16μπα dialysis upper pilot hole 168 (see paragraph 3 3 3 4 picture). The remainder of the sample (red blood cells, etc.) will remain in the top cover channel 94. Downstream of the pathogen dialysis zone, the canopy channel 94 becomes a waste channel 72 that directs waste to the waste reservoir 76. For a type of biological sample that will produce a substantial amount of waste, the bubble illustration or other apertured element 49 in the test module 1 〇 housing 13 can be configured to be in fluid communication with the waste reservoir 76 (see paragraph 1) Figure). -52- 201209404 The pathogen dialysis zone 70 is fully functional by the capillary action of the fluid sample. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis zone 70 has capillary actuation features (CIFs) 166 (see Figure 33) so that fluid can be pulled down to the dialysis MST channel 204 below. The first upper pilot hole 198 of the target channel 74 also has a CIF 202 (see Figure 15) to avoid fluid flow to simply secure the meniscus to the orifice of the entire dialysis upper pilot hole 168. The small component dialysis zone 682, shown schematically at Figure 79, can have a configuration similar to the pathogen dialysis zone 70. The small component dialysis zone can separate all small target cells or molecules from the sample by small holes of different sizes (if desired, by different shapes), which allow small target cells or molecules to pass through. And enter the target channel and continue for further analysis. Larger cells or molecules are moved to waste reservoir 766. Thus, the LOC device 30 (see Figures 1 and 112) is not limited to isolation of pathogens less than 3 microns in size, and can be used to isolate cells or molecules of any desired size. Cytolysis zone Referring back to Figures 7, 11, and 13, the ruminant material in the sample can be released from the cell by chemical cytolysis. As discussed above, the lysing reagent from the lysis reservoir 56 will mix with the sample stream within the target channel 74 downstream of the surface tension valve 128 of the lysate reservoir 56. However, some diagnostic tests are more suitable for the treatment of target cells by thermocytolysis, even in combination with chemical cytolysis and thermocytolysis. The LOC device 301 can accommodate this problem by the heated microchannel 210 of the incubation zone 114. The sample stream was filled 1 in the incubation zone and stopped at the boiling-starting valve 1〇6. Incubating the microchannel 210 heats the -53-201209404 sample to the temperature at which the cell membrane collapses. In some thermocytolytic applications, the enzyme reaction is not required in the chemical cytolysis zone 130 and the thermocytolysis completely replaces the enzyme reaction in the chemical cytolysis zone 130. Boiling Start Valve As discussed above, the LOC unit 301 has three boiling start valves 126, 106 and 108. The location of these valves is shown in Figure 6. Figure 31 shows an enlarged plan view of the boiling start valve 108 at the end of the heated microchannel 158 of the amplification zone 112. The sample stream 1 19 is pulled by the capillary action along the heated microchannel 158 to the boiling start valve 108 to stop. The meniscus 丨2〇 before the sample flow is fixed to the meniscus anchor 98 of the valve inlet 146. The geometry of the meniscus anchor 98 stops the forward meniscus and blocks capillary flow. As shown in Figures 31 and 3, the meniscus anchor 98 is an aperture provided from the MST channel 90 to the conduit opening above the canopy channel 94. The surface tension of the meniscus 1 2 令 causes the valve to close. An annular heater 152 surrounds the valve inlet 146. The annular heater 152 is CMOS-controlled through the boiling start valve heater contact 153. To open the valve, the CMOS circuit 86 sends a current pulse to the valve heater contact 153. The annular heater 152 is resistively heated until the liquid sample 119 is boiled. Boiling disintegrates the meniscus 120 of the valve inlet 146 and causes the cap passage 94 to wet. . Once the top cover passage 94 begins to wet, the capillary flow resumes. The fluid sample 1 1 9 is filled with the top cover channel 94 'flows through the valve down conduit 1 50 to the valve outlet 1 4 8 where the capillary drive flow continues along the amplification zone exit channel 160 into the hybrid and detection zone 52. The liquid sensor 174 is placed before or after the valve for diagnosis. It should be understood that once the boiling start valve is opened, it cannot be closed. However, since the LOC device 310 and the test module 10 are used and lost, it is not necessary to close the valves. Incubation area and nucleic acid amplification area Figures 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubation area 1 14 and the amplification area 1 1 2 . The incubation zone 141 has a single heated incubation microchannel 210 etched into a sputum pattern that is located in the lower conduit opening 134 leading to the MST channel layer 100 of the boiling starter valve 106 (see Figures 13 and 14). Temperature control of the incubation zone 1 14 allows the enzyme reaction to proceed with better performance. Similarly, the amplification zone 112 has a heated amplifying microchannel 158 that is connected to the boiling starter valve 108 from the boiling start valve 1〇6 in a 蜿蜒 configuration (see Figures 6 and 14). These valves block the flow so that the target cells remain in the heat-cultured or amplified microchannels 210 or 158 for mixing, incubation, and nucleic acid amplification. The ruthenium pattern of the microchannel also accelerates (to some extent) the mixing of target cells with reagents. The sample cells and reagents in the incubation zone 1 14 and the amplification zone 1 1 2 are heated by a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each of the meandering configurations of the heated incubation microchannel 210 and the amplification microchannel 158 has three independently operable heaters 154 extending between their individual heater contacts 156 (see Figure 14). These contacts provide two-dimensional regulation of the input heat flux density. As best shown in Figure 51, the heater 1 54 is supported by the top wall layer 66 and embedded in the lower seal 64. The heater material is TiA1 -55- 201209404, but other conductive metals can also be used. The elongated heaters 154 are parallel to the longitudinal portions of the respective channel regions which form the wide meandering shape of the meandering shape. In the amplification zone 112, each wide stream can be manipulated as a separate PCR chamber by individual heaters. The size of the amplicon required for an assay system employing a microfluidic device, such as LOC device 301, is so small that the desired volume of the amplification mixture of the amplification zone 112 is also small. This volume can easily be less than 400 nanoliters, and in most cases it is less than 170 nanoliters, typically less than 70 nanoliters, and in the case of LOC device 310, it is 2 nanoliters to 30 nanoliters. between. Higher heating rates and better diffusion mixing Small cross-sections of each channel zone increase the heating rate of the augmentation fluid mixture. All fluids are kept at a fairly short distance from the heaters 154. Reducing the cross-section of the channel (this is the cross-section of the augmented microchannels 158) to less than 100,000 square microns is much higher than that of devices with more "large size". The etch fabrication technique allows the ablation microchannels 158 to have a cross-sectional area perpendicular to the fluid-flow path of less than 1 6,000 square microns, which provides a substantially higher heating rate. The feature members of the size of about 1 micrometer are easily provided by etching techniques. If a very small amplicon is required (as in the case of the LOC device 310), the cross-sectional area can be reduced to less than 2,500 square microns. For diagnostic assays containing 1, 〇〇〇 to 2,000 probes on the LOC device and requiring less than 1 minute from "sample placement" to "result output", preferably perpendicular to the flow The size of the area should be between 400 square microns and 1 square micron. -56- 201209404 The heating element in the amplification microchannel 158 can be heated at a rate of more than 80 Kelvin (K) per second, and in most cases at a rate of more than 1 sec per second. sequence. Typically, the heating element can heat the nucleic acid sequences at a rate of more than one, 〇〇〇 K per second, and in many instances the heating elements heat the nucleic acid sequences at a rate of more than 10,000 K per second. Frequently, based on the requirements of the calibration system, the heating element can be used in excess of 100,000 K per second, over 1,000,000 K per second, and over 10. per second. 000.  000 K, more than 20,000,000 K per second, more than 40 per second. 000.  000 K, over 80,000,000 K per second and over 160 per second. 000.  A speed of 000 K to heat the nucleic acid sequences. The passage of the small cross-sectional area is also advantageous for diffusion mixing of all reagents with the sample fluid. Before the diffusion mixing is completed, the diffusion of one liquid to the other is the fastest near the junction. The concentration decreases as the distance from the interface increases. By using a microchannel with a smaller cross-section perpendicular to the flow direction, both fluids can flow close to the interface and diffuse and mix more quickly. Shrinking the cross-section of the channel to less than 100,000 square microns provides a much higher mixing rate than a device that uses a larger "large size". The etch fabrication technique produces microchannels having a cross-sectional area perpendicular to the flow path of less than 1600 square microns, which significantly provides a higher mixing rate. If a small volume is required (as in the case of LOC device 301), the cross-sectional area can be reduced to less than 2,500 square microns. For diagnostic tests that have 1,000 to 2,000 probes on the LOC device and must be less than 1 minute from "sample placement" to "result-output", the cross-sectional area of the channel perpendicular to the flow is preferably 400 Between square microns and 1 square micron. -57- 201209404 Short Thermal Cycle Time Keeping the sample mixture close to the heater and using a very small fluid volume allows for rapid thermal cycling during nucleic acid amplification. For a target sequence of up to 150 base pairs (bp) long, each regenerative cycle (i.e., denaturation, binding, and primer extension) is completed in less than 30 seconds. In most diagnostic tests, 'individual thermal cycling time is less than 11 seconds, and most will be less than 4 seconds. The LOC device 30, which contains some of the most common diagnostic tests, has a thermal cycle time of between 145 and 1.25 seconds for a target sequence of up to 150 bp. This rate of thermal cycling allows the test module to complete the nucleic acid amplification process in less than 10 minutes, usually in less than 22 seconds. For most assays, the amplification zone produces sufficient amplicons from the sample fluid entering the sample inlet in less than 80 seconds. For many verifications, enough amplification can be generated in 30 seconds. After completing the current number of amplification cycles, the amplicon can be fed to the hybrid and detection zone 52 via the boiling start valve 1 〇 8. Hybrid Chambers Figures 52, 53, 54, 56, and 57 show the hybrid chamber 180 of the hybrid chamber assay 110. The hybrid and detection zone 52 has a 24x45 array of hybrid chambers. The array of arrays 1 10 ′ each has a hybrid reactive FRET probe 186, a heating element 182, and an integrated photodiode 184. The photodiode 184 is inserted to detect fluorescence generated by the target nucleic acid sequence or protein hybridized with the F R E T probe 186. Each photodiode 184 is independently controlled by a CMOS circuit 86. Any substance between the -58-201209404 FRET probe 186 and the photodiode 184 must be transparent to the radiation. Accordingly, the wall region 97 between the probe 186 and the photodiode 184 is also optically transparent to the emitted light. In the LOC device 301, the wall region 97 is a thin layer of cerium oxide (about 0. 5 microns). Incorporating the photodiode 184 directly under each of the hybrid chambers 180 allows the probe-target hybrid to be small in size while still producing a detectable fluorescent signal (see Figure 54). The small amount allows the hybrid chamber to have a small volume. The detectable amount of probe-target hybrid requires a probe before the hybridization, which is easily less than 270 pg (equivalent to 900,000 cubic microns), in the vast majority of cases Less than 60 picograms (equivalent to 200,000 cubic micrometers), typically less than 12 picograms (equivalent to 40,000 cubic micrometers), and the LOC device 301 shown in the following figures is less than 2. 