TW201209158A - LOC device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification - Google Patents

Abstract

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

Description

201209158 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of microsystem technology (MST). In addition, the present invention relates to microfluidic analysis for molecular diagnostics. [Prior Art] Molecular diagnosis has emerged as a hot field. Before the symptoms are revealed, disease detection brings hope. For detection: • Hereditary diseases • Acquired disorders • Infectious diseases • Genetic susceptibility molecular diagnostics related to health status Highly accurate and rapid treatment of less effective health care services, improved patient outcomes and realization The possibility of patient care personalization. The molecular system is based on the detection and identification of specific nucleic acids derived from biological samples (eg, blood and amplified, including both deoxyribonucleic acid (RNA). Short sequences of complementary days (oligonucleotides) of nucleic acid bases) The specific nucleic acid sequence can be used for nucleic acid testing. If a heterozygous reaction occurs, it is in a complementary sequence. This makes it possible, for example, to predict the nature and pathogenicity of an individual's infectious agent in the future or to measure the individual's diagnostic device. In the early stage (many molecular diagnostic test system, and many techniques or saliva with reduced fruit, improved disease tube diagnosis), extracting acid (DNA) and nuclear nature allow synthetic DNA to bind (heterozygous), indicating that it is stored in the sample. The disease and the response to the drug will be possible. The nucleic acid-based molecular diagnostic test has four different steps: 1. Sample preparation 2. Nucleic acid extraction 3 Nucleic acid amplification (optional) 4. Detection of many sample types for genetic analysis, such as blood, urine, saliva and tissue samples. The type of sample required for the test decision, not all samples are representative of the disease course. These samples have various components, but usually only one of these components is the target of interest. For example, in the blood, high concentrations of red blood cells may inhibit the pair. Detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required in the early stages of nucleic acid testing. Blood is one of the most commonly sought sample types. Blood has three main components: white blood cells, red blood cells, and platelets. The activity can be maintained in vitro. To inhibit coagulation, the sample is mixed with a reagent (eg, ethylenediaminetetraacetic acid (EDTA)) prior to purification and concentration. The red blood cells are typically removed from the sample to concentrate the target cells. Among them, the number of red blood cells accounts for about 99% of the cell material, but since the red blood cells do not have a nucleus, the red blood cells do not carry DN A. Furthermore, the red blood cells contain various components such as hemoglobin, and hemoglobin may interfere with downstream nucleic acid amplification processing (will explain In the next section. By dissolving red blood differentially in the lysis solution The ball can achieve red blood cell removal and retain the remaining cellular material intact, and then the remaining cell material can be separated from the sample by centrifugation from June to 201209158. This provides concentration of the target cells to extract nucleic acids from the target cells. The extraction method used to extract nucleic acids depends on the sample and the diagnostic assay to be performed. For example, the method of extracting the virus RN A will be quite different from the method of extracting the gene DN A. However, in the self-targeting cells Extraction of the nucleic acid typically involves a lysis step and subsequent purification of the nucleic acid following the lysis step. The lysis step ruptures the cell with the nuclear membrane to release the genetic material. A lytic detergent (eg, sodium lauryl sulfate) is typically used. To achieve this step, the lytic detergent also denatures a large amount of protein present in the cell.

The nucleic acid is then purified in an alcohol precipitation step (usually using ice-cold ethanol or isopropanol) or purified by a solid phase purification step, usually in the presence of a high concentration of chaotropic salt in the column ( The silica matrix), the resin or the solid phase purification step on the paramagnetic beads, followed by a washing step, and subsequent elution with a low ionic strength buffer. The optional step performed prior to precipitation of the nucleic acid is the addition of a protein-degrading protease to further purify the sample. Other lysis methods include mechanical lysis by ultrasonic vibration and thermal lysis of the cell membrane by heating the sample to 94 °C. The target DN A or RN A present in the extracted material may be extremely small, especially if the target is the DNA or RNA of the pathogenic microorganism. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a low concentration of a particular target nucleic acid to a detectable concentration. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The 201209158 PCR method is well known in the art and is based on "Principles and Technical Aspects of PCR amplification" by E. Van Pelt-Verkuil et al., published by Springer Press in 2008. A comprehensive description of such reactions is provided in the book. PCR is a powerful technique for amplifying target DNA sequences from complex DNA backgrounds. If RNA is to be amplified (by PCR), it is necessary to first transcribe RNA into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the generated cDNA was amplified by PCR. PCR is an exponential method and can be continued as long as the conditions for maintaining the reaction are acceptable. The components of the reaction are as follows: 1 - Primer pair ~ Short single-stranded DNA of about 10 to 30 nucleotides complementary to the region flanking the target sequence. 2. DN A polymerase ~ a thermostable enzyme that synthesizes DNA. 3. Deoxyribonucleoside triphosphates (dNTPs) ~ provide nucleotides that are incorporated into the newly synthesized DNA strand. 4. Buffer ~ provides the best chemical environment for DNA synthesis. PCR generally involves placing such reactants in a small tube containing the extracted nucleic acid (about 1 〇 to 15 μl). The tube is placed in a temperature circulator; the temperature circulator is an instrument that allows the reaction to be carried out at a range of different temperatures for different periods of time. The standard procedure for each temperature cycle involves the denaturation phase, the annealing phase, and the extension phase. The extension phase is sometimes referred to as the primer extension phase. In addition to this three-step procedure, a two-step temperature program can be used to combine the bonding phase with the extended step 201209158 in a two-step temperature program. The denaturation phase generally involves raising the reaction temperature to 90 to 95 ° C to denature the DNA strand; in the bonding phase, the temperature is lowered to about 50 to 60 ° C to bond the primer; and then in the elongation phase, the temperature is made. The DNA polymerase optimal activity temperature was raised to 60 ° 7 to 2 ° C to carry out the primer extension reaction. This process is repeated for about 20 to 40 cycles, with the end result being the creation of millions of copies of the target sequence between the primers. Standard PCR programs are available in a variety of variations, such as for molecular diagnostics, multiplex PCR, linker-primer PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR, etc.

Multiplex PCR uses multiple sets of primers in a single PCR reaction mixture to create different sizes of amplicon that are specific for different DNA sequences. By locking multiple genes at once, additional information can be obtained from a single test round, otherwise additional experiments may be required to obtain additional information. However, optimization of multiplex PCR is difficult and requires selection of primers with similar binding temperatures' and the amplicon has similar length and base composition to ensure equal amplification efficiency for each amplicon. Linker-initiator PCR, also known as ngation adaptor PCR, is a method that allows nucleic acid amplification of virtually all DNA sequences in a complex DNA mixture without the use of a target-specific primer. The method first involves the cleavage of the marker DNA population using an appropriate restriction endonuclease (enzyme). The double-stranded oligonucleotide linker (also known as a adaptor) with the appropriate cantilever ends is then ligated to the ends of the target DNA fragment using a ligase (iigase enZyme). Nucleic acid amplification is then performed using oligonucleotide primers that are specific for the 201209158 subsequence. Thus, all fragments of the DN A source that are pinched by the linker oligonucleotides can be amplified. Direct PCR describes a method of performing PCR directly on a sample without any nucleic acid extraction or minimal nucleic acid extraction. It has long been believed that many components present in unpurified biological samples (e.g., blood red components in blood) inhibit the PCR reaction. Traditionally, PCR requires extensive purification of the target nucleic acid prior to preparation of the reaction mixture. However, by appropriately changing the chemical reagent and the sample concentration, it is possible to perform PCR with minimal DNA purification or directly perform PCR. Modification of PCR chemistries for direct PCR involves increasing buffer strength, using a polymerase with high activity and persistence, and additives that can be chelated with potential polymerase inhibitors. Tandem PCR utilizes two different rounds of nucleic acid amplification reactions to increase the probability of amplifying the correct amplicon. One form of tandem PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in two different rounds of nucleic acid amplification reactions. The first pair of primers is hybridized to a nucleic acid sequence located at an exosome region of the target nucleic acid sequence. A second pair of primers (nested primers) for use in the second round of nucleic acid amplification reaction binds within the first PCR product and produces a second PCR product comprising the target nucleic acid sequence, the second PCR product Will be shorter than the first PCR product. The rationale behind this method is that if the wrong locus is amplified during the first round of nucleic acid amplification, the second amplification of the second pair of primers will also amplify the possibility of the wrong locus. Extremely low to ensure specificity. -10- 201209158 Real-time PCR (or quantitative PCR) is used to measure the amount of a PCR product in real time. The initial amount of nucleic acid in the sample can be quantified by using a fluorescent dye or a probe containing a fluorescent luminescent group in the reaction in conjunction with a set of reaction standards. This method is especially useful for molecular diagnostics where processing options vary with the source of the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. A reverse transcriptase is an enzyme that reversely transcribes RNA into a complementary DNA (cDNA) and then amplifies the complementary DNA by PCR. RT-PCR is widely used in gene expression studies to determine the expression of a gene or to identify sequences of RNA transcripts including the start and end positions of transcription. Reverse transcriptase PCR is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. The thermostatic nucleic acid amplification method does not require denaturation of the template DNA by continuous heating to produce a single-stranded molecule for use as a template for further amplification, and thus does not require a sophisticated machine. Therefore, the thermostatic nucleic acid amplification method can be performed at the original location or easily operated outside the laboratory environment. At present, various thermostatic nucleic acid amplification methods have been disclosed, including strand displacement amplification, transcription-mediated amplification, nucleic acid sequence-dependent amplification, recombinase polymerase amplification, rolling circle amplification, and branching. Amplification method, helicase-dependent constant temperature Dn A amplification method and circular nucleic acid-mediated amplification method. The constant temperature nucleic acid amplification method does not rely on continuous heating to denature the template DNA to produce a single-stranded molecule as a template for further amplification, instead of using an alternative method, such as enzymatic cleavage using a specific restriction endonuclease The gap is formed on the DN A molecule or the enzyme is used to untwist the -11 - 201209158 DNA double strand. Chain displacement amplification (SDA) relies on the ability of certain restriction enzymes to form gaps in the unmodified strand of semi-modified DNA and the ability of 5'_3f endonuclease-deficient polymerase to extend and replace downstream DNA strands. ability. Exponential nucleic acid amplification is then achieved by combining a synonymous reaction with an antisense reaction (a strand displacement system derived from synonymous reactions in these reactions as a template for an antisense reaction). Instead of cutting DN A in a conventional manner, it is useful for such reactions to use nicking enzymes (e.g., N. Alwl, N. BstNBl, and Mlyl) that create a gap in one of the DN A duplexes. The strand displacement amplification method has been improved by using a combination of a thermostable restriction enzyme (Χναΐ) and a thermostable outer polymerase (5^ polymerase). This combination has been shown to increase the amplification efficiency of the reaction from 1-8 fold amplification to 1 〇IQ fold amplification, and thus it may be possible to amplify a particular single replica molecule using this technique. Transcription-mediated amplification (TMA) and nucleic acid sequence-dependent amplification (NASBA) use RNA polymerase to replicate the RNA sequence 歹IJ instead of replicating the corresponding genomic DNA. This technique uses two primers and two or three enzymes, RN A polymerase, reverse transcriptase, and (if the reverse transcriptase does not have rn A hydrolase activity) RNA hydrolase H (RNase H). One of the primers contains a promoter sequence for use by RN A polymerase. In the first step of nucleic acid amplification, the primer is hybridized to the target ribosomal RNA (rRNA) at a defined position. The reverse transcriptase creates a DNA replica of the target rRNA by prolonging from the 3' end of the promoter primer. RN A of -12-201209158 in RNA:DNA double-stranded complex produced by reverse transcriptase RNA hydrolase activity (if the reverse transcriptase has RNA hydrolase activity) or added RNase Η degradation. The second primer is then combined with the 〇ΝA replica. A double-stranded DNA molecule is created by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon is re-entered into the above procedure and used as a template for a new round of replication reactions. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a particular DNA fragment is achieved by binding the opposite sequence of the oligonucleotide primer to the template DNA and using the DNA polymerase to extend the primer. The double-stranded DNA (dsDNA) template is denatured without the use of heat. Instead, RPA uses a recombinase-introduction complex to scan double-stranded DN A and aid in strand exchange at homologous positions. The resulting structure is stabilized by interaction of the single-stranded DNA binding protein with the displaced template strand, thereby avoiding the priming of the primer due to branch migration. Disassembly of the recombinase allows the 3' end of the oligonucleotide to be contacted with a strand displacement DNA polymerase (a large fragment of the strand displacement DNA polymerase such as Bacillus subtilis polymerase 1 (5^)) And -φ is followed by an extension of the primer. Exponential nucleic acid amplification is achieved by repeating this procedure. The helicase-dependent thermoregulating DN Α amplification method (HD A) is a single-strand template in which the DNA helicase is used to generate a heterozygous reaction for primer hybridization and then the primer-extension reaction is carried out by DN A polymerase. In the first step of the HD A reaction, the helicase travels along the target DN.A and breaks the hydrogen bond connecting the two DNA strands, and then the two DNA strands bind to the single-stranded binding protein. Exposure of the single-stranded target region by helicase allows the primer to bind to the single-strand target region. The DNA polymerase then extended the 3' end of each primer using free deoxyribose-13-201209158 nucleoside triphosphates (dNTPs) to generate two DNA copies. The two replicated double-stranded DNAs each enter the next HDA cycle, resulting in exponential nucleic acid amplification of the target sequence. Other DNA-dependent thermostat techniques include rolling circle amplification (RCA). In rolling circle amplification, DNA polymerase continuously extends the primer around a circular DNA template, and the replication is replicated by multiple replicates of the circular template. The long DN A product consisting of the material. Until the end of the reaction, the polymerase produces replicas of thousands of circular templates, and the chain length of the replicas is related to the original target DN A . This method allows for spatial resolution of the target and rapid nucleic acid amplification of the message. This method can generate up to 1012 template replicas in one hour. The branching amplification method is a variant of the RCA method, and the branching amplification method utilizes a closed circular probe (C-probe) or a padlock probe and a high persistence The DNA polymerase exponentially amplifies the C_probe under constant temperature conditions.

The circular nucleic acid-mediated amplification (LAMP) provides high selectivity and employs a DNA polymerase and a set of four specially designed primers that recognize a total of six different sequences on the target DN A. LAMP is initiated with an internal primer containing the sequence of the syntactic and antisense strands of the target DNA. The subsequent strand displacement DNA synthesis reaction guided by the outer primer releases a single-stranded DNA. The single-stranded DNA system serves as a template for DNA synthesis reaction guided by the second inner primer and the second outer primer, and the second inner primer and the second outer primer are hybridized with the other end of the target to generate a dry - a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer is hybridized to the loop on the product and the pro-DNA synthesis reaction is initiated from -14 to 201209158 to obtain the original dry-loop enthalpy and one having a double stem length. New stem-cyclic DNA. This cyclic reaction continues at a rate that accumulates 1 to 9 target replicas in one hour. The final product is a 'target sequence with several inverted repeats, and the final product is cross-linked by the reverse repeating target sequences in the same chain to form a dry-loop of broccoli-like structure with multiple loops. Form DN A. After completion of the nucleic acid amplification reaction, it is necessary to analyze the amplification products to determine whether or not the expected amplicon (amplification amount of the target nucleic acid) is generated. The method of analyzing the product can range from simply judging the size of the amplicon by gel electrophoresis to identifying the nucleotide composition of the amplicon using the DN A hybrid method. Gel electrophoresis is one of the simplest methods for confirming whether a nucleic acid amplification process produces an expected enhancer. Gel electrophoresis separates DNA fragments by applying an electric field on a gel matrix. Negatively charged DNA fragments move through the matrix at different rates, which are primarily dependent on the size of the DNA fragments. After the electrophoresis is completed, the fragments in the gel can be dyed with -φ lines to see the fragments. It is usually dyed with ethidium bromide, which emits fluorescence under ultraviolet light. The DN Α size marker and the amplicon can be electrophoresed side by side on the gel, and the size of the fragments can be judged by comparison with DNA size markers (stepped DNA bands) containing DNA fragments of known size. Since the oligonucleotide primers bind to a specific position on both wings of the target DN A, the size of the amplification product can be predicted, and the amplification product is detected on the gel to exhibit a strip of a known size. band. In order to confirm the identity of the amplicon or if several amplicons are generated, the amplicon is usually subjected to DN A probe hybridization -15-201209158. DNA hybridization refers to the formation of double-stranded DNA by complementary base pairing. The DN A hybridization reaction for positive identification of specific amplification products requires the use of a DN A probe of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DN A sequence, a heterozygous reaction will occur under conditions of suitable temperature, pH 値 and ion concentration. If a heterozygous reaction occurs, it indicates that the gene of interest or the DN A sequence is present in the original sample. Optical detection is the most common method for detecting heterozygous reactions. Any of the amplicons or probes are labeled to emit light by means of fluorescence luminescence or electrochemiluminescence (ECL). The difference in these methods is that the means for generating the luminescent group in the excited state are different, but both methods can covalently label the nucleoside chain. In electrochemiluminescence (ECL), current is stimulated to cause luminescent molecules or luminescent complexes to illuminate. In the luminescence method, irradiation is performed by laser light which causes luminescence. The detection unit and the illumination source for providing excitation light (the wavelength of which is absorbed by the fluorescent molecules) are used to detect fluorescence. The detecting unit includes a photo sensor for detecting a transmitted signal (for example, a photomultiplier tube or a charge coupled device (CCD) array) and a mechanism for preventing the excitation light from being incorporated into the photosensor output signal (for example) , wavelength selective filter). The fluorescent molecules should be excited to emit light to release stokes shifted light, and the emitted light is collected by the detecting unit. The Stokes shift is the difference in frequency between the light emitted and the absorbed excitation light -16- 201209158 or wavelength difference. The EC L luminescence is detected using a light sensor that is sensitive to the luminescence wavelength of the EC L species employed. For example, transition metal-ligand complexes emit light of visible wavelengths, so conventional photodiodes and charge coupled devices (CCDs) can be used as photosensors. The advantage of ECL is that if the ambient light is excluded, the ECL light may be the only light that appears in the detection system, thus

Improve sensitivity. Microarrays allow hundreds of thousands of DNA hybrid experiments to be performed simultaneously. Microarrays have powerful tools for screening thousands of genetic diseases or detecting the potential of many infectious agents in a single test. A microarray consists of a number of different probes that are spotted on a substrate. The target DNA (amplicon) is first labeled with a fluorescent molecule or a luminescent molecule during or after the nucleic acid amplification reaction, and then the labeled target DNA (amplifier) is subsequently applied to the probe array. The microarray is incubated for a period of hours or days in a temperature controlled and humid environment during which a heterozygous reaction occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound DNA strands. After cleaning, the microarray is dried using an air stream (usually nitrogen). The severity of the heterozygous reaction and cleaning is very important. Not being harsh enough can lead to a high degree of non-specificity. Excessive rigor may result in inability to properly combine to reduce sensitivity. The heterozygous reaction can be identified by detecting the light emanating from the labeled amplicon that has formed a hybrid with the complementary probe. Fluorescence from a microarray is detected using a microarray scanner, which is typically a computer-controlled inverted scanning fluorescence confocal microscpe (inverted scanning fluorescence confocal microscpe) A laser is typically used to excite the fluorescent dye) and a light sensor (eg, a photomultiplier tube or CCD) is used to detect the luminescent signal. The fluorescent molecules emit Stokes shifted light as described above, and the Stokes shifted light is collected by the detecting unit. The released fluorescence must be collected, separated from the unabsorbed excitation wavelength and transmitted to the detector. In microarray scanners, a conjugate focal configuration is typically used to eliminate out-of-focus information by placing conjugated focal pinholes at the imaging plane. This mode allows only the focused portion of the light to be measured. Light from above and below the focus plane of the target cannot enter the detector, increasing the signal to noise ratio. The detector is used to convert the measured fluorescent photons into electrical energy and then convert the electrical energy into a digital signal. This digital signal is translated into a number representing the intensity of the fluorescence from the specified pixel. Each feature of the array consists of one or more such 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 heterozygous target sequences can be identified and analyzed simultaneously. More information on fluorescent probes is available at: http://www.premierbiosoft.com/tech_notes/FRET_probe.ht ml ; and http://www.invitrogen.com/site/us/en/home /References/

Molecular-Probes-The-Handbook/Technical-Notes-and-

Product-Highlights/Fluorescence-Resonance-Energy- -18- 201209158

Transfer-FRET.html. Molecular Diagnostics for Key Care Although molecular diagnostic tests offer many advantages, the development of such tests in clinical testing is less than expected and remains a small practical application of laboratory medicine. The main reason for this is that nucleic acid testing is more complicated and costly than testing that relies on methods that do not involve nucleic acids. The universal adaptability of molecular diagnostic tests in clinical disposition and the development of instruments and equipment (significantly reducing costs, providing rapid and automated analytical assays from the beginning (sample processing) to the end (generating results) and can operate without human intervention) It is closely related. Key care technologies for use in clinics, hospital clinics, or even consumers at home can bring many benefits, including: • Quick results to facilitate immediate treatment and improved care quality

• Ability to obtain inspections by examining very few samples. • Reduce clinical workload • Reduce administrative workload and increase office efficiency by reducing administrative work. • Improve the cost per patient by reducing the length of hospital stay, summarizing the consultations of newly diagnosed outpatients, and reducing the handling, storage, and delivery of samples. • Helps make clinical management decisions such as infection control and antibiotic use. 0 -19- 201209158 On-wafer laboratory (LOC)-based molecular diagnostics Microfluidic-based molecular diagnostic systems provide an automated and accelerated molecule A tool for diagnostic analysis testing. The primary reason for the faster detection time of this system is the diagnostic method steps of a built-in tandem device that can be implemented in a microfluidic device with minimal volume, automation, and low cost. The volume of nanoliters and microliters also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are a common form of microfluidic devices. The LOC device has a plurality of MS T structures integrated into a single carrier substrate (typically helium) for use in fluid processing of the MS T layer. Fabrication of the LOC device using the ultra-large integrated circuit (VSLI) lithography technology of the semiconductor industry allows each LOC device to maintain an extremely low unit cost. However, large external piping systems and electronics are required to control the flow of fluid through the LOC unit, to add reagents, to control reaction conditions, and to perform such operations. Efficiently connecting the LOC device to such external devices limits the use of the L0C device for molecular diagnostics in a laboratory environment. The cost of external equipment and the complexity of the operation of such equipment hamper L0C molecular diagnostics as a practical option for a focused care environment. For the above reasons, there is a need for a molecular diagnostic system based on a wafer-on-a-lab (L0C) device that can be used for focused care. SUMMARY OF THE INVENTION Various systems of the present invention will now be described in the following paragraphs. GCF011.1 The system of the present invention provides a wafer-on-lab (L〇c) device for biological analysis of -20-201209158 gene analysis, the L〇c device comprising an inlet for receiving the sample; a carrier substrate ;

a dialysis section, wherein the cells in the sample having a size greater than a predetermined threshold are separated from the smaller component, wherein the cells having a size greater than a predetermined threshold include target cells containing genetic material for analysis; a reagent storage tank; a lysis section located downstream of the dialysis section and configured to dissolve the target cells to release genetic material in the cell, the lysing section and the containing One of the reagent storage tanks for dissolving the lysis reagent of the target cells in the lysing section is in fluid communication; the incubation section is located downstream of the lysis section, and the cultivating section is And one of the reagent storage tanks containing a plurality of enzymes for enzymatically reacting with the genetic material: and a nucleic acid amplification section located downstream of the incubation section for use in genetic material The nucleic acid sequence is amplified; wherein the dialysis section, the lysis section, the incubation section, and the nucleic acid amplification section are all carried on the carrier substrate. GCF01 1.2 Preferably, the first nucleic acid amplification segment is a polymerase chain reaction (PCR) segment. GCF0 11.3 Preferably, the LOC device further comprises a hybrid segment located downstream of the PCR segment, and the hybrid segment has a probe array and a photo sensor, the probe array is used Hybridization with the target nucleic acid sequence-21 - 201209158 in the sample, and the photosensor is used to detect the heterozygous reaction of any of the probes in the array. GCF0 1 1.4 Preferably, the dialysis section has a first passage, a second passage and a plurality of holes, the first passage being in fluid communication with an inlet at an upstream end, the second passage being connected to a waste liquid passage at a downstream end Fluidly connected, and the pores are smaller than the target cells and larger than the smaller components, and the second channel is in fluid communication with the first channel by the pores such that the target cells remain in the first channel and At the same time, the smaller components flow into the second channel GCF01 1.5. Preferably, the first channel and the second channel are constructed to fill the sample by capillary action. GCF0 11.6 Preferably, the lysis zone has an active valve that retains the target cells within the lysis section during lysis so that when the active valve is opened, capillary drive to the incubation section The fluid can continue. GCF0 1 1 _7 Preferably, the nucleic acid amplification segment is a thermostatic nucleic acid amplification segment. GCF011.8 Preferably, the reagent storage tanks each have a surface tension valve for allowing the reagent to stay in the reagent storage tank, the surface tension valve having a meniscus, the meniscus anchor system for holding (pi The meniscus of the reagent is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow reagent to flow out of the reagent reservoir. GCF 011.9 Preferably, the LOC device also has a flow path from the inlet to the hybrid section, wherein the flow path is configured to direct the sample from the inlet to the hybrid section by capillary action. Preferably, the LOC device also has a complementary metal oxide-22-201209158 semiconductor (CMOS) circuit, a temperature sensor and a microsystem technology (MST) layer, the MST layer comprising the PCR segment, wherein the a CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to achieve feedback control of the PC R segment using the output of the temperature sensor 〇GCF011.il preferably, the PCR region The segment has a PCR microchannel for thermal cycling of the sample to amplify the nucleic acid sequence, ^ the PCR microchannel defines a portion of the sample flow path, the PCR microchannel having less than 1 000 square microns The flow cross-sectional area. GCF01 1.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequence in the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel. Preferably, at least one segment of the PCR microchannel is shaped into a PCR chamber. Preferably, the PCR segment has a plurality of elongate PCR chambers and each elongate PCR chamber is formed by an individual segment of the PCR microchannel having a series of A wide curved channel formed by a meandering configuration, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. GCF0 11.15 Preferably, the incubation section has a heater to heat the genetic material and the plurality of enzymes to a predetermined enzyme catalytic reaction temperature. GCF011.16 preferably, the LOC device also includes an array of hybrid chambers for housing probes such that the probes within each hybrid chamber are configured to interact with one of the target nucleic acid sequences mixed. -23- 201209158 GCFO 1 1 .1 7 Preferably, the photosensor is an array of photodiodes arranged in a compartment. GCFO 1 1.18 Preferably, the CMOS circuit has a data interface for storing the hybrid data originating from the output, and the data interface is the heterogeneous device. Preferably, the PCR section has a main valve that maintains a liquid during the thermal cycle to maintain a start signal of the CMOS circuit in the PCR section to allow liquid to flow to the boast GCF01 1.20 preferably, the active valve The boiling boiling start valve has a meniscus anchor and a heater, and the curved heat exchanger for holding the capillary driving flow for blocking the liquid is used to boil the liquid to disengage the meniscus so that the capillary drive flows Can continue. The easy-to-use, mass-produced and low-cost gene set receives the white blood cells contained in the dialysis section separation sample by receiving the reagent stored in the reagent storage tank of the LOC device by the sample placement tank of the LOC device. The genetic material of the sample in the culture section of the LOC device that dissolves the white blood cells in the lysis chamber to release the white blood cells, and the target gene sequence is amplified by the LOC. The integrated imaging array of the device senses the hybrid reaction of the probes to analyze the core of the sample. The function of the dialysis section extracts the attached force from the sample. The hybrid cavity digital memory and light perception The data of the detector is transmitted to the external moving valve, and the response is derived from the acoustic hybrid chamber. The starter valve, the liquid level anchor is constructed through the liquid level, and the meniscus anchor is added, and the LOC is loaded with the sample, and the material is transferred by the LOC device: the LOC device, and the line is pretreated and expanded. Hybrid and borrow the acid sequence of the imaging array. Message and improve the score -24- 201209158 Analysis of the sensitivity, signal noise ratio and dynamic range of the inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysing subunit integrated in the unit provides a simple analytical test procedure, low system component count and simple system manufacturing procedures for inexpensive analysis

