TW201209407A - Microfluidic device with reagent mixing proportions determined by number of active outlet valves - Google Patents

Abstract

A microfluidic device for testing a fluid, the microfluidic device having an inlet for receiving the fluid, a reservoir containing a reagent, a flow-path extending from the inlet, a plurality of outlet valves for fluid communication between the flow-path and the reservoir, each of the outlet valves having an actuator for opening the outlet valve in response to an activation signal, wherein during use, a number of the outlet valves are selectively opened such that the reagent flows into the flow-path to combine with the fluid from the inlet to produce a combined flow having a proportion of the reagent, the proportion of the reagent in the combined flow being determined by the number of the outlet valves opened.

Description

201209407 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to microfluidic and biochemical treatments and assays for molecular diagnostics. [Prior Art] Molecular diagnosis has been used to provide an area for early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the occurrence and improvement of ineffective health care Patient outcome, improved disease management, and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobases allows the short sequence of synthetic DNA (oligonucleotides) to bind (hybridize) to a particular nucleic acid sequence for use in nucleic acid assays. If a hybrid occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will get in the future, to determine the type and pathogen of the infectious pathogen, or to determine the individual's response to the drug. 201209407 Nucleic Acid-Based Molecular Diagnostic Tests Nucleic acid-based assays have four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine , sputum and tissue samples are used for genetic analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required at the outset. Blood is one of the more frequently requested sample types. It has three main components: white blood cells, red blood cells, and thrombocytes (platelets). Thrombotic cells promote agglutination and maintain activity in vitro. To inhibit coacervation, the sample is mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of the cellular material but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). The red blood cells can be removed by differentially lysing the red blood cells in the lysate, leaving the remaining intact cellular material, which can then be separated from the sample using centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. The exact procedure used to extract nucleic acids depends on the sample and the diagnosis to be performed -6 - 201209407 Analysis. For example, the protocol used to extract viral RNA is quite different from the protocol used to extract the genome DN A. However, self-targeting cell extraction of nucleic acids typically involves a cell lysis step and subsequent nucleic acid purification. The cell lysis step ruptures the cell and nuclear membrane and releases the genetic material. This is often accomplished using a lyophilized detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The nucleic acid is then purified in a precipitation step with alcohol (usually ice ethanol or isopropanol) or via a solid phase purification step prior to washing in the presence of a high concentration of chaotropic salt, usually in a fractionation column. The matrix, resin or paramagnetic beads are then eluted with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protein-cleaving protease to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to disrupt thermal lysis of the cell membrane. The target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogen source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target (in the case of a low concentration in terms of detectability). The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The PCR is known in the art and provides a comprehensive comprehensible description of such reactions in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008. PCR is a useful technique for amplifying a target DNA sequence relative to a complex DNA background. If RNA is to be amplified (by PCR), it is first necessary to transcribe 201209407 into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the obtained cDNA was amplified by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is:

1. Primer pair - a short single strand of DNA having approximately 1〇_3〇 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis of DNA thermostable enzymes 3. Deoxyribose Nucleoside triphosphate (dNTP) - provides nucleotides for integration into newly synthesized DNA strands. 4. Buffer - the best chemical environment for DN A synthesis. PCR typically involves placing these reagents in small tubes containing extracted nucleic acids ( ~10-50 microliters). The tube is placed in a polymerase chain cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocols for each thermal cycle include the denaturing phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denaturation phase generally involves heating the reaction temperature to 90-95 ° C to denature the DNA strand: in the adhesive phase, the temperature is lowered to ~50-60 ° C for adhesion of the primer; then in the extended phase, the temperature is raised to The optimal DNA polymerase activity temperature is 60-72 °C for extension of the primer. This method repeats the cycle for about 20-40 times, with the end result being a target sequence between millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multi-initiator PCR, linker-primed 201209407 PCR, direct PCR, tandem PCR, real-time PCR, and Reverse transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single experiment by targeting multiple genes at once (in other ways, several trials are required). It is more difficult to optimize multi-primer PCR because it requires the selection of primers with approximate adhesion temperature and amplicon with approximate length and base composition to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR, is a method for enabling nucleic acid amplification of virtually all DNA sequences in complex DNA mixtures without the need for target-specific Sexual introduction. This method first digests the target DNA population with a suitable restriction endonuclease. Using a zymase enzyme, a double-stranded oligonucleotide linker (also known as a zygote) having a suitable overhanging end is then ligated to the terminal of the target DNA fragment. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. By this, all fragments of the DNA source adjacent to the linker oligonucleotide can be amplified. 〇 Direct PCR describes a system that performs PCR directly on the sample without any nucleic acid extraction (or minimal nucleic acid extraction). It has long been believed that PCR reactions are inhibited by many components present in unpurified biological samples, such as the protohemoglobin component in the blood. Traditionally, PCR requires enhanced purification of target nucleic acids prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentration, DNA purification can be minimized and PCR can be performed at 201209407 or direct PCR. Modifications in PCR chemistry for direct PCR include potentiation of buffer strength, use of high activity and processivity polymerases, and additions to potential polymerase inhibitors. Tandem repeat PCR utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. One type of tandem repeat PCR is nested PCR in which two pairs of PCR primers are used to perform single locus amplification for separate nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence of the outer region of the target nucleic acid sequence. The second pair of primers (nested primers) used in the second amplification binds to the first PCR product and produces a second PCR product (short in the first PCR product) containing the target nucleic acid. The rationale used in this strategy is that if the wrong genomic locus is amplified due to a mistake during the first nucleic acid amplification, the probability of re-amplifying the erroneous locus by the second pair of primers is very low, thus ensuring specificity. The amount of PCR product was measured in real time using either real-time PCR or quantitative PCR. The initial amount of nucleic acid in a sample can be determined by using a fluorophore containing a probe or a fluorescent dye and a reference standard in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample.

Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. » Reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), followed by PCR amplification of cDNA. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to amplify the R N A -10- 201209407 virus, such as the human immunodeficiency virus or the hepatitis C virus. Constant temperature amplification for another type of nucleic acid. Amplification, which does not rely on thermal denaturation of the target DN A during the amplification reaction, does not require complex machinery. The thermostatic nucleic acid amplification method can thus be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Some thermostated nucleic acid amplifications of Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification The method has been described. The constant temperature nucleic acid amplification method does not rely on the continuous heat denaturation of the template DNA to produce a single strand of the molecule as a template for further amplification, but relies on an enzymatic cleavage such as a DNA molecule which specifically limits the endonuclease at a normal temperature, or Other methods of using enzymes to separate DNA strands" The ability of femoral substitution amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi-modified DNA, and to rely on 5'-3' exonuclease - Lack of ability of the polymerase to extend and replace downstream stocks. Exponential nucleic acid amplification is then achieved by a reaction between sense and antisense, wherein the strand of the sense reaction is substituted as a template for the antisense reaction. Use a nicking enzyme (such as N.) that does not cut DNA in the usual way but produces a nick on one of the strands of DNA.  Alwl, N.  BstNBl and Mlyl) are available for -11 - 201209407. SDA has been improved by the combination of heat stable restriction enzyme Μναΐ and thermostable exo-polymerase polymerase. This combination appears to increase the amplification efficiency of the reaction from 1-8 fold amplification to 1〇1 (fold fold) amplification so that this technique can be used to amplify unique single copy molecules. Transcription-mediated amplification (ΤΜΑ) and nucleic acid sequence-dependent amplification (NASBA) use RNA polymerase to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase, and any RNase Η (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, the primer hybridizes to a target ribosomal RNA (rRNA) at a defined location. The reverse transcriptase produces a DN A copy of the target rRN A by extension from the 3' end of the promoter primer. If another RNase is present, the RNA in the obtained RNA:DNA double strand is decomposed by the RNase activity of the reverse transcriptase. Next, the second primer binds to the DNA copy. A double-stranded DNA molecule is produced by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RNA polymerase recognizes the promoter in the DNA template and initiates transcription. Each newly synthesized RNA amplicon re-enters the process and serves as a template for new replication. In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a specific DNA fragment is achieved by binding the opposite oligonucleotide to the template DNA and extending it by the DNA polymerase. Denaturation of the double-stranded DNA (dsDNA) template does not require heat. Conversely, RPA uses recombinant enzyme-primer mismatches to scan dsDNA and promote share exchange at cognate locations. The resulting -12-201209407 structure is stabilized by the interaction of a single strand of DNA binding protein with a substituted template strand' thus preventing primers from being released due to branch migration. The recombinant enzyme decomposes close to the y-end of the oligonucleotide that replaces the DNA polymerase (such as a large fragment of Pol I (hM)), and the primer then begins to extend. This step is repeated by cycling to achieve exponential nucleic acid amplification. Helicase amplification (HDA) mimics the in vivo system, using DNA helicase in an in vivo system to generate a single strand template for primer hybridization and then extending the primer with a DNA polymerase. In the first step of the HDA reaction, the helicase traverses the target DNA, destroying the hydrogen bonds that bind the two strands, which are then bound by a single-stranded binding protein. The single-strand target area exposed by the helicase allows the primer to adhere. DN A polymerase uses free deoxyribonucleoside triphosphate (dNTP) to subsequently extend the 3' end of each primer to create two DNA replicas. Two replicated dsDNA strands enter the next HDA cycle independently' resulting in exponential nucleic acid amplification of the target sequence. Other DNA-based thermostating techniques include rolling cycle amplification (RCA) in which a DNA polymerase continuously extends a primer around a circular DNA template to produce a long DNA product consisting of a number of circular repeat copies. By terminating the reaction, the polymerase produces thousands of copies of the circular template with a copy strand tethered to the original target DNA. This results in a spatial resolution of the target and rapid nucleic acid amplification of the signal. A template of up to 1〇12 copies can be produced in one hour. Branch-type amplification is a variant of RCA, and a circular probe (C-probe) or a latching probe and a highly progressive DNA polymerase are used to exponentially expand C at room temperature. - Probe. Circular Thermostat Amplification (LAMP) provides high selectivity and utilizes DNA polymerase and a primer set containing four specially designed primers. The primer set identifies a total of six different sequences on the target -13-201209407 DNA. The inner primer, which contains the sense strand of the target DNA and the antisense strand sequence, initiates the LAMP. The subsequent strand-derived DNA synthesis initiated by the external primer releases a single strand of DNA. This serves as a template for DNA synthesis initiated by the second internal and external primers, and the second internal and external primers hybridize to the other end of the target to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer hybridizes to the loop on the product and initiates the replacement of DNA synthesis' to produce the original stem-loop DNA and the new stem-loop DNA with twice the stem length. The cycle reaction was continued for one hour to accumulate 1 〇 9 copies of the target. The final product is a stem-loop DNA with several inverted repeat targets and a broccoli-like structure with a plurality of loops (formed alternately with inverted repeat targets in the same strand). After completion of the nucleic acid amplification, the amplified product must be analyzed to determine whether the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product is to simply measure the size of the amplicon by colloidal electrophoresis and use DNA hybridization to identify the nucleotide composition of the amplicon. Colloidal electrophoresis is one of the simplest ways to check the nucleic acid amplification step to produce the desired amplicon. Colloidal electrophoresis utilizes an electric field applied to a colloidal matrix to separate DNA fragments. Negatively charged DNA fragments will move through the matrix at different rates (mainly depending on their size). After the electrophoresis is completed, the fragments in the colloid can be made visible. Brominated fluorinated fluorene fluorene under UV light is the most commonly used dye. The size of the fragment is judged by comparison with a DNA size marker (DNA ladder). The D N A size marker contains a DNA fragment of a known size which is run along with the amplicon. Since the oligonucleotide primer binds to a specific position adjacent to the target DNA of -14-201209407, the size of the amplified product can be predicted and detected using a band of a known size on the colloid. In order to confirm why an amplicon or a plurality of amplicons are generated, a DNA probe is often used to hybridize with an amplicon. DNA hybridization means the formation of double-stranded DNA by complementary base pairing. DNA hybridization for the positive recognition of a particular amplification product requires the use of a DNA probe of about 20 nucleotides in length. If the probe has a sequence complementary to the amplicon (target) DNA sequence, hybridization will occur under conditions of favorable temperature, pH and ion concentration. If hybridization occurs, the gene or DNA sequence of interest is present in the original sample. Optical detection is the most common method of detecting hybridization. The amplicon or probe is labeled to emit light via fluorescing or electrochemiluminescence. These methods have different ways of inducing the excited state of the luminescent moiety, but both are equally capable of covalent labeling of the nucleoside stock. In electrochemiluminescence (ECL), when excited by an electric current, light is generated by a luminophore molecule or a complex. When the fluorescent light is emitted, the excitation light is emitted to emit light. The fluorescent light source is used to detect the fluorescent light, and the light emitting source provides the excitation light having the wavelength absorbed by the fluorescent molecules and the detecting unit. The detection unit includes a photo sensor (such as a photomultiplier tube or a charge coupled device (CCD) array) to detect the transmitted signal' and a mechanism to prevent excitation light from being included in the photosensor output (such as a wavelength selective filter). Back to stress luminescence, the fluorescent molecules emit Stokes-shifted light, and the emitted light is collected by the detection unit. Stokes converts the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. -15- 201209407 Use a light sensor to detect ECL emissions, and the light sensor is sensitive to the emission wavelength of the ECL type used. For example, a transition metal coordination complex emits light of a visible wavelength, and thus a conventional photodiode and a CCD are used as photosensors. The advantage of ECL is that if ambient light is excluded, the ECL emission can be the only light present in the detection system, thus increasing sensitivity. Microarrays allow hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are useful molecular diagnostic tools that can screen thousands of genetic diseases or detect the presence of several infectious pathogens in a single assay. The microarray consists of a number of different DNA probes that are fixed to the substrate and are spotted. The target DNA (amplicon) is first labeled with fluorescent or luminescent molecules (during or after nucleic acid amplification) and then applied to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment where hybridization occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. Less stringent may result in highly non-specific binding. Excessive rigor may result in inability to combine properly resulting in reduced sensitivity. The hybridization is identified by detecting the light emission from the labeled amplicon that hybridizes with the complementary probe. Fluorescence from the microarray is detected using a microarray scanner, typically a computer-controlled, inverting scanning fluorescent conjugated focus microscope that typically uses a laser and photosensor that excites the fluorescent dye ( Such as a photomultiplier tube or CCD) to detect the emitted signal. The fluorescent molecules emit light converted by Stokes (as described above), and the light is collected by the detecting unit. -16- 201209407 The emitted fluorescence must be collected, separated from the unabsorbed excitation wavelength, and transmitted to the detector. A conjugate focal configuration is often used in microarray scanners to remove out-of-focus information by conjugated focal pinholes located in the image plane. This makes it possible to detect only the focused portion of the light. Light that is prevented above or below the focal plane of the object enters the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, which is then converted into a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. More information about fluorescent probes can be found below: http : //www. Premierbiosoft. Com/tech_notes/FRET_probe. Html and http : //www. Invitrogen. Com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET. Html In-situ Nursing Molecular Diagnostics Despite the advantages offered by molecular diagnostic tests, this type of test in clinical tests has grown less than expected and still accounts for only a small portion of the implementation of laboratory medicine. This is mainly due to the complexity and cost associated with nucleic acid testing compared to experiments based on non-amino acid methods. The broad applicability of molecular diagnostic tests to clinical treatment is associated with rapid and automated analysis that can significantly reduce costs, from initial (sample processing) to end (resulting in results), and instruments that do not require extensive human intervention. -17- 201209407 Development It is closely related. Local healthcare technologies for physicians' clinics, proximity or user-based hospitals, and homes offer the following benefits: • Quickly get results that quickly promote treatment and improve care quality. • Ability to test sputum by testing a very small number of samples. • Reduce clinical workload. • Reduce lab workload and increase productivity by reducing management effort. • Improve the cost per patient by reducing hospital stays, getting results from outpatient visits at the first visit, and simplifying the handling, storage, and delivery of samples • Promoting clinical management decisions such as vaccination control and antibiotic use. On-wafer laboratory (LOC)-based molecular diagnostics is based on methods for providing automated and accelerated molecular diagnostic analysis for molecular diagnostic systems for fluid technology. The shorter detection time is mainly due to the use of very small amounts of automation, built-in and low overhead cascades in the diagnostic method steps in the microfluidic device. The use of nanoliters and micro-upgrades also reduces reagent consumption and cost. On-wafer laboratory (LOC) devices are a common form of microfluidic devices. The on-wafer laboratory apparatus has an MST structure in the MST layer to integrate fluid processing into a single-support substrate (usually helium). The manufacture of VLSI (Ultra Large Integrated Circuit) technology in the semiconductor industry makes the unit cost of each LOC device very low. However, controlling the flow of fluid through the LOC device, adding reagents, controlling reaction conditions, etc. requires a large volume of external tubing and electronics. Connecting the LOC device to these external devices actually limits the use of the LOC device for molecular diagnostics to inspection processing. The cost of external equipment and its operational complexity -18-201209407 eliminates the use of LOC-based molecular diagnostics as a practical option for in-situ treatment. In view of the above, there is a need for a molecular diagnostic system based on LOC devices for in situ care. SUMMARY OF THE INVENTION Various aspects of the present invention will be described in the following paragraphs. GB S0 02. 1 This invention provides a microfluidic device comprising: a support substrate; a microsystem technology (MST) layer covering the support substrate, the MST layer defining an MST channel for fluid flow in the MST layer; a cover covering the MST layer, The cover defines a cover passage for fluid to flow within the cover, and a sump for retaining fluid; wherein the MST passage is in fluid communication with the cover passage. GBS002. 2 Preferably, the sump is in fluid communication with the MST channel. GBS002. Preferably, at least one of the sump is configured to retain liquid in the sump by securing the meniscus such that during use, the liquid is retained in the sump until fluid flow in the MST channel contacts and is removed Meniscus. GBS002. Preferably, the cover has a lower seal to close the cover passage and the lower seal has a plurality of openings to provide fluid communication between the cover passage and the MST passage. GBS0 02. Preferably, at least some of the openings are configured to hold the bend -19-201209407 level until the meniscus is removed from contact with the fluid stream. GBS002. Preferably, the microfluidic device also has a CMOS circuit between the support substrate and the MST layer, the CMOS circuit having a feedback sensor to sense the characteristics of the fluid flow through the MST channel. GBS002. Preferably, the MST layer has heater elements to heat the fluid stream. GBS002. Preferably, the lid has a seal formed over the outer layer and the upper seal has a through hole that is dimensioned to allow the air to flow in when the tank empties the liquid (but still retains liquid in the sump). GBS002. Preferably, the microfluidic device also has an actuator valve having an inlet and an outlet configured to draw fluid from the inlet to the outlet by capillary action along the flow direction: and, the actuator The valve also has a movable member intermediate the inlet and the outlet, the movable member being configured for movement between the static position and the actuated position (moving from the static position); and the orifice is configured to lend The flow of liquid is stopped by the fixed meniscus at the orifice; wherein, in use, the movable member is displaced to the actuating position to create a pressure pulse to break the meniscus and force the liquid through the orifice. GBS002. Preferably, the movable member at least partially defines the aperture 〇 GBS002. Il Preferably, the actuator valve has an actuator for moving the movable member, the actuator having a resistive element to initiate differential thermal expansion to move the movable member. -20- 201209407 GBS 00 2. Preferably, the actuator valve reciprocates the movable member between the rest and displacement positions until the outlet immediately downstream of the orifice is sufficiently filled for capillary action to reestablish the flow of liquid in the direction of flow. GBS002. Preferably, the CMOS circuit operatively controls the actuator valve. GBS002. Preferably, the microfluidic device also has a nucleic acid amplification portion for amplifying a target nucleic acid sequence in the biological sample. GBS002. Preferably, the microfluidic device also has a dialysis section for removing some cells from the biological sample depending on the size of the cells. GBS002. Preferably, the microfluidic device also has a probe array for hybridization to the target acid sequence to form probe-target hybridization. GBS002. Preferably, the CMOS circuit has an array of photodiodes for detecting probe-target hybridization. GBS 002. Preferably, the storage tank stores an analytical reagent selected from the group consisting of: an anticoagulant; a lysis reagent; dNTP, a buffer and a primer; a polymerase; and, water. GBS002. Preferably, the actuator valve is located at the downstream end of the PCR portion such that upon predetermined thermal cycling by the heater element, the CMOS circuit opens the actuator valve to cause flow into the probe array. GBS002. Preferably, the CMOS circuit has a memory for storing a series of reagents in the loading reservoir and a probe type at its location within the probe array. -21 - 201209407 The surface micromachined layer provides a small feature of high density. The cover provides the larger features required. The surface micromachined layer and cover together provide the necessary plural features. GVA000. 1 This invention provides a microfluidic device comprising: a reservoir for containing a reagent; an outlet valve in fluid communication with the main reservoir, the outlet valve having a meniscus holder and an actuator, a meniscus holder Configured to form a meniscus that holds the reagent in the sump, the actuator is configured to receive an activation signal and actuate in response to the activation signal such that the meniscus is released from the meniscus holder and the reagent is allowed to flow out Storage tank. GVA006. Preferably, the outlet valve has a moveable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the moveable member to generate a pulse in the reagent to move the meniscus. GVA006. Preferably, the movable member is configured for movement between the static position and the actuated position (moving from the static position), and the meniscus holder is configured to secure the meniscus to the hole Stop the mouth of the liquid flow. GVA006. 4 Preferably, the movable member at least partially defines the aperture. GVA006. Preferably, the thermal actuator has a resistive element to initiate differential thermal expansion to move the movable member. GVA006. Preferably, during use, the activation signal is a series of electrical pulses and is responsive to an actuator valve that moves the movable member back and forth between the rest and displacement positions. GVA006. Preferably, the movable member is configured to move from the rest position -22-201209407 to the actuated position such that displacement from the movable member to the actuating position causes the meniscus to extend and downstream of the outlet valve The surface contacts, so capillary action drives the reagent out of the sump. GVA006. Preferably, the surface downstream of the outlet valve is a capillary action initiation feature for reconstituting the meniscus and moving the meniscus away from the outlet valve. GVA006. Preferably, the thermal expansion actuator is configured to initiate differential thermal expansion to move the movable member. GVA006. Preferably, the actuator has a valve heater for heating the meniscus to release the meniscus by the meniscus holder. GVA00 6.  Preferably, the meniscus holder is configured to stop the flow of reagent out of the reservoir by fixing the meniscus to the orifice and the valve extends around the periphery of the orifice. GVA006. Preferably, the valve heater is configured to cause the reagent at the meniscus to boil and release the meniscus from the meniscus holder. GVA00 6. Preferably, the microfluidic device of claim 1 further comprises a liquid sensor to sense the presence or absence of liquid adjacent the outlet valve. GVA006. Preferably, the microfluidic device of claim 13 further comprises a support substrate and a CMOS circuit on the support substrate to operatively control the outlet valve" GVA006. Preferably, the sensor provides feedback to the CMOS circuit for operative control of the outlet valve. The easy to use, mass-produced and inexpensive microfluidic device receives liquid samples for processing and analysis. If necessary, add -23-201209407 reagent to the liquid with some active valves to utilize the reagents stored in the reagent reservoir of the device. The reagent sump is integrated into the unit to provide reagent storage for itself, thus providing a low system component volume and a simple process for an inexpensive analytical system. GVA008. 1 The present invention is directed to a microfluidic device for testing a fluid, the microfluidic device comprising: an inlet for receiving a fluid; a reservoir containing a reagent: a flow path extending from the inlet; and a fluid between the flow path and the storage tank a plurality of outlet valves connected, each outlet valve having an actuator for opening the outlet valve in response to the activation signal; wherein, in use, selectively opening the valves such that the reagent flows into the flow path to merge with the fluid from the inlet The resulting turbulent flow with the reagent ratio is determined by the number of open outlet valves. GVA008. 2 Preferably, the microfluidic device also has a plurality of channels extending between the sump and the flow path, and the outlet valve is disposed in each channel, and the channel is configured to attract the reagent to the flow path from the sump by capillary action, wherein The outlet valves each have a meniscus holder, and at the meniscus holder, the capillary flow toward the flow path drives the reagent flow to be stopped and forms a meniscus ^ GVA008. Preferably, there is more than one outlet valve in each channel. GVA008. Preferably, the actuator is a heater to release the meniscus from the meniscus holder in response to the activation signal. GVA008. 5 Preferably, each outlet valve has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the movable member 24 to 201209407 to generate a pulse in the reagent to move the movable member In order to restore the flow of reagents towards the flow path. GVA008. Preferably, the movable member is configured for movement between the static position and the actuated position (moving from the static position), and the meniscus holder is configured to secure the meniscus to the hole The orifice of the reagent flow is stopped and the thermal actuator is configured to move the movable member back and forth between the static position and the actuated position to force the reagent through the orifice. GVA008. Preferably, the aperture is defined in the movable member. GVA0 0 8. Preferably, the meniscus holder is configured to stop the orifice of the reagent stream by fixing the meniscus to the orifice, and the thermal actuator is configured to cause a portion of the reagent at the orifice to boil. The meniscus is released from the meniscus holder. GVA0 0 8. Preferably, each outlet valve has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the movable member to move the meniscus to be downstream of the meniscus holder The surface is in contact whereby the meniscus holder releases the meniscus and resumes the capillary action drive flow toward the flow path. GVA008. Preferably, the surface downstream of the meniscus holder is a capillary action initiation feature configured to direct the meniscus to the channel wall. GVA00 8. Preferably, the microfluidic device of claim 1 further comprises a support substrate for the sump, the outlet valve, the inlet and the flow path, and a CMO S circuit for operatively controlling the outlet valve. GVA008. Preferably, the microfluidic device of claim 11 further comprises at least one liquid responsive sensor to provide feedback to the -25-201209407 CMOS circuit for operative control of the respective valves. GVA008. Preferably, at least one of the sensors is a liquid sensor for sensing the presence or absence of liquid at a location in one of the channels. GVA008. Preferably, the microfluidic device of claim 1 further comprises a plurality of storage tanks each containing different reagents and outlet valves each having a plurality of storage tanks and individual flow paths, wherein the combined flow is The ratio of any different reagents is related to the number of separate valves that are open in the corresponding valve configuration. GVA008. Preferably, the CMOS circuit selects the number of outlet valves that are open for each sump based on the type of reagent and the ratio desired for the turbulent flow. GVA008. 16. Preferably, the microfluidic device of claim 15 further comprises a polymerase chain reaction (PCR) portion for amplifying a target nucleic acid sequence in a fluid" GVA008. Preferably, the different reagents in the sump comprise one or more of the following: a polymerase; a restriction enzyme; a dNTP and a primer in the buffer; a lysis reagent; and an anticoagulant. GVA008. Preferably, the microfluidic device of claim 15 further comprises a hybridization portion having a probe array for hybridizing to a target nucleic acid sequence in the fluid, wherein the CMOS circuit has a detection -26- 201209407 Sensor array for hybridization of any probe within the probe array. The easy to use, mass-produced and inexpensive microfluidic device receives liquid samples for processing and analysis. Reagents are added to the liquid by some active valves to utilize the reagents stored in the reagent reservoir of the device, as needed. During the operation of the apparatus, the necessary mixing ratio of the reagent to the other liquid components is determined by the number of valves that are opened, so that it is possible to reliably and reliably achieve the difficult control target of the microfluidic contents. [Embodiment] This general specification indicates a main component of a molecular diagnostic system including a specific example of the present invention. The details of the system structure and operation are discussed in the following description. 0 Referring to Figures 1, 2, 3, 104 and 105, the system has the following most important components: Test modules 10 and 11 are of the size of a conventional USB flash drive and can be manufactured inexpensively. . The test modules 10 and 11 each contain a microfluidic device, which is generally in the form of a laboratory on-wafer (LOC) device 30 and preloaded with reagents, and generally has more than 1000 probes for molecular diagnostic analysis (see Figure 1). And 104). The test module 10 illustrated in Figure 1 uses a fluorescence-based detection technique to identify target molecules, while the test module 11 of Figure 104 is based on electrochemiluminescence detection techniques. The LOC device 30 has an integrated photosensor 44 (described in detail below) for fluorescence or electrochemiluminescence detection. The test modules 10 and 11 all use the standard for power, data and control -27-201209407 micro-USB connector 14, each has a printed circuit board (PCB) 57' and a capacitor 32 and an inductor 15 both having external power supply . The test modules 10 and 11 are both for single use for mass production and are distributed in sterile packaging for use. The outer casing 13 has a large container 24 for receiving biological samples and a removable sterile sealing strip 2 2 It is preferred to have a low viscosity adhesive to cover the large container prior to use. The membrane seal 408 having the membrane guard 410 forms part of the outer casing 13 to reduce the moisture resistance in the test module, while the pressure drop is provided by the small pressure fluctuation. The membrane guard 410 protects the membrane seal 40 8 from damage. The test module reader 1 2 is powered to the test module 1 〇 or 1 1 via the micro-USB 埠1. The test module reader 12 can be in many different forms, and its selection is described below. The version of the reader 12 shown in Figures 1, 3 and 104 is a specific example of a smart phone. A block diagram of the reader 12 is shown in FIG. The processor 42 executes application software from the program storage 43. The processor 42 is also interfaced with a display screen 18 and a user interface (UI) touch screen 17 and button 19, a cellular radio 2, a wireless network connection 23, and a satellite navigation system 25. Honeycomb radio 2 1 and wireless internet connection 2 3 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location data. Alternatively, the position data can be input by the touch screen 17 or the button 19. The data store 27 holds genetic and diagnostic information ' ^ M is m ' information about the patient, the analysis used to identify each probe and the probe data and its array position. The data store 27 and the program store 43 can be shared by a common memory device. The application software installed in the test module reader 12 provides results analysis and additional test and diagnostic information. -28- 201209407 To perform a diagnostic test, the test module 1〇 (or test module 11) is inserted into the micro-USB port 16 on the test module reader 12. The sterile sealing strip 22 is turned up and the biological sample (in liquid form) is loaded into the large sample container 24. The start button 20 is pressed to initiate the test by applying the software. The sample is flowed into the LOC device 30 and analyzed for extraction, culture, amplification, and hybridization of the pre-synthesized hybrid-reactive oligonucleotide probe to the sample nucleic acid (target). In the case of the test module 1 ( (which uses fluorescence-based detection), the probe is fluorescently labeled and the LED 26 placed in the housing 13 provides the necessary excitation light to induce fluorescence from the hybridized probe. Launch (see Figures 1 and 2). In test module 11 (which uses electrochemiluminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to produce a photoluminescent firefly. Conversely, electrodes 8 60 and 8 70 provide an excitation current (see Figure 105). A light sensor 44 integrated with a CMOS circuit on each LOC device is used to detect the emission (fluorescence or photoluminescence). The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the results. The test module 10 or 11 can be removed from the test module reader 12 by locally storing the data and/or uploading the data to a web server containing the patient record and processing them appropriately. Figure 1 '3 and 1 〇 4 show the test module reader 1 2 configured as a mobile phone/smart phone 2 8 . In other forms, the test module reader is a laptop/notebook 101, a dedicated reader 103, an e-book reader 107, a tablet 109, or a desktop computer used in a hospital, private clinic, or laboratory. 105 (see Figure 106). The reader can interface with some additional -29-201209407 applications, such as patient records, accounting, and online actor environments. It can also be used with some local or remote peripheral printers and patient smart cards. Referring to FIG. 107, the data generated by the reader 12 and the network 125 can be used to update the epidemiological database maintained by the epidemiological data system and the genetic database retained by the genetic data system. The electronic health record maintained by the host system for the electronic health record, and the electronic system for the electronic medical record (PHR) 123 maintained by the host system for the electronic record (EMR) 121 recording. On the contrary, through the network 1 25 and the reader 12, the genetic data held by the host system of the epidemiological data held by the host system of the data 1 1 1 is used for recording (EHR) 15 The medical record maintained by the host system and the personal medical record maintained by the electronic medical record (EMR) 121 and the personal health record maintained by the personal health record (PHR) 123 can be used to update the test module 10 Digital memory. Referring again to Figures 1, 2, 104 and 105, battery power is used in the mobile power reader 12. Test and diagnostic information contained in the mobile phone reader. Data is uploaded via some network or contact interface to enable connection to a peripheral or device, computer or online mini-USB port 16 to connect a computer or main power supply pool. The library and the multi-connection, such as the host system of the test module 1 1 (EHR) l 15 medical record, and some individuals for epidemics, for genetic electronic In the health configuration record, in the electronic configuration of the host system LOC 30, all preloads can also be loaded or connected. Recharger -30- 201209407 Figure 78 shows a specific example of the test module 10 for testing that only needs to know the presence or absence of a specific target, such as whether the test individual is exposed to, for example, a sputum influenza virus. Η1Ν1 infection. It is only suitable as a built-in module 47 for USB power/indicator only. No other readers or application software is required. The indicator 45 on the module 47 of the USB power/indicator only shows a positive or negative result. This configuration is ideal for large screenings. Additional items supplied to the system may include test tubes containing reagents for pre-treating a particular sample, and a tongue depressor and lancet containing sample collection. For convenience, the test module of the specific example shown in Fig. 78 includes a spring-loaded retractable lancet 3 90 and a lancet release button 392. Satellite phones can be used in remote areas. Test Module Electronics Figure 2 and Figure 05 are block diagrams of the electronic components in test modules 10 and 11. The CMOS circuit integrated in LOC device 30 has USB device driver 36, controller 34, USB compatible LED driver 29, clock 3 3, power conditioner 3 1 , RAM 3 8 and program and data flash memory 40. These are provided for test module 10 or 1 1 integral and associated driver 3 including light sensor 44, temperature sensor 170, liquid sensor 174, and various heaters 152, 154, 182, 234 7 and 3 9 and the control and memory of registers 3 5 and 41. Only LED 26 (in the case of test module 10), external power supply capacitor 32 and micro-USB connector 14 are external to LOC device 30. The LOC device 30 includes an adhesive pad for attachment to these external components. RAM 3 8 and program and data flash memory 40 have application software and diagnostic and experimental information (flash/guaranteed storage, such as via encryption) for over 200009407 over 1 000 probes. In the case of the test module 11 configured for E C L detection, there is no LED 26 (see Figs. 104 and 105). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 is loaded with electrochemiluminescent probes and hybridization chambers each having an ECL excitation electrode pair 860 and 870. Many types of test modules 10' are manufactured in a number of test formats that are ready for ready-to-use users. The test format differs in the reagents and probes of the on board assay. Some examples of infectious diseases that are quickly identified by this system include: Influenza and influenza viruses A, B, C, infectious salmon anemia virus, tocovirus, pneumonia-respiratory fusion virus (RSV), adenovirus, interstitial pneumonia, pneumococci, Staphylococcus aureus • Tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africana, Mycobacterium vaccae and Mycobacterium vaccae • Plasmodium falciparum, Toxoplasma gondii and other parasitic protozoa • Typhoid-cold typhoid • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever – Yellow fever virus • Hepatitis (A to E) • Iatrogenic infections – such as Clostridium difficile, vancomycin-resistant enterococci and drug-resistant golden yellow Staphylococcus aureus-32- 201209407 • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) • Encephalitis - Japanese encephalitis virus, Zhangdipula virus • Pertussis-B. pertussis • Measles-paramyxovirus • Meningitis – Streptococcus pneumoniae and meningococcus • Anthracnose – Some examples of hereditary diseases identified by this system include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black Mongolian idiots • Hemochromatosis • Cerebral arterial disease • Crohn's disease • Polycystic kidney disease • Congenital heart disease • A few options for the disease identified by the diagnostic system : • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinoblastoma - 33 - 201209407 • Turcot syndrome The above list is not exhaustive and the diagnostic system can be configured to use nucleic acids and protein bodies Analyze to detect many different diseases and symptoms. Detailed Structure of System Components LOC Device The LOC device 30 is the center of the diagnostic system. It uses a microfluidic platform to rapidly perform the four main steps of nucleic acid-based molecular diagnostic analysis, ie sample preparation, nucleic acid extraction, nucleic acid amplification and detection. The LOC device also has alternative uses and will be described in more detail below. As discussed above, test modules 10 and 11 can take many different configurations to detect different targets. As such, the LOC device 30 has a number of different embodiments for targeting the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a pathogen of a whole blood sample. For the purpose of the description, the structure and operation of the LOC device 301 are described in detail with reference to Figs. 4 to 26 and 27 to 57. 4 is a schematic diagram showing the structure of the LOC device 301. For convenience, the processing stages shown in FIG. 4 are represented by the component symbols corresponding to the functional portions of the LO C device 301 that implements the processing stage. The processing stages associated with the major steps in each of the nucleic acid-based molecular diagnostic assays also represent: sample input and preparation 288, extraction 290, culture 291, amplification 292, and detection 294. The various reservoirs, chambers, valves, and other components of the LOC device 301 will be described more closely below.

