TW201211243A - Microfluidic device with dialysis section having stomata tapering counter to flow direction - Google Patents

Abstract

A microfluidic device for removing cell debris from a biological sample, the microfluidic device having a dialysis section with a large constituent channel and a small constituent channel, and a series of stoma for fluid communication between the large constituent channel and the small constituent channel, the large constituent channel having an upstream end for receiving the biological sample, the biological sample being a liquid carrying a mixture of cell debris and target molecules, the small constituent channel having a downstream end for connection to a hybridization section with an array of probes for reaction with the target molecules to form probe-target complexes, wherein, each of the stoma is tapered in a counter-flow direction such that each have a small opening to the large constituent channel and a large opening to the small constituent channel, the small openings being sized to allow the target molecules to flow into the small constituent channel but retain the cell debris larger than a threshold size in the large constituent channel.

Description

201211243 VI. Description of the Invention: [Technical Field to Which the Invention pertains] The present invention relates to a diagnostic apparatus using Microsystems Technology (MST). In particular, the present invention relates to the processing and analysis of microfluidics and biochemicals for molecular diagnostics. [Prior Art]

Molecular diagnostics have been used to provide an early indication of disease detection 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 DNA and nucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobases allows for the binding (hybridization) of synthetic DNA (oligonucleotide) short sequences to specific nucleic acid sequences for nucleic acid assays. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will receive in the future, determine the type and pathogenicity of an infectious pathogen, or determine the individual's response to the drug. 201211243 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

Many sample types, such as blood, urine, sputum, and tissue samples, are used for genetic analysis. Diagnostic tests determine the type of sample required 'because 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 beginning of a nucleic acid assay.

Blood is one of the more commonly sought 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 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 lysis solution, 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 protocol used to extract the nucleic acid depends on the sample and the diagnostic analysis to be performed. For example, the protocol used to extract viral RNA is quite different from the protocol used to extract genomic DNA. 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 done using a lysing detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells.

The step of precipitating with an alcohol (usually ice ethanol or isopropanol) in the presence of a high concentration of chaotropic salt, usually in a cerium oxide matrix, resin or paramagnetic beads in a fractionation column, prior to washing Alternatively, the nucleic acid is purified via a solid phase purification step followed by elution with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protein cleavage 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. Target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogenic 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 system is known in the industry and is also available from E. van Pelt-Verkuil et al.

A comprehensive comprehensible description of such reactions is provided in 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 must first be transcribed 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 DN A having about 10-30 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis DNA thermostable enzyme 3. Deoxyribose nucleus Glycoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - the best chemical environment for DNA synthesis. PCR generally involves placing these reagents in small tubes containing extracted nucleic acids (~ 1〇-5〇 microliters). The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. Standard procedures for each thermal cycle include the denatured phase, the adhesive phase, and the extended phase. The extension phase sometimes represents the primer extension phase. In addition to this three-step procedure, a two-step thermal procedure can be employed in which the adhesion and extension are combined. The denatured phase generally comprises heating the reaction temperature to 90-9 5 ° 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 Warm up to an optimal DNA polymerase activity temperature of 60-72 ° C for extension of the primer. This method repeats the cycle for about 20-40 times with the end result of generating 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, multiple primer set PCR, linker-primed PCR, direct PCR 'tandem PCR, ie 201211243 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). Optimizing multiple primer set PCR is more difficult because it requires the selection of primers with approximate adhesion temperatures and amplicon with approximate length and base composition to ensure equal amplification efficiency of each amplicon.

Linker-initiated PCR, also known as ligation adaptor PCR, is a method for enabling nucleic acid amplification of substantially all DNA sequences in complex DNA mixtures without the need for target-specific primers. This method first digests the target DNA population with a suitable restriction endonuclease (enzyme). Using a zymase enzyme, a double stranded oligonucleotide linker (also known as a zygote) with a suitable overhanging end is then ligated to the terminal of the target DNA fragment. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. Thereby, all fragments of the DNA source adjacent to the linker oligonucleotide can be amplified. Direct PCR describes a system that performs PCR directly on a sample without any nucleic acid extraction (or minimal nucleic acid extraction). It has long been believed that the PCR reaction is 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, PCR can be performed or direct PCR can be performed with minimal DNA purification. PCR chemistry for direct PCR 201211243 Qualitative adjustments include enhanced buffer strength, the use of highly active and processivity polymerases, and additions to potential polymerase inhibitors.

Repeated sequence PCR utilizes two independent nucleic acid amplifications to increase the probability of amplifying the correct amplicon. One type of repeat PCR is nested PCR in which two pairs of PCR primers are used to perform single 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 locus is amplified by mistakes during the first nucleic acid amplification, the probability of re-amplifying the wrong locus by the second pair of primers is very low, thus ensuring specificity.

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 probe containing a fluorophore or a fluorescent dye and a set of reference standards in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the pathogen contained in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. A reverse transcriptase is an enzyme that reverse transcribes RNA into a complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used in expression profiling to determine the expression of a gene or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It is also used to augment RNA viruses such as human immunodeficiency virus or hepatitis C virus. -10- 201211243

Constant temperature amplification is another type of nucleic acid amplification that does not rely on thermal denaturation of the target DNA during the amplification reaction, and thus does not require complicated machinery. The thermostatic nucleic acid amplification method can be performed at the original location or easily outside the laboratory environment. Including Strand Displacement Amplification, Transcription Meditation Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Some of the constant temperature nucleic acid amplification of Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification, and Loop-Mediated Isothermal Amplification The method of addition 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 It is another way to separate DNA strands using enzymes. The ability of strand-substituted amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi-modified DNA, and 5'-3' exonuclease-deficient polymerases to extend and replace Downstream stocks. Exponential nucleic acid amplification is then achieved by a reaction between the sense and the antisense, wherein the strand of the sense reaction is substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl -11 - 201211243) which cleaves DNA on one of the DNA strands without cutting the DNA in a conventional manner is useful for this reaction. SDA has been improved by the use of a combination of a thermostable restriction enzyme (J να 1 ) and a thermostable exo-polymerase (polymerase). This combination appears to increase the amplification efficiency of the reaction from 1 to 8 fold amplification to 101 (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 the corresponding genomic DN Α. 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 position. The reverse transcriptase produces a DNA 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 sequence in the DNA template and initiates transcription. Each newly synthesized RNA amplicon is reintroduced into the process and serves as a template for new replication. In recombinant enzyme polymerase amplification (RPA), constant binding amplification of specific DNA fragments is achieved by binding the opposite oligonucleotide primer to the template DNA and extending it by DN A polymerase. Denatured double-stranded DNA (dsDNA) templates do not require heat. Conversely, RPA uses recombinant enzyme-primer mismatches to scan dsDNA and promote share exchange at homeostatic (COgnate) positions. The structure obtained by the -12-201211243 is stabilized by the interaction of the single-stranded DNA-binding protein with the substituted template strand, thus preventing the primer from being released due to branch migration. The recombinant enzyme decomposes and can be replaced by a DNA polymerase (such as the 3' end of the oligonucleotide close to Bacillus whi/k Pol I (large fragment), and the primer then begins to extend. Repeat this step by loop to reach the index Amplification of nucleic acids.

The 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 followed by a DNA polymerase extension primer. In the first step of the HD A reaction, the helicase passes through the target DNA, damaging the two hydrogen bonds, which are then bound by a single-stranded binding protein. The single-strand target region exposed by the helicase allows the primer to adhere. The DNA polymerase uses free deoxyribonucleoside triphosphate (dNTP) to subsequently extend the 3' end of each primer to produce two DNA replicas. Two replicated dsDNA strands enter the next HDA cycle independently, causing exponential nucleic acid amplification of the target sequence. Other DNA-based thermostating techniques include rolling cycle amplification (RCA) in which DNA polymerase surrounds a circular DNA template The primer is continuously extended to produce a long DNA product consisting of a number of circular repeat copies. Thousands of copies of the circular template are generated by terminating the reaction ' polymerase, which has 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 an hour. Branch-type amplification is a variant of RC A, and uses a closed circular probe (C-probe) or a latching probe and a highly progressive 〇ΝΑ polymerase' to expand exponentially at normal temperature. Increase c _ probe. -13- 201211243 Circular Thermostat Amplification (LAMP) provides high selectivity and utilizes DNA polymerase and a primer set containing four specially designed primers. The primer set recognizes a total of six different sequences on the target DNA. The inner primer, which contains the sense strand of the target DNA and the antisense strand sequence, initiates the LAMP. Subsequent strands initiated by exogenous substitutions replace DNA synthesis to release single-stranded DNA. This serves as a template for DNA synthesis initiated by the second internal and external primers, and the second inner and outer primers hybridize to the other end of the target to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the inner primer hybridizes to the loop on the product and initiates the replacement DNA synthesis, producing the original stem-loop DNA and the new stem-loop DNA with twice the stem length. Continuously circulate the reaction in less than one hour to accumulate 1 〇 9 copies of the target. The final product is 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 determine 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 if a nucleic acid amplification step produces 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 determined by comparison with a DNA size marker (DNA ladder 201211243) which 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, the size of the amplified product can be predicted and detected as a band of 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 a current, light is generated by a luminophore molecule or a complex. When the fluorescent light is emitted, the emitted light is emitted to emit light. The illuminating source is used to detect fluorescence, and the illuminating source provides excitation light having a wavelength absorbed by the fluorescent molecules and a detecting unit. The detection unit includes a photosensor (such as a photomultiplier tube or a charge coupled device (CCD) array) to detect the transmitted signal and a mechanism (such as a wavelength-select filter) that prevents excitation light from being included in the output of the photosensor. Back-stress luminescence, fluorescent molecules emit Stokes-shifted light, and this emitted light is collected by the -15-201211243 unit. The Toks shift is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. A light sensor is used to detect the ECL emission, 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 the 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 detect thousands of genetic diseases or detect the presence of several infectious pathogens in a single experiment. 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 (either 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 microarray surface is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. Less stringent may result in highly non-specific binding. Excessive rigor may result in inability to properly combine and result in reduced sensitivity. Hybridization is identified by detecting the light emission from the labeled amplicon that hybridizes to the complementary probe. Fluorescence from a microarray is detected using a microarray scanner, typically a computer-controlled, inverting-scanning fluorescent conjugated focus microscope, -16-201211243, which typically uses lasers and light that excite fluorescent dyes. A sensor (such as a photomultiplier tube or CCD) detects the emitted signal "fluorescent molecules emit down-converted light (as described above), while light is collected by the detection unit.

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. Prevents light above or below the focal plane of the object from entering the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, which is then converted into a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be identified and analyzed simultaneously. More information on fluorescent probes can be found below: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References / Molecular-probes-The-Handbook/Technical-Notes-and- product-Highlights/Fluorescence-Resonance-Energy-

Transfer-FRET.html In-situ Care Molecular Diagnostics Despite the advantages of molecular diagnostic tests, the growth of this type of test in clinical tests is not as good as expected and still only accounts for a small portion of the implementation of laboratory medicine. This is primarily due to the complexity and cost associated with 'nucleic acid assays' compared to assays based on non-closed nucleic acid methods. The broad applicability of molecular diagnostic tests to clinical treatment is closely related to the rapid and automated analysis that can significantly reduce costs, provide initial (sample processing) to final (results), and the development of instruments that do not require extensive human manipulation.

For physicians' clinics, proximity or user-based hospitals, home-based in-home healthcare technologies offer the following benefits: • Quick results to enable rapid treatment and improved care quality • Ability to test sputum by testing a very small number of samples . • Reduce clinical workload. • Reduce laboratory workload and increase work efficiency by reducing management efforts.

• Improve the cost per patient by reducing hospital stays, getting results from first visits to clinic patients, and simplifying the handling, storage and delivery of samples. • Promote clinical management decisions such as infection control and antibiotic use. Molecular Diagnostics Based on On-Wafer Laboratory (LOC) Molecular diagnostic systems based on microfluidics provide devices that automate and accelerate molecular diagnostic analysis. The shorter detection time is primarily due to the fact that the required sample volume is less active, automated, and low-cost built-in cascaded diagnostic method steps within the microfluidic device. The volume of the scale of the nanoliter and microliter is also -18- 201211243

Reduce reagent consumption and cost. On-wafer laboratory (LOC) devices are in the form of common microfluidic devices. The LOC device has an MST structure within the MST for integrating fluid processing onto a single support substrate (usually 矽). Manufactured by VLSI (Ultra Large Integrated Circuit) lithography technology for the semiconductor industry to make each LOC device The unit cost is very low. However, large amounts of external piping and electronics are required to control the flow of fluid through the LOC unit, to add reagents, to control reaction conditions, and the like. Connecting the LOC devices to these external devices greatly limits the molecular diagnostic use of the LOC devices in the laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as a practical option in a local healthcare setting. 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 The dissimilarities of the present invention are now described in the following paragraphs. GDI0 3 9.1 The aspect of the invention provides a microfluidic device for removing cell debris from a biological sample. The microfluidic device comprises: a dialysis portion having a large component channel, a group channel, and a plurality of holes for fluid communication between the large component channel and the component channel, the large component channel having a An upstream end of the biological sample, the biological sample being a liquid with a mixture of cell debris and a target molecule, the set of channels having a link for reacting with the target molecule to form a probe-target complex The downstream end of the hybridization portion of the probe array; wherein the pores are spaced along the large component channel at intervals of 1 micrometer and 1 〇 micrometer -19-201211243, and the size of each of the pores can allow the label The target molecule flows into the panel channel, but cell debris larger than the 闳 size is retained in the large component channel. GDI03 9.2 Preferably, the large component channel and the component channel have a common sidewall, and the plurality of holes extend through a series of tapered holes of the common sidewall, each tapered hole having a plurality of channels to the component channel The small opening and the large opening to the opposite narrowing of the channel of the group.

GDI03 9.3 Preferably, the microfluidic device also has a plurality of the plurality of component channels each sharing a common sidewall with the plurality of component channels and in fluid communication via a series of tapered bores. GDI 039.4 Preferably, each of the small openings of the tapered bore has a height and width dimension between 1 and 8 microns. GDI 039.5 Preferably, the microfluidic device also has a waste sump, wherein the large component passage has a downstream end connected to the waste sump.

GDI039.6 preferably, the microfluidic device also has a lysis portion upstream of the dialysis portion, wherein the target molecule is a target nucleic acid sequence and the lysate portion is configured to dissolve cells in the biological sample and The target nucleic acid sequence therein is released. GDI039.7 Preferably, the microfluidic device also has a nucleic acid amplification unit for amplifying the target nucleic acid sequence. G D10 3 9.8 Preferably, the probe system is configured in a line to hybridize to the target nucleic acid sequence to form a probe-target hybrid. The probe-target hybrid can react to the excitation light. Light. G D10 3 9.9 Preferably, the microfluidic device also has a CMOS circuit for controlling the nucleotide amplification section for operability -20-201211243, the CMOS circuit also having a hybridization from the probe-target Photosensing Fluorescence Emission Photosensor GDI039.1 0 Preferably, the hybridization portion has an array of hybridization chambers comprising probes for hybridization to the target nucleic acid sequence. Preferably, the photosensor is located adjacent to the respective array of photodiodes of each of the hybridization chambers.