7 picograms (equivalent to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybrid chamber allows for a higher density of hybrid chambers and thus more probes can be placed on the LOC device. In LOC device 301, the hybrid region has more than one chamber (i.e., less than 2250 square microns per chamber) in an area of 1500 microns by 15 microns. The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of the small amount of probe required for each chamber is that only a small amount of probe solution needs to be added to each chamber during manufacture of the LOC device. In the specific example of the LOC device of the present invention, a probe solution of 1 picoliter or less may be added. After nucleic acid amplification, the boiling start valve 1 〇 8 is activated and the amplicon flows along the flow path 176 into each of the hybrid chambers 18 (see Figures 52 and 56). When the hybrid chamber 18 is filled with amplicons, an endpoint liquid sensor 178 will display this phenomenon and -59-201209404 heater 1 8 2 will be activated. After ample mixing time, the LED 26 (see Figure 2) will be activated. The opening of each of the hybrid chambers 180 provides an optical window 136 for exposing the FRET probe 186 to excitation radiation (see Figures 52, 54 and 56). The LED 26 is illuminated for a period of time sufficient to cause the probe to emit a high intensity fluorescent signal. During the excitation, the photodiode U4 is short-circuited. After a period of time delay of 300 (see Figure 2), the photodiode 184 is activated and detects fluorescence emission when there is no excitation light. The incident light that is incident on the active region 185 (see Fig. 54) of the photodiode 184 is converted into a photocurrent, which is then measured by a CMOS circuit 86. Each of the hybrid chambers 180 is equipped with a probe for detecting a single target nucleic acid sequence. If necessary, each of the hybrid chambers can be equipped with probes capable of detecting more than one different target. Alternatively, the same probe can be loaded into many or all of the hybrid chambers to repeatedly detect the same target nucleic acid. Repeating the loading of such probes in this manner across the array of hybrid chambers 1 10 can increase the confidence of the results obtained; if desired, such results can be obtained by light diodes in close proximity to such hybrid chambers. Combine into a single result. Those skilled in the art will be aware of the specifications of the inspection. There may be from 1 to more than one different probes on the array 11 of the hybrid chamber. Humidifier and Humidity Sensor The illustration AG of Figure 6 indicates the location of the humidifier i96. The humidifier prevents reagents and probes from evaporating during operation of the L Ο C unit. The magnified view of Figure 5 is best shown as a water reservoir! 8 8 will be connected to the three evaporators 1 9 〇 with -60- 201209404 fluid. The reservoir i 8 8 is loaded with molecular biology-grade water and sealed during manufacture. As best shown in Figures 55 and 68, water is drawn into the three lower conduits 194 and flows into the three upper conduits 193 of the evaporator 190 along the individual water supply passages i92 by capillary action. - The meniscus is fixed to each of the upper ducts 193 to retain water. The evaporator has an annular heater 191 that encloses the upper conduit 193. The ring heater 191 is connected to the CMOS circuit 86 by a conductive stem 768 connected to the metal top layer 195 (see Fig. 37). When the ring heater 191 is activated, the heater heats the water to cause evaporation and humidify the surrounding portion of the device. The position of the humidity sensor 232 is also shown in Figure 6. However, as best shown in the enlarged view of the illustration AH of Fig. 63, the humidity sensor has a capacitive comb structure. A first electrode 296 etched by an etching technique and another second electrode 298 etched by an etching technique face each other and the comb teeth are interleaved. The opposite electrode forms a capacitor having a capacitance that can be monitored by CMOS circuitry 86. As the humidity increases, the dielectric constant of the air gap between the electrodes increases, so the capacitance also increases. The humidity sensor 23 2 is adjacent to the hybrid chamber array 110, where humidity measurement is of the utmost importance to mitigate evaporation of the solution containing the exposed probe. The feedback sensor insulates the temperature and liquid sensors into the entire L0C device 3〇1 to provide feedback and diagnostics during operation of the device. Referring to Figure 35, there are nine temperature sensors 170 scattered throughout the amplification zone 112° similarly. The incubation zone 114 also has nine temperature sensors 1 70 ° These sensors all use a 2x2 array of bipolar-61 - 201209404 Contact Junction Transistors (BJTs) to monitor fluid temperature and provide feedback to CM〇s circuit 86. The CMOS circuit 86 uses this feedback to precisely control the thermal cycling during thermal amplification and the thermal lysis and incubation during incubation. In the hybrid chamber 180, the CMOS circuit 86 uses a hybrid heater 182 as a temperature sensor (see Fig. 56). The resistance of the hybrid heater 182 is temperature dependent and the CMOS circuit 86 utilizes this characteristic to derive temperature readings for each of the hybrid chambers 180. The LOC device 301 also has a plurality of MST channel liquid sensors 174 and a cap channel liquid sensor 208. Figure 35 shows a row of M S T channel liquid sensors 丨 74 at one end of each of the spaced meanders in the heated microchannel 158. As best shown in FIG. 3, the MST channel liquid sensor 1 74 is a pair of electrodes formed by exposed regions of the metal top layer 195 within the CMOS structure 86. The liquid closes the circuit between the electrodes and indicates the presence of liquid at the location of the sensor. Figure 25 shows an enlarged perspective view of the top cover channel liquid sensor 208. Opposite electrical TiAl electrode pairs 218 and 22 are deposited on top wall layer 66. Between the electrodes 2 18 and 2 2 0 is a gap 2 2 2 which keeps the circuit clear when there is no liquid. The presence of liquid shuts down this circuit and CMOS circuit 86 uses this feedback to monitor the flow. Gravity Irrelevance Test Module 1 is not affected by orientation. They do not need to be fixed to operate on a smooth surface. The capillary drive flow flows and no external tubing is connected to the auxiliary device, making the modules truly portable and simply plugging in a portable handheld reader such as a mobile phone like -62 - 201209404. Operations that are not related to gravity mean that the test modules are also unrelated to acceleration at all utility levels. They are resistant to violent vibrations and vibrations and can be operated on mobile vehicles or when carrying mobile phones with them. Dialysis-Modified White Blood Cell Target The dialysis design described above in the LOC device 30 1 targets the pathogen. Figure 64 is a schematic cross-sectional view of a dialysis zone 328 designed to concentrate white blood cells from a blood sample for human DNA analysis. It should be understood that this structure is substantially identical in structure to the pathogen dialysis zone 70 described above, but with a diameter of 7. A small hole in the form of a 5 micron hole 165 limits the white blood cell from flowing from the canopy channel 94 to the dialysis MST channel 204. In the case where the analysis sample is a whole blood sample and the hemoglobin from the red blood cells interferes with the subsequent reaction step, addition of the red blood cell cytolysis buffer (see Fig. 22) in addition to the anticoagulant added to the reservoir 54 will be available. It is ensured that most of the lysed red blood cells (and thus their heme) can be removed from the sample during the dialysis step. A commonly used red blood cell lysis buffer is 0. 15M NH4CL, 10mM KHC03, O. lmM EDTA, pH 7. 2-7. 4. However, those skilled in the art know that any buffer that is effective for lytic red blood cells can be used. Downstream of the leukocyte dialysis zone 328, the cap channel 94 becomes the target channel 74, so the white blood cells continue to remain as part of the assay. Further, in this example, the dialysis upper tube aperture 168 will lead to the waste channel 72 to remove all of the smaller cells and components within the sample. It should be noted that this dialysis -63-201209404 variant will only reduce the concentration of unwanted samples in the target channel 74. Fig. 80 is a schematic view showing a large component dialysis zone 668 which can also separate any large target component from the sample. The pores of this dialysis zone are sized and shaped to retain the large target component of interest in the target channel for further analysis. When using the leukocyte dialysis zone described above, most, but not all, of the smaller cells, organisms or molecules will flow to the waste reservoir 768. Therefore, other specific examples of the LOC device are not limited to being able to separate only larger than 7. A 5 micron white blood cell can also be used to isolate cells, organisms or molecules of any desired size. Dialysis zone for preventing trapped air bubbles by flow channels The following is a specific example of a LOC device called LOC variant VIII 518 and is shown in Figures 73, 74, 75 and 76. The LOC device has a dialysis zone filled with a fluid sample and without trapped bubbles within the channel. The LOC variant VIII 518 also has an additional layer of material known as interface layer 594. The interface layer 594 is located between the top pass channel layer 80 and the MST channel layer 100 of the CMOS + MST device 48. The interfacial layer 594 allows for more complex fluid interconnections between the reagent reservoir and the M S T layer 87 without increasing the size of the crucible substrate 84. Referring to Fig. 74, the bypass passage 600 is designed to introduce a time delay when the fluid sample stream flows from the interface waste passage 604 to the interface target passage 602. This time delay allows the fluid sample to flow through the dialysis MST channel 240 to the dialysis upper conduit 168 where a meniscus is secured. The sample fluid from the dialysis MST channel 204 will flow from all of the dialysis upper conduits 1 68 by capillary actuation of the conduits located in the bypass passage 600 to the conduit above the interface target passage 602 (CIF) 202. One point begins to gradually fill the interface target channel 602. Without the bypass channel 6 00, the interface target channel 62 will still be charged from the upstream end, but the forward-moving meniscus will eventually reach and pass over the unfilled MST channel. And trap the air at this point. The trapped air reduces the flow rate of the sample stream through the white blood cell dialysis zone 3 28 . Pre-hybridization filtration - a variant of the LOC device, LOC Variant XII 75 8 uses a small component dialysis zone 682 placed at the exit of the amplification zone 112 (see Figures 96-103). The small component dialysis zone 682 provides a pre-hybrid filtration purification period of 293 (see Figure 96). Filtration before hybridization removes cell debris that remains in the sample stream after cell lysis. Hybridization is affected by cell debris, so it is useful to reduce the concentration of cell debris before mixing. Referring to Figures 101, 102 and 103, the small component dialysis zone 682 has three adjacent channels built into the bottom channel layer 100: a large component channel 760 sandwiched between two small component channels 762. Along the sides of the large component channel 760, there are a series of apertures in the form of inverted tapered openings 764 that allow the large component channels 760 to be fluidly coupled to the small component channels 762. In most practical applications, the orifice is 1 to 8 microns wide and 1 to 8 microns high. When the sample flows down to the large component channel 760, particles that are small enough to pass through the inverted cone opening (eg, the amplicon) will flow into the small component channels 762-65-201209404' while larger particles (eg, cells) The residue is left in the Dacheng large component channel and ends at a closed end 766. The smaller component channels continue to flow to the hybrid chamber array 1 〇 and the opposite ones follow the 蜿蜒 path through the array to their respective 妾 11 3). These small component amplicons will be in the hybrid chamber 180 before detection.