FIG. 5 is a perspective view of the LOC device 301. It is manufactured using high volume CMOS -34-201209407 and MST (Microsystem Technology) manufacturing technology. The layered configuration of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) of FIG. 12. The LOC device 301 has a germanium substrate 84 supporting a COMS + MST wafer 48, including a CMOS circuit 86 and an MST layer 87, to cover 46 covers the MST layer 87. For the purposes of this patent specification, the term "MST layer" relates to a collection of structures and layers of a sample treated with different reagents. Accordingly, these structures and components are configured to define a flow path having a characteristic size that supports a capillary action driven liquid stream having physical properties similar to the physical properties of the sample during processing. Accordingly, MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing methods can also produce structures and components that are sized for liquid flow driven by capillary action and that are processed to very small volumes. The particular embodiment described in this specification shows that the MST layer is a structure and active component supported on CMOS circuitry 86, but excludes the features of cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample. The overall dimensions of the LOC device shown in the following figures are 1760 microns χ 58 24 microns. Of course, LOC devices made for different applications can have different sizes. Figure 6 shows the features of the MST layer 87 overlaid with the cover features. The illustrations AA to AD, AG and AH shown in Fig. 6 are individually enlarged in Figs. 13, 14, 35, 56, 55 and 63, and a sufficient understanding of the respective structures in the LOC device 301 will be described in detail below. When FIG. 11 independently shows the structure of the CMOSS + MST device 48, FIGS. 7 through 10 independently show the features of the cover 46. -35- 201209407 Layered Structure Figures 12 and 22 are schematic representations of the layered construction of the CMOS + MST device 48, the cover 46 and the fluid interaction therebetween. The drawings are not drawn to scale for the purpose of illustration. Figure 12 is a cross-sectional view through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12, CMOS+MST device 48 has a germanium substrate 84 that supports CMOS circuitry 86 that operates the active components within MST layer 87 described above. Passivation layer 88 seals and protects CMOS layer 86 from fluid flow through MST layer 87. The fluid flows through both the cap layer 46 and the MST channel layer 100 and the M ST channel 90 (see, for example, Figures 7 and 16). When biochemical treatment is performed on the smaller MST channel 90, cell delivery occurs in the larger channel 94 made in the cover 46. The cell delivery channels are sized to deliver cells in the sample to predetermined locations in the MST channel 90. Cells that deliver a size greater than 20 microns (e. g., certain white blood cells) require channel sizes greater than 20 microns, and thus cross-sectional areas across the flow are greater than 400 square microns. The MST channel, particularly at locations in the LOC that do not require delivery of cells, can be significantly smaller. It will be understood that the cover channel 94 and the MST channel 90 are common references and that the particular M S channel 90 can also be, for example, a heated microchannel or a dialysis MST channel due to its particular function. The MST channel 90 is formed by etching through the MST channel layer 100 deposited on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is formed by the top layer 66 around the top layer forming the top of the CMOS + MST device 48 (relative to the orientation shown in the figure). Although sometimes shown as a separate layer, the cover channel layer 80 and the sump layer -36 - 201209407 78 are formed from a single piece of material. Of course, the piece of material can also be non-unitary. The sheets of material are etched from both sides to form a cover channel layer 80 and a sump layer 78, and a cover channel 94 is etched in the cover channel layer 80, and sumpes 54, 56, 58, 60 and 02 are etched in the sump layer 78. Alternatively, the sump and cover channel are formed by a micro-formation process. Both etching and microforming techniques are used to fabricate channels having traverse fluids of up to 2 Å, 〇〇〇 square microns and up to 8 s 2 microns. There are suitable choices for the cross-sectional area of the passage across the fluid at different locations in the LOC device. A large number of samples or samples having a large component are accommodated in the channel, and a cross-sectional area of up to 20,000 square micrometers (e.g., a 200 micrometer wide channel in a layer of 100 micrometers thick) is suitable. A small amount of liquid or a mixture free of large cells is contained in the channel, preferably a very small cross-sectional area across the fluid. The lower seal 64 surrounds the lid passage 94 and the upper seal layer 82 surrounds the sump 54, 56, 58, 60 and I 62 » five tanks 54, 56, 58, 60 and 62 are preloaded with the reagent of the particular analysis. In the embodiments described herein, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant, the selectivity of which includes red blood cell lysis buffer • storage tank 5 6 : lysis reagent • Slot 58: Restriction enzymes, ligases, and junctions (for junction initiation PCR) (see Figure 77, excerpt from T.  .  Staehan et al. , Human Molecular Genetics 2, Garland Science, NY and London, -37- 201209407 1999)) • Storage tank 60: amplification mixture (deoxyribonucleoside triphosphate (dNTP), primer, buffer), and • storage tank 62: DNA polymerase. Cover 46 and CMOS + MST layer 48 are in fluid communication via respective openings in lower seal 64 and top layer 66. The upper conduit 96 and the lower conduit 92 are representative of whether the fluid flows from the MST passage 90 to the cover passage 94 or vice versa. LOC Device Operation The operation of LOC device 301 is described step by step with reference to the analysis of pathogenic DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using a suitable kit or combination of reagents, test protocols, LOC variants, and detection systems. Referring to Figure 4, analyzing a biological sample involves five major steps, including: sample input and preparation 288, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294 ° sample input and preparation steps 2 8 8 mixed blood The pathogen is separated from the white blood cells and red blood cells by the anticoagulant 1 16 and then by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, blood samples enter the device via sample inlet 68. The capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood stream opens its surface tension valve 1 18, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants prevent the formation of blood clots that can block flow. -38- 201209407 As best shown in Figure 22, the anticoagulant 116 is withdrawn from the sump 54 by capillary action and enters the MST channel 90 via the lower conduit 92. The conduit 92 has a capillary action initiation feature (CIF). 102 to form a meniscus geometry that is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the reservoir 54, the vents 122 in the upper seal 82 allow air to replace the anticoagulant 116. The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 is filled with a surface tension valve 118 and secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. Prior to use, the meniscus 120 remains fixed to the upper conduit 96 such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the cover channel 94 to the upper conduit 96, the meniscus 110 is removed and the anticoagulant is drawn into the fluid. Figures 15 through 21 show an inset AE which is part of the inset AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 18 has three separate MST passages 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent entering the sample mixture. When the sample mixture and reagents are mixed by diffusion, the flow rate away from the sump determines the concentration of the reagent in the sample stream. Therefore, the surface tension valve of each tank is configured to meet the desired reagent concentration. Blood is passed to the pathogen dialysis section 70 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of orifices 1 64 of a predetermined size. Cells smaller than the valve stomata pass through the orifice, while large cells cannot pass through the orifice. While the target cells continue to be part of the analysis, the undesired cells -39 - 201209407 are reintroduced into the waste unit 76. Undesired cells are large cells blocked by an array of orifices 164 or small cells that pass through the orifice. In the pathogen dialysis section 70 described herein, the pathogen from the whole blood sample is concentrated for microbial DNA analysis. The array of orifices is formed by a plurality of 3 micron diameter orifices 1 64 that fluidly communicate with the input flow in the lid passage 94 to the target passage 74. The 3 micron diameter orifice 1 64 and the dialysis extraction orifice 168 for the target channel 74 are connected by a series of dialysis MST channels 206 (best shown in Figures 15 and 21). The pathogen is small enough to pass through the dialysis MST channel 206 through the 3 micron diameter orifice 164 and to fill the target channel 74. Cells larger than 3 microns, such as red blood cells and white blood cells, are retained in the waste channel 72 of the lid 46, which leads to the waste reservoir 76 (see Figure 7). Other orifice shapes, sizes, and aspect ratios can be used to isolate specific pathogens or other target cells, such as white blood cells for human DNA analysis. More detailed details of the dialysis section and the dialysis variant are provided later. Referring again to Figures 6 and 7, fluid is drawn through target channel 74 to surface tension valve 128 in lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending between the lysis reagent reservoir 56 and the target channel 74. When the meniscus is removed from the sample stream, the flow rate of all seven MST channels 90 will be greater than the flow rate of the anticoagulant reservoir 54, wherein the surface tension valve 118 has three MST channels 90 (assuming the physical properties of the fluid are approximately equal). Therefore, the proportion of the lysis reagent in the sample mixture is greater than the ratio of the anticoagulant. The lysis reagent and the target cells are mixed by diffusion in the target channel 74 within the chemical lysis unit 130. The boiling priming valve 126 stops the flow until the expansion and lysis are performed for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). Referring to Figures 31 and 3, the structure and operation of the boiling pilot valve will be described in detail below. Other active valve types (as opposed to passive valves, such as surface tension valve 118) have also been developed by the applicant, which can be used in place of the boiling pilot valve. These alternative valve designs are also described below. When the boiling pilot valve 126 is turned on, the lysed cells flow into the mixing portion 131 to pre-amplify restriction digestion and linker ligation. Referring to Figure 13, when the fluid is removed from the meniscus on the surface tension valve 1 32 at the beginning of the mixing portion 131, the restriction enzyme, linker and ligase are released from the reservoir 58. The mixture flows through the length of the mixing portion 131 for diffusion mixing. At the end of the mixing portion 131 is a lower duct 134 (see Fig. 13) leading to the incubator inlet passage 133 of the culture portion 114. The incubator inlet channel 133 feeds the mixture into the crucible structure of the heated microchannel 210, which provides a culture chamber for retaining the sample during restriction enzyme cleavage and junction ligation (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the inset AB of Figure 6. The figures show successive layers to form a layer of CM0S+MST layer 48 and cover 46 structures. The inset AB shows the end of the culture section 114 and the start of the amplification section 112. As best shown in Figures 14 and 23, the fluid fills the microchannel 210 of the culture portion 114 until it reaches the boiling pilot valve 106, where the fluid stops when diffusion occurs. As discussed above, the microchannel 210 upstream of the boiling pilot valve 106 becomes a culture chamber containing a sample, a restriction enzyme, a ligase, and a linker. Heater 154 then activates -41 - 201209407 and maintains at a stable temperature for the restriction enzyme shear and junction bonding to occur for a specific period of time. Those skilled in the art will appreciate that this incubation step 291 (see Figure 4) is arbitrary and is only required for some types of nucleic acid amplification assays. Further, in some instances, it may be desirable to have a heating step at the end of the incubation period to increase the temperature above the culture temperature. The temperature is increased to inactivate the restriction enzyme and ligase before entering the amplification unit 112. Limiting the inactivation of enzymes and ligases has a specific effect when amplified with isothermal acid. After the incubation, the boiling start valve 106 is activated (opened) and the fluid re-enters the amplifying portion 112. Referring to Figures 31 and 32, the mixture is filled with the structure of the microchannels 1 58 until it reaches the boiling pilot valve 1 〇 8, which forms one or more amplification chambers. As best shown in the cross-sectional view of Fig. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and the polymerase is then released from the storage tank 62 to enter the junction culture section and the amplification section (1 1 4 and 112) The intermediate MST channel 212. Figures 35 through 51 show the layers of the LOC device 301 in the inset AC of Figure 6. The figures show layers that are continuously stacked to form the CMOS + MST device 48 and cover 46 structures. The illustration AC shows the end of the amplification section 112 and the onset of the hybridization and detection section 52. The cultured sample, amplification mixture, and polymerase flow through the microchannel 1 58 to the boiling pilot valve 108. After diffusion mixing for a sufficient time, the heater 1 54 in the microchannel 1 58 is activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification procedure, the boiling pilot valve 108 is turned on and the fluid re-enters the hybridization and detection portion 52. The operation of the boiling pilot valve -42- 201209407 is described in more detail below. As shown in Figure 52, hybridization and detection portion 52 has an array 110 of hybridization chambers. Figures 52, 53, 54 and 56 show the hybridization chamber array 11 and individual hybridization chambers 180 in detail. The entrance to hybridization chamber 180 is a diffusion barrier 175 that prevents diffusion of the target nucleic acid, probe strands, and hybridization probes between hybridization chambers 180 during hybridization to prevent erroneous hybridization assay results. The flow path length of the diffusion barrier 175 is long enough to prevent the target sequence and probe from diffusing out of one chamber and contaminating the other during the time the probe and nucleic acid hybridize and detect the signal, thus avoiding erroneous results. Another mechanism to prevent erroneous reading is to have the same probe in some hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 184 corresponding to the hybridization chamber 180 containing the same probe. The results of the anomalies in the derived single result can be ignored or given different weights. The thermal energy required for hybridization is provided by a CMOS controlled heater 182 (described in more detail below). Hybridization occurs between the complementary target probe sequences after the heater is activated. The LED driver 29 in the CM〇S circuit 86 transmits a message to cause the LED 26 located in the test module 1 to emit light. These probes only fluoresce when hybridization occurs, thereby eliminating the cleaning and drying steps that are often required to remove unbound strands. The stem and loop structure of the hybrid forced FRET probe 186 is opened, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as detailed below. Fluorescence is detected by photodiode 184 located in CMOS circuitry 86 under each hybridization chamber 180 (see the description of the hybridization chamber below). The photodiode 184 and associated electronics for all of the hybridization chambers collectively form a photosensor 44 (see Figure 70). In other embodiments, the photosensor can be a charge coupled device array (CCD array) from -43 to 201209407. The signal detected by the photodiode 1 84 is amplified and converted to a digital output that can be analyzed by the test module reader 12. Further details of the detection method are described below. Other Detailed Description of the LOC Device The modular design LOC device 301 has a number of functional components including reagent reservoirs 54, 56' 58, 60 and 62, dialysis section 70, lysis section 130, culture section 114, and amplification section 1 1 2 In the LOC device of another specific example, the functional portions may be omitted, but another functional portion or a functional portion different from the use of the above-described device may be added. For example, the culture portion 1 14 can be used as the first amplification portion 112 of the tandem repeat amplification analysis system, and the lysis reagent reservoir 56 can be used to add the first amplification mixture of the primer, the dNTP, and the buffer, and use The reagent reservoir 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 56, or alternatively, the sample may be heated for a predetermined period of time to cause thermal lysis in the culture. In some embodiments, if chemical lysis is required and the chemical lysis reagent is mixed and separated therefrom, additional sump may be combined upstream of the adjacent sump 58 for the introduction of the primer, dNTP, and buffer. In some cases, steps such as incubation step 291 are omitted. In this case, the LOC device may be specially manufactured to avoid the reagent storage tank 58 and the culture portion 1 14 or the storage tank may only carry the reagent, or when the active valve is present, it is not activated to dispense the reagent into the sample stream. The culture unit is simply a passage for transferring the sample-44-201209407 from the lysis unit 130 to the amplification unit 112. The heaters operate independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the fluid system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after amplification phase 292, prior to hybridization and detection step 294, dialysis is performed to remove cell debris. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplifications 112 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using a particular nucleic acid probe in the hybrid array 110. To analyze, for example, a sample of whole blood in which dialysis is not required, the dialysis portion 70 is simply omitted from the sample input and preparation portion 288 of the LOC design. In some cases, even if the analysis does not require dialysis, it is not necessary to omit the dialysis section 70 from the LOC device. » If the presence of the dialysis section does not cause geometrical obstruction, the sample input and preparation section can have the LOC of the dialysis section 70. Will not lose the required functionality. In addition, the detection portion 294 can include a protein body array array that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to a protein present in the non-amplified sample, rather than being designed to A nucleic acid probe that hybridizes to a target nucleic acid sequence. It will be appreciated that the LOC device manufactured for use with this diagnostic system is different from the combination of functional components selected for the particular LOC application. Most of the functional parts are common to many LOC devices, and the design of additional LOC devices for new applications is well organized in the large functional options used in the existing LOC installations -45-201209407. The functional part of the combination. Only a few LOC devices are shown in this description and some others are shown to illustrate the design flexibility of the LOC devices manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices shown herein are not exhaustive' and that many additional LOC designs are related to the combination of appropriate functional components. Sample Type LOC Variants can accept and analyze a variety of nucleic acid or protein contents in liquid form, including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord Blood, breast milk, sweat, pleural effusion, tears, pericardial fluid, peritoneal fluid, environmental water samples and beverage samples. Amplicon derived from meganucleic acid amplification can also be analyzed using a LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, and only use dialysis, lysis The culture and amplification section is used to transfer the sample from the sample inlet 68 to the hybridization chamber for nucleic acid detection 180, as described above. For some sample types, a pre-treatment step is required, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and possibly pre-treat the mucus with enzyme to reduce stickiness. Sample Input Referring to Figures 1 and 12, a sample is added to the large container 24 of the test module 10. The large container 24 is a truncated cone that is fed into the inlet 68 of the LOC unit 301 by capillary action. Here, it flows into the 64 μπι χ 60 μιη deep cover channel -46 - 201209407 94 and is also attracted to the anticoagulant sump 54 by capillary action. Reagent Tanks A microfluidic device, such as LOC unit 301, is used to analyze the system with a small amount of reagent such that the reagent reservoir contains all of the necessary reagents for biochemical treatment and each reagent reservoir is in a small volume. This volume is indeed less than 1, 〇〇〇, 〇〇〇, 〇〇〇 cubic micron, in most cases less than 300, 〇〇〇, 〇〇〇 cubic micron, usually less than 70,000,00 〇 cubic micron, And in the case of the LOC device 301 shown in the drawings, it is less than 20,0 〇〇, 〇〇〇 cubic micron. Dialysis section Referring to Figures 15 to 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate the pathogen target cells from the sample. As previously described, a plurality of apertures in the top layer 66 are apertures 164 having a diameter of 3 microns, filtering the target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice 164, the microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via a 16 [mu] dialysis extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) is retained in the cover channel 94. Downstream of the pathogen dialysis section 70, the cover channel 94 becomes a waste channel 72 to the waste reservoir 76. Foam illustrations or other porous elements 49 in the outer casing 13 of the test module 10 are configured to be in fluid communication with the waste reservoir 76 (see Figure 1) for a biological sample type that produces a substantial amount of waste. The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. -47- 201209407 A 3 micron diameter orifice 164 located at the upstream end of the pathogen dialysis section 7 has a capillary action initiation feature (CIF) 166 (see Figure 33) such that fluid is pulled down to the dialysis MST channel 204 below. in. The first extraction aperture 198 for the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus over the dialysis extraction aperture 168. The small component dialysis section 682 shown in Fig. 8 2 may have a structure similar to that of the pathogen dialysis section 70. The small component dialysis section separates any small target cells or molecules from the sample by size (and shaping, if necessary) suitable for the orifices that allow the small target cells or molecules to pass to the target channel and continue to be further analyzed. Large size cells or molecules are removed to the waste reservoir 766. Therefore, the LOC device 30 (see Figures 1 and 104) is not limited to isolation of pathogens having a size of less than 3 μm, but can be used to separate cells or molecules of any desired size. Lysis section Referring again to Figures 7, 11 and 13, the chemical species in the sample are released from the cells by chemical lysis. As described above, the cytolytic reagent from the lysis tank 56 is mixed with the sample flowing in the target channel 74 downstream of the surface tension valve 128 of the lysis tank 56. However, some diagnostic assays preferably use hot lysis, or even a combination of chemical and thermal lysis of the target cells. The LOC device 301 houses the heated microchannels 210 of the culture portion 1 14 . The sample stream is flooded with the culture portion 114 and stopped at the boiling pilot valve 106. The culture microchannel 210 heats the sample to the temperature at which the cell membrane ruptures. In some hot lysis applications, the enzyme -48-201209407 reaction is not required in the chemical lysis unit 130, and the hot lysis completely replaces the enzyme reaction in the chemical lysis unit 130. Boiling Pilot Valve As discussed above, LOC unit 301 has three boiling pilot valves 126, 106 and 108. The position of these valves is shown in Figure 6. Figure 31 is an enlarged plan view of the independent boiling pilot valve 108 on the heated microchannel 158 side of the amplifying portion 112. By capillary action, the sample stream 1 1 9 is drawn along the heated microchannel 1 58 until it reaches the boiling pilot valve 108. The meniscus 120 at the leading edge of the sample stream is secured to the meniscus holder 98 of the valve inlet 146. The meniscus holder 98 geometry stops the meniscus from moving forward and prevents capillary flow. As shown in Figures 31 and 32, the meniscus holder 98 is a conduit on the orifice provided by the opening of the pipe from the MS T passage 90 to the cover passage 94. The surface tension of the meniscus 120 keeps the valve closed. Ring heater 152 is located around valve inlet 1 46. The ring heater 1 52 is CMOS controlled by boiling the valve heater contact 153. To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater contact 153. The ring heater 152 is resistively heated until the liquid sample 119 is boiled. Boiling removes meniscus 120 from valve inlet 146 and begins to wet cover passage 94. Once the lid passage 94 is wetted, the capillary action is restored. The fluid sample 119 is filled with the passage 94 and flows through the valve down conduit 150 to the valve outlet 148, wherein the capillary driven liquid flow advances along the expansion outlet passage 160 into the hybridization and detection portion 52. The liquid sensor 174 is placed before and after the valve for diagnosis. -49- 201209407 It will be understood that once the boiling pilot valve is opened, it is impossible to close it again. However, since the LOC device 301 and the test module 10 are single-purpose devices, it is not necessary to close the valve. Culture section and nucleic acid amplification section The culture section 114 and the amplification section 112 are shown in Figs. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51. The culture portion 114 has a single, heated culture microchannel 210 that is etched to form a serpentine pattern in the MST channel layer 100 from the lower conduit opening 134 to the boiling pilot valve 106 (see Figures 13 and 14). Controlling the temperature of the culture section 114 enables a more efficient enzyme reaction. Similarly, the amplifying portion 112 has a heating microchannel 158 that is heated from the boiling pilot valve 106 to the boiling pilot valve 108 (see Figs. 6 and 14). Upon mixing, culture, and nucleic acid amplification, the valves stop flow to retain the target cells in the heated culture or amplification microchannels 2 1 or 158. The microchannel 蜿蜒 pattern also promotes (to some extent) the target cells to mix with the reagents. In the culture unit 114 and the amplification unit 112, the sample cells and reagents are heated by the heater 154 controlled by the pulse width modulation (PWM) CMOS circuit 86. The heated culture microchannel 210 and the amplification microchannel 158 are heated. Each of the turns has three independently operable heaters 154 (extending between the individual heater contacts 156 (see Figure 14)) which provide two-dimensional control of the input heat flux density. As best shown in Figure 51, heater 154 is supported on top layer 66 and buried in lower seal 64. The heater material is TiAl, but many other conductive metals are also available -50- 201209407. The elongated heater 154 is parallel to the longitudinal length of each channel portion forming a wide meandering meandering flow. In the amplification unit 1 1 2, each wide stream is operated as an independent PCR chamber via individual heater control. The use of a microfluidic device, such as the LOC device 301, requires a small volume of amplicons required by the analysis system to allow for the amplification of a small volume of amplification mixture in the amplification portion 1 1 2 . This volume is less than 400 nanoliters, and in most cases less than 170 nanoliters, ordinary. Less than 70 nanoliters, and in the case of LOC device 301, this volume is between 2 nanoliters and 30 nanoliters. Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a relatively short distance from the heater 154. The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 100,000 square microns, while the "high scale" equipment has a significantly higher heating rate. The lithography manufacturing technique allows the amplifying microchannel 158 to have a cross-section that provides a higher heating rate across less than 1 6,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only a very small amount of amplicons are required (as is the case in LOC device 301), the cross section can be reduced to less than 2,500 square microns. For the diagnostic analysis required for "sample entry, answer out" in 1 minute on a LOC device, the appropriate cross-sectional area across the fluid is 400 square microns and 1 square micron. The heater element in the 〇 amplification microchannel 158 heats the nucleic acid sequence at a rate greater than 80 absolute temperatures (K) per second, in most cases a rate of -51 - 201209407 per second greater than 100 K. The heater element heats the nucleic acid sequence at a rate greater than 1000 Torr per second, and in many cases, the heater element heats the nucleic acid sequence at a rate greater than 10,000 κ per second. Typically, based on the needs of the analytical system, the heater element is greater than 100,000 sec per second, greater than 1,000,000 sec per second, greater than 1,000,000 sec per second, and greater than 20. per second. 000.  000 Κ, greater than 40,000,000 每秒 per second, greater than 80 per second. 000.  The nucleic acid sequence is heated at a rate of 000 Κ and greater than 1 60,000,000 每秒 per second. A small cross-sectional area channel is also beneficial for the diffusive mixing of any reagent with the sample fluid. The diffusion of one liquid to another is most pronounced near the interface between the two liquids before the diffusion mixing is completed. The density of the phenomenon decreases with distance from the interface. A microchannel with a relatively small cross-sectional area across the direction of the fluid is used while maintaining the flow of the two fluids at the interface for rapid diffusion mixing. Reducing the channel cross-section to less than 100,000 square microns results in a significantly higher diffusion rate for "large scale" equipment. The lithography manufacturing technique allows the microchannels to have cross sections that traverse less than 1 6,000 square microns and substantially provide a higher mixing rate. If only a very small amount of amplicons are required (as in the LOC device 310), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1 to 2,000 probes on a LOC device and "sample entry, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and 1 Between square microns. Short thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid, resulting in a rapid thermal cycle during the nucleic acid amplification process. Each thermal cycle (i.e., adhesion and primer extension) is completed in 30 seconds for a target sequence of up to 150 (bp) in length. In most diagnostic analyses, the cycle time is less than 11 seconds and most is less than 4 seconds. For some of the base pair (bp) long target sequences, the thermal cycle time for some of the most common diagnostic LOC devices 30 is 〇·45 seconds to 1. A thermal cycle of between 5 seconds allows the test module to be acid-expanded for much less than 1 minute; often within 220 seconds. For most of the analysis, an accumulator is generated by the sample fluid entering the sample inlet within 80 seconds. For most of the analysis, sufficient yield is generated in 30 seconds. Upon completion of the predetermined number of amplification cycles, the amplicon is fed to the hybridization and detection portion 52 via the boiling priming valve. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chamber array 11 compartments 180. The hybridization and detection unit 52 has 24 > column 1 10 ' of the hybridization chamber 180 each having a hybrid-reactive FRET probe 186, an addition member 182, and an integrated photodiode 184. The intrusion photodiode 184 is derived from a target nucleic acid sequence or protein that hybridizes to the FRET probe 186. The material between the FRET probe 186 and the photodiode 184 must be transparent by independently controlling the light emitted by each photodiode U4 by the CMOS circuit 86. Therefore, the probe 186 and the wall portion 97 of the photodiode 184 must also be optically transparent to the emitted light. At l〇C, the base pair was denatured and the individual heat was 150. At this rate, the nucleus is amplified, and the amplified amplicon 108 is amplified by the fluorescent element in the fluorescent device. Between 301 - 53 - 201209407, the wall portion 97 is a thin layer of cerium oxide (about 5 μm) directly incorporated into each of the hybrid chambers 180. The needle-target hybridization still produces a plaque (see Figure 5 4). A small amount of detectable probe-target hybridization requires 270 picograms (corresponding to 900,000 cubic milligrams less than 60 picograms (corresponding to 200,000) ; less than 2. in the case of the LOC device 301 shown in less than 12 picograms (corresponding to 40,000 cubic micrometers).  To a volume of 9,000 cubic microns). Of course, shrinking allows for higher chamber densities and therefore more LOC devices in LOC device 301, having more than 1, 1 room at 1,500 microns by 1,500 micro-hybrids (ie, each room is less than meters) . The smaller volume also reduces the reaction time, making the speed faster. Another advantage of the small number of probes required for each chamber is that during manufacture, only a very small amount of probe solution needs to be configured to a probe solution that can be configured with a specific example of the LOC device of the present invention. After the nucleic acid amplification, the boiling priming valve 108 is flowed by the opening flow path 176 and flows into each of the hybridization chambers 180 (see FIG. 52 and the body sensor 187 indicating that the hybridization chamber is filled with the amplicon 182). After a sufficient hybridization time, the LED 26 is activated (see the opening in Fig. 180 to provide an optical window 136 to allow the FRET probe 1 8 4 to allow the detected fluorescent signal to pass through the chamber. The amount of the hybrid needle is less than a few meters. ), in most δ: square micron), ρ and picogram in Figure 7 (probe corresponding to the size of the hybridization chamber. Within the area of the meter, 2,250 square micro-crossing and detection is faster in each chamber of the LOC device) Genner ML or less and the amplicon along: 56). End point fluid and startable heating 2). Each hybrid cell leaf 186 was exposed to -54-201209407 excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a sufficient period of time to induce a high intensity fluorescent signal from the probe. The photodiode 184 is shorted during excitation. After a preprogrammed delay of 300 (see Figure 2), the photodiode 184 is enabled and the fluorescent emission is detected in the absence of excitation light. The incident light on the active region 185 of the photodiode 184 (see Figure 54) is converted to a photocurrent that can be measured using the CMOS circuit 86. Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. If desired, each hybridization chamber 180 can carry probes that detect more than 1,000 different targets. Alternatively, many or all of the hybridization chambers may carry the same probe that repeatedly detects the same target nucleic acid. Copying the probes in this manner in the hybridization chamber array 110 increases the confidence in the results obtained, and if desired, all results can be combined by photodiodes in adjacent hybridization chambers to obtain a single result. Those skilled in the art will appreciate that there may be from 1 to over 1,000 different probes on the hybrid chamber array 110, depending on the analytical details. Humidifier and Humidity Detector The inset AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of evaporation of reagents and probes during operation of the LOC device. As best shown in the enlarged view of Fig. 55, the water sump 188 is fluidly connected to the three evaporators 190. The water storage tank 188 is filled with molecular biological grade water and is sealed during manufacture. As best shown in Figures 55 and 75, by capillary action, water is drawn to the three lower tubes 1 94 and along the individual water supply channels 92 to the three upper tubes 193 of the evaporator 190. The meniscus is fixed to each of the upper ducts 193 to retain water. The evaporator has a ring heater -55 - 201209407 191' which surrounds the upper pipe 193. The annular heater 191 is connected to the CMOS circuit 86 to the top metal layer 195 (see Fig. 37) by the heat conducting column 3 76. At startup, the annular heater 191 heats the water causing the water to evaporate and wet the surrounding device. The position of the humidity sensor 232 is also shown in FIG. However, the best is shown in Figure 63. In the magnified image of the illustration AH in the middle, the humidity sensing device has a capacitive comb structure. The lithographically etched first electrode 296 and the lithographically etched second electrode 298 are opposed to each other such that their teeth are interleaved. The opposing electrodes form a capacitor having a capacitance that can be monitored by CMOS circuitry 86. As the humidity increases, the dielectric constant of the air gap between the electrodes increases, causing the capacitance to increase. Humidity Sensor 2 3 2 Adjacent to the hybridization chamber array 1 1 〇 (most important humidity measurement location) to slow the evaporation of the solution containing the exposed probe. The feedback sensor temperature and liquid sensors are integrated into the LOC device 301 as a whole to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors 170 are assigned to all of the amplifying portion 112. Similarly, the culture portion 114 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BjT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control thermal cycling during nucleic acid amplification as well as thermal lysis and any heating during incubation. In the hybridization chamber 180, the CMOS circuit 86 uses the hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of the hybrid heater 182 is -56 - 201209407 dependent, and the CMOS circuit 86 uses this to drive each hybrid chamber read. The LOC device 301 also has some MST channel liquid sensation and cover channel liquid sensors 208. Figure 35 shows the MST channel liquid sensing line at one end of each of the spaced turns in the heating 158. Preferably, as shown in Figure 37, the MST channel liquid sense is formed by the exposure of the top metal layer 195 in the CMOS structure 86 to form a counter electrode. The liquid closes the current between the electrodes to indicate the position of the sensor. Figure 25 shows that the enlarged transmissive TiAl electrode pairs 218 and 220 of the cap channel liquid sensor 208 are deposited between the top layers 66 2 18 and 220 as a gap 222 to open the circuit lacking liquid. The circuit is closed when the liquid is present and the CMOS is used to monitor the flow. The GRAVITATIONAL INDEPENDENCE test module 10 is self-directed. It does not need to be fastened to operate. The fluid flow driven by the capillary action and the lack of access to the auxiliary external conduit make the module a portable and easily portable portable reader such as a mobile phone. The gravity autonomous test module is also acceleration independent of all practical ranges. The area of the detector 174 that vibrates and can be used on a moving vehicle or on a mobile phone that is carried by the carrier _ valve is selected from the field of the detector 174 of the microchannel 174 of the temperature detector 174 of the 180. In contrast. Maintaining the 86 in the electrode condition smooth surface Help the device into a similar operation Resistant to impact and tampering. -57- 201209407 Hot Bend Actuated Valve Variant 1 Figures 64 and 65 show the first variation of the hot bend actuated valve. The hot bend actuated pressure pulse valve. Figure 65 is a schematic cross-sectional view taken along line 70-70 of Figure 64. The first variant hot bending actuated valve TiAl, TiN or similar electrically resistive heater material is constructed as a movable member in the form of a thermal bending actuator 404. The valve inlet 146 in the MST passage 90, however, stops at the meniscus orifice 306. The embodiment shown in Figures 64 and 65 has an orifice outside the member, but it can also be at least a movable member. In this state, the valve is closed. The CMOS circuit 86 is rushed to the thermal bending actuator 304 to open the valve. The CMOS actuator 304 is coupled to the cantilever portion 162 of the top layer 66 (fixed and free at the inlet end). The differential thermal expansion bends the cantilever portion toward the passivation layer 88 to move rapidly. The fluid draw prevents the liquid sample 3, and the liquid 119 is ejected to the cover via the orifice 306. The cantilever portion 1 62 moves back and forth to the static and displaced position to ensure that the meniscus 120 is released from the orifice 306. Sample 1 19 is in 94, until its surface is wetted and capillary action drives the flow to the top cover channel 94, then passes through the valve down tube 150 and 148 ° hot bends to actuate the valve variant 2 Figures 66 and 67 show thermal bending A second thermal bending actuated surface tension valve of the movable valve 308. 67 is an ί 302 in FIG. 66, which is shown along line 032 having a CMOS start sample stream entering 120 fixed to the display movable portion to define a series of electrical pulses that are thermally bent at the end of the aperture to be 162 such that they are long 1 1 9 reflux: 94. Allow a period of time to accumulate in the cover channel. The sample flow to the valve outlet variant is shown in cross section along the line -58- 201209407 72-72. The second variant hot bend actuated valve 308 has a CMOS activated thermal bending actuator 304 constructed of TiAl, TiN or similar resistive heater material. The sample flows along the MST passage 90 and flows into the valve inlet 146 of the valve 151. The liquid sample 119 is filled with the passage 94 but stops when the meniscus 120 is fixed to the orifice 306. In this state, the valve closes. To open the valve, the CMOS circuit 86 transmits a series of electrical pulses to the thermal bending actuator 304. The CMOS activated thermal bending actuator 304 is coupled to the top layer 66 (which is fixed at the inlet end and free at the orifice end). 162. The differential thermal spread bends the cantilever portion 162 such that the aperture 306 moves toward the passivation layer 88. The orifice 306 attracts the meniscus 120 from the lid channel 94 into the MST channel 90 to reestablish the capillary flow. To reestablish the capillary flow, the opposing sidewalls of the channel converge into a narrow portion immediately downstream of the movable member such that when the movable member is moved to the actuated position, the meniscus contacts the narrow portion. The liquid sensor 1 74 is placed before or after the debug valve. Hot Bend Actuated Valve Variant 3 Figures 68 and 69 show a third variation of the hot bend actuated valve 312, 'which is a hot bend actuated surface tension valve' and for the liquid sample to be retained in the cover channel 94 Time. Figure 69 is a schematic cross-sectional view along line 74_74 shown in Figure 68. The third variation of the thermal bend actuated valve 312 is similar to the second variant hot bend actuated valve 308 except that the valve inlet 146 is located in the cover passage 94. The sample flows along the cover channel 94 and flows into the valve inlet 146° upstream of the CMOS-activated thermal bending actuator 304. The liquid sample 119 is filled with the channel 94, but stops at the meniscus 120 and is fixed to the orifice 3〇6. Time. In this state -59- 201209407, the valve is closed. To open the valve, the CMOS circuit 86 is transferred to the thermal bending actuator 3 〇4. The cantilever portion of the thermal bending actuator 3〇4 (66 is fixed at the inlet end and free at the orifice end) thermally expands the cantilever portion 162 such that the orifice 306 is blunt. The orifice 306 draws the meniscus 120 from the lid passage 94 into the 90 to reestablish the capillary flow along the valve outlet 148. The name 174 is placed behind the debug valve.

