TW201209405A - Microfluidic device with flow-channel structure having active valve for capillary-driven fluidic propulsion without trapped air bubbles - Google Patents

Abstract

A microfluidic device for processing a fluid sample, the microfluidic device having a first channel configured to fill with the sample by capillary action, a second channel configured to fill with the sample by capillary action, a plurality of fluid connections between the first channel and the second channel, one of the fluid connections having an active valve to arrest the sample flow into the second channel and the remaining fluid connections each being configured to pin a meniscus of the sample that arrests capillary flow between the first channel and the second channel, the fluid connection with the active valve being upstream of the remaining fluid connections, wherein during use, the sample flow into the second channel is initiated by activation of the active valve such that the sample flow in the second channel progressively removes the meniscus at each of the meniscus anchors.

Description

201209405 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to 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 get in the future, to determine the type and pathogenicity of an infectious pathogen, or to determine an individual's response to a drug. 201209405 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, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are often required at the 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 humans, red blood cells account for about 99% of cellular material, but they do not carry DN A 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

-6- S 201209405 Diagnostic analysis to be performed. For example, the protocol used to extract viral RN A is quite different from the protocol used to extract genomic DN A. However, self-targeting cell extraction of nucleic acids typically involves a cell lysis step followed by 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 precipitation step is followed by 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-cleaving proteinase 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., 'copy) a particular target (in the case of a low concentration in terms of detectability). The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). The PCR is known in the art and provides a comprehensive and comprehensible description of such reactions in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008. PCR is a useful technique to amplify target DNA sequences against a relatively complex DNA background. If RNA is to be amplified (by PCR), it must first be transcribed into cDNA (complementary DNA) using the 201209405 enzyme called reverse transcriptase. Subsequently, the obtained cDNA was amplified by PCR. PCR is an exponential method which can be continued as long as the conditions for maintaining the reaction are acceptable. The composition of the reaction is ·’

1. Primer pair - a short single strand of DNA having about 10-30 nucleotides complementary to the flanking target sequence region 2. DNA polymerase-synthesis DNA thermostable enzyme

3. Deoxyribonucleoside triphosphate (dNTP) - provides nucleotides integrated into newly synthesized DNA strands. 4. Buffer - provides the best chemical environment for DN A synthesis

PCR generally involves placing these reactants in vials (~10-50 microliters) containing the extracted nucleic acids. 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 90-95 ° C to denature the DN A-strand; in the adhesive phase, lowering the temperature to ~50-6 (TC for adhesion of the primer; then in the extended phase, The temperature is raised to an optimum DNA polymerase activity temperature of 60-72 ° C for extension of the primer. This method repeats the cycle for about 20-40 times, and the final result is to generate a target sequence between millions of copies of the primer. Variants of many standard PCR protocols for molecular diagnostics, including, for example, multiple primer set PCR, linker-primed PCR, direct PCR, repeat sequence IJ (tandem) PCR, real-time PCR to • 8 -

S 201209405 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 sub-targeting multiple genes (in other ways, several trials are required). It is more difficult to optimize multi-initiator PCR because of the need to select primers with approximate adhesion temperature and amplicons 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 conjugate) with a suitable overhanging end is then ligated to the terminal of the target DNA fragment. Nucleic acid amplification is next carried out using oligonucleotide primers specific for the linker sequence. Thereby, all fragments of the DN A 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 P C R reaction is inhibited by many components present in unpurified biological samples, such as the original heme 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. Modification of PCR chemistries for direct PCR involves potentiating buffer strength, using high activity and progressive (processivity 201209405) polymerases and additives chelated with potential polymerase inhibitors.

Repetitive 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-nucleotide 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: If the wrong locus is amplified due to a mistake 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.

Instant PCR or quantitative PCR was used to measure the amount of PCR product in real time. The initial amount of nucleic acid in the 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 particularly 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 amplify RN A viruses, such as human immunodeficiency virus or hepatitis C virus. 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. Constant

-10- S 201209405

The warm nucleic acid amplification method can be performed at the original location or outside the laboratory environment. Including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Cycle Expansion Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent I sotherm DNA A mp 1 ificati ο η and Loop-Mediated Isothermal Amplification Some methods of constant temperature nucleic acid amplification have been described. The thermostatic nucleic acid amplification method does not rely on the continuous heat denaturation of the template DN A to produce a single-stranded molecule as a template for further amplification, but relies on an enzymatic cleavage such as a DNA molecule that specifically limits an endonuclease at a normal temperature, Or other methods of using enzymes to separate DNA strands. 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. An exponential nucleic acid amplification is then achieved by a coupling sense and an antisense reaction in which the strands from the sense reaction are substituted as a template for the antisense reaction. A nicking enzyme (such as N. Alwl, N. BstNBl and Mlyl) which produces a nick on one of the strands of DNA using a DNA which is not cleaved 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 -11 - 201209405 The amplification efficiency of the reaction is increased from 10 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 the RN Α sequence rather than the corresponding genomic DN A. 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 the target ribosomal RNA (rRNA) at a defined position. The reverse transcriptase produces a DNA copy of the target rRNA 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 DN A molecule is produced by synthesizing a new DNA strand from the end of the primer by reverse transcriptase. RN A polymerase recognizes the promoter sequence in the DN A template and initiates transcription. Each newly synthesized RNA amplicon re-enters the process and serves as a template for new replication.

In recombinant enzyme polymerase amplification (RPA), constant temperature amplification of a particular DNA fragment is achieved by binding the opposite oligonucleotide primer to template DN A and extending it by DNA polymerase. Denatured double-stranded DNA (dsDNA) template does not require heat. Instead, RP A uses recombinant enzyme-primer mismatches to scan dsDNA and promote share exchange at cognate locations. The resulting structure is stabilized by the interaction of a single DN A binding protein with a substituted template strand, thus preventing the primer from being released due to branch migration. The recombinant enzyme decomposes to the 3' end of the oligonucleotide to which the strand replaces the DNA polymerase (such as a large fragment of Pol I -12-s 201209405 (hw)), and the primer then begins to extend. This step is repeated by cycling to achieve exponential nucleic acid amplification.

The helicase amplification (HDA) mimics the in vivo system, using DN Α helicase in an in vivo system to generate a single-strand template for primer hybridization and then extending the primer with a DNA polymerase. In the first step of the HDA reaction, the helicase passes through the target DNA, destroying the hydrogen bonds that bind the two strands, which are then bound by a single-stranded binding protein. The single-strand target region exposed by the helicase allows the primer to adhere. DN A polymerase uses free deoxyribonucleoside triphosphate (dNTP) to subsequently extend the 3' end of each primer to produce two DNA replicas. The two replicated ds D N A strands independently enter the next HDA cycle, causing exponential nucleic acid amplification of the target sequence. Other DNA-based thermostating techniques include rolling cycle amplification (RCA) in which a DNA polymerase continuously extends a primer around a circular DNA template to produce a long DN A product consisting of a number of circular repeat copies. By terminating the reaction, the polymerase produces thousands of copies of the circular template with a copy strand tethered to the original target DN A . This results in a spatial resolution of the target and rapid nucleic acid amplification of the signal. A template of up to 1 copy can be produced in one hour. Branch-type amplification is a variant of RCA, and a circular probe (C-probe) or a latching probe and a highly progressive DNA polymerase are used to exponentially expand C at room temperature. - Probe. Circular thermostat amplification (LAMP) provides high selectivity and utilizes DMA polymerase and a primer set containing four specially designed primers to identify 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 stocks triggered by external primers 201209405

Generation of DNA is released by generation of DNA. This serves as a template for the synthesis of DN A initiated by the second internal and external primers, and the second internal and external primers hybridize to the other end of the target to produce a stem-loop DNA structure. In the subsequent LAMP cycle, the 'inner primer hybridizes to the loop on the product and initiates the replacement DNA synthesis' to produce the original stem-loop DNA and the new stem-loop DNA with twice the stem length. The cyclic reaction was continued for less than one hour to accumulate 109 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 nucleic acid amplification, the amplified product must be analyzed to determine whether the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product is to simply measure the size of the amplicon by colloidal electrophoresis and use DNA hybridization to identify the nucleotide composition of the amplicon.

Colloidal electrophoresis is one of the simplest ways to check if a nucleic acid amplification step produces the desired amplicon. Colloidal electrophoresis utilizes an electric field applied to the colloidal matrix to separate the DN A fragment. Negatively charged DN A fragments will move through the matrix at different rates, depending primarily on their size. After the electrophoresis is completed, the fragments in the colloid can be stained to make them visible. Brominated phenanthrenequinone, which is fluorescent 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) 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 DN A, the size of the amplified product can be predicted and detected as a band of known size on the colloid. To confirm why the amplicon or • 14-

S 201209405 When several amplicon are produced, the DN A probe is often used to hybridize with the amplicon. DNA hybridization means the formation of a double-stranded ruthenium by complementary base pairing. A DN A probe of about 20 nucleotides in length is required for DN A hybridization for positive recognition of a particular amplification product. If the probe has a sequence complementary to the amplicon (target) DN A sequence, hybridization will occur at favorable temperature, pH and ion concentration conditions. If hybridization occurs, the gene or DN A 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 the excitation light from being included in the output of the photosensor. Back stress illuminates, the fluorescent molecules emit Stokes-shifted light, and the emitted light is collected by the detection unit. The Tox shift is the frequency difference or wavelength difference between the emitted light and the absorbed excitation light. A light sensor is used to detect ECL emissions, and the light sensor is sensitive to the emission wavelength of the ECL type employed. For example, transition metal coordination complexes emit light of visible wavelengths, thus using conventional photodiodes and CCDs as -15-201209405 photosensors. The advantage of ECL is that if ambient light is excluded, the ECL emission can be the only light present in the detection system, thus increasing sensitivity.

Microarrays allow hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are useful molecular diagnostic tools that can screen thousands of genetic diseases or detect the presence of several infectious pathogens in a single experiment. The microarray consists of a number of different DN A probes that are attached to the substrate and are in the form of dots. The target DNA (amplicon) is first labeled with fluorescent or luminescent molecules (during or after nucleic acid amplification) and then applied to the probe array. The microarray is cultured for several hours or days at a controlled temperature in a humid environment where hybridization occurs between the probe and the amplicon. After incubation, the microarray must be washed with a series of buffers to remove unbound strands. Once cleaned, the surface of the microarray is dried with a stream of air (usually nitrogen). The stringency of hybridization and cleaning is important. Less stringent may result in highly non-specific binding. Excessive rigor may result in inability to properly combine and result in reduced sensitivity. Identification of hybridization by detecting light emission from a labeled amplicon that hybridizes to a complementary probe

Fluorescence from the microarray is detected using a microarray scanner, typically a computer-controlled, inverting scanning fluorescent conjugated focus microscope that typically uses a laser and photosensor that excites the fluorescent dye ( Such as a photomultiplier tube or CCD) to detect the emitted signal. The fluorescent molecules emit down-converted light (as described above) and the 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

S •16- 201209405 makes it possible to detect only the focused portion of the light. Light that is prevented above or below the focal plane of the object enters the detector, thereby increasing the signal-to-noise ratio. The detector converts the detected fluorescent photons into electrical energy, which is then converted into a digital signal. This digital signal is converted to a number representing the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the hybrid target sequence can be simultaneously identified and analyzed.