Nucleic Acid Amplification Variants Direct PCR Traditionally, PCR requires thorough purification prior to preparation of the reaction mixture. However, nucleic acid amplification or amplification can be performed after purification of some microDNA by appropriately changing the chemicality. This method is used when the nucleic acid amplification process is PCR. When the nucleic acid amplification in the LOC device is at a controlled constant temperature, this method is a direct isothermal amplification method. In the case of direct nucleic acid amplification techniques, it is particularly a simplified aspect of the desired fluid design. Direct P C R or direct isothermal amplification increases the buffer strength for amplification chemistry, uses additives with high activity and continuous efficacy, and sequesters potential polymerase inhibitors. Trial dilution is also important. In order to take advantage of the direct nucleic acid amplification technology, the program incorporates two additional features. The first feature register (such as the reservoir 58 of Figure 8), which is just the size of the amplification mixture or diluent, may therefore dry in the channel, the particles along the small side, here two ί Closed end 768 (see 塡 所有 将 将 将 将 将 将 将 将 将 将 将 将 将 将 将 将 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 768 The polymerase, the component of the LOC device of the intra-inhibitor is a reagent that can be supplied with sufficient scrambling chemistry. 66-201209404 The final concentration of the sample component will be low enough for successful nucleic acid amplification. The dilution required for the sample component is in the range of 5X to 20X. Different LOC structures such as the fourth can be used in cases where it is possible to appropriately determine that the concentration of the target nucleic acid sequence is maintained at a sufficient level for amplification and detection. The pathogen dialysis zone 70. In this specific example (further shown in Figure 6), there is a dialysis zone upstream of the sample extraction zone 290 which is effective to concentrate pathogens small enough to enter the amplification zone 292. And reject The large cells are sent to the waste storage tank 76. In another embodiment, the dialysis zone is used to selectively remove proteins and salts from the plasma while retaining the cells to be studied. The second one can support The LOC structural property of direct nucleic acid amplification is designed by adjusting the aspect ratio of the channel to adjust the mixing ratio of the sample and the components of the amplification mixture. For example, to ensure a single mixing step, the accompanying inhibitor in the sample can be diluted to Preferably, the range of 5X to 20X, so the length and cross section of the sample and reagent channels are designed such that the flow impedance of the sample channel upstream of the point at which mixing begins to occur is higher than the flow impedance of the reagent mixture flow channel. 4X to 1 9X. The control of the flow impedance in the microchannel can be easily achieved by the control of the design geometry. When the cross-section size is constant, the flow impedance of the microchannel increases linearly with the increase of the channel length. Very importantly, the flow impedance of the microchannel is more primarily dependent on the size of the smallest cross-section. For example, when the aspect ratio is very inconsistent, The flow impedance of a microchannel with a rectangular cross section is inversely proportional to the cube of the smallest orthogonal size. -67- 201209404 Reverse transcriptase PCR (RT-PCR) When the sample nucleic acid species being analyzed or extracted is RNA, for example from RNA of RNA virus or messenger RN A must be reverse transcribed into complementary DNA (cDNA) before PCR amplification. The reverse transcription reaction can be performed in the same chamber for PCR (single-step RT-PCR) or It can be performed by a separate initial reaction (two-step RT-PCR). In the LOC variants described herein, single-step RT-PCR can be performed simply by adding reverse transcriptase and polymerase to the reagent reservoir. The heater 1 54 is programmed and controlled in a manner such that the temperature cycle is first adapted to the reverse recording step and then to the nucleic acid amplification step. Two-step RT-PCR can also be used to store and dispense buffer 'primers, dN TPs, and reverse transcriptases by using reagent reservoir 58 and perform reverse transcription steps in incubation zone 114 followed by amplification in zone 1 1 2 amplification is achieved. Isothermal Nucleic Acid Amplification For some applications, isothermal nucleic acid amplification is a preferred method of nucleic acid amplification that avoids the need to repeatedly circulate and heat the reaction components at different temperature cycles. The build-up zone is maintained at a constant temperature (typically about 37 ° C to 41 ° C). Several isothermal nucleic acid amplification methods have been described, including strand displacement amplification (SDA), transcription vector amplification (TMA), and nucleic acid sequence-based amplification (NASBA) 'recombinase polymerase amplification. Method (RPA), helicase-dependent isothermal DNA amplification (HDA), rolling circle amplification (RCA), reticular amplification (RAM) and circular medium isothermal amplification (LA) Μ P ), any of these isothermal amplification methods or other isothermal amplification methods can be used in the detailed description of the LOC device described herein - 68-201209404. For isothermal nucleic acid amplification, the PCR amplification mixture and polymerase are no longer loaded into the reagent reservoirs 60 and 62 adjacent to the amplification zone, and the appropriate reagents for the particular isothermal method are instead loaded. For example, in the case of the SDA method, the reagent reservoir 60 contains amplification buffers, primers, and dNTPs, while the reagent reservoir 62 contains appropriate nicking enzymes and Exo-DNA polymerase. In the case of the RPA method, the reagent reservoir 60 contains amplification buffer, primers, dN TPs, and recombinase proteins, while the reagent reservoir 62 contains a stock-replacement DNA polymerase, for example. Similarly, in the case of the HDA method, the reagent reservoir 60 contains amplification buffer, primers and dNTPS and the reagent reservoir 62 contains the appropriate DNA polymerase and helicase to replace the heat to unwind the double stranded DNA strand. . Those skilled in the art will appreciate that the reagents required can be placed in two reagent reservoirs in any manner suitable for the nucleic acid amplification method. For the amplification of viral nucleic acids from RN A viruses such as HI V or hepatitis C virus, the 'NASB A or TM A method is well suited because they do not require transcription of the RNA into cDNA first. In this example, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, while reagent reservoir 62 contains RNA polymerase, reverse transcriptase, and optionally RNase®. For some forms of isothermal nucleic acid amplification, a preliminary denaturation cycle may be required to separate the double-stranded DN Α template prior to maintaining the temperature for isothermal nucleic acid amplification. This object can be readily achieved in the specific examples of all of the LOC devices described herein because the temperature of the mixture within the amplification zone 112 can be carefully controlled by the heater 154 within the amplification microchannel 158 (see Figure 14). ). -69- 201209404 Isothermal nucleic acid amplification is more tolerant to possible inhibitors in the sample, so these methods are generally suitable when direct nucleic acid amplification from the sample is required. Therefore, isothermal nucleic acid amplification methods are sometimes used in LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677, which are found in Figures 81, 82 and 84, respectively. Direct isothermal amplification may also be combined with one or more pre-amplification dialysis steps 70, 686 or 682 (as shown in Figures 81 and 84) and/or prehybridization dialysis step 682. (as shown in Figure 82), respectively, to facilitate partial concentration of target cells in the sample prior to nucleic acid amplification or cell debris removed prior to entry of the sample into the array of hybrid chambers. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be employed. Isothermal nucleic acid amplification can also be performed in parallel amplification regions as shown schematically in Figures 72, 77, and 78. Multiple isothermal nucleic acid amplification methods and some isothermal nucleic acid amplification methods such as LAMP can be used with the initial The reverse transcription step is compatible to amplify the RNA. Further details of the fluorescence detection system Figures 58 and 59 show the hybrid-reactive FRET probe 2 3 6 . These probes are often referred to as molecular indicators and are stem-loop probes produced from single-stranded nucleic acids that fluoresce when hybridized to a complementary nucleic acid. Figure 58 shows a single FRET probe 23 6 prior to hybridization with the target nucleic acid sequence 2 3 8 . The probe has a ring 240, a stem 242, a fluorophore 246 at the 5' end, and a quencher 248 at the 3' end. Loop 240 is composed of a sequence complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are joined to each other to form stem 24 2 . -70- 201209404 When there is no complementary target sequence, the probe remains closed as shown in Figure 58. The stems 242 are such that the fluorophore-quenching species are in close proximity to each other, so that significant resonance energy transfer occurs between the two, substantially eliminating the ability of the fluorophore to emit fluorescence upon exposure to the excitation light 244. Figure 59 shows the FRET probe 236 in an open or hybrid configuration. After hybridization to the complementary target nucleic acid sequence 23 8 , the stem-loop structure collapses, and the fluorophore and quencher are spatially separated, so that the fluorophore 246 regains the ability to emit fluorescence. Fluorescent emission 250 is optically detectable as an indicator that the probe has been hybridized. Since the stem helix of the probe is designed to be more stable than the probe-target helix of a non-complementary single nucleotide, these probes will hybridize to the complementary target with very high specificity. Since the double-stranded DNA is relatively rigid, it is spatially impossible to coexist with the probe-target helix and the stem helix. Primer-Linked Probes Primer-linked stem-loop probes and primer-linked linear probes (also known as 蝎 probes) are another molecular indicator and can be used for both real-time and quantitative nucleic acid amplification of LOC devices. The immediate amplification is performed directly in the hybrid chamber of the LOC device. The advantage of using a primer to link the probe is that the probe element is physically linked to the primer, so that only a single hybrid event occurs during nucleic acid amplification without the need to separately mix the primer and the probe. This ensures that the reaction occurs more efficiently and with greater efficiency, shorter reaction times, and better discrimination than with the use of separate primers and probes. The probe -71 - 201209404 needle (and polymerase and amplification mixture) are deposited in the hybrid chamber 180 during manufacture, so that no separate amplification regions are required on the LOC device. Alternatively, the amplification region can be left unused or used in other reactions. Primer-Linked Linear Probes Figures 8 6 and 8 7 show the primer-ligated linear probe 692 and the probe in a heterozygous configuration during subsequent nucleic acid amplification, respectively, during the first round of nucleic acid amplification. Referring to Fig. 86, the primer-linked linear probe 6 92 has a double-stranded stem segment 242. One of them will break into a probe sequence 696 that has been linked to a primer, which is homologous to a region of the target nucleic acid 696 and has been labeled with a fluorophore 246 at its 5' end and amplifying at the 3' end. The fragment 694 is coupled to an oligonucleotide primer 700. The other strand of the stem 242 is labeled with a quencher group 248 at the 3' end. Upon completion of the first round of nucleic acid amplification, the probe will be looped again and hybridized to the extended strand with the now complementary sequence 69 8 . During the first round of nucleic acid amplification, the oligonucleotide primer binds to the target DNA 23 8 (see Figure 86) and then extends to form a DN A strand that contains both the probe sequence and the amplified product. Amplification blocker 694 prevents the polymerase from reading and copying probe region 696. During subsequent denaturation, the extended oligonucleotide primer 700/template hybrid will detach, and likewise the double stem stem 242 of the primer-ligated linear probe will also detach and release the quencher 248. Once the temperature is lowered for the binding and extension steps, the primer-ligated linear probe primer-linking probe sequence 696 is curled and hybridized to the amplified complementary sequence 6.8 on the stretched strand and detected Fluorescent shows the presence of target DNA. Linear probes with unextended primers retain their twin stems -72 - 201209404 and the fluorescence remains quenched. This detection method is especially suitable for fast detection systems because it relies on a single-molecule process. Primer-Linked Stem Ring Probes Figures 88A through 88F show the operation of the primer-ligated stem loop probe 7〇4. Referring to Fig. 88A, the primer-ligated stem-loop probe 7〇4 has a stem portion 242 of complementary double-stranded DNA and a loop portion 240 having a probe sequence. One of the stem strands 70 8 is marked with a fluorophore 246 at the 5' end. Another strand 710 is labeled with quencher 248 at the 3' end and carries amplification blocker 694 and oligonucleotide primer 700. During the initial degeneration period (see 88B), the double strands of the target nucleic acid 23 8 are separated from each other, as is the stem portion 242 of the primer-linked stem-loop probe 704. When the temperature is cooled during the binding phase (see Figure 88C), the oligonucleotide primer 700 on the primer-ligated stem loop probe 704 will hybridize to the target nucleic acid sequence 23 8 . During extension (see Figure 88D), the complement 706 of the target nucleic acid sequence 238 is synthesized and forms a DNA strand containing both the probe sequence 704 and the amplified product. The amplification blocker 694 prevents the polymerase from reading and copying the probe region 704. Upon subsequent binding of the probe after denaturation, the probe sequence of the link segment 240 of the primer-ligated stem-loop probe (see Figure 88F) will bind to the complementary sequence 706 on the extended strand. This configuration leaves the fluorophore 246 relatively remote from the quencher 248, resulting in a significant increase in fluorescence emission. Control Probes The Hybrid Chamber Array 11 contains a number of hybrid chambers 180 with positive control probes and negative control probes -73-201209404 for quality control. Figures 108 and 109 schematically show the negative control probes without fluorophores <796, and the 11th and in maps are thumbnails of the positive control probes 798 without quenching. The positive and negative control probes have a stem-loop structure similar to the FRET probe described above. However, regardless of whether the probes are in a heterozygous open configuration or remain locked, the fluorescent signal 250 will always be emitted by the positive control probe 798 and the negative control probe 796 will never emit the fluorescent signal 250. Referring to Figures 108 and 109, the negative control probe 796 does not have a fluorophore (and may or may not contain quencher 248). Thus, regardless of whether the target nucleic acid sequence 238 is hybridized to the probe (see Figure 109), or the probe remains in the stem-loop configuration (see Figure 108), the response to the excitation ray 244 is negligible. Alternatively, the negative control probe 796 can be designed to remain in a quenched state at all times. For example, by synthesizing the loop portion 24 into a contained probe sequence, it does not hybridize to any of the nucleic acid sequences in the sample, such that the stem portion 2 42 of the probe molecule is hybridized with itself and the fluorophore The quenching material is kept in close proximity without a noticeable fluorescent signal being emitted. This negative control signal is equivalent to the low amount of emission in the hybrid chamber 180 where the probes are not hybridized but the quencher does not quench the emission of all of the reporters. Conversely, the positive control probe 798 can be constructed to be free of quenching as shown in Figures 1 1 and 1 1 1 . Regardless of whether the positive control probe 7 98 is heterozygous to the target nucleic acid sequence 23 8 , nothing will quench the fluorescent emission 250 produced by the reaction of the fluorophore 246 with the excitation light 244. Figure 5 2 shows the possible distribution of the positive and negative control probes (378 and 380, respectively) throughout the hybrid chamber array 110. The control probes -74 - 201209404 378 and 380 are placed in the hybrid chamber 180 in a row across the array of hybrid chambers 110. However, the configuration of the control probes within the array is arbitrary (as in the configuration of the hybrid chamber array. 1 10). The fluorophore design requires a long fluorescent lifetime fluorophore so that the excitation light has sufficient time to decay below the intensity of the fluorescent emission, during which time the photo sensor 44 is activated to provide sufficient Signal to noise ratio. Similarly, a longer fluorescence lifetime can be converted to a larger integrated fluorescence photon count. The fluorescence lifetime of the fluorophore 246 (see Figure 59) will be greater than 100 nanoseconds, typically greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and greater than 400 nanoseconds in most instances. Metal-ligand complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal, chemical, and photochemical stability. It is a beneficial property of the need for a fluorescent detection system. A metal-ligand complex which is mainly composed of transition metal ruthenium ions (Ru(II)) and which has been studied in detail is tris(2,2'-bipyridyl) ruthenium (II) ([Ru(bpy)3) ]2+), which has a lifetime of about 1 microsecond (μβ). This complex is available from Biosearch Technologies under the trade name Pulsar 650 -75- 201209404 Table 1: Photophysical properties of Pulsar 6 50 (ruthenium chelate)