Inspection system. In the culturing section, the genetic material undergoes various pretreatments, such as DNA restriction shearing and conjugation of the primers, to provide the most appropriate or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to improve the analysis. Verify system sensitivity, signal-to-noise ratio, and reliability. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely compact. Easy to carry system. The integrated imaging sensor increases read sensitivity and eliminates the need to use multiple optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks that are integrated into the LOC device and that hold all of the reagents for the analysis require -25-201209158 provide a low system component count and a simple manufacturing procedure, resulting in an inexpensive analytical inspection system. GCF 0 1 2.1 This system of the invention provides a wafer-on-lab (LOC) device for genetic analysis of a biological sample, the LOC device comprising an inlet for receiving the sample; a carrier substrate; a dialysis section, the dialysis zone The segment separates cells of the sample having a size greater than a predetermined threshold 与 from the smaller component, wherein the cells having a size greater than the predetermined threshold 包含 comprise target cells containing genetic material for analysis; a plurality of reagent storage tanks; a segment, the lysis segment being located downstream of the dialysis section and for dissolving the target cells to release genetic material within the cell, the lysing segment being associated with the lysing segment One of the reagent storage tanks of the lysis reagent of the target cell is in fluid communication: a cultivation section, the cultivation section is located downstream of the lysis section, and the cultivation section is associated with the genetic material a reagent storage tank for performing a plurality of enzymes for enzymatically catalyzing fluid communication; a first nucleic acid amplification section, the first nucleic acid amplification section being located downstream of the incubation section for amplifying the genetic material And a second nucleic acid amplification segment downstream of the incubation segment for amplifying the genetic material in parallel with the first nucleic acid amplification segment a nucleic acid sequence; wherein the dialysis section, the lysis section, the incubation section 'the first nucleic acid-26-201209158 amplification section and the second nucleic acid amplification section are all carried on the carrier substrate. GCF0 12.2 Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment and the second nucleic acid amplification segment is a second polymerase chain reaction (PCR) segment. Preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs. And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. GCF012.4 Preferably, the first PCR segment and the second PCR segment are constructed and operable with different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Polymerase species; concentration of deoxyribonucleoside triphosphate;

Thermal cycle time; number of thermal cycle repetitions; and temperature during a particular phase of PCR. Preferably, the LOC device also includes a first hybrid segment and a second hybrid segment and a photo sensor, the first hybrid segment being located downstream of the first PCR segment and the first hybrid The segment has a first probe array for hybridization with the first target nucleic acid sequence, and the second hybrid segment is located downstream of the second PCR segment and the second hybrid segment has a second and second hybrid segment Target Nucleic Acid-27-201209158 A heterozygous second probe array, and the photosensor is used to detect the heterozygous reaction of any of the probes in the first array and the second array. GCF0 12.6 Preferably, the dialysis section has a first passage, a second passage and a plurality of orifices, the first passage being in fluid communication with an inlet at an upstream end, the second passage being associated with a waste liquid passage at a downstream end Connecting 'and the holes are smaller than the target cells and larger than the smaller components'. The second channel is in fluid communication with the first channel by the holes such that the target cells remain in the first channel At the same time, the smaller components flow into the second channel GCF 012.7. Preferably, the first channel and the second channel are configured to fill the sample by capillary action. GCF 0 1 2.8 Preferably, the lysis section has an active valve that retains the target cells within the lysis section during lysis, such that when the active valve is opened, flow to the incubation section The capillary drive fluid continues. Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment. Preferably, the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system for holding The meniscus of the reagent is removed until the meniscus of the reagent contacts the sample fluid to remove the meniscus to allow reagent to flow out of the reagent reservoir. GCF012.il preferably, the LOC device also has a complementary metal oxide semiconductor (CMOS) circuit, a temperature sensor and a microsystem technology (-28-201209158 MST) layer, the MST layer comprising the first PCR segment and The second PCR segment, wherein the CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to use the output of the temperature sensor to achieve the first PCR segment and the second Feedback control of the PCR segment. Preferably, the PCR segment has a PCR microchannel for thermal cycling of the sample, the PCR microchannel defining a flow path having a cross-sectional cross-sectional area of less than 100,000 square microns. GCF012.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel. Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The channel formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers.

GCF0 12.15 Preferably, the incubation section has a heater to heat the genetic material and enzyme to a predetermined enzyme catalyzed reaction temperature. Preferably, the LOC device also includes a first hybrid chamber array for accommodating the first probes such that the first probes within each hybrid chamber are configured to One of the first target nucleic acid sequences is heterozygous. Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF0 12.18 Preferably, the CMOS circuit has a digital interface and a -29-201209158 data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the Hybrid data to an external device. GCF012.19 Preferably, the first PCR section has an active valve that retains liquid within the first PC R section during thermal cycling and allows liquid in response to an activation signal originating from the CMOS circuit Flow to the first hybrid chamber array. GCF012.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor so that the capillary drive flow continues. The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and pretreats the genetic material of the sample in the incubation section of the LOC device, Amplifying the target gene sequence and analyzing the nucleic acid sequence of the sample by heterozygous with the oligonucleotide probe and sensing the heterozygous reaction of the probes by the integrated imaging array of the L Ο C device . The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process -30-201209158 order to obtain an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system.

In the culturing section, the genetic material undergoes various pretreatments, such as DNA restriction shearing and conjugation of the primers, to provide the most appropriate or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to improve the analysis. Verify system sensitivity, signal-to-noise ratio, and reliability. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the parallel amplification chambers allow for different targets or target groups 1 and each suitably uses a different primer pair or group of primers and also allows the use of different optimal amplification parameters, thereby allowing Improve sensitivity, signal noise ratio and reliability of analytical tests. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, productive, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. -31 - 201209158 The reagent storage tanks integrated in the LOC device and holding the analysis can provide a low system of metaprogramming, thereby obtaining an inexpensive analytical test system GCF013.1. The system of the present invention provides a genetic analysis. a wafer on-lab (LOC) device for receiving an inlet of the sample; a carrier substrate: a dialysis section that separates cells of the sample boundary from smaller components, wherein the cells of the cells include a plurality of reagent storage tanks for analysis: a lysis section, the lysis section is located in the dialysis solution to the target cells to release the genetic material in the cells and the contents are used to dissolve the lysed section One of the target reagent storage tanks is in fluid communication; the incubation section is located in the lysate section and is in fluid communication with one of the reagent storage tanks containing the enzyme for seeding the genetic material a first nucleic acid amplification section downstream of the first nucleic acid expansion section for amplifying a second nucleic acid amplification section in the genetic material, the second nucleic acid being expanded downstream of the nucleic acid amplification section for use Expansion Derived from the second subset of the nucleic acid sequence of the amplification; wherein all reagents required parts' number of inspection systems and simple system. For use in a biological sample, the LOC device comprises a target cell having a medium size greater than a predetermined proximity size greater than a predetermined threshold; downstream of the segment and for a solute, the lysis segment being downstream of a segment of the cell lysing reagent, and The multi-increasing segment of the enzyme-catalyzed reaction is located in the incubation nucleic acid sequence; and the extension segment is located in the first nucleic acid amplification segment -32 - 201209158, the dialysis section, the lysis segment, the incubation The segment, the first nucleic acid amplification segment, and the second nucleic acid amplification segment are all carried on the carrier substrate. Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second polymerase chain reaction (PCR) segment. . GCF013.3 preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. GCF013.4 Preferably, the first PCR segment and the second PCR segment are constructed and operable with different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Type of polymerase;

Deoxyribonucleoside triphosphate concentration: buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. GCF013.5 Preferably, the LOC device also has a hybrid segment located downstream of the second PCR segment, and the hybrid segment has a probe array and a photo sensor 'the probe The array is used to hybridize to the target nucleic acid sequence and the photosensor is used to detect heterozygous anti-33-201209158 of any probe in the array. Preferably, the dialysis section has a first passage, a second passage, and a plurality of holes, the first passage being in fluid communication with the inlet at the upstream end, the second passage being associated with the waste at the downstream end The liquid channel is in fluid communication, the holes being smaller than the target cells and larger than the smaller components, the second channel being in fluid communication with the first channel by the holes to leave the target cells in the first The smaller components are introduced into the second channel within the channel and simultaneously. GCF0 13.7 Preferably, the first channel and the second channel are constructed to fill the sample by capillary action. GCF0 13.8 Preferably, the lysis section has an active valve that retains the target cells in the lysis section during lysis, such that when the active valve is opened, the flow is directed to the culturing zone The capillary drive flow of the segment continues. Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment. _ GCF0 1 3 . 1 0 Preferably, the reagent storage tanks each have a surface tension valve for leaving reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system The meniscus of the reagent is retained until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. GCF013.il Preferably, the LOC device also has a CMOS circuit, a temperature sensor and a micro system technology (MST) layer, the MST layer comprising the -34-201209158-PC R segment and the second PC R region a segment, wherein the CMOS circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to use the output of the temperature sensor to achieve the first PCR segment and the second PCR segment Feedback control. GCF013.12 Preferably, the first PCR segment has a PCR microchannel that thermally cycles the sample, the PCR microchannel defining a flow having a cross-sectional cross-sectional area of less than 100,000 square microns Path 〇GCF013.13 Preferably, the PCR microchannel has at least one elongated heater element that extends parallel to the PCR microchannel.

Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The crucible is formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers -φ. GCF0 13.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzyme catalyzed reaction temperature. Preferably, the LOC device also includes an array of hybrid chambers for holding the probes such that the probes within each hybrid chamber are configured to interact with the target nucleic acid sequences One is heterozygous. G C F 0 1 3 · 1 7 Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF013.18 Preferably, the CMOS circuit has a digital memory and a data interface of -35-201209158, the digital system is used for storing the hybrid data originating from the output of the photo sensor, and the data interface is transmitted. The hybrid data is sent to an external device. GCF013_19 preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid to flow to the activation signal in response to the activation signal from the CMOS circuit The first hybrid chamber array. GCF013.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid The heater is used to boil the liquid to disengage the meniscus from the meniscus anchor so that the capillary drive flow continues. The genomic analysis L0C device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and pretreats the genetic material of the sample in the incubation section of the LOC device, The target gene sequence is amplified and the nucleic acid sequence of the sample is analyzed by heterozygous with the oligonucleotide probe and sensing the heterozygous reaction of the probes by an integrated imaging array of the LOC device. The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system. In the culturing section, the genetic material undergoes various pretreatments, such as DNA φ restriction shearing and conjugation of the primer primers, to provide the most appropriate or necessary conditions for subsequent analysis stages, to increase the message content of the analysis results, and to increase the Analyze the sensitivity, signal-to-noise ratio, and reliability of the inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the cascaded amplification chambers allow for segmental local optimization of the early and late cycles of the amplification process, thereby increasing sensitivity, signal to noise ratio and reliability of the assay. The probe hybrid segment achieves the analysis of the target -φ by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks that are integrated into the LOC device and that hold all of the reagents for the analysis require -37-201209158 provide a low system component count and a simple manufacturing procedure to obtain an inexpensive analytical inspection system. GCF014.1 This system of the invention provides a wafer on-lab (LOC) device for genetic analysis of a biological sample, the LOC device comprising an inlet for receiving the sample; a carrier substrate; a dialysis section, the dialysis section The cells in the sample greater than the predetermined threshold are separated from the smaller components, wherein the cells greater than the predetermined threshold include a plurality of reagent storage tanks containing the target cells for analysis of the genetic material; a lysis segment, a lytic segment located downstream of the dialysis section for dissolving the target cells to release genetic material within the target cells, the lysing segments being associated with the lysate for dissolving the lysing zone One of the reagent storage tanks of the lysis reagent of the target cells in the segment is in fluid communication; and the nucleic acid amplification segment is located downstream of the lysis segment for use from the genetic material Amplifying the nucleic acid sequence; wherein the dialysis section, the lysis section, and the nucleic acid amplification section are all carried on the carrier substrate. GCF0 14.2 Preferably, the nucleic acid amplification segment is a polymerase chain reaction (P C R ) segment. GCF014.3 Preferably, the LOC device also has a hybrid segment located downstream of the PCR segment, and the hybrid segment has a probe array and a photo sensor, the probe array For hybridization with a target nucleic acid sequence, -38-201209158 and the photosensor is used to detect a heterozygous reaction of any probe in the array. GCF014.4 preferably, the dialysis section has a first a channel, a second channel, and a plurality of holes, the first channel being in fluid communication with the inlet at an upstream end, the second channel being in fluid communication with a waste channel at a downstream end, the holes being smaller than the target cells And greater than the smaller components, the second channel is in fluid communication with the first channel by the holes to leave the target cells in the first channel while allowing the smaller components to flow into the second aisle. GCF 014.5 Preferably, the first channel and the second channel are constructed to fill the sample by capillary action. Preferably, the lysis zone has an active valve that retains the target cells within the lysis zone during lysis so that when the active valve is opened, the flow is directed to the incubation The capillary drive flow of the section continues.

GCF014.7 Preferably, the nucleic acid amplification segment is a thermostatic nucleic acid amplification segment. GCF014.8 Preferably, the reagent storage tanks each have a surface tension valve for leaving reagents in the reagent storage tanks. The surface tension valve has a meniscus anchor, and the meniscus anchor is used to hold the reagent. The meniscus is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. GCF014.9 preferably, the L〇C device also has a flow path from the inlet to the hybrid section, wherein the flow path is constructed to direct the sample from the inlet to the capillary by capillary-39-201209158 Hybrid section. GCF014.10 Preferably, the device also has a CMOS circuit, a temperature sensor and a micro system technology (MST) layer, the MST layer includes the PCR segment, wherein the CMOS circuit is disposed on the carrier substrate and The CMOS circuit is constructed between the MST layers to achieve feedback control of the PCR segment using the output of the temperature sensor. GCF014.il preferably, the PCR segment has a PCR microchannel that thermally cycles the sample to amplify a nucleic acid sequence, the PCR microchannel defining a portion of the sample flow path, and the pCR microchannel Has a cross-sectional cross-sectional area of less than 100,000 square microns. Preferably, the PCR microchannel has at least one elongate heater element for heating the nucleic acid sequence within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel. GCF014.13 Preferably, at least one segment of the PCR microchannel is shaped into a growth PCR chamber. Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The crucible configuration formed by the flow channels, each wide curved channel being a channel section for forming the elongate PCR chamber.

GCF014.15 Preferably, the LOC device also has a reagent storage tank containing reagents for PCR: and a surface tension valve having a hole, the surface tension valve having a hole configured to hold the meniscus of the reagent' Thus, the meniscus retains the reagent in the reagent storage tank from -40 to 201209158 until the meniscus contacts the fluid sample to remove the meniscus and the reagent flows out of the reagent reservoir. Preferably, the LOC device also has an array of hybrid chambers for holding the probes such that the probes within each hybrid chamber are configured to interact with the target nucleic acid sequences One is heterozygous. GCF014.17 Preferably, the photosensor is associated with an array of photodiodes arranged by the hybrid chambers. GCF014.18 Preferably, the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the hybrid data to External device.

GCF014.19 preferably, the PCR section has an active valve that retains liquid within the PCR section during thermal cycling and allows liquid to flow to the impurities in response to an activation signal originating from the CMOS circuit Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the capillary drive to prevent liquid The flowing meniscus is used and the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor so that the capillary drive flow continues. The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, amplifies the sequence of the -41 - 201209158 target gene, and uses the oligomeric nucleus The glycoside probe is hybridized and the hybridization reaction of the probes is sensed by the integrated imaging array of the L0C device to analyze the nucleic acid sequence of the sample. The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysate unit integrated in the unit provides a simple analytical inspection program, low system component count, and minimal system manufacturing procedures for an inexpensive analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks that are integrated into the LOC device and that hold all of the reagents for the analysis require -42-201209158 provide a low system component count and a simple manufacturing procedure, resulting in an inexpensive analytical inspection system. GCF01 5.1 This system of the invention provides a wafer on-lab (LOC) device for 'gene analysis of biological samples, the LOC device comprising an inlet for receiving the sample; a carrier substrate; a dialysis section, the dialysis section The cells in the sample greater than the predetermined threshold 分离 are separated from the smaller components, wherein the cells greater than the predetermined threshold 包括 include target cells containing genetic material for analysis; a plurality of reagent storage tanks; a lysis section, The lytic segment is located downstream of the dialysis section for lysing cells to release genetic material within the cell, the lysing segment being associated with the target for dissolving the lysed segment One of the reagent storage tanks of the lysis reagent of the cells is in fluid communication;

a first nucleic acid amplification segment, the first nucleic acid amplification segment being located downstream of the lysis segment for amplifying a nucleic acid sequence within the genetic material; and a second nucleic acid amplification segment, the second nucleic acid An amplification segment is located downstream of the lysis segment for amplifying a nucleic acid sequence in the genetic material in parallel with the first nucleic acid amplification segment; wherein the dialysis section, the lysis segment, the The first nucleic acid amplification segment and the second nucleic acid amplification segment are both carried on the carrier substrate. GCF015.2 Preferably, the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment, and the second nucleic acid amplification segment is a second poly-43-201209158 synthase chain reaction (PCR) section. GCF015.3 Preferably, the first PCR segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second PCR segment has a second set of primer pairs And the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. GCF015.4 Preferably, the first PCR segment and the second PCR segment are constructed to operate using different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. Preferably, the LOC device further comprises a first hybrid segment and a second hybrid segment and a photo sensor, the first hybrid segment being located downstream of the first PCR segment and the first hybrid The segment has a first probe array for hybridization with the first target nucleic acid sequence, and the second hybrid segment is located downstream of the second PCR segment and the second hybrid segment has a second and second hybrid segment A second probe array that is hybridized to the target nucleic acid sequence, and the photosensor is used to detect a heterozygous reaction of any of the probes in the first array and the second array. Preferably, the dialysis section has a first passage, a second -44 - 201209158 passage, and a plurality of holes, the first passage being in fluid communication with the inlet at the upstream end - the second passage is downstream The waste channel of the end is in fluid communication, the holes are smaller than the target cells and larger than the smaller components, and the second channel is in fluid communication with the first channel by the holes to leave the target cells in The first channel and the second channel are simultaneously configured to fill the sample by capillary action. Preferably, the lysis zone has an active valve that retains the target cells within the lysis zone during lysis so that when the active valve is opened, the flow is directed to the culturing zone The capillary drive flow of the segment continues. Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment.

Preferably, the reagent storage tanks each have a surface tension valve for retaining the reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system for holding the reagent The meniscus is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. Preferably, the LOC device also has a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer, the MS T layer comprising the first PCR segment and the second PCR segment, wherein the CMOS a circuit is disposed between the carrier substrate and the MST layer, the CMOS circuit is constructed such that -45 - 201209158 uses the output of the temperature sensor to achieve feedback of the first PCR segment and the second PCR segment control. GCF015.12 Preferably, the first PCR segment has a PCR microchannel that thermally cycles the sample, the PCR microchannel defining a cross-sectional area of flow having a flow cross-section of less than 1 000 000 micrometers Flow Path 〇 GCF015.13 Preferably, the PCR microchannel has at least one elongated heater element that extends parallel to the PCR microchannel. GCF015.14 Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PC R microchannel having a series of widths The meandering path formed by the meandering channel, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. Preferably, the LOC device also has a reagent storage tank containing reagents for PCR; and a surface tension valve having a hole configured to hold the meniscus of the reagent, The meniscus thus retains the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. Preferably, the LOC device also has a first hybrid chamber array for accommodating the first probes such that the first probes within each hybrid chamber are configured to One of the first target nucleic acid sequences is heterozygous. -46- 201209158 G CFO 15. 17 Preferably, the photo sensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF0 15.18 Preferably, the CMOS circuit has a digital memory and a data interface, the digital memory system is configured to store the hybrid data originating from the output of the photo sensor, and the data interface transmits the hybrid data To an external device. GCF015.19 Preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid flow in response to an activation signal originating from the CMOS circuit To the first hybrid chamber array. GCF015.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents capillary movement of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, such that the capillary drive flow continues.

The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and amplifies the target gene sequence' and by using the oligonucleotide probe Hybridization is performed and the hybridization reaction of the probes is sensed by the integrated imaging array of the 'LOC device to analyze the nucleic acid sequence of the sample. The function of the dialysis section is to extract additional information from the sample and to improve the sensitivity, signal-to-noise ratio and dynamic range of the test system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. The lysis method extracts analytical and diagnostic targets from cells in the sample and provides continuity processing and analysis of the targets. The lysed subunit elements integrated into the unit provide a simple analytical inspection program, low system component count, and simple system manufacturing procedures for an inexpensive analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the parallel amplification chambers allow different target or target groups to suitably use different primer pairs or primer groups and also allow for the use of different optimal amplification parameters, thereby Improve sensitivity, signal noise ratio and reliability of analytical tests. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent storage tanks integrated into the LOC device and holding all of the reagents required for the analysis can provide a low number of system components and a simple system.