Find out more about fluorescent probes here: http://www. premierbiosoft.com/tech_notes/FRETjprobe_ 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 medical molecular diagnostics Although molecular diagnostic tests provide advantages, this type of test in clinical tests Growth is not as good as expected and still only accounts for a small portion of the implementation of laboratory medicine. This is mainly due to the complexity and cost associated with nucleic acid testing compared to experiments based on non-amino acid methods. The broad applicability of molecular diagnostic tests to clinical treatment is 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: • Quickly get results that quickly promote treatment and improve care quality -17- 201209405 • Get a very small sample by experiment The ability to test defects. • 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 outpatient visits at the first visit, and simplifying the handling, storage and delivery of samples. • Promote clinical management decisions such as infection control and antibiotic use. Molecular diagnosis 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 nanoliters and microliters also reduces 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 layer for integrating fluid processing onto a single support substrate (typically helium). Utilizing the VLSI (Ultra Large Integrated Circuit) lithography technology of the semiconductor industry, the unit cost of each LOC device is very low. However, controlling the flow of fluid through the LOC unit, adding reagents, controlling reaction conditions, and the like requires large external piping and electronics. Connecting the LOC devices to these external devices substantially limits the molecular diagnostic use of the LOC devices in a laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as a basis for

S -18- 201209405 Practical choice in a local healthcare environment. 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 different states of the invention are now described in the following paragraphs. GVA022.1 provides a microfluidic device comprising: a passage having an inlet, an outlet, and a bore between the inlet and the outlet; wherein the orifice has a geometry that prevents the meniscus of the liquid flow from being fixed to the orifice, such that The flow of liquid from the inlet to the outlet is not interrupted. GVA 022.2 Preferably, the passage is configured to draw liquid from the inlet to the outlet by capillary action. GVA022.3 Preferably, the aperture has a capillary initiation feature to form a meniscus in the aperture such that surface tension draws at least a portion of the meniscus through the aperture and

GVA022.4 Preferably, the microfluidic device also has: a support substrate: a microsystem technology (MST) layer on the support substrate; and a cover overlying the MS T layer, the cover having a plurality of layers between the cover and the MST layer The fluid is in fluid flow from the MST layer to the cap and the liquid stream is flowed from the cap to the MST layer; wherein at least one of the fluid communication between the cap and the MST layer is a hole. GVA022.5 Preferably, the channel extends through the MST layer and the cover is connected to -19-201209405 with less fluid communication. GVA 022.6 preferably has a top layer and a hole portion formed in the top layer. GVA022.7 Preferably, the top layer is less than 5 microns thick. GVA 022.8 Preferably, the channel portion in the MST layer has a cross-sectional area across the fluid direction of less than 400 square microns.

GVA 022.9 Preferably, the liquid comprises a biological sample comprising cells of different sizes, and at least one of the fluid communication is an array of pores sized to avoid passage of cells larger than a predetermined threshold. GVA022.1 0 Preferably, the array of holes is a portion of the dialysis section, and the dialysis section is configured to separate cells larger than a predetermined threshold to a portion of the sample, and the cells containing only less than a predetermined threshold The remaining samples are processed separately. GVA022.il Preferably, the orifices are configured such that cells less than a predetermined threshold include pathogens.

GVA022.1 2 Preferably, the microfluidic device also has a lysis unit configured to lyse the pathogen and release the genetic material therein. GVA022.1 3 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) moiety to amplify the nucleic acid sequence in the liquid. GVA022.1 4 Preferably, the microfluidic device also has a CMOS circuit disposed between the support substrate and the MST layer to operatively control the PCR portion. GVA022.1 5 Preferably, the microfluidic device also has a culture section to culture the liquid prior to amplifying the nucleic acid sequence in the liquid. GVA022.1 6 Preferably, the microfluidic device also has a hybridization portion,

-20-S 201209405 has a probe array GVA022.1 7 for hybridization with a target nucleic acid sequence amplified by a PCR portion. Preferably, the microfluidic device also has a hybridization method for detecting a probe within the probe array. Photodiode array. GVA022.1 8 Preferably, the microfluidic device also has a bond pad and is configured to deliver the hybridization data to an external device. GVA022.1 9 Preferably, the PCR section has a thermal cycle time of less than 4

second. GVA022.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds. The microfluidic holes are easily fabricated and precisely lithographically stepped to provide a reliable liquid unfixed flow path between the two microfluidic stages. GDI014.1 Aspects of the present invention provide a microfluidic device for processing a liquid sample, the microfluidic device comprising: a first channel configured to be filled with a sample by capillary action; and a second channel configured to be filled with a sample by capillary action; A plurality of fluid communication 'one of the fluid communication between the first channel and the second channel has an active valve to prevent sample flow to the second channel' and the remaining fluid communication configurations are configured to fix the meniscus of the sample, which blocks In the capillary flow between the first channel and the second channel, the fluid communication with the active valve is upstream of the remaining fluid communication; wherein during use, the sample flow entering the second channel is initiated by activation of the active valve, such that The sample stream in the second channel is gradually removed from the meniscus of each meniscus holder. -21 - 201209405 GDI014.2 Preferably, one of the remaining fluid connections has a liquid sensor for triggering activation of the active valve. GDI014.3 Preferably, the sample comprises biological samples of different size components, and the plurality of fluid communication holes having a size according to a predetermined size threshold, such that the components flowing into the second channel are less than a predetermined size threshold. The smaller component, while the component remaining in the first channel includes a larger component that is greater than a predetermined size threshold.

GDI014.4 Preferably, the active valve is a boiling initiation valve of a meniscus holder having a meniscus of a fixed liquid, the meniscus blocks fluid, and the heater is disposed adjacent to the meniscus holder such that the heater The liquid on the boiling meniscus holder is activated to remove the meniscus to reflow the fluid. GDI014.5 Preferably, the active valve has a meniscus holder to fix the meniscus of the liquid, the meniscus blocks fluid, and the bending actuator is configured to bend at startup and from the meniscus The retainer removes the meniscus to restore fluid.

GDI 014.6 Preferably, the first channel, the second channel, and the plurality of fluid communication portions are part of the dialysis unit for separately processing the smaller component and/or the larger component. GDI014.7 Preferably, the biological sample is blood, and the larger components include white blood cells, while the smaller components include red blood cells. GDI 014.8 Preferably, the microfluidic device also has an MST layer for analyzing genetic material in the white blood cells, wherein the MS T layer has a lysis unit for lysing the white blood cells to release the target nucleic acid sequence therein. GDI014.9 Preferably, the MST layer has a molecule for amplifying the target nucleic acid

-22- S 201209405 Nucleic Acid Amplification Department. GDI014.10 Preferably, the microfluidic device also has a hybridization portion having a probe array for hybridization with the target nucleic acid sequence, the target nucleic acid sequence being amplified by the nucleic acid amplification portion. GDI014.il Preferably, the probe system is configured to form a probe-target hybrid with the target nucleic acid sequence, and the probe-target hybrid system emits fluorescence in response to the excitation light.

GDI0 1 4.1 2 Preferably, the microfluidic device also has a CMOS circuit for operative control of the PCR portion, the CMOS circuit having a photosensor for sensing the fluorescent emission from the probe-target hybrid. GDI014.13 Preferably, the hybridization portion has a hybridization array chamber comprising a probe for hybridizing to a target nucleic acid sequence. GDI014.14 Preferably, the photosensors are arranged in an array of photodiodes adjacent to the respective hybridization chambers. GDI014.15 Preferably, the photodiode is less than 24.9 microns from the corresponding hybridization chamber. GDI014.16 Preferably, the probe is a fluorescence resonance energy transfer (FRET) probe. GDI014.17 Preferably, the hybridization chamber has an optical window arranged to expose the FRET probe to excitation light. GDI0M.18 Preferably, the FRET probes each have a fluorophore and a quencher. When the FRET probe has formed a probe-target hybrid, the fluorophore is configured to emit a fluorescent signal to the photodiode. Back to stress illumination, the CMOS circuit is configured to enable the photodiode after a predetermined delay, with a predetermined delay occurring at -23-201209405. After the excitation light is extinction, the digital memory contains a predetermined delay. GDI0 1 4.1 9 Preferably, the CMOS circuit has a bond pad electrically connected to the external device, and is configured to convert the output from the photodiode into a signal indicative of a fret probe that hybridizes to the target nucleic acid sequence, and provides a signal To the bond pad for transfer to an external device. GDI014.20 Preferably, the microfluidic device also has a plurality of reservoirs for retaining liquid reagents for incorporation into the sample.

The microfluidic device, which is simple to use, can be mass-produced and inexpensive, receives the biological sample, uses the dialysis section to separate cells of different sizes, and separately processes the nucleic acid contents of the cells separated by their size. The dialysis function extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the analysis system. The dialysis unit is a device that provides low system components and simple manufacturing steps, resulting in an inexpensive analytical system. A fluid-channel structure, particularly with an active valve, provides a capillary phenomenon of the dialysis section that drives the start without trapping bubbles.

[Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Overview This general specification indicates the main components of a molecular diagnostic system incorporating a specific embodiment of the present invention. The details of the construction and operation of this system are described in the following description. Referring to Figures 1, 2, 3, 118 and 119, the system has the following most important components: -24- 201209405

Test modules 10 and 11 are typical USB flash drives and are very inexpensive to manufacture. The test modules 10 and 1 each comprise a microfluidic device, typically in the form of a lab-on-lab (LOC) device 30, preloaded with reagents and typically more than 1000 probes for diagnostic analysis of the molecule. (See Figures i and 1 18). When the test module 1 1 in Fig. 118 uses an electrochemiluminescence-based detection technique, the test module 10 shown in Fig. 1 uses a fluorescence-based detection technique to identify the target. molecule. The LOC device 30 has an integrated light sensor 44 for fluorescence or electrochemiluminescence detection (described in more 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 32 and an inductor 15. Both test modules 10 and 11 are single use for mass production only and are distributed in sterile packaging for use. The outer casing 13 has a large container 24 for receiving biological samples and a removable sterile sealing strip 22 which preferably has a low viscosity adhesive to cover the large container prior to use. The membrane seal 408 having the membrane shield 410 forms part of the outer casing 13 to reduce the moisture resistance in the test module while providing a pressure relief effect by small pressure changes. Membrane guard 4 10 protects membrane seal 408 from damage. The test module reader 12 supplies power to the test module 1 or 11 via the micro-USB port 16. The test module reader 12 can be in many different forms, and its selection is described later. The reader 12 version shown in Figures 1, 3 and 118 is a specific embodiment of a smart phone. A block diagram of the reader 12 is shown in FIG. The processor 42 executes application software from the program storage 43. The processor 42 is also interfaced with the display screen 18 and the user interface (UI) touch screen 17 and buttons 19-25-201209405, the cellular radio 2 1 , 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 data. Alternatively, the location data can be manually entered using touch screen 1 7 or button 1 9 . The data store 27 holds genetic and diagnostic information, test results, patient information, analysis and probe data for identifying each probe, and its array position. The gastric reservoir 27 and the program storage 43 can be shared by a common device. The application software installed in the test module reader I2 provides results analysis and additional test and diagnostic information. To perform a diagnostic test, the test module 10 (or test module 1 1) is inserted into the micro-USB port 16 on the test module reader 12. The sterile sealing strip 22 is turned up and the biological sample (in liquid form) is loaded into the large sample container 24. The start button 20 is pressed to initiate the test by applying the software. The sample is passed to LOC unit 30 and extracted by on-board assay, cultured, amplified and hybridized with sample nucleic acid (target) using a pre-synthesized hybrid-reactive oligonucleotide probe. In the case of the test module 1 ( (which uses fluorescence-based detection), the probe is fluorescently labeled and the LEDs 26 placed in the shell 13 provide the necessary excitation light to induce the self-hybridization probe. Light emission (see Figures 1 and 2). In test module 11 (which uses electrochemiluminescence (ECL) based detection), LOC device 30 carries an ECL probe (as described above) and LED 26 is not necessary to produce illumination. Conversely, electrodes 8 60 and 8 70 provide an excitation current (see Figure 119). The light sensor 44 integrated with the CMOS circuitry on each LOC device is used to detect the emission (fluorescence or photoluminescence). The detected signal is amplified and converted into a digital transmission 201209405 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 1 1 is removed from the test module reader 1 2 and processed as appropriate.

Figures 1, 3 and 1 18 show a test module reader 12 that is configured as a mobile/smartphone 28. In other forms, the test module reader is a laptop/notebook computer 101 used in a hospital, private clinic or laboratory, a dedicated reader 103, an e-book reader 107, a tablet 109 Or desktop computer 105 (see Figure 120). 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 peripherals, such as printers and patient smart cards. Referring to FIG. 121, the data generated by the test module 1 through the reader 12 and the network 125 can be used to update the epidemiological database contained in the host system for the epidemiological data 111 for genetic data. 1 1 3 The host system φ contains the genetic database, the electronic health record contained in the host system for the electronic health record (EHR) 1 1 5, for the electronic medical record (EMR) 121 The electronic medical records contained in the host system and the personal health records contained in the host system for personal health records (PHR) 123. Conversely, via the network 125 and the reader 12, the epidemiological data contained in the host system for the epidemiological data 1 1 1 , the genetic data contained in the host system for the genetic data 113, The electronic health record contained in the electronic health record (EHR) U 5 host system, the host system for electronic medical record (EMR) 121 contains electronic medical records of -27-201209405, and A personal health record contained in the host system of the Personal Health Record (PHR) 123 can be used to update the digital memory in the test module 10 LOC 30.

Referring again to Figures 1, 2, 1 18 and 119, the reader 12 uses battery power in a mobile telephone configuration. The mobile phone reader contains all preloaded 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 Micro-USB port 16 to connect the computer or main power supply to recharge the battery. Figure 71 shows a specific embodiment of the test module 10 for testing that only requires the presence or absence of a particular target, such as whether the test individual is infected with, for example, influenza A virus H1N1. It is only suitable as a built-in module 47 for USB power/indicator only. No other readers or application software is required. The indicator 45 on the module 47 of the USB power/indicator only shows a positive or negative result. This configuration is ideal for large screenings.

Additional items supplied to the system may include test tubes containing reagents for pre-treating a particular sample, and a tongue depressor and lancet containing sample collection. For convenience, the test module of the embodiment shown in Fig. 71 includes a spring-loaded retractable lancet 390 and a lancet release button 3 92. Satellite phones can be used in remote areas. Test Module Electronics Figure 2 and 1 1 9 are block diagrams of the electronic components in Test Modules 1 and 11. The CMOS circuit integrated in the lab device 30 on the wafer has a USB device driver 36, a controller 34, a USB compatible LED driver 29,

S -28- 201209405

Timer 3 3, power conditioner 3 丨, RAM 3 8 and program and data flash 400 million. These are provided for test modules 1 or 11 and associated drivers 37 including the photosensor 44, the temperature sensor 170, the liquid sensor 174 and the various heaters 152, 154, 182, 234. And 39 and the control and memory of the registers 35 and 41. Only the LED 26 (in the example of the test module 1), the external power supply capacitor 32, and the micro USB connector 14 are external to the lab device 30 on the wafer. The on-wafer laboratory device 30 includes an adhesive pad for joining to these external components. The RAM 38 and the program and data flash memory 40 have application software and diagnostic and detection information for 1 000 probes (flash/security storage, such as via encryption). In the example of the test module 1 1 configured with ECL detection, there is no LED 26 (see Figures 1 18 and 1 19). The data is encrypted by the lab device 30 on the wafer to preserve storage and communicate with external devices. The lab device on the wafer is loaded with electrochemiluminescent probes and the hybridization chamber, each having ECL excitation electrode pairs 860 and 870. Many types of test modules 1 are manufactured in some form of inspection and are ready for ready use. These assay formats differ in the onboard analysis of reagents and probes. Some examples of infectious diseases that are rapidly identified by this system include: Influenza-influenza virus A, B, C, infectious salmon anemia virus, Tossin virus • Pneumonia-respiratory fusion virus (RSV), gland Virus, interstitial pneumonia virus, pneumococci, staphylococcus aureus, tuberculosis, mycobacterium tuberculosis, mycobacterium bovis, mycobacteria, mycobacteria, and mycobacteria;, > -29 - 201209405 • Plasmodium falciparum, Toxoplasma gondii and other parasitic protozoa • Typhoid- typhoid bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever - flavivirus • Hepatitis (A to E)

• Iatrogenic infections - such as Clostridium pneumoniae, vancomycin-resistant enterococci, and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Giant cell virus (CMV) • Epstein-Barr virus (EBV) • Encephalitis - Japanese Encephalitis Virus, Zhangdipula Virus • Pertussis - Pertussis • Hemp - Paramyxovirus • Meningitis • Streptococcus pneumoniae and Meningococcus. • Anthracnose - Bacillus anthracis Some examples of hereditary diseases include: • spastic fibrosis • hemophilia • sickle cell anemia • sable idiots • hemochromatosis • cerebral arterial disease • Crohn's disease

S -30-

201209405 • Polycystic kidney disease • Congenital heart disease • Leier's disease A few options for cancer identified by this diagnostic system include: • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinoblastoma • Turkic disease (Turcot syndrome) The above list is not exhaustive, and the diagnostic system can be configured to detect the structure of many different diseases and symptomatic system components using nucleic acid and proteomic analysis. The LOC device LOC device 30 is the center of the diagnostic system. It uses microfluidic flow to perform four important steps in nucleic acid-based molecular diagnostic analysis: sample preparation, nucleic acid extraction, nucleic acid amplification, and detection. The LOC device is also an alternative use' and will be described in detail below. As discussed above, the test module 1 1 can take many different configurations to detect different targets. Similarly, device 30 has a number of different implementations for the target of interest. One form of LOC device 30 is a LOC device 301 for fluorescence detection of nucleic acid sequences in a pathogen of a whole blood sample. The structure and operation of the LOC device 301 for purposes of illustration are described with reference to Figures 4 through 26 and 27 through 57. Set to stand fast, that is, with 10 and LOC. Target, detail -31 - 201209405 Figure 4 is a schematic diagram of the LOC device 3 01 structure. For the sake of convenience, the processing stages shown in Fig. 4 are denoted by the component symbols corresponding to the functional portions of the LOC device 3〇1 that implements the processing stage. The processing stages associated with the main steps of each of the nucleic acid-based molecular diagnostic assays also represent: sample input and preparation 28, extraction 290, culture 291, amplification 292, and detection 294. The various reservoirs, chambers, valves, and other components of the LOC device 301 will be described more closely below.

FIG. 5 is a perspective view of the LOC device 301. It is manufactured using high volume CMOS and MST (microsystem technology) manufacturing techniques. The layered construction of the LOC device 301 is illustrated in a partial cross-sectional view (not to scale) of Figure 12. The LOC device 301 has a germanium substrate 84 supporting a COMS + MST wafer 48, including a CMOS circuit 86 and an MST layer 87, covering 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. Accordingly, 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 manufacturing methods can also produce structures and assemblies that are sized for liquid flow driven by capillary action and that are processed to very small volumes. The particular embodiment described in this specification shows that the MST layer is a structural and active component that is supported on CMOS circuitry 86, but excludes the features of cover 46. However, those skilled in the art will appreciate that the MST layer does not require the underlying CMOS or even the overlying cover to handle the sample. The overall size of the LOC device shown in the following figure is 1760 microns x • 32-

S 201209405 5824 microns. Of course, LOC devices made for different applications can have different sizes. Figure 6 shows the features of the M ST layer 87 that overlaps the cover feature. The illustrations AD to AD, AG and ΑΗ 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 is 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 are schematic views showing the layered configuration of the CMOS + MST device 48, the cover 46, and the fluid interaction therebetween. The figures are not drawn to scale for illustrative purposes. Figure 12 is a cross-sectional view through the sample inlet 68 and Figure 22 is a cross-sectional view through the sump 54. As best shown in FIG. 12, the CMOS + MST device 48 has a germanium substrate 84 that supports the φ CMOS circuit 86 that operates the active components in the MST layer 87 described above. Passivation layer 88 seals and protects CMOS layer 86 from fluid flow through the M S T layer 87. Fluid flows through both the cover channel 94 and the MST channel 90 in the cap layer 46 and the MST channel layer 100 (see, for example, Figures 7 and 16). While biochemical treatment is being performed on the smaller MST channel 90, cell transport occurs in the larger channel 94 made in the cover 46. The cell transport channel is sized to carry the cells in the sample to a predetermined point in the MST channel 90. Transporting cells larger than 20 microns in size (e.g., certain white blood cells) requires channel sizes greater than 20 microns, and thus the cross-sectional area across the flow is greater than -33 - 201209405 400 square microns. The MST channel, particularly at locations in the LOC that do not need to transport cells, can be significantly smaller.