Parameter symbol number 値 unit absorption wavelength ^abs 460 nm emission wavelength ^em 650 nm extinction coefficient E 14800 M-1cm-1 fluorescence lifetime Tf 1.0 μs quantum yield Η 1 (deoxidation) N/A bismuth chelate, a kind Lanthanide metal-ligand complexes have been successfully shown as fluorescent reporters for the FRET probe system and have a long lifetime of ι 600 μ3.

Table 2: 铽 chelation; kiss light physical property parameter number of symbols 値 unit absorption wavelength ^abs 330-350 nm emission wavelength λ έ πι 548 nm extinction coefficient Ε 13800 (kbs and ligand dependence, up to 30000 @ λε = 340 nm ) Fluorescence lifetime Xf 1600 (hybrid probe) Ms Quantum yield Η 1 (ligand dependent) N/A The fluorescent system used in the LOC device 3 01 does not use filters to remove unwanted background fluorescence. Therefore, in order to increase the signal-to-noise ratio, it would be advantageous if the quencher 248 had no native emission. Quenching 248 does not provide background fluorescence when there is no intrinsic emission. High quenching efficiency is also important, so that there is no fluorescence until a heterozygous event occurs. Black Hole Quenchers (BHQ) from Biosearch Technologies, Inc. of Novato California, with no organic emission and high quenching efficiency, is suitable for quenching of this system. The maximum absorbance of BHQ-1 is 534 nm and the quenching range is 480-580 nm, making it suitable as a quencher for Tb-chelate fluorophores. BHQ-2 has a maximum absorbance of 579 nm and a quenching range of 560-670 nm, making it suitable as a quencher for Pulsar 650. The Iowa Black Quenchers (Iowa Black FQ and RQ) supplied by Integrated DNA Technologies of Coralville, Iowa, have little or no background emission and are another suitable quencher. Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorbance of 531 nm and is therefore suitable for quenching of Tb-chelate fluorophores. The Iowa Black RQ has a maximum absorbance of 65 6 nm and a quenching range of 500-700 nm, making it an ideal quench for the Pulsar 650. In the specific examples described herein, the quencher 248 is a functional group that is initially attached to the probe, although in other embodiments the quencher may be an individual molecule that is free in solution. The excitation is based on the specific example of the fluorescence detection described herein. Based on the consideration of low-calorie power, low cost and small volume, the LED is used as the excitation source instead of the laser diode and the high-power electric lamp. Or a laser. Referring to Fig. 89, the LED 26 is placed directly on the outer surface of the LOC device 3〇1 above the array of hybrid cells 1 1〇. On the opposite side of the hybrid cell array 1 1 is a photo sensor 44 consisting of an array of photodiodes 184 (see Figures 53, 54 and 65) to detect from each -77-201209404 Fluorescent signal of the room. Figures 90, 91 and 92 schematically show other specific examples of exposing the probe to excitation light. In the LOC device 30 shown in Fig. 90, the excitation light 20 generated by the excitation LED 26 is directed through the lens 254 to the array of hybrid cells n. The excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent emission. In the LOC device 30 shown in FIG. 91, the excitation light 244 generated by the excitation LED 26 is directed to the hybrid chamber array 110 by the lens 254, the first optical 稜鏡 712, and the second optical 稜鏡 714. The excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent emission. Similarly, in the LOC device 30 shown in Fig. 92, the excitation light 244 generated by the excitation LED 26 is guided to the hybrid chamber array 110 by the lens 254, the first mirror 716 and the second mirror 71 8 . . Again, the excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent emissions. The excitation wavelength of LED 26 depends on the fluorescent dye selected. Philips LXK2-PR14-R00 is an exciting source for Pulsar 6 5 0 dyes. SET UVT0P 3 3 5 T039BL LED is an excitation source suitable for Tb-chelate labeling. Table 3: Philips LXK2-PR14-R00 LED Specifications

Parameter symbol number 値 unit wavelength ^ex 460 nm Transmit frequency Vem 6.52(10)14 Hz Output power Pi 0.515(min) @ ΙΑ W Radiation pattern Lambertian profile N/A -78- 201209404 Table 4: SET UVT0P334T039BL LED Specifications

Parameter symbol number 値 unit wavelength λο 340 nm Transmitting frequency Ve 8.82(10)14 Hz Power Pi 0.000240 (min) @ 20mA W Instantaneous forward pulse current I 200 mA Radiation pattern Lambertian N/A Ultraviolet excitation light 矽Absorbs light from the UV spectrum very little. Therefore, it is useful to use UV to excite light. A UV LED excitation source can be used, but the broad spectrum of the LED 26 reduces the efficiency of the method. To handle this problem, a filtered UV LED can be used. Arbitrarily, UV lasers can be used arbitrarily as an excitation source unless the higher cost of the laser is made difficult for a particular test module market. The LED driver LED driver 29 will drive the LED 26 for a desired period of time at a steady current. A low-power USB 2.0-certifiable device (USB 2.0-certifiable device) can achieve a maximum of 1 unit load (1 〇〇 mA) with a minimum operating voltage of 4.4V. A standard power conditioning circuit can be used for this purpose. Photodiode Figure 54 shows the photodiode 184 integrated into the CMOS circuit 86 of the LOC device 301. The photodiode 184 is fabricated as part of the CMOS circuit 86 from -79 to 201209404 without additional masking or steps. This is a significant advantage of CM OS photodiodes over CCDs, another sensing technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard processing steps. The cost of detection on the wafer is low and the size of the assay system can be reduced. The shorter optical path reduces noise in the surrounding environment to efficiently collect fluorescent signals and eliminate the need for conventional optical components for lenses and filters. The quantum efficiency of the photodiode 184 is a fraction of photons that are effectively converted into photo-electrons by impinging on the effective region 185. For the standard tantalum process, the quantum efficiency is in the range of 0.3 to 0.5 under visible light, depending on the method parameters such as the amount of the cover layer and the light absorbing properties. The detection threshold of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection 闽値 can also determine the size of the photodiode 128, and thus the number of hybrid chambers 180 in the hybrid and detection zone 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 1760μηιχ 5 824 μιη), and the actual estimates are in other functional modules such as the pathogen dialysis zone 70. The amplification zone 112 (one or more) is not known until it is inserted. For standard 矽 process, photodiode 1 84 can detect a minimum of 5 photons. However, to ensure reliable detection, the minimum amount can be set to 10 photons. Therefore, when the quantum efficiency is in the range of 0.3 to 0.5 (as discussed above), the fluorescent emission of the probe should be at least 17 photons, but it should be treated as 30 photons to break into the appropriate reliable detection. Error margin. -80- 201209404 Calibration Chamber The inconsistent electrical characteristics of the photodiode 184, the spontaneous excitation of the photon flux that is not completely attenuated, etc., introduce a background shift into the output signal. This background noise can be removed using one or more calibration outputs. The calibration signal can be generated by exposing the calibration photodiode 184 within the array to a respective calibration source. A quasi-source is used to determine the negative result of the target not reacting with the probe. Using the quasi-source as a positive result indicating the presence of the probe-target complex, in the specific example described herein, the low calibration source is provided by a calibration chamber 382 in the hybrid 110, which: Any probe; the probe contained does not have a fluorescent reporter; or contains a probe with a sniffer and quencher, but is designed to quench. The output signals from these calibration chambers 382 are very closely offset from the noise to the output signals of all of the hybrid chambers within the LOC device. Deducting the calibration signal from the output signal generated by the combination will substantially distort the noise and leave the signal generated by the fluorescent emission (if any). Signals generated by the ambient light around the room are also deducted. It should be understood that the negative control described above (refer to Figures 1 to 8 to 111) is detected in the calibration chamber. However, as shown in Figures 94 and 95 (which are magnified views of the illustrations DG and DH of the LOC change g) and Fig. 93, the other is to separate the calibration chamber 382 from the ampersons by fluid separation. And the measurement of the offset can be made by leaving the fluid-isolated rooms vacant and also noise and quasi-signal one or more low-school college indicators. The array of chambers is always close and will be miscellaneous. The back array area is available for needles ί X 728 schemes. Background, or -81 - 201209404 Let chambers containing probes without a sniffer, or chambers containing any "normal" probes with both a sniffer and quencher, isolate the heterozygous condition by fluid isolation get on. The calibration chamber 382 can provide a high calibration source to generate a strong signal in the corresponding photodiode. This strong signal corresponds to all probes that have been hybridized to each chamber. A playback probe with a broadcaster but no quenching or only a broadcaster can continuously provide a signal that approximates the signal from a hybrid hybrid chamber in which a dominant number of probes have been hybridized. It will be appreciated that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and configuration of the calibration chambers 3 82 throughout the array of hybrid chambers are arbitrary. However, if the photodiode 184 is calibrated by a closer calibration chamber 382, the calibration will be more accurate. Referring to Fig. 56, the hybrid chamber array 110 has one calibration chamber 382 in every eight of the hybrid chambers 180. That is, the calibration chamber 382 is located at the center of the hybrid chamber 180 which is arranged in a 3 X 3 square shape. In this configuration, the hybrid chamber 180 is calibrated by the calibration chamber 382 in the immediate vicinity. FIG. 07 shows a differential imager circuit 788 which can subtract the signal emitted by the light-emitting diode 184 corresponding to the calibration chamber 382 from the fluorescent signal emitted from the surrounding hybrid chamber 180 by the stress light-emitting line. . The differential imager circuit 788 samples the signal from the pixels 790 and the "virtual" pixels 792. In one embodiment, the "virtual" pixel 792 is obscured from illumination, so its output signal can be used as a dark reference. Alternatively, the "virtual" pixel 792 can be exposed to the excitation light with the remainder of the array. In the specific case where the "virtual" pixel 7 9 2 is subjected to -82-201209404 illumination, the signal generated by the ambient light in the array area of the room is also deducted. The signal from pixel 790 is very weak (i.e., close to the background dark signal), and it is difficult to distinguish between the background signal and the very weak signal when there is no reference signal to the darkness level. During use, the "read column (read_ro w)" 794 and "read column d (read_row - d)" 795 are activated and the M4 797 and MD4 801 transistors are activated. Switches 807 and 809 are turned off such that the outputs from pixel 790 and "virtual" pixel 792 are stored in pixel capacitor 803 and virtual pixel capacitor 805, respectively. After the pixel signal is stored, switches 807 and 809 are deactivated. The "read line (read_C〇l)" switch 81 1 and the virtual "read line" switch 813 are then turned off and the output switched capacitor amplifier 815 amplifies the differential signal 8 1 7 . The suppression and activation of the photodiode requires suppression of the photodiode 184 during excitation with the LED 26 and activation of the photodiode 184 during the illumination period. Fig. 66 is a circuit diagram of a single photodiode 184 and Fig. 67 is a timing diagram of the photodiode control signal. The circuit has a photodiode 184 and six MOS transistors - MShunt 394, Mtx 3 96, Mreset 3 98, Msf 400, Mread 402 and Mbias 404. At the beginning of the excitation cycle (tl), the transistors MShUnt 3 94 and Mreset 3 9 8 are activated by pulling the Mshunt gate 384 and the reset gate 3 88 high. During this period, the excitation photons will generate carriers in the photodiode 184. These carriers must be removed because the amount of carriers produced is sufficient to satisfy the photodiode 184. During this cycle, Mshunt 3 94 will directly remove the carriers generated by photodiodes 184 -83 - 201209404, while Mreset 3 98 will reset all the carriers accumulated in node 'NS' 406. Leakage of the crystal or due to diffusion of the carrier generated by the excitation in the substrate. After excitation, the capture cycle begins at t4. During this cycle, the emission response of the fluorophore is captured and integrated into the circuit of node 'NS' 406. This can be achieved by pulling the gate 386 high, which activates the transistor Mtx 3 96 and transfers all of the carriers accumulated on the photodiode 184 to the node 'NS' 406. The capture cycle can last as long as the fluorophore emission. The output of all of the photodiodes 184 in the hybrid cell array 11 can be simultaneously captured. There is a time delay between the end of the capture cycle t5 and the beginning of the read cycle t6. This delay is due to the fact that each photodiode 184 within the hybrid cell array 110 (see Figure 52) must be read separately after the capture cycle is completed. The first read photodiode 1 8 4 has the shortest time delay before the read cycle, while the last photodiode 184 has the longest time delay before the read cycle. During reading, the transistor Mread 402 is activated by pulling the read gate 3 93 high. The 'NS' node 406 voltage can be buffered and read using a source-follower transistor MSf 400. More alternative methods for activating or suppressing such photodiodes are discussed below: 1. Suppression methods