GDI03 9.1 2 Preferably, the CMOS circuit has digital memory for storing data relating to processing the fluid, the data including probe details and the location of each probe in the hybridization chamber array. GDI03 9.1 3 Preferably, the CMOS circuit has at least one temperature sensor for sensing temperature in the hybridization chamber array. GDI03 9.1 4 Preferably, the microfluidic device also has a heater controlled by the CMOS circuit using feedback from the temperature sensor for maintaining the probe and the target nucleic acid sequence at the hybridization temperature. GDI03 9.1 5 Preferably, the photodiode system is less than 249 microns from the corresponding hybridization chamber. GDI03 9.1 6 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GDI 039.1 7 Preferably, the hybridization chamber has an optical window disposed to expose the FRET probe under excitation light. GDI039.1 8 preferably, the FRET probes each have a fluorophore and a quenching agent, and when the FRET probe has formed a probe-target hybrid, the fluorophore is configured to emit a fluorescent signal to The photodiode is responsive to excitation light-21 - 201211243, the CMOS circuit being arranged to energize the photodiode after a predetermined delay after extinction of the excitation light, the digital memory comprising the predetermined delay. GDI039.19 Preferably, the CMOS circuit has a bonding pad for electrically connecting to an external device, and is configured to convert the output from the photodiode into a FRET probe representative of hybridization with the target nucleic acid sequence. a signal, and the signal is supplied to the bonding pad for transmission to the external device.

GDI03 9.20 Preferably, the microfluidic device also has a plurality of reservoirs for retaining fluid reagents for addition to the sample. The easy-to-use, mass-produced, and inexpensive microfluidic device accepts a biological sample, uses a dialysis section for separating the different sized sample components, and separately processes sample components separated based on their size.

The dialysis section directly selects a component comprising the sample of the target, removing unwanted components of the treated mixture, which may interfere with subsequent detection of the target, inhibit subsequent analysis steps, or may block the chamber or be in a microfluidic device and degrade The connection within the operation. The dialysis functionality also extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the analysis system. The dialysis section, which is indispensable for the device, provides an inexpensive analysis system for low system component counting and simple preparation procedures. The dialysis section, which is manufactured only by surface micromachining, is also provided for this simple and inexpensive preparation procedure, resulting in a further reduction in the cost of the analytical system. GDI040.1 The aspect of the invention provides a microfluidic device for removing cell debris from a biological sample. The microfluidic device comprises: a dialysis portion having a large component channel, a component channel, and a series of tapered holes for fluid communication between the large component channel and the component channel, the large component channel having a -22 - 201211243 to receive an upstream end of the biological sample, the biological sample being a liquid with a mixture of cell debris and a target molecule having a channel for attachment to react with the target molecule to form a probe a downstream end of the hybridization portion of the probe array of the target complex; wherein

Each of the tapered holes is tapered in a direction opposite to the flow such that each has a small opening to the large component channel and a large opening to the group of channels, the small openings being sized to allow the target The target molecule flows into the panel channel, but cell debris larger than the threshold size is retained in the large component channel. Preferably, the GDI 040.2 is spaced along the large component channel by a spacing between 1 micrometer and 10 micrometers. Preferably, the microfluidic device also has a plurality of the plurality of channels, wherein the plurality of channels share the same side wall with the large component channel and are in fluid communication via the series of tapered holes. GDI040.4 preferably. The small opening of each of the tapered holes has a height and a width of between 1 micrometer and 8 micrometers. GDI 040.5 Preferably, the microfluidic device also has a waste sump, wherein the large component passage has a downstream end connected to the waste sump. GDI040.6 preferably, the microfluidic device also has a lysis portion upstream of the dialysis portion, wherein the target molecule is a target nucleic acid sequence and the lysis portion is configured to lyse cells in the biological sample and The target nucleic acid sequence therein is released. GDI040.7 Preferably, the microfluidic device also has a nucleic acid amplification unit for amplifying the target nucleic acid sequence. -23- 201211243 GDI040.8 Preferably, the probe is constructed to hybridize to the target nucleic acid sequence to form a probe-target hybrid that reflects fluorescence of the excitation light . GDI040.9 preferably, the microfluidic device also has a CMOS circuit for operatively controlling the nucleic acid amplification portion, the CMOS circuit also having light for sensing fluorescence emission from the probe-target hybrid Sensor

Preferably, the hybridization portion has a hybridization chamber array comprising probes for hybridization to the target nucleic acid sequence. Preferably, the photosensor is located adjacent to the respective array of photodiodes of each of the hybridization chambers. GD 1040.1 2 Preferably, the CMOS circuit has a digital memory for storing data relating to processing the fluid, the data including probe details and the location of each probe in the array of hybridization chambers. GDI040.1 3 Preferably, the CMOS circuit has at least one temperature sensor for sensing temperature in the hybridization chamber array.

GDI040.1 4 Preferably, the microfluidic device also has a heater controlled by the CMOS circuit using feedback from the temperature sensor for maintaining the probe and the target nucleic acid sequence at the hybridization temperature. GDI040.1 5 Preferably, the photodiode system is less than 249 microns from the corresponding hybridization chamber. GD 10 40.1 6 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GDI040.1 7 Preferably, the hybridization chamber has an optical window disposed to expose the FRET probe under excitation light. -24- 201211243 GDI040.1 8 Preferably, the FRET probes each have a fluorophore and a quenching agent, and when the FRET probe has formed a probe-target hybrid, the fluorophore is set to emit A fluorescent signal is applied to the photodiode to illuminate the light. The CMOS circuit is configured to energize the photodiode after a predetermined delay after extinction of the excitation light, the digital inclusion comprising the predetermined delay.

GDI040.1 9 preferably, the CMOS circuit has a bonding pad for electrically connecting to an external device, and is configured to convert the output from the photodiode into the FRET probe representative of hybridization with the target nucleic acid sequence. The signal is provided to the bonding pad for transmission to the external device. GDI 040.20 Preferably, the microfluidic device also has a plurality of reservoirs for retaining fluid reagents for addition to the sample. The easy-to-use, mass-produced, and inexpensive microfluidic device accepts a biological sample, uses a dialysis section for separating the different sized sample components, and separately processes sample components separated based on their size. The dialysis section directly selects a component comprising the sample of the target, removing unwanted components of the treated mixture, which may interfere with subsequent detection of the target, inhibit subsequent analysis steps, or may block the chamber or be in a microfluidic device and degrade The connection within the operation. The dialysis functionality also extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the analysis system. The dialysis section contains a tapered bore based on a trapezoidal flat column; this design is designed to reduce the size of the tapered bore required for many applications for a given photoetching space width. The dialysis section, which is indispensable for the device, provides an inexpensive analysis system for low system component counting and simple preparation procedures. The dialysis section, which is manufactured only by surface micromachining, is also provided for this simple and inexpensive preparation procedure -25-201211243, resulting in a further reduction in the cost of the analytical system. [Embodiment] Detailed Description of Preferred Embodiments

This general specification identifies the major components of a molecular diagnostic system incorporating a particular embodiment of the invention. The details of the construction and operation of this system are described in the following description. Referring to Figures 1, 2, 3, 108 and 109, the system has the following most important components:

Test modules 10 and 11 are typical USB flash drives and are very inexpensive to manufacture. The test modules 1 and 11 each comprise a microfluidic device, typically in the form of a laboratory on-wafer (LOC) device 30, preloaded with reagents and typically more than 1 000 probes for diagnostic analysis of the molecule. Needle (see Figures 1 and 108). While the test module 1 1 in Fig. 108 uses an electrochemiluminescence-based detection technique, the test module 1 shown in Fig. 1 uses a fluorescence-based detection technique to identify the target molecules. The LOC device 30 has an integrated light sensor 44 for fluorescence or electrochemiluminescence detection (described in detail below). Both test modules 10 and 1 1 use standard micro USB connectors 14 for power, data, and control, each having a printed circuit board (PCB) 57, and each having an externally powered capacitor 3 2 and an inductor 15 . Both test modules 1 and 11 are for single use in large quantities and are distributed in sterile packaging for use. The outer casing 13 has a large container 24 for receiving biological samples and a sterile sealing tape 22 of removable -26-201211243, which preferably has 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 4 10 protects the membrane seal 408 from damage. The test module reader 12 supplies power to the test module 10 or 11 via the micro-USB port 16. The test module reader 12 can be described in a number of different forms' and its selection. The reader 12 version shown in Figures 1, 3 and 1 is a specific embodiment of a smart phone. A block diagram of the reader 12 is shown in FIG. The processor 42 executes the application software from the program storage 43. The processor 42 is also interfaced with the display honey screen 18 and the user interface (UI) touch screen 17 and buttons 19, the cellular radio 21, the wireless network connection 23', and the satellite navigation system 25. The cellular radio 21 and the wireless network connection 23 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location information. Alternatively, the location data can be manually entered using the touch screen 17 or button 19. The data store 27 maintains φ transmission and diagnostic information, test results, patient information, analysis and probe data for identifying each probe, and its array position. The data store 27 and the program store 43 can be shared by a common memory device. Test Module. The application software installed in the reader 12 provides results analysis and additional test and diagnostic information. To perform a diagnostic test, the test module 1 〇 (or test module i i ) is inserted into the micro_USB port 16 on the test module reader 12. The sterile world seal 22 is turned up and the biological sample (in liquid form) is loaded into the sample trough 24 . Press the start button 2〇 to start the test by applying the software. The sample flows into the LOC device 30 and is analyzed onboard (〇n_b〇ard -27- 201211243

Assays are extracted, cultured, amplified and hybridized with a pre-synthesized hybrid-reactive oligonucleotide probe and a sample nucleic acid (target). In the case of the test module 10 (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 emission from the hybridized probe. (See Figures 1 and 2). In test module U (which uses electrochemiluminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to generate photoluminescent fire. Conversely, electrodes 860 and 870 provide an excitation current (see Figure 109). The emission (fluorescence or photoluminescence) is detected using a photo sensor 44 integrated with a CMOS circuit on each LOC device. The detected signal is amplified and converted to a digital output analyzed by the test module reader 12. The reader then displays the result. Data can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 11 is removed from the test module reader 1 2 and processed as appropriate.

1, 3 and 108 show a test module reader 12 configured as a mobile/smartphone 28. 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 table used in a hospital, private clinic or laboratory. The upper computer is 1〇5 (see Figure 110). The reader can be interfaced with additional applications such as patient records, accounting, online databases and multi-user environments. It can also interface with some local or remote peripheral devices, such as printers and patient smart cards. Referring to Figure 1 1 1, through the reader 1 2 and the network 1 25, by the test module -28- 201211243

10 The data generated can be used to update the epidemiological database contained in the host system for epidemiological data 111, the genetic database contained in the host system for genetic data, and for electronic health records. (EHR) 115 host computer system with electronic health records, electronic medical records contained in the host system for electronic medical record (EMR) 121, and host for personal health record (PHR) 123 Personal health records contained in the system. Conversely, via the network 125 and the reader 12, the epidemiological data contained in the host system for the epidemiological data 111, the genetic data contained in the host system for the genetic material 113, and the electronic An electronic health record contained in the host system of the Health Record (EHR) 1 15 , an electronic medical record contained in the host system for the Electronic Medical Record (EMR) 121, and a personal health record (PHR) The personal health record contained in the host system of 123 can be used to update the digital memory in the test module 10 LOC 30. Referring again to Figures 1, 2, 108 and 109, the reader 12 uses battery power in a mobile telephone configuration. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be loaded or updated via some wireless or contact interface to enable communication with peripheral devices, computers or online servers. Set the mini-USB port 16 to connect to the computer or main power supply to recharge the battery. Figure 7A shows a specific embodiment of the test module 1 for testing only the presence or absence of a particular target, such as whether the test individual is infected with, for example, influenza A virus Η 1 N 1 . It is only suitable as a built-in module 47 for USB power/indicator only. No other readers are required • 29 - 201211243 or application software. 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 embodiment shown in Fig. 7A includes a spring-loaded retractable lancet 390 and a lancet release button 392. Satellite phones can be used in remote areas.

Test module electronic device

Figures 2 and 1 09 are each a block diagram of the electronic components of test modules 1 and 11. The CMOS circuit integrated in the on-wafer laboratory device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a timer 33, a power conditioner 31, a RAM 38, and a program and data flash memory. . These are provided for the entire test module 1 or η including the photo sensor 44, the temperature sensor 1 74 and the various heaters 152, 154, 182, 234 and associated Drivers 3 7 and 3 9 and the control and memory of registers 35 and 41. Only the LED 26 (in the example of the test module 10), the external power supply capacitor 32, and the micro USB plug 14 are external to the lab device 30 on the wafer. The on-wafer laboratory device 30 includes an adhesive pad for joining to the outer components. The RAM 38 and the program and data flash memory 40 have application software and diagnostic and detection information (flash/security storage, such as via encryption) for 1 000 probes. In the example of the test module 11 configured with ECL detection, there is no LED 26 (see Figures 1〇8 and 1〇9). The data is encrypted by the Crystal -30-201211243 on-chip laboratory unit 30 to preserve storage and communicate with external devices. The on-wafer laboratory device 30 is loaded with electrochemiluminescent probes and the hybridization chamber, each having an ECL excitation electrode pair 860 and 870. Many types of test modules 1 are manufactured in some form of inspection and are ready for ready use. These test formats differ in the onboard analysis of reagents and probes.