It will be understood that the cover channel 94 and the MST channel 90 are generic referenced. The special M S T channel 90 can also be referred to, for example, as a heated microchannel or a dialysis MST channel, depending on its particular function. The MST channel 90 is formed by etching through the MS channel layer 100 deposited on the passivation layer 88 and patterned with a photoresist. The MST channel 90 is surrounded by a top layer 66 that forms the top of the CMOS + MST device 48 (relative to the orientation shown in the figure).

Although sometimes shown as a separate layer, the cover channel layer 8 and the reservoir layer 78 are formed from a single piece of material. 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 80 and a reservoir layer 78'. The lid channel 94 is etched in the lid channel layer 80, and the reservoirs 54' 56, 58, 60 and 62 are etched in the reservoir layer 78. Further, the sump and the cover passage are formed by a micro-forming process. Both etching and microforming techniques are used to fabricate channels having a cross-sectional area across the fluid that is as large as 20,000 square microns and as small as 8 square microns. There are a series of suitable choices for one of the cross-sectional areas of the channels 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 contained 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° - 34 - 201209405. The five sump 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: sump 54: anticoagulant' which has the option of including red blood cell lysis buffer. • Storage tank 56: lysis reagent

• Storage tank 5 8 : Restriction of enzymes, ligases and junctions (for junction-priming PCR (see Figure 70, from t. Stachan et al, Human Molecular Genetics 2, Garland Science, NY and London, 1 9 9 9) Excerpt) • Slot 60: amplification mixture (deoxynucleotide triphosphate (dNTPs), primer, buffer) and • sump 62: DNA polymerase the cover 46 and the CMOS + MST layer 48 via the lower seal 64 is in fluid communication with a corresponding opening in the top layer 66. The openings represent the upper conduit 96 and the lower conduit 92 depending on whether the fluid φ flows from the MST passage 9 to the cover passage 94 or vice versa. The LOC device operates The operation of the LOC device 301 is described step by step with reference to the analysis of the pathogen DNA in the blood sample. Of course, other biological or non-biological liquid species also use appropriate reagents, detection protocols, LOC variants, and sets or combinations of detection systems. Analysis. Referring again to Figure 4, the analysis of the biological sample involves five major steps, including: sample input and preparation 288, nucleic acid extraction 290, nuclear-35-201209405 acid culture 291, nucleic acid amplification 292, and detection and analysis 294.

The sample input and preparation step 28 8 involves mixing the blood with the anticoagulant 1 16 and then separating the pathogen from the white blood cells and the 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 action and enters the MST channel 90 via the lower conduit 92. The lower conduit 92 has a capillary initiation configuration feature (CIF) 102 to form the meniscus geometry such that it is not fixed to the edge of the lower conduit 92. When the anticoagulant 116 is withdrawn from the sump 54, the vent 122 in the upper seal 82 allows air to replace the anticoagulant 116.

The MST channel 90 shown in Figure 22 is part of the surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and is secured to the meniscus 120 of the upper tube 96 to the meniscus holder 98. Prior to use, the meniscus 120 remains fixed to the upper conduit 96 such that the anticoagulant does not flow into the lid passage 94. As the blood flows through the cover channel 94 to the upper conduit 96, the meniscus 120 is removed and the anticoagulant is drawn into the fluid. 15 to 21 show an illustration AE ' which is a portion of the illustration AA shown in Fig. 13. As shown in Figures 15, 16 and 17, the surface tension valve ι 18 has three separate MST passages 90 extending between the individual lower conduits 92 and the upper conduits 96. These MST channels 90 in the surface tension valve can be varied to change into -36-

S 201209405 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 of 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 use an array of holes 1 64 sized according to a predetermined valve.

The sample is concentrated. Cells smaller than the valve pass through the pores, and large cells cannot pass through the pores. At the same time as the target cells continue to be part of the analysis, the unwanted cells are reintroduced into the waste unit 76. The unwanted cells are large cells blocked by the array of holes 1 64, or small cells through the wells are in the pathogen dialysis section 70 described herein, and the pathogens from the whole blood sample are concentrated for microbial DNA analysis. . The array of holes is formed by fluidly communicating the input 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 φ 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 well 164 via the dialysis MST channel 206 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 department and dialysis variants are provided later. -37- 201209405

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 MST 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 ( It is assumed that the physical properties of the fluid are approximately equal). Thus the proportion of cytolysis agent in the sample mixture is greater than the ratio of the anticoagulant. The lysis reagent and target cells are mixed by diffusion in the target channel 74 in 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 shown in Figures 31 and 3 Detailed description is given below. Other active valve types (as opposed to passive valves such as the surface tension valve 1 18) 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 1 26 is opened, the lysed cells flow into the mixing portion 131 for pre-amplification restriction digestion and linker ligation. Referring to Figure 13, when the fluid is removed from the meniscus on the surface tension valve 132 initiated by the mixing portion 131, the restriction enzyme, linker and ligase are released from the sump 58. The mixture flows through the length of the mixing portion 133 for diffusion mixing. At the end of the mixing portion 131 is a lower duct 134 (see Fig. 13) leading to the incubator inlet passage 133 of the cultivating portion 114. The incubator inlet passage 133 feeds the mixture into the crucible configuration of the heated microchannel 210, which is -38-

S 201209405 provides a culture chamber that retains the sample during restriction enzyme digestion and junction ligation (see Figures 13 and 14).

Figures 23, 24, 25, 26, 27, 28 and 29 are shown in Figure AB of Figure 6.

The layers of the LOC device 301 within. The various figures show the continuous addition of the layers forming the CMOS + MST layer 48 and the structure of the cover 46. The illustration AB shows the end of the culture portion 114 and the start of the amplification portion 112. As best shown in Figures 14 and 23, the fluid breaks into the microchannels 2 10 of the culture portion II4 until the boiling initiation valve 106 is reached, wherein the fluid ceases 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 stable temperature for 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 291 (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 increase in temperature before entering the amplification section 112 renders the restriction enzyme and ligase inactive. There is a specific association between restriction enzyme and ligase inactivation when using thermostated nucleic acid amplification. 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 crucible structure of the heated microchannels 158 until the boiling initiation valve 1〇8 is reached, which form one or more amplification chambers. As shown in the cross-sectional schematic view of Fig. 30, the amplification mixture (dNTP, primer, buffer) is released from the storage tank 60 and the polymerase is then released from the storage tank 62 into the culture section and the amplification -39-201209405 (each The intermediate MST channel 212 is 114 and 112).

Figures 35 through 51 show the layers of the LOC device 301 in the inset AC of Figure 6. 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 1 12 and the start of the hybridization and detection unit 52 are shown. The cultured sample, amplification mixture, and polymerase flow through the microchannel 1 58 to the boiling initiation valve 108. After diffusion mixing for a sufficient time, the heater 154 in the microchannel 158 is activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification procedure, the boiling initiation valve 1〇8 is opened and the fluid re-enters the hybridization and detection unit 52. The operation of the boiling initiation valve is described in more detail below.

As shown in Figure 52, hybridization and detection portion 52 has an array 110 of hybridization chambers. Figures 52, 53, 54 and 56 show the hybridization chamber array 11 and individual hybridization chambers 180 in detail. The entrance to hybridization chamber 180 is a diffusion barrier 175 that prevents diffusion of the target nucleic acid, probe strands, and hybridization probes between hybridization chambers 18 during hybridization to prevent erroneous hybridization assay results. The flow path length of the diffusion barrier 175 is long enough to prevent the target sequence and probe from diffusing out of one chamber and contaminating another chamber during probe and nucleic acid hybridization and detection signals, 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 different weights. The thermal energy required to supply hybridization is provided by the CMOS controlled heater 182. -40-

S 201209405

(described in more detail below). Hybridization occurs between the complementary target probe sequences after activation 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-loop structure of the F R E T probe 186 to open, which allows the fluorophore to emit back the fluorescent energy that should be LED-excited, as detailed below. Fluorescence is detected by photodiode 184 in the CMOS circuit 86 located below each hybridization chamber 180 (see the hybrid chamber description below). The photodiode 18 4 and associated electronics for all of the hybridization chambers together form the photosensor 44 (see Figure 65). In other embodiments, the photosensor can be a charge coupled device array (CCD array). The signal detected from the photodiode 184 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 φ 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 functional portions or functional portions for alternative uses of the above-described devices may be added. For example, the culture portion 1 14 can be used as the first amplification portion of the repeated sequence amplification analysis system, and the chemical lysis reagent storage tank 56 can be used to add the first amplification mixture of the introduction -41 - 201209405, dNTP, and buffer. And reagent reservoir 58 is used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with amplification mix) may be added to the sump 5, or alternatively, the sample may be heated for a predetermined period of time to generate thermal lysis in the culture. In some embodiments, if chemical lysis is desired and the chemical lysis reagent is mixed therewith, additional sump can be combined upstream of the sump 58 for mixing the primer, dNTP, 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 1M 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 heaters operate independently, so when the reaction relies on heat, such as hot lysis, the heater is not activated during this step, ensuring that hot lysis does not occur in LOC devices that do not require hot lysis. The dialysis section 70 can be located at the beginning of the fluid system within the microfluidic device as shown in Figure 4, or can be located at any other location within the microfluidic device. In some cases, for example, after the amplification phase 292, prior to the hybridization and detection step 2 94, dialysis is performed to remove cell debris. Alternatively, two or more permeable sections can be combined at any location on the LOC device. Similarly, additional amplifications 112 can be combined to enable simultaneous or sequential amplification of multiple targets prior to detection using a particular nucleic acid probe in hybridization chamber array 110. To analyze, for example, a sample of whole blood in which dialysis is not required, the dialysis section 7 is simply omitted from the sample input and preparation section 288 of the LOC design. In some cases, even if the analysis does not require dialysis, -42-

S 201209405 It is not necessary to omit the dialysis section 70 from the LOC device. If the presence of the dialysis section does not create a geometrical obstruction, the sample input and preparation section can still have the LOC of the dialysis section without losing the desired function. In addition, 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 a sample target protein present in the non-amplified sample, rather than a design Nucleic acid probe for hybridization 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. The vast majority of functional units are common in many LOC devices, and the design of additional LOC devices for new applications has a combination of appropriate functional components in the bulk 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 other 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

-43- 201209405 is configured to release its contents and use only the dialysis, lysis, culture and amplification sections to transfer 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, for example, prior to input into the LOC device, it may be necessary to liquefy the semen and possibly pre-treat the mucus with enzyme to reduce stickiness.