Figures 104, 105 and 1-6 show three possible configurations 77, 780 and 782 of Mshunt transistor 394. Mshunt transistor 3 94 has a very high off ratio of -84 - 201209404 when the maximum 値|FCi| = 5 V (activated during excitation). As shown in Fig. 104, the Mshunt gate 3 84 can be configured to be at the edge of the photodiode 184. Optionally, the Mshunt gate 3 84 can be configured to be around the photodiode 184 as shown in FIG. As shown in Fig. 106, the third scheme is to arrange Mshun々1 3 84 inside the photodiode 184. In the third scheme, the active area 185 of the photodiode will be small. These three configurations 778, 780, and 782 can reduce the average path length between all locations within the photodiode 184 to the Mshunt gate 384. In Figure 1, Figure 4, the Mshunt Gate 384 is located on one side of the photodiode 184. This configuration is the simplest to manufacture and has the least impact on the active area 185 of the photodiode. However, it takes a long time for all the carriers staying on the far side of the photodiode 1 84 to be spread to the ^^^ gate 3 84. In Fig. 105, the Mshunt gate 384 surrounds the photodiode 184» which further reduces the average length of the path between the carrier in the photodiode 184 to the Mshunt gate 3 84. However, extending the Mshunt gate 3 84 along the perimeter of the photodiode 1812 3 will greatly reduce the active area 185 of the photodiode. The configuration 782 of Fig. 106 places the Mshunt gate 3 84 within the active region 185. This provides the shortest path average length to Mshunt Gate 3 84 and thus the shortest migration time. However, it has the greatest impact on the active area 185. It also provides a wider leak path. -85- 1 Activated Method. Method 2 a. Drive the shunt transistor with a time delay of a fixed length of the trigger photodiode. 3 b. Drive the shunt transistor with the delay time of the trigger photodiode controlled by the program length of time 201209404. C. The shunt cell system is pulsed with an LED driver for a fixed length of time delay. d. The shunt transistor is driven as described in 2c but with a delay time of program control time. Fig. 69 is a schematic cross-sectional view showing the photodiode 184 and the trigger diode 187 embedded in the CMOS circuit 86 through the hybrid chamber 180. A small area at the corner of the photodiode 184 is replaced with a trigger photodiode 187. - A small area of the triggering light diode 187 is sufficient because the intensity of the excitation light is already high compared to the amount of fluorescent emission. The trigger photodiode 1 8 7 is sensitive to the excitation light 244. The triggering photodiode 18 7 automatically records that the laser beam 244 has disappeared after a brief time delay At 3 00 and activates the photodiode 184 (see Figure 2). This time delay allows the fluorescent photodiode 184 to detect fluorescent emissions from the FRET probe 186 without the excitation light 244. This allows detection to be implemented and improves the signal-to-noise ratio. Both the photodiode 184 and the triggering photodiode 187 are within the CMOS circuit 86 below each of the hybrid chambers 180. The array of photodiodes and appropriate electronic components are combined to form a photosensor 44 (see Figure 65). The photodiode 184 is fabricated without the use of an additional mask or step with a pn_ junction when fabricating the CMOS structure. During the MST manufacturing process, the dielectric layer (not shown) above the photodiode 184 can be arbitrarily thinned using standard MST photolithography techniques to make the target illumination line more than 4 hairs. Body fit very two objects cursor needle probe 18 area within the area 80 Sexual Firefly above the table 8 1 The collar chamber feels the miscellaneous to the 1 body to enter the pole to make one by one nickname light ^ letter the light In the case of -86-201209404, the light is converted into a photocurrent, which can then be detected by the CMOS circuit 86. Alternatively, one or more of the hybrid chambers 180 can be provided with a triggering photodiode 187. These schemes can be used in the combination of the above 2a and skillfully. Fluorescence Delay Detection The following derivation is a description of the time-delayed fluorescence detection using a long-lived fluorophore for the above LED/fluorescent combination. As shown in Fig. 60, the fluorescence intensity is derived as a function of time after excitation from the time 〇 to the ideal pulse of the fixed intensity ie. Let [S1] (0 is equal to the excimer density of time t, and then during excitation and after excitation, the number of excitations per unit volume per unit time can be explained by the following differential equation: Back (7) + Legs... (!) Dt tf hve where C is the molar concentration of the fluorophore, ε is the molar extinction coefficient, is the extinction frequency, and h = 6.62606896(1 0)-34, Js is the Braun constant. This differential equation has the general formula: ^-+p(x)y = g(x) The solution is: ..•(2) | P(x)dx q(x)dx + k ΛΧ) = —— Now solve the equation (1) using this equation -87- 201209404 [sim=l^LL+ke-^ ...(3) hVe and from equation (3) now at time ί i, [1 ] (i,) Ι^εοτ f . /rk = - ~~f-e'lTf ...(4) hv· Substituting equation (4) into [magic] (1) K£cxf lescTj _(ί_, ι)/Τ(. ---g hve hve at time O : ...(5) hve hvc When GO, the excitatory state decays exponentially and it can be expressed as [phantom] (0 = [magic] (6) 〆... (6) Substituting equation (5) into equation (6): _) = And 2[1- β-(/2-«,)/Γ/]β-(/-ί2)/ν (7) kVe Fluorescence intensity can be provided by the following equation:

If{t) = -^^-hv/nl (8) αχ where V/ is the fluorescence frequency, η is the quantum yield and 1 is the optical path length. Now from equation (7): 蚵 S1K0 dt

...(9) Substituting equation (9) into equation (8):

If (〇 = Ι(εαΙη-^[\ -e'Vl"')lTr ...(10)

For ^ 〇〇, I At) -> ΙεεαΙη^β'(,~,ι),Τί -88- 201209404 Therefore, we can write the following approximate equation to illustrate the egg after the 'sufficiently long excitation pulse stimulation Light intensity time delay phenomenon: /, (1)=/e£c/;7"^ (' '2>/r/ for QG ...(11) ^ e In the previous paragraph, we concluded that when (a)-ii>&gt ;Tf, for id2, heart W = person ^ e From the above equation, we can introduce the following formula: nf{t) = ne£c/77e~(,~,2>/r/ ...( 12) where ^ (0 = :^ is the number of fluorescent photons per unit area per unit time, and hvf & =·^- is the number of excitation photons per unit area per unit time, Αν, thus, 00 \(, ) = ]·~(_ ...(13) h where seven is the number of fluorescent photons per unit area, ο is the instantaneous time at which the photodiode is activated. Substituting equation (1 2 ) into equation (1 3 ): Βθ nf =^neec^e~{l~h)lTfdt (14) >} Now, the number of fluorescent photons of the photodiode is reached per unit area per unit time, seven (0, which can be provided by: Ns(〇= ^fm -(15) where nine are the light of the optical system Efficiency. Substituting the formula (1 2 ) into the formula (i 5 ) we find that -89 - 201209404 η!{ΐ) = φύηίεοΙηβ~{ι^)Ι^ (16) Similarly, the fluorescence area per unit reaches the light II The number of fluorescent photons of a polar body will be as follows: 屺=(ί) and substituting (1 6 ) and integrating: hs =φ0η(,εαΙητ/β~(1ί~'ι)/Τ/ Therefore, ns =φ0ήίεοΙητ /β^'Τί (17) Since the excitation photon flow is attenuated much faster than the fluorescent photon flow, when the photodiode 184 is excited by the photon due to the photon, the photodiode 184 is excited by the photodiode 184. The photon is the best when the photon generation rate is the same. The electron output rate of the sensor per unit of fluorescence area produced by the fluorescence is: where is the quantum efficiency of the inductor at the wavelength of the fluorescence. Substitution (1 7) We can get: β/(ί) = φ/φ0ή€εαΙηβ-{,-,ΐ)ΙΤ/ (18) Similarly, the sensor electron output rate per unit of fluorescence area due to excitation photons is : 9 is the quantum efficiency of the inductor at the excitation wavelength and is the time constant equivalent to the "off" characteristic of the excited LED. For the time between t2 'attenuation of the photon flux of the LED will increase the intensity of the fluorescence signal and to extend its decay time, but we assume that this line to the If (the square only a negligible impact, so a conservative policy. -90 - 201209404 Now, as mentioned earlier, when Ά(ί3)=·έ·β(ί3), there is the best ί3値. So 'from equations (18) and (1 9 ) we get: ΦΑΙεοΙψ” :ne-ih_hVr. After rearranging we found:

In (ring M), 3-, 2=-i~f— (20)

Xf Xe From the first two paragraphs, we get the following two equations of operation: ns =ΦΰΚΡτίβ~6"Τ/ ...(21) φ \ / -(22)