Nucleic Acid Amplification Variants Direct PCR Traditionally, PCR requires target DNA prior to preparation of the reaction mixture. However, when the chemical and sample concentrations are appropriately changed, a small amount of DNA is purified to perform nucleic acid amplification, or a direct amplification PCR is used for nucleic acid amplification, the method is referred to as a controlled direct nucleic acid amplification at a normal temperature in a direct PCR device. This side is thermostatically amplified. When used in LOC devices, especially with regard to the simplification of the calculations, the direct nucleic acid amplification technique has considerable optimization PCR or direct constant temperature amplification. The amplification chemical adjustment includes increased degree, high activity and high progress. A polymerase and an additive that chelate with a potential polymeric agent. Inhibitors in diluted samples are also important for the design of LOC devices using direct nucleic acid amplification techniques. The first feature is a reagent reservoir (eg, in Figure 8, which is appropriately sized to supply a sufficient amount of amplification reaction mixture such that the final connotation of the sample components that may affect the amplification chemistry is pulsed to the top layer 162. Show: layer 88 shift MST channel body sensor can be used in the largest amount of purification. When the LOC method is directly required for fluid potential. Direct buffer strong enzyme inhibition. Into two amounts] storage tank 5 8 The combination or dilution is sufficiently low -60-201209407 to successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LO C structure, such as the pathogen dialysis section 70 of Figure 4, is used. In this specific example (further illustrated in Figure 6), dialysis is performed upstream of the sample extraction section 290 by effectively concentrating the concentration of the pathogen that is sufficiently small to enter the amplification section 292 and discharging the larger cells to the waste container 76. unit. In another embodiment, a dialysis unit is used to selectively remove proteins and salts in the plasma to retain the cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of the channel to adjust the mixing ratio between the sample and the amplified mixture. For example, to ensure that the dilution of the inhibitor associated with the sample via a single mixing step is in the range of preferably 5 to 20 times, the length and cross section of the sample and reagent channels are designed such that the sample channel upstream of the mixing start position The composition of the flow group is 4 to 19 times higher than that of the channel through which the reagent mixture flows. The flow group resistance in the microchannel is easily controlled by controlling the design. For a constant cross-sectional area, the flow resistance of the microchannel increases linearly with the length of the channel. It is important for the hybrid design that the flow group resistance in the microchannel is more dependent on the minimum cross-sectional area size. For example, when the aspect ratio is extremely heterogeneous, the flow resistance of the microchannel of the square cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RNA, For example, from RNA virus or messenger RNA, RNA trans-61 - 201209407 must be transcribed into complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction (one-step RT-PCR) can be carried out in the same chamber as the PCR, or it can be a separate initial reaction (two-step RT-PCR). In the LOC variant described herein, the reverse transcription step and the subsequent step of performing the nucleic acid amplification step can be performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the stylized heater 1 54 to simply perform a nucleic acid amplification step. Step RT-PCR. The storage and distribution of the buffer, the primer, the dNTP, and the reverse transcriptase by the reagent storage tank 58 and the use of the culture portion 114 for the reverse transcription step, followed by amplification in the ordinary portion of the amplification portion 112 may be simple. The two-step RT-PCR was completed. Constant temperature nucleic acid amplification For some applications, the preferred nucleic acid amplification method is constant temperature nucleic acid amplification'. Therefore, it is not necessary to repeatedly circulate the reaction components in various temperature cycles, but the amplification portion is maintained at normal temperature, usually about 37 ° C. To 41. (: Some thermostatic nucleic acid amplification methods have been described, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid-dependent sequence amplification (NASBA), recombinant enzyme polymerase amplification (RPA), and solutions. Spin-regulated DNA amplification (HDA), rolling-cycle amplification (RCA), branched-type amplification (RAM), and circular thermostat amplification (LAMP)', and any or other isothermal amplification methods of this type can be specifically used in this paper. In a specific example of the LOC device, in order to perform constant temperature nucleic acid amplification, the reagent storage tanks 60 and 62 adjacent to the amplification unit will carry an appropriate reagent for a specific constant temperature method instead of carrying the PCR amplification mix and the polymerase. For example, For SDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reagent reservoir 62 contains the appropriate -62-201209407 endonuclease and exo-DNA polymerase. For RPA, reagent reservoir 60 contains amplification buffer , primer, dNTP and recombinase protein, and reagent reservoir 62 contain a stock-substituted DNA polymerase, such as, for HDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reservoir 62 contains the appropriate DNA polymerase. Reconciliation The rotase (rather than the heat) is used to unravel the double-stranded DNA. Those skilled in the art will appreciate that the necessary reagents can be dispensed into two reagent reservoirs in any manner suitable for nucleic acid amplification. For self-RNA viruses, such as HIV Or amplification of viral nucleic acid of hepatitis C virus, NASBA or TMA is suitable because it does not need to first transcribe RNA into cDNA. In this example, reagent storage tank 60 is filled with amplification buffer, primer and dNTP, and The reagent storage tank 62 is filled with RNA polymerase, reverse transcriptase and any RNase Η. For some types of thermostatic nucleic acid amplification, an initial denaturation cycle must be used to separate the double before the temperature of the constant temperature nucleic acid amplification is maintained to facilitate the reaction. Strand DNA template. Since the temperature of the mixing in the amplification section 1 1 2 can be tightly controlled by the heater 154 in the amplification microchannel 158, this can be easily accomplished in all of the specific examples of the LOC apparatus described herein. Denaturation cycle (see Figure 14) ° Constant temperature nucleic acid amplification is more tolerant to potential inhibitors in the sample and is therefore generally suitable for direct nucleic acid amplification from a desired sample. Therefore, thermostatic nucleic acid amplification is especially useful. LOC variant XLIII 67 3, LOC variant XLIV 674 and LOC variant XLVII 677 shown in Figures 83, 84 and 85, respectively. Direct thermostatic amplification can also be as shown in Figures 83 and 85 - or multiple The preamplification dialysis step 7〇, 686 or 682 and/or the pre-hybridization dialysis step 682 shown in Fig. 84-63-201209407 is combined to facilitate the expansion of the nucleic acid to facilitate the portion of the target cell in the sample. The fractions are concentrated or the unwanted cell debris is removed before the sample enters the chamber array 110. Those skilled in the art will be able to use any combination of pre-amplification dialysis and pre-hybrid dialysis. Thermostatic nucleic acid amplification can also be performed in parallel amplifications, such as those described in Figures 79, 80 and 81. Multiplex and some thermostatic nucleic acid amplifications, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA. Other Design Variants A specific embodiment of a reagent sump LOC device with active (mechanical) valves uses a hot bend actuated valve (see Figure 64 69) instead of a surface tension valve (see Figures 15 and 22) to hold the reagents In the slot. Figure 71 shows the agent sump 344 in fluid communication with the target channel 74 via the three thermal bend actuated valves of the third variant 312 shown in Figures 68 and 69. The reagent reservoir 344 is filled with reagents that flow into the cover channel to a third variant of the thermal bend actuated valve 312, wherein the meniscus forms an aperture 306 of each CMOS actuated thermal bending actuator 304. The fluid sample flows through the extraction aperture 96 along the target channel 74. Before the sample stream reaches the draw 96, the third variant of the hot bend actuated valve 3 1 2 opens and the reagent flow MST channel 90 to the draw hole 96. When the sample stream reaches the extraction well 96, the reagent begins to fill the target channel 74. The merged flow continues along the target channel 74. Additional details of the fluorescence detection system are complicated by the enthalpy method to the storage test. 94 - 201209407 Figure 58 and 59 show the hybridization-reactive FRET probe 236. These are often referred to as molecular beacons and are stem-and-loop probes produced from single-stranded nucleic acids and fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization to the target nucleic acid sequence 238. The probe has a ring 240, a stem 242, a fluorophore 2M at the 5' end, and a quencher 24 8 at the 3' end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are joined together to form stem 242. In the absence of a complementary target sequence, as shown in Figure 58, the probe remains closed. The stems 242 maintain the fluorophore-quenching agents relatively close to each other such that a large amount of resonant energy can be transmitted between each other, while substantially eliminating the ability of the fluorophore to emit light rays when illuminated by the excitation light 244. Figure 59 shows the FRET probe 236. in an open or hybridized configuration. Upon hybridization to the complementary target nucleic acid sequence 238, the stem-and-loop structure is disrupted, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to fluoresce. The fluorescent emission 250 is optically detected as an indicator that the probe has hybridized. The probe hybridizes to the complementary target with very high specificity, since the stem helix of the probe is designed to be stable to the probe-target helix with a single non-complementary nucleotide. Because the double-stranded DNA is relatively robust, the stereoscopic probe-target helix and the stem helix cannot coexist. Primer-coupled probe