Sample Input Referring to Figures 1 and 12, a sample is added to the large container 24 of the test module 1 . 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 μηι wide 60 μπη deep cover channel 94 and is also attracted to the anticoagulant storage tank 5 by capillary action. Reagent storage tank

Using a microfluidic device, such as LOC device 301, the small amount of reagent required by the analytical 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 less than 300,000,000 cubic micrometers, typically less than 70,000,000 cubic micrometers, and in the case of the LOC device 301 shown in the figures is less than 20,000,000 cubic micrometers. Dialysis Section Referring to Figures 15 through 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate the pathogen target cells from the sample. As mentioned above, the top layer 6 6 -44-

S 201209405

i is a plurality of orifices of orifice 164 having a diameter of 3 microns, filtering target cells from a large number of samples. As the sample flows through a 3 micron diameter orifice, the 1 4 4 'microbial pathogen passes through the well into a series of dialysis MST channels 204 and is returned to the target channel 74 via the 16μηι dialysis extraction well 168 (see Figures 33 and 34). The remaining sample (red blood cells, etc.) is retained in the cover channel 94. Downstream of the pathogen dialysis section 70, the cover channel 94 becomes a waste channel 72 to the waste reservoir 76. Foam illustrations or other porous elements 49 in the outer casing 13 of the test module 10 are configured to be in fluid communication with the waste reservoir 76 (see Figure 1) for a biological sample type that produces a substantial amount of waste. The pathogen dialysis unit 70 operates with the capillary action of the fluid sample. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis section 70 has a capillary action initiation feature (CIF) 166 (see Figure 33) such that fluid is drawn down into the underlying dialysis MST channel 204. The first suction aperture 198 for the target passage 74 also has a CIF 202 (see Figure 15) to prevent fluid from easily securing the meniscus above the dialysis suction aperture 168. The panel dialysis section 682, which is schematically shown in Fig. 79, may have a structure similar to that of the pathogen dialysis section. By sizing (and shaping, if necessary) suitable for orifices that allow small target cells or molecules to pass to the target channel and continue to be further analyzed, the panel dialysis section separates the sample from any small target cells or molecules. Large size cells or molecules are removed to waste reservoir 766. Thus, LOC device 30 (see Figures 1 and 18) is not limited to isolation of pathogens having a size less than 3 μηι, but can be used to isolate cells or molecules of any desired size. Lysis Department -45- 201209405

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 for 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 filled with the culture portion 1 14 and stopped at the boiling initiation valve 106. The culture microchannel 210 heats the sample to a 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 trigger valve

As discussed above, LOC device 301 has three boiling initiation valves 126, 106, and 108. The position of these valves is shown in Figure 6. Figure 31 is a plan view showing the enlargement of the independent boiling initiation valve 108 at the end of the heated microchannel 158 of the amplifying portion 112. By capillary action, sample stream 119 is attracted along heated microchannels 158 until it reaches 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 causes the meniscus to stop moving forward to prevent capillary flow. As shown in Figures 31 and 3, the meniscus holder 98 is provided by an orifice from the upper opening of the pipe from the MST passage 90 to the cover passage 94. The surface tension of the meniscus 120 keeps the valve closed. A ring heater 152 is located around the valve inlet 146. The annular heater 152 is CMOS controlled via a boiling induced valve heater contact 153. -46-

S 201209405 To open the valve, CMOS circuit 86 sends an electrical pulse to valve heater contact 1 5 3 . The ring heater 125 is heated in a resistive manner until the liquid sample 1 19 is boiled. Boiling removes meniscus 120 from valve inlet 146 and begins to wet cover passage 94. Once the wet cover channel 94 is started, 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 16 to the hybridization and detection portion 52. The liquid sensor 1 7 4 is placed for diagnosis

Before and after the valve. It will be understood that once the 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 Figs. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51 show the culture unit 114 and the amplification unit 112. The culture portion 114 has a single, heated φ culture microchannel 210 that is etched to form a ruthenium pattern in the MST 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 1?6 to the boil-inducing 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 submarine pattern of the microchannel also promotes (to some extent) the mixing of the target cells with the reagents.

-47- 201209405

In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated via a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. 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 1 5 6 (see Figure 1 4). )) 'It 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 Π 2, each of the wide zigzags 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, typically less than 70 nanoliters, and in the case of LOC device 301, this volume is between 2 nanoliters and 30 nanoliters. . Increased heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the amplification fluid mixture. All fluids are kept at a relatively short distance from the heater 154. The channel cross-sectional area (i.e., the cross section of the augmented microchannel 158) is reduced to less than 100,000 square microns, while the "high scale" equipment has a significantly higher heating rate. The lithography manufacturing technique allows the amplifying microchannel 158 to have a cross-section that provides a higher heating rate across substantially less than 16,000 square microns. Made with lithography -48-

S 201209405 technology easily achieves 1 micron size features. If only a very small amount of amplicons are required (as in the case of L Ο C device 301), the cross section can be reduced to less than 2,500 square microns. For a diagnostic analysis of 1 to 2,000 probes on a LOC device and "sample entry, answer out" within 1 minute, the appropriate cross-sectional area across the fluid is 400 square microns and Between 1 square micron.

The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate of greater than 80 absolute temperature (K) per second, in most cases at a rate 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 heats the nucleic acid sequence at a rate greater than 1 000 000 K per second. Typically, based on the requirements of the analytical system, the heater elements are greater than 1 每秒 per second, 〇〇〇 K, greater than 1,000,000 K per second, greater than 1 0,000,000 K per second, greater than 20.000 000 K per second, greater than A temperature of 40,000,000 K, greater than 80.000. 000 K per second, and a rate of greater than 160,000,000 K per second heating the edge acid channel small cross-sectional area channel is also beneficial for 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 channel cross-section to less than 100, 〇〇〇 square microns results in a significantly higher diffusion rate than "large-scale" devices. The lithography manufacturing technique allows the microchannels to have a cross-section that spans less than 16,000 square microns and substantially provides a higher flow path of -49-201209405. If only a very small amount of amplicons are required (as is the case in LOC unit 301), the cross section can be reduced to less than 2,500 square microns. For the diagnostic analysis required for "sample entry, answer out" in 1 minute on a LOC device, the appropriate cross-sectional area across the fluid is 400 square microns and 1 square micron. between. 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., denaturation, 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 thermal cycle time of the LOC device 30 for some of the most common diagnostic assays is between 0.45 seconds and 1.5 seconds. This rate of thermal cycling allows the test module to complete the nucleic acid amplification procedure in less than one minute; often within 220 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 amplicons were generated in 30 seconds. Upon completion of the predetermined number of amplification cycles, the amplicon is fed to the hybridization and detection portion 52 via the boiling initiation valve 1 〇8. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show the hybrid chamber 180 in the hybridization chamber array 110. Hybridization and detection section 52 has 24 X 45 arrays of hybridization chambers 180

S -50- 201209405

Column 1 ίο each has a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. Photodiode 184 is incorporated to detect fluorescence from a target nucleic acid sequence or protein that hybridizes to 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 184 must also be optically transparent to the emitted light. In LOC device 301, wall portion 97 is a thin layer of ruthenium dioxide (about 0.5 microns). 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 a detectable fluorescent signal (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 amount of probe-target hybrid is greater than 270 pg (1^(:(^^111) (corresponding to 900,0〇0 cubic microns), In most cases less than 60 picograms (corresponding to 200,000 cubic micrometers), typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and less than 2.7 picograms in the case of the LOC device 301 shown in the figures. (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chamber allows for higher chamber densities and therefore more probes on the LOC device. In the LOC device 301, at 1,5〇0 In the micron by 1,500 micron area, the hybrid has more than 1,000 chambers (ie, less than 2,250 square microns per chamber). 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 LOC device, 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 can be used with 201209405 1 nanoliter or less probe solution configuration After nucleic acid amplification, the boiling initiation valve 1 〇 8 is activated and the amplicon flows along the flow path 176 and flows into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 indicates hybridization. The chamber is filled with amplicon and the time at which the heater 182 can be activated.

After sufficient hybridization time, LED 26 is activated (see Figure 2). The opening in each hybridization chamber 180 is provided with an optical window 136 to expose the FRET probe 186 to excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a sufficient period of time to induce a high intensity fluorescent signal from the probe. During the excitation, the photodiode 1 84 is shorted (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. The incident light on the active region 185 of the photodiode 184 (see Figure 54) is converted to a photocurrent that can be measured using a CMOS circuit 86.

Each hybridization chamber 180 carries a probe for detecting a single target nucleic acid sequence. If desired, each hybridization chamber 180 can carry a probe that detects more than one of the different targets. Alternatively, many or all of the hybridization chambers may carry the same probe that repeatedly detects the same target nucleic acid. Copying the probes in this manner in the hybridization chamber array 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 there may be from 1 to over 1,000 different probes on the hybrid chamber array 110, depending on the analytical details. Proteomic analysis chamber - some LOC variants such as LOC variant L 729, configured to be protein -52 - 201209405

A homogeneous proteosome analysis of the crude cell lysate is performed in the plastid analysis chamber array (see, for example, Figures 124.1 to 124.3 of Figures 125 and 126) for detection of host cells and/or pathogen proteins. The proteomic analysis chamber arrays 124.1 - 1 24.3 were made and configured in exactly the same manner as the hybrid chamber array 1 10 (see Figures 52, 53, 54 and 56). Each protein body analysis chamber has a diffusion barrier 175 at the inlet to prevent diffusion of the sample and reagents between the chambers, thus avoiding erroneous results (see Figures 1A and 1〇1, which are diagrams DC and DD of Figure 97). . When required for protein hybridization or conjugation, thermal energy is provided by CMOS controlled heaters 182 in each chamber. In some embodiments, the endpoint liquid sensor 1 78 is used to indicate when the proteomic analysis chamber has been primed by the sample, such that the heater 128 can be activated. After a sufficient period of time, the fluorescent or electrochemiluminescent signal generated by protein recognition is detected by the photo sensor 44. Humidifier and Humidity Detector The inset AG of Figure 6 indicates the position of the humidifier 196. The humidifier is free of evaporation of reagents and probes during operation of the LOC device. As best shown in the enlarged view of Fig. 55, the water sump 188 is fluidly connected to the three evaporators 190. The water storage tank 188 is filled with molecular bio-grade water and is sealed during manufacture. As best shown in Figures 55 and 68, by capillary action, water is drawn to the three lower conduits 194 and along the individual water supply passages 192 to the three upper conduits 193 of the evaporator 190. The meniscus is fixed to each of the upper ducts 193 to retain water. The evaporator has a ring heater 191 that surrounds the upper pipe 193. With the thermally conductive column 376, the ring heater 191 is connected to the CMOS circuit -53 - 201209405 86 to the top metal layer 195 (see Figure 3.7). At startup, the ring heater 197 heats the water causing the water to evaporate and wet the surrounding equipment.