Some examples of infectious diseases that are quickly identified by this system include: • Influenza-influenza virus A, B, C, infectious salmon anemia virus, tocovirus • pneumonia-respiratory fusion virus (RSV), gland Virus, interstitial pneumonia virus, pneumococci, Staphylococcus aureus • tuberculosis - Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africana, Mycobacterium vaccae and Mycobacterium vaccae • Malignant sore, Toxoplasma and other parasitic protozoa • Typhoid- typhoid bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever - flavivirus • Hepatitis (A to E) • Iatrogenic infections – such as refractory spores Bacteria, vancomycin-resistant enterococci, and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) -31 - 201211243 • Encephalitis - Japanese brain Inflammatory virus, Zhangdipula virus • Pertussis-pertussis • Hemp pain • Paramyxovirus • Meningitis – Streptococcus pneumoniae and meningitis • Anthracnose – Anthrax Some examples of sexually transmitted diseases include: • Cystic fibrosis • Hemophilia

• sickle cell anemia • black scrotic idiotism • hemochromatosis • cerebral arterial disease • Crohn's disease • polycystic kidney disease • congenital heart disease • a few of the cancers identified by the diagnostic system Stomach: • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinoblastoma • Turcot syndrome The above list is not exhaustive' and the diagnostic system can be configured to detect many using nucleic acid and protein analysis Different g & and ^ $ 爿 dogs. -32-

201211243 Detailed structure of system components LOC device LOC device 30 is the center of the diagnostic system. It uses microfluidics to rapidly perform four important steps in nucleic acid-based molecular diagnostic analysis: sample preparation, nucleic acid extraction, nucleic acid amplification, and detection. The LOC device has an alternative use and will be detailed below. As discussed above, Trials 1 and 11 can take many different configurations to detect different targets. The same ' LOC device 30 has many different embodiments for targeting the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of a target nucleic acid sequence in a body of a whole blood sample. For the purposes of this description, the structure and operation of LOC device 301 are described in detail with reference to Figures 4: and 27 through 57. 4 is a schematic diagram showing the structure of the LOC device 301. The processing stage shown in Fig. 4 is represented by the component symbols corresponding to the functional portions of the apparatus 301 that implements the processing stage. The stages of processing associated with each of the major steps in the diagnostic analysis of nucleic acids are also indicated: Input and Preparation 288, Extraction 290, Culture 291, and Expansion 292 to determine 294. The various reservoirs, chambers, valves, and components of the LOC device 301 will be described more closely below. Figure 5 is a perspective view of the LOC device 301. It is manufactured using high CMOS and MST (microsystem technology) manufacturing techniques. The layered structure of LOC 3 〇1 is a partial cross-sectional view of Figure 12 (not to scale) LOC device 301 has a platform that supports COMS + MST wafer 48, and also has a specific pathogen for the module. Advantages of LOC basic sample and inspection group volume device) 矽基-33- 201211243

The board 84, which includes a CMOS circuit 86 and an MST layer 87, covers the MST layer 87 with a cover 46. For the purposes of this patent specification, the term "MST layer" relates to a collection of structures and layers of a sample treated with different reagents. Thus, these structures and components are configured to define a flow path having a characteristic size that supports a capillary action driven liquid flow 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 fabrication methods can also produce structures and assemblies that are sized for capillary action and that are processed to very small volumes. . The particular embodiment described in this specification shows that the MST layer is a structural and active component supported on CMOS circuitry 86, but excluding the features of cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample.

The overall dimensions of the LOC device shown in the following figures are 1 760 microns X 5 824 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 CMOS+MST device 48, FIGS. 7 through 10 independently show the features of the cover 46. Layered structure Figures 12 and 22 show the layered shape of the CMOS + MST device 48 -34-

201211243 Construction, the cover 46, and the fluid interaction between the two are omitted. For the purposes of illustration, the drawings are not drawn to scale for a cross-sectional view through the sample inlet 68 and FIG. 22 is a sump 54. The outline is to be a sectional view. As best shown in FIG. 12, CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86 that operates the active components in the MST layer 87. Passivation 密封 seals and protects the CMOS layer 86 from fluid flow through the MST layer 87 fluid through both the cap layer 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 100 (see, eg, Figure 7). While performing the biochemical treatment on the smaller MST channel 90, the fine delivery occurs in the larger channel 94 made in the cover 46. The fine delivery channel is sized to carry the cells in the sample to the A predetermined point in channel 90. Cells carrying a size greater than 20 microns (some white blood cells) require a channel size greater than 20 microns, and thus the cross-sectional area of the stream is greater than 40 0 square microns, particularly in LOCs that do not require shipping. The MST channel can be significantly smaller. It will be understood that the cover channel 94 and the MST channel 90 are versatile, and the particular MS T channel 90 can also be referred to as a heated microchannel or dialysis MST channel depending on its particular function. . The MST channel 90 etch is formed by a channel layer 1 沉积 deposited on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is formed by a top layer 66 around the top layer to form the top of the CMOS + MST device 48 (relative to the orientation in the display). Although sometimes shown as a separate layer, the cover channel layer 80 and the figure. . </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Of course, the sheet of material may also be non-unitary. The sheet of material is etched from both sides to form a lid channel layer and a reservoir layer 78'. The lid channel 94 is etched in the lid channel layer 8 and the reservoir 54 is engraved in the reservoir layer 78. 56, 58, 60, and 62. Additionally, the sump and the cover channel are formed by a micro-shaping process. Both etching and micro-shaping techniques are used to fabricate a cross-sectional area across the fluid. It is as large as a square micron and has a small channel of 8 square microns.

There are a series of suitable choices for one of the cross-sectional areas of the channel 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 more than 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 accommodated in the channel' preferably a very small cross-sectional area across the fluid. A lower seal 64 surrounds the cover channel 94 and the upper seal layer 82 surrounds the sump 54' 56, 58, 60 and 62.

The five reservoirs 54, 56, 58, 60 and 62 are preloaded to analyze specific reagents. In the particular embodiment described, the reservoir is preloaded with the following reagents, but can be easily replaced with other reagents: • Storage tank 54: anticoagulant with selection of red blood cell lysis buffer • Storage tank 56: lysis reagent

• Slots 58: Restriction of enzymes, ligases, and junctions (for junction-priming PCR (see Figure 69, from T. Stachan et al., Human Molecular Genetics 2, Garland Science, NY-36-201211243 and London, 1 999 Excerpts • Storage tank 60·Amplification mixture (deoxynucleotide triphosphate (dNTp), primer, buffer) and • Storage tank 62: DNA polymerase

The cover 46 and the CMOS + MST layer 48 are in fluid communication via respective openings in the lower seal 64 and the top layer 66. The openings represent the upper conduit 96 and the lower conduit 92 depending on whether fluid flows from the MST passage 90 to the cover passage 94 or vice versa. LOC Device Operation The operation of the LOC device 301 is described step by step with reference to the analysis of pathogen DNA in a blood sample. Of course, other types of biological or non-biological fluids are also analyzed using kits or combinations of appropriate reagents, assay protocols, LOC variants, and detection systems. Referring again to Figure 4, the analysis of the biological sample involves five major steps, including: sample input and preparation 288, nucleic acid extraction 2 90, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294. The sample input and preparation step 288 involves mixing the blood with the anticoagulant 116 and then separating the pathogen from the white blood cells and red blood cells by the pathogen dialysis unit 70. As best shown in Figures 7 and 12, the blood sample enters the device via the sample inlet 68. Capillary action draws the blood sample along the lid channel 94 to the sump 54. When the sample blood flow opens its surface tension valve 118, the anticoagulant is released from the sump 54 (see Figures 15 and 22). The anticoagulant prevents the formation of blood clots that can block flow. As best shown in Figure 22, the anticoagulant 116 is withdrawn from the sump 54 by capillary -37-201211243 and enters the MST channel 90 via the lower conduit 92. The lower duct 92 has a capillary initiation configuration feature (CIF) 102 to define the geometry of the meniscus such that it is not fixed to the edge of the lower duct 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent hole I22 in the upper seal portion 82 allows air to replace the anticoagulant 166.

The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and is secured to the meniscus 120 of the upper conduit 96 to the meniscus holder 98. The meniscus 120 remains fixed to the upper conduit 96 prior to use 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 120 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 118 has three separate MST passages 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can be varied to vary the flow rate of the reagent entering the sample mixture. The flow rate of the sample mixture in the sample stream is determined by the flow rate of the sample mixture as it is mixed by diffusion and the reagents. Thus, the surface tension valve for each of the sump is configured to meet the desired reagent concentration. The blood passes through the pathogen dialysis section 70 (see Figures 4 and 15), wherein the target cells are concentrated from the sample using an array of wells 1 64 sized according to a predetermined valve. Cells smaller than the valve pass through the holes, and large cells cannot pass through the holes. When the target cell continues to be part of the analysis -38-201211243, the unwanted cells are reintroduced into the waste unit 76. The unwanted cells are large cells that are blocked by the array of such holes 1 64, or small cells that pass through the holes.

In the pathogen dialysis section 7 described herein, the pathogen from the whole blood sample is concentrated for microbial DNA analysis. The array of holes is formed by fluidly communicating the input flow in the cover channel 94 to a plurality of 3 micron diameter holes 164 of the target channel 74. The 3 micron diameter aperture 164 and the dialysis suction aperture 168 for the target channel 74 are connected by a series of dialysis MST channels 204 (best shown in Figures 15 and 21). The pathogen is small enough to pass through the 3 micron diameter aperture 164 via the dialysis MST channel 204 and to fill the target channel 74. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 of the lid 46, which leads to the waste reservoir 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be used to isolate specific pathogens or other target cells such as white blood cells for human DNA analysis. More detailed details of the dialysis section and dialysis variants are provided at the tip. Referring again to Figures 6 and 7, the fluid is drawn through the target passage 74 to the surface tension valve 128 in the 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 from all seven of the MS T channels 90 will be greater than the flow rate from the anticoagulant reservoir 54, wherein the surface tension valve 118 has three MST channels 90. (Assume that the physical properties of the fluid are approximately equal). Therefore, the proportion of the lysing reagent in the sample mixture is greater than the ratio of the anticoagulant -39 - 201211243.

The lysis reagent and target cells are mixed by diffusion in the target channel 74 within the chemical lysis unit 130. The boiling initiation valve 126 stops the flow until diffusion and lysis occur for a sufficient time to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling initiation valve are described in detail below with reference to Figs. 31 and 32. Other active valve types (as opposed to passive valves such as the surface tension valve 118) have also been developed by the Applicant, which can be used in place of the boiling initiation valve. These alternative valve designs are also described below. When the boiling initiation valve 126 is opened, the lysed cells flow into the mixing portion 131 for pre-amplification restriction digestion and linker ligation.

Referring to Fig. 13, when the fluid is removed from the surface of the bay on the surface tension valve 132 which is initiated by the mixing portion 131, the restriction enzyme, the linker and the ligase are released from the sump 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 leading to the incubator inlet passage 133 of the culture portion 114 (see Fig. 13). The culturer inlet channel 133 feeds the mixture into the crucible configuration of the heated microchannel 210, which provides a culture chamber that retains the sample during restriction digestion 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 various figures show the continuous addition of the layers forming the CMOS + MST layer 48 and the structure of the cover 46. The inset A B shows the end of the culture portion 1 14 and the start of the amplification portion 1 1 2 . As best shown in Figures 14 and 23, the fluid breaks into the microchannels 210 of the culture section 1 - 4 - 201211243 until the boiling initiation valve 106 is reached, wherein the fluid stops while diffusion occurs. As discussed above, the microchannel 210 upstream of the boiling initiation valve 106 becomes a culture chamber containing the sample, restriction enzymes, ligase, and linker. The heater 154 is then activated and maintains a steady temperature for the occurrence of restriction enzyme digestion and junction bonding for a specific period of time.

Those skilled in the art will appreciate that this incubation step 29 1 (see Figure 4) is selected and only requires some type of nucleic acid amplification analysis. Again, in some instances, it may be desirable to have a heating step at the end of the incubation period to increase the temperature above the culture temperature. This temperature increase before entering the amplification section 112, so that the restriction enzyme and the ligase are not activated. When using thermostated nucleic acid amplification, there is a specific association between restriction enzymes and ligase inactivation. After the incubation, the boiling initiation valve 106 is activated (turned on) and the fluid is returned to the amplification portion 112. Referring to Figures 31 and 32, the mixture fills the heated microchannel 1 58 structure until the boiling initiation valve 108 is reached, which form one or more amplification chambers. As best shown in the cross-sectional view of Figure 30, the amplification mixture (dNTPs, primers, buffers) is released from the reservoir 60 and the polymerase is then released from the reservoir 62 into the culture section and the amplification section (114 and 112, respectively). The intermediate MST channel 212 of FIGS. 35 through 51 shows the layers of the LOC device 301 in the inset AC of FIG. The figures show successive layers of layers forming the CMOS + MST device 48 and cover 46 structures. The end of the AC-based amplification unit 112 and the start of the hybridization and detection unit 52 are shown. The cultured sample, amplification mixture, and polymerase flow through microchannel 158 to boiling initiation valve 1〇8. After diffusion mixing for a sufficient time -41 - 201211243, the heater 154 in the microchannel 158 is activated for thermal cycling or constant temperature 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 initiation valve 108 is opened and the fluid re-enters the hybridization and detection portion 52. The operation of the boiling pilot valve is described in more detail below.

As shown in Figure 52, hybridization and detection portion 52 has an array of hybridization chambers 110» Figures 52, 53, 54, and 56 show hybridization chamber array 110 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 from 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 of the hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 184 of the hybridization chamber 180 containing the same probe. The results of exporting anomalies in this single result can be ignored or given a different weight. The thermal energy required to supply hybridization is provided by a CMOS controlled heater 128 (described in more detail below). Hybridization occurs between the complementary target probe sequences after initiation of the heater. The LED driver 29 in the CMOS circuit 86 transmits a message to cause the leD 26 located in the test module 10 to illuminate. These probes only fluoresce when hybridization occurs to avoid the cleaning and drying steps typically required to remove unbound strands. Hybridization forces the stem and/or ring structure of the FRET probe 186 to open, which allows the fluorophore to be emitted back -42-201211243

The fluorescent energy of the LED excitation light should be detailed below. Fluorescence is detected by photodiode 184 in the CMOS circuit 86 located below each hybrid cavity 180 (see hybrid cell description below). The photodiode 1 84 and associated electronics for all of the hybridization chambers together form the photosensor 44 (see Figure 64). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected from 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 this 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, a dialysis section 70, a lysis section 130, a culture section 114, and an amplification section 112, Valve type, humidifier and humidity sensor. In other embodiments of the LOC device, such functional portions may be omitted, and additional φ functional portions or functional portions for alternative uses of the above devices may be added. For example, the culture portion 1 14 can be used as the first amplification portion 112 of the repetitive sequence amplification analysis system, and the chemical lysis reagent reservoir 56 can be used to add the first amplification mixture of the primer 'dNTP and the buffer, and the reagent is used. Sump 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 5 6 ' or alternatively, thermal lysis may occur in the culture by heating the sample for a predetermined period of time. In some embodiments, if chemical lysis is desired and the chemical lysis reagent is mixed therewith, additional reservoirs can be combined upstream of the sump 58 for the mixing of the primers, -43-201211243 dNTPs, and buffer.

In some cases, steps such as incubation step 291 are omitted. In this case, the LOC device can be specially manufactured to prevent the reagent storage tank 58 and the culture portion 114 or the storage tank from carrying more than the reagent, or if there is an active valve, it is not activated to dispense the reagent into the sample flow, and the culture portion It is simply a channel for transferring the sample from the lysis unit 130 to the amplification unit 112. The heater operates 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 flow system within the microfluidic device, as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after the amplification phase 292, prior to the hybridization and detection step 294, dialysis is performed to remove the cell debris system. Alternatively, two or more dialysis sections can be combined at any location on the LOC device. Similarly, additional amplifications 1 1 2 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using a particular nucleic acid probe in hybridization chamber 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 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 portion can still have the LOC of the dialysis section 70 without losing the desired function. Further, the detection portion 294 can include a protein body array array that is identical to the hybridization chamber array but carries a probe that is designed to conjugate or hybridize to the -44 - 201211243 sample target protein present in the non-amplified sample. Instead of a nucleic acid probe designed to hybridize to a target nucleic acid sequence. It will be appreciated that the LOC devices manufactured for use with this diagnostic system are different combinations of functional components selected for particular LOC applications. Most of the functional components are common to many LOC devices, and the design of additional LOC devices for new applications has a combination of appropriate functional components in the bulk of the functional options used in existing LOC devices.