Xf Xe where corpse = £C/; 7 and ΔΠ3-ί2. We also know the truth. The best time to detect fluorescence using the Philips LXK2-PR14-ROO LED and Pulsar 650 dye and the number of measured photons are determined as follows: The best detection time is determined using equation (22): ' The concentration of the amplicon is called, and assuming that all the amplicons are heterozygous, the concentration of the fluorescent fluorophore is: c = 2.89 (l〇V6mol/L, the height of the chamber is the length of the optimal path 1 = 8 ( 1〇Γό m. We see that the area of the fluorescent light is equal to the area of our photodiode, but our actual fluorescent area is basically larger than the area of our photodiode; thus we roughly assume the light collection efficiency of the optical system used. =0.5. From the characteristics of the photodiode, f = i 〇 is very conservative for the quantum efficiency of the photodiode at the fluorescence wavelength of -91 - 201209404. Number 値. Under the typical LED attenuation lifetime of '=0.5 ns and using the Pulsar 650 specification, Δί : F = [1.48(10)6][2.89(10)-6][8(10)'6]( l) = 3.42(10)5 _ ln([3.42(10)-5](10)(0.5)) _ _1___1_ 1(10)-6 ~ 0.5(10)-9 =4.34(1 〇y9s Detected The number of photons is determined by equation (2 1 ). First, the number of excitation photons emitted per unit time is determined by detecting the following emission geometry. The Philips LXK2-PR14-R00 LED has a Lambertian radiation pattern. Therefore: Η·, = )ϊ/0 cos (6) (23) where h• is the number of photons emitted per unit time from the unit solid angle of the front axial angle of the LED, and ^ is the front axle To β値 The total number of photons emitted by the LED per unit time is: «, = Π =Jnl0 cos(9)dQ η Now, -92- ...(24) 201209404 ΑΩ = 2π[1 — cos(0 + A0)] - 2/τ[1 — cos(0)] ΔΩ = 2^-[cos(0) - cos(^ + Δ^)] sin[specific]+ 4;r cos(0) sin2 (specifically dfl = 2Trsin( 0)d0 Substituting this formula into equation (24) π 2 ή, =j2ml0cos(9)sin(e)de 0 = ^/0 Rearrange, we get: «,0=^ ...(26) π This LED The output power is 0.515 W and ve= 6.52(10) 14 Hz, so: =_0.515_ ~[6.63(1〇Γ34][6.52(10)14] = 1.19(10)18 photon/s For equation (2 6 ), we get: ...1.19(10)18 71,0 = π = 3.79(10)17 photons/s/sr refer to Fig. 61, which schematically shows the LED Optical center 252 and lens 254 of 26. The photodiode is 16 μηιχ16 μηι, and for the photodiode at the center of the array, the solid angle (Ω) of the light emitted from the LED 26 to the photodiode 184 is approximately: Ω = sensor area /r2 93 - 201209404 [16 (10)-6][16(10)-6] 2.825(10)'3]2 = 3.21(l〇)'5 sr It should be understood that the center of the photodiode array 44 is used. The photodiode 184 is calculated here. Sensors located at the edge of the array receive only less than 2% of photons at the Langber's excitation source intensity distribution when heterozygous. The number of excitation photons emitted per unit time is: ne = ή, Ω ... (28) = [3.79(10)17][3.21(1〇Γ5] =1.22(10)13 Photon/s Reference now (2 9): ns=</>0neFTfe~^ ns = (0.5)[1.22(10),3][3.42(10)-5][l(10)-6]eJ,34(,〇r, /l(10)"6 = 2 0 8 photon number/sensors. When using the Philips LXK2-PR14-R00 LED^ Pulsar 650 fluorophore, we can easily detect the emission of this number of emitted photons. Any of the hybrid events. The SET LED emission geometry is shown in Figure 62. At ID = 20 mA, the LED has a minimum optical power output centered at λε = 340 nm (the absorption wavelength of the ruthenium chelate) pi = 240 μ\ν. Driving the LED with ID = 200 mA linearly increases the output power to Pl = 2.4 mW by placing the LED optical center 2 5 2 about 1 from the hybrid array i丨0 At 7.5 mm, we can concentrate this output flux to a circle with a diameter of up to 2 mm. The photon flux of 2 mm-diameter spots away from the hybrid plane can be seen by equation (27). -94- 201209404 « /= Ρι Ave 2.4(10)-3 ~[6.63(10)-34][8.82(10)14] = 4.10(10)15 Photon /s From equation (28) we know: he = =4·10(10)15·)72_ 4i(i〇r3]2 = 3.34(10)11 photons/s Now, call up equation (22) and use the previously listed Tb chelate property ln[(6.94(10)-s)(10)(0.5)] _1___1_ 1(10)-3 _ 0.5(10)-9 = 3.98(10)'9s Now, from equation (21) : ns = (0.5)[3.34(10)u][6.94(10)-5][l(10)"3>-J-98(1〇r,/,(I〇rl =1 1,600 The photon/sensor can easily detect that the number of photons emitted by the SET LED and the 铽 chelating system in a hybrid event can reach its theoretical number and far exceed the 30 photons required for the above-mentioned trusted detection. The minimum number of probes and the maximum pitch between the photodiodes is detected on the wafer to avoid the need to detect via confocal microscopy (see background of the invention). This is a saving from the traditional detection technology. An important factor in time and cost. Traditional detection requires the use of a lens or curved mirror for optical imaging. By using non-optical imaging, the diagnostic system avoids the need for complex and bulky optical components. Keeping the photodiode close to the probe provides the advantage of providing extremely high collection efficiency: when the material thickness between the probe and the light is at the 1 micron level, the angle of the emitted light can be as high as 173°. This angle is calculated by considering the light emitted from the probe at the centroid of the hybrid chamber closest to the photodiode having a flat active surface parallel to the surface of the hybrid chamber. The emission angle cone in which light can be absorbed by light is defined as the perimeter of the emission probe and the perimeter of the flat surface. For a 16 micron χ 16 micrometer sense, the apex angle of the cone is 170°; in the extreme case (where the light body is spread out so that its area matches the 29 micron χ 19.75 micron hybrid area), the top The angular angle is 173°. It is easy to separate the chamber surface from the polar active surface by 1 micron or less. The use of a non-optical imaging scheme requires that the photodiode 184 be in close proximity to the hybrid chamber to collect sufficient fluorescent emission photons. The large spacing between the photodiode and the probe can be determined in the following manner with reference to Figure 54. Using a chelating chelate fluorophore and SET UVTOP3 3 5T039BL, we calculated that 1 1 600 photons were arriving from my individual hybrid chambers 18 〇 to my micron X 16 micron photodiode 184. In doing this calculation, the collection area of our hybrid chamber 180 has the same bottom area as our photodiode 185, and one-half of all the hybrid photons have the photodiode 1 84. That is, the light collection of the optical system & = 0 · 5. More precisely, we can write out = [(the bottom of the divergent chamber light collection area) / (photodiode area)] [Ω / 4π], where Ω = the second pole collection on the base of the hybrid chamber (the surface Polar body detector - the light of the pole room is the most LEDs 16 The hypothetical surface reaches the generation of the efficiency area -96 201209404 The solid angle of the light diode of the point. For the regular pyramid geometry: Q = 4arcsin ( A2/(4d〇2 + a2)), where d〇=the distance between the chamber and the photodiode, and a is the size of the photodiode. Each of the hybrid chambers can release 23,200 photons. The detection threshold of the polar body is 11 photons; therefore, the minimum optical efficiency required is: φΰ = 1 7/23 200 = 7.3 3 x 1 0'4 The bottom area of the collection area of the hybrid chamber 180 is 29 micron χ 19.75 micron 〇 solve do, we get the maximum limit distance between our hybrid chamber bottom and the light diode 1 84 d 〇 = 249 microns. Within this limit, the above defined cone angle is only 〇.8 °. It should be noted that this analysis ignores the slight effects of refraction. LOC variants all LOC devices 3 described and above. 01 is just one of many possible LOC device designs. LOC device changes using different combinations of the different functional areas described above will be described by placing the sample into the detection and/or displaying it in a schematic flow chart to show some Possible combinations. Where appropriate, the flow chart will be divided into sample insertion and preparation stages 28 8 , extraction stage 290, incubation stage 291, amplification stage 292, pre-hybrid stage 293, and detection stage 294 For all of the LOC variants described above or only in schematic form, for the sake of brevity and clarity, not all of the accompanying complete layouts are shown. Also for clarity, no smaller functional units are shown. For example, liquid sensors and temperature sensors, it should be understood that these units have been incorporated into the appropriate "97 - 201209404 position in each of the following LOC device designs.

The LOC variant XII LOC variant XII 758 is shown in Figures 96 to 103. The LOC device extracts 290, culturing 291, amplifying 292 and detecting 294 pathogen DNA, and using a pre-hybridization purification step 293 to increase hybrid efficiency. The sample (eg whole blood) It is applied to the sample inlet 6 8 (see Figure 9 8) and the capillary action pulls the sample to the surface tension valve 118 where the anticoagulant from the reservoir 54 is added. The sample will continue to flow in the cap channel 94 to the pathogen dialysis zone 70. The dialysis zone 70 has a bypass passage 600 to prevent entrapment of air bubbles (see Figure 98). After dialysis by the pathogen dialysis zone 7 〇, the red blood cell and white blood cell streams will lead to the waste reservoir 76 and the pathogens continue to flow in the sample stream to the surface tension valve 1 2 8 where they are added from the reservoir 5 6 cytosolic reagent. The sample is left in the chemical cell chamber 130 by the boiling-start valve 206 and fills the chemical cell chamber until the lysing reagent has diffused throughout the sample and releases most, if not all, of the pathogen DNA. When the boiling start valve 206 is opened, the sample will flow to the surface tension valve 132 where the restriction enzyme, ligase and linker primer from the reservoir 58 are added. The sample will fill the incubation zone 1 14 and be heated while performing restriction enzyme digestion of the pathogen DN A and linker ligation (see Figure 98). After restriction enzyme digestion and ligation of the linker, the boiling starter valve 72 is opened to allow the sample to flow into the amplification zone Π2. The amplification mixture from reservoir 60 is added through surface tension valve 138 and polymerase from reservoir 62 passes through surface tension valve 140 as it flows to amplification zone -98-201209404112. The pathogen DNA is first amplified by thermal cycling before the boiling start valve 1 〇 8 is opened to allow the amplicon to flow to the small component dialysis zone 682 and the large component is removed (see Figure 98). As shown in Figures 101 and 102, the small component dialysis zone 682 has a large component channel 760 between the two small component channels 762 built into the bottom channel layer 100 (see Figure 97). The large component channel 760 is coupled to the small component channels 762 through a series of apertures in the form of inverted cone openings 764. In most practical applications, the orifice is 1 to 8 microns wide and 1 to 8 microns high. As the amplicon flows to the large component channel 760, small components (smaller than the inverted cone opening 764) begin to diffuse into the small component channels 762. As the sample stream continues to flow to the downstream end of the small component dialysis zone 682, the concentration of small components in the large component channel 760 decreases. Other advantages of microfabricated orifices are that the number of orifices per unit length along the channel is very high, so the separation is more efficient. In order to effectively separate the components having the desired size, the distance between adjacent orifices is between 1 micrometer and 10 micrometers; in the specific example shown in Fig. 102, the distance between adjacent orifices is 8 micrometers. Figure 103 shows the downstream end of the small component dialysis zone 682. The large component channel 760 branches into a wide meandering tip with a closed end 766 that acts as a waste reservoir. The two small component channels 762 lead to the opposite side of the hybrid array 1 1 'where the two channels follow the path through the array to the respective closed ends 768. The small component amplicon first fills all individual hybrid chambers 180' and then periodically starts the hybrid heater and performs subsequent probe-target hybrid detection (as previously described). •99- 201209404 This small component dialysis zone 6 82 removes cell debris that remains in the sample stream after cell lysis. Cell debris can interfere with heterozygous efficiency.

LOC Variant XLIV Figure 82 shows LOC variant XLIV 674, which can detect pathogens via nucleic acid amplification. The sample is applied to the sample inlet 68 and the capillary action pulls the sample to the surface tension valve 1 18 . The reagent in reservoir 54 is mixed with the sample via surface tension valve 187 and the flow continues to flow to phase 292. The reagent reservoir 54 may contain a chemical cytolytic reagent if desired. The amplification mixture of reservoir 60 is added via surface tension valve 138 and the polymerase of reservoir 62 is added via surface tension valve 140. The thermal cycling of the amplification zone 112 will amplify genetic material from any cytolytic cell. Chemical cytolysis and/or thermocytolysis can be carried out in the amplification zone 112 prior to thermal cycling. When sufficient amplicons are generated, the boiling start valve 108 opens to allow the capillary drive flow to flow into the small component dialysis zone 682. Small sample components such as dissolved molecules and amplified nucleic acids are left in the sample. Large components such as cell membrane debris and any uncytosoluble pathogens will flow into the waste reservoir 766. The small components in the purified sample continue to flow into the hybrid chamber array 11 for the photodetector 44 to be detected by the hybrid.