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 sensor 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 2 3 2 abuts the hybridization chamber array 1 1 〇 (mostly the humidity measurement position) to slow the evaporation of the solution containing the exposed probe. Feedback sensor

The temperature and liquid sensor system is integrated into the L Ο C unit 3 0 1 to provide feedback and diagnostics during device operation. Referring to Fig. 35, nine temperature sensors 170 are distributed to the entire amplification unit 1 12 . Similarly, the culture unit 14 also has nine temperature sensors 170. These sensors each use a 2 x 2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The C Μ Ο S circuit 8 6 utilizes this to accurately control thermal cycling during the nucleic acid amplification process as well as any lysis during hot lysis and culture. In the hybridization chamber 180, the CMOS circuit 86 uses the hybridization heater 182 as a temperature sensor (see Figure 566). The resistance of the hybrid heater 182 is temperature dependent' and the CMOS circuit 86 utilizes this to obtain a temperature reading of each hybridization chamber 18 。. -54-

S 201209405 LOC device 301 also has some 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 blocks the circuitry between the electrodes to indicate that they are not present at the sensor.

Figure 25 shows an enlarged perspective view of the lid channel liquid sensor 208. Opposite TiAl electrode pairs 218 and 220 are deposited on top layer 66. A gap 222 is provided between the electrodes 218 and 220 to keep the circuit open in the absence of liquid. The circuit is closed when the liquid is present and the CMOS circuit 86 utilizes this feedback to monitor the flow. The GRAVITATIONAL INDEPENDENCE test module 10 is self-directed. It does not need to be fastened to a smooth surface to operate. The fluid flow driven by capillary action and the lack of Φ 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. It is shock and vibration resistant and can be operated on mobile vehicles or on mobile phones. Dialysis variants White blood cell targets The dialysis design described above in LOC device 301 targets pathogens. Figure 64 shows the enrichment of white blood cells from blood samples designed for human DNA analysis -55-201209405

A schematic cross-sectional view of the dialysis section 3 28 . In addition to restricting leukocytes in the form of orifices in the form of 7.5 micrometer diameter wells 165 from the dialysis MST channel 204 from the capping channel 94, it will be understood that the structure is substantially the same as that of the pathogen target dialysis section 70 described above. In the case where the sample being analyzed is a blood sample and the presence of hemoglobin from the red blood cells interferes with the subsequent reaction steps, the addition of red blood cell lysis buffer and anticoagulant to the reservoir 54 (see Figure 2 2) will ensure the majority The lysed red blood cells (and therefore hemoglobin) will be removed from the sample during this dialysis step. The commonly used erythrocyte lysis buffer is 5.1 5M NH4CL, 10 mM KHC03, O.lmM EDTA, pH 7.2-7.4, but those skilled in the art will recognize any buffer that can use effective lysed red blood cells. The leukocyte dialysis section 328, downstream of the cap channel 94, becomes the target channel 74, causing the leukocytes to continue to be part of the analysis. Again, in this case, the dialysis suction port 168 leads to the waste channel 72 such that all of the smaller cells and components in the sample are removed. It should be noted that this dialysis variant only reduces the concentration of unwanted samples in the target channel 74.

Figure 80 shows schematically a large component dialysis section 686 which also separates any large target components from the sample. The orifice fabrication in this dialysis section is modified in size and shape to block large target components of interest in the target channel for further analysis. Most of the (but not all) smaller sized cells, organisms or molecules flow into the waste sump 768, as in the leukocyte dialysis section described above. Thus, other specific examples of LOC devices are not limited to separating leukocytes that are larger than 7.5 microns in size but can be used to separate cells, organisms or molecules of any desired size.

S-56-201209405 Dialysis section with fluid passage to avoid trapped bubbles The following is a specific embodiment of the LOC device of LOC variant VIII 5 18 shown with reference to Figures 73, 74, 75 and 76. This LOC device has a dialysis section that is trapped in 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

It is disposed between the cover channel layer 80 and the MST channel layer 100 of the CMOS + MST device 48. Interfacial layer 594 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 74, the bypass passage 600 is designed to introduce a time delay from the fluid sample from the interface waste channel 604 to the interface target channel 602. This time delay causes the fluid sample to flow through the dialysis M S T channel 240 to the dialysis draw 168 of the fixed meniscus. Capillary action initiation feature (CIF) 202 from the bypass channel 600 at the upper conduit to the interface target channel 602, upstream of all dialysis suction holes 168 from the dialysis MST channel 204, sample fluid entanglement interface target Channel 602. Without the bypass channel 600, the interface target channel 206 still begins to charge from the upstream end, but eventually, the traveling meniscus reaches and passes through the tube above the MST channel that is not charged, leading to this point Captured air. The trapped air reduces the sample flow rate through the leukocyte dialysis unit 3 28 . Dialysis section with active valve and liquid sensor Figure 91 illustrates an alternative dialysis section 722 having air bubbles designed to avoid trapping. The replacement dialysis section has the same structure as shown in Fig. 74, but does not have the bypass passage 600. The liquid sample enters the interface waste-57-201209405 material channel 604 from the cover channel 94. The foreign body sample flows into the dialysis MST channel 204 through a 3 micron lower tube opening 164. Cells larger than 3 microns remain in the interface waste channel 604 and ultimately lead to the waste sump 76. The dialysis upper pipe port 168 is a surface tension valve that holds the meniscus to stop the flow of the liquid sample. Instead of the bypass passage 600 (see Fig. 74), an active valve such as a boiling initiation valve 724 is located at the dialysis upper conduit port 168 at the upstream end of the dialysis MST passage 204. A liquid sensor 174 is incorporated in the dialysis MST channel 204 at the downstream end. The liquid sensor 174 triggers the boiling initiation valve 724 when all of the dialysis MST channels 204 have been charged. The sample flows into the upstream end of the interface target channel 022, and finally opens the surface tension valve at each dialysis upper pipe port 168 in the downstream direction. Without the boiling initiation valve 724, the interface target channel 602 (assuming that the panel channel contains target cells, such as pathogens) begins to fill from the upstream end, but the next meniscus is easily overlooked. , causing the air to catch at that point. Any active valve variant may be replaced by a boiling initiation valve 724, and in further embodiments, all of the dialysis upper orifices 168 may have an active valve to ensure that the sample flows from all of the dialysis MST channels 204 into the interface target channel 602.

Nucleic Acid Amplification Variants Direct PCR Traditionally, PCR required a large amount of purification target D N A prior to preparation of the reaction mixture. However, by appropriately changing the chemical and sample concentrations, nucleic acid amplification can be performed using a minimum amount of DN A purification, or direct amplification can be performed. When nucleic acid amplification is performed by PCR, this method is called direct PCr. When the nucleic acid amplification was carried out at a controlled normal temperature in a l〇c pack 201209405, the method was 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 increasing buffer strength, using highly active and highly progressive polymerases, and additions to potential polymerase inhibitors. It is also important to dilute the inhibitor 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, sump 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 is sufficiently low To successfully perform nucleic acid amplification. The desired dilution of the non-cellular sample component is 5 to 20 times. When the concentration of the target nucleic acid sequence is moderately confirmed to be sufficiently high for amplification and detection, a different 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 that is sufficiently small enough to enter the amplification portion 292 and the larger cells are discharged to the waste storage tank is used upstream of the sample extraction portion 290. 76 dialysis department. In another 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 anti-59-201209405 is 4 times to 19 times higher than the flow group of the flow channel of the reagent mixture. The flow group resistance in the microchannel is easily controlled by controlling the design. The flow group resistance for a constant cross-sectional area 'microchannel' increases linearly with channel length. It is important for the hybrid design that the flow group resistance in the microchannel depends more on the minimum cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of the microchannels of 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) before PCR amplification. Implementing the opposite in the same chamber of PCR

Transcription reaction (one-step RT-PCR), or it may be a separate initial reaction (two-step RT-PCR). In the LOC variant described herein, the reverse transcription step and the subsequent step of performing the nucleic acid amplification step can be performed by adding a reverse transcriptase and a polymerase to the reagent storage tank 62 and the stylized heater 1 54 to simply perform a nucleic acid amplification step. Step RT-PCR. The buffer, the primer, the dNTP, and the reverse transcriptase are stored and distributed by the reagent storage tank 58, and the culture unit 14 is used for the reverse transcription step, and then amplified in the amplification unit 112 in a normal manner. The two-step RT-PCR is simply done. 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. Has described some

S-60- 201209405 Constant temperature nucleic acid amplification methods, including strand-substituted amplification (SDA), transcription-mediated amplification (Τ Μ A ), nucleic acid sequence-dependent amplification (ΝΑ SBA ), recombinant enzyme polymerase amplification (RPA) , untwisted constant temperature DNA amplification (HDA), rolling cycle amplification (RCA), branched amplification (RAM), and circular thermostat amplification (LAMP), and any or other isothermal amplification methods of this can be used in this paper. In a specific embodiment of the LOC device.