Only a few LOC devices are shown in this description, and some others are shown to illustrate the design flexibility of the LOC devices manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices shown herein are not exhaustive, and that many 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 dips. 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 delivers the sample from the sample inlet 68 to the hybridization chamber 180 for nucleic acid detection, as described above. For some sample types, a pre-treatment step is required&apos;, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and may need to treat the mucus with enzyme pre-45-201211243 to reduce stickiness. Sample Input Referring to Figures 1 and 12, a sample is added to the large container 24 of the test module 10. The large container 24 is a truncated cone that is fed into the inlet 68 of the LOC unit 301 by capillary action. Here, it flows into the 64 μm wide 60 μηι deep cover channel 94 and is also attracted to the anticoagulant reservoir 54 by capillary action.

Reagent storage tank

Using a microfluidic device, such as the LOC device 301, the small amount of reagent required for the analysis system allows the reagent reservoir to contain all of the necessary reagents for biochemical treatment, and each reagent reservoir is in a small volume. This volume is indeed less than 1,000,000,000 cubic microns. In most cases, it is less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns, and in the case of the LOC device 301 shown in the figures is less than 20,000,000 cubic microns. 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 orifice into a series of dialysis MST channels 206 and is returned to the target channel 74 via the 16 μιη dialysis suction port 168 (-46-201211243 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. The foam illustration or other porous element 49 in the outer casing 13 of the test module 10 is configured to be in fluid communication with the waste reservoir 76 for a biological sample type that produces a substantial amount of waste (see Figure 1). .

The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. An orifice 164 having a diameter of 3 microns at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Fig. 33) such that the fluid is pulled downward into the dialysis MST channel 204 below. The first suction aperture 198 for the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus above the dialysis suction aperture 168. The small component dialysis section 682, which is schematically shown in Fig. 78, may have a structure similar to that of the pathogen dialysis section 70. The small component dialysis section separates the sample from any small target cells or molecules by sizing (and shaping, if necessary) to allow small target cells or molecules to pass to the target channel and continue to further analyze the well. Large size cells or molecules are removed to waste reservoir 766. Thus, the LOC device 30 (see Figures 1 and 108) is not limited to isolation of pathogens smaller than 3 μηη, but can be used to isolate cells or molecules of any desired size. Lysis Department 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 lysing reagent from the lysate reservoir 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 128 of the lysis tank 56. However, some diagnostic assays are preferably suitable for hot lysis treatment, or even a combination of chemical and thermal lysis of target cells. The LOC device 301 houses the heated microchannels 210 of the culture portion 114. The sample stream is flooded with the culture portion 114 and stopped at the boiling initiation 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 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 Initiating Valve As discussed above, LOC unit 310 has three boiling inducing 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 initiation valve 108 at the end of the heated microchannel 158 of the amplification section 112.

By capillary action, the sample stream 1 1 9 is drawn along the heated microchannel 1 58 until it reaches the boiling initiation 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 an orifice provided by the opening of the pipe from the MST passage 90 to the cover passage 94. The surface tension of the meniscus 12 使 keeps the valve closed. A ring heater 152 is located around the valve inlet 146. The ring heater 1 52 is controlled by C Μ Ο S via a boiling induced valve heater contact 53. To open the valve 'CMOS circuit 86, send an electrical pulse to the valve heater to connect -48- 201211243 point 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 wet cover channel 94' begins to recover, the capillary action is restored. The fluid sample 119 is filled with the passage 94 and flows through the lower valve line 150 to the valve outlet 148, wherein the capillary driven liquid flow advances along the expansion outlet passage 160 into the hybridization and detection portion 52. The liquid sensor 174 is placed before and after the valve for diagnosis.

It will be understood that once the boiling trigger 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 ruthenium pattern in the MS T channel layer 100 from the lower conduit opening 134 to the boiling initiation valve 106 (see Figures 13 and 14). . Controlling the temperature of the culture portion 114 enables a more efficient enzyme reaction. Similarly, the amplifying portion 112 has an amplifying microchannel 158 (see Figs. 6 and 14) which is heated from the boiling inducing valve 106 to the boiling initiation valve 108. Upon mixing, culture, and nucleic acid amplification, the valves stop flow to retain the target cells in the heated culture or amplification microchannels 210 or 158. The microchannel 蜿蜒 pattern also promotes (to some extent) the target cells to be mixed with the reagents. In the culture unit 141 and the amplification unit 丨i 2, the sample cells and reagents are heated via a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86 at -49-201211243. Each of the meandering structures of the heated culture microchannel 210 and the amplification microchannel 158 has three independently operable heaters 154 extending between the individual heater contacts 156 (see Figure 14).

), which provides 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 suitable. The elongated heater 154 is parallel to the longitudinal length of each of the channel portions forming the meandering meandering. In the amplification section 112, each of the wide meanders can be operated as an independent PCR chamber via individual heater control. The use of a microfluidic device, such as LOC device 301, requires a small volume of amplicons required for the analysis system to allow amplification of the small volume of amplification mixture in amplification portion 112. This volume is less than 400 nanoliters, in most cases less than 170 nanoliters, less than 70 nanoliters, and in the case of LOC device 310, this volume is between 2 nanoliters and 30 nanoliters. between.

Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a fairly short distance from the heater 1 5 4 . The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 1 〇〇, 〇〇〇 square micron, and has a significantly higher heating rate than the "large scale" device. The lithography manufacturing technique allows the amplifying microchannel 158 to have a cross-section that spans a flow path that provides a relatively high heating rate of less than 1 6,000 square microns. The 1 micron size feature is easily achieved with lithography manufacturing techniques. If only very small amplicons of -50-201211243 (as is the case in LOC unit 301) are required, the cross-section can be reduced to less than 2,500 square microns. For a diagnostic analysis required to perform a "sample entry, answer out" within 1 minute on a LOC device, the appropriate cross-sectional area across the fluid is 400. Between square micron and 1 square micron.

The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate of greater than 80 absolute temperatures (K) per second, which in most cases is greater than 100 K per second. Typically, the heater element heats the nucleic acid sequence at a rate greater than 1 〇〇〇 K per second, and in many cases, the heater element is heated at a rate greater than 10,000 K per second. Typically, based on the needs of the analytical system, the heater elements are greater than 100,000 K per second, greater than 1,000,000 K per second, greater than 10,000,000 K per second, greater than 20,000,000 K per second, greater than 40,000,000 K per second, and greater than 80,000,000 per second. K and the nucleic acid sequence are heated at a rate greater than 1 60,000,000 K per second. A small cross-sectional area channel is also beneficial for the diffusive mixing of any reagent with the sample fluid. 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 flow direction is used while keeping the two fluids flowing through the interface for rapid diffusion mixing. Reducing the cross-section of the channel to less than 1 〇〇, 〇〇〇 square micron, results in a significantly higher diffusion rate than the "large scale" devices. The lithography manufacturing technique allows the microchannels to have a cross-section that spans a flow path that provides a higher mixing rate of less than 16,000 square microns. If only a very small amount of amplicons -51 - 201211243 (as in L O C device 301) is required, the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1 to 2,000 probes on the 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

Keeping the sample mixture close to the heater and using a very small amount of fluid results in rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e., variability, adhesion, and primer extension) was completed in 30 seconds for a target sequence of up to 150 base pairs (bp) long. In most diagnostic analyses, individual thermal cycle times are less than 11 seconds and most are less than 4 seconds. For a target sequence of up to 150 base pairs (bp) long, the LOC device 30 for some of the most common diagnostic assays has a thermal cycle time between 0.45 seconds and 1.5 seconds. This rate of thermal cycling allows the test module to perform nucleic acid amplification procedures in far less than 10 minutes; often within 22 seconds. For most analyses, the amplification section produces sufficient amplicons from the sample fluid entering the sample inlet within 80 seconds. For most of the analysis, sufficient amplicon is generated in 30 seconds. Upon completion of the predetermined number of amplification cycles, the amplicon is fed to the hybridization and detection section 52 via the boiling initiation valve 108. Hybridization Chambers Figures 52, 53, 54, 56, and 57 show hybridization chamber arrays in 11〇 -52- 201211243

Hybridization chamber 180. The hybridization and detection section 52 has a 24 X 45 array 110 of hybridization chambers 180 each having a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184» into the photodiode 184 for detection. Fluorescence from a target nucleic acid sequence or protein that hybridizes to the FRET probe 186. Each of the photodiodes 184 is independently controlled by the CMOS circuit 86. For the emitted light, any material between the FRET probe 186 and the photodiode 184 must be transparent. Therefore, the wall portion 97 between the probe 186 and the photodiode 1 8 4 must also be optically transparent to the emitted light. In the L〇C device 301, the wall portion 97 is a thin layer of cerium oxide (about 0.5 μm). Direct intrusion of photodiode 184 beneath each hybridization chamber 180 allows the use of a very small volume of probe-target hybrid, yet still produces detectable fluorescent signals (see Figure 54). A small volume of hybridization chamber can be used because of the small amount. Prior to hybridization, the amount of probe required to detect the probe-target hybrid is greater than 270 picograms (corresponding to 900,000 cubic micrometers), and in most cases less than 60 picograms (corresponding to Up to 200,000 cubic micrometers, typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and in the case of the LOC device 301 shown in Figure 1 is less than 2.7. picograms (corresponding to a chamber volume of 9,000 cubic micrometers) ). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes on the LOC device. In LOC unit 301, the hybridization portion has more than 1,000 chambers (i.e., less than 2,250 square microns per chamber) in an area of 1,500 microns by 1,500 microns. The smaller volume also reduces reaction time, making hybridization and detection faster. Another advantage of the small number of probes required for each chamber is that during the manufacture of the L0C device -53-201211243, only a very small amount of probe solution needs to be configured into each chamber. A specific embodiment of the LOC device according to the present invention may be configured using a probe solution of 1 nanoliter or less. After nucleic acid amplification, the boiling initiation valve 108 is activated and the amplicon flows along the flow path 176 and into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates the point at which the hybridization chamber 1 1000 is charged with the amplicon and the heater 182 can be activated.

After sufficient hybridization time, LED 26 is activated (see Figure 2). The opening in each of the hybrid chambers 180 is provided with an optical window 136 to expose the FRET probe 186 to the excitation radiation (see Figures 52, 54 and 56). The LED 26 illuminates for a sufficiently long period of time to induce a high intensity fluorescent signal from the probe. During the excitation, the photodiode 184 is shorted. After a preprogrammed delay of 300 (see Figure 2), the photodiode 184 is enabled and the fluorescent emission is detected under no excitation light. Incidence of the active region 185 of the photodiode 184

Light (see Figure 54) is converted to a photocurrent that can be measured using CMOS circuitry 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 hybrid chamber array 1 1 使得 increases the confidence of the results obtained, and if desired, all results can be combined by photodiodes of adjacent hybridization chambers to obtain a single result. Those skilled in the art will appreciate that depending on the analysis details, there may be from 1 to over 1,000 different probes in the hybrid chamber array U0. Humidifier and humidity sensor

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 301. 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 67, by capillary action, water is drawn to the three lower tubes 1 94 and along the individual water supply channels 192 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 an annular heater 191 that surrounds the upper conduit 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 ring heater 191 heats the water causing the water to evaporate and wet the surrounding devices. The position of the humidity sensor 232 is also shown in FIG. However, preferably, as shown in the enlarged view of the illustration AH shown in Fig. 63, 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. The humidity sensor 23 2 abuts the hybridization chamber array 1 1 〇 (the most important humidity measurement location) to slow the evaporation of the solution containing the exposed probe. -55- 201211243 Feedback Sensor

The temperature and liquid sensor system is integrated into the LOC device 310 to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors 170 are assigned to the entire amplification unit 1 1 2 . Similarly, the culture unit 1 14 also has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (B)T to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 utilizes this to accurately control the thermal cycling during the nucleic acid amplification process as well as any heating during the thermal lysis and incubation in the hybridization chamber 180, which uses the hybridization heater 1 8 2 as a temperature sensor ( See Figure 5 6). The resistance of the hybrid heater 108 is temperature dependent and the CMOS circuit 86 utilizes this to obtain a temperature reading of each of the hybridization chambers 180.

The LOC device 301 also has a number of MST channel liquid sensors 174 and a cover channel liquid sensor 208. Figure 35 shows the line of the MST channel liquid sensor 174 at one of the ends of each of the heated microchannels 158. Preferably, as shown in FIG. 37, the MST channel liquid sensor 174 is a pair of electrodes formed by the exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the circuit between the electrodes to indicate where they are located in the sensor. Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. Pairs of TiAl electrode pairs 218 and 220 are deposited on top layer 66. Between the electrodes 2 1 8 and 2 2 0 is a gap 2 22 to keep the circuit open in the absence of liquid -56-201211243. The circuit is closed when the liquid is present and the CMOS circuit 86 uses this feedback to monitor the flow. Gravity autonomous (GRAVITATIONAL INDEPENDENCE)

The test module 10 is directional and operates without being fastened to a smooth surface. The fluid flow driven by capillary action and the lack of external tubing to the auxiliary device make the module truly portable and easily plugged into a similar portable handheld reader, such as a mobile phone. The gravity autonomous operation represents that the test module is also acceleration independent of all practical ranges. The dialysis unit which is resistant to impact and vibration and can operate the dialysis variant on a moving vehicle or on a mobile phone to have a fluid passage to avoid trapped bubbles is described below with reference to Figures 72, 73, 74 and 75. A specific embodiment of the LOC device of LOC variant VIII 518 is shown. The LOC device has a dialysis section that is filled with a fluid sample and trapped in a channel without bubbles. LOC Variant VIII 518 also has an additional layer of material, referenced to interface layer 594. Interface layer 594 is disposed between cover channel layer 80 and MST channel layer 100 of CMOS + MST device 48. Interfacial layer 5 94 causes a more complex fluid interconnection between the reagent sump and MST layer 87 without increasing the size of ruthenium substrate 84. Referring to Figure 73, bypass channel 600 is designed to introduce a time delay from the fluid sample from interface waste channel 604 to interface target channel 602. This 201211243 time delay causes the fluid sample to flow through the dialysis MST channel 204 to the dialysis draw 168 of the fixed meniscus. The capillary action initiation feature (CIF) 2 02 of the bypass channel 600 to the interface target channel 2020 at the upper conduit, the point upstream of all the dialysis suction holes 168 of the dialysis MST channel 204, the sample fluid filling interface Target channel 602. Without the bypass channel 600, the interface target channel 602 still begins to charge from the upstream end, but eventually, the traveling meniscus arrives and passes through the untwisted MST channel above the pipe, leading to the point capture air. The trapped air reduces the sample flow rate through the leukocyte dialysis section 328. A pre-hybridization filter variant of the LOC device, LOC variant XII 758, uses a small component dialysis section 682 located at the outlet of the amplification section 112 (see Figures 9 2 to 9 9). The small component dialysis section 682 provides a pre-hybridization filtration purification stage 29 3 (see Figure 92). Pre-hybridization filters remove cell debris that remains in the sample stream after cell lysis. Hybridization efficiency can be affected by cell debris, so it is advantageous to reduce the concentration of cell debris prior to hybridization. Referring to Figures 97, 98 and 99, the small component dialysis section 68 2 has three adjacent channels in the bottom channel layer 100; the two small component channels 762 are adjacent to both sides of the large component channel 760. A series of tapered bores along the large component passage 760 are provided with a series of tapered bores that provide fluid connection with the small component passages 762. In most practical applications, the taper is 1 to 8 microns wide and 1 to 8 microns high. When the sample flows down to the large component channel 760, particles that are small enough to pass through the inverted tapered opening (eg, amplicons) flow through the small component channels 762-58-201211243, while larger particles (eg, cells) The fragments are left in the large component channels that end up at the blind terminal 766. The smaller particles continue to flow along the small component channels to the opposite side of the hybridization chamber array 110, and the two smaller particle streams all travel along the enthalpy path through the array to the respective blind terminals 768 (see Figure 99). The small component amplicons fill all of the individual hybridization chambers 180 prior to detection. Nucleic acid amplification variant