201209158 Created a program to obtain an inexpensive analytical inspection system. GCF0 16_1 This invention provides a wafer-on-lab (LOC) device for biological sample gene analysis, the LOC device for receiving an inlet of the sample; a carrier substrate; a dialysis section, the dialysis section The cells in the sample larger than the predetermined cells are separated from the smaller components, wherein the cells are larger than the predetermined threshold, including the target cells containing the genetic material for analysis; a plurality of reagent storage tanks; a lysis section, the lysis zone a segment is located downstream of the dialysis section to dissolve the target cells to release a genetic material lysing segment within the target cells and the cells for dissolving the cells in the lysing segment One of the reagent storage tanks of the lysis reagent is in fluid communication; a first nucleic acid amplification section, the first nucleic acid amplification section being located downstream of the cell section for amplifying the first nucleic acid sequence in the genetic material a second nucleic acid amplification segment, the second nucleic acid amplification segment being located downstream of a nucleic acid amplification segment for amplifying a second nucleic acid sequence derived from the amplicon of the first nucleic acid amplification; The dialysis section, the Cell segments, the first nucleic acid amplification of a second nucleic acid segment amplification segments are supported on the carrier substrate. Preferably, the first nucleic acid amplification segment is a first enzyme chain reaction (PCR) segment and the second nucleic acid amplification segment is a synthase chain reaction (PCR) segment. The inclusion of the boundary cell for the target is: and the first segment and the polymeric dimerization -49 - 201209158 GCF016.3 preferably, the first PCR segment has a first set of primer pairs and the The first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the second set of primers has a second set of primer pairs and the second set of primer pairs is used to bind the second set of complementary nucleic acid sequences, the first A set of complementary nucleic acid sequences differs from the second set of complementary nucleic acid sequences. GCF016.4 Preferably, the first PCR segment and the second PCR segment are constructed to operate with different amplification parameters, the amplification parameters being at least one of the following parameters: a reverse transcriptase species; Polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of cycles of thermal cycling; and temperature during a particular phase of PCR. GCF016.5 Preferably, the LOC device also has a hybrid segment located downstream of the second PCR segment, and the hybrid segment has a probe array and a photo sensor, the probe The array is used to hybridize to a target nucleic acid sequence and the photosensor is used to detect a heterozygous reaction of any of the probes in the array. GCF016.6 preferably, the dialysis section has a first passage, a second passage, and a plurality of holes, the first passage being in fluid communication with the inlet at the upstream end, the second passage being associated with the waste at the downstream end The liquid channel is in fluid communication, and the holes are smaller than the target cells and larger than the smaller components, and the second channel of the -50-201209158 is in fluid communication with the first channel to allow the target cells to remain Simultaneously flowing the smaller components into the second channel 〇GCF016.7 in the first channel. Preferably, the first channel and the second channel are configured to fill the sample by capillary action. Preferably, the lysis zone has an active valve that retains the target cells in the lysis zone during lysis so that when the active valve is opened, the flow is directed to the culturing zone The capillary drive flow of the segment continues. GCF0 16.9 Preferably, the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment. GCF0 16.10 Preferably, the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the meniscus anchor system for holding the reagent The meniscus is removed until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. GCF016.il preferably, the LOC device also has a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer, the MST layer comprising the first PCR segment and the second PCR segment, wherein the CMOS circuit And being disposed between the carrier substrate and the MST layer, the CMOS circuit being configured to use the output of the temperature sensor to achieve feedback control of the first PCR segment and the second PCR segment. GCF01 6.1 2 Preferably, the first PCR segment has PCR microchannels -51 - 201209158, the PCR microchannels thermally cycling the sample, the PCR microchannel defining a flow cross-section having less than 100,000 square microns Flow path of the cross-sectional area 〇GCF016.13 Preferably, the PCR microchannel has at least one elongated heater element that extends parallel to the PCR microchannel. GCF016.14 Preferably, the PCR segment has a plurality of elongate PCR chambers, and each elongate PCR chamber is formed by individual segments of the PCR microchannel having a series of wide curves The channel formed by the flow path, and each wide curved channel is a channel section for forming one of the elongate PCR chambers. Preferably, the LOC device also has a reagent storage tank containing reagents for PCR; and a surface tension valve having a hole configured to hold the meniscus of the reagent, The meniscus thus retains the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. Preferably, the LOC device also has an array of hybrid chambers for accommodating the probes such that the probes within each hybrid chamber are configured to interact with the target nucleic acid sequences One is heterozygous. GCF0 16.17 Preferably, the photosensor is associated with an array of photodiodes arranged in the hybrid chambers. GCF016.18 Preferably, the CMOS circuit has a digital memory and a data interface, and the digital system is used to store the hybrid data derived from the optical sensor - 52 - 201209158, and the data interface is The hybrid data is transmitted to an external device. GCF016.19 preferably, the first PCR section has an active valve that retains liquid within the first PCR section during thermal cycling and allows liquid flow in response to an activation signal originating from the CMOS circuit To the first hybrid chamber array. GCF016.20 Preferably, the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the meniscus that prevents the capillary drive flow of the liquid And the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor, such that the capillary drive flow continues. The genomic analysis LOC device that is simple to use, mass-produced, and inexpensive is to receive a biological sample by using a sample placement tank of the LOC device, and using the reagent stored in the reagent storage tank of the LOC device to use the LOC device. The dialysis section separates the white blood cells contained in the sample, dissolves the white blood cells in the chemical lysis chamber of the LOC device to release the genetic material of the white blood cells, and pretreats the genetic material of the sample in the incubation section of the LOC device, Amplifying the target gene sequence and analyzing the nucleic acid sequence of the sample by heterozygous with the oligonucleotide probe and sensing the heterozygous reaction of the probes by the integrated imaging array of the L Ο C device . The function of the dialysis section is to extract additional information from the sample and to increase the sensitivity, signal to noise ratio, and dynamic range of the analytical inspection system. The dialysis section integrated into the unit provides a low number of system components and a simple manufacturing process, resulting in an inexpensive analytical inspection system. -53- 201209158 This lysis method extracts the target to be analyzed and diagnosed from the cells in the sample and provides continuity processing and analysis of the targets. The lysed subunit elements integrated in the device provide a simple analytical test procedure, a low number of system components, and a simple system manufacturing process to obtain an inexpensive analytical inspection system. The amplification reaction of the target gene sequence can increase the sensitivity and signal to noise ratio of the analytical test system. Furthermore, the cascaded amplification chambers allow for segmental local optimization of the early and late cycles of the amplification process, thereby increasing sensitivity, signal to noise ratio and reliability of the assay. The probe hybrid segment achieves analysis of the targets by providing a hybrid reaction. The integrated probe hybrid section provides an integrated solution that is easy to use, mass-produced, inexpensive, and has a low number of system components. The integrated imaging sensor eliminates the need for expensive external imaging systems and offers an integrated solution with low system component count, mass production and low cost. This integrated solution is compact, lightweight and extremely convenient. Carrying system. The integrated imaging sensor increases read sensitivity and eliminates the need to use many optical components in an optical collection system by benefiting from large light collection angles. The reagent reservoirs integrated into the LOC device and holding all of the reagents required for the assay can provide a low number of system components and a simple manufacturing procedure' to obtain an inexpensive analytical inspection system. [Embodiment] Summary • 54-201209158 This review indicates the main components of the molecular diagnostic system incorporating the specific embodiment of the present invention. The full details of the structure and operation of the system will be described later in this specification. Referring to Figures 1, 2, 3, 139 and 140, the system has the following primary components:

The inspection module 10 and the inspection module 1 1 are typical USB memory keys and the cost of such modules is extremely low. The inspection module 10 and the inspection module 1 1 each comprise a microfluidic device, typically in the form of a wafer-on-lab (LOC) device 30, which has been pre-loaded for analysis of molecular diagnostics. The reagents tested and usually more than 1 000 probes (see Figures 1 and 139). The test module 10 to be displayed in Fig. 1 uses a fluorescence-based detection technique to identify target molecules, and the test module 1 in Fig. 39 uses detection based on electrochemiluminescence. technology. The LOC device 30 has an integrated photo sensor 44 for fluorescence detection or electrochemiluminescence detection (the detection methods will be described in detail in both the lower inspection module 1 and the inspection module 1 1). Both standard micro USB plugs 14 are used for power supply, data transfer and control. Both modules have a printed circuit board (PCB) 5 7 ' and both modules have an external supply capacitor 32 and an inductor 15. Both the Group 1 and the inspection module 11 are single-use for mass production and sale in a ready-to-use sterilization package. The housing 13 has a large placement slot 24 for receiving biological samples and covers the large area prior to use. The tearable sterilizing sealing tape 22 of the groove 24 is placed (the tape 22 preferably has a low-adhesive adhesive). The sealing film 408 having the film protective sheet 410 forms part of the outer casing 13 to reduce dehumidification in the inspection module. Acting and simultaneously releasing -55-201209158 due to the pressure caused by small fluctuations in air pressure. The film protector 410 protects the sealing film 408 from damage. The test module reader 12 is powered by the micro USB slot 16 to the same. Inspection module 1 检验 or inspection module 1 1. Inspection The module reader 12 can take many different forms, and the selection of such forms will be described later. The versions of the reader 12 shown in Figures 1, 3 and 139 are a smart phone implementation. The block diagram of the reader 12 is shown in Fig. 3. The processor 42 runs the application software from the program storage 43. The processor 42 also connects the display screen 18 and the user interface (UI) touch screen. 17 and a button 19, a cellular radio 21, a wireless network connector 23, and a satellite navigation system 25. The cellular radio 21 and the wireless network connector 23 are used for communication. The satellite navigation system 25 is used to update the attached location data. Epidemiological database. Alternatively, the location data can be manually entered via touch screen 17 or button 19. Data storage 27 holds genetic and diagnostic information, test results, patient data, and is used to identify each probe and probe. The analysis of the array position of the needle and the probe data. The data storage 27 and the program storage 43 can be shared to a shared memory device. The application software installed on the inspection module reader 12 provides result analysis. Additional diagnostic and diagnostic messages are included. For diagnostic testing, the test module 10 (or test module 1 1) is inserted into the micro USB slot 16 of the test module reader 12. The sealing tape 2 2 is sterilized and a biological sample (in liquid form) is injected into the sample large placement slot 24. Pressing the start button 20 begins the inspection by the application software. The sample flows into the LOC device 30, and the carrier plate The assay is performed by extracting, culturing, and amplifying the sample nucleic acid (the target) and hybridizing the sample nucleic acid with a hybrid reactive oligonucleotide probe synthesized in advance-56-201209158. In the case of the inspection module 10 (the inspection module 1 1 uses an electrochemiluminescence-based detection method), the probes are fluorescently labeled and housed in the housing 13 Body (LED) 26 provides the necessary excitation light to induce the heterozygous probe to emit fluorescence (see Figures 1 and 2). In the case of the inspection module 1 1 (the inspection module 1 1 uses a detection method based on electrochemiluminescence), the LOC device 30 is equipped with the above ECL probe, and the light emitting diode must be used for the φ φ (LED) 26 produces a luminescent effect of luminescence. Switching electrode 860 and electrode 870 are used to provide an excitation current (see Figure 140). The illuminating effect (fluorescent or luminescent) is detected using a photo sensor 44 integrated in a CMOS circuit of each LOC device. The measured signal is amplified and converted to a digital output 値, and the digital output 値 is analyzed by the test module reader 12. The reader then displays the result. This information can be stored locally and/or uploaded to a web server containing patient records. The inspection module 10 or the inspection module 1 1 is removed from the inspection module reader 1 2 and the inspection module is properly disposed. Figures 1, 3, and 139 show the construction of a test module reader 12 that constitutes a mobile phone/smartphone 28. In other aspects, the test module reader can be used in a portable personal computer/notebook computer in a hospital, a private practice clinic or a laboratory, a dedicated reader 101, an electronic book reader 1 〇7, tablet 109 or desktop computer 105 (see Figure 141). The reader can be used to link a variety of additional application uses, such as patient records, bills, online databases, and multi-person environments. The reader can also be connected to a variety of local peripherals or remote peripherals such as printers and patient smart cards. • 57- 201209158 Referring to Figure 142, the epidemiological database built into the host system 1 1 1 that can update epidemiological data via the reader 12 and the network 125 using the data generated by the test module 1 〇 The host system of the genetic data system 1 1 3 built-in genetic database, electronic health record (EHR) host system 115 built-in electronic health record, electronic medical record (EMR) host system 121 built-in electronic medical records and A personal health record built into the personal health record (PHR) host system 123. On the other hand, the epidemiological database built into the host system 1 1 1 of the epidemiological data, the genetic database built in the host system 113 of the genetic data, and the electronic health record can be used via the network 125 and the reader 12. The electronic health record built in the host system 115 of the EHR), the electronic medical record built into the host system 121 of the electronic medical record (EMR), and the personal health record built into the host system 123 of the personal health record (PHR) update the test mode. The digits in the LOC device 30 of group 10 are recorded in billions. Returning to the 1, 2, 1 3 9 and 1 4 0 diagrams, the reader 1 2 is powered by the battery in the mobile phone configuration. The mobile phone reader contains all the inspection and diagnostic information that has been pre-uploaded. It can also be loaded or updated via some wireless interface or contact interface to communicate with peripheral devices, computers or online servers. The micro USB slot 16 is for connecting to a computer or to a mains power supply for charging the battery. Figure 7 1 shows a specific embodiment of the test module 1 , for testing that only needs to obtain positive or negative results for a particular target, such as for testing whether an individual is infected, for example, H1N1 Influenza A virus. Modules 47 powered solely by USB and as indicators for specific purposes are sufficient for this task. No other readers or application software is required. USB-only -58-201209158 and the indicator 45 on the module 47 as an indicator signal a positive or negative result. This configuration is ideal for large-scale screening. Additional tools provided with the system may include test tubes containing reagents (for the pretreatment of certain samples) with a spatula and lancet for collecting samples. Fig. 7 shows a specific embodiment of an inspection module including a compression telescopic lancet 3 90 and a lancet release button 3 92 for convenience of use. Satellite phones can be used in remote areas. Inspection module electronic component

Figures 2 and 140 are block diagrams of the electronic components inside the inspection module 10 and the inspection module 1 1 , respectively. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a clock 3 3, a power conditioner 31, a random access memory (RAM) 38, and a program. Data flash memory 40. These components are the entire inspection module 10 or inspection module 1 1 (including photosensor 44, temperature sensor 170, liquid sensor 174, various heaters 152, 154, 182, 234 along with associated drivers) 37 and 39 and recorders 35 and 41) provide control and memory functions. Only the light emitting diode (LED) 26 (as in the example of the test module 10), the external power supply capacitor 32 and the micro USB plug 14 are located outside of the LOC device 30. The LOC device 30 includes a plurality of bond pads for connection to such external components. The random access memory (RAM) 38 and the program and data flashing body 40 have the application software and diagnostic and verification information for more than one probe (eg, encrypted flash/safe Sexual storage). No illumination is required in the case of an inspection module 1 1 constructed for ECL detection -59- 201209158 Diode (LED) 26 (see Figures 139 and 140). The data is encrypted by the LOC device 30 to achieve secure storage and secure communication with external devices. The LOC device 30 is equipped with electrochemiluminescent probes, and each of the hybrid chambers has a pair of ECL excitation electrodes 860 and 870. A variety of inspection modules 1 are manufactured in a variety of inspection types for off-the-shelf use. The difference between these assays depends on the assays on the plates performed by the reagents and probes. Some examples of infectious diseases that are rapidly identified using this system include • Influenza A, Influenza B, Influenza C, Isavirus, Thogotovirus • Pneumonia – Respiratory Cell Fusion Virus (R s V ), Adenovirus, Interstitial Pneumonia Virus, Streptococcus pneumoniae, Staphylococcus aureus • Tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium tuberculosis Mycobacterium vaccae • Plasmodium falciparum, Toxoplasma gondii and other protozoan parasites • Salmonella enteric serovar typhi • Ebola virus • Human immunodeficiency virus (HIV) • Dengue-flavor virus • Type A, B, C, D, and E hepatitis • In-hospital infections – such as Clostridium difficile, vancomycin resistant bowel -60- 201209158 Cocci and xylene penicillin resistant golden yellow Staphylococcus • Herpes Simplex Virus (HSV) • Cytomegalovirus (CMV) • Epstein-Barr's Virus (EBV) • Encephalitis – Japanese Brain Inflammatory virus, Chandipura virus • Pertussis I. pneumoniae • Hemp pain - paramyxovirus • Meningitis - Streptococcus pneumoniae and meningococcus • Anthrax _ Anthrax can be identified by this system Specific examples of hereditary diseases include: • Cystic fibrosis • Hemophilia • Sickle anemia • Tay-Sachs disease (also known as familial sputum dementia) • Hemochromatosis • White encephalopathy ( Cerebral arteriopathy) • Crohn's disease • Polycystic kidney disease • Congenital heart disease • Rett syndrome -61 - 201209158 A few cancer options that can be identified by this diagnostic system include: • Ovary Tumors • Colorectal cancer • Multiple endocrine neoplasia • Retinal blastoma • Turcot syndrome The above options are not exhaustively listed and the diagnostic system can be constructed to detect distant using nucleic acid and proteomic analysis techniques. It is better than the various diseases and physical conditions of the above categories. Detail Construction of System Components L0C Device The LOC device 30 is the core of the diagnostic system. The LOC device uses a microfluidic platform to rapidly perform four major steps in nucleic acid-dependent molecular diagnostic assays: preparing samples, extracting nucleic acids, amplifying nucleic acids, and detecting. The LOC device also has different uses and will be described in detail later. As described above, the inspection module 10 and the inspection module 1 1 can be configured in different configurations to detect different targets. As such, the LOC device 30 has a number of different specific embodiments tailored to the target of interest. One of the L0C devices 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a pathogen within a whole blood sample. For the purpose of explanation, the structure and operation of the LOC device 301 will now be described in detail with reference to Figures 4 to 26 and 27 to 57. Fig. 4 is a schematic diagram showing the construction of the LOC device 301. For the sake of the method -62-201209158, the method symbols corresponding to the functional sections of the LOC device 310 for performing the method stages are labeled with the method stages shown in Fig. 4. The method stages associated with each of the major steps in the diagnostic assay of nucleic acid molecules are also indicated as: sample insertion and preparation stage 288, extraction stage 290 'cultivation stage 291, amplification stage 292, and detection stage 294. The various reservoirs, chambers, valves and other components of the LOC device 301 will be described in more detail later. 5 is a perspective view of the LOC device 301. The LOC device 301 is manufactured using mass production techniques of CMOS and MST (Microsystem Technology). The layered structure of the LOC device 301 is illustrated in a partial cross-sectional view of Fig. 12 (not to scale). The LOC device 301 has a germanium substrate 84 for carrying a CMOS + MST wafer 48, the CMOS + MST wafer 48 comprising a complementary metal oxide semiconductor (CMOS) circuit 86 and a microsystem technology (MST) layer 87, and having A cap layer 46 on the MST layer 87. For the purposes of this patent specification, the term "microsystem technology (MST) layer" refers to an assembly of structures and layers that process samples using various reagents. Thus, the structures and components are constructed to define a plurality of flow paths having characteristic dimensions that support the liquid (which has similar physical properties to the sample) for capillary drive flow during processing. In view of this, the MST layers and members are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and components that are sized to achieve capillary drive flow and that can handle very small volumes. The specific embodiment described in the description of the present invention shows that the MST layer is carried on the CMOS circuit 86 but excludes a plurality of structures and active components other than the features of the cover layer 46 of the -63-201209158. However, it will be understood by those skilled in the art that in order for the MST layer to be able to process samples, the MST layer does not have to have an underlying CMOS or indeed does not have an overlying cap layer. The overall dimensions of the LOC device shown in the following figures It is 1760 microns x 5 8 24 microns. Of course, LOC devices made for different applications may have different sizes. Figure 6 shows the features of the MST layer 87 that overlap the features of the cap layer. The illustrations AA to AD and the illustrations AG to AH shown in Fig. 6 are enlarged and displayed in the figures 13, 13, 35, 56, 55 and 63, respectively, and the illustrations AA to AD and the illustrations AG to AH are detailed. The following is provided for a comprehensive understanding of the various structures in the LOC device 301. The features of the cap layer 46 are shown separately in Figures 7 through 10, while the structure of the CMOS + MST device 48 is shown separately in Figure 11. Layered Structures Figures 12 and 22 are schematic diagrams showing the layered structure of the CMOS + MST device 48, the cap layer 46, and the fluid interaction between the two. These drawings are not drawn to scale and are intended to be illustrative. Fig. 12 is a cross-sectional view through the sample inlet 68, and Fig. 22 is a cross-sectional view through the storage tank 54. Preferably, as shown in Fig. 12, the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86 and that operates the active components located above the MST layer 87. The passivation layer 88 seals and protects the CMOS layer 86 from the fluid flowing through the MST layer 87. The fluid flows through the capping channel 94 of the -64-201209158 and the microsystem technology (MST) channel 90 located in the cap layer 46 and the MS T channel layer 100, respectively (see Figures 7 and 16). Cell transport occurs in the larger channels 94 fabricated in the cap layer 46 while biochemical processing is performed within the smaller MST channels 90. The cell transport channel is sized to transport cells in the sample to predetermined locations within the MST channels 90. Cells that are larger than 20 microns in size (e.g., certain white blood cells) require channel sizes greater than 20 microns, so the flow cross-sectional area is greater than 400 square microns. The MST channel (especially the MST channel located at the location within the LOC device where no cells need to be transported) can be significantly smaller. It will be understood that the capping channel 94 and the MST channel 90 system, and in particular the M ST channel 90, may also be referred to as a heated microchannel or a dialysis MST channel depending on the particular function of the channel. The MS Τ channel 90 is formed by patterning with a photoresist and etching through the MS Τ channel layer 100 deposited on the passivation layer 88. The MST channels 90 are covered by a top layer 66 and the top layer 66 forms the top of the CMOS + MST device 48 (refer to the orientation shown in the figures).

Although the cover channel layer 80 and the reservoir layer 78 are sometimes shown as different film layers, the cover channel layer 80 and the reservoir layer 78 are formed from a single piece of material. Of course, the piece of material may not be a whole piece. Both sides of the sheet of material are etched to form a capping channel layer 80 (and the capping channels 94 are etched in the layer) and a reservoir layer 7 8 (the reservoirs 54, 56 are etched in this layer, 58, 60 and 62). Alternatively, the storage tanks and the cover channels may be formed by micro-molding. Both the etching technique and the micro-molding technique are used to fabricate channels having a flow cross-sectional area of up to about 2,000 square microns and as small as about 8 square microns. -65- 201209158 At different locations in the LOC device, the flow cross-sectional area of the channels may have an appropriate range of choice. When the channel contains a large number of samples or a large number of components contained in the channel, it is suitable to use a cross-sectional area of 20,000 square micrometers of high stomach (for example, a channel having a width of 2 μm in a thick layer of 100 μm). When the channel contains a small amount of liquid or a mixture containing no large cells, it is preferred to use a very small flow cross-sectional area. The lower sealing layer 64 seals the capping channels 94 and the upper sealing layer 82 covers the reservoirs 54, 56, 58, 60 and 62. The five storage tanks 54, 56, 58, 60, and 62 are pre-loaded with reagents for analysis and inspection. In the specific embodiments described in the present invention, the storage tanks are pre-filled with the following reagents, but can be easily replaced with other reagents, such as: • Storage tank 5 4: anticoagulant and optionally Contains red blood cell lysis buffer • Storage tank 5 6 : Lysis reagent • Storage tank 5 8 : Restriction enzyme, ligase and linker (for PCR of linker primer, see Figure 70 and see T. Stanchan et al. And in 1999, in New York and London, published by Gariancj Science, "Human Molecular Genetics 2"). • Storage tank 60: amplification mixture (deoxyribonucleoside triphosphates (dNTPs), primers, buffer), and • storage tank 62: DNA polymerase. The cap layer 46 and the CMOS + MST layer 48 are in fluid communication by corresponding openings in the lower sealing film 64 and the top layer 66. These openings are referred to as upper suction holes 96 and lower suction holes 92 ° LOC devices depending on whether the fluid flows from the MST passages 90 to the cover channels 94 or in the opposite direction depending on whether the fluid is -66 - 201209158.