LOC Variant XLVI Figure 83 shows schematically LOC Variant XLVI 676, which can use a large component dialysis zone 686, incubation zone 114, amplification zone 112, hybrid chamber array 11 and photosensor 44 to detect pathogens. . The large component dialysis zone 68 6 is designed to retain components (including pathogens) that are larger than a particular threshold size. Components smaller than the threshold are transferred to the waste reservoir 768. The purified sample continues to flow into the incubation phase 291 where it is introduced into the restriction enzyme, ligase and linker primer from the reservoir 58 via the surface tension valve 132 and subjected to restriction enzyme digestion and linker ligation in the incubation zone 114. After digestion, the boiling start valve 108 opens and the sample enters the expansion zone 112 where it is added to the amplification mixture of reservoir 60 via surface tension valve 138 and to the polymerase of reservoir 62 via surface tension valve 140. After amplification of the nucleic acid, the amplicon enters the pre-hybridization purification phase 293. The small component dialysis zone 682 will remove large components from the amplicon from the amplification zone 1 12 . The large components such as cells, pathogens, particles and debris are transferred to the second waste reservoir 766 and left in the sample to eat smaller components such as amplicons which will be filled with the hybrid chamber array 110. The hybrid systems are detected by photosensor 44.

LOC Variant XLVIII Figure 85 schematically shows LOC variant XLVIII 67 8, which can use restriction enzymes, ligase and linker incubation zone 1 14 , amplification zone 1 1 2, small component dialysis zone 682, hybrid compartment Array 1 10 and light sensor 44 are used to detect pathogens. The sample is applied to the sample inlet 6 8 and the capillary action pulls the sample to the surface t tension valve 118. The reagent in the reservoir 54 containing the chemical cytosol is mixed with the sample through the surface tension valve 186 and the flow continues into the incubation phase 291. The sample is passed through a surface tension valve 1 32 to bind the restriction enzyme, ligase and linker from the reservoir 58 and subjected to restriction enzyme digestion and linker ligation in the incubation zone 1 14 until exiting at the incubation zone 1 14 The boiling start valve 1〇8 is opened and the capillary-101 - 201209404 fine drive flow re-enters the amplification phase 2 9 2 . The amplification mixture of reservoir 60 is introduced through surface tension valve 138 and polymerase of reservoir 62 is passed through surface tension valve 140. The thermal cycle of the amplification region Π2 amplifies the genetic material from any cytolytic cell. When sufficient amplicons are generated, the boiling start valve 108 opens to allow the capillary drive flow to flow into the small component dialysis zone 68 2 . Small sample components such as dissolved molecules and amplified nucleic acids are left in the sample. Large components such as cell membrane debris and any non-lytic pathogens will flow into the waste reservoir 766. The small components in the purified sample continue to flow into the hybrid chamber array 1 1 for the photodetector 44 for hybrid detection. Conclusion The devices, systems, and methods described herein enable people to perform molecular diagnostic tests at a care location and at a low cost. The above-described systems and their components are purely for display and can be easily understood by those skilled in the art from the numerous variations and modifications of the spirit and scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will now be described with reference to the following drawings, wherein: FIG. 1 shows a test module and a test module reader using fluorescence detection; Figure 2 is a schematic view of the electronic components in the test module using the fluorescence detection: Figure 3 is a schematic view of the electronic components in the test module reader -102-201209404 Figure 4 is the LOC device A schematic representation of the overall structure; Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device and its features and structures of all layers laminated to each other; Figure 7 is a separate view of the top cover structure Figure 8 is a top perspective view of the top cover and the internal passages and reservoirs are indicated by dashed lines; Figure 9 is an exploded top perspective view of the top cover and the internal passages and reservoirs are indicated by dashed lines; The figure shows a bottom perspective view of the channel configuration above the top cover; Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device separately; Figure 12 is a schematic cross-sectional view of the LOC device at the sample inlet. Figure 13 is Figure 6 is an enlarged view of the illustration AA shown in Figure 6; Figure 14 is an enlarged view of the illustration AB shown in Figure 6; Figure 15 is an enlarged view of the illustration AE shown in Figure 13; A partial perspective view of the layered structure of the LOC device in the inset AE; Figure 17 is a partial perspective view showing the layered structure of the LOC device in the inset AE; Figure 18 is a layer showing the LOC device in the inset AE Partial perspective view of the structure; Figure 19 is a perspective view showing the layered structure of the LOC device in the illustration AE -103 - 201209404; Figure 20 is a view showing the layered structure of the LOC device in the illustration AE Fig. 21 is a partial perspective view showing the layered structure of the LOC device in the inset AE; Fig. 22 is a schematic sectional view of the lysing reagent reservoir shown in Fig. 2; Fig. 23 is a view A partial perspective view of the layered structure of the LOC device in the inset AB; Fig. 24 is a partial perspective view showing the layered structure of the LOC device in the inset AB; Fig. 25 is a view showing the LOC device in the illustration AI Partial perspective view of the layered structure; Figure 26 shows the illustration of the LOC device Partial perspective view of the layered structure in AB; Figure 27 is a partial perspective view showing the layered structure of the LOC device in the inset AB; Figure 28 is a layered structure showing the l〇C device in the inset AB Partial perspective view: Figure 29 is a partial perspective view showing the layered structure of the LOC device in the inset ab; Section 30I® is a schematic sectional view of the amplification mixture reservoir and the polymerase reservoir; The figure shows a characteristic component of a boiling start valve separately; -104- 201209404 Figure 3 2 is a schematic sectional view of the boiling start valve of the 3 3 -3 3 line shown in Figure 31; Figure 33 shows the 15th figure Fig. 34 is a schematic cross-sectional view showing the upstream end of the dialysis zone of the line 35-35 of Fig. 33; Fig. 35 is an enlarged view of the illustration AC of Fig. 6, the 36th The figure is a further enlarged view of the amplification region inside the illustration AC; Fig. 37 is a further enlarged view of the amplification region inside the illustration AC; Fig. 38 is a further enlarged view of the amplification region inside the illustration AC; Figure 38 shows the further enlargement of the inside of the illustration AK Figure 1 Figure 40 is an illustration of AC Further enlarged view of the amplification chamber; Fig. 41 is a further enlarged view of the amplification region inside the illustration AC; Fig. 42 is a further enlarged view of the amplification chamber inside the illustration AC; Fig. 43 is a diagram showing the 42 A further enlarged view of the inside of the illustration AL; Fig. 44 is a further enlarged view of the amplification area inside the illustration AC; Fig. 45 is a further enlarged view of the inside of the illustration AM shown in Fig. 44. Fig. 46 is an enlarged view of the interior of the illustration AC. Further enlarged view of the expansion chamber; Fig. 47 is a further enlarged view of the inside of the illustration AN shown in Fig. 46. ^ Fig. 48 is a further enlarged view of the amplification chamber inside the illustration AC; Fig. 49 is an enlarged view of the interior of the illustration AC A further enlarged view of the expansion chamber; Figure 50 is a further enlarged view of the amplification region inside the illustration AC; -105- 201209404 Figure 51 is a schematic sectional view of the amplification region; Figure 52 is the hybrid region A magnified plan view; Fig. 53 is a further enlarged plan view of the two separate compartments; Fig. 54 is a schematic sectional view of a single hybrid chamber; and Fig. 55 is a humidifier of the illustration AG shown in Fig. 6. Magnified image; Figure 56 is the illustration of Figure 52 showing AD Figure 57 is an exploded perspective view of the LOC device in the illustration AD; Figure 58 is a diagram of the FRET probe in a closed configuration; Figure 59 is a diagram of the FRET probe in an open and hybrid configuration; The figure shows the intensity of the excitation light as a function of time; Figure 6 is the excitation illuminating geometry of the hybrid chamber array; Figure 62 is the sensor electronic technology (Sensor Electronic Technology) LED illuminating geometry; The figure is an enlarged plan view of the humidity sensor shown in the illustration AH of Fig. 6; Fig. 64 is a schematic sectional view of a white blood cell target dialysis area; Fig. 65 is a view showing a part of the photodiode of the light sensor Figure 66 is a circuit diagram of a single photodiode; Fig. 67 is a timing diagram of the photodiode control signal; and Fig. 68 is an enlarged view of the evaporator shown in the illustration AP of Fig. 55; Figure 69 is a schematic cross-sectional view through a hybrid chamber with a detection photodiode and a trigger photodiode; Figure 70 is a diagram of linker-primed PCR-106-201209404 Figure 7 1 shows a test module for a lancet Figure 72 is a graphical representation of the overall structure of the LOC variant VII; Figure 73 is a plan view of the LOC variant VIII and the features and structures of all layers laminated to each other; Figure 74 is shown in Figure An enlarged view of the illustration CA of Fig. 73;

Figure 75 is a partial perspective view showing the layered structure of the LOC variation type VIII inside the inset CA of Fig. 73; Fig. 76 is an enlarged view of the illustration CE shown in Fig. 74; Fig. 77 is the LOC variation type VIII. A schematic diagram of the overall structure; Figure 78 is a schematic diagram of the overall structure of the LOC variant XIV; Figure 79 is a schematic diagram of the overall structure of the LOC variant XLI; Figure 80 is a schematic diagram of the overall structure of the LOC variant XLII; Figure 81 is a schematic diagram of the overall structure of the LOC variant XLIII; Figure 82 is a schematic diagram of the overall structure of the LOC variant XLIV; Figure 83 is a schematic diagram of the overall structure of the LOC variant XLVI; Figure 84 is a LOC variant XLVII Schematic diagram of the overall structure; Figure 85 is a schematic diagram of the overall structure of the LOC variant XLVIII; Figure 8 is a linear fluorescent probe map of the primer-linker during the initial amplification. Figure 8 7 shows the subsequent amplification cycle. During the introduction of the primer-linker linear luminescence probe map; the 88A to 88F diagram shows the thermal cycle change of the primer-linker luminaire ring probe; -107- 201209404 Figure 89 is relative to the miscellaneous Array of rooms and such light II Schematic diagram of the excited LED of the body; Figure 90 is a schematic diagram of the excitation LED and the optical lens guiding the light toward the array of hybrid chambers of the LOC device; Figure 91 is the excitation of the guiding light toward the array of hybrid chambers of the LOC device Schematic diagram of LEDs, optical lenses, and optical cymbals; - Figure 92 is a schematic diagram of the arrangement of the excitation LEDs, optical lenses, and mirrors that direct the light toward the array of hybrid chambers of the LOC device; Figure 93 shows all of the stacked layers. The feature member and the plan view showing the position of the illustration DA to DK; Fig. 94 is an enlarged view of the illustration DG shown in Fig. 93; Fig. 95 is an enlarged view of the illustration DH shown in Fig. 93; Fig. 96 is the LOC Graphical representation of the overall structure of the variant XII; Figure 97 is a perspective view of the LOC variant XII; Figure 98 is a plan view showing the FA to FC position of all the features and illustrations of the LOC variant XII; The figure is a plan view showing the top cover feature of the LOC variant XII separately; Fig. 100 is a plan view showing the structure of the CMOS + MST device of the LOC variant XII alone; · Fig. 101 is an illustration FA shown in Fig. 98 Put Figure 102 is an enlarged view of the illustration FB shown in Figure 98; Figure 103 is an enlarged view of the illustration FC shown in Figure 98; Figure 1 04 is the shunt transistor for the photodiode Specific examples; -108- 201209404 Figure 105 is a specific example of the shunt transistor for the photodiode; Figure 106 is a specific example of the shunt transistor for the photodiode; Figure 107 is a differential imager (differential imager) circuit diagram, FIG. 108 schematically shows a negative control fluorescent probe in a stem-loop configuration; and FIG. 109 is a schematic view showing the negative control fluorescent probe of the 108th aspect in an open configuration; Fig. 110 is a view schematically showing a positive control fluorescent probe in a stem-and-loop configuration; FIG. 11 is a view schematically showing a case where the positive-controlled fluorescent probe of the first aspect is in an open configuration; The figure shows the test module and test module reader using ECL detection; the first 1 3 is a schematic view of the electronic components in the test module using ECL detection; the U 4 figure shows a test module And another test module reader; Figure 115 shows a test module and a host system with different Test module reader of the database; [Key component symbol description] I 〇: Test module II: Test module 1 2: Test module reader 13: Shell 14: Micro_USB plug 109 201209404 1 5 : Inductor 1 6 : Micro-USB埠17: User Interface (UI) Touch Screen 18: Display Screen 19: Button 2 0: Start Button 2 1 : Honeycomb Radio 22: Aseptic Sealing Tape 2 3 : Wireless Network Connection Line 2 4: Large tank 25: satellite navigation system