Direct PCR Traditionally, PCR requires extensive purification of target DNA prior to preparation of the reaction mixture. However, by appropriately changing the chemical and sample concentrations, nucleic acid amplification can be performed using minimal amount of DNA purification, or direct amplification can be performed. When nucleic acid amplification is performed by PCR, this method is called direct PCR. When nucleic acid amplification is carried out in a LOC apparatus at a controlled normal temperature, the method is direct constant temperature amplification. Direct nucleic acid amplification techniques have considerable advantages when used in LOC devices, especially with regard to the simplification of the desired fluid design. Amplification chemical adjustments for direct PCR or direct isothermal amplification include increased buffer strength, the use of highly active and highly progressive polymerases, and additions to potential polymerase inhibitors. It is also important to dilute the inhibitors present in the sample. To exploit direct nucleic acid amplification techniques, LOC devices are designed to incorporate two additional features. The first feature is a reagent reservoir (eg, 'storage tank 58 in Figure 8) that is appropriately sized to supply a sufficient amount of amplification reaction mix or diluent such that the final concentration of sample components that may interfere with the amplification chemistry may be Low enough to successfully perform nucleic acid amplification. Desirable dilution of non-cellular sample components -59- 201211243

The degree is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different LOC structure, such as the pathogen dialysis section 70 of Figure 4, is used. In this particular embodiment (further illustrated in Figure 6), the concentration of the pathogen sufficient to enter the amplification portion 292 is effectively concentrated upstream of the sample extraction portion 290 and the larger cells are discharged to the waste storage tank 76. Dialysis department. In another specific embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest.

A second LOC structural feature that supports direct nucleic acid amplification is to design the aspect ratio of 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 1 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 non-uniform, the flow resistance of the microchannels 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, such as from RNA virus or messenger RNA, RNA must be reverse transcribed into complementary DNA (cDNA) prior to PCR amplification. Can be used in the same chamber as PCR -60- 201211243

A reverse transcription reaction (one-step RT-PCR) is applied, or it may be a separate initiation reaction (two-step RT-PCR). In the LOC variant described herein, a step can be simply performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the staging heater 154 to circulate the reverse transcription step and continue the nucleic acid amplification 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, so that it is not necessary to repeatedly circulate the reaction components at various temperature cycles, but the amplification portion is maintained at normal temperature, usually about 37 ° C. To 41 ° C. Some methods for thermostatic nucleic acid amplification have been described, including strand-substituted amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-dependent amplification (NASBA), recombinant enzyme polymerase amplification (RPA), uncoupling thermostated DNA Amplification (HDA), rolling cycle amplification (RCA), branched amplification (RAM), and circular thermostat amplification (LAMP) and any other or other isothermal amplification methods available for use in the LOC devices herein In a specific embodiment. To perform a constant temperature nucleic acid amplification, reagent reservoirs 60 and 62 adjacent to the amplification section will carry appropriate reagents for a particular constant temperature method rather than carrying PCR amplification mixes and polymerases. For example, for SDA, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, and reagent reservoir 62 contains appropriate endonucleases and exo-DNA polymerases. For rpa, reagent storage -61 - 201211243 tank 60 contains amplification buffer, primer, dNTP and recombinase protein, and reagent reservoir 62 contains a stock-substituted ruthenium polymerase, such as. Similarly, the HDA&apos; reagent reservoir 60 contains amplification buffer, primer and dNTP&apos; and reagent reservoir 62 contains appropriate DN A polymerase and helicase (without heat) to unwind 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 amplification of viral viruses, such as HIV or hepatitis C virus nucleic acids, NASB A or TMA is appropriate because it does not require the transcription of RNA into cDNA. In this example, the reagent reservoir 60 is filled with amplification buffer, primer, and dNTP, and the reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNaseH.

For some types of thermostatic nucleic acid amplification, an initial denaturation cycle must be employed to separate the double-stranded DNA template before maintaining the temperature of the thermostated nucleic acid amplification for continued reaction. Since the temperature of the mixing in the amplification section 112 can be tightly controlled by the heater 154 in the amplification microchannel 158, this denaturation cycle can be easily accomplished in all of the specific embodiments of the LOC device described herein (see Figure 1). 4). Thermostatic nucleic acid amplification is more tolerant to potential inhibitors in the sample and is therefore generally suitable for direct nucleic acid amplification from a desired sample. Thus, constant temperature nucleic acid amplification is particularly useful for LOC variant XLIII 6 73, LOC variant XLIV 674 and LOC variant XLVII 677, respectively, shown in Figures 79, 80 and 81. Direct® temperature amplification can also be as shown in Figures 79 and 81—or multiple pre-amplification dialysis steps 7〇, 68 6 or 6 8 2 and/or as shown in Figure 80-62-201211243 Hybridization dialysis step 682 combines to facilitate partial concentration of target cells in the sample prior to nucleic acid amplification, respectively, or to remove unwanted cell debris prior to entry of the sample into hybridization chamber array 110. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be used. Constant temperature nucleic acid amplification can also be performed in parallel amplification sections, such as those described in Figures 71, 76 and 77. Multiplex and some constant temperature nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA.

Additional Details of the Fluorescence Detection System Figures 58 and 59 show hybridization-reactive FRET probes 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 246 at the 5' end, and a quencher 248 at the 3' end. Loop 240 comprises a sequence that is complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are bonded 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 in close proximity to each other such that a large amount of resonant energy can be transmitted between each other and substantially eliminate the ability of the fluorophore to emit fluorophores when illuminated by the excitation light 244. Figure 59 shows a FRET probe 23 6 in an open or hybridized configuration. Upon hybridization with the complementary target nucleic acid sequence 23 8 , the stem-and-loop structure is disrupted, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to fluoresce. Optical detection of fluorescence emission 250 as a probe has been hybridized -63-201211243 Index" probes are highly specific to hybridization with complementary targets, because the probe stem system is designed to have a single non-complementary nucleotide The probe-target helix is stable. Because the double-stranded DN A is relatively strong, the stereo-top probe—the target helix and the stem helix cannot coexist.” The primer-coupled probe

Primer-linked stem-and-loop probes and primer-linked scorpion probes, also known as scorpion-type probes, are alternatives to molecular beacons and can be used for both immediate and quantitative nucleic acid amplification of LOC devices. 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, thus requiring only a single hybridization event during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures immediate and efficient response and produces stronger signals and shorter reaction times when using separate primers and probes' for better recognition. 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 82 and 83 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 82, the primer-coupled linear 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 nucleus-64-201211243 acid 696 and is labeled with the fluorophore 246 at its 5' end, and linked via amplification blocker 694 Its 3' end to oligonucleotide primer 700. The 3' end of the other strand of stem 242 is labeled with quenching portion 248. After completion of the first round of nucleic acid amplification, the currently complementary sequence 698&apos; probe can be used to wrap around and hybridize to the extended strand. During the first round of nucleic acid amplification, oligonucleotide primer 700 is attached to target DN A 238 (Fig. 82) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker

694 prevents the polymerase from reading through and copying the probe region 6 96 . Upon subsequent denaturation, the hybridized extended oligonucleotide primer 700/template and the primer-linked linear probe of the double stem 242 are separated, thereby releasing the quencher 248. Once the temperature for the adhesion and extension steps is reduced, the primer-linked probe sequence 696 of the primer-joined linear probe is crimped and hybridized to the amplified complementary sequence 698 on the extended strand, and the detected fluorescent light indicates the target. DNA exists. 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 84A through 84F show the operation of the primer-coupled stem-and-loop probes 704. Referring to Figure 84A, the primer-and-loop probe 7〇4 has a complementary double-stranded DNA stem 242 and a loop-like probe sequence loop 240» labeled with one of the stem strands 708 by egg light 246. end. Another 71 标记 is labeled with 3,-end quencher 248, and the other 71 〇 carries both amplification blocker 694 and oligonucleotide primer 700. In the initial degeneration phase (see Figure 84B), -65- 201211243

The stem of the target nucleic acid 23 8 and the stem 242 to which the primer is ligated are separated from the stem-and-loop probe 704. When the temperature is cooled for the adhesive phase (see Figure 84C), the oligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 hybridizes to the target nucleic acid sequence 23 8 . During extension (see Figure 84D), the complement 706 of the target nucleic acid sequence 23 is synthesized to form a DNA strand containing both the probe sequence 704 and the amplified product. The amplification blocker 694 prevents the polymerase from being read through and the probe region 704 is denatured, and when the probe is subsequently attached, the probe sequence of the primer-linked stem-and-loop probe loop segment 240 (See Figure 84F) A complementary sequence 706 adhered to the extended strand. This configuration leaves the fluorophore 246 far from the quencher 248, resulting in a significant increase in fluorescence emission. Control probe

Hybridization chamber array 110 includes a number of hybridization chambers 180 having positive and negative control probes for analytical quality control. Figures 104 and 105 summarize the negative control probe 796 without the fluorophore, and Figures 106 and 107 depict the positive control probe 798 without the extinguishing agent. The positive and negative control probes have a stem-and-loop structure as described above for the F R E T probe. However, regardless of whether the probe hybridizes to an open configuration or remains closed, the fluorescent signal 2 50 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 104 and 105, the negative control probe 796 does not have a fluorophore (and may or may not have a quencher 2 4 8 ). Thus, regardless of whether the target nucleic acid sequence 238 hybridizes to the probe (see Figure 1-5) or the probe maintains its stem-and-loop configuration (see Figure 104), the response to the excitation light 244 can be ignored. Alternatively, the -66-201211243' can be designed with a negative control probe 796 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 Stay in close proximity 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 is not quenching all emissions from the indicator.

Conversely, a positive control probe 7 8 8 without quenching is constructed as shown in FIGS. 106 and 107. Back to the stress luminescence 244, whether or not the positive control probe 798 is hybridized to the target nucleic acid sequence 238, the no substance quenches the fluorescent emission 250 from the fluorophore 246. Figure 52 shows a possible distribution of positive and negative control probes (378 and 380, respectively) in the hybridization chamber array 110. Control probes 3 78 and 380 are placed in hybridization chamber 180 and positioned across the line of hybridization chamber array 1 1 0 . However, the configuration of the control probes within the array is arbitrary (as is the configuration of the hybrid chamber array 110). The fluorophore design requires a fluorophore with a long fluorescence lifetime to allow the excitation light to have sufficient time to decay to a lower intensity than the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal For the noise ratio. Moreover, a longer fluorescence lifetime represents a larger integrated fluorescence count. Fluorescence lifetime 246 (see Figure 59) has a fluorescence lifetime greater than 1 nanosecond, often greater than 200 nanoseconds, more commonly greater than 300 nanoseconds, and greater than 400 nanoseconds in most cases of 67-201211243. . 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. It is a beneficial feature related to the needs of fluorescent detection systems.

A particularly thorough study of the metal-coordination complex based on the transition metal ion ruthenium (Ru(II)) is ginseng (2,2'-bipyridyl) ruthenium (II) ([Ru(bpy)3) ]2+), the lifetime of which is about 1Ms»This complex is available from Biosearch Technologies &gt; under the trade name Pulsar 650 »

Table 1: Photophysical properties of Pulsar 650 (钌 chelate)

Parameter symbol 値 unit absorption wavelength ^abs 460 nm emission wavelength λβπι 650 nm absorption coefficient Ε 14800 M—1 cm·1 egg light lifetime Xf 1.0 μδ quantum yield Η 1 (deoxidized) N/A lanthanide metal-coordination The subcomplex, ruthenium chelate, has been successfully shown as a fluorescent indicator in the FRET probe system and has a long lifetime of 1 600 μ5. -68- 201211243 Table 2: Photophysical Properties of Bismuth Constituents Parameter Symbol 値 Unit Absorption Wavelength 330-350 nm Emission Wavelength λέπι 548 nm Absorption Coefficient Ε 13800 (with Us, and ligand-dependent, up to 30000 (3⁄4) Λβ=,40 nm) Egg photo lifetime Tf 1600 MS (hybridization probe) Quantum yield Η 1 N/A (coordination dependent) LO C device 3 0 1 Fluorescence detection system does not use filters To remove unwanted background fluorescence. If quencher 248 has no natural emission to increase the signal-to-noise ratio, then there is an advantage. Without natural emission, quencher 248 does not contribute to background fluorescence. Quenching efficiency is also important, which results in no fluorescence before hybridization. The black hole quencher (BHQ) from Biosearch Technologies, Inc., Novato, Calif., does not have natural emission and high quenching efficiency, and is used. A suitable quencher for the system. The maximum absorption 値 of φ BHQ-1 occurs at 534 nm and the quenching range is 480-580 nm, making it a suitable quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 5 79 nm and the quenching range is 560_ 6 70 nm makes it a suitable quencher for Pulsar 650. Iowa Black FQ and RQ from Intergrated DNA Technologies, Coralville, Iowa, suitable for a little or no An alternative quencher for background emission. Iowa Black FQ has a quenching range of 42 0-620 nm with a maximum absorption enthalpy at 531 nm and is therefore a suitable quencher for Tb-chelating fluorophores. -69- 201211243

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 embodiments described herein, quenching agent 248 is a functional portion that is initially attached to the probe, but in other embodiments, the quenching agent can be a separate molecule that is free of solution. Excitation source

In the specific embodiment based on the fluorescence detection described herein, the LED is selected as an alternative to the excitation source of the laser diode, high power lamp or laser because of low power consumption, low cost, and small size. Referring to Figure 85, LEDs 26 are disposed directly on the hybrid chamber array 110 from each chamber on the exterior surface of LOC device 301. Opposite the hybrid cell array array is a photosensor 44 consisting of an array of photodiodes 1 84 for detecting fluorescent signals (see Figures 53, 54 and 64).

Figures 86, 87 and 88 summarize other specific embodiments for exposing the probe to excitation light. In the LOC device 30 shown in FIG. 86, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 onto the hybridization chamber array 110. The pulse excitation excites the LED 26 and is detected by the photo sensor 44. In the LOC device 30 shown in FIG. 87, the excitation light 244 generated by the excitation LED 26 is composed of a lens 254, a first aperture 712, and a first A second aperture 714 is directed over the array 11 of hybridization chambers. The pulse excitation excites the LED 20 and the photodetector 44 detects the fluorescent emission. Similarly, the excitation light 244 produced by the LOC device 30' shown in Fig. 88 from the excitation -70-201211243 LED26 is directed by the lens 254, the first mirror 716 and the second mirror 718 onto the hybridization chamber array 110. Re-pulsing the excitation LED 26 again and detecting the fluorescence emission by the photo sensor 44. The excitation wavelength of the LED 26 relies on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for the Pu 1 sar 650 dye. SET UVT0P3 3 5T039BL LED is the appropriate excitation source for the ruthenium chelate label. Table 3: Philips LXK2-PR14-R00 LED Specifications

Parameter Symbol 値 Unit Wavelength λβχ 460 nm Transmit frequency Vem 6.52(10)14 Hz Output power Pi 0.515 parts) @ ΙΑ W Radiation pattern Lambertian data sheet N/A Table 4 : SET UVT0P334T039BL LED Specifications

Parameter Symbol 单位 Unit Wavelength K 340 nm Transmit frequency Ve 8.82(10)14 Hz Power Pi 0.000240 (minutes) @ 20mA W Pulse forward current I 200 mA Radiation pattern Lambertian N/A

Ultraviolet light

Helium absorbs a small amount of light in the UV spectrum. Therefore, it is advantageous to use UV excitation light. A UV LED excitation source can be used, but the broad spectrum of LED 26 reduces the effectiveness of this method. To illustrate this, a filtered UV LED -71 - 201211243 is used. Optionally, a UV laser can be an excitation source unless a relatively high laser cost is not practical for a particular test module market. LED driver

LED driver 29 drives LED 26 at a fixed current for the desired duration. The low-power USB 2.0 certified device is available with a minimum operating voltage of 4.4 volts for up to 1 unit load (1 〇〇 mA). Standard power conditioning circuit is used for this purpose" Light Diode

Figure 54 shows photodiode 184 that is integrated into CMOS circuit 86 of LOC device 301. Light diode 184 is formed as part of CMOS circuit 86 without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCDs, another sensing technique that can be integrated onto the same wafer or fabricated on adjacent wafers using non-standard processing steps. The on-wafer detection system is inexpensive and reduces the size of the analysis system. The shorter optical path length reduces noise from the surrounding environment to effectively collect the fluorescent signal, and suppresses the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the photon collision with its effective area 185. Rate, the photon system is effectively converted into photoelectrons. For standard enthalpy treatment, the quantum efficiency is in the range of 〇·3 to 〇·5 depending on processing parameters such as the number of coating layers and the absorbing properties for visible light. The detection valve of the photodiode 1 84 determines the minimum intensity of the -122-201211243 of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and the number of hybridization chambers 180 in the hybridization and detection section 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (in the example of LOC device 301, which is 1760 microns x 5824 microns) and incorporates other functional modules (such as pathogen dialysis unit 70). And the size of the non-animal parts that can be used after the expansion part 112)

For standard 矽 processing, photodiode 184 detects at least 5 photons. However, in order to confirm reliable detection, the minimum 値 can be set to 〇 a photon. Thus, with quantum efficiency ranging from 0. 3 to 0.5 (as discussed above), the fluorescence emission from the iso probe is a minimum of 17 photons, and a suitable margin of error for reliable detection of 30 photons is included. Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, autofluorescence, and residual excitation photon flux that is not fully attenuated by φ φ introduces background noise and shifts to the output signal. Use one or more calibration signals to remove the background from each output signal. The calibration signal is generated by exposing one or more of the calibration photodiodes 184 in the array to respective calibration sources. A low calibration source is used to determine the negative result of the target 尙 not reacting with the probe. A high calibration source represents a positive result from the probe-target complex. In the specific embodiment described herein, the low calibration source is provided by a calibration chamber 382 in the hybridization chamber array 110, which does not contain any probes; -73-201211243 contains a probe that does not have a fluorescent indicator; Or _ contains a probe with an indicator and a configuration such that quenching agents are always expected to quench.

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 hybridization chambers in the LOC device. The output signal from the other hybridization chamber minus the calibration signal substantially removes the background and leaves the signal generated by the fluorescent emission (if any signal is generated). Signals generated by ambient light in the area of the array of chambers are also removed.

It will be appreciated that the negative control group probe described above with reference to Figures 104 through 107 can be used to calibrate the chamber. However, as shown in Figures 90 and 91, which is an enlarged view of the inset DG and DH of the LOC variant X728 shown in Figure 89, another option is to isolate the calibration chamber 382 from the amplicon fluid. When hybridization is prevented by fluid isolation, background noise and compensation can be cleared by chambers that isolate the fluid or by any probe that contains a missing indicator or any "standard" probe that has both an indicator and a quencher. Judge. The calibration chamber 382 can provide a high calibration source to produce a high signal for the corresponding photodiode. The high signal corresponds to all probes in the hybridized chamber. The indicator, with no quencher or only the indicator spotting probe, will consistently provide a signal that approximates the hybridization chamber signal, and the majority of the probes have been hybridized within the hybridization chamber. It will be appreciated that the calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 throughout the array of hybrid chambers is arbitrary. However, if the photodiode 184 is calibrated by the relatively closest calibration chamber 382-74-201211243, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has a calibration chamber 3 82 for every eight hybridization chambers 180. That is, calibration chamber 382 is positioned in the middle of each three by three square hybridization chamber 180. In this configuration, the hybridization chamber 180 is calibrated by the adjacent hybridization chambers 382.

Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, FIG. 103 shows a differential imager circuit 78 8 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. Differential imager circuit 78 8 samples the signal from pixel 790 and "virtual" pixel 792. In one embodiment, the "virtual" pixel 792 is shielded from light illumination, so its output signal provides a dark reference pixel. Alternatively, the "virtual" pixel 792 can be exposed to the excitation light and the remainder of the array. In one embodiment, the "virtual" pixel 792 is light permeable and the signal from ambient light in the area of the array of cells can also be subtracted. The signal from pixel 790 is weak (eg, close to the dark signal). It is difficult to distinguish between background 値 and very weak signals without reference to the dark signal level. During use, "read_row" 794 and "read_row_d" 795 are enabled and the M4 797 and MD4 801 transistors are turned on. Switches 807 and 809 are turned off such that the outputs from pixel 790 and "virtual" pixel 792 are each stored on pixel capacitor 803 and virtual pixel capacitor 805. Switches 807 and 809 are disabled after the pixel signals are stored. The "read_col" switch 811 and the dummy "read_col" switch 813 are then turned off, and the converted capacitor amplifier 815 is amplified at the output of the differential signal 817. -75- 201211243 Inhibition and activation of light diodes

The photodiode 184 must be suppressed during the excitation of L E D 2 6 and the photodiode 184 must be enabled during the luminescence. 65 is a circuit diagram of a single photodiode 184 and FIG. 66 is a timing diagram of an optical diode control signal. The circuit has a photodiode 184 and six MOS transistors 'Mshunt 3 94, Mtx 3 96, Mreset 398, Msf 400, Mread 402 and Mbias 04. 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 38 8 high. During this period, the photons are excited in the photodiode 184. Generate a carrier. When the amount of carrier generated is sufficient to saturate the photodiode 184, the carriers must be removed. During this cycle, Mshunt 394 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or due to excitation-generated carrier diffusion in the substrate, while Mreset 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 circuit on node 'NS' 608. This is achieved by dragging the tx gate 386 high, which turns on any accumulated carrier on the transistor Mtx 3 96 and the transfer photodiode 184 to node 'NS' 406. The capture cycle can be as long as a fluorescent emission. The outputs from all of the photodiodes 184 in the hybrid chamber array 110 are simultaneously captured. There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. This delay is due to the need to read the respective photodiodes 184 in the array of hybridization chambers 1 1 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, -76-201211243 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 3 93 high. The source-slaffer transistor Msf 400 is used to buffer and read the voltage of the 'NS' node 406. Any other method of enabling or suppressing a photodiode is discussed below: 1 · Suppression method

Figures 100, 101 and 102 show three possible configurations 778, 78 0, 782 for Mshunt transistor 3 94. The Mshunt transistor 3 94 has a very high turn-off ratio at the maximum 値5 V that is enabled during the excitation period. As shown in FIG. 100, the Mshunt Gate 3 84 is configured to be located on the edge of the photodiode 184. Optionally, as shown in Figure 1-1, the Mshunt gate 3 84 can be configured to surround the photodiode 1 84 » The third option is to fabricate the Mshunt gate 384 within the photodiode 184. As shown in FIG. According to the third option, the photodiode effective area 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 384. In FIG. 100, 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 area of the photodiode 185. However, any carrier remaining at the distal end of the photodiode 184 requires a longer time to diffuse through the Mshunt gate 3 84. In FIG. 101, the Mshunt gate 3 84 surrounds the photodiode 184. This further reduces the average path length of the carrier in the photodiode 184 to the Mshunt gate 384. However, extending the Mshunt gate around the photodiode 184 -77 - 201211243 pole 384 causes the optical diode active region 185 to be substantially reduced. Configuration 782 in FIG. 102 positions Mshunt gate 3 84 in active area 185. This provides the shortest average path length to the Mshunt gate 3 84 and thus the shortest transition time. However, the impact on the active area 185 is greatest. It also creates a wide leak path. 2. Method of enabling

a. The flip-flop photodiode drives the parallel transistor with a fixed delay. b. The flip-flop photodiode drives the parallel electro-crystal body with a programmable delay. C. Driving the parallel transistor with a fixed delay by the LED drive pulse d. Drive the parallel transistor as a 2c but with a programmable delay.

68 is a schematic cross-sectional view showing the photodiode 184 and the flip-flop photodiode 187 buried in the CMOS circuit 86 through the hybridization chamber 180. The small area in the corner of the photodiode 184 is replaced by the flip-flop photodiode 187. The trigger photodiode 187 having a small area is sufficient because the intensity of the excitation light is higher than that of the fluorescent emission. The flip-flop photodiode 187 is sensitive to the excitation light 244. The flip-flop photodiode 187 shows that the excitation light 2 44 is extinguished and the photodiode 184 is activated after a brief delay of At 300 (see Fig. 2). This delay allows the fluorescent photodiode 1 84 to detect the fluorescent emission from the F R E T probe 186 without the excitation light 24 4 . This enables detection of -78- 201211243 and enhanced signal-to-noise ratio.

Under each hybridization chamber, both photodiode 184 and flip-flop photodiode 1 87 are located in CMOS circuit 86. The photodiode array is combined with appropriate electronic components to form a photosensor 44 (see Figure 64). The photodiode 184 is a pn junction made during the fabrication of the CMOS structure without the need for additional masking or steps. During MST fabrication, the dielectric layer (not shown) over the photodiode 184 is arbitrarily thinned using standard MST photolithography techniques to cause more phosphor to illuminate the active region 185 of the photodiode 184. The photodiode 184 has a field of view such that fluorescent signals from the probe-target hybrid within the hybridization chamber 180 are incident on the surface of the sensor. The converted fluorescence becomes a photocurrent that can then be measured using the CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 may be dedicated only to the trigger photodiode 187. These choices can be used in combination with 2a and 2b above. Fluorescent Delay Detection The following derivation shows the use of long-life fluorophore fluorescence delay detection for the above LED/fluorescent combination. After the ideal pulse excitation of the fixed intensity Ie between time h and Ο shown in Fig. 60, the fluorescence intensity is derived as a function of time. [^1] (1) is equal to the intensity of the excited state at time t, and then during the excitation period. And then, the number of excited states per unit volume per unit time is described by the following differential equation: Back (0 + calendar = plex... (1) dt tf hve -79- 201211243 where C is the molar concentration of the fluorophore, ε is the molar Extinction coefficient, Ve is the excitation frequency, and h = 6.62606896 (l〇r34Js is the Planck constant^. This differential equation has the general formula: ^- + p{x)y = q{x) The solution is: fe^p {x)dx q{x)dx + k —...(2)

Now use this to solve equations (1) ... (3) and then at time h0,: from equation (3), get k (4) hve substituting (4) into (3) [51] (0 IΑ Tf IΑ τf c_U -hV τί hve hve

At time i2, ..(5) hve hve at d 〇, the excited state decays exponentially and is described by equation (6): [51](〇= [51](/2)e-(,-,l)/ r^ (6) Substituting (5) into (6): hVe The fluorescence intensity is obtained by the following equation: -80- 201211243 I/(0 = -d[S£(t)hv/^ (8) where V/ For the fluorescence frequency, η is the quantum yield 'and 1 is the optical path length. Then we can see from (7): d[S\](t) ^ Ieec dt hve Substituting (9) into (8) ··

If{t) = Ieecln^-[\-eH,1&quot;'),T^ ...(10) because iizilyoo, / (7)- people

Tf Ve Therefore, we can write the following approximation' which describes the decay of the luminous intensity after a sufficiently long excitation pulse (ί2-ίι &gt;&gt;rf): when i then /〆〇= /#/;; ...(11) In the previous section, we summarized the situation for &gt;&gt; rf, and then Κ. From the above equation, we can derive the following formula: nf{t) = nesc^e~{,~,l)lT/ (12) where is the number of fluorescent photons per unit area per unit time and

..._ K n* =7— is the number of excitation photons per unit area per unit time. Therefore, -81 - 00 201211243 nf{t) = [nf{t)dt (13) b where is the number of fluorescent photons per unit area and ^ is the time point at which the photodiode is turned on. Substituting (12) into (13): 00 nf =^riesc^e~{,'ll)lTfdt (14) h At present, the number of fluorescent photons reaching the photodiode per unit area per unit time, %( 0, obtained by:

Nine of them are the light collection efficiency of the optical system. Substituting (12) into (1 5 ) we find that ηΙ(ί) = Φ〇^£ΐ:ιψ'(,',ι)ΙΤ/ &quot;.(16) Similarly, each unit of fluorescent area reaches the photodiode The number of fluorescent photons will be as follows: 〇〇n, =\ns(t)dt , 3 and substituted (1 6 ) and integrated:

Ns = φ^εοίητ fe~〇i~&lt;l)lTf Therefore, the ideal 値 of ns ^Φο^εοΙητ^1^ (17) ί3 is due to the generation of photons in the photodiode 184 due to fluorescence photons. The electron rate is equal to the electron rate generated by the excitation photons in the photodiode 184 because the excitation photon flux decays more rapidly than the fluorescence photon flux. ° Sensor output electrons per unit of fluorescence area due to fluorescence The rate is: βί{ί)=φίη!{1) where &amp; is the quantum efficiency of the sensor at the fluorescent wavelength. -82- 201211243 Substituting (1 7 ) We get: ef{t) = φ^εοίηβ'^'^ (18) Similarly, the sensor output electron rate per unit of fluorescence area of the excited photons : ee(t) = (19) where is the quantum efficiency of the sensor at the excitation wavelength and I is

The time constant of the "cut off" characteristic of the excited LED" After time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and prolongs its decay time, but we assume a negligible effect on If ( t ) So we take a conservative approach. At present, as mentioned earlier, the ideal ί of ί3 is: pure) = Di) Therefore, from (1 8 ) and (1 9 ) we get: = φ-a^h-hVr, and after reforming we get : ΗεοΙηίίΐ) ...(20)

Tf re From the above two paragraphs, we get the following two working equations: .&quot;(21)

...(22) We also know that in fact F = sclrj R = , »τί 0 -83- 201211243 ideal time for fluorescence detection and use Philips LXK2-PR1 4- R00 LED and pulsar 650 dye detection The number of fluorescent photons measured is determined as follows: The ideal detection time is determined using equation (22): recall the concentration of the amplicon, and assuming that all amplicons are hybridized, then the fluorescent concentration of the fluorescent is :c = 2.89(10)_6mol/L. The height of the chamber is the optical path length 1 = 8 (10) · 6ιη. The fluorescent region has been considered to be equivalent to the photodiode region, but the actual fluorescent region is substantially larger than the photodiode region: therefore, it is assumed that nine = 0.5 is the optical collection efficiency of the optical system. The characteristics of the photodiode, $1 = 10 fe, are extremely conservative in the 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 attenuation life cycle &amp; = 0.5 nanoseconds and use

Pulsar650 specifications, can decide the weeping: corpse = [1.48 (10) 6 ] [2.89 (10 wide) [8 (10) _6 ] (1) = 3.42 (10) '5 .. ln ([3.42 (10)-5 ](10)(0.5)) Δί= i 1 1(10)-6 ~ 0.5(10)-9 =4.34(10)'9 s The number of detected photons is determined using equation (2 1 ). The number of excitation photons emitted per unit time is determined by the detection illumination geometry.