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 and polymerase. 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 reservoir 60 contains amplification buffer, primers, dNTPs, and recombinase proteins, and reagent reservoir 62 contains strand-substituted DNA polymerase, such as. Similarly, for HDA, reagent reservoir 60 contains amplification buffer 'primer and dNTP, and reagent reservoir 62 contains the appropriate DNA polymerase and helicase (instead of using heat) to unravel the double stranded DNA. Those skilled in the art will appreciate that the necessary reagents can be dispensed into two reagent reservoirs in any manner suitable for nucleic acid amplification. For the amplification of viral nucleic acids from RN A viruses, such as HI V or hepatitis C virus, NASBA or TMA is appropriate because it does not require the transcription of RNA into cDNA first. In this example, the reagent reservoir 60 is filled with amplification buffer, primers, and dNTPs, and the reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase, and any RNase H. For some thermostatic nucleic acid amplification types, an initial denaturation cycle must be employed to separate the double-61 - 201209405 strand DN A template before maintaining the temperature of the thermostated nucleic acid amplification for the reaction to continue. 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 14). )°

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 673, LOC variant XLIV 6 74 and LOC variant XLVII 677, respectively, shown in Figures 81, 82 and 83. Direct thermostatic amplification can also be combined with one or more preamplification dialysis steps 70, 686 or 682 as shown in Figures 81 and 83 and/or pre-hybridization dialysis step 682 as shown in Figure 82, respectively. Concentration of the target cells in the sample prior to nucleic acid amplification or removal of unwanted cell debris before the sample enters the 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 72, 77 and 78. Multiplex and some constant temperature nucleic acid amplification methods, such as LAMP, are compatible with the initial reverse transcription step to amplify RNA. Other Design Variant Flow Sensors In addition to temperature and liquid sensors, the LOC device can also be combined with a CMOS-controlled flow rate sensor 740, as illustrated in Figure 17 and in the LOC variant X 728. (See Figures 92 to 108). These sensors are used to determine the flow rate in the two steps. In the first step, the heater element 814

S -62- 201209405

The temperature is determined by applying a low current and measuring the voltage to determine the resistance of the heater element 814, and thus the temperature of the element 814 is determined using the known relationship between the resistance of the heater element and the temperature. At this stage, the heat dissipated in element 814 is minimal and the temperature of the liquid in the channel is equal to the temperature of the calculated element 814. In the second step, a higher current is applied to the helium heater element 8 1 4 such that the temperature of element 814 increases and some of the heat is lost by the flowing liquid. While applying a higher voltage, the new resistance of element 814 is determined and the increased temperature is again calculated by CMOS circuit 86 by again measuring the voltage across element 814. The flow rate of the liquid is determined using the new temperature of the crucible heater element 81 4 and the known sample liquid temperature calculated in the first step. The flow rate of the liquid in the channel is calculated from the known channel cross-section geometry and flow velocity. Additional Details of the Fluorescence Detection System Figures 58 and 59 show hybridization-reactive FRET probes 23 6 . These are often referred to as molecular beacons by φ and stem-and-loop probes produced by 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 23 8 . The probe has a ring 240, a stem 242, a bluing 246 at the 5' end, and a quench 2 at the 3' end. Loop 2, 40 contains a sequence that is complementary to the target nucleic acid sequence 2W. The complementary sequences flanking the probe sequences are bonded together to form stems 242. In the absence of a complementary target sequence, as shown in Figure 58, the probe remains closed. The stem 242 maintains the fluorophore-quenching agent relatively close to each other, so that a large amount of resonance energy can be transmitted between each other, and when irradiated with the excitation light 244, -63-201209405 substantially eliminates the ability of the fluorescent fluorophore . Figure 59 shows the F R E T probe 2 3 6 in an open or hybridized configuration. Upon hybridization to the complementary target nucleic acid sequence 238, the stem-and-loop structure is disrupted, and the fluorophore and quencher are spatially separated, thereby restoring the ability of the fluorophore 246 to fluoresce. Optical detection of the fluorescence emission of 250 to serve as an indicator that the probe has hybridized

The probe hybridizes to the complementary target with very high specificity, since the stem helix of the probe is designed to be stable to the probe-target helix with a single non-complementary nucleotide. Due to the relatively strong double-stranded DNA, it is impossible for the probe-target helix and the stem helix to coexist. Primer-Linked Probe Primers-Linked Stem-and-Ring Probes and Primers-Linked Linear Probes, also known as scorpion-type probes, are alternatives to molecular beacons and can be used for immediate and quantitative LOC devices. Nucleic acid amplification. 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 an immediate and effective response and produces a stronger signal, shorter reaction times, and better recognition when using separate primers and probes. During manufacture, the probe (mixed with the polymerase and amplification) will be deposited in the hybridization chamber 180 without the need for a separate amplification portion on the LOC device. Alternatively, the amplification portion is not used or used for other reactions.

S -64- 201209405 Introduction-Linked Linear Probe

Figures 84 and 85 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 84, 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 nucleic acid 696 and is labeled with the 5' end of the fluorophore 2M, and the 3' end thereof is coupled via the amplification blocker 694 to Oligonucleotide primer 700. The other 3' end of the stem 242 is labeled with a quencher portion 248. After completion of the first round of nucleic acid amplification, using the currently complementary sequence 698, the probe can surround and hybridize to the extended strand. During the first round of nucleic acid amplification, the oligonucleotide primer 700 is attached to the target DNA 23 8 (Fig. 84) and then extended to form a DNA strand containing both the probe sequence and the amplification product. Amplification blocker 694 prevents the reading of polymerase through and copying probe region 696. 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 lowered, 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 86A through 86F show the operation of the primer-coupled stem-and-loop probes 7〇4. Referring to Fig. 86A, the primer-linked stem-and-loop probe 7〇4 has a complementary double -65-201209405

The stem 242 of the stranded DNA and the loop 240 of the combined probe sequence. The 5' end of one of the stem strands 708 is marked with a fluorophore 24 6 . The other strand 710 is labeled with a 3' end quencher 248 and the other strand 710 carries both an amplification blocker 694 and an oligonucleotide primer 700. In the initial denaturing phase (see Figure 86B), the strands of the target nucleic acid 23 8 and the primer-linked stem 242 are separated from the stem-and-loop probe 704. When the temperature is cooled for the adhesive phase (see Figure 86C), 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 86D), the complement 706 of the target nucleic acid sequence 238 is synthesized to form a DN A strand containing both the probe sequence 704 and the amplified product. Amplification blocker 694 prevents the polymerase from reading through and copying probe region 74. After denaturation, when the probe is attached, the probe sequence of the primer-linked stem-and-loop probe loop region (see Figure 86F) is adhered to the complementary sequence 706 on the extended strand. This configuration leaves the fluorophore 246 at a great distance from the quencher 248, resulting in a significant increase in fluorescence emission.

Control Probes The hybridization chamber array 110 includes a plurality of hybridization chambers 180 having positive and negative control probes for analytical quality control. Figures 113 and 114 illustrate a negative control probe 796 without a fluorophore, and Figures 1 1 5 and 11 6 depict a positive control probe 798 without a quencher. The positive and negative control probes have a stem-and-loop structure as described above for the FRET probe. However, whether the probe hybridizes to an open configuration or remains closed, the fluorescent signal 250 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 3 and 1 14, the negative control probe 796 does not have a fluorophore (and

S-66- 201209405 with or without quenching agent 248). Thus, regardless of whether the target nucleic acid sequence 238 hybridizes to the probe (see Figure 114) or the probe maintains its stem-and-loop configuration (see Figure 113), the response to the excitation light 244 can be ignored. Alternatively, it can be designed

The negative control probe 7 9 6 keeps it 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 of interest, the stem 242 of the probe molecule will rehybridize with itself, and the fluorophore and quench The agent will remain 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 does not quench all of the emission from the indicator. Conversely, a positive control probe 798 without quenching is constructed, as shown in Figures 115 and 116. The back-stress luminescence 244, whether or not the control probe 798 is hybridized to the target nucleic acid sequence 23, is free of material to quench the fluorescent emission 25 from the fluorescent 246. Figure 52 shows a possible distribution of positive and negative control probes in the hybrid chamber array 110 (divided into 3 7 8 and 3 80 ). Control probes 3 7 8 and 380 are placed in the hybrid chamber 180 and positioned to traverse the line of the hybrid chamber array 11 . However, the configuration of the control probes within the array is arbitrary (as is the configuration of the hybrid chamber array 1 10). Protein Detection Variants Some specific examples of L Ο C devices use homogeneous protein detection assays to detect specific proteins in crude cell lysates. Many homogeneous protein detection assays have been developed for these specific embodiments of LOC devices. Usually this -67- 201209405 These analyses use antibodies or aptamers to capture target proteins.

In one type of analysis, aptamer 141 bound to a particular protein 142 is labeled with two different fluorophores or luminescent groups 143 and 144, which can be used as fluorescence resonance energy transfer (FRET) or electrochemiluminescence resonance energy transfer ( ERET) Hosts and receptors in the reaction (see Figures 122A and 122B). Both the precursor 143 and the receptor 144 are linked to the same aptamer 141, and when bound to the target protein 142, the change in conformation causes a change in the spacing. For example, in the absence of a target aptamer 141, the conformation of the donor and acceptor is located at the proximal end (see Figure 1 22 A): when bound to the target, the new configuration results in a larger between the donor and the receptor. The interval (see Figure 1 22B). When the acceptor is a quencher and the precursor is a luminophore, the effect of binding to the target is to increase the light emission by 250 or 8 62 (see Figure 122B).

The second type of analysis uses two antibodies 145 or two aptamers 141 (see Figures 123A, 123B, 124A and 124B) that must be ligated independently to different and non-overlapping epitopes or target protein 142 regions. These antibodies 145 or aptamers 141 are labeled with different fluorophores or luminescent groups 143 and 144, which serve as precursors and receptors in fluorescence resonance energy transfer (FRET) or electrochemiluminescence resonance energy transfer (ERET) reactions. Fluorescent or luminous group! 43 and 144 form a portion of a short complementary oligonucleotide pair 147 that is joined to the antibody or aptamer via a long elastic linker 1 49 (see Figures 123A and 124A). Once antibody 145 or aptamer 141 is linked to target protein 142, complementary oligonucleotides 147 collide with each other and hybridize to each other (see Figures 123B and 124B). Thus, the donor and acceptors 143 and 144 are brought close to each other, resulting in an effective FRET 250 or ERET 862 that can be used as a signal for target protein detection.

S -68- 201209405

In order to determine that there is no, or very little, background signal due to the hybridization of oligonucleotides 147 ligated to two antibodies 145 or aptamer 141 to each other without ligating to protein 142, careful selection of complementary oligonucleotides 147 is required. The sequence and length ' makes the dissociation constant (kd) of the double strands quite high (~5μΜ). Therefore, 'when the free antibodies or aptamers labeled with these oligonucleotides are mixed at a nanomolar concentration, it happens to be lower than the molar concentration of kd, the probability of double-strand formation and the FRET 250 or ERET 862 signal system produced. Ignorable. However, when both antibody 145 or aptamer 141 are conjugated to target protein 142, the regional concentration of oligonucleotide 147 will be much higher than its level, resulting in almost complete hybridization and producing a detectable FRET 250 or ERE T 862 signal. The choice of fluorophores or luminophores is an important consideration when designing a homogeneous protein assay. Crude cell lysates are typically turbid and may contain substances that autofluorescent. In this case, it is necessary to use molecules with long-acting fluorescence or electrochemiluminescence as well as donor-acceptor pairs 143 and 144 optimized to emit maximum FRET 25 0 or ETER 8 62. One such φ-body-receptor pair is a ruthenium chelate and Cy5, which, when compared to other host-receptor pairs, has previously been shown to significantly enhance the signal in such systems. - Background ratio, which causes the signal to be read by attenuating background fluorescence, electrochemiluminescence or scattered light attenuation. The ruthenium chelate and the AlexaFUor 647 or ruthenium chelate and the Fluorescein FRET or ERET pair also work well. The sensitivity and specificity of this method is similar to that of enzyme-linked immunosorbent assay (ELISA), but does not require manipulation of the sample. In some embodiments of the LOC device, one of the antibodies 145 or one of the aptamers 141 is ligated to the proteomic analysis chamber 124 (see, for example, Figure-69-201209405 125 and 126), and the protein lysate is During lysis, it is combined with another antibody 145 or aptamer 141 within chemical lysis portion 130 to facilitate binding to first antibody 丨45 or aptamer M1 prior to entry into proteomic analysis chamber 124. This requires an increase in the speed of the connection and the resulting detectable signal when only one junction or hybridization event is required in the proteomic analysis chamber. Fluorescent group design

A fluorophore having a long fluorescent lifetime is required to allow the excitation light to have sufficient time to decay to a lower intensity of the fluorescent emission when the photosensor 44 is enabled, thereby increasing the sufficient signal-to-noise ratio . Moreover, longer fluorescence lifetimes represent larger integrated fluorescence counts. The fluorescence lifetime of fluorophore 246 (see Figure 59) is greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and in most cases greater than 400 nanoseconds.