Primer-linked stem-and-loop probes and primer-linked linear probes, also known as scorpion-type probes, are alternatives to molecular beacons and can be used for real-time and quantitative nucleic acid amplification in LOC-65-201209407 devices. increase. Timely amplification can be performed directly in the hybridization chamber of the LOC device. The advantage of using a primer-ligated probe is that the probe element is physically linked to the primer, so that only a single hybridization is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an immediate and efficient response and produces a stronger signal, shorter reaction times, and better discrimination when using separate primers and probes. During manufacture, the probe (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 180 without the need for a separate amplification portion on the LOC device. Alternatively, the amplification portion is not used or used for other reactions. Primer-Linked Linear Probes Figures 86 and 87 show the configuration of the primer-ligated linear probe 692 during the first round of nucleic acid amplification and the hybridization during subsequent nucleic acid amplification, respectively. Referring to Figure 86, the primer-coupled probe 692 has a double stem section 242. One of the probe sequences 696, which binds to the primer, is homologous to the region on the target nucleic acid 696 and is labeled with its 5' end by fluorophore 246, and its 3' end is coupled via amplification blocker 694 to Oligonucleotide primer 700. The other 3' end of the stem 242 is labeled with a quencher portion 248. After completion of the first round of nucleic acid amplification, using the currently complementary sequence 698, the probe can wrap around and hybridize to the extended strand. During the first round of nucleic acid amplification, oligonucleotide primer 700 is attached to target DNA 23 8 (Fig. 86) and then extended to form a DNA strand containing both the probe sequence and the amplification product. The amplification blocker 6P4 prevents the reading of the polymerase through and copying the probe region 696. Upon subsequent denaturation, the hybridized extended oligonucleotide primer 700/template and the primer-linked strand-66-201209407 probe of the double stem 242 are separated' thus releasing the butyl 248. - but the temperature for the adhesion and extension steps is lowered 'the primer-linked linear probe primer-linked probe sequence 696 is curled and hybridized to the amplified complementary sequence 698 on the extended strand, and the detected fluorescent indicator Target DNA is present. The unextended primer-linked linear probe retains its double stem and the fluorescence remains quenched. This test method is particularly suitable for rapid detection systems' because it relies on a single molecular process. Primer-Linked Stem-and-Ring Probes Figures 88A through 88F show the operation of the primer-coupled stem-and-loop probes 704. Referring to Fig. 88A, the primer-ligated stem-and-loop probe 704 has a stem 240 that complements the double stranded DNA and a loop 240 of the combined probe sequence. The 5' end of one of the stem strands 708 is labeled with a fluorophore 246. Another 71 标 is labeled with the 3'-end quencher 248, and the other 710 carries both the amplification blocker 694 and the oligonucleotide primer 700. In the initial denaturing phase (see Figure 8B), the strands of the target nucleic acid 23 and the primer-coupled stem 242 separate the stem-and-loop probe 704. When the temperature is cooled for the adhesive phase (see Figure 88C), the oligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 is hybridized to the target nucleic acid sequence 23 8 . During extension (see Figure 88D), complement 706 of the target nucleic acid sequence 238 is synthesized to form a DNA strand containing both the probe sequence 704 and the amplified product. Amplification blocker 694 prevents the polymerase from reading through and copying the probe region 7〇4. After denaturation, the probe sequence of the loop-coupled stem-and-loop probe loop segment 240 (see Figure 88F) is adhered to the complementary sequence of the extended strand 766 when the probe is subsequently attached. This configuration makes the fluorophore 246 far removed from the quencher -67-201209407 248, resulting in a significant increase in fluorescence emission. Control Probes The hybridization chamber array 1 1 includes some hybridization chambers 180 with positive and negative control probes for analytical quality control. Figures 100 and 1B illustrate negative control probes without fluorophore 796, and Figures 1A and 103 depict positive control probes without quencher 798. The positive and negative control probes have a stem-and-loop structure as described above for the FRET probe. However, whether the probe hybridizes to an open configuration or remains closed, the fluorescent signal 25 will always be emitted from the positive control probe 798 and the negative control probe 796 will never emit the fluorescent signal 250. Referring to Figures 1A and 101, the negative control probe 796 does not have a fluorophore (and may or may not have a quencher 248). Thus, regardless of whether the target nucleic acid sequence 23 8 hybridizes to the probe (see Figure 101) or the probe maintains its stem-and-loop configuration (see Figure 100), the response to excitation light 244 can be ignored. Alternatively, the negative control probe 7 96 can be designed such that it remains quenched forever. For example, by synthesizing loop 240, a probe sequence will be obtained that will not hybridize to any of the nucleic acid sequences in the sample in question, and stem 242 of the probe molecule will rehybridize with itself, and the fluorophore and quencher Will remain close to each other and will not emit visible fluorescence. This negative control signal corresponds to a low order emission from the hybridization chamber 180, where the probe is not hybridized but the quencher does not quench all of the emission from the reporter. Conversely, a positive control probe 798 without quenching agent was constructed, as shown in Figures 102 and 103. Back to stress luminescence 244, regardless of whether positive control probe 7 98 hybridizes to target nucleic acid sequence 238, no material quenches the fluorescent emission 250 from fluoro-68-201209407 cluster 246. Figure 52 shows a possible distribution of positive and negative control probes (3 78 and 380, respectively) in hybridization chamber array 110. Control probes 378 and 380 are placed in hybridization chamber 180 and positioned across the line of hybridization chamber array 110. However, the configuration of the control probes within the array is arbitrary (like the configuration of the hybrid chamber array Π 0). The fluorophore design requires a fluorophore with a long fluorescence lifetime to allow the excitation light to have sufficient time to decay to a lower intensity than the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal. For the noise ratio. Moreover, a longer fluorescence lifetime represents a larger integrated fluorescence count. Fluorescence lifetime 246 (see Figure 59) has a fluorescence lifetime greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and in most cases greater than 4 nanoseconds. Metal-coordination complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal, chemical, and photochemical stability. Both are advantageous features related to the needs of fluorescent detection systems. The particularly complex metal-coordination complex based on the transition metal ion ruthenium (Ru(II)) is ginseng (2,2'-bipyridyl) ruthenium (II) ([Ru(bpy) 3] 2 + ), and its lifetime is about 1 μ§. This complex is commercially available from Biosearch Technologies under the trade name Pulsar 650. -69- 201209407 Table 1: Photophysical properties of Pulsar 65 0 (钌 chelate)

Parameter symbol 値 unit absorption wavelength ^abs 460 nm emission wavelength λβπι 650 nm absorption coefficient Ε 14800 M·1 cm·1 fluorescence lifetime Xf 1.0 μδ quantum yield Η 1 (deoxidized) N/A lanthanide metal-coordination The sub-complex, ruthenium chelate, has been successfully shown as a fluorescent reporter in the FRET probe system and has a long lifetime of Ι600μ5. Table 2: Photophysical properties of ruthenium chelate

Parameter symbol 値 unit absorption wavelength ^abs 330-350 nm emission wavelength λέπι 548 nm absorption coefficient Ε 13800 (Xabs, and ligand-dependent, up to 30000 (fll λβ=340ηηι) M.W1 fluorescence lifetime Tf 1600 (hybridization Probes) μδ Quantum Yield Η 1 (Coordination Dependent) N/A LOC Device 3 0 1 The fluorescence detection system used does not use filtering to remove unwanted background fluorescence. If quencher 2 4 8 There is no natural emission to increase the signal-to-noise ratio, so it has an advantage. Without natural emission, the quencher 24 does not contribute to background fluorescence. High quenching efficiency is also important, which makes hybridization happen before No camp light. Biosearch from Novato, California -70- 201209407

Technologies, Inc.'s black hole quencher (BHQ) does not have natural emission and high quenching efficiency, and is suitable for the system's quenching agent. The maximum absorption of BHQ-1 occurs at 534 nm and quenching range. It is 480-580 nm, making it a suitable quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 579 nm and the quenching range is 560-670 nm making it a suitable quencher for Pulsar 650. Iowa Black FQ and RQ from Integrated DNA Technologies, Coralville, Iowa, are suitable alternative quenchers with little or no background emission. The Iowa Black FQ has a quenching range of 420-620 nm with a maximum absorption enthalpy at 531 nm and is therefore a suitable quencher for Tb-chelating fluorophores. Iowa Black RQ has a maximum absorption enthalpy at 656 nm and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 650. In the specific examples described herein, the quencher 248 is a functional portion that is attached to the probe initially, but in other embodiments, the quencher can be a separate molecule that is free of solution. Excitation Sources In the specific example based on the fluorescence detection described herein, LEDs are selected to replace laser diodes, high power lamps, or laser excitation sources because of low power consumption, low cost, and small size. Referring to Figure 89, LEDs 26 are placed directly on hybrid array 110 on the exterior surface of LOC device 301. Opposite the hybridization cell array 110 is a photosensor 44 consisting of an array of photodiodes 1 84 from each chamber for detecting fluorescent signals (see Figures 5 3, -71 - 201209407 54 and 70). . Figures 90, 91 and 92 illustrate other specific examples for exposing the probe to excitation light. In the LOC device 30 shown in Fig. 90, the excitation light 244 generated by the excitation LED 26 is directed by the lens 2 54 over the hybrid array 810. The LED 26 is pulsed and the fluorescent emission is detected by the photo sensor 44. In the LOC device 30 shown in Fig. 91, the excitation light 2 44 generated by the excitation LED 26 is directed by the lens 254, the first aperture 712 and the second aperture 714 over the hybridization chamber array 110. The LED 2 6 is pulsed and the fluorescent emission is detected by the photo sensor 44. Similarly, the LOC device 30 shown in FIG. 92, the excitation light 2 44 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 over the hybridization chamber array 110. The LED 26 is pulsed again and the fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 is dependent on the choice of fluorescent dye. Philips LXK2-PR1 4-R00 is a suitable excitation source for the P u 1 s a r 6 5 0 dye. SET UVT0P 3 3 5 T039BL LED is a suitable excitation source for the ruthenium chelate label. Table 3: Philips LXK2-PR14_R00 LED Specifications

Parameter Symbol 値 Element Wavelength λεχ 460 nm Transmit frequency Vem 6,52(10)14 Hz Output power Pi 0.515(min)@ ΙΑ W Transmit mode Lambertian data sheet N/A -72- 201209407 Table 4 : SET UVT0P334T039BL LED Specifications

Parameter symbol 値cell wavelength λβ 340 nm emission frequency Ve 8.82(10)14 Hz power Pi 0.000240(min) (% 20mA W pulse forward current I 200 mA emission mode Lambertian N/A ultraviolet excitation pupil absorbs a small amount in the UV spectrum Light. Therefore, it is advantageous to use UV excitation light. A UV LED excitation source can be used, but the broad spectrum of LED 26 reduces the effect of this method. For this, a filtered UV LED can be used. Optionally, UV laser can be used. The excitation source is not practical for a particular test module market due to the relatively high cost of the laser. The LED driver LED driver 29 drives the LED 26 at a fixed current for the desired duration. The low power USB 2.0 certified device can A maximum operating voltage of 4.4 volts is obtained at a maximum of 1 unit load (10 mA). A standard power conditioning circuit is used for this purpose. Photodiode Figure 54 shows photodiode 184, which is integrated into the CMOS circuit of LOC device 301. 86. The photodiode 184 is part of the CMOS circuit 86 without additional masking or steps. This is a significant advantage of the CMOS photodiode over the -73-201209407 CCD, CCD Another sensing technique that can be integrated onto the same wafer or fabricated on an adjacent wafer using non-standard processing steps. On-wafer inspection is inexpensive and reduces the size of the array system. Shorter optical path lengths are reduced from the surrounding environment. The noise is used to efficiently collect the fluorescent signal' and reduce the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the fraction of photons colliding with its active region 185, and the photon is efficiently converted into photoelectrons. In standard 矽 processing, the quantum efficiency of visible light is in the range of 0.3 to 0.5 depending on the processing parameters (such as the number of cladding layers and absorption characteristics). The detection threshold of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and the number of hybrid chambers 180 in the hybridization and detection portion 52 (see Figure 52). The size and number of chambers are technical parameters and are determined by the size of the LOC device. (In the example of LOC device 301, its size is 1*760 microns X 58 24 microns), and it is subject to other functional modules (such as diseases) The body dialysis unit 70 and the amplification unit 1 12) are limited by the size of the non-animal parts that can be used. For the standard 矽 treatment, the photodiode 184 detects a minimum of 5 photons. However, in order to confirm the reliable detection, the minimum 値 can be set to 1 光 a photon. Therefore the quantum efficiency range is 〇·3 to 〇.5 (as discussed above), the fluorescence emission from the probe is a minimum of 17 photons' and the 30 photons contain a suitable margin for the error of reliable detection. Calibration Room Photodiode 184 Inhomogeneous Electrical Characteristics, Automated Egg Light, and Unfinished -74- 201209407 The residual attenuated photon flux of full attenuation introduces and shifts background noise to the output signal. The background is removed from each output signal using one or more calibration signals. A calibration signal is generated by exposing one or more of the calibration photodiodes 1S4 in the array to respective calibration sources. A low calibration source is used to determine the negative result of the target 尙 non-probe reaction. A high calibration source represents a positive result from the probe-target complex. In the specific example described herein, the low calibration source is provided by a calibration chamber 382 in the array of cells 110, which: does not contain any probes; includes probes that do not have a fluorescent reporter; or contains reports The probe is configured and configured to expect quenching quenching agents to occur forever. The output signal from such a calibration chamber 382 is very close to the noise and bias in the output signal from all of the hybrid chambers in the LOC. The output signal from the other chambers is subtracted from the calibration signal, substantially removing the back and leaving the signal generated by the fluorescent emission (if any signal is generated) from the ambient light in the area of the chamber array. Was removed. It will be appreciated that the negative control group probe described above with reference to Figures 1 00 through 031 is used in the calibration chamber. However, as shown in Figures 94 and 95, which are enlarged views of the inset DG and DH of the LOC variant X 728 shown in the figure, the other is selected to fluidly isolate the calibration chamber 382 from the amplicons. When hybridization is prevented by fluid barriers, background noise and bias can be judged by a chamber that is fluidly isolated or by a probe containing a lack of reporter or any "standard" probe that does have both a reporter and a quencher. The calibration chamber 3 82 provides a high calibration source to produce a high signal at the corresponding output of the intersection of the white and the illuminator. 93 - 201209407 Diode. The high signal corresponds to all probes in the chamber that has been hybridized. Reporting with no quenching agent or only with a reporter spotting probe will consistently provide a signal that a large number of probes in the hybridization chamber have hybridized within the hybridization chamber. It is also understood that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 for the entire array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by a relatively close calibration chamber 382, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has one calibration chamber 382 for every eight hybridization chambers «180. That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybrid chambers 180. In this configuration, the hybridization chamber 180 is calibrated by the immediately adjacent calibration chamber 382. Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, Figure 99 shows a differential imager circuit 878 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. The differential imager circuit 7 8 8 samples the signal from the pixel 790 and the "virtual" pixel 792. In one embodiment, the "virtual" pixel 792 is shielded from light illumination, so its output signal provides a dark reference. Alternatively, the "virtual" pixel 792 can be exposed to the laser with the remainder of the array. In the specific example where the "virtual" pixel 792 is light accommodating, the signal produced by ambient light in the region of the self-chamber array is also subtracted. The signal from pixel 709 is weak (for example, close to a dark signal), and it is difficult to distinguish between background 非常 and very weak signals because there is no reference to dark signal levels. During use, the "read_column" 794 and "read-column_d" 795 are enabled and the M4 797 and MD4 80 1 transistors are turned on. Turning off switches 807 and 809 causes the outputs from pixel 790 and "virtual" pixel 792 to be stored on pixel capacitor 803 and virtual pixel capacitor 805, respectively. After the pixel -76-201209407 signal is stored, switches 807 and 809 are disabled. The "read_row" switch 81 1 and the virtual "read_row" switch 813 are then turned off, and the outputted switched capacitor amplifier 815 amplifies the differential signal 817. The suppression and enabling of the photodiode must be such that the photodiode 184 must be inhibited during excitation of the LED 26 and the photodiode 184 must be enabled during the fluorescence period. Figure 73 is a circuit diagram of a single photodiode 1 84 and Figure 74 is a timing diagram of an optical diode control signal. The circuit has a photodiode 184 and six MOS transistors, Mshunt 394, Mtx 396, Mreset 3 98, Msf 40 0, Mread 402 and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 394, and Mreset 398 are turned on by pulling the Mshunt gate 384 and resetting the gate 3 8 8 high. During this period, the photons are excited to generate carriers in the photodiode 184. When the amount of carriers generated is sufficient to saturate the photodiode 184, the carriers must be removed. During this cycle, Mshunt 394 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or due to excitation-generated carrier diffusion in the substrate, while Mreset 3 98 resets to accumulate at the node' Any carrier of NS' 406. After excitation, the capture cycle begins at t4. In this loop, the response from the emission of the fluorophore is captured and integrated into the circuitry on node WS' 406. This is achieved by dragging the tx gate 386 high, which turns on the transistor Mtx 396 and any accumulated carrier on the transfer photodiode 184 to the node WS' 406. The capture cycle can be as long as a fluorescent emission. The output from all of the photodiodes 184 in the array of hybrid cells 110 is simultaneously captured. There is a delay between the end capture cycle t5 and the start of the read cycle t6 -77- 201209407. This delay is due to the need to read the respective photodiodes 184 in the hybridization cell array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle and the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Mread 402 is turned on by dragging the gate 393 high. The source-slaffer transistor Msf 400 is used to buffer and read the voltage of the 'NS' node 406. Any other method of enabling or suppressing a photodiode is discussed below: 1. Inhibition method

Figures 96, 97 and 98 show possible configurations 778, 780, 782 for Mshunt transistor 394. Mshunt transistor 394 has a very high turn-off ratio when the maximum 値|FC5|=5 V is enabled during excitation. As shown in Figure 96, the Mshunt Gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in Figure 97, the Mshunt Gate 3 84 can be configured to surround the photodiode 184. A third option is to place the Mshunt gate 3 84 within the photodiode 184, as shown in FIG. According to the third option, the photodiode active region 185 is less. These three configurations 778, 780, and 782 reduce the average path length from all locations in the photodiode 184 to the Mshunt gate 3 84. In Fig. 96, the Mshunt gate 3 84 is attached to one side of the photodiode 184. This is the simplest configuration to make and the smallest impact on the active region of the photodiode 1 85. However, any carrier remaining at the distal end of the photodiode 184 requires a longer time to diffuse through the Mshunt gate 384. -78- 201209407 In Figure 97, the Mshunt gate 384 surrounds the photodiode 184. This further reduces the average path length of the carriers in the photodiode 184 to the Mshunt gate 3 84. However, extending the Mshunt gate 384 around the photodiode 184 causes the photodiode active region 185 to be substantially reduced. The configuration 7 82 in Figure 98 positions the Mshunt gate 384 in the active region 185. This provides the shortest average path to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active zone 185 is greatest. It also creates a wide leak path. 2. Enabling method a. The flip-flop photodiode drives the parallel transistor with a fixed delay. b. The flip-flop photodiode drives the shunt transistor with a programmable delay. c. The parallel drive transistor is driven by the LED drive pulse with a fixed delay. d. Drive the shunt transistor as a 2 c but with a programmable delay. Fig. 76 is a schematic view showing the photodiode 184 and the flip-flop photodiode 187 buried in the CMOS circuit 86 through the hybridization chamber 180. The small area in the corner of the photodiode 184 is replaced by the trigger photodiode 187. Since the intensity of the excitation light is higher than that of the fluorescent emission, the photodiode 187 having a small area is sufficient. The flip-flop photodiode 187 is sensitive to excitation light 244. The flip-flop photodiode 187 shows that the excitation light 244 has extinguished and activates the photodiode 184 (see Fig. 2) after a brief delay of At 300. This delay allows the fluorescent photodiode 1 84 to detect the fluorescent emission from the FRET probe 186 without the excitation light 244. This enables detection and enhancement of the signal to noise ratio. -79- 201209407 In each hybrid cell 180, both photodiode 184 and flip-flop photodiode 187 are located in CMOS circuit 86. The photodiode array is combined with an appropriate electronic component to form a photosensor 4 4 (see Figure 70). Photodiode] A pn junction made during the fabrication of a CMOS structure without the need for additional masks or steps. During MST fabrication, the dielectric layer (not shown) over the photodiode 184 is arbitrarily thinned by standard MST photolithography techniques to illuminate the active region 185 of the photodiode 184 with more phosphor. The photodiode has a field of view such that an optical signal from the probe-target hybridization within the hybridization chamber 180 is incident on the surface of the sensor. The converted fluorescence becomes the photocurrent that can then be measured by the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to the trigger diode 187. These choices can be used in combination with 2a and 2b above. Fluorescence Delay Detection The following derivation is based on the above-mentioned LED/fluorescent combination using the fluorescence luminescence detection of the lifetime fluorophore. Fluorescence intensity is a function of time after an ideal pulse excitation of the fixed intensity [e] between time and ί2 shown in Figure 60. Let [Sl] (when the time t is equal to the intensity of the excited state, and then between and after the excitation, the number of excited states per unit volume per unit time is described by the following equation = d[S\] { [^1] (〇 _ Iesc dt tf ~ hve body 84 when the electricity is used to make 84 the length of the fire and the light of the micro-80--(1) 201209407 where C is the molar concentration of the fluorophore, ε is the molar quenching coefficient, Ve To excite the frequency, and h = 6.62606896(10)·34 Js is the Planck constant. This differential equation has the general formula: It has a solution: y(x) = J e^p(x)dx q{^x)dx + k ^p{x)dx ...(2) Now use this to solve equation (1), [S\m = I^TL + ke-'^ ...(3) hve then at time h, [ S1](7))= 〇, and from (3): k = J^LLe, 'lTf ...(4) hve Substituting (4) into (3): [sm=

IeSCTf IeSCTf 广!',丨" hve hve at time ί2,: -81 - 201209407 [增2) 8gCT/ (5) hve hve at / 2 G, the excited state is exponentially decayed and described by equation (6) (5 Substituting (6): -(7) l.SCX r [just ="^[1- hve The fluorescence intensity is obtained by the following equation: j {t) = _d[srmhv t (8) αχ where V/ is The fluorescence frequency, η is the quantum yield, and 1 is the optical path length from (7): <^[51](^) _ Iesc _,^-(t-i2)/Tf ( 9 ) ""dt~ ~h^~ Substituting (9) into (8): -82- 201209407 Because 4 〇〇, I εαΙηΥΑίιντ' τί ve Therefore 'we can write the following approximation, which is described in full-length excitation Fluorescence intensity decay after pulse (Q-hATf): For /^, (0 = 7e£c/^^-e-(,-,j)/r/ ...(1 1)

K In the previous section, we summarize the situation, and for tt ti If{f) = Iεεα1η丄e%1,1 ”. From the above equation, we can derive the following expression: nf{t) = nesc ^e~{t'hVTf (12) where h•(7) is the number of fluorescent photons per unit area per unit time and hvf 屺 is the number of excitation photons per unit area per unit time ° kVe Therefore, 〇0 iif{t ) = \nf{t)dt ...(13) -83- 201209407 Seven of the diodes are turned on as the number of fluorescent photons per unit area and ^ is the light time point. Substituting (12) into (13): 〇〇af = jyiesclne-"-h, lr/dt ... ( 1 4) ti The fluorescent light of the polar body currently reaches the number of sub-numbers of light per unit area per unit time, Η·, (〇, by the following formula Obtain: ns(t) = nfm -(15) where is the light collection efficiency of the optical system. Substituting (12) into (15) we find that ηί{ί) = φϋηίεαΙηβ'(,~,ι)ΙΤ/ ... (16) Fluorescent photons of the body Similarly, the unit of light per unit of fluorescence area 乂 reaches the number of light poles as follows: 〇〇圮=ί\(〇Α and substituting (16) and integrating: ns =φ^ηΐεοΙητ fe(h ~hVlf Therefore, η,^ΦΧ εοΙητ〆611" (17) -84 - 201209407 ί3 is ideal when the electron rate generated in the photodiode 184 due to the fluorescent photon is equal to the electron rate generated by the excitation photon in the photodiode 184 Because the excitation photon flux attenuation is much faster than the fluorescence photon flux decay. The output electron rate of the sensor per unit of fluorescence area of the fluorescence is: )) = (4)) where 0 / is at the wavelength of the fluorescence Quantum efficiency of the sensor. Substituting (17) we get: έ·ί{ί) = φίφΰηΐεοΙηβ-{ί-,ι),^ (1 8) Similarly, due to the per-unit fluorescence area of the excited photons The output electron ratio is: «咖·(19) where is the quantum efficiency of the sensor at the excitation wavelength and is the time constant relative to the "off" characteristic of the excited LED. After time t2, The attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends its decay time, but we assume that this is a negligible effect on If(t), so we take a conservative approach. -85 - 201209407 Currently, As mentioned earlier, the ideal g of g is: 4(6)=3⁄4(6) From (18) and (19) we get: and after reforming we get: ...(20) \η{εαΙη^φ-) τ/ From the above two paragraphs, we get the following two expressions (kKFi - Δί/ι At =

Ίί Te ...(2 1 ) -(22) where corpse = «τ/;; and Δί = ί3 - ί2 ‘We also know that, in fact, q » Γ/. Ideal time for fluorescence detection and use Ρ h i 1 i ρ s L X Κ 2 - Ρ R 1 4 -R00 The number of fluorescent photons detected by the LED and Pulsar 650 dye is determined as follows. -86- 201209407 The ideal detection time is determined by equation (22): Recall the concentration of the amplicon, and assuming that all the amplicons are hybridized, the fluorescence concentration of the fluorescing is: c = 2.89(10)_6mol/L . The height of the chamber is the optical path length l = 8 (l 〇) _ 6 m. The fluorescent region has been regarded as equivalent to the photodiode region, but the actual fluorescent region is substantially larger than the photodiode region; therefore, it can be assumed that nine = 0.5 is the optical collection efficiency of the optical system. The characteristic of the photodiode, | = 10 < Pe is the extremely conservative ratio of the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength. With a typical LED decay lifetime re = 0.5 nanoseconds and using the Pulsar650 specification, it is possible to determine Δί : F = [1.48(10)6 ][2.89(10广][8(10)"6 ](1) =3.42( 10)5 ln([3.42(10)-5](10)(0.5)) 1 1 1(10)-6 ~ 0.5(10)-9 -4.34(10)-9 s Number of detected photons It is determined using equation (2 1 ). First, the number of excitation photons emitted per unit time is largely determined by examining the illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian emission mode, so: -87- ... (23) 201209407 where & is the photon emitted per unit time per unit solid angle from the direction of the forward axis of the LED The number, and & is ^, in the direction of the forward axis. The total number of photons emitted by the LED per unit time is:

ή[ = j h'/dQ Ω (24)

=I nIQ cos(0)dQ Ω Now, △Ω = 2;r[l - cos(0 + Δ0)] - 2;r[l - cos(0)] ΔΩ = 2n[cos{0) ~ cos( ^ + Δ0)] (Αθλ . —sin l 2 J l 2 J H-4^cos(^)sin2 =4;rsin(0)cos dQ = 2π3ΐη(θ)άθ Substitute (24 wide 2ml0 cos(0)sin (0)d9

Rearranged, we get: ...(26) -88- ...«/ n!0: — 201209407 The output power of the LED is 0.515 watts and ve = 6.52 (10) 14 Hz.

Pi Ave ... (27) _0.515_ [6.63(10)-34][6.52(10)14] = 1.19(10)18 Photons/sec Bring this 値 into (26) We get: ... 1.19 (10)18 ”/〇=- π = 3.79(10)17 photons/sec/sphericity Referring to Fig. 61, the optical center 2 52 and the lens 254 of the LED 26 are shown schematically. The photodiode is 16 micrometers χ 16 micrometers. And for the photodiode in the middle of the array, the solid angle (Ω) of the light cone emitted from the LED 26 to the photodiode 184 is approximately: Ω = sensor area / r2 [16 (10 wide) [16 (10) -6] 2.825(10)-3 f =3.21(1〇)·5 Sphericality It will be understood that the central photodiode 184 of the photodiode array 44 is used for these calculations. The sensors at the edge of the array are hybridized. Event only -89 - 201209407 Receive low 2% photons for Lambertian excitation source intensity distribution. Number of excitation photons emitted per unit time: he = «;Ω ...(2 8) =[3.79(10)17] [3.21(10)-5] =1 .22(10)13 Photon/sec Now refer to equation (29): / · -t—t —Δί I τ f ns =(0.5)[1.22(10)13] [3.42(10)-5][l(10)-6]e'434(1〇r9/1(1°)'6 = 208 Photon/Sensor So, use Philips LXK2-PR14-R00 LED and With the Pulsar 650 fluorophore, we can easily detect any hybridization events that cause these numbers of photons to be excited. The SET LED illumination geometry is shown in Figure 62. The LED has a minimum optical power output when ID = 20 mA. 240 microwatts with a wavelength center at λβ = 340 nm (absorption wavelength of ruthenium chelate). Drive the LED at Id = 200 mA, linearly increasing the output power to p1 = 2.4 mW. By placing the optical center of the LED 252 Placed at a distance of 17.5 mm from the array of hybridization chambers 11 'We concentrated the output flux on a dot size with a maximum diameter of 2 mm. The photon flux in the 2 mm diameter point of the hybrid array plane is determined by the equation - 90- 201209407 2 7Get. «/=

Pi hVe 2.4(10)-3 [6.63(10)-34][8.82(10)14] = 4.10(10)15 photons/second using equation 2 8, we get he = η, Ω 4.10(10)15 [16(10)-6]2 4i(i〇)·3]2 : 3.34(10)11 Photons/sec Now, return to Equation 22 and use the previously listed Tb chelate properties, ln[(6.94( 10)-5)(10)(0.5)] _1___1_ 1(10)-3 ~ 0.5(10)-9 = 3.98(10)-9 seconds Now from the equation 21: ns = (0.5)[3.34(10) 1,][6.94(10)-5][1(10)-3>-3'98(,0>', /1(>〇)'3 = 11,600 photon/sensor. By hybridization The event uses the SET LED and the ytterbium chelate system to emit light -91 - 201209407 The sub-theoretical number system can be easily detected and far exceeds the lower limit of 30 photons, which is used for the light sensation indicated by the above The reliable detection required by the detector. The on-wafer detection of the maximum spacing between the probe and the photodiode avoids the need for detection by a conjugate focal microscope (see prior art). This departure from conventional detection techniques is system dependent. Time and cost savings are important factors. Traditional inspection requires the use of lens and curved mirror imaging optics. By using non-imaging optics, diagnosis The break system avoids the need for complex and cumbersome strings of optical components. The placement of the photodiode in close proximity to the probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode is 1 micron, the emission The light collection angle is as high as 173. This angle is calculated by considering the light emitted from the probe closest to the center of the surface of the hybrid chamber of the photodiode, which has a planar active surface area parallel to the chamber surface. The cone of emission in which light can be absorbed by the photodiode is defined as having a transmitting probe at its apex and at the sensor corners around its plane. For a 16 micron χ 16 micron sensor, this cone The apex angle is 1 70°; in the case where the photodiode is expanded such that its area conforms to the 29 micron χ19·75 micron hybrid chamber area, the apex angle is 1 to 3°. On the chamber surface and the photodiode It is easy to achieve a separation between the active surfaces of 丨 micron or less. Applying a non-imaging optical method requires the photodiode 184 to be very close to the hybridization chamber to collect sufficient photons of the fluorescent emission. Photodiodes and probes Maximum interval-based decision reference to the following FIG. 54 92--. 201209407

Using the ruthenium chelate fluorophore and the SET UVTOP3 3 5T039BL LED, we calculated 16,000 photons from individual hybridization chambers 180 to 16 micron x 16 micron light diodes 184. In carrying out this calculation, we assume that the light collection region of the hybridization chamber 180 has the same bottom area as the photodiode active region 185, and that half of the total number of hybrid photons reaches the photodiode 184. That is, the light collection efficiency of the optical system is nine = 0.5. More precisely, we can write nine = [(the bottom area of the light collection area of the hybridization chamber) / (photodiode area)] [Ω / 4π], where Ω = solid angle is opposite to the substrate of the hybridization chamber The light diode of the representative point. For the right square pyramid geometry: 0 = 4^^402/(4(1()2 + 32)), where d0 = the distance between the chamber and the photodiode, and α is the photodiode size . Each hybrid cell releases 23,200 photons, and the selected photodiode has a lower detection limit of 17 photons. Therefore, the minimum optical efficiency required is: ^0=1 7/23200 = 7.33 χ1〇·4 Hybridization chamber The bottom area of the light collection area of 180 is 29 microns χ 19.75 microns. Solving do, the maximum limit distance between the hybridization chamber and the photodiode 184 is dG = 249 microns. In this limitation, the collection cone angle as defined above is only 0.8°. It should be noted that this analysis ignores the negligible effect of refraction. -93- 201209407

LOC Variant V The LOC variant V 362 shown in Figure 72 was extracted 290, cultured 291, expanded 292, and tested 294 (using the leukocyte dialysis unit 3 28) human DNA. The reagent reservoirs 54, 56, 58, 60 and 62 use the third embodiment of the thermal bending actuated valve 312 at their outlets. Conclusion The devices, systems, and methods described herein facilitate molecular diagnostic testing at low cost and high speed and in situ care. The above-described system and its components are merely illustrative, and many variations and modifications will be readily apparent to those skilled in the art without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will be described with reference to the accompanying drawings, in which: FIG. 1 shows a test module configured for fluorescence detection and a test module read. Figure 2 is a schematic diagram of the electronic components in the test module configured for fluorescence detection; Figure 3 is a schematic view of the electronic components in the test module reader; Figure 4 is a representation Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device 94-201209407 with all layer structures and features superimposed on each other; Figure 7 is an independent display Plan view of the LOC device of the cover structure» Fig. 8 is a perspective view of the top surface of the inner passage and the sump shown by the broken line. Fig. 9 is a perspective view of the exploded top surface of the inner passage and the sump shown by the broken line. Fig. 10 is a view A bottom perspective view of the cover of the upper channel configuration; Figure 11 is a plan view of the LOC device showing the structure of the CMOS + MST device; Figure 12 is a schematic view of the sample inlet of the LOC device; Figure 13 is a view of the sample shown in Figure 6. An enlarged view of the illustration AA; Figure 14 is a representation of Figure 6. Figure 15 is an enlarged view of the illustration AE shown in Figure 13; Figure 6 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE; Figure 17 is an illustration of the illustration AE Partial perspective view of the laminated structure of the LOC device; Figure 18 is a partial perspective view illustrating the laminated structure of the L0C device in the illustration AE; Figure 19 is a portion illustrating the laminated structure of the L0C device in the illustration AE Figure 20 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE; -95-201209407 Figure 21 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AE; Figure 23 is a schematic view of the lysing reagent sump shown in Figure 21; Figure 23 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AB; Figure 24 is a elaboration of the lamination of the LOC device in the illustration AB Partial perspective view of the structure; Figure 25 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AI; Figure 26 is a partial perspective view illustrating the laminated structure of the LOC device in the illustration AB: Figure 27 is Explain a partial perspective view of the laminated structure of the LOC device in the illustration AB; Figure 28 illustrates the insertion Partial perspective view of the laminated structure of the LOC device in AB: Figure 29 is a partial perspective view of the laminated structure of the LOC device in the illustration AB; Figure 30 is a schematic diagram of the amplified mixing tank and the polymerase storage tank Figure 31 shows the characteristics of the independent boiling pilot valve; Figure 32 is a schematic view of the boiling pilot valve taken along line 3 3 - 3 3 shown in Figure 31; Figure 33 is the same as shown in Figure 15. Figure 34 is a schematic view of the upstream end of the dialysis section taken along line 3 5 - 3 5 shown in Figure 33; 96 - 201209407 Figure 35 is an enlarged view of the illustration AC shown in Figure 6. Figure 36 is a further enlarged view showing the amplification portion in the illustration AC; Figure 37 is a further enlarged view showing the amplification portion in the illustration AC; Figure 38 is a further enlarged view showing the amplification portion in the illustration AC; Further enlarged view of the illustration AK shown in Fig. 38; Fig. 40 is a further enlarged view showing the amplification portion in the illustration AC; Fig. 41 is a further enlarged view showing the amplification portion in the illustration AC; A further enlarged view of the amplification section is shown; FIG. 43 is a further enlargement of the illustration AL shown in FIG. Figure 44 is a further enlarged view showing the amplification portion in the illustration AC; Figure 45 is a further enlarged view of the illustration AM shown in Figure 44; Figure 46 is a further enlarged view showing the amplification portion in the illustration AC; 47 is a further enlarged view of the illustration AN shown in FIG. 46; FIG. 48 is a further enlarged view showing the amplification section in the illustration AC; FIG. 49 is a further enlarged view showing the amplification section in the illustration AC; Further enlarged views of the amplification section are shown in AC; Fig. 51 is a schematic view of the amplification section; Fig. 52 is an enlarged plan view of the hybridization section; Fig. 53 is a further enlarged view of two independent hybridization chambers; Figure 5 is an enlarged view of the humidifier illustrated in the inset AG shown in Figure 6; Figure 56 is an enlarged view of the inset AD shown in Figure 52; Explosive perspective view of the LOC device; -97- 201209407 Figure 58 is a diagram of the FRET probe in a closed configuration; Figure 59 is a diagram of the FRET probe in an open and hybrid configuration; Figure 60 is the excitation light versus time Figure 61; Excitation illumination geo of the parent-parent array Figure 62 is a diagram of the LED illumination geometry of the sensor electronics; Figure 63 is an enlarged plan view of the humidity sensor shown in the inset AH of Figure 6: Figure 64 shows the thermal bending actuation valve Figure 65 is a schematic illustration of a first variation of the thermal bending actuated valve taken along line 70-70 of Figure 64; Figure 66 shows the features of a second variant of the thermal bending actuated valve; A schematic view of a second variant of the thermally bent actuated valve taken along line 72-72 of Figure 66; Figure 68 shows the features of a third variant of the hot bend actuated valve; Figure 69 is taken along line 74-74 of Figure 68. Schematic diagram of a third variant of the thermal bending actuated valve; Fig. 70 shows a partial photodiode array of a portion of the photosensor shown in Fig. 71. Fig. 71 shows a reagent reservoir with a hot bending actuated valve. Figure 72 is a graphical representation of the structure of the fifth LOC variant; Figure 73 is a circuit diagram of a single photodiode; Figure 7 is a time diagram of the photodiode control signal; Figure 75 is the evaporator shown in Figure 55 Magnified view of AP: •98- 201209407 Figure 76 is a schematic diagram of hybridization chamber and detection photodiode and flip-flop photodiode; Figure 7 is a schematic representation of a test module with a lancet; Figure 79 is a graphical representation of the structure of the LOC variant VII; Figure 80 is a graphical representation of the structure of the LOC variant VIII Figure 81 is a graphical representation of the structure of the LOC variant XIV; Figure 82 is a graphical representation of the structure of the LOC variant XLI; Figure 83 is a graphical representation of the structure of the LOC variant XLIII; Figure 84 is the structure of the LOC variant XLIV Graphical representation; Figure 85 is a graphical representation of the structure of the LOC variant XLVII; Figure 86 is a diagram of the primer-linked linear fluorescent probe during initial amplification, and Figure 87 is the linearity of the primer-join during the subsequent amplification cycle. Figure of the fluorescent probe; Figures 88A to 88F graphically illustrate the thermal cycling of the primer-linked fluorescent stem_and _ring probe; Figure 89 is related to the hybrid chamber array and the light diode excitation LED Figure 90 is a schematic illustration of an excitation LED and an optical lens for directing light onto a hybrid chamber array of a LOC device: Figure 91 is an excitation LED, optical lens for directing light onto a hybrid chamber array of a LOC device And the light is to be explained; Figure 92 is used to light guide A schematic diagram of LEDs, optical lenses, and mirror configurations on the array of hybrid chambers of the LOC device; Figure 93 is a plan view showing all features superimposed on each other and showing the positions of the insets DA to DK; An enlarged view of the inset DG shown in Fig. 93; Fig. 95 is an enlarged view of the inset DH shown in Fig. 93; Fig. 96 shows an embodiment of a parallel transistor for the photodiode; Fig. 97 shows an example for the photodiode Embodiment of a parallel parallel transistor; FIG. 98 shows an embodiment of a parallel transistor for a photodiode; FIG. 99 is a circuit diagram of the differential imager; FIG. 1 schematically depicts a stem-and-ring structure Negative control fluorescent probe

I Figure 101 schematically depicts the negative control fluorescent probe of Figure 100 in an open configuration; Figure 102 schematically depicts a positive control fluorescent probe in the stem-and-loop configuration. Figure 103 is a brief description of the open The positive control fluorescent probe of Figure 102 of the structure; Figure 1 04 shows the test module and test module reader configured for use with EC L detection: Figure 105 shows the test module used with ECL detection The schematic diagram of the electronic components; Figure 1 06 shows the test module and the alternative test module reader; and Figure 1 07 shows the test module and the alternative test module reader and the host system for storing various databases. -100- 201209407 [Explanation of main component symbols] I 〇: Test module II: Test module 1 2 : Test module reader 1 3 : Case 14 : Micro-USB connector 15 : Sensor 1 6 : Micro-USB埠17: Touch screen 18: Display screen 19: Button 20: Start button 2 1 : Honeycomb radio 22: Aseptic sealing tape 23: Wireless network connection 24: Large container 2 5: Satellite navigation system 26: Light-emitting diode 27 : Data storage 2 8 : Telephone 29 : LED driver 30 : LOC device 3 1 : Power conditioner - 101 201209407 32 : Capacitor 33 : Clock 34 : Controller 35 : Register 36 : USB device driver 3 7 : Driver 3 8 : Random Access Memory 39 : Driver 40 : Flash Memory 41 : Register 42 : Processor 43 : Program Memory 44 : Light Sensor 45 : Indicator 46 : Cover 47 : Module 48 : CMOS + MST device 49: porous element 52: detection portion 54: sump 56, 56.1, 56.2, 56.3: sump 5 7: printed circuit board 58, 58.1, 58.2: sump 60, 60.1-60.12, 60.X: sump - 102 201209407 62,: 62.1, 62.2, 62.3, 62.4, 62.X: sump 6 4 : lower seal 66: top layer 6 8 : Present inlet 70: dialysis section 72: waste channel 74: target channel 76: waste reservoir 7 8: sump layer 80: cover channel layer 8 2: upper sealing layer 84: 矽 substrate 86: CMOS circuit 87: MST layer 8 8 : Passivation layer 90 : MST channel 92 : Lower pipe 94 : Cover channel 96 : Upper pipe 97 : Wall 98 : Meniscus holder 1 00 : MST channel layer 101 : Laptop / Notebook 102 : Capillary action Starting feature -103 - 201209407 103 : Dedicated reader 105 : Desktop computer 106 : Boiling pilot valve 107 : E-book reader 108 : Boiling pilot valve 109 : Tablet computer 110 , 110.1-110.12, 110.X: Hybrid room Array 1 1 1 : Epidemiological data 112, 112.1-112.12, 112.X: Amplification section 1 1 3 : Genetic data 114.1-114.4: Culture section 1 1 5: Electronic health record 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 1 9 : Liquid sample 1 2 0 : Meniscus 1 2 1 : Electronic medical record 1 2 2 : Through hole 1 2 3 : Personal health record 125 : Network 126 : Boiling pilot valve 128, 128.2, 128.3: Surface tension valve 130, 130.1-130.3: Lysis part 1 3 1 : Mixing section -104- 201209407 132, 132.1, 132.3: Surface tension valve 1 3 3 : incubator inlet channel 134: lower tube 136: optical window 138, 138.1, 138.2, 138.X: surface tension valve 140, 140.1, 140.2, 140.X: surface tension valve 146: valve inlet 1 48: valve outlet 150: valve under the pipe 1 5 1 : valve upper pipe 152: ring heater 1 5 3 : valve heater contact 1 5 4 : heater 1 5 6 : heater contact 158 : micro channel 160 : outlet channel 162 : Cantilever portion 1 64 : orifice 166 : capillary action initiation feature 1 6 8 : dialysis extraction aperture 170 : temperature sensor 174 : liquid sensor 175 : diffusion barrier 176 : flow path -105 - 201209407 1 7 8 : liquid Sensor 180: hybridization chamber 1 8 2 : heater 1 84 : photodiode 1 85 : active region 1 86 : probe 187 : photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : annular heating 192: water supply channel 193: upper pipe 194: lower pipe 1 9 5: top metal layer 196: humidifier 1 9 8 : extraction hole 202: capillary action starting feature 204: MST channel 206: boiling pilot valve 207: Boiling start valve 208: liquid sensor 210: microchannel 2 1 2 : MST channel 2 1 8 : electrode -106 - 201209407 220 : electrode 222 : gap 23 2 : humidity sensing 2 3 4 : Heater 236 : FRET probe 23 8 : Target nucleic acid sequence 240 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 2 4 8 : Quencher 25 0 : Fluorescence signal 2 5 2 : Optical Center 254: Lens 28 8 : Sample Input and Preparation 290: Extraction Stage 291: Culture Stage 292: Amplification Stage 293: Pre-Hybridization Filtration Purification Stage 294: Detection Phase 296: First Electrode 298: Second Electrode 3 0 0 : Delay 301 : LOC device - 107 201209407 302 : Variant 3 04 : Actuator 306 : Port 3 08 : Valve 3 1 2 : Valve 328 : Leukocyte dialysis unit 344 : Slot 3 62 : LOC Variant 3 76 : Heat conduction Column 3 78 : Positive control probe 3 80 0 : Negative control probe 3 82 : Calibration chamber 384 : Gate 3 8 6 : Gate 3 8 8 : Gate 3 9 0 : Retractable lancet 3 92 : Sting Blood needle release button 3 9 3 : Wenji 394: MOS transistor 396: MOS transistor 398: MOS transistor 400: MOS transistor 402: MOS transistor 404: MOS transistor 201209407 4 0 6 : Node 408: film Seal 4 1 0 : Membrane guard 492 : LOC variant 5 1 8 : L Ο C Variant 594 : Interfacial layer 600 : Bypass channel 602 : Interface target channel 604 : Scrap channel 6 3 8: hot lysate 641 : LOC variant 673 : LOC variant 674 : LOC variant 6 8 2 : dialysis section 6 8 6 : dialysis step 692 : primer-linked linear probe 694 : amplification blocker 6 9 6 : probe region 698 : complementary sequence 700 : oligonucleotide primer 704 : stem-and-loop probe 706 : complementary sequence 708 : stem 7 1 0 : strand -109 - 201209407 7 1 2 : first light稜鏡 7 1 4 : second aperture 7 1 6 : first mirror 7 1 8 : second mirror 740 : flow velocity sensor 746 : LOC variant 75 8 : LOC variant 7 6 0 : large component channel 7 6 2 : Small component channel 764 : Opening 766 : 肓 Terminal 7 6 8 : Blind terminal 7 7 8 : Configuration 7 8 0 : Configuration 7 8 2 : Configuration 788: Differential imager circuit 7 9 0 : Pixel 7 9 2 : Virtual Pixel 7 94 : Read - Column 795 : Read - Column 796 : Negative Control Probe 797 : (Crystal) 798 : Positive Control Probe 8 0 1 : (Crystal) -110- 201209407 8 03 : Pixel capacitor 805: Virtual pixel capacitor 807: Switch 8 0 9 : Switch 8 1 1 : Switch 8 1 3 : Switch 815: Capacitor amplifier 8 1 7 : Differential signal 8 60 : ECL excitation electrode 8 70 : ECL excitation electrode

Claims (1)

201209407 VII. Patent application scope: 1. A microfluidic device for testing a fluid, the microfluidic device comprising: an inlet for receiving a fluid; a storage tank containing a reagent; a flow path extending from the inlet; and a flow path and a storage tank a plurality of outlet valves in fluid communication, each outlet valve having an actuator for opening the outlet valve in response to the activation signal; wherein, in use, selectively opening some of the outlet valves such that the reagent flows into the flow path to and from the inlet The fluid is combined to produce a turbulent flow having a ratio of reagents, and the proportion of the reagents in the combined turbulent flow is determined by the number of open outlet valves. 2 The microfluidic device of claim 1 is further included in a plurality of channels extending between the storage tank and the flow path, the outlet valve is disposed in each channel, and the channel is configured to attract the reagent to the flow path from the storage tank by capillary action, wherein the outlet valves each have a meniscus fixer, At the meniscus holder, the capillary action toward the flow path drives the reagent flow to be stopped and forms a meniscus 〇3. The microflow as in claim 2 It means, wherein each channel has more than one outlet valve. 4. The microfluidic device of claim 2, wherein the actuator is a heater to release the meniscus from the meniscus holder in response to the activation signal. 5. The microfluid according to claim 4 Means, wherein each outlet-112-201209407 valve has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator 'which is used to displace the movable member to generate a pulse in the reagent to move the meniscus, This allows the reagent flow towards the flow path to be restored. 6. The microfluidic device of claim 5, wherein the movable member is configured for movement between a static position and an actuated position (moving from a static position), and a meniscus holder set The state stops the orifice of the reagent stream by fixing the meniscus to the orifice, and the thermal actuator is configured to move the movable member back and forth between the static position and the actuated position to force the reagent through the orifice. 7. The microfluidic device of claim 6, wherein the orifice is defined in the movable member. 8. The microfluidic device of claim 4, wherein the meniscus holder is configured to stop the reagent flow orifice by fixing the meniscus to the orifice, and the thermal actuator is configured The meniscus is released from the meniscus holder by boiling a portion of the reagent at the orifice. 9. The microfluidic device of claim 4, wherein each outlet valve has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the movable member and moving the meniscus The surface is in contact with the surface downstream of the meniscus holder, thereby releasing the meniscus and restoring the capillary action driving flow toward the flow path. The microfluidic device of claim 4, wherein the meniscus is fixed The surface downstream of the device is a capillary action initiation feature that is configured to direct the meniscus to the channel wall. 1 1. A microfluidic device as claimed in claim 1 further comprising - 113 - 201209407 comprising a support substrate for a sump, an outlet valve, an inlet and a flow path' and a CMOS circuit for operatively controlling the outlet valve. 12. The microfluidic device of claim 11, further comprising at least one liquid responsive sensor to provide feedback to the CMOS circuit for separate valve operability control. 13. The microfluidic device of claim 12, wherein at least one of the sensors is a liquid sensor for sensing the presence or absence of a liquid at a location in one of the channels. The microfluidic device of claim 1, further comprising a plurality of storage tanks each containing different reagents and a plurality of outlet valves each having a storage tank and a flow path, wherein any different reagents in the combined flow The ratio is related to the number of separate valves that are open in the corresponding valve configuration. 1 5 . The microfluidic device of claim 14, wherein the CMOS circuit selects the number of outlet valves that are opened for each of the storage tanks according to a different reagent type and a ratio equal to that of the combined flow. 16. The microfluidic device of claim 15, further comprising a polymerase chain reaction (PCR) portion for amplifying a target nucleic acid sequence in the fluid. 17. The microfluidic device of claim 16, wherein the different reagents in the sump comprise one or more of the following: a polymerase; a restriction enzyme; a dNTP and a primer in the buffer; a lysis reagent: and, - 114- 201209407 Anticoagulant. 18. The microfluidic device of claim 15, further comprising a hybridization portion having a probe array for hybridizing to a target nucleic acid sequence in the fluid, wherein the CMOS circuit has a probe for detecting A sensor array that hybridizes to any probe within the array. -115-