The Philips LXK2-PR1 4-R00 LED has a Lamb erti an radiation mode, so: n, = nl0 cos(9) ...(23) where % is the angle to the forward axis of the LED is 每 every 201211243 units The number of photons emitted per unit time by the solid angle, and ^ is the β in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: ή, =|η; ί/Ω η Ω ... (24) Now,

△Ω = 2;τ[1 - cos(0 + Δ0)] - 2;τ[1 - cos(0)] △Ω = 2;r[cos(0) - cos(0 + A0)] dQ. = 2π3ΐη(θ)(ίθ

Substituting (24): π 2 ή, = J 2m)0 cos(e)sin(9)d0 o = ^10 Rearrange, we get:

=^L ΊΟ ...(26) The output power of the LED is 0.515 watts and ν, 6.52 (10) 14 Hz, therefore:

Pi hve ..彳27) __0.515_ _[6.63(10)·34][6.52(10)μ] = 1.19(10)18 Photon 揪 Bring this 値 into (26) We get: -85- 201211243 . .. 1.19(10)18 «; 〇 = - π = 3.79 (10) 17 photons / sec. Referring to Figure 61, the optical center 252 and the lens 254 of the LED 26 are shown in the schematic. The solid angle of the photodiode is 16 micrometers χ 16 micrometers and the solid angle (Ω) of the light cone emitted from the LED 26 to the photodiode 184 for the photodiode in the middle of the array is approximately: Ω = sensor area / r2

[ΙόΟΟ^ΐαόρΟ)&quot;6] ~[2.825(10) 3| =3_21(10)_5 Sphericality The central photodiode 184 of the photodiode array 44 will be understood for use in these calculations. Photosensors located at the edge of the array receive only 2% of the photons for the Lambertian excitation source intensity distribution during the hybridization event. Number of excitation photons emitted per unit time: he = n, Cl ... (28)

=[3.79(10)17][3.21(10)·5] =1.22(10)13 Photon 渺 Now refer to equation (29): η, =Φ〇^τ/β'ΜΙΤ/ ns = (0.5)[ 1.22(10)'3][3.42(10)·5][1(10)-6&gt;-434(1〇γ, /1(ΙΟ)'&lt;= 208 photons per sensor Therefore, use Philips LXK2-PR14-R00 LED and Pulsar 6 50 fluorophore, we can easily detect any hybridization events that cause the number of excited photons. The SET LED illumination geometry is shown in Figure 62. At ID = 20 mA-86-

4.10(10)15 201211243 Ampere, the LED has a minimum optical power output of Pl = 240 microwatts, and the center of the wave is λβ = 3 40 nm (the absorption wavelength of the ruthenium chelate). Drive the ID = 00 mA linearly to increase the output power to ρι = 2.4 m by placing the optical center 252 of the LED at 17.5 mm from the hybridization chamber array 1 and we are concentrating the output flux to a maximum of 2 The dot size of millimeters] The photon flux in the 2 mm diameter point of the hybrid array plane is obtained from (2 7 ). 2.4(10)~3 ~ [6.63(10)'34][8.82(10)14] = 4.10(10)15 Photon 渺 Using equation (28), we get: he = η; Ω [16(10) -6] 2 pairs of 1(10)-3]2 3.34(10)11 photon 渺 long medium LED watts. 0 distance diameter equation

Chelation now, call equation (22) again and use the previously listed Tb properties, A_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 equality 2 1 : ns = (0.5)[3.34(10)11][6.94(10)-5][l(10)-3]e-398&lt;l0r , /, (,o)'3 = 11,600 photons per sensor 87 - 201211243 The number of photons theoretically emitted by the SET LED and the ruthenium chelate system from the hybridization event can be easily detected and far exceeds 3 The minimum number of zero photons is 'this minimum size' is required for reliable detection of the photosensors indicated above. Maximum spacing between the probe and the photodiode

Detection on hybrid wafers avoids the need for detection by a confocal microscope (see the background of the present invention). Deviating from traditional detection techniques is an important factor in saving time and cost associated with the system. Conventional detection requires imaging optics that must use lenses and curved mirrors. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. Placing the photodiode in a very close proximity to the probe has the advantage of extremely high collection efficiency. When the material thickness between the probe and the photodiode is 1 micron, the collection angle of the emitted light is as high as 173°. This angle is calculated by considering the light emitted from the probe closest to the center of the hybrid chamber surface of the photodiode, which has a planar active surface region parallel to the surface of the chamber. The emission pyramid system in which light can be absorbed by the photodiode is defined as having a radiation probe at its apex and at the sensor corners around its plane. For a 16 micron χ 16 micron sensor, the apex angle of the cone is 170°; in the case where the photodiode is expanded such that its area conforms to the hybridization chamber area of 29 μm χ19·75 μm, the apex angle is 】 73 °. It is easily achieved that the separation between the chamber surface and the photodiode active surface is i microns or less. The application of a non-imaging optical method requires that the photodiode 184 be in close proximity to the hybridization chamber to collect sufficient photons of the fluorescent radiation. The maximum spacing between -8 - 201211243 between the photodiode and the probe is determined as shown in Figure 54 below. Using the ruthenium chelate fluorophore and the SET UVTOP33 5T039BL LED, we calculated 11600 photons from individual hybridization chambers 180 to 16 micron x 16 micron photodiodes 184. In carrying out this calculation, we assume that the light collecting 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 out = [(the bottom area of the light collection area of the hybrid chamber) / (photodiode area)] [Ω / 4τι], which is the light diode from the base of the hybridization chamber at the representative point The opposite Ω = solid angle. For the correct square pyramid geometry: Ω = 4a^csin ( a2/(4d〇2 + a2)), where d〇 = the distance between the chamber and the photodiode, and α is the size of the photodiode. Each hybridization chamber releases 23,200 photons, and the selected photodiode has a detection of a minimum of 17 photons, thus requiring minimal optical efficiency.

Φ0 = 1 7/23200 = 7.3 3 χ10·4 The bottom area of the light collection region of the hybridization chamber 180 is 29 μm X 1 9.75 μm. Solving ", the maximum limit distance between the hybridization chamber and the photodiode 1 84 will be dG = 249 microns. In this limitation, the collection cone angle as defined above is only 〇·8°. It should be noted This analysis ignores the negligible effect of refraction. -89- 201211243 LOC variant

The LOC device 301, described and illustrated in detail above, is only one of many possible LOC device designs. Some possible combinations will now be set forth in the context of a flow chart (from sample input to detection) and/or display of LOC device variants using different combinations of the various functional aspects described above. The flow chart is appropriately divided into a sample input and preparation stage 2 8 8 , an extraction stage 290, a culture stage 291, an amplification stage 292, a pre-hybridization stage 293, and a detection stage 294. For the sake of clarity and conciseness, only a brief description or summary of all LOC variants is shown and no detailed configuration is shown. Also for clarity, smaller functional units, such as liquid sensors and temperature sensors, are not shown, but it should be understood that such functional units have been incorporated into the proper location of each of the following LOC device designs.

LOC variant XII

The LOC variant XII758 is shown in Figures 92-99. This LOC device extracts 290, cultures 291, expands 292, and detects 294 pathogen DNA' and uses a pre-hybridization purification step to increase hybridization efficiency. A sample, such as a whole blood sample, is applied to the sample inlet 68 (see Figure 94)&apos; and capillaryly draws the sample to the surface tension valve 118&apos; where the anticoagulant is added from the reservoir 54. The sample continues to the cover channel 94 to the pathogen dialysis section 70. The dialysis section 70 has a bypass passage 600 to avoid trapped air bubbles (see FIG. 94). After the dialysis of the pathogen dialysis section 70, the red blood cells and white blood cell flow are introduced into the waste storage tank 76 while continuing to flow the sample to the surface tension valve 128'. The lysis reagent is added from the storage tank 56. The sample is filled with a chemical lysis chamber 130-90-201211243 in which the sample is retained by the boiling initiation valve 206 until the lysis reagent diffuses through the sample to release most, if not all, of the pathogen DNA. When the boiling initiation valve 206 is opened, the sample flows to the surface tension valve 132 where the restriction enzyme, ligase, and linker primer are added from the reservoir 58. The sample is filled with culture 114 and the sample is heated as the pathogen DN A restriction enzyme and junction junctions occur (see Figure 94).

After the restriction enzyme digestion and the junction bonding, the boiling initiation valve 207 is opened to allow the sample to flow into the amplification portion 112. As the sample flows into the amplification section 112, the amplification mixture from the sump 60 is added via the surface tension valve 138 and the polymerase from the sump 62 passes through the surface tension valve 140. Before the boiling initiation valve 108 is opened to allow the amplicon to flow to the panel dialysis section 682, the pathogen DNA is amplified by thermal cycling, with the large components removed (see Figure 94). Most preferably shown in Figures 97 and 98, the small component dialysis section 682 has a large component channel 760 (see Figure 93) between the two component channels 762 formed in the bottom channel layer 100. The bulk component channel 760 is connected to the sub-channel 762 (smaller at the end of the large component channel) by a hole in the form of a series of reverse tapered openings 764. In most practical applications, the pores are 1 to 8 microns wide and 1 to 8 microns high. As the amplicon flows into the large component channel 760, the population (less than the reverse tapered opening 764) begins to diffuse into the component channel 762. When flowing to the downstream end of the panel dialysis section 682, the concentration of the panel in the bulk channel 760 is lowered. An additional advantage of microfabricated holes is that the number of holes per unit length along the channel is very high, making separation more efficient. For effective separation of components of size by interest, the spacing between adjacent wells is between 1 micron and 10 micrometers; in the specific embodiment -91 - 201211243 shown in Figure 98, between adjacent wells The interval is 8 microns. Figure 9 shows the downstream end of the panel dialysis section 682. The large component channel 760 is turned to terminate at the wide track of the blind terminal 766, and the blind terminal provides a waste sump. Two sub-channels 762 are passed to the opposite side of the hybrid chamber array 1 1 , wherein both pass the array along the meandering path to the respective blind terminals 7 6 8 . The panel amplicon fills up all individual hybridization chambers 1 80 prior to the start of the timing of the hybridization heater and subsequent probe-target hybridization detection.

The panel dialysis section 682 removes cellular debris that may remain in the sample stream after the cells have been lysed. Cell debris can interfere with hybridization efficiency. in conclusion

The devices, systems, and methods described herein facilitate molecular diagnostic testing as a low cost, rapid, and focused care test. The system and its components described above are fully described, and those skilled in the art will be able to readily recognize many variations and modifications without departing from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which: FIG. 1 shows a test module and a test module reader configured for fluorescence detection. . Figure 2 is a schematic representation of the electronic components in a test module configured for fluorescence detection. -92- 201211243 Figure 3 is a schematic diagram of the electronic components in the test module reader. Figure 4 is a schematic view of the structure of the LOC device. Figure 5 is a perspective view of the LOC device. Figure 6 is a plan view of an LOC device having features and structures from all of the layers superimposed on each other. Figure 7 is a plan view of a LOC device having a cover structure that is independently shown

Figure 8 is a top perspective view of the cover with the inner channel and the sump shown in phantom. Figure 9 is an exploded top perspective view of the cover with the inner passage and the sump shown in phantom. Figure 10 is a bottom perspective view showing the cover of the top channel configuration. Figure 11 is a plan view showing the LOC device independently showing the structure of the CMOS + MST device. Figure 12 is a schematic cross-sectional view of the device at the inlet of the sample. Figure 13 is an enlarged view of the illustration aa shown in Figure 6. Figure 14 is an enlarged view of the inset ab shown in Figure 6. Figure 15 is an enlarged view of the inset AE shown in Figure 13. Figure 16 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset ae. Figure 17 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset ae. Figure 18 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset ae. -93- 201211243 Figure 19 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 20 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AE. Figure 21 is a partial perspective view showing the layered structure of the LOC device inside the inset AE. Figure 22 is a schematic cross-sectional view showing the lysing reagent storage tank of Figure 21.

Figure 23 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 24 is a partial perspective view illustrating the layered configuration of the LOC device inside the illustration. Figure 25 is a partial perspective view illustrating the layered configuration of the LOC device inside the illustration AI.

Figure 26 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 27 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 28 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 29 is a partial perspective view illustrating the layered configuration of the LOC device inside the inset AB. Figure 30 is a schematic cross-sectional view of an amplification mixing tank and a polymerase reservoir. Figure 31 shows the characteristics of a separate boiling initiation valve. -94- 201211243 Figure 32 is a schematic cross-sectional view showing the boiling initiation valve of the line 3 2-3 2 of Figure 31. Figure 33 is an enlarged view of the inset AF shown in Figure 15. Figure 3 is a schematic cross-sectional view of the boiling initiation valve shown in Figure 3, line 3, line 4 4-3. Figure 35 is an enlarged view of the inset AC shown in Figure 6. Figure 36 is a further enlarged view showing the inside of the illustration AC of the amplification section

37 is a further enlarged view showing the inside of the illustration AC of the amplification section. FIG. 38 is a further enlarged view showing the inside of the illustration AC of the amplification section. FIG. 39 is a further enlarged view of the inside of the illustration AK shown in FIG. 38. Illustration of the chamber is further enlarged inside the AC

Fig. 41 is a further enlarged view showing the inside of the illustration AC of the amplification section. Fig. 42 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 43 is a further enlarged view of the inside of the illustration AL which is not shown in Figure 42. Figure 44 is a further enlarged view showing the inside of the illustration AC of the amplification section.