The following is an example of analyzing the operation of the LOC device 301 by analyzing the pathogenic DNA in the blood sample as an example. Of course, other types of biological or abiotic fluids can also be analyzed using a suitable set of reagents or reagent combinations, assay procedures, LOC device variants and detection systems. Returning to Figure 4, the analysis of the biological sample involves five major steps, including sample insertion and preparation steps 288, nucleic acid extraction step 290, nucleic acid incubation step 291, nucleic acid amplification step 292, and detection and analysis. Step 294. The sample placement and preparation step 28 8 involves mixing the blood with the anticoagulant 1 16 and then using the pathogenic dialysis section 70 to separate the pathogen from the white blood cells and red blood cells. Preferably, as shown in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action draws the blood sample along the cover channel 94 to the reservoir 54. When the blood sample fluid causes the surface tension valve 118 to open, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants avoid the formation of agglomerates that may block fluid flow. Preferably, as shown in Fig. 22, the anticoagulant 116 is aspirated from the reservoir 54 by capillary action and the anticoagulant 116 is introduced into the M S T channel 90 via the lower suction port 92. The lower suction aperture 92 has a capillary action priming feature (CIF) 102 for shaping the geometry of the meniscus such that the meniscus does not park at the edge of the lower suction aperture 92. When the anticoagulant 116 is aspirated from the reservoir 54, the vent 22 in the upper sealing layer 82 allows air to enter instead of the anticoagulant -67-201209158 116° MST channel 90 shown in Fig. 22. A portion of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve U8 and positions the meniscus 120 at the meniscus anchor 98 of the upper suction hole 96. Prior to use, the meniscus 120 remains settled at the upper suction aperture 96 such that the anticoagulant does not flow into the cover layer passage 94. When the blood flows through the cap channel 94 to the upper suction port 96, the meniscus 120 is removed and the anticoagulant is drawn into the blood fluid. Figures I5 to 21 show an illustration AE which is part of the insert A A shown in Fig. 13. As shown in Figures 15, 5 and 17 , the surface tension valve 1 18 has three separate MST channels 90 extending between the respective lower suction holes 92 and the upper suction holes 96. . The number of MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent flowing into the sample mixture. When the sample mixture is mixed with the reagents by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample body stream. Therefore, the surface tension valve of each storage tank is constructed to meet the desired reagent concentration. Blood flows into the source dialysis section 70 (see Figures 4 and 15) in which the target cells are concentrated using an array of a plurality of wells 1 64 of a predetermined threshold size. Cells smaller than the critical enthalpy can pass through the holes' while larger cells cannot pass through the holes. Unwanted cells may be any of the larger cells that are blocked by the array of holes or smaller cells that pass through the holes, and direct the unwanted cells to turn to the waste unit 7 6 'at the same time The target cells continue to be analyzed for analysis. In the pathogenic dialysis section 70 described herein, the source derived from the whole blood sample is concentrated for microbial D N A analysis. The array of holes is formed by a plurality of -68-201209158 3 micron diameter holes 164 that connect the feed fluid in the cap layer channel 94 to the target channel 74 in fluid communication. The 3 micron diameter holes 164 are coupled to the dialysis uptake holes 168 of the target channel 74 through a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The source of the disease is small enough to pass through the 3 micron diameter 1 64 and the source is injected into the target channel 74 via the dialysis MST channel 204. Cells larger than 3 microns (e.g., red blood cells and white blood cells) remain in the waste channel 72 in the cover layer 46, which leads to the waste storage tank 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate a particular source or other target cell', such as leukocytes for human DNA analysis. A more detailed description of the dialysis section and variations of the dialysis method is provided later. Referring again to Figures 6 and 7, the fluid is drawn through the target passage 74 to the surface tension valve 128 of the lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending from the

The lysis reagent reservoir 56 is between the target channel 74. When the meniscus is removed by the sample fluid, assuming that the physical properties of the fluids are substantially equal, the flow rate from all seven MST channels 90 will be greater than the source of anticoagulant storage 54 (the surface tension) Valve 118 has a flow rate of three MST passages 90). Therefore, the proportion of the lysing reagent in the sample mixture is greater than the proportion of the anticoagulant. The lysis reagent is mixed with the target cells by diffusion in the target channel 74 of the chemical lysis section 1130. The boiling start valve 1 2 6 stops the fluid for a period of time sufficient for diffusion and lysis - 69-201209158 to release the genetic material within the target cells (see Figures 6 and 7). The structure and operation of the boiling start valve will be described in more detail below with reference to Figures 31 and 32. The Applicant has also issued other active valve types that can be used in the present invention to replace the boiling start valve (active valve trains are relative to passive valves such as surface tension valve 118). These alternative valve designs are also described later. When the boiling start valve 126 is opened, the dissolved cells flow into the mixing section 133 to perform the limiting shear reaction and the linker engagement reaction before amplification. Referring to Fig. 13 3, when the fluid releases the meniscus at the surface tension valve 132 at the starting point of the mixing section 133, the restriction enzyme, linker and ligase are released from the storage tank 58. The mixture flows through the entire length of the mixing section 133 for diffusion mixing. The lower suction hole 134 at the end of the mixing section 133 is the cultivating chamber inlet passage 1 3 3 leading to the cultivating section 141 (see Fig. 13). The chamber inlet channel 33 feeds the mixture into a crucible configuration consisting of a plurality of heated microchannels 210, the crucible configuration of which provides an incubation chamber for limiting shearing reactions And the linker is used to accommodate the sample during the junction reaction (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the inset AB of Figure 6. The 23th to 29th views each show the case where the film layers forming the CMOS + MST layer 48 and the cap layer 46 are sequentially increased. Panel AB shows the endpoint of the incubation section 1 14 and the beginning of the amplification section 112. Preferably, as shown in Figures 14 and 23, the liquid stream is injected into the microchannels 210 of the incubation section 1 14 until the fluid reaches the boiling start valve 1〇6' at the boiling start-70- At 201209158, the fluid stops flowing at the valve 106 and simultaneously diffuses. As described above, the microchannel 210 upstream of the boiling start valve 106 becomes an incubation chamber containing a sample, a restriction enzyme, a ligase, and a linker. The heaters 154 are then activated and maintained at a constant temperature for a specified period of time for limiting the shear reaction and the linker ligation reaction. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is an optional step and that only certain types of nucleic acid amplification assays require this φ incubation step 291. In addition, in some cases, a heating step may be required at the end of the incubation phase to raise the temperature above the incubation temperature. Increasing the temperature before the fluid enters the amplification section 112 deactivates the restriction enzymes and the junction 'enzyme. When a thermostatic nucleic acid amplification method is to be employed, there is a close correlation between the restriction enzyme and the inactivation of the binding enzyme. After incubation, the boiling start valve 106 is activated (turned on) and the fluid continues to flow into the amplification section 112. Referring to Figures 31 and 32, a plurality of microchannels 158 form one or more amplification chambers, and the mixture 'φ is injected into the crucible configuration of the heated microchannels 158 until the mixture. Arrives at the boiling start Valve 108. Preferably, as shown in the cross-sectional view of Fig. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and then the polymerase is released from the storage tank 62 to flow into the incubation section 1 14 And the intermediate MST channel 2 1 2 of the amplification section 1 12 . Figures 35 through 51 show the layers of the LOC device 301 in the illustration AC of Figure 6. Figures 35 through 51 each show the case where the layers of the CMOS + MST device 48 and the cap layer 46 are sequentially incremented. The illustration AC is located at the end of the amplification section 112 and at the beginning of the hybrid and detection section 52. The incubated -71 - 201209158 sample, amplification mixture and polymerase flow through the microchannels 158 to the boiling start valve 108. After sufficient time has elapsed for diffusion mixing, the heaters 154 in the microchannels 158 are activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification process, the boiling start valve 108 is opened and fluid continues to flow into the hybrid and detection section 52. The operation of these boiling start valves will be further detailed later. As shown in Fig. 52, the hybrid and detection section 52 has a hybrid chamber array 110. Figures 52, 53, 54 and 56 show details of the hybrid chamber array 110 and the respective hybrid chambers 180. A diffusion barrier 1 75 is located at the entrance of the hybrid chamber 180, and the diffusion barrier 175 prevents the occurrence of the target nucleic acid, the probe strand and the already between the hybrid chambers 180 during hybridization. The diffusion of heterozygous probes to avoid erroneous hybrid detection results. The diffusion barriers 175 provide a length of flow path that is long enough to prevent hybridization of the probes to the nucleic acid molecules and to prevent diffusion of the target sequences and probes during the detection of signals. One chamber is left and the other chamber is contaminated to avoid erroneous results. Another mechanism for preventing erroneous readings is to have the same probe in a plurality of such hybrid chambers. A plurality of photodiodes 1 84 correspond to the hybrid chambers 180' containing the same probes and the CMOS circuit 86 obtains a single result from the photodiodes 1 84. Outlier results in the derivation of a single result can be ignored or not included in the calculation or differential weighting calculation. Providing the heat required for the hybrid reaction by the CMOS controlled heater 182 -72-201209158

Yes (the heaters 182 are described in further detail below). After activation of the heaters, a hybrid reaction occurs between the complementary target sequence and the probe sequence. The LED driver 29 in the CMOS circuit 86 sends a signal to a light emitting diode (LED) 26 located in the test module 1 to cause the LED to emit light. These probes only illuminate when a heterozygous reaction has taken place, thus eliminating the cleaning and drying steps typically used to remove unbound probe strands. The hybrid reaction forces the dry-loop structure of the fluorescent resonance energy transfer (FRET) probe 186 to open, allowing the fluorescent luminescent group to emit fluorescent energy in response to the excitation light of the LED, which will be performed later. More detailed description. Fluorescence is detected by photodiodes 184 located in CMOS circuitry 86 below each of the hybrid chambers 180 (see below for a description of hybrid chambers). The photodiode 184 and associated electronic components for all of the hybrid chambers collectively form the photosensor 44 (see Figure 65). In other embodiments, the photosensor may be a charge coupled device array (CCD array). The signal measured from the photodiode 184 is amplified and converted to a digital output 値, and the digital output 値 is analyzed by the test module reader 12. Further details of this detection method are given later. Modularization of additional details of the LOC device The LOC device 301 has a number of functional sections including the reagent reservoirs 54, 56, 58, 60 and 62, the dialysis section 70, the lysis section 130, the incubation section 1 1 4 and the amplification section 1 1 2. Various types of valves, humidifiers and humidity sensors. In other embodiments of the LOC device, such functional areas -73-201209158 may be omitted and additional functional sections may be added' or such functionality may be used to achieve different uses than those described above. For example, the incubation section 1 14 can serve as the first amplification section 1 1 2 of the tandem amplification assay assay system, and the chemical lysis reagent storage tank 56 is used for the addition of primers, dNTPs, and buffers. The first amplification mixture, and reagent storage tank 58, is used to add reverse transcriptase and/or polymerase. If chemical lysis of the sample is desired, the chemical lysis reagent may also be added to the storage tank 56 along with the amplification mixture, or by heating the sample for a predetermined period of time in the incubation section. Hot lysis. In some embodiments, if chemical lysis is desired and a mixture of the primer, dNTP, and buffer is desired to be separated from the chemical lysis reagent, an additional storage tank can be inserted immediately upstream of the storage tank 58 Used to hold a mixture of the primer, dNTP and buffer. In some cases, it may be desirable to omit a certain step, such as incubation step 291. In this case, the LOC device may be specially manufactured to omit the reagent storage tank 58 and the incubation section 112, or may simply not load the reagent into the storage tank, or may have such an active valve if it has an active valve The active valve is not activated without dispensing the reagents into the sample fluid, and at this point the incubation section is simply turned into a channel to deliver the sample from the lysis section 130 to the amplification section 112. The heaters can operate independently, and thus the reaction proceeds depending on thermal energy (eg, the thermal lysis reaction depends on thermal energy), the heaters are programmable such that the heaters do not start during this step to ensure that they are not A hot lysis reaction does not occur in a LOC device that requires a hot lysis reaction. The dialysis section 70 can be located at a starting point -74 - 201209158 of the fluid system within the microfluidic device as shown in Figure 4, or the dialysis section 70 can be located anywhere else within the microfluidic device. For example, in some cases it may be beneficial to perform dialysis after the amplification phase 292 to remove cell debris prior to the hybridization and detection step 294. Alternatively, two or more dialysis sections can be inserted at any location throughout the LOC device. Similarly, the LOC device can incorporate a plurality of additional amplification segments 112 to ensure amplification of multiple targets in parallel or in series, followed by utilization of specific nucleic acid probes in the hybrid chamber array 1 1 0 The targets were measured. When analyzing a sample that does not require dialysis (e.g., a whole blood sample), the dialysis section 70 can simply be omitted from the placement and preparation section of the sample designed for the LOC device. In some cases, the dialysis section 70 need not be omitted from the LOC device even if the analysis does not require dialysis. If the presence of the dialysis section does not create a geometrical impediment to the analytical test, the LOC device with the dialysis section 70 in the insertion and preparation sections of the sample can still be used without compromising the necessary function. Furthermore, the detection section 294 can comprise a plurality of protein body chamber arrays that are identical to the hybrid chamber array, but are loaded in the protein body chamber array to be designed to exist The probe in the unamplified sample binds or hybridizes to the probe, rather than the nucleic acid probe designed to hybridize to the target nucleic acid sequence. It will be appreciated that the LOC device manufactured for use in this diagnostic system selects different combinations of multiple functional segments depending on the particular LOC application. Most of the LOC devices in these LOC devices have most of the functional segments, and are suitably composed of multiple functional segments by extensive options from the functional segments used within existing LOC devices. The combination can be designed with additional LOC units for new applications from -75 to 201209158. Only a few LOC devices are shown in this specification, and a device is only shown to illustrate the design flexibility created for this system. Those skilled in the art will readily appreciate that the L0C devices shown in the description of the present specification are not eliminated, and that a plurality of functional regions are merged from items of functional segments used in existing L0C devices. An appropriate combination of segment components can be used to make an external L0C design. Sample types A variety of LOC device variants are acceptable and analyze nucleic acid components or protein components in various classes of liquids. Such liquid sample types are not limited to blood and blood products, saliva, cerebrospinal fluid, urine, amniotic fluid, cord blood, breast milk. , sweat, pleural exudate, tears, ascites, environmental water samples and beverage samples. The LOC can also be used to amplify amplicon obtained from a large amount of nucleic acid amplification; in this case, the reagent storage tank will be emptied or configured so as not to release the fraction in the storage tank and the dialysis section is dissolved Cell segments, incubation segments, and amplification are only used to deliver samples from the sample inlets 6 to the hybrid chambers 1 for nucleic acid detection as described above. For some sample types, a pretreatment step is required, such as placement in the L0C device. Semen may need to be liquefied, and multiple enzymes are required to pretreat the mucus to reduce viscosity. Some L0C LOC packs will be available in a wide range of sample sizes, but semen and pericardial fluids will be analyzed. All included segments will be included in the sample and possibly -76-201209158 samples. Figures 1 and 12 show the sample being added to the large placement slot 24 of the inspection module. The large placement slot 24 is a frustoconical cone for feeding the sample into the inlet 68 of the LOC device 301 by capillary action. The sample is here introduced into a 64 μm wide X 60 μm deep capping channel 94, and the sample is attracted to the anticoagulant storage tank 54 by capillary action in the capping channel 94. Reagent storage tanks Analytical inspection systems using microfluidic devices (eg, LOC devices 3〇1) require small volumes of reagents, allowing the reagent storage tanks to contain all reagents required for biochemical processing, each reagent storage tank Has a small volume. This volume is less than about 1,000,000,000 cubic microns, and in most cases the volume is less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns, and in the example of LOC device 301 shown in the figures, this volume is less than 20,000,000. Cubic micrometers. Dialysis section Referring to Figures 15 to 21, 33 and 34, the pathogenic dialysis section 70 is designed to concentrate pathogenic target cells from the sample. As previously described, there are a plurality of apertures in the top layer 66 that are shaped as apertures 164 having a diameter of 3 microns that filter the target cells from a large number of samples. As the sample flows through the 3 micron diameter holes 164, microbial pathogens pass through the holes into the series of dialysis MST channels 204, and the microbial sources flow upwardly back to the target via the 16 micron dialysis uptake 168. In channel 74 (see Figures 33 and 34 -77 - 201209158). The remainder of the sample (e.g., red blood cells, etc.) remains in the cap layer channel 94. Downstream of the pathogenic dialysis section 70, the cap channel 94 is turned into a waste channel 72 to the waste reservoir 76. For the type of biological sample that produces a significant amount of waste, a foam-like insert or other porous element 49 is disposed in the outer casing 13 of the test module 10 to be in fluid communication with the waste storage tank 76 (see paragraph 1). Figure). The operation of the source dialysis section 70 relies entirely on the capillary action of the fluid sample. The 3 micron diameter holes 164 at the upstream end of the source dialysis section 70 have a capillary action priming feature (CIF) 166 (see Figure 33) to attract the fluid down into the dialysis MST channel 204 below. . The first upper suction aperture 198 for the target passageway 74 also has a capillary action priming feature (CIF) 202 (see Figure 15) to prevent the fluid from easily resting the meniscus on the dialysis suction orifice. 1 68 on. The small component dialysis section 682 to be shown in Fig. 5 may have a structure similar to the pathogenic dialysis section 70. The small component dialysis section separates the small target cells from the sample by shaping the size of the pores (and, if necessary, shaping the shape of the pores) such that the pores are adapted to allow small target cells or molecules to pass through or Molecules, such that the small target cells or molecules enter the target channel and continue for further analysis. Larger sized cells or molecules are sent to waste storage tank 766. Thus, the LOC device 30 (see Figures 1 and 39) is not limited to isolation of pathogens having a size of less than 3 microns, but can also be used to isolate cells or molecules of any desired size. Lysis segment -78- 201209158 Returning to Figures 7, 1 and 1 3, the genetic material in the sample is released from the cell by chemical lysis. As described above, the lysis reagent from the lysis reagent reservoir 56 is mixed with the sample fluid in the target channel 74 downstream of the surface tension valve 128 of the lysis reagent reservoir 56. However, some diagnostic assays are more suitable for use with the hot lysis method, or even for the target cells using the chemical lysis method and the hot lysis method. The LOC device 301 provides heated microchannels 210 of the incubation section 14 for thermal lysis. The sample fluid is injected into the incubation section 14 and stops at the boiling start valve 106. The microchannel 210 is incubated to heat the sample to reach a temperature at which the cell membrane is broken. In some hot lysis applications, the enzymatic reaction in the chemical lysis section 130 is not necessary, and the hot lysis reaction completely replaces the enzyme catalyzed reaction within the chemical lysis section 130. Boiling start valve

As discussed above, LOC device 301 has three boiling start valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of a boiling start valve 1 〇 8 located at the end of the heated microchannels 158 of the amplification section 112, respectively. The sample fluid 1 1 9 is attracted by capillary action along the heated microchannels 1 58 until the sample fluid reaches the boiling start valve 108. The leading meniscus 120 of the sample fluid settles at the meniscus anchor 98 at the valve inlet 146. The geometry of the meniscus anchor 98 stops the leading meniscus to stop the capillary flow. As shown in Figures 31 and 32, the meniscus anchor 98 is provided by an opening provided from the MST passage 90 to the upper suction opening of the cover passage 94 -79-201209158. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 152 is located at the peripheral edge of the valve inlet 146. The ring heater 152 is CMOS controlled by the heater contact point 153 of the boiling start valve. To open the valve, CMOS circuit 86 delivers an electrical pulse to heater contact 153 of the valve. The annular heater 152 heats the liquid sample 1 1 9 until the liquid sample boils. The boiling action causes the meniscus 1 20 to disengage from the valve inlet 1 46 and begin to wet the cap channel 94. Once the cover channel 94 begins to wet, capillary flow is again performed. A fluid sample 119 is injected into the capping channel 94 and through the lower suction port 150 to the valve outlet 148 where the capillary drive fluid continues to flow along the adiabatic section away from the channel 160. The hybrid and detection section 52. A plurality of liquid sensors 1 74 are provided at the front and rear of the valve for judgment. It will be appreciated that once the boiling start valves are opened, the boiling start valves can no longer be closed. However, when the LOC device 301 and the test module 1 are single-use devices, it is not necessary to close the valves again. The culture section and the nucleic acid amplification section 6, 6, 7, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubation section 114 and the amplification section 112. The incubation section 114 has a single heated incubation microchannel 210 that is located in the MST channel layer 100 and etched from the lower suction port 134 to the boiling start The 蜿蜒 pattern of the valve 1〇6 (see 13 and )). Controlling the temperature of the culture section 112 can enable an enzymatic reaction with higher efficiency. Similarly, the amplification section Π2 has heated amplifying microchannels 158 from the boiling start valve 1〇6 to the boiling start-80-201209158 valve 108 and in a 蜿蜒 configuration (see section 6). With 1 4 figure). These valves terminate the flow of the fluid to allow the target cells to remain in the heated incubation microchannel 210 or amplification microchannel 158 while mixing, culturing, and nucleic acid amplification. The enthalpy pattern of the microchannels also facilitates mixing of the target cells and reagents to some extent. In the incubation section 112 and the amplification section 112, the heaters 154 are controlled by CMOS circuitry 86 to heat the sample cells and reagents using pulse width modulation (PWM). Each wide curved flow path of the heated incubation microchannel 210 and the amplification microchannel 158 has three independently operable heaters 1 54 extending from the heater 1 54 Between the respective heater contact points 156 of the heaters (see Figure 14), the heater contacts 156 provide two-dimensional control of the input heat flux density. Preferably, as shown in Fig. 51, the heaters 154 are carried on the top layer 66 and embedded in the lower sealing layer 64. The heater material is titanium aluminum alloy (TiAl), but a variety of other conductive materials are also suitable. The elongate heaters 154 are parallel to the length of each of the channel sections (each of which forms the wide curved path of the profile). In the amplification section 112, each of the wide flow channels can be operated as a separate PCR chamber by individual heater control. This analytical assay system using a microfluidic device (e.g., LOC device 301) requires a small volume of amplicons, thus allowing amplification reactions to be performed in the amplification section 1 1 2 with a low volume of amplification mixture. This volume is less than about 400 microliters (Nanoliter), in most cases less than 170 microliters, typically less than 70 microliters and in the case of LOC device 301 this volume is between 2 microliters and 30 microliters. -81 - 201209158 Increased heating rate and higher diffusion mixing The small cross section of each channel section increases the heating rate of the augmented fluid mixture. All fluids are at a relatively short distance from the heater 154. It can be seen that the heating rate achieved by reducing the cross-section of the channel (i.e., the cross-section of the amplifying microchannel 158) to less than 100,000 square microns is greater than that of utilization. Large format equipment provides a higher heating rate. The lithography manufacturing technique allows the fabrication of an augmented microchannel 158 having a cross-sectional cross-sectional area of a flow path of less than 16,000 square microns that provides a substantially higher heating rate across a cross-sectional profile of a flow path of 16,000 square microns. Feature sizes of up to 1 micron can be easily achieved using lithography. If very few amplicons are required (as is the case with LOC unit 301), the cross-sectional area can be reduced to less than 2,500 square microns. In order to achieve the diagnostic analysis test using 1000 to 2000 probes on the LOC device and complete the "implant sample, output result" requirement within one minute, the flow cross between 400 square micrometers and 1 square micrometer The cross-sectional area meets this requirement. The heater elements within the amplification microchannel 158 heat the nucleic acid sequences at a rate above 80 Kelvin (K) per second, and in most cases heat the rate at a rate greater than 100 K per second. And other nucleic acid sequences. In general, the heater element heats the nucleic acid sequences at a rate of greater than 1, 〇〇〇K per second, and in many cases, the heater element heats the nucleic acid sequences at a rate of more than 10,000 K per second. . Usually according to the requirements of the analysis and inspection system, the heater element is at a rate of more than 100,000 K per second, a rate of 1,000,000 K per second or more, a rate of more than 10,000,000 K per second -82-201209158 rate, per second. 20,000,000 1 <: above rate, 40,000,000 per second 〖the above rate, a rate of 80,000,000 1 C per second or more and a rate of 1 60,000,000 K per second or more to heat the nucleic acid sequences. The passage of the small cross-sectional area is also beneficial for the diffusion mixing of any reagent with the sample fluid. The diffusion of one liquid into the other prior to the diffusion mixing is maximized by the diffusion near the interface between the two liquids. The concentration will decrease with distance from the interface. The use of microchannels with a relatively small cross-sectional area of flow direction ensures that the two fluids flow next to the interface for faster diffusion mixing. It can be seen that the cross-section of the channel is reduced to less than 100, and the mixing rate achieved by 〇〇〇 square micron is higher than the mixing rate provided by a larger specification device. The lithography manufacturing technique allows the fabrication of microchannels having a cross-sectional area of the flow path of less than 1 60 square microns, which provides a significantly higher mixing rate than the cross-sectional area of 1 60 square microns. If a very small volume is required (as is the case with LOC device 301), the cross-sectional area can be reduced to less than 20,000 square microns. In order to achieve a diagnostic analysis test using 1000 to 2000 probes on the LOC device and complete the "put sample, output result" requirement within one minute, the flow between 400 square microns and 1 square micron The cross sectional area can meet this requirement. A short thermal cycle time keeps the sample mixture close to the heaters and uses a very small fluid volume to allow for rapid thermal cycling during the nucleic acid amplification process. For target sequences up to 150 base pairs (bp) in length, each thermal cycle (83 - 201209158, denaturation, adhesion, and primer extension) is completed in 30 seconds. In most diagnostic assays, individual thermal cycling times are within 11 seconds' and most of the thermal cycling time is within 4 seconds. The LOC device 30 for performing a portion of the most commonly used diagnostic assays has a thermal cycle time of between about 0.45 seconds and 1.5 seconds for a target sequence of up to 150 base pairs in length. Thermal cycling at this rate allows the assay module to complete the nucleic acid amplification procedure for much less than 10 minutes, and typically less than 220 seconds. For most analytical assays, the amplified segment can produce sufficient amplicons within 80 seconds from the entry of the sample fluid into the sample inlet. For most analytical tests, sufficient amplicons are generated within 30 seconds. After completing the predetermined number of amplification cycles, the amplicon is fed into the hybrid and detection section 52 via a boiling start valve 108. Hybrid Chambers Figures 52, 53, 54, 56 and 57 show the hybrid chambers 180 in the array of hybrid chambers 110. The hybrid and detection section 52 has an array 110 of 24x25 composed of a hybrid chamber 180, and each hybrid chamber has a hybrid sensitive FRET probe 186, a heater element 182, and an integrated photodiode ι 84 . The photoreceptor 1 8 4 is introgressed for detecting fluorescence generated by a heterozygous reaction of the target nucleic acid sequence or protein with the FRET probes 186. Each of the photodiodes 184 is independently controlled by a CMOS circuit 86. Any material between the F R E T probes 1 8 6 and the photodiodes 1 8 4 must allow light to pass through. Therefore, the partition wall section 97 between the probes 186 and the photodiode I" is also permeable to the emitted light. In the apparatus 301 of the L〇c-84-201209158, the partition section 97 is a thin layer of cerium oxide (about 0.5 micrometer), and a photodiode 184 is inserted directly under each of the hybrid chambers 180 to allow The volume of the probe-target hybrid is minimal while still producing a detectable fluorescent signal (see Figure 54). A small amount of probe-target hybrid allows the use of a small volume of hybrid chamber. The amount of probe required to achieve a detectable amount of probe-target hybrid prior to hybridization is less than about 270 picograms (equivalent to 900,000 cubic micrometers), and in most cases less than about 60 skins. Grams (equivalent to 200,000 cubic microns), typically less than about 12 picograms (equivalent to 40,000 cubic micrometers) and less than about 2.7 picograms in the case of the L〇c device 301 shown in the figures (equivalent to 9,000 cubic micron chamber volume). Of course, reducing the size of the hybrid chambers allows for a higher chamber density on the LOC device and thus more probes. In the LOC device 301, the hybrid section has more than 1 000 chambers in an area of 1500 microns by 1500 microns (i.e., the area of each chamber is less than 225 0 φ square microns). Smaller volumes also reduce reaction time, making hybridization and detection faster. An additional advantage of requiring a small amount of probe in each chamber is that only a minimal amount of probe solution needs to be dispensed into each chamber during the manufacturing process of the LOC device. A specific embodiment of the LOC device of the present invention is made using a volume of probe solution of 1 picoliter or less than 1 picoliter. After the nucleic acid amplification, the boiling start valve 108 is activated, and the amplicon flows along the flow path 176 and flows into each of the hybrid chambers 1 8 〇 (see Figures 5 and 5) ). The endpoint liquid sensor 178 indicates when the amplicon is injected into the hybrid chambers 180 and when the heaters 182 can be activated. -85- 201209158 After enough mixing time, the light-emitting diode (LED) 26 (see Figure 2) is activated. The openings in each of the hybrid chambers 180 provide an optical window 136 for exposing the FRET probes 186 to lasing (see Figures 52, 54 and 56). The light emitting diode (LED) 26 continues to illuminate for a sufficient period of time to induce the probes to emit high intensity fluorescent signals. The photodiodes 184 are shorted during the excitation. After a pre-programmed (p re-pro gemmed) delay time of 300 (see Figure 2), the photodiode 184 operates and detects fluorescent light emission without excitation light. Incident light (see Figure 5 4) on the active region 185 of the photodiode 184 is converted to photocurrent, which can then be measured using a c Μ 0 S circuit 8.6. The hybrid chambers 180 are each equipped with a probe for detecting a single target nucleic acid sequence. If desired, probes can be placed in each of the hybrid chambers 180 to detect more than 100 different targets. Alternatively, the same probe can be loaded into multiple hybrid chambers or all hybrid chambers to repeatedly detect the same target nucleic acid. Repeated loading of the probes throughout the hybrid chamber array 110 in this manner results in increased confidence in the results obtained, and if desired, can be adjacent to the hybrid chambers. The multiple results measured by the photodiodes are combined to provide a single result. Those skilled in the art will appreciate that depending on the specifications of the analytical test, the hybrid chamber array 110 may have from one to more than one different probes. Humidifier and Humidity Detector The illustration AG of Figure 6 indicates the location of the humidifier 196. The humidifier -86-201209158 prevents evaporation of the reagents and probes during operation of the L O C device 310. Preferably, the water storage tank 18 8 is in fluid communication with the three evaporators 190 as shown in the enlarged view of FIG. During the manufacturing period, molecular water-grade water is injected into the water storage tank 1 8 8 and the water storage tank 1 8 8 is sealed. Preferably, as shown in Figures 5 and 6 8, water is drawn into the three lower suction holes 1 94 by capillary action and flows along the respective water supply channels 1 92 to the evaporators 1 90 at three A set of upper suction holes 1 93 » The meniscus is fixed at each upper suction hole 1 93 to retain water. The evaporators have annular heaters 191 that surround the upper suction holes 193. The ring heaters 191 are connected to the CMOS circuit 86 by conductive posts 3 76 to the top metal layer 195 (see Figure 37). When activated, the ring-shaped force [] heater 191 heats the water, causing moisture to evaporate and humidifying the device environment. The position of the humidity sensor 232 is also shown in Figure 6. However, as shown in the enlarged view of the illustration in Fig. 63, the humidity sensing device has a capacitive comb structure. The lithographically etched first electrode 2 96 and the microetched second electrode 298 face each other such that the teeth of the first electrode 296 and the second electrode 298 are interleaved. The opposing electrodes form a capacitor having a capacitance' and the capacitor can be controlled by CMOS circuitry 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, resulting in an increase in capacitance. Humidity sensor 2 3 2 is adjacent to the hybrid chamber array 1 1 〇, and humidity measurement at the hybrid chamber array 1 1 极为 is extremely important for retarding the evaporation of the solution (containing the exposed probe). Feedback Sensor -87- 201209158 Temperature sensor and liquid sensor are included in the LOC unit 301 to provide feedback and diagnosis during device operation. Referring to Fig. 35, nine temperature sensors 1 70 are distributed throughout the amplification section 112. Similarly, the incubation section Π 4 also has nine temperature sensors 170. Each of these sensors uses a 2x2 bipolar junction transistor (BJT) to monitor the temperature of the fluid and provide feedback to the CMOS circuit 86. The CMOS circuit 86 uses this feedback to precisely control the thermal cycling during the nucleic acid amplification procedure and to precisely control any heating during hot lysis and incubation. In the hybrid chamber 180, the CMOS circuit 86 uses the hybrid heaters 182 as temperature sensors (see Figure 56). The resistance of the hybrid heaters 182 is temperature dependent, and the CMOS circuit 86 derives the heater readings for each of the hybrid chambers 180 using the resistance to be temperature dependent. The LOC device 301 also has a plurality of MST channel liquid sensors 174 and a capping channel liquid sensor 208. Figure 35 shows a list of MST channel liquid sensors 1 74 located at one of the ends of every other meandering channel of the heated microchannel 158. Preferably, as shown in Fig. 37, the MST channel liquid sensors 174 are a pair of electrodes formed by exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid breaks the circuit between the electrodes to indicate that liquid is present at the location of the sensor. Figure 25 shows an enlarged perspective view of the capping channel liquid sensor 208. A plurality of pairs of opposing titanium aluminum (TiAl) electrodes 21 8 and 220 are disposed on the top layer 66. A gap 222 is interposed between the electrodes 21 8 and 220 to keep the circuit open without liquid -88 - 201209158. The presence of liquid breaks the circuit and the CMOS circuit 86 uses this feedback signal to monitor the fluid. Gravity-independent test module 1 〇 system position independent. The inspection module 10 is not required to be fixed to a flat, stable surface for operation. The capillary drive fluid flows without external piping to the accessory, thereby allowing the modules to be easily carried and easily inserted into a portable portable reader (e.g., a mobile phone). A gravity-independent operation means that the test module is also acceleration-independent in all practical situations. The inspection modules are shock and shock resistant, and the inspection modules will operate on the moving vehicle or while holding the mobile phone. Dialysis-modified white blood cell target cell

The dialysis design within the LOC device 301 described above is designed for the source of the disease. Figure 64 is a cross-sectional view of the dialysis section 3 2 8 designed to concentrate white blood cells in a blood sample for human DN A analysis. It will be understood that the structure of the dialysis section 328 is substantially identical to the dialysis section 70 of the above-described pathogenic target cells 'only the holes 165 of the dialysis section 328 are holes of 7.5 micrometers in diameter' to limit white blood cells from The capping channel 94 enters the dialysis MST channel 204. When the sample to be analyzed is a blood sample and the hemoglobin derived from the red blood cells interferes with the subsequent reaction step, the anti-coagulant in the storage tank 54 (see Fig. 22) is added together with the red blood cell lysis-89-201209158 The flushing will ensure that most of the lysed red blood cells in the sample are removed during the dialysis step (thus removing hemoglobin). The commonly used red blood cell lysis buffer is a solution containing 0.15M ammonium chloride (NH4C1), 10 mM potassium hydrogencarbonate (KHC03), O.lmM ethylenediaminetetraacetic acid (EDTA) and pH 7_2~7_4, but belongs to Those skilled in the art will understand that any buffer that effectively dissolves red blood cells can be used. The capping channel 94 downstream of the leukocyte dialysis section 328 is converted to the target channel 74 such that the leukocyte continues the assay. Further, in this case, the dialysis upper suction port 68 leads to the waste channel 72' to remove all of the smaller cells and components in the sample. It should be noted that this dialysis variant only reduces the concentration of undesired samples in the target channel 74. Figure 116 schematically illustrates a large component dialysis section 686 which also separates large target components from the sample. The pores in the dialysis section are manufactured to have a sized and shaped shape to block the large target component of interest within the target channel for further analysis. If a leukocyte dialysis section as described above is used, most, but not all, of the smaller cells, organisms or molecules flow into the waste reservoir 768. Thus, other embodiments of the LOC device are not limited to separating white blood cells having a size greater than 7.5 microns, but can also be used to separate cells, organisms or molecules of any desired size. ο 8 segment L 7 zone 4 analysis IIII7 V V type in the Daohua show change 1 set 18 3M3 flow pack 5 0C fall L trap 0 0 i said j gas system stop description There are examples of this and the installation of the real map 3) 3 with 1 set and -90- 201209158. This LOC device has a dialysis section that can inject a fluid sample without leaving air bubbles trapped within the channel. The LOC device variant VIII 518 also has a layer of additional material referred to as the interface layer 5 94. The interfacial layer 594 is between the capping channel layer 80 and the MST channel layer 100 of the CMOS+ MST device 48. The interfacial layer 594 allows for a more complex fluid interconnect structure between the reagent reservoirs and the MST layer 87 without increasing the size of the crucible substrate 84. Referring to Fig. 78, the bypass channel 600 is designed to introduce a time delay as the fluid sample flows from the interface waste channel 206 to the interface target channel 602. This time delay allows the fluid sample to flow through the dialysis MST channel 204 to the dialysis uptake 168 that holds the meniscus. The sample fluid is passed from all of the dialysis uptake holes 168 originating from the dialysis MST channel 204 using a capillary action priming feature (CIF) 202 located at the suction port from the bypass channel 600 to the interface target channel 602. The upstream end is injected into the interface target channel 602. Without the bypass channel 600, the sample fluid will still be injected into the interface target channel 602 from the upstream end, but the final advancing meniscus arrives and passes through the upper suction hole belonging to the unfilled MS T channel. Causes air to fall at this point. This trapped air reduces the sample flow rate through the leukocyte dialysis section 328. Nucleic Acid Amplification Variants Parallel Polymerase Chain Reactions Several variants of the LOC device have multiple extensions operating in parallel - 91 - 201209158. For example, the LOC device variant VII 492 shown in Fig. 72 has a plurality of parallel amplification segments 1 1 2 2 . 1 to 1 1 2.4, and the parallel amplification segments 112.1 to 112.4 allow simultaneous execution of multiple nucleic acid amplifications. Increase analytical testing. The LOC device variant XI 746 shown in Fig. 130 also has a plurality of parallel amplification sections 112.1 to 112.4, but additionally has a plurality of parallel incubation sections 114.1 to 114.4, so that the sample can be expanded prior to amplification. Treated differently. Other LOC device variants (eg, LOC device variant XIV 641 to be displayed in Figure 1-5) indicate that the plurality of parallel amplified segments may be "X", which is limited only by the size of the LOC device. size. The larger the LOC device, the more multiple amplification reaction segments can be accommodated in parallel. The separate amplification segments can be configured to perform different number of cycles and/or temperatures for a particular target size or a particular amplification mixture composition. The L0C device can perform a multiplex nucleic acid amplification method or a single nucleic acid amplification method in each segment by a plurality of amplification sections operating in parallel. In multiplex nucleic acid amplification, more than one target sequence is amplified using one or more primers. A parallel nucleic acid amplification system having "m" chambers can perform an amplification reaction equivalent to η weight, where n = n(l) + n(2) + _..n(i) + ...+ n(m), and n(i) represents the primer number to be used for the chamber "i" in the different primer pairs used for the multiplex amplification reaction, and it is necessary to keep in mind that the parallel amplification system The signal-to-noise ratio (SNR) is higher than the signal-to-noise ratio of the η-type amplification reaction performed in a single-chamber system. In the special case of n ( i ) =1, the amplification reaction in the chamber [i] just becomes a single-fold amplification reaction. -92 - 201209158 Cascading Polymerase Chain Reactions Figures 106, 107, 108, 111 and 114 in these figures illustrate the amplification sections 112.1 and 11 2.2 in the device and the L0C devices operating in series. The first amplification section 1 12.1 includes a reagent storage tank 60.1 of the amplification mixture and a reagent storage tank of the polymerase 62.1 °. Each amplification section after the initial section also includes two reagent storage tanks. The storage tank 60.2 of the mixture and the storage tank 62.2 of the polymerase are added. Multiple amplification segments of φ in series allow for tandem PCR analysis to be performed such that the first amplification segment 11 2 1 is used to perform a pre-amplification reaction to enhance subsequent nucleic acid execution in segment 1 1 2.2 Sensitivity of the amplification reaction. Multiplexed segments can also be used for nested polymerase chain reaction (PCR). In cascade PCR for preamplification, the first amplified segment 112.1 is amplified. A nucleic acid sequence within a sample of the target sequence. This amplification reaction does not require specificity for the target sequence (e.g., amplification of the whole genome)' but this amplification reaction does increase the concentration of the target sequence. After the pre-amplification reaction, the sample is mixed with the reagents from the storage tank 6 0.2 and the storage tank 6 2.2 and then the sample mixture is flowed into the second amplification section 1 1 2.2. The reagents stored in reservoir 60<2> contain specific probes that only amplify the target sequences in the pre-amplified sample mixture. It should be noted that • a similar method of replacing PCR with a constant temperature technique in the first amplification stage or the second amplification stage can be used to achieve the advantages of pre-amplification. Nested PCR is a special form of tandem PCr method. This method has the added advantage of high target specificity. In the nested PCR method, the first amplification-93-201209158 segment 1 1 2.1 nucleic acid amplification step is performed by using a primer to amplify a sequence having a length greater than the final target sequence, and the primers are formed. A portion of the amplification mixing reagent stored in the reservoir 6 0.1, and the primers are complementary to the region at the outside of the target sequence. The reaction in the first amplification segment 1 1 2. 1 generates an amplicon consisting of the target sequence plus a flanking section. This amplified mixture is mixed with a reagent derived from storage tank 60.2 and a polymerase derived from storage tank 62.2. The reagents stored in storage tank 60.2 comprise primers that are complementary to the position at both ends of the target sequence (i.e., the sequence of the sub-regions derived from the amplicon of the first amplification stage). When the nucleic acid amplification reaction is performed in the second amplification section 11 2.2, since the concentration of the amplicon derived from the first amplification stage is much larger than the concentration of the original sample molecule, the sequence position unrelated to the target The probability of an amplification reaction occurring is greatly reduced. The sensitivity and specificity of the nested PCR method can also be achieved when the sequence-specific thermostatic amplification technique is used to replace one or both of these PCR amplification stages. The advantage of separately storing the polymerase and separately adding the polymerase to the sample mixture is that different polymerases can be selected for the pre-amplification step and the final nucleic acid amplification step. For example, this approach allows for a low error rate (eg, readable) polymerase for the preamplification step to prevent the creation of a faulty target sequence or an incorrect target sequence while allowing for the final amplification step. Use a higher speed or temperature resistant polymerase. Direct Polymerase Chain Reactions Traditionally, PCR requires extensive purification of the standard -94-201209158 DN A prior to preparation of the reaction mixture. However, by appropriately changing the chemical reagent and sample concentration, it is possible to perform a nucleic acid amplification reaction or directly perform amplification by minimal DNA purification. When the nucleic acid amplification method is a PCR method, this method is called direct PCR. In a LOC device that performs nucleic acid at a controlled constant temperature, the method is a direct isothermal amplification method. The use of direct nucleic acid amplification techniques in L(R) devices, particularly with respect to simplifying the required fluid design, has considerable advantages. Adjustments to amplification chemistries for direct PCR or direct isothermal amplification include increasing buffer strength, using polymerases with high activity and high persistence, and additives that can be chelated with polymerase inhibitors. It is also important to dilute the inhibitor present in the sample. To take advantage of direct nucleic acid amplification techniques, the L O C device design incorporates two additional features. The first feature is a reagent storage tank (e.g., storage tank 58 of Figure 8) having an appropriate size to supply a sufficient amount of amplification reaction mixture or diluent to cause interference with the sample components of the amplification chemical agent. The concentration is low enough to allow for successful nucleic acid amplification. The desired dilution of the non-cellular sample components is between 5 and 20 times. Different LOC structures (e. g., the source dialysis section 70 in Figure 4) can be used at the appropriate time to ensure that the concentration of the target nucleic acid sequence is maintained at a sufficiently high concentration for amplification and detection. In this particular embodiment (further depicted in FIG. 6), a dialysis section is employed upstream of the sample extraction section 290, the dialysis section being effective to concentrate the small enough to enter the amplification section. The source of 2 and the larger cells are removed to allow the larger cells to enter the waste storage tank 76. In another embodiment, the dialysis section is used to selectively reject proteins and salts in the plasma while leaving the cells of interest. -95- 201209158 This second LOC structural feature (characteristic for supporting direct nucleic acid amplification) is a channel aspect ratio design to adjust the mixing ratio between the sample and the components of the amplification mixture. For example, to ensure that the inhibitor of the sample is preferably diluted 5 to 20 times by a single mixing step, the length and cross section of the sample channel and the reagent channel are designed such that the mixing action is initiated. The flow impedance of the upstream sample channel is 4 to 19 times greater than the flow impedance of the channel through which the reagent mixture flows. The flow impedance in the control microchannel can be easily achieved by controlling the geometry of the design. For a constant cross section, the flow impedance of the microchannel increases linearly with the length of the channel. The focus of the hybrid design is that the flow impedance in the microchannel is more dependent on the smallest cross-sectional dimension. For example, when the aspect ratios of the microchannels are inconsistent, the flow impedance of the microchannel having a rectangular cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse transcriptase-polymerase chain reaction (RT-PCR) When the sample nucleic acid species RN A to be analyzed or extracted, such as RNA or RNA derived from RNA virus, prior to PCR amplification, the RNA is first Reverse transcription into complementary DNA (cDNA). The reverse transcription reaction (single-step RT-PCR) can be performed in the same chamber as the PCR, or the reverse transcription reaction can be performed as an independent initial reaction (two-step RT-PCR). In the variants of the LOC device described in the present application, the reverse transcriptase can be added to the reagent storage tank 6 2 together with the polymerase, and the heaters 1 54 can be programmed to perform the reverse transcription step first and then RT_PCR can be simply performed by performing a cycle of the nucleic acid amplification step. The reverse transcription step is carried out by using the -96-201209158 incubation section 1 14 and using the reagent storage tank 58 to store and dispense the buffers' primers, dNTPs and reverse transcriptase, and then using the general method for amplification Amplification in the segment 1 1 2 can also easily achieve two-step RT-PCR. Thermostatic Nucleic Acid Amplification Method For certain applications, the constant temperature nucleic acid amplification method is a preferred nucleic acid amplification method, thereby eliminating the need to repeatedly cycle the reaction components through various temperature cycles, and instead, the amplification region is replaced. The section is maintained at a constant temperature (typically about 3 7 ° C ~ 4 1 ° C). A variety of thermostatic nucleic acid amplification methods have been disclosed, including strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), and recombinant enzyme polymerase amplification (RPA). ), helicase-dependent® warm DNA amplification (HDA), rolling circle amplification (RCA), ramification amplification (RAM), and circular nucleic acid-mediated amplification (LAMP), and Any of these methods or other isothermal amplification methods can be employed in particular embodiments of the LOC device described herein. To perform thermostatic nucleic acid amplification, the reagent storage tank 60 and reagent storage tank 62 adjacent to the amplification section will be loaded with a reagent 's suitable for performing the constant temperature method' in place of the PCR amplification mixture and the polymerase. For example, to perform strand displacement amplification (SDA), reagent storage tank 60 contains amplification buffer, primers, and dNTP' and reagent storage tank 62 contains appropriate nickase and exo-DNA polymerase (exo- DNA polymerase). For recombinase polymerase amplification (RPA), reagent storage tank 60 contains amplification buffer -97 - 201209158, primer, dNTP, and recombinase, and reagent storage tank 62 contains strand displacement dna polymerase (eg, 10,000 μ). The sample 'for the helicase-dependent' region temperature DNA amplification method (HDA) 'reagent storage tank 60 contains amplification buffer, primer and dNTP' and reagent storage tank 62 contains suitable DNA polymerase and substituted heating To unwind the helicase of the double stranded DNA strand. Those skilled in the art will appreciate that such necessary reagents can be dispensed between any of the two reagent storage tanks in any manner suitable for nucleic acid amplification. Since nucleic acid sequence-dependent amplification (NASBA) or transcription-mediated amplification (TMA) does not require transcription of RNA into cDNA first, the NASBA method or the TMA method is suitable for amplifying RNA viruses (eg HIV viruses or Viral nucleic acid of hepatitis C virus). In this example, reagent reservoir 60 is loaded with amplification buffer, primers, and dNTPs, and reagent reservoir 62 is loaded with RNA polymerase 'reverse transcriptase and optional RNA hydrolase H (RNase®). For some forms of thermostatic nucleic acid amplification, it may be desirable to have an initial denaturation cycle to separate the double-stranded DN Α template prior to maintaining the temperature for the thermostatic nucleic acid amplification process. Since the temperature of the mixture within the amplification section 112 can be carefully controlled by the heaters 154 in the amplification microchannels 158 (see Figure 14), in all of the specific embodiments of the LOC apparatus described herein This step can be easily reached. The thermostatic nucleic acid amplification method is more tolerant of potential inhibitors in the sample, and as described above, the thermostatic nucleic acid amplification method is generally suitable when it is desired to perform a direct nucleic acid amplification reaction with the sample. Therefore, the thermostatic nucleic acid amplification method is sometimes particularly suitable for the LOC device variant XLIII 673, the LOC device variant XLIV 674, and the LOC device variant XLVII 677, which are separately 673, 674, and -98-201209158 6 7 7 respectively. Shown on pages 1 1 7 , 1 1 8 and 1 1 9 . The direct isothermal amplification method can also be combined with one or more pre-amplification dialysis steps 70, 686 or 682 as shown in Figures 117 and 119 and/or a pre-hybridization dialysis step 682 as shown in Figure 118. The hydrazine is used to help partially concentrate the target cells in the sample prior to performing the nucleic acid amplification reaction or to remove unwanted cell debris prior to entering the hybrid chamber array 110. Those skilled in the art will appreciate that any combination of the pre-amplification dialysis step described above and the pre-hybridization dialysis step can be used. Thermostatic nucleic acid amplification, multi- or some thermostatic nucleic acid amplification methods can also be performed in multiple amplification sections in parallel (such as the amplification sections to be depicted in Figures 72, 104, and 105) (eg, Circular Nucleic Acid Mediated Amplification (LAMP) is compatible with the initial reverse transcription step for amplification of RNA. Additional details of the fluorescence detection system