26 : LED 2 7 : Data memory 29 : USB-compatible LED driver 30 : LOC device 3 1 : Power conditioner 3 2 : Power supply capacitor 3 3 : Clock 3 4 : Controller 3 5 : Register 36: USB Device driver 3 7 : drive 38 : RAM 3 9 : drive - 110 - 201209404 40 : program and data flash memory 41 : register 42 : processor 43 : program memory 44 : light sensor 45 : indicator 46: Top cover 47: _ USB power/indicator module 48: CMOS + MST chip (device) 49: Foam illustration or other perforated element 54: (anticoagulant) reservoir 56: (cytosol) storage 5 7 : Printed circuit board (PCB) 5 8 : reservoir 60 : reservoir 62 : reservoir 64 : lower seal 66 : top wall layer 6 8 : sample inlet 7 0 : dialysis zone 72 : waste channel 74 : target channel 7 6 : waste unit 78 : reservoir layer - 111 - 201209404 8 0 : top cover channel layer 8 2 : upper seal layer 8 4 : tantalum substrate 86 : CMOS circuit 87 : MST layer 8 8 : passivation Layer 90: MST channel 92: Downcomer 94: Top cover channel 96: Upper tube 9 7. Wall area 9 8: Meniscus anchor 1 00: MST channel layer 101: Laptop/note Computer 102: Capillary start feature 103: Dedicated reader 105: Desktop computer 1 〇 6: Boiling-start valve 107: E-book reader 1 〇 8: Boiling-starting valve 1 〇 9: Tablet 1 1 〇: Hybrid chamber array 1 1 1 : Epidemiological data 1 1 2 : Amplification area -112 - 201209404 1 13 : Genetic data 1 1 4 : Cultivation area 1 15 : Electronic health record (HER) 1 1 6 : Resistance Coagulant 1 18 : Surface tension valve 1 1 9 : Sample flow 1 2 0 : Meniscus 121 : Electronic medical record (EMR ) 122 : Vent hole 123 : Personal health record (PHR) 125 : Network 1 2 6: Boiling-starting valve 1 2 8 : Surface tension valve 1 3 0 : Chemical cytolytic zone 1 3 1 : Mixing zone 132 : Surface tension valve 1 3 3 : Incubation zone inlet channel 1 3 4 : Downcomer (opening) 1 36: Optical window 1 4 6 : Valve inlet 1 5 0 : Valve down conduit 1 5 2 : Heater 153 : Boiling - Start valve heater contact 1 5 4 : Heater-113- 201209404 1 5 6 : Heater connection Point 158: Microchannel 160: Amplification zone outlet channel 1 6 4 : Small hole 1 6 6 : Capillary starting characteristic member 1 6 8 : Dialysis upper catheter hole 170: Temperature sensor 174: Liquid sensor 1 7 5 : Diffusion barrier 1 7 6 : Flow path 1 7 8 : Liquid sensor 1 80 : Hybrid chamber 1 8 2 : Heater 1 84 : Light diode 1 8 5 : Active area 186: FRET Probe 187 : Trigger light diode 1 8 8 : Water reservoir 190 : Evaporator 1 9 1 : Heater 192 : Water supply channel 1 93 : Upper pipe 1 94 : Down pipe 1 9 5 : Metal top layer - 114 - 201209404 1 9 6 : Humidifier

198: first upper conduit hole 202: CIF 204: dialysis MST channel 206: boiling start valve 207: boiling start valve 208: top cover channel liquid sensor 210: heated microchannel 212: intermediate MST channel 2 1 8 : T i A1 electrode 220: TiAl electrode 222: gap 23 2 : humidity sensor 2 3 4 : heater 23 6 : FRET probe 23 8 : target nucleic acid sequence 240 : ring 242 : stem segment 244 : excitation light 246 : fluorescence Group 248: Quenching 25 0 : Fluorescence emission 2 5 4 : Lens 28 8 : Sample placement and preparation -115- 201209404 290 : Nucleic acid extraction 2 9 1 : Nucleic acid incubation 292 : Nucleic acid amplification 2 94 : Detection And analysis 2 9 6 : first electrode 29 8 : second electrode 3 00 : pre-programming delay 301 : LOC device 328 : white blood cell dialysis zone 3 7 6 : conductive column 3 7 8 : forward control probe 3 8 0 : Reverse control probe 3 8 2 : Calibration chamber 3 8 4 : Mshunt gate 3 8 8 : Reset gate 3 9 0 : Lancet 3 92 : Lancet release button 3 9 3 : Read gate 3 94 : MOS transistor Mshun 396 : MOS transistor Mtx 3 98 : MOS transistor Mrese 400: MOS transistor Msf 402 : MOS transistor Mread 404: MOS transistor Mbias 201209404 4 0 6 : node, N S ' 408 : film seal 4 1 0 : film cover

518 : LOC variant VIII 5 9 4 : Interfacial layer 600 : Bypass channel 602 : Interface target channel 6 04 : Interface waste (abandoned cell) channel

673 : LOC variant XLIII 674 : LOC variant XLIV 6 76 : LOC variant XLVI 677 : LOC variant XLVII 678: LOC variant XLVIII 6 8 2 : small component dialysis zone 686: large component dialysis zone 6 9 2 : linear Probe 694: amplification blocker 696: probe sequence 698: complementary sequence 700: oligonucleotide primer 704: stem loop probe 7 0 8 : stem strand 7 1 〇: another strand 7 1 2 : An optical prism-117- 201209404

7 1 4 : second optical 稜鏡 7 1 6 : first mirror 7 1 8 : second mirror 728 : LOC variant X 75 8 : LOC variant XII 760 : large component channel 7 6 2 : small component channel 764: opening 766: closed end 76 8 : closed end 7 7 8 : configuration 780 : configuration 782 : configuration 7 8 8 : differential imager circuit 7 9 0 : pixel 792 : "virtual" pixel 794 : "read Column" 79 5 : "Read column d" 7 9 6 : Negative control probe 797 : M4 transistor 7 9 8 : Positive control probe 8 0 1 : MD 4 transistor 8 0 3 : Pixel capacitor 805: Virtual pixel Capacitor 201209404 8 〇7: Switch 8 〇9: Switch 81 1 : "Read Row" Switch 8 13 : Virtual "Read Row" Switch 8 1 5 : Capacitor Amplifier 8 1 7 : Differential Signal 860: ECL Excitation Electrode (Cathode 8 70 : ECL excitation electrode (anode) -119-

Claims (1)

201209404 VII. Patent Application Range: 1. A wafer-on-lab (LOC) device for genetic analysis of biological samples, the LOC device comprising: a receiving inlet for receiving the sample; a supporting substrate; a reagent reservoir; a nucleic acid amplification region located downstream of the incubation region for amplifying a nucleic acid sequence in the sample; and an amplification located downstream of the nucleic acid amplification region for filtering the nucleic acid amplification region before hybridization a dialysis zone of the extensor, the dialysis zone being configured to remove cell debris from the amplicon: wherein both the nucleic acid amplification zone and the dialysis zone are carried on the support substrate 〇2. The LOC device of item 1 further comprises a photo sensor and a hybrid region downstream of the dialysis zone, the hybrid region having a hybrid probe array to form a probe-target with the target nucleic acid sequence in the sample A hybrid, wherein the photosensor is configured to detect the probe-target hybrid. 3 _ The LOC device of claim 2, wherein the volume of each hybrid chamber is less than 900,000 cubic microns. 4. The LOC device of claim 3, wherein each of the hybrid chambers has a volume of less than 200,000 cubic microns. 5. The LOC device of claim 4, wherein each of the hybrid chambers has a volume of less than 40,000 cubic microns. -120-201209404 6. The LOC device of claim 1, wherein the dialysis zone has a large component channel, a small component channel, and a plurality of small holes capable of fluidly connecting the large component channel to the small component channel The pores are sized to allow a nucleic acid sequence to flow from the large component channel to the small component channel, while cell debris larger than the small pore is retained in the large component channel, the small component channel and the hybrid region Fluid communication. 7. The LOC device of claim 1, wherein the nucleic acid amplification region is an isothermal nucleic acid amplification region. 8. The LOC device of claim 7, further comprising a reagent reservoir for containing reagents for isothermal nucleic acid amplification; and a surface tension valve having a small aperture configured to immobilize the reagent The meniscus is such that the meniscus allows the reagent to remain in the reagent reservoir prior to contact with the fluid sample to remove the meniscus. 9. The LOC device of claim 1, wherein the nucleic acid amplification region is a polymerase chain reaction (PCR) amplification region. 10. The LOC device of claim 9 which further comprises a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer having the PCR region, wherein the CMOS circuit is located on the support base Between the material and the MST layer, the CMOS circuit is configured to use the temperature sensor output to feedback control the PCR region. 11. The L0C device of claim 10, wherein the PCR region has a PCR microchannel, and during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining the test Part of the flow path, and its cross-sectional area perpendicular to the flow is less than 100, 00 square microns. -121 - 201209404 12. The LOC device as claimed in claim 1 wherein the PCR region further comprises at least one elongated heating element to heat the nucleic acid sequence in the elongated PCR microchannel 'the elongated heating element system It extends in parallel with the PCR microchannel. 13. The LOC device of claim 12, wherein at least one of the PCR microchannels forms an elongated PCR chamber. 14. The LOC device of claim 13, wherein the PCR region has a plurality of elongated PCR chambers respectively formed by respective blocks of the PCR microchannel, the PC R microchannel having a series of wide meandering flows The respective configuration of each of the wide curved streams is a channel block that forms the elongated PCR chamber. 1. The LOC device of claim 14, further comprising a reagent reservoir for containing the reagent for the CR; and a surface tension valve having a small aperture configured to secure the bend of the reagent The liquid level 'Therefore, the meniscus can leave the reagent in the reagent reservoir before contacting the fluid sample to remove the meniscus. 16. The device of claim 15, wherein the hybrid region has an array of hybrid chambers containing probes such that probes within each hybrid chamber are configured to be capable of interacting with target nucleic acid sequences A sequence of heterozygous. 1 7 · If the device is in the 〇c device, the photo sensor is a photodiode array, and the position of the photodiode is registered with the hybrid chamber. The L0C device of claim 16, wherein the CMOS circuit has a digital memory for storing the hybrid data from the photosensor output and a data interface for transmitting the hybrid data to an external device. -122-201209404 19. The LOC device of claim 16, wherein the PCR zone has an active valve to allow liquid to remain in the PCR zone during thermal cycling and to react to activation signals from the CMOS circuit The liquid flow to the hybrid chamber 〇20. The LOC device of claim 19, wherein the active valve is a boiling start valve having a meniscus anchor and the meniscus anchor is configured to be fixed The meniscus drives the capillary drive flow of the liquid and a heater heats the liquid to boiling to release the meniscus from the meniscus anchor, causing the capillary drive flow to resume. -123-