-95 - C 201211243 Figure 45 is an enlarged view of the inside of the illustration AM shown in Figure 44. Figure 46 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Figure 47 is a further enlarged view of the inside of the illustration AN shown in Figure 46. Figure 48 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 50 is a further enlarged view showing the inside of the illustration AC of the amplification section. Fig. 51 is a schematic cross-sectional view showing the amplification section. Figure 52 is an enlarged plan view of the hybridization section. Figure 53 is a further enlarged plan view of two separate hybridization chambers. Figure 54 is a schematic cross-sectional view of a single hybridization chamber. Figure 55 is an enlarged view of the humidifier illustrated in the inset AG of Figure 6. Figure 56 is an enlarged view of the inset AD shown in Figure 52. Figure 57 is an exploded perspective view of the LOC device in the inset AD. Figure 58 is a diagram of a FRET probe in a closed configuration. Figure 59 is a diagram of a FRET probe in an open and hybrid configuration. Figure 60 is a graph of excitation light density as a function of time. Exc

Figure 61 is a graph of the excitation illumination geometry (-96 - 201211243 illumination geometry) of the hybrid chamber array. Figure 62 is a graphical representation of the sensor electronics electronics LED illumination geometry. Figure 63 is an enlarged plan view showing the humidity sensor of Figure AH of Figure 6. Figure 64 is a schematic diagram showing an array of photodiodes of a portion of the photosensor. Figure 65 is a circuit diagram of a single photodiode.

Figure 66 is a timing diagram of the photodiode control signal. Figure 67 is an enlarged view of the evaporator of the illustration AP shown in Figure 55. Figure 68 is a schematic cross-sectional view showing the detection of the photodiode and the triggering photodiode through the hybridization chamber. Figure 69 is a diagram of junction-priming PCR. Figure 70 is a schematic view showing a test module having a lancet. Figure 71 is a graphical representation of the structure of LOC Variant VII. Figure 72 is a plan view of a LOC variant VIII having features and structures of all layers stacked one on another. Figure 73 is an enlarged view of the inset CA shown in Figure 72. Figure 74 is a partial perspective view illustrating the layered configuration of the LOC variant VIII in the inset CA shown in Figure 72. Figure 75 is an enlarged view of the inset CE shown in Figure 73. Figure 76 is a diagram showing the construction of LOC variant VIII. Figure 77 is a graphical representation of the structure of the LOC variant XIV. Figure 78 is a graphical representation of the structure of the LOC variant XLI. Figure 79 is a graphical representation of the structure of the LOC variant XLIII. -97- 201211243 Figure 80 is a graphical representation of the structure of the LOC variant XLIV. Figure 81 is a graphical representation of the structure of the LOC variant XLIVII. Figure 82 is a diagram of a linear fluorescent probe linked by a primer during the initial amplification. Figure 83 is a diagram of a linear fluorescent probe coupled to a primer during a subsequent amplification cycle.

Figures 84A through 84F illustrate the thermal cycling of the primer-linked fluorescent stem-and-loop probe. Figure 85 is a schematic illustration of the excitation LEDs for the hybrid chamber array and photodiode. Figure 86 is a schematic illustration of the excitation LED and optical lens that direct light into the hybrid chamber array of the LOC device. Figure 87 is a schematic illustration of an excitation LED, an optical lens, and an optical raft for directing light into the hybrid chamber array of the LOC device.

Figure 88 is a schematic illustration of the excitation LED, optical lens and mirror arrangement for directing light into the hybrid chamber array of the LOC device. Figure 89 is a plan view showing all the features overlapping each other and showing the positions of the insets DA to DK. Figure 90 is an enlarged view of the illustration DG which is not shown in Figure 89. Figure 91 is an enlarged view of the inset DH shown in Figure 89. Fig. 92 is a diagram showing the configuration of the L0C variant XII. Figure 93 is a perspective view of the LOC variant XII. Figure 94 is a plan view showing all the features overlapping each other and showing the positions of the insets FA to FC. -98- 201211243 Figure 95 is a plan view of the LOC variant XII showing only the features of the respective covers. Figure 96 is a plan view showing the LOC variant XII of the respective CMOS + MST device. Figure 97 is an enlarged view of the illustration FA shown in Figure 94. Figure 98 is an enlarged view of the inset FB shown in Figure 94. Figure 99 is an enlarged view of the illustration FC shown in Figure 94.

Fig. 1A shows a specific embodiment of a parallel transistor for an optical diode. Fig. 1 is a specific embodiment of a parallel transistor for an optical diode. Figure 102 is a specific embodiment of a parallel transistor for an optical diode. Figure 103 is a circuit diagram of the differential imager. Figure 104 schematically illustrates the negative control fluorescent probe in the stem-and-loop structure

Figure 105 schematically illustrates the negative control fluorescent probe of Figure 104 in an open configuration. Figure 106 schematically illustrates the positive control fluorescent probe in the stem-and-loop configuration. Figure 107 schematically illustrates the positive control fluorescent probe of Figure 1-6 in an open configuration. Figure 1 08 shows the test module and test module reader that are configured for use with ECL detection. -99- 201211243 Figure 119 is a graphical representation of the electronic components in the test module that are configured for use with ECL detection. Figure 1 1 shows the test module and the alternative test module reader. Figure 1 1 1 shows the test module and test module reader and the host system that houses the various databases. [Main component symbol description] 1 〇: Test module 1 1 : Test module 1 2 : Test module reader 13 : Case 14 : Micro-USB connector 15 : Sensor 1 6 : Micro-USB 埠 17 : Touch Control Screen 18: Display Screen 19: Button 2 0: Start Button 2 1 : Honeycomb Radio 22: Aseptic Sealing Band 2 3: Wireless Network Connection 24: Large Container 25: Satellite Navigation System 26: Light Emitting Diode-100- 201211243 27 = Data Storage 2 8 : Telephone 29 : LED Driver 30 : LOC Device 3 1 : Power Regulator 32 : Capacitor

3 3 : Timer 34 : Controller 35 : Register 36 : Micro USB device 1. 1 or 2.0 3 7 : Driver 3 8 : Random access memory 39 : ECL excitation driver 40 : Program and data cache 41 : ECL excitation register

43 : Program Memory 44 : Light Sensor 45 : Indicator 46 : Cover 47 : Module 48 : CMOS + MST Device 49 : Porous Element 52 : Hybridization and Detection Department -101 201211243

54: Anticoagulant storage tanks 56, 56.1, 56.2, 56.3: Storage tank 57: Printed circuit board 58' 5 8 . 1 , 5 8 · 2 : Storage tank 60, 6 0.1 - 6 0 . 1 2, 6 0 . X : Sumps 62, 62.1, 62.2, 62.3, 62.4, 64: Lower seal 66: Top layer 68: Sample inlet 70: Dialysis section 72: Waste channel 74: Target channel 76: Waste tank 78: Tank layer 80: Cover channel layer 82 : Upper sealing layer 84 : 矽 substrate 86 : CMOS circuit 87 : MST layer 88 : passivation layer 90 : MST channel 92 : lower pipe 94 : cover channel 96 : upper pipe 6 2 . X : sump - 102 - 201211243 97 : wall Part 98: Meniscus Holder 1 00 : MST Channel Layer 101: Laptop/Notebook 102: Capillary Action Start Feature 103: Dedicated Reader 105: Desktop Computer

106: boiling initiation valve 107: e-book reader 108: boiling initiation valve 109: tablet 110, 110.1-110.12, 110.X: hybridization chamber array 1 1 1 : epidemiological data 112, 112.1-112.12, 112. X: Amplification part 1 1 3 : genetic data

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 : Vent 123: Personal Health Record-103-201211243 1 2 5 : Network 126: boiling initiation valve 128, 128.2, 128.3: surface tension valve 130, 130.1-130.3: lysis unit 1 3 1 : mixing unit 132, 132.1, 132.3: Surface tension valve 1 3 3 : incubator inlet channel

134: Lower pipe 1 36 : Optical window 1 3 8 : Surface tension valve 1 40 : Surface tension valve 1 46 : Valve inlet 1 48 : Valve outlet 150 : Under-valve pipe 152 : Ring heater

1 5 3 : Valve heater contact 1 5 4 : Heater 1 5 6 : Heater contact 1 5 8 : Microchannel 1 60 : Outlet channel 1 6 4 : Orifice 166 : Capillary action starting feature 1 6 8 : Dialysis suction port 170 : Temperature sensor - 104 - 201211243 174 : Liquid sensor 175 : Diffusion barrier 176 : Flow path 178 : Liquid sensor 180 : Hybridization chamber

1 8 2 : heater 184 : photodiode 1 8 5 : effective area 1 8 6 : probe 187 : photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : ring heater 192 : water supply 逋Lane 193: Upper Pipe 194: Lower Pipe 1 9 5: Top Metal Layer 196: Humidifier 1 9 8 : Suction Hole 202: Capillary Action Starting Feature 204: MST Channel 206: Boiling Initiating Valve 207: Boiling Initiating Valve 208: Liquid sensor -105 201211243 210 : Microchannel 2 1 2 : MST channel 2 1 8 : Electrode 22 0 : Electrode 222 : Gap 2 3 2 : Humidity sensor 2 3 4 : Heater 2 3 6 : FRET probe 2 3 8 : Target nucleic acid sequence 240 : Ring 242 : Stem 244 : Excitation light 246 : Fluorescent group 2 4 8 : Quencher 2 5 0 : Fluorescence signal 2 5 2 : Optical center 2 5 4 : Lens 2 8 8 : sample input and preparation 2 9 0 : extraction stage 2 9 1 : culture stage 2 9 2 : amplification stage 29 3 : pre-hybridization stage 294 : detection stage 29 6 : first electrode 201211243 2 9 8 : second electrode 3 00 : Programmable delay 301 : L Ο C Device 328: White blood cell dialysis unit 3 76 : Thermally conductive column

3 7 8 : Positive control probe 3 8 0 : Negative control probe 3 82 : Calibration chamber 3 8 4 : Wide pole 3 8 6 : Gate 3 8 8 : Gate 3 9 0 : Retractable lancet 3 92 : Lancet release button 3 9 3 : Idle 394: MOS transistor 396: MOS transistor 3 9 8 : Μ Ο S transistor 400: MOS transistor 402: MOS transistor 404: MOS transistor 406: node 40 8 : film seal

4 1 0 : Membrane guard 5 1 8 : LOC variant VIII 201211243 5 94 : Interfacial layer 600 : Bypass channel 602 : Interface target channel 604 : Interface waste channel

673 : LOC variant XLIII

674 : LOC variant XLIV 677 : LOC variant XLVII 682 : 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 710: strand 7 1 2: first pupil 7 1 4: second pupil 7 1 6 : A mirror 7 1 8 : second mirror 7 5 8 : LOC variant XII 7 6 0 : large component channel -108 - 201211243 762 : small component channel 764 : opening 766 : waste storage tank 768 : blind terminal 7 7 8 : group State 7 8 0 : Configuration 7 8 2 : Configuration

788: Differential Imager Circuit 7 9 0 : Pixel 792 : Virtual Pixel 7 94 : Read_Column 795 : Virtual Read_Column 796 : Negative Control Probe 797 : (Crystal) 7 9 8 : Positive Control Probe

803: pixel capacitor 805: virtual pixel capacitor 807: switch 8 0 9 : switch 8 1 1 : switch 8 1 3 : switch 8 1 5 : capacitor amplifier 8 1 7 : differential signal -109 - 201211243 8 60 : ECL excitation electrode 870 : ECL excitation electrode

Claims (1)

201211243 VII. Patent Application Range: 1. A microfluidic device for removing cell debris from a biological sample, the microfluidic device comprising: a dialysis section having a large component channel, a group channel, and a series of tapered holes for fluid communication between the large component channel and the component channel, the large component channel having an upstream end for receiving the biological sample 'The biological sample is a liquid with a mixture of cell debris and a target molecule having a probe array for attachment to a probe array having a reaction to react with the target molecule to form a probe-target complex. a downstream end of the hybrid portion; wherein each of the tapered holes is tapered in a direction opposite to the flow such that each has a small opening to the large component channel and a large opening to the group of channels, the small openings The size may allow the target molecule to flow into the panel channel 'but retain cell debris larger than the size limit in the large component channel. 2. The microfluidic device of claim 1, wherein the cone φ hole is spaced along the large component channel by a distance between 1 micrometer and 1 〇 micrometer 〇 3 as in the second aspect of the patent application scope The fluidic device, further comprising a plurality of the plurality of sub-channels each sharing a common sidewall with the large component channel and in fluid communication via the series of tapered apertures. 4. The microfluidic device of claim 2, wherein the small opening of each of the tapered holes has a height and a width between 1 micrometer and 8 micrometers. 5. The microfluidic device of claim 4, further comprising -111 - 201211243 comprising a waste storage tank, wherein the large component passage has a downstream end connected to the waste storage tank. 6 - The microfluidic device of claim 2, further comprising a lysis portion upstream of the dialysis portion, wherein the target molecule is a target nucleic acid sequence and the lysis portion is configured to dissolve in the organism The cells in the sample release the target nucleic acid sequence therein. 7. The microfluidic device of claim 6, further comprising a nucleic acid amplification portion for amplifying the target nucleic acid sequence. 8. The microfluidic device of claim 7, wherein the probe is configured to hybridize to the target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid reacting to the excitation light Fluorescent. 9. The microfluidic device of claim 8, further comprising a CMOS circuit for operatively controlling the nucleotide amplification portion, the CMOS circuit also having sensing for hybridization from the probe-target Light sensor for body emission. 10. The microfluidic device of claim 9, wherein the hybrid has an array of hybridization chambers comprising probes for hybridizing to the target nucleic acid sequence. 11. The microfluidic device of claim 10, wherein the photosensor is located adjacent to an array of respective photodiodes of each of the hybridization chambers. 12. The microfluidic device of claim 11, wherein the CMOS circuit has a digital memory for storing data relating to processing the fluid, the data comprising probe details and individual probes in the hybridization chamber array s position. The microfluidic device of claim 12, wherein the CMOS circuit has at least one temperature sensor for sensing temperature in the hybridization chamber array. 14. The microfluidic device of claim 13, further comprising a heater controlled by the CMOS circuit using feedback from the temperature sensor for maintaining the probe and the target nucleic acid sequence at a hybridization temperature . 15. The microfluidic device of claim 14, wherein the photodiode system is less than 249 microns from the corresponding hybridization chamber. 16. The microfluidic device of claim 15 wherein the probe is a fluorescence resonance energy transfer (FRET) probe. 17. The microfluidic device of claim 16, wherein the hybridization chamber has an optical window disposed to expose the FRET probe to excitation light. 18. The microfluidic device of claim 17, wherein the FRET probes each have a fluorophore and a quencher, and when the FRET probe has formed a probe-target hybrid, The fluorophore is configured to emit a fluorescent signal to the photodiode to return the excitation light, and the CMOS circuit is configured to energize the photodiode after a predetermined delay after extinction of the excitation light, the digital memory including the Scheduled delay. 19. The microfluidic device of claim 18, wherein the CMOS circuit has a bond pad for electrically connecting to an external device, and is configured to convert the output from the photodiode into a representative and the target nucleic acid sequence. Hybridizing the signal of the FRET probe and providing the signal to the bond pad for delivery to the external device. -113- 201211243 20. A microfluidic device as claimed in claim 13 further comprising a plurality of reservoirs for retaining a liquid reagent for addition to the sample. -114-