Figures 58 and 59 show the hybrid sensitive FRET probe 236. Such probes are commonly referred to as molecular beacons and are dry-loop probes that are produced from single-stranded nucleic acids and that emit fluorescence when the probes are hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization with the target nucleic acid sequence 23 8 . The probe has a ring portion 240, a stem 242, a fluorescent luminescent group 246 at the 5' end, and a matting group 248 at the V end. The loop portion 240 is composed of a sequence complementary to the target nucleic acid sequence 238. Complementary sequences located on either side of the probe sequence are bonded together to form the stem 242. In the absence of a complementary target sequence, the probe remains closed as shown in Figure 58. The stem 242 maintains the phosphorescent group and the matting group-99-201209158 pair close to each other, so that significant resonance energy transfer can occur between the fluorescent group and the extinction group, and substantial elimination occurs. The luminescent group of the luminescent group is illuminated by the excitation light 244. Figure 59 shows the structure in which the FRET probe 2 3 6 is open or hybrid. When hybridized with the target nucleic acid sequence 23 8 , the dry-loop structure is collapsed, and the fluorescent luminescent group 246 and the extinction group 2 48 are spatially separated, thereby restoring the camping luminescent group 246 The ability to emit fluorescent light. Optically measuring the fluorescence release 250 indicates that the probe has been hybridized. Since the backbone helical structure of the probe is designed such that the backbone helical structure is more stable than the probe-target helical structure that is not complementary to a single nucleic acid, the probes have very high specificity and complementarity The target sequence is heterozygous. Since the double-stranded DNA system is relatively rigid, it is unlikely that the double-stranded DNA is stereostructured to coexist with the probe-target helix structure and the backbone helix structure. The probe with the primer is followed by a dry-loop probe with a primer and a linear probe (or 蝎 probe) with a primer, which is a substitute for the molecular beacon, and the probe can be used for the L Ο C device for immediate and quantitative nucleic acid amplification. The instant amplification method can be performed directly in the hybrid chambers of the LOC device. The benefit of using a probe with a primer is that the probe molecule is physically linked to the primer, so that only a single heterozygous event occurs during the nucleic acid amplification period, without the need for a hybrid reaction of the primer and the probe. The heterozygous reaction proceeds separately. This ensures that the reaction is carried out efficiently and in real time, and that the probe with the primer gives a stronger signal, shorter reaction time and better discrimination than the separate use of the primer and the probe -100-201209158 . The probes (along with the polymerase and the amplification mixture) can be placed into the hybrid chambers 180 during manufacture without the need for separate amplification sections on the LOC device. Alternatively, the amplified segment can be retained but not used or used for other reactions. The linear probes with primers are shown in Figures 120 and 121, respectively, showing a linear probe 692 with primers during the initial round of the nucleic acid amplification reaction and the linear probe during the subsequent round of nucleic acid amplification reactions. Structure. Referring to Figure 1 20, the linear probe 692 with the primer has a double-stranded stem portion 242. One of the double strands comprises the probe sequence 696 with the primer, the probe sequence 696 is identical to a region on the target nucleic acid sequence 696, and the fluorescent sequence is labeled on the V-terminus of the probe sequence 696. The group 246 and the Y-terminus of the probe sequence 696 are ligated to the oligonucleotide primer 700 via amplification blocker 694. A matting group 248 is marked at the Y end of the other chain of the backbone 242. Upon completion of the nucleic acid amplification reaction of the initial round, the probe can be looped and hybridized to the extended nucleic acid strand using the now complementary sequence 698. During the nucleic acid amplification reaction of the initial round, the oligonucleotide primer 700 is adhered to the target DNA 23 8 (see FIG. 120) and then the primer 700 is extended to form the probe sequence and the extension. The DNA strand of both products is increased. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 696. When the subsequent denaturation reaction is carried out, the extended oligonucleotide primer 700 is dissociated from the hybrid system of the template, and the double-stranded backbone-101 - 201209158 242 with the linear probe of the primer is also dissociated. The matting group 248 is exited. Once the temperature is lowered for the binding step and the extension step, the primer-like probe sequence 696 of the primer-attached linear probe is rolled up and the amplified complementary sequence 6 on the extended nucleic acid strand 9 8 is heterozygous, and the measured fluorescence indicates the presence of the standard DN A. The unexpanded linear probe with primers retains its own double-stranded backbone and remains in a matte state. Since this detection method relies on a single molecular process, this detection method is particularly suitable for fast detection systems. Dry-ring probe with primers The 122A to 122F diagrams show the operation of the stem-loop probe 704 with the primer attached. Referring to Fig. 122A, the stem-loop probe 7 〇4 with primer has a stem 242 composed of complementary double-stranded DNA and a loop 240 containing the probe sequence. Fluorescent luminescent groups 246 are labeled on the 5' end of one of the backbone chains 708. The other backbone strand 710 is linked to a V-terminally labeled extinction group 248, and the backbone strand 710 carries both the amplification blocker 694 and the oligonucleotide primer 700. During the initial denaturation phase (see Figure 122B), the nucleic acid strands of the target nucleic acid 238 are separated as the stem 242 of the stem-loop probe 7 〇4 with the primer introduced. When the temperature is cooled to carry out the bonding stage (see Figure 122C), the oligonucleotide primer 700 on the stem-loop probe 704 with the primer is hybridized to the target nucleic acid sequence 2 3 8 . During the elongation phase (see Figure 122D), the complementary sequence 760, which is complementary to the target nucleic acid sequence 298, is synthesized to form a DN A chain containing both the probe sequence 7 〇4 and the amplification product. . Amplification blocker 6 94 prevents the polymerase from reading and replicating the probe region -102- 201209158 domain 7 〇4. When the probe is followed by a denaturation step (see Figure 122E) followed by adhesion, the probe sequence of the loop portion 240 of the stem-loop probe with the primer (see Figure 122F) is complementary to the extension chain. Sequence 706 is bonded. This configuration allows the fluorescent luminescent group 246 to be relatively far from the extinction group 248 to significantly enhance luminescence. Control Probes φ Hybrid Chamber Array 110 contains a number of hybrid chambers 1 80 containing positive control probes and negative control probes for quality control of analytical assays. Sections 135 and 136 illustrate a negative control probe 796 that does not contain a fluorescent luminescent group, and lines 137 and 138 are schematic representations of a positive control probe 798 without an extinction group. The positive control probe and the negative control probe have a dry-loop structure similar to the above FRET probe. However, regardless of whether the probes are hybridized to become an open structure or remain closed, the positive control probe 79 8 will always emit a fluorescent signal 25 0, and the negative control probe 796 will always emit a fluorescent signal 250. . Referring to Figures 135 and 136, the negative control probe 796 does not have a fluorescent luminescent group (and may or may not have an extinction group 248). Thus, whether or not the target nucleic acid sequence 23 8 is hybridized to the probe (see Figure 136), or the probe maintains its own dry-loop structure (see Figure 135), the negative The response of the control probe to excitation light 244 was negligible. Alternatively, the negative control probe 796 can be designed to keep the negative control probe 796 permanently matted. For example, by artificially synthesizing the loop 240, the loop has a probe sequence that will not be hybridized to any nucleic acid sequence in the sample under investigation -103-201209158, and the backbone of the probe molecule 2 42 will be re It is hybridized to the probe molecule itself, and the fluorescent luminescent group and the extinction group will remain close and no visible fluorescent signal will be detected. This negative control signal can correspond to a low release of light originating from the hybrid chamber 180 (the probes in the chambers are not heterozygous but the extinction group fails to eliminate all of the reporters from the reporter) Release light). Conversely, as shown in Figures 137 and 138, the positive control probe 798 is constructed to contain no matting groups. Whether or not the positive control probe 798 is heterozygous to the target nucleic acid sequence 238 does not eliminate the fluorescent light 250 released by the fluorescent luminescent group 246 back to the stress luminescence 244. Figure 5 2 shows the possible distribution of the positive control probe and the negative control probe throughout the hybrid chamber array 110 (3 7 8 and 380, respectively). The control probes 3 7 8 and 380 are placed in a plurality of hybrid chambers 180 arranged in a row across the hybrid chamber array 110. However, the arrangement of the control probes in the array is arbitrarily configured (depending on the configuration of the hybrid chamber array 1 1 〇 at the time). The design of the fluorescent luminescent group is necessary to have a long-term luminescent lifetime of the fluorescent luminescent group to allow sufficient time for the excitation light to decay below the intensity of the fluorescent luminescence (the light sensing is now performed) The device 44 operates) to provide sufficient signal to noise ratio. In addition, the longer the egg light luminescence life cycle, the greater the conversion to the integral camp photon count. Fluorescent luminescent group 246 (see Figure 59) has a fluorescence lifetime of greater than 100 nanoseconds, typically greater than 200 nanoseconds, more often greater than 300 nanoseconds, and most often -104 to 201209158 is greater than 400 nanoseconds. Metal-ligand complexes based on transition metals or lanthanides have long lifetimes (from hundreds of nanoseconds to hundreds of milliseconds), sufficient quantum yield and high temperature stability, chemical stability and photochemistry Stability 'These properties are of an advantageous nature in relation to the requirements of the fluorescence detection system. Metal-ligand complex based on transition metal ion ruthenium (Ru(II)) is fully studied. Tris(2,2f-bipyridyl) ruthenium (Π) ( [Ru ( bpy) 3] 2+ The complex has a life cycle of about 1 microsecond. This complex is a product of Biosearch Technology under the name Pulsar 650. Table 1 , Pulsar 650 (钌 chelate) photophysical property parameter number 値 unit absorption wavelength λ abs 460 nm emission wavelength λ em 650 nm Extinction coefficient E 1 4800 Fluorescence lifetime Tf 1.0 Microsecond quantum yield H 1 (deoxidation) None (n/a) Terbium chelate is a lanthanide metal-ligand complex that has been successfully demonstrated The ruthenium chelate can serve as a fluorescent reporter group in the FRET probe system, and the ruthenium complex also has a long life cycle of 1600 microseconds. -105- 201209158 Table 2. Photophysical properties of ruthenium chelate parameters Symbol number 値 unit absorption wavelength Labs 330-350 nm luminescence wavelength ^em 548 nm extinction coefficient E 13800 (according to xabs and ligand, at Xe=340 At the nanometer, the extinction coefficient can be as high as 30,000) NfW1 Fluorescence lifetime Tf 1600 (probe of hybrid probe) Microsecond quantum yield Η 1 (depending on the ligand) None (N/A) used by LOC device 301 The fluorescence detection system does not use a filter to remove unwanted background fluorescence. Therefore, it is beneficial if the extinction group 24 8 does not have a native emission, thereby increasing the signal to noise ratio. Without native light, there is no background fluorescence from the extinction group 248. High extinction is also important to prevent fluorescence from occurring before a heterozygous reaction occurs. The black hole type matting group (BHQ) does not have primary light and has a high extinction ratio (black hole type extinction group available from Biosearch Technologies of Novato, California, USA), which is an extinction group suitable for the system. . The black hole type matting group BHQ-1 has a maximum absorption wavelength of 534 nm, and the extinction range is from 480 nm to 580 nm, making BHQ-1 suitable as a fluorescent luminescent group for Tb-chelate. Extinction group. The black hole type matting group BHQ-2 has a maximum absorption wavelength of 5 79 nm, and the extinction range is from 560 nm to 670 nm, making BHQ-2 an extinction group suitable for the fluorescent luminescent group Pulsar 650. . Iowa Black Matting Group (Iowa Black FQ and RQ, available from US-106-201209158, Integrated DNA Technologies, Coralville, Iowa) with little or no background light Suitable alternative extinction groups. Iowa Black FQ has an extinction range of 420 nm to 620 nm and has a maximum absorption wavelength of 531 nm, so Iowa Black FQ is suitable for the extinction base of Tb-chelate fluorescent group. group. Iowa Black RQ has a maximum absorption wavelength of 65 6 nm, and the extinction range is from 500 nm to 700 nm, making Iowa Black RQ an ideal extinction group for the fluorescent luminescent group Pulsar 650. In the embodiments described herein, the matting group 248 is a functional group that is initially attached to the probe, but in other feasible embodiments the matting group is freely present in solution. molecule. Source of excitation light

In the fluorescence detection based embodiment described in the present application, since the light emitting diode (LED) has low power consumption, low cost, and small size, the LED is selected to replace the laser diode, the high power lamp or the laser. As a source of excitation light. Referring to Fig. 123, LEDs 26 are disposed on the outer surface of the LOC device 301 and directly above the hybrid chamber array 110. The photo sensor 44 is located on the opposite side of the hybrid chamber array 110. The photo sensor 44 is an array of a plurality of photodiodes 184 (see Figures 53, 54 and 65) for detection. The fluorescent signal originating from each chamber is measured. Embodiments 124, 125, and 126 illustrate other embodiments for exposing the probe to excitation light. In the LOC device 30 shown in Fig. 124, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 to the hybrid cavity - 107 - 201209158 chamber array 1 10 . The excitation LED 26 is pulsed' and the photodetector 44 is used to detect the fluorescence release light. In the LOC device 30 shown in FIG. 125, the excitation light 2 44 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 71 4 . on. The excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent light. Similarly, in the LOC device 30 shown in FIG. 126, the excitation light 244 generated by the excitation LED 26 is guided to the hybrid chamber by the lens 2 54 , the first mirror 716 and the second mirror 7 18 . Array 1 1 0. Similarly, the excitation LED 26 is pulsed and the photodetector 44 is used to detect the fluorescent light release. The wavelength of the excitation light of LED 26 varies depending on the choice of fluorescent dye. Philips LXK2-PR14-R00 LED system is suitable for Pulsar 650 dyeing! J excitation source. SET UVT0P 3 3 5 The T039BL LED is suitable for the excitation source of the Tb-chelate label. Table 3, Fei〗 FIJ Pu LXK2-PR14-R00 type LED specification parameter number 値 unit wavelength λ e X 460 nm emission frequency Vem 6.52 (10) 14 Hz (Hz) output power Pl per 1 amp 0.5 1 5 minutes Watt (w) Radiation Profile Lambert Distribution Profile None (N/A) -108- 201209158 Table IV, SET UVT0P334T039BL LED Specifications Parameter Number 値 Unit Wavelength λε 340 Nano Transmit Frequency Ve 8.82(10)14 Hertz (Hz Power Pi per 20 milliamperes 0·000240 minutes watts (w) pulse forward current I 200 milliamperes (mA) radiation profile Lambertian distribution no (N/A) UV excitation pupils absorb ultraviolet (UV) spectra Very little light. Therefore, it is advantageous to use ultraviolet light to excite light. Although UV LEDs can be used to excite the light source', the broad spectrum of LED 26 reduces the effectiveness of this method. To solve this problem, a filtered UV LED can be used. Unless the relatively high cost of the laser cannot be implemented in the specific inspection module market, the ultraviolet laser can be used as the excitation source at will. LED Driver The time required for the LED driver 29 to drive the LED 26 at a fixed current for a sustained period of time. The low-power USB 2.0 certified driver provides up to 1 unit load (100 mA) with a minimum operating voltage of 4.4 volts. Standard power conditioning circuitry is used for this purpose. Photodiode Figure 54 shows the photodiode 184 integrated in the CMOS circuit 86 of the L〇C device 301. Light diode I84 is made part of CMOS circuit 86 without the need for additional masking or steps. This is the advantage of CMOS photodiodes over the -109-201209158 CCD (CCD is an alternative sensing technique that uses non-standard processing steps to integrate CCDs on the same wafer or on adjacent wafers). On-wafer detection is low cost and reduces the size of the analytical inspection system. The shorter optical path length reduces noise from ambient environments for efficient collection of fluorescent signals and eliminates the need for conventional optical components consisting of lenses and filters. The quantum efficiency of the photodiode 184 is caused by photons impinging on the active region 185 of the photodiode 184 to be efficiently converted into photoelectrons. For standard tantalum processes, the quantum efficiency for visible light ranges from 0.3 to 0.5 depending on process parameters (for example, the number of overlays and the absorptive properties). The detection threshold of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection threshold also determines the size of the photodiode 184 and thereby determines the number of hybrid chambers 180 in the hybrid and detection section 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (in the case of LOC device 301, which is 1 760 microns X 5 8 24 microns) and incorporated into other functional modules. The actual footprint available after (eg, pathogenic dialysis section 70 and amplification section 11 2 ). For standard 矽 process, photodiode 1 84 detects a minimum of 5 photons. However, to ensure reliable detection, the minimum 値 can be set to 1 光 photons. Therefore, as described above with a quantum efficiency of 0.3 to 0.5, the fluorescence-derived light from the probes must have a minimum of 17 photons, but 30 photons can be included in the appropriate error range for reliable detection. . -110- 201209158 Calibration Chamber The inconsistencies in the residual excitation photon flux of the electrical characteristics, spontaneous fluorescence, and incomplete decay of the photodiode 184 introduce background noise and offset into the output signal. The background 値 in each output signal is removed using one or more calibration signals. A calibration signal is generated by exposing one or more of the calibration photodiodes 184 in the array to respective calibration sources. A low calibration source is used to determine the negative result (the negative result is that the target is not reacting with the probe). A highly calibrated light source indicates a positive result derived from the probe-target complex. In the embodiment of the present invention, the low calibration light source is provided by a plurality of calibration chambers 382 in the hybrid chamber array 110, the calibration chambers 3 8 2: without any probes; A probe of a light reporter group; or a probe comprising a reporter group and an extinction group but the matte group is configured to anticipate that matting will occur all the time.

The output signals from such calibration chambers 3 82 are nearly as close to the noise and offset from the output signals of all of the hybrid chambers in the LOC device. Subtracting the calibration signal from the output signal produced by the other hybrid chamber substantially removes the background and leaves the signal produced by the fluorescent light (if there is fluorescence). It is also possible to subtract the signal caused by the ambient light in the array of cells. It will be understood that the negative control probes shown in Figures 135 to 138 above can be used in the calibration chamber. However, as shown in Figures 128 and 129, '128 and 129 are diagrams 127 of the LOC device variant X 728 in Fig. 127 and an enlarged view of Fig. DH. Another option is to make these The calibration chamber 382 is fluidly isolated from the amplicon. The chambers are indeed emptied by emptied the fluid-isolated chambers, or the fluid-isolated chambers contain probes that do not have a reporter group, or because the fluidization does not allow the hybridization reaction to occur. The inclusion of any "normal" probe with both a reporter group and a matting group can determine the background noise and offset. The calibration chambers 382 can provide a high calibration source to produce a high signal in the corresponding photodiode. This high signal corresponds to all probes in the chamber that have been hybridized. Pointing a probe with a reporter group but no extinction group or a dot-only matting group will constantly provide a signal near the time when most of the probes in the hybrid chamber have been hybridized. It will also be appreciated that the calibration chambers 382 can be used in place of the control probes or that the calibration chambers 382 can be additionally used in addition to the control probes. The number and configuration of the calibration chambers 382 in the entire array of hybrid chambers can be determined at will. However, if the photodiodes 184 are calibrated by the relatively closest calibration chamber 382, the calibration is more accurate. Referring to Fig. 56, the hybrid chamber array 110 has a calibration chamber 382 for use by all eight hybrid chambers 180. That is, the calibration chamber 382 is located in the center of the 3 x 3 square array comprised of the hybrid chambers 180. In this configuration, the hybrid chambers 180 are calibrated by the adjacent calibration chamber 382. Figure 134 shows a differential imaging circuit 788 that subtracts the excitation light from the fluorescent signals surrounding the hybrid chambers 80 to cause the photodiodes corresponding to the calibration chambers 382. Signal sent by 184. The differential imaging circuit 768 samples the letter from the pixel 709 and the "false" pixel 7 9 2 -112-201209158. In a specific embodiment, the "false" pixel 7 9 2 is shielded from light, so the output signal of the dummy pixel 792 provides a dark reference. Alternatively, the "false" pixel 792 will be exposed to the excitation light along with the rest of the array. In a particular embodiment where the "false" pixel 792 is exposed to light, the signal caused by ambient light in the array of cells is also subtracted. The signal from pixel 790 is small (ie, close to the dark signal), and it is difficult to distinguish between background 値 and very small signal without reference to dark signal 「 during use, "read line (read_r〇w)" 794 and "false pixel" The read row line (read_row_d) 795 is activated, and the M4 transistor 797 and the MD4 transistor 801 are turned on. Switch 807 and switch 809 are turned off so that the output 源自 from pixel 790 and "false" pixel 792 are stored on pixel capacitor 803 and dummy pixel capacitor 805, respectively. After the pixel signals are stored, the switch 807 and the switch 809 are stopped. Subsequently, the "read column line (read_C)" switch 811 and the dummy pixel "read column line" switch 813 are turned off, and the switched capacitor amplifiers 8 1 5 at the output end amplify the differential Signal 8 1 7. Inhibition and Activation of the Photodiode The photodiode 184 is inhibited by the LED 26 during excitation and the photodiode 184 is activated during the illumination. Figure 66 is a circuit diagram of a single photodiode 184, and Figure 67 is a timing diagram for a diode control signal. The circuit has a photodiode 184 and six metal oxide semiconductor (MOS) transistors Mshunt 394, Mtx 396, Mreset 398, Msf 400, Mread 402-113-201209158 and Mbias 4 04. During the initial period t1 of the excitation cycle, the shunt transistor Mshunt 3 94 and the reset transistor Mreset 3 98 are activated by pulling up the shunt transistor (Mshunt) gate 3 84 and the reset gate 38 8 . During this period, the excitation photons generate carriers in the photodiode 184. When the number of carriers produced may be sufficient to saturate the photodiode 1 84, these carriers need to be removed. The transistor Mshunt 394 directly removes the carrier generated in the photodiode 184 during the cycle due to leakage inside the transistor or diffusion due to the excitation of the carrier generated in the substrate, while the reset of the transistor Mreset 3 98 is accumulated. Any carrier on node "NS" 4 06. After the excitation, a capture cycle is started at time t4. During this capture cycle, the response from the luminescent group is captured and integrated in the circuit on node "NS" 406. This operation can be achieved by pulling the transistor Mtx gate 3 86 to activate the transistor M, x3 96 and transferring any carriers accumulated on the photodiode 184 to the node "NS" 406. The period of the capture cycle may be as long as the luminescence time of the fluorescent luminescent group. The output chirp from all of the photodiodes 184 in the hybrid chamber array 1 10 is delayed by simultaneously capturing the end of the capture cycle t5 and the beginning of the read cycle t6. This delay is caused by the individual reading of each photodiode 184 in the hybrid chamber array 11 (see Figure 52) after the capture cycle needs to be continued. The first photodiode 184 to be read will have the shortest delay before the read cycle, while the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Μ "ad 4 0 2 is activated by pulling up the read gate 3 3 3 . The source is followed by the transistor - 114 - 201209158 body Msf 400 buffer and read out" NS" node 406 voltage.尙 has an additional method for starting or suppressing the photodiode, and the methods are discussed as follows: 1 · Suppression method Figures 13 1 , 132 and 133 show three feasible methods for shunting transistor (Mshun ) 3 94 Structure configuration. (778, 780, 782) shunt transistors (Mshunt) 394 have a very high off-rate ratio at the maximum |Κω| = 5 volts used during excitation. As shown in Figure 13, a shunt transistor (Mshunt) gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in FIG. 132, the shunt transistor (Mshunt) gate 3 84 can be configured to surround the photodiode 184. The third option is to divide the shunt transistor as shown in FIG. The (Mshunt) gate 3 84 is constructed at the inner side of the photodiode 1 84. When the third option is selected, there will be fewer photodiode active regions 185. These three structural configurations 7 7 8 ' 7 8 0 and 7 8 2 shorten the average path length from all positions in the photodiode 184 to the shunt transistor (Mshunt) gate 3 84. In Fig. 131, a shunt transistor (Mshunt) gate 3 84 is located on one side of the photodiode 184. This configuration is configured to be the simplest and least encroaching of the photodiode active region 丨85. However, any carriers remaining on the far side of the photodiode 1 84 may take longer to propagate to the shunt transistor (Mshunt). In Fig. 132, a shunt transistor (Mshunt) gate 3 84 surrounds the photodiode 184. This configuration further shortens the average path length of the carrier in the photodiode 184 from -115 to 201209158 to the shunt transistor (Mshunl) gate 3 84. However, extending the shunt transistor (Msh unt) gate 3 8 4 to the vicinity of the perimeter of the photodiode 1 8 4 forces the photoreceptor active region 185 to be more substantially reduced. The configuration 782 in Figure 133 is such that the shunt transistor (Mshunt) gate 3 84 is located within the active region 185. This configuration provides the shortest path length to the shunt transistor (Mshunt) gate 384 and thus provides the shortest transition time. However, the impact on the active zone 185 is greatest. This configuration 782 also has a wider leakage path. 2. Startup Method a. The trigger photodiode system uses a fixed delay to drive the shunt transistor. The _b. trigger photodiode system uses a programmable delay to drive the shunt transistor. c. The shunting electron crystal is driven by the LED segment pulse with a fixed delay. d. Drive the shunt transistor as shown in 2 c but with a programmable delay. Figure 69 is a cross-sectional view of the cross-hybrid cavity 1 80 showing the photodiode 184 and the triggering photodiode 187 embedded in the CMOS circuit 86. A small area in the corner of the photodiode 184 is replaced with a trigger photodiode 187. A small-area triggering photodiode 18 7 is sufficient for when the intensity of the laser is high enough to be comparable to the intensity of the fluorescent light. The trigger photodiode 187 is sensitive to the excitation light 244. The trigger photodiode 187 records that the excitation -116-201209158 light 244 has subsided and the photodiode 184 is activated after a short delay Δί 3 00 (see Figure 2). This delay allows the fluorescent photodiode 184 to detect fluorescent light emission from the FRET probes 186 without excitation light 244. This configuration allows for detection and improves signal to noise ratio. Both photodiode 184 and trigger diode 187 are located within CMOS circuitry 86 below each hybrid cavity 180. An array of a plurality of photodiodes is combined with appropriate electronic components to form the photosensor 44 (see Figure 65). The photodiodes 184 do not require the use of additional masks or pn-junctions formed during the fabrication of the CMOS structure. During the MST fabrication, a standard MST photolithography technique is used to thin the dielectric layer (not shown) above the photodiode 184 to allow more phosphor to illuminate the photodiode 184. Active area 185. The photodiode 184 has a field of view such that fluorescent signals originating from the probe-target hybrids within the hybrid chamber 180 are incident on the sensing surface of the sensor. The fluorescent light is converted to a photocurrent, and the photocurrent can then be measured using a CMOS circuit 86. Alternatively, one or more of the hybrid chambers 180 may only exclusively trigger the photodiode 187. These choices can be used in these 2c systems and in combination with the above 2a and 2b systems. Fluorescence Delay Detection The following derivative functions illustrate the use of delayed fluorescence detection of long lifetime fluorescent light-emitting groups in the above LED/fluorescent group combination. As shown in Fig. 60, the time function of the intensity of the fluorescence after excitation is derived by the ideal constant intensity pulse /e between time & time > -117- 201209158 Let [phantom] (〇 is equal to the density of the excited state at time t, then the number of excited states per unit volume per unit volume is described by the following differential equation during and after excitation. (1) dt tf hve where m is the molar concentration of the fluorescent luminescent group, ε is the molar extinction coefficient ve is the excitation frequency, and the Planck constant A = 6.62606896 (1 〇 yyyy joules sec ( ) ° The differential equation has the following General formula: + p(x)y = Φ) αχ The solution of this equation: -.(2) [e^P()dx q{x)dx +k ΛΧ) = —— Now use this formula to solve the equation ( 1) '[sm)=l^iL+ke-^ ... ο) hVe is at time 6, and is obtained by equation (3): k = -i^lLe', ... (4) hve Substituting (4) into equation (3): at time: ...(5) hve hve -118- 201209158 For &匕, the excited states are exponentially decayed and described by the following equation = [51] (〇= [51](i2)e-(,-,2)/^ (6) Substituting equation (5) into equation (6):

J FCT

[51](〇 = -~^[l-e-(,2',l)/r/]g-(^)/r/ (7) hve The fluorescence intensity is written as the following equation:

Where v/ is the fluorescence frequency, 7 is the quantum yield, and / is the optical path length. Now obtained by equation (7): 4^1](〇_ Ieec 己-('2-'1)"/ 1己-(卜,2)"/ dt hve Substituting equation (9) into equation (8) ), get: IAt) = Iesc^^[\-e~{,1-h)lTf ]eHt~,l)lTf ...(10) For / (〇_>/〆咖', call) &quot ;/

Xf Ve Therefore, we can write the following approximate equation after a sufficiently long excitation pulse (, 2 - G » Γ,): For, /,(〇=/e£c/77Ye_('-'2)/ r/ ...(11) In the previous paragraph, we can infer when »,, for, /, (i) = /esc/7-^e_('_'2)/r/. From the above equation, We can obtain the following formula: -119- 201209158 h'f(t) = nesc^e'(l',l)lT, ...(12) where, I Μ) = is the unit time per unit area The number of fluorescent photons, and \ is the number of excitation photons per unit area per unit time. The hVe result is cc nf{t) = \nf{t)dt (13) the number of fluorescent photons per unit area of the center system, and the instantaneous time when the ί3 light diode is turned on. Substituting equation (1 2 ) into equation (1 3 ):

CO At this time, the number of fluorescent photons per unit area of the photodiode is Ϊ, (0 is written as: «; (0 = «/(0^〇...(15) where What is the light collection efficiency of the optical system. Substituting equation (12) into equation (15), we can get: ns{t) = φ0ήεεεΙηβ'(,~'ι)ΙΤί (16) Similarly, arrival The number of fluorescent photons per unit of fluorescence area of the photodiode will be as follows: co 纪 = ί & (〇 and substituted into equation (16) and the integral is obtained: '3

Hs = φ^η(εα1ητ fe~(h~,:t)lTf Therefore, we can get: ~= recognize εάητ〆1^” (17) -120- 201209158 If the rate of decay of the excited photon flux is much faster than The rate of decay of the fluorescent photon flux, which is the time at which the rate at which the fluorescent photons generate electrons in the photodiode 184 is equal to the rate at which the excited photons generate electrons in the photodiode 184. Caused by fluorescence The rate at which the sensor outputs electrons per unit of fluorescence area is: '^) = φ/ήχί) I where ^ is the quantum yield of the sensor at the wavelength of the fluorescence. Substituting the above formula into (1 7 ), we get: ^(0 = ii^〇«>/77^(W2,/r/ -(18) Similarly, the rate of electron output from the sensor per unit of fluorescence area caused by the excitation photons The system: ... (π) where the quantum yield of the sensor at the wavelength of the excitation light 'and & corresponds to the time constant of the "off" property of the excitation LED. After the time interval #ί2, the The photon flux in the decay of the LED may increase the intensity of the fluorescent signal and extend the decay time of the photon flux', but we assume the effect of this condition on If (t) It is very small' therefore we have adopted a conservative approach. At this time, as mentioned earlier, 'the best of 3's: therefore' by equations (18) and (19) 'we have: and after rearranging Wait until: -121 - ...(20)201209158

\η{εεΙη

Te

Tf is explained by the two paragraphs above. We get the following two expressions: ns = φ^ι^τ!...(21)

At =

In F Φ / Φο y -- (22) where = Jiang / 77 and = 6 _ G. We also know that 丨2 — (1 » Γ/. The best time for fluorescent detection and the use of Philips [乂]<:2-?1114-11〇〇 LEDs and Pulsar 650 Fluorescent Dyestuffs The measured number of fluorescent photons is determined by the following method: The optimal detection time is determined using equation (2 2 ): recalling the concentration of the amplicon, and assuming that all amplicons are heterozygous, the fluorescent emission The concentration of the group is: c = 2.89 (10) 4 m / liter. The height of the chamber is the optical path length / = 8 (10) _ 6 m. We make the area of the phosphor equal to the area of the photodiode However, the actual area of the phosphor is substantially larger than the area of the photodiode; therefore, we can roughly assume the light collection efficiency of our optical system & = 〇.5. From the characteristics of these photodiodes, f = l〇 The ratio of the quantum yield of the photodiode at the fluorescence wavelength is much more conservative than the quantum yield at the wavelength of the excitation Φα. The decay life cycle of the general LED is re=0.5 nanoseconds and the Pulsar 650 is used. Specifications can be determined △ /: •122- 201209158 F = [1.48(10)6 ][2.89(10) *6 ][8(10)'6 ] (1) = 3.42(10)-5 A. ln( [3.42(10) -5](10)(0.5)) 1 1 1(10)-6 —0.5(10)-9 = 4.34(10)-9 seconds Use equation (21) to determine the number of photons measured. First, by Check the illumination geometry to determine the number of excitation photons emitted per unit time. The Philips LXK2-PR14-R00 LED has a Lambertian # radiation pattern, so: n, = nl0 cos(0) -.-(23) % is the number of photons emitted per unit time of the solid angle of the off-axis angle of the LED, and is the valve of the & in the direction of the axis. The photon emitted by the LED per unit time The total number is: ή, =j η,c/Ω Ω ...(24)

=J )ϊ/0 cos(^)JQ Ω

At this time, ΑΩ = 2π[1 - cos(0 + △ 0)] — 2 river 1 - cos(0)] ΔΩ = 2^[cos(0) - cos(^ + Δ0)] :4;rsin(0 )cos .(ΑΘ) L 2 夕 l 2 J + 4; rcos(0)sin2 ΆΘ' dQ. = 2nsm{6)de Substituting this formula into equation (24) yields: 123 201209158 ή, = j 2τίηΙΰ cos( 0) sin(9)d0 ο = monument 0 */0 After rearranging, we get: ' ...(26) The output power of this LED is 0.515 watts (W) and K = 6.52 (10) 14 Hz.

Ave ..•(27) __0.515_ ~ [6.63(10)-34 ][6.52(10)14] = 1.19(10)18 Photon 渺 Substituting this 入 into equation (2 6 ), we get: . .. 19.19(10)18 ηιο = π =3.79(10)17 Photons/sec Refer to Figure 61 for the optical center 252 and lens 254 of the LED 26. The photodiode system is 16 micrometers χ 16 micrometers, and for the photodiode in the center of the array, the cone solid angle (Ω) of the light emitted from the LED 26 and reaching the photodiode 1 84 is about: Ω = sensing Area /r2 [16(10)~6][16(10)~63 [2.825(10)-3]2~ = 3·21(10)·5 seconds will understand the central light of the photodiode array 44 The purpose of the polar body 184 is for such calculations. When a heterodyne of the Lambertian excitation source intensity distribution occurs -124-201209158, the sensor at the edge of the array may only receive less than 2% of the photons. The number of excitation photons emitted per unit time: ne = ήμ ... (28) = [3.79(10), 7] [3.21(10)'5] = 1.22(10)13 Photon /# 2 9 ): ns =Φ〇ήβΓτ/β'Δ, /Τ/ π, = (0.5)[1.22(10)l3][3.42(10)-5][l(10)_6]e'434(l 〇r, /1 (1〇r6 = 208 photons per sensor) Therefore, using Philips LXK2-PR14-R00 LED and Pulsar 650 fluorescent illuminating group, we can easily detect any generated photons Number of hybrid events. The illumination geometry of the SET-type LED shown in Figure 62. At ID = 20 mA, the LED has a minimum optical power of center wavelength = 340 nm (absorption wavelength of ruthenium chelate) Output Pi = 240 microwatts (μΨ). Driving the LED at ID = 200 mA can linearly increase the output power to Pl = 2.4 milliwatts (mW) by setting the optical center 252 of the LED to The chamber arrays 110 are spaced 17.5 mm apart, and we can concentrate this output flux in a circular point having a maximum diameter of 2 mm. The 2 mm located at the hybridization away plane The photon flux in the point of the diameter is The program 27 indicates. ή _ Pi 1 hve _ 2.4(10)-3 [6.63(10)-34 ][8.82(10)14] -125- 201209158 = 4.10(10)15 photons/second using equation 2 8, Etc. get: he = η, Ω. = 4.10(10)15 [16(10)~6]2 4K10)'3]2 = 3.34(10)"Photons/sec is now back to Equation 22 and is listed previously After the (Tb) chelate @ nature, the following formula can be obtained: 1η[(6·94(10)-5)(10)(0.5)]

Af = -Γ i 1(10)·3 ~ 0.5(10)-9 =3.98(10)·9 seconds is now obtained from Equation 2 1 : ns = (0.5)[3.34(10)M][6.94(10) -5][l(10)-3]e_3 98<1〇r,/I(I〇r3 = 11600 photon/sensor can be easily detected using SET type LE D and ruthenium chelate system The number of photon theories emitted by the event, and the number of photon theories exceeds the minimum of 30 photons required for reliable detection by the light sensor. The maximum spacing between the probe and the photodiode is heterozygous on the wafer. The detection method eliminates the need for detection by a conjugate focal microscope (see the paragraph of the prior art). This detection method from the conventional detection technology is an important factor saving time and cost in the system of the present invention. The measurement method requires the use of imaging optics of many lenses or curved mirrors. By using non-imaging optics, the diagnostic system eliminates the need for complex and bulky optical component strings. The photodiode -126 - 201209158 The advantage of having very high collection efficiency when placed very close to the probes: when the probes and the photodiode When the thickness of the material is 1 micron, the collection angle of the released light is up to 173. This angle is based on the probe located at the centroid of the surface of the hybrid chamber closest to the surface of the photodiode. The light is calculated, wherein the photodiode has a flat active surface region parallel to the surface of the chamber. The light in the cone of the illuminating angle can be absorbed by the photodiode, and the cone of the illuminating angle is defined as the flat a luminescent probe at the sensor corner on the perimeter of the chamber face and at the apex of the cone. For a 16 micron X 16 micron sensor, the apex angle of the cone is 170. The pole body is enlarged to a limit equivalent to a hybrid chamber of 29 micrometers and 19.75 micrometers, which is 173 degrees. It can be easily achieved between the surface of the chamber and the active surface of the photodiode. The separation distance is 1 micron or less. The system using non-imaging optical technology must have the photodiode 184 very close to the hybrid chamber to collect sufficient fluorescent photons. Refer to Figure 54 to determine the light II as follows. Polar body and probe The maximum spacing. Using the ruthenium chelate luminescent group and the SET UVT0P3 3 5T039BL type LED, we calculated that 11600 photons from the respective hybrid chamber 180 reached the 16 micron x 16 micron photodiode. 184. When performing this calculation, we assume that the collection region of the hybrid chamber 180 has a substrate area equal to the photodiode active region 185, and that half of the total number of such photons arrive at the photodiode 184. That is, the light collection efficiency of the optical system is nine = 0.5. More precisely, we can write a formula = [(light collecting area of the hybrid chamber) / (photodiode area)] [Ω / 4π] , where Ω = the solid angle directly opposite the photodiode at a representative point on the hybrid chamber substrate -127-201209158. For the geometry of a regular pyramid: n = 4arcsin(fl2/(4i^2 + fl2)) 'where <= the distance between the chamber and the photodiode, and the size of the photodiode . Each hybrid chamber releases 23,200 photons. The selected photodiode has a detection threshold of 17 photons; therefore, the minimum optical efficiency required is ^0 = 17/23200 = 7.33 x1ο-4 the substrate area of the collection region of the hybrid chamber 180 29 microns χ 19.75 microns. Solution 4, we will get the maximum limit distance between the bottom of the hybrid chamber and the photodiode 1 8 4 is 4 = 249 microns. Under this constraint, the above-defined light collecting cone angle is only 0 · 8 degrees. It should be noted that this analysis ignores this negligible refraction effect. LOC Device Variations The LOC device 301, described and illustrated above, is only one of many possible L Ο C device designs. A variety of LOC device variations of the various functional segments described above using different combinations will now be described and/or illustrated in a schematic flow diagram (from sample entry to detection) to illustrate some of the combinations of such possible combinations. The flow diagrams are suitably divided into sample placement and preparation stages 28 8 , extraction stage 290, incubation stage 291, amplification section 2 92, pre-hybridization stage 2 93, and detection stage 294. For all LOC devices that are briefly described above or only in the form of abbreviations, for the sake of clarity and conciseness, -128-201209158 does not show the drawings of the complete layout. Also for clarity, the smaller functional units (eg, liquid sensors and temperature sensors) are not shown, but it will be appreciated that such smaller functional units have been incorporated into each of the following LOC devices. At the right place in the design.

LOC Device Variant VIII Figures 73-77 and 78-104 show LOC device variant VIII 518. The features and structures corresponding to the equivalent features and structures shown in LOC device 301 are denoted by the same reference numerals in the drawings. Features that do not correspond to the previously described features are denoted by new component symbols. As shown schematically in Figure 104, the variant 518 of the LOC device uses 12 different amplification segments 112.1 to 112.12 to extract 290, incubate 291, expand 292, and detect 294 human DNA. The LOC device variant VIII 518 uses multiple amplification segments to increase the sensitivity of the assay and to increase the signal to noise ratio of the detected fluorescence. Referring to Figures 73, 74 and 75, the blood sample enters the sample inlet 68 and capillary action draws the blood sample along the cap channel 94 to the anticoagulant surface tension valve Π 8. The cover layer 46 is manufactured to have an alternative layer for replacing the lower seal layer 64. In this design, interface layer 594 is located between capping channel layer 80 and MST channel layer 100 of the CMOS + MST device 48. The interface layer 594 allows for a more complex fluid interconnect structure between the reagent reservoirs and the MST layer 87 without increasing the size of the germanium substrate 84. Figure 75 overlays the storage tanks, the top channels, and the interface channels to illustrate the more sophisticated piping -129 - 201209158 project achieved with the interface layer 594. The preferred system, as shown in Figure 103, of the interface layer 594 requires the anticoagulant surface tension valve 118 to have two interface channels 5 96 and 5 98. The storage tank side interface 596 connects the storage tank outlet to the lower suction holes 92, and the sample side interface passage 598 connects the upper suction holes 96 with the cover layer passage 94. The anticoagulant from the reservoir 5 4 flows through the MST channel 90 via the interface channel 596 on the reservoir side to position the meniscus at the upper suction holes 96. The sample fluid flowing along the capping channel 94 wets the interface channel 594 on the sample side to remove the meniscus such that when the sample fluid continues to flow to the white blood cell dialysis section 3 2 8 , the anticoagulant is Blood samples are combined. • Referring to Figures 78 and 103, the white blood cell dialysis section 3 28 includes a bypass channel 600 for accommodating the fluid channel structures without capturing air bubbles. The blood sample flows through the capping channel 94 to the upstream end of the interface target channel 602. The interface target channel 602 is in fluid communication with a plurality of dialysis MST channels 204 via a plurality of apertures 165 that are shaped like holes having a diameter of 7.5 microns. Each of the dialysis MST channels 204 leads from a 7.5 micron diameter orifice 165 to a respective dialysis uptake orifice 168. The dialysis upper suction holes 168 are open to the interface waste liquid passage 604. However, the upper suction holes are configured to hold the meniscus without allowing the capillary drive flow to continue. Conversely, the bypass channel 600 at the extreme upstream end of the leukocyte dialysis section 3 28 has a capillary priming feature (ciF) 202 to facilitate capillary flow of fluid from the bypass channel 600 into the interface waste channel 604 (see pages 78 and 103). Figure). The bypass channel also has a wide curved flow path to extend the flow path from the interface target channel 602 to the interface waste channel 604. The sample fluid is delayed by a longer flow path than -130-201209158 such that the sample fluid is injected into the interface waste channel 604 after the meniscus is formed at the most upstream dialysis MST channel 204. The sample fluid begins at the upstream end, and as the sample fluid moves downstream along the interface waste channel 604, the sample fluid releases the meniscus at each dialysis upper suction port 168. This ensures that all dialysis MST channels are filled with sample fluid when the sample fluid is injected into the dialysis section. Some dialysis MST channels 204 may not be full when there is no bypass channel 600 or no dialysis upper suction port 168 configured to hold the meniscus. Similarly, air bubbles may form in the interface waste channel 604. In either case, it is possible to stop the flow through the dialysis section. Returning to Figures 74 and 75, the interface waste channel 604 feeds into the waste channel 72 that flows to the waste reservoir 76. Interface target channel 602 is fed to the target to 74. A sample flow system containing target cells is drawn along the target channel 74 to the surface of the lysate reagent. Tension valve 128. If the anticoagulant surface tension valve 118 is provided, the lysis reagent surface tension valve 128 has an interface channel 606 on the lysis reagent storage tank side and an interface channel 608 on the lysis sample side (see Fig. 75). The lysis reagent flows from the reservoir 56 through the capping channel 94 to the interface channel 606 on the lysis reagent storage tank side. The reagent flows into the lower suction holes 92, through the MST passage 90, to the upper suction holes 96, and the reagents hold the meniscus at the upper suction holes 96 (see Fig. 74). A sample fluid originating from the target channel 74 is injected into the interface channel 608 on the lysis sample side. The sample fluid removes the meniscus at the upper suction holes 96, and when the sample fluid flows into the chemical lysis section 130, the lysis reagent is combined with the sample. In the chemical lysis section 130, a lysis reagent is diffused and mixed throughout the sample fluid to dissolve the target cells and release the genetic material within the target cells. The sample fluid stops at the mixing section outlet valve 206. Best Modes As shown in Figures 78 and 103, the mixing section inlet valve is a boil-start valve 206. The sample of the lysed cells flows into the mixing section outlet lower suction hole 6 1 2 via the interface conduit 61. The sample continues to flow along the MST channel 90 to the boiling start valve 206, and when the meniscus is positioned at the valve upper suction port 151 in the top layer 66, the sample stops at the boiling start valve 206 (see especially 80th and 81A)). A liquid sensor 1 74 located upstream of the valve provides feedback that the sample flow system generally reaches the suction port 151 of the valve. If the CMOS circuit 86 is programmed to write a delay time to ensure that the target cells are completely dissolved, the feedback from the liquid sensor will begin to perform the delay time. After the delay time, the ring heater 152 is activated by the heater contact points 156 (see Figures 81A and 81B). The sample liquid located at the suction hole 151 of the valve boils and the meniscus is released. The sample flows into the boiling start valve interface groove 616 (see Figure 82) and out of the valve lower suction hole 150 (see Figure 8 1 B). The downstream liquid sensor 174 indicates that the fluid has started to flow again along the μ S T channel 90. The lysed sample fluid continues to flow to the upper suction port 96 of the restriction enzyme, ligase, and linker surface tension valve 132 (see Figure 78). Referring to Figures 80, 81, 82, 83 and 84, the restriction enzymes, ligase and linker-introducers in the reservoir 58 flow into the capping channel 94 and proceed to the valve interface channel of the restriction enzyme, ligase and linker. 6 1 4. The restriction enzyme, ligase, and valve interface channel 614 of the -132-201209158 primer are open to the three upper suction holes 96 (the enzyme and linker-inducers are retained at the three upper suction holes 96 by the meniscus). The lysed sample fluid in the MST channel 90 passes through the upper suction holes 96 and removes the meniscus' such that the restriction enzyme, ligase, and linker-introduction are mixed with the sample fluid. Referring to Figure 78, the sample, restriction enzyme, ligase, and linker-primer flow through the MST channel mixing section 丨3丨 for diffusion mixing and then into the heated microchannel of the incubation section 112. The incubation section n 4 is comprised of a microchannel 210 of tantalum (see Figure 79) and is heated by a respective heater 154 (see Figure 81A) carried on the upper top layer 66 (see Figure 81A). 210. The heaters 154 extend between respective pairs of heater contacts 156 connected to the CMOS circuit 86. Referring to Figure 85, the sample flow system is stopped at the incubation chamber outlet valve 207. The chamber exit valve 207 is a boil-start valve that is similar to the mixing section outlet valve 206. A liquid sensor 1 74 immediately upstream of the chamber exit valve 207 indicates when the sample fluid will stop at the valve upper suction port 151 (see Figures 85, 87 and 88B). The CMOS circuit 86 begins to implement the incubation time delay (if needed) in response to the liquid sensor. After sufficient incubation, the ring heater 152 boils the liquid at the suction port 157 of the valve to escape the meniscus. The fluid again begins to flow into the boiling start valve interface groove 616 (see Figure 89) and out of the valve lower suction port 150 (see Figure 87). Samples from the lower valve suction port 150 flow along the MST culture outlet channel 630 (see Figure 85) to the polymerase surface tension valve 140 (see Figures 74 and 75). When the sample fluid travels through the -133-201209158 蜿蜒 path of the amplification input channel 632, the polymerase originating from reservoir 6 is fluidly conjugated to the sample. Returning to Fig. 85, the amplification injection channel 63 2 directs the sample fluid through the twelve amplification mixture surface tension valves 138. The amplification mixture in each of the amplification mixture storage tanks 60.1 -6 0. 1 2 (see Figure 75) flows through the respective capping channels 94 (see Figures 90 and 91) and the respective augmentation interface conduits 6 18 ~ 629 (see Figure 89) to position the meniscus at the surface tension valve 138 of the amplification mixture (see Figure 95). The sample fluid sequentially opens each of the surface tension valves, and the amplification mixture from the individual amplification mixture storage tanks 60.1 to 60.12 (see Figure 75) and the sample fluid enter the 12 amplification zones. Each of the segments 112.1 to 112.12 is heated by a microchannel 1 5 8 . Referring to Fig. 92, each of the twelve amplification sections 112.1 to 112.12 has one of the amplification outlet valves 108. The sample fluid is stopped at the valve upper suction hole 151 of each of the expansion outlet valves 108. After thermal cycling, the valve heater 152 boils the liquid at the suction port 151 of the valve (best shown in Figure 96B) and the sample flows toward the valve interface groove 616 (see Figure 97). And flowing out from the i-port lower suction hole 150. Located downstream of the amplification outlet valve 108 is a plurality of independent hybrid chamber arrays composed of a plurality of hybrid chambers iso, the hybrid chamber arrays 110.1 to 110.12 are used for the 12 different types, respectively. Of the amplicon (see Figures 9 2 and 1 0 0). The sample flows along the flow path 167 through each of the individual hybrid chamber arrays 1 1 〇 1 1 1 1 〇 1 2 and flows into the individual hybrid chambers via respective diffusion barrier inlets 175. In the room 180. Refer to Figure 1 'When the sample fluid reaches the endpoint liquid sensor〗 78, energizing the hybrid-134-201209158 heater 182 for the most appropriate probe-target hybrid reaction. In the 77th and 100th to 102th, the titanium nitride (TiN) strip deposited on the top layer 66 surrounds the humidity sensor 2 32 and the hybrid and detection section 52. The titanium nitride strip provides an LED wafer carrier surface 634 for the excitation LED 26 (see Figure 3). The excitation LEDs are sealed to the LED wafer carrier surface 634, and the air pressure in the hybrid chambers 180 is equal to atmospheric pressure through the vents 122 and venting channels 636 located in the MST layer 87 (see 00 and 00 1 02 picture). Referring to Fig. 104, the hybrid chamber arrays 110.1 to 110.12 for each of the 12 ampersons have a single photosensor 44 within the lower CMOS circuit 86 (see Fig. 12). The probe-target hybrid in any of the hybrid chambers 180 emits a fluorescent signal and the fluorescent signal is detected by the corresponding photodiode 184. Each of the hybrid chamber arrays 110.110.12 has at least one calibration chamber 382 that is fluidly isolated from the sample such that no amplicon enters the calibration chamber 382. As described elsewhere in this specification, the calibration chambers 3 82 are used to calibrate the output ports of the photodiodes to adjust for system noise based on read errors.

LOC Device Variant XXIII The LOC Device Variant XXIII 650 of Figure 109 is used for genetic analysis and uses a white blood cell dialysis section 328 to substantially reduce the red blood cell concentration in the sample. In the chemical lysis section 130, the lysate-135-201209158 agent from the storage tank 56 releases the genetic material in the white blood cells. Downstream of the chemical lysis section 130, the sample is mixed with restriction enzymes, ligase and linker-derived from storage tank 58 and injected into the incubation section 1 14 . After incubation, the boiling start valve 108 immediately downstream of the incubation section 1 ! 4 opens to allow the sample to flow into the amplification section 1 1 2 and ultimately into the hybrid chamber array 1 1 〇.

LOC Device Variant XXIV LOC Device Variant XXIV 651 is a genetically analyzed LOC device having a white blood cell dialysis section 328, a chemical lysis section 1 30 and a restriction enzyme, ligase and linker incubation section 114 ( See picture 11). The LOC device variant XXIV 651 uses a plurality of parallel amplification sections 112. ι, 1 12.2...1 12·X and individual hybrid chamber arrays 1 lo.i, 1 丨 0.2... 1 10.X.

LOC Device Variant XXV LOC Device Variant XXV 652 (see Figure 111) is a specific example of a genetic analysis LOC device. The LOC device variant XXV 652 uses a white blood cell dialysis section 3 28, a chemical lysis section 1 30 and a restriction enzyme, ligase and linker incubation section 114. The sample is then fed into the cascaded amplification chamber 112.1 and the amplification chamber 112.2, and then detected in a single hybrid chamber array 1 1 .

LOC Device Variant XXVI The LOC Device Variant XXVI 653 shown in Figure 112 is used for genetic analysis and uses a white blood cell dialysis section 328 to substantially reduce the red blood cell concentration of -136 - 201209158 in the sample. In the chemical lysis section 130, the lysis reagent derived from the storage tank 56 releases the genetic material in the white blood cells. After chemical lysis, the boiling start valve 1 〇 8 immediately downstream of the chemical lysis section 130 is opened to cause the sample to flow into the amplification section Π 2 and ultimately into the hybrid chamber array 丨丨〇.

LOC device variant XXVII

The LOC device variant XXVII 654 shown in Fig. 113 is a genetic analysis LOC device having a white blood cell dialysis section 328 and a chemical lysis zone 130. The LOC device variant XXVII 654 then uses a plurality of parallel expansion sections 112.1, 112.2...112.X and individual hybrid chamber arrays nn ' 1 1 0.2 ... 1 1 0.X.

LOC Device Variant XXVIII LOC Device Variant XXVIII 65 5 Line Genetic Analysis Example 114 shows a specific embodiment of the LOC device. The LOC device variant XXVIII 655 uses a white blood cell dialysis section 328 and a chemical lysis zone 130. The sample is then fed into the converging amplification chamber 112.1 and the amplification chamber 112.2, and then detected in a single hybrid chamber array 1 1 . Conclusion The devices, systems, and methods described in this case facilitate molecular diagnostic testing at low cost, high speed, and focused care. The above-mentioned system and the components of the system are purely illustrative, and those skilled in the art will be able to easily comprehend many changes and modifications of the spirit and scope of the general inventive concept without deviating from the present case-137-201209158 Aspect. BRIEF DESCRIPTION OF THE DRAWINGS A number of preferred embodiments of the present invention, which are described with reference to the drawings, are for illustrative purposes only, and the drawings are as follows: Figure 1 shows the construction for fluorescence detection. Inspection module and inspection module reader. Figure 2 is a summary view of the electronic components within the inspection module constructed for fluorescence detection. Figure 3 is a schematic overview of the electronic components within the test module reader. Figure 4 is a schematic representation of the construction of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of the LOC device having features and structures derived from all of the overlapping film layers. Figure 7 is a plan view of the LOC device showing the cover structure separately. Figure 8 is a top perspective view of the cover layer having internal passages and storage tanks, the internal passages and storage tanks being shown in dashed lines. Figure 9 is a top exploded perspective view of the cover layer having internal passages and storage tanks, the internal passages and storage tanks being shown in dashed lines. The first drawing is a bottom perspective view of the cover, the bottom perspective showing the structural configuration of the top channels. Figure 11 is a plan view of the LOC device showing the structure of the -138-201209158 CMOS + MST device separately. Fig. 12 is a schematic cross-sectional view of the LOC device at the entrance of the sample. Fig. 13 is an enlarged view of the figure AA of τκ in Fig. 6. Figure 14 is an enlarged view of the illustration AB of Figure 6. Fig. 15 is an enlarged view of the illustration AE shown in Fig. 13. Figure 16 is a partial perspective view showing 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 partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 19 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 20 is a partial perspective view showing the layered structure of the LOC device in the illustration AE. Figure 21 is a partial perspective view showing the layered structure of the LOC device in the inset AE. Figure 22 is a schematic cross-sectional view of the lysis reagent storage tank shown in Figure 21 〇 Figure 23 is a partial perspective view showing the layered structure of the LOC device in the illustration AB. Fig. 24 is a partial perspective view showing the layered structure of the L Ο C device in the illustration A B. Figure 25 is a partial perspective view of the layered structure of the LOC device in the illustration AI - 139 - 201209158. Figure 26 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 27 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 28 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 29 is a partial perspective view showing the layered structure of the LOC device in the inset AB. Figure 30 is a cross-sectional view of the amplification mixture storage tank and the polymerase storage tank. Figure 31 is a separate illustration of the characteristics of a boiling start valve. Figure 3 is a schematic cross-sectional view of the boiling start valve taken along line 3 3 - 3 3 shown in Figure 31. Fig. 33 is an enlarged view of the illustration AF shown in Fig. 15. Figure 34 is a cross-sectional view of the upstream end of the dialysis section taken along line 35-35 of Figure 33. Fig. 35 is an enlarged view of the illustration AC shown in Fig. 6. Figure 36 is a further enlarged view showing the inside of the illustration AC of the amplified section. Figure 37 is a further enlarged view of the inside of the illustration AC of the amplification section. Figure 38 is a further enlarged view showing the ACR portion of the illustration of the amplified segment. -140- 201209158 Figure 39 is a further enlarged view of the illustration AK shown in Figure 38. Figure 40 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 41 is a further enlarged view showing the inside of the illustration AC of the enlarged section. Figure 42 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 43 is a further enlarged view of the inside of the illustration AL shown in Fig. 42. Fig. 44 is a further enlarged view showing the inside of the illustration AC of the enlarged section. Fig. 45 is a further enlarged view of the illustration AM shown in Fig. 44. Figure 46 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 47 is a further enlarged view of the illustration AN shown in Fig. 46. Figure 48 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 50 is a further enlarged view showing the inside of the illustration AC of the enlarged section. Fig. 51 is a schematic cross-sectional view of the enlarged section. Fig. 52 is an enlarged plan view of the hybrid section. Figure 53 is a further enlarged cross-sectional view showing two hybrid chambers separately -141 - 201209158 Figure 54 is a cross-sectional view of a single hybrid chamber. Fig. 55 is an enlarged view of the humidifier shown in the illustration AG shown in Fig. 6. Fig. 56 is an enlarged view of the illustration AD shown in Fig. 52. Figure 5 is an exploded perspective view of the LOC device in the illustration AD. Figure 58 is a FRET probe diagram in a closed configuration. Figure 59 is a diagram of the FRET probe in an open and heterozygous structure. Figure 60 is a line graph of excitation light intensity over time. Figure 61 is a graphical representation of the excitation light geometry of a hybrid chamber array. Figure 62 is an illustration of the LED illumination geometry of the sensor electronics. Figure 6 is an enlarged plan view of the humidity sensor shown in Figure A of Figure 6. Figure 64 is a schematic cross-sectional view of the dialysis section of the white blood cell target. Figure 65 is a schematic partial view of an optical diode array of a photosensor. Figure 66 is a circuit diagram of a single photodiode. Figure 67 is a time diagram of the photodiode control signal. Figure 68 is an enlarged view of the evaporator shown in the illustration of Figure 55. Figure 69 is a cross-sectional view through a hybrid chamber having a detection photodiode and a trigger photodiode. Figure 70 is a linker-primed PCR. Figure 7 is a schematic representation of a test module equipped with a lancet. Figure 72 is a graphical representation of the construction of the LOC device variant VII. Figure 73 is a perspective view of a variation VIII of the LOC device. - 142 - 201209158 Figure 74 is a plan view of a LOC device variant VIII having all of the features and structures of the overlapping layers. Fig. 75 is a plan view showing the LOC device variation type VHI of the cap layer structure alone. Figure 76 is a bottom perspective view of the cover channels for the LOC device variant VIII. Figure 77 is a plan view of the LOC device variant VIII, which shows the structure of the CMOS + MST device separately. Fig. 78 is an enlarged view of the illustration CA shown in Fig. 74. Fig. 79 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 80 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 81 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 82 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 83 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 84 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CA shown in Fig. 74. Fig. 85 is an enlarged view of the illustration CB shown in Fig. 74. Fig. 8 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. - 143 - 201209158 Figure 8 7 is a partial perspective view showing the layered structure of the LOC device variant VIII in the inset CB shown in Figure 74. Parts 88A and 88B are partial perspective views showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 8 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 90 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 91 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CB shown in Fig. 74. Fig. 92 is an enlarged view of the illustration CC shown in Fig. 74. Fig. 9 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 94 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 95 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Sections 96A and 96B are partial perspective views showing the layered structure of the LOC device variant VIII in the inset CC shown in Fig. 74. Fig. 97 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 98 is a partial perspective view showing the layered structure of the LOC device variation VIII in the inset CC shown in Fig. 74. Fig. 99 is a partial perspective view showing the layered structure of the LOC device variation VIII in the illustration CC of Fig. 74-144-201209158. Figure 100 is an enlarged view of the illustration CD shown in Figure 74. Figure 101 is an enlarged perspective view of the illustration CD shown in Figure 74. Figure 102 is an exploded view of the illustration CD shown in Figure 74. Fig. 103 is an enlarged view of the illustration CE shown in Fig. 78. Figure 104 is a graphical representation of the construction of the LOC device variant VIII. Figure 105 is a schematic diagram of the construction of the variable XIV of the LOC device. Fig. 106 is a schematic diagram showing the construction of the variable XV of the LOC device. Figure 107 is a schematic diagram of the construction of the LOC device variant XVIII. Figure 108 is a schematic diagram of the construction of the LOC device variant XXII. Figure 109 is a schematic diagram of the construction of the LOC device variant XXIII. Figure 10 is a schematic diagram of the construction of the LOC device variant XXIV. Figure 111 is a schematic diagram of the construction of the LOC device variant XXV. Figure 112 is a schematic diagram of the construction of the LOC device variant XXVI. Figure 113 is a schematic diagram of the construction of the LOC device variant XXVII. Figure 114 is a schematic diagram of the construction of the LOC device variant XXVIII. Figure 115 is a schematic diagram of the construction of the LOC device variant XLI. Figure 116 is a schematic diagram of the construction of the LOC device variant XLII. Figure 117 is a schematic diagram of the construction of the LOC device variant XLIII. Figure 118 is a schematic diagram of the construction of the LOC device variant XLIV. Figure 119 is a schematic diagram of the construction of the LOC device variant XLVII. Figure 20 is a graphical representation of the linear probe attached to the primer during the initial round of the amplification reaction. Figure 1 2 1 is an illustration of a linear probe with primers during the subsequent amplification cycle -145 - 201209158. Panels 122A through 122F illustrate the thermal cycling of a fluorescent dry-loop probe with primers attached thereto. Figure 123 is a schematic diagram of the excitation LED associated with the array of hybrid chambers and the photodiodes. Figure 124 is a schematic diagram of an excitation LED and an optical lens for directing light onto a hybrid chamber of the LOC device. Figure 125 is a schematic diagram of an excitation LED 'optical lens and optical aperture' for directing light onto a hybrid chamber of the LOC device. Figure 126 is a schematic diagram of the excitation LED, optical lens, and mirror configuration for directing light onto the hybrid chamber of the LOC device. Fig. 127 is a plan view 'The plan view shows all the features overlapping each other and the positions of the illustrations DA to DK are displayed. Figure 128 is an enlarged view of the illustration DG shown in Fig. 127. Fig. 129 is an enlarged view of the illustration DH shown in Fig. 127. Figure 130 is a graphical representation of the construction of the LOC device variant XI. Figure 131 is a specific embodiment of a shunt transistor for the photodiodes. Fig. 1 2 2 is a specific embodiment of a shunt transistor for the photodiodes. The first 133 diagram is a specific embodiment of the shunt transistor for the photodiodes. Figure 134 is a circuit diagram of a differential imager. Figure 135 shows a negative control fluorophore -146-201209158 probe in a dry-loop configuration. Figure 1 3 6 illustrates the negative control set of fluorescent probes in Figure 135 of the open configuration. Figure 1 3 7 illustrates the positive control camp light probe in a dry-loop configuration. Figure 1 3 8 illustrates the positive control set of fluorescent probes in Figure 137 of the open configuration. Figure 139 shows the test module and test module reader for ECL detection. Figure 1 40 is an overview of the electronic components in the inspection module for fluorescence detection. Figure 1 4 1 shows the inspection module and a variety of optional inspection module readers. Figure I42 shows the inspection module and inspection module reader of the host system with various databases. [Main component symbol description] 1 〇: inspection module 1 1 : inspection module 1 2 : inspection module reader 13 : housing 1 4 : micro USB plug 15 : inductor 16 : micro USB slot - 147- 201209158 1 7 : Touch screen 18 : Display screen 1 9 : Button 20 : Start button 2 1 : Honeycomb radio 22 : Tornable sealing tape 23 : Wireless network connector 24 : Large slot 25 : Satellite navigation system 26: Light-emitting diode (LED) 2 7 : Data storage 28: Mobile phone/Smartphone 29: LED driver 30: On-wafer laboratory (LOC) device 3 1 : Power conditioner 32: Capacitor 3 3: Clock 3 4 : Controller 3 5 : Recorder 36 : USB device driver 3 7 : Drive 38 : Random access memory (RAM ) 3 9 : Driver 40 : Program and data flashing billion body -148 - 201209158 4 1 : Record 42: Processor 43: Program Memory 44 _· Light Sensor/Photo Sensor Array 4 5: Indicator 46: Cover 47: Powered by USB and as a module 48: CMOS + MST Wafer

49: foam insert/porous element 5 2 : hybrid and detection section 5 4 : storage tank 5 6 : storage tank 5 7 : printed circuit board 5 8 : storage tank 60 : storage tank 60.1 : storage tank 60.2 : Storage tank 60.3 : Storage tank 6 〇. 4 : Storage tank 6 0.5 : Storage tank 6 0.6 : Storage tank 6 〇. 7 : Storage tank 6 〇. 8 : Storage tank 60.9 : Storage tank -149- 201209158 60.10 : Storage Tank 6 〇. 1 1 : Storage tank 6 0 . 1 2 : Storage tank 6 0 . X : Storage tank 6 2 : Storage tank 6 2 . 1 : Storage tank 6 2.2 : Storage tank 6 2.3 : Storage tank 6 2.4 : Storage Slot 6 2 . X : Storage tank 6 4 : Lower sealing layer 66 : Top layer 6 8 : Sample inlet 70 : Dialysis section / Dialysis step 72 : Waste liquid channel 74 : Target channel 76 : Waste unit / waste storage tank 7 8 : Storage tank layer 80: Cover channel layer 8 2: Upper sealing layer 8 4: 矽 substrate (CMOS) circuit 86: Complementary metal oxide semiconductor 87: Microsystem technology (MST) layer 8 8 : Passivation layer - 150- 201209158 90 : Microsystem Technology (MST) Channel 92: Lower Suction Hole 94: Cover Channel 96: Upper Suction Hole 97: Partition Wall Section 9 8: Meniscus Anchor 100: Microsystem Technology (MST) Channel Layer 1 01 : Portable Human Computer/Notebook 102: Capillary Actuation Feature (CIF) 103: Dedicated Reader 105: Desktop Computer 106: Boiling Start Valve 107: E-book Reader 108: Boiling Start Valve/Amplification Exit Valve 109: Tablet PC 1 1 〇: Hybrid Chamber Array 1 1 〇. 1 : Hybrid Chamber Array 1 1 〇. 2 : Hybrid Chamber Array 1 1 0.3 : Hybrid Chamber Array I 10.4 : Hybrid Chamber Array II 〇. 5: Hybrid Chamber Array 1 10.6: Hybrid Chamber Array 1 10.7: Hybrid Chamber Array 1 1 〇. 8: Hybrid Chamber Array - 151 201209158 1 1 ο . 9 : Miscellaneous Pool array 1 1 0 . 1 0 : Hybrid chamber array 1 1 〇. 1 1 : Hybrid chamber array 1 1 0.1 2 : Hybrid chamber array 1 1 0 . X : Hybrid chamber array 1 1 1 : host system of epidemiological data 1 1 2 : amplification section 1 1 2 2 : amplification section 1 1 2.2 : amplification section 1 1 2.3 : amplification section 1 12.4 : amplification zone Segment 1 1 2.5: Amplified segment 1 1 2.6 : Amplified segment 1 1 2.7 : Amplified segment 1 1 2.8 : Amplified segment 1 1 2.9 : Amplified segment 1 1 2.1 0 : Amplified segment 1 1 2.1 1 : Amplification section 112.12: Amplification section 1 1 2 . X : Amplification section 1 1 3 : host system of genetic data 1 1 4 : incubation section 1 1 4 · 1 : cultivation section 1 1 4.2 : cultivation section -152 - 201209158 1 1 4.3 : cultivation section 1 1 4.4 : cultivation section 1 1 5 : Host system of electronic health record (EHR) 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Sample fluid 1 2 0 : Meniscus 121 : Host of electronic medical record (EMR) System 122: Vent 123: Personal Medical Record (PHR) Host System 125: Network 126: Boiling Start Valve 1 2 8 : Surface Tension Valve 1 3 0 : (Chemical) Cell Segment 1 3 1 : Mixing Zone Section 1 3 2 : Surface tension valve 1 3 3 : Incubator inlet channel 1 3 4 : Lower suction hole 136: Optical window 1 3 8 : Amplification mixture surface tension valve 1 3 8 . 1 : Amplification mixture surface tension valve 138.2 : Amplification mixture surface tension valve 1 3 8.3 : Amplification mixture surface tension valve 1 3 8.4 : Amplification mixture surface tension valve -153- 201209158 1 3 8 · X : Amplification mixture surface tension valve 140: Polymerase surface tension valve 140.1: Polymerase Surface Tension Valve M0.2: Polymerase Surface Tension Valve 140.3: Polymerase Surface Tension Valve 140.4: Polymerase Surface Sheet Valve 140.X: Polymerase surface tension valve 1 4 6 : Valve inlet 1 4 8 : Valve outlet 1 5 0 : Lower suction hole 1 5 1 : Valve upper suction hole 1 5 2 : Heater 1 5 3 : Boiling start type Valve heater contact point 1 5 4 : Heater 1 5 6 : Heater contact point 158 : Microchannel 160 : Amplification section leaves channel 164 : Hole 165 : Hole 166 : Capillary action priming feature (CIF ) 1 6 8 : Dialysis upper suction hole 170: temperature sensor 174: liquid sensor 175: diffusion barrier -154 - 201209158 1 7 6 : flow path 178: end point liquid sensor 180: hybrid chamber 1 8 2 : heater 184 : photodiode 18 5 · active area 186 : fluorescent resonance energy transfer (FRET) probe 187 : triggered photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : ring heater 192: water supply channel 1 9 3 : upper suction hole 194 : lower suction hole 1 9 5 : exposed area of the top metal layer 1 9 6 : humidifier 197 : cover channel liquid sensor 1 9 8 : first on Suction hole 202: capillary action priming feature (CIF) 204: dialysis MST channel 2 06: mixing section outlet valve/boiling start valve 2 07: incubation chamber outlet valve 2 0 8 : mixing section outlet valve 2 1 0 : plus Microchannel-155- 201209158 2 1 2 : Intermediate MST channel 2 1 8 : Chin Ming electrode 2 2 0 : Chin Ming electrode 222 : Gap 23 2 : Humidity sensor 2 3 4 : Heater 23 6 : Fluorescence resonance Energy Transfer Probe (FRET) Probe

2 3 8 : Target nucleic acid sequence / target DNA 240 : Ring portion 242 : stem 244 : excitation light 246 : fluorescent luminescent group 248 : extinction group 25 0 : release fluorescent / fluorescent signal 2 5 2 : optical Center 2 5 4 : Lens 2 8 8 : Sample placement and preparation stage 290 : Extraction stage (extraction stage) 291 : Cultivation stage / incubation step 292 : Amplification stage / amplification step / amplification section 293 : Pre Hybridization phase 294: detection phase/detection section 296: first electrode 2 9 8 : first electrode - 156 - 201209158 3 00: preset delay time 301: on-wafer laboratory (LOC) device 3 2 8 : Dialysis section 3 7 6 : Conductive column 3 78 : Control probe 3 80 : Control probe 3 82 : Calibration chamber 3 84 : Shunt transistor (Mshunt) gate 3 86 : Transistor Mtx gate 3 8 8 : Reset gate 3 90: spring-loaded telescopic needle 3 92 : lancet release button 3 93 : read gate 394: metal oxide transistor, Mshunt 396: metal oxide transistor, Mtx 3 98: metal Oxide transistor, Mreset 400: source follower transistor, Msf 402: metal oxide transistor, Mread 404: metal oxide transistor, Mbias 4 0 6 : node 408: dense Film 410: protective film sheet

492: LOC device variant VII 5 1 8 : L Ο C device variant V 111 -157- 201209158 594 : interface layer 5 96 : interface channel 5 9 8 : interface channel 600 : bypass channel 602 : interface target channel 604: interface waste liquid channel 606: interface channel 608 on the side of the lysis reagent storage tank: interface channel 610 on the side of the lysis reagent storage tank: interface conduit 6 1 2: mixing section outlet lower suction hole 614: valve interface channel 6 1 6: valve interface groove 6 1 8 : amplification interface conduit 619 : amplification interface conduit 620 : amplification interface conduit 621 : amplification interface conduit 622 : amplification interface conduit 623 : amplification interface conduit 624 : amplification interface conduit 62 5 : Amplification interface catheter 626 : amplification interface catheter 627 : amplification interface catheter 62 8 : amplification interface catheter 629 : amplification interface catheter -158 - 201209158

630: MST incubation outlet channel 63 2 : Amplification injection channel 634 : LED wafer carrier surface 63 6 : Ventilation channel 6 3 8 : Thermal lysis chamber 641 : LOC device variant XIV 650 : LOC device variant XXIII 651 : LOC Device variant XXIV 652: LOC device variant XXV 65 3 : LOC device variant XXVI 654: LOC device variant XXVII 655: LOC device variant XX VIII

673 : LOC device variant XLIII

674: LOC device variant XLIV 677: LOC device variant XLVII 6 82: dialysis section / dialysis step / hybrid pre-dialysis step 686: pre-amplification dialysis step 692: linear probe 694 with primer: amplification resistance Breaker 696: Probe sequence with primers 69 8 : Amplified complementary sequence 700: Oligonucleotide primer 7 04: Dry-loop probe/probe region 706 with complementary primers: Complementary sequence - 159- 201209158 708: trunk chain 710: trunk chain 712: first optical 稜鏡 714: second optical 稜鏡 7 1 6 : first mirror 7 1 8 : first mirror

728: LOC device variant X

746: LOC device variant XI 766: waste storage tank 76 8 : waste storage tank 7 8 2 : configuration 788: differential imaging circuit 7 9 0 : pixel 792 : dummy pixel 794 : read line 7 9 5 : dummy pixel read line Line 796: Negative control probe 797: M4 transistor 798: Positive control probe 8 0 1 : MD 4 transistor 8 〇 3 : Pixel capacitor 8 0 5 : Fake pixel capacitor 8 0 7 : Switcher 80 9 : Switcher -160- 201209158 8 1 1 : Read line switch 8 1 3 : Fake pixel read line switch 815 : Capacitor amplifier 8 1 7 : Differential signal 860 : Electrode 870 : Electrode

-161 -

Claims (1)

201209158 VII. Application for Patent Park: 1. A wafer-on-lab (LOC) device for genetic analysis of biological samples, the LOC device comprising: an inlet for receiving the sample; a carrier substrate; a dialysis section, The dialysis section separates cells in the sample larger than the predetermined threshold 与 from the smaller components, wherein the cells larger than the predetermined threshold 包括 include target cells containing the genetic material for analysis; a plurality of reagent storage tanks; lysis a segment, the lysis segment being located downstream of the dialysis section for dissolving the target cells to release genetic material within the target cells, the lysing segments being associated with the cells for dissolution One of the reagent storage tanks of the lysis reagent of the target cells in the lysing section is in fluid communication; the first nucleic acid amplification section, the first nucleic acid amplification section is located downstream of the lysis section For amplifying a first nucleic acid sequence within the genetic material; and a second nucleic acid amplification segment downstream of the first nucleic acid amplification segment for amplification derived The first nucleic acid amplification a second nucleic acid sequence in the amplicon of the segment: wherein the dialysis section, the lysis section, the first nucleic acid amplification section, and the second nucleic acid amplification section are all carried on the carrier substrate . 2. The LOC device of claim 1, wherein the first nucleic acid amplification segment is a first polymerase chain reaction (PCR) segment and the second nucleic acid amplification segment is a second PCR segment . 3. The LOC device of claim 2, wherein the first PCR-162-201209158 segment has a first set of primer pairs and the first set of primer pairs is used to bind the first set of complementary nucleic acid sequences, and the The second PCR segment has a second set of primer pairs and the second set of primer pairs is used to bind a second set of complementary nucleic acid sequences that differ from the second set of complementary nucleic acid sequences. 4. The LOC device of claim 3, wherein the first PCR segment and the second PCR segment are constructed to operate with different amplification parameters, the amplification parameters being at least one of : reverse transcriptase species; polymerase species; deoxyribonucleoside triphosphate concentration; buffer solution; thermal cycle time; number of thermal cycle repeats; and temperature during a particular phase of PC R. 5. The LOC device of claim 4, further comprising a hybrid segment located downstream of the second PCR segment, the hybrid segment having a probe array and light A sensor, the probe array is for hybridization with a target nucleic acid sequence and the photosensor is for detecting a heterozygous reaction of any of the probes in the array. 6. The LOC device of claim 3, wherein the dialysis section has a first channel, a second channel, and a plurality of holes, the first channel being in fluid communication with the inlet at the upstream end, the second channel And being in fluid communication with the waste liquid channel at the downstream end, wherein the holes are smaller than the target cells and larger than the smaller components, and the second channel is connected to the first channel by the holes -163-201209158 Connected to allow the target cells to remain in the first channel while the smaller components flow into the second channel. 7. The LOC device of claim 6, wherein the first channel and the second channel are configured to fill the sample by capillary action. 8. The apparatus of claim 1, wherein the lysis section has an active valve that causes the target cells to remain in the lysis section during lysis, such that When the active valve is opened, the capillary drive flow to the incubation section continues. 9. The LOC device of claim 1, wherein the first nucleic acid amplification segment is a first thermostatic nucleic acid amplification segment and the second nucleic acid amplification segment is a second thermostatic nucleic acid amplification segment . 10. The LOC device of claim 1, wherein the reagent storage tanks each have a surface tension valve for retaining reagents in the reagent storage tanks, the surface tension valve having a meniscus anchor, the bend The liquid level anchor is used to hold the meniscus of the reagent until the meniscus of the reagent contacts the sample stream to remove the meniscus to allow the reagent to flow out of the reagent reservoir. 1 1. As claimed in the patent application a LOC device of 4, the LOC device further comprising a CMOS circuit, a temperature sensor and a microsystem technology (MST) layer, the MST layer comprising the first PCR segment and the second PCR segment, wherein the CMOS circuit system And disposed between the carrier substrate and the MST layer, the CMOS circuit is configured to use the output of the temperature sensor to achieve feedback control of the first PCR segment and the second PCR segment. 12. The LOC device of claim 11, wherein the first PCR segment has a PCR microchannel for thermal cycling of the sample -164 - 201209158 'the PCR microchannel system A flow path having a cross-sectional cross-sectional area of less than 100,000 square microns. 13. The LOC device of claim 12, wherein the PCR microchannel has at least one elongated heater element' that extends parallel to the PCR microchannel. 14. The LOC device of claim 13, wherein the PCR segment has a plurality of elongate PCR chambers, and each of the elongate PCR chambers is formed by an individual segment of the PCR microchannel, the PCR The microchannel has a crucible configuration formed by a series of wide curved channels, and each of the wide curved channels forms a channel section of one of the elongate PCR chambers. 15. The LOC device of claim 14, wherein the LOC device further comprises: a reagent storage tank containing a reagent for PCR; and a surface tension valve having a hole, the surface tension valve having the hole being constructed to be fixed The meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until the meniscus contacts the fluid sample to remove the meniscus and the reagent exits the reagent reservoir. 16. The LOC device of claim 14, wherein the LOC device further comprises an array of hybrid chambers for receiving the probes such that the probes within each hybrid chamber are configured to One of the target nucleic acid sequences is heterozygous. 1 7 · The L Ο C device of claim 16 of the patent application, wherein the photo sensor is associated with an array of photodiodes arranged by the hybrid chambers. 18. The LOC device of claim 16, wherein the CMOS-165-201209158 circuit has a digital memory and a data interface for storing hybrid data originating from the photosensor output, and The data interface transmits the hybrid data to an external device. 19. The LOC device of claim 16, wherein the first PCR segment has an active valve that retains liquid within the first PCR segment during thermal cycling and is responsive to the CMOS circuit The activation signal allows liquid to flow to the first hybrid chamber array. 20. The LOC device of claim 19, wherein the active valve is a boiling start valve having a meniscus anchor and a heater, the meniscus anchor being constructed to hold the block The capillary of the liquid drives the meniscus of the flow, and the heater is used to boil the liquid to disengage the meniscus from the meniscus anchor such that capillary drive flow continues. -166 -