Metal-coordination complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal, chemical, and photochemical stability. It is a beneficial feature related to the needs of fluorescent detection systems. A metal-coordination complex based on the transition metal ion ruthenium (Ru(II)), which is particularly thoroughly studied, is ginseng (2,2'-bipyridyl) ruthenium (II) ([Ru(bpy)3) ] 2+), the lifetime of which is about this complex is available from Biosearch Technologies under the trade name Pulsar 650. -70-

S 201209405 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 _ Fluorescence lifetime Tf 1.0 Δδ a quantum yield Η 1 (deoxygenated) Ν/Α a lanthanide metal-coordination complex, a ruthenium chelate, has been successfully shown as a fluorescent indicator in the FRET probe system with 1 Long life of 600μ5

Life. Table 2: Photochemical stagnation of ruthenium chelate a parameter symbol 値 unit-absorption wavelength ^abs 330-350 nm emission wavelength λέπι 548 nm absorption coefficient Ε 13800 (depending on, bs and ligand, up to 30000 @ Λε=340 nm) M*1 cm'1 璧 light lifetime Tf 1600 (hybridization probe) μδ quantum yield Η 1 (coordination dependent) Ν/Α The fluorescence detection system used in the LOC device 301 is not utilized. Filter to remove unwanted background fluorescence. It is therefore advantageous if the quencher 248 has no natural emission to increase the signal-to-noise ratio. Without natural emission, the quencher 248 does not contribute to background fluorescence. High quenching efficiency is also important, which results in no fluorescence before hybridization occurs. Black Hole Quencher (BHQ), available from Biosearch Technologies, Inc. of Novato, Calif., does not have natural emissions and has high quenching efficiency, as well as suitable quenchers for use in systems. The maximum absorption enthalpy of BHQ-1 occurs at 534 nm and the quenching range is 480-580 nm, making it a suitable quencher for Tb-chelate fluorophores. -71 - 201209405 The maximum absorption enthalpy of BHQ-2 occurs at 579 nm and the quenching range is 560-670 nm making it a suitable quencher for Pulsar 6 50.

Iowa Black FQ and RQ from Integrated DNA Technologies of Coralville, Iowa, are suitable alternative quenchers with little or no background emission. Iowa Black FQ has a quenching range of 420-620 nm' with a maximum absorption enthalpy at 531 nm and is therefore a suitable quencher for Tb-chelate fluorophores. The i〇wa 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 6 50. In the specific embodiments described herein, the quencher 248 is a functional portion that is initially attached to the probe, but in other embodiments, the quencher 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 excitation source instead of a laser diode, a high power lamp or a laser because of low power consumption, low cost, and small size. Referring to Fig. 87, LEDs 26 are directly disposed on the outer surface of the LOC device 301 from the hybrid chamber array 110 of each chamber. Opposite to the hybridization chamber array 1 1 为 is a photo sensor 44 consisting of an array of photodiodes 1 8 4 for detecting fluorescent signals (see Figures 53, 54 and 65). Figures 88, 89 and 90 summarize other specific embodiments for exposing the probe to excitation light. In the LOC device 30 shown in FIG. 88, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 to the hybridization chamber array 110-72-

S 201209405. The pulse excitation excites the LED 26 and the photodetector 44 detects the fluorescent emission. In the LOC device 30 shown in Fig. 89, the excitation light 2 44 generated by the excitation LED 26 is directed by the lens 254, the first aperture 712 and the second aperture 714 over the hybridization chamber array 110. The pulse excitation excites the LED 26 and is detected by the photo sensor 44.

Similarly, the LOC device 30 shown in FIG. 90, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254, the first mirror 716, and the second mirror 718 over the hybridization chamber array 110. The LED 26 is excited by the pulse excitation again and the fluorescent emission is detected by the photo sensor 44. The excitation wavelength of LED 26 relies on the choice of fluorescent dye. Philips LXK2-PR14-R00 is a suitable excitation source for Pulsar 65 0 dye. SET UVT0P 3 3 5 T039BL LED is a suitable excitation source for the ruthenium chelate label. Table 3: Philips LXK2-PR14-R00 LED Specifications

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

Parameter symbol 値 unit wavelength Xe 340 nm Transmitting 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 -73- 201209405 Ultraviolet excitation pupil in uv A small amount of light is absorbed in the 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 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 The LED driver 29 drives the 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 mA). Standard power conditioning circuits are 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 forms 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. On-chip inspection is inexpensive and reduces the size of the analytical system. The shorter optical path length reduces noise from the surrounding environment to efficiently collect the fluorescent signal and to suppress the need for conventional optical assemblies for lenses and filters. The quantum efficiency of photodiode 184 is the fraction of photons colliding with its effective region 185. The photonic system is efficiently converted into photoelectrons. For standard 矽 processing, for -74-

S 201209405 Seeing the quantum efficiency is based on processing parameters such as the number of cap layers and absorption properties in the range of 0.3 to 0.5.

The detection valve of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184 and the number of 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 (1 760 microns χ 5 824 microns in the example of LOC device 301) and incorporate other functional modules (such as pathogens) The dialysis portion 70 and the amplification portion 1 1 2) are limited by the size of the non-animal pieces that can be used thereafter. For standard 矽 processing, photodiode 184 detects at least 5 photons. However, in order to confirm reliable detection, the minimum chirp can be set to ten photons. Thus, with quantum efficiencies ranging from 0.3 to 0.5 (as discussed above), the fluorescence emission from the iso probe is a minimum of 17 photons, and the appropriate error bounds for reliable detection of 30 photons are included. φ Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, autofluorescence, and residual excitation photon flux that are not fully attenuated 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 11A, which: -75-201209405 does not contain any probes; includes probes that do not have a fluorescent indicator A needle; or a probe comprising an indicator and a configuration such that quenching agents are always expected to undergo quenching.

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 probes described above with reference to Figures 113 through 116 can be used in the calibration chamber. However, as shown in Figures 104 and 105, which is an enlarged view of the inset DG and DH of the LOC variant X728 shown in Figure 85, another option is to isolate the calibration chamber 382 from the amplicon fluid. When hybridization is prevented by fluid isolation, background noise and bias 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. Judgment The calibration chamber 382 can provide a high calibration source to generate 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

S -76- 201209405 Meaning. However, if the photodiode 184 is calibrated by the relatively closest calibration chamber 382, the calibration is more accurate. Referring to Figure 56, hybridization chamber array 110 has a calibration chamber 382 for every eight hybridization chambers 18 0 . That is, the calibration chamber 382 is disposed in the middle of each of the three by three square hybridization chambers 180. In this configuration, the hybridization chamber is calibrated by the adjacent hybridization chamber 382.

Due to the excitation light from the fluorescent signal from the surrounding hybridization chamber 180, FIG. 112 shows a differential imager circuit 78 8 for subtracting the signal from the photodiode 184 of the corresponding calibration chamber 382. The differential imager circuit 7 8 8 samples the signal from the pixel 790 and the "virtual" pixel 792. In one embodiment, the "virtual" pixel 792 is shielded from light illumination, so its output signal provides dark reference pixels. Alternatively, the "virtual" pixel 792 can be "exposed to the excitation light" with the rest of the array. In one embodiment, the "virtual" pixel 792 is light-receiving, and signals from ambient light in the area of the array of cells can also be subtracted. The signal from pixel 790 is weak (e.g., close to a dark signal), and it is difficult to distinguish between background and very weak signals without reference to the dark signal level. During use, "Read_Column" 794 and "Read_Column_(1" 795 are enabled and M4 797 and MD4 8 0 1 transistors are turned on. Off switches 807 and 809 are turned off from pixel 790 and "virtual" pixel 792 The outputs are each stored on pixel capacitor 803 and virtual pixel capacitor 805. After the pixel signal is stored, switches 807 and 809 are disabled. Then the "read_row" switch 81 1 and the virtual "read_row" switch are turned off. 8 13, and the output of the converted capacitor amplifier 81 5 amplifies the differential signal 817. -77- 201209405 Light diode suppression and activation

The photodiode 184 must be suppressed during the excitation of the LED 26 and the photodiode 184 must be enabled during the fluorescent period. Fig. 66 is a circuit diagram of a single photodiode 184 and Fig. 67 is a timing diagram of an optical diode control signal. The circuit has a photodiode 184 and six MOS transistors, Mshun, 394, Mtx 3 96, Mreset 3 98, Msf 400, Mread 402, and Mbias 404. At the beginning of the excitation cycle, tl, transistor Mshunt 3 94 and Mreset 398 are turned on by Mshunt gate 3 84 plus high voltage and reset gate 38 8 for high voltage. During this period, the photons are excited to generate carriers in the photodiode 184. These carriers must be removed when the amount of carrier produced is sufficient to saturate the photodiode 184. During this cycle, Mshunt 3 94 directly removes the carriers generated in photodiode 184 due to leakage of the transistor or diffusion of the carrier due to excitation in the substrate, while Mreset 3 9 8 is reset. Any carrier of node 'NS' 406. After the excitation, the capture cycle begins at t4. In this loop, the response from the emission of the fluorophore is captured and integrated into the circuitry on node 'NS' 406. This is achieved by applying a high voltage to the tx gate 386, which turns on the transistor Mtx 396 and any accumulated carrier on the transfer photodiode 184 to the node 'NS' 406. The capture cycle can be as long as the campfire launch. The output from all of the photodiodes 184 in the hybrid chamber array 110 is 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 hybridization chamber array 110 after the capture cycle (see Figure 52). The first photodiode 184 to be read will have the shortest delay before the read cycle, and finally

S -78- 201209405 Light diode 184 will have the longest delay before the read cycle. During the read cycle, the transistor Mread 402 is turned on by applying a high voltage to the read gate 393. The source-slaffer transistor Msf 400 is used to buffer and read the voltage of the 'NS' node 406. Any other method of enabling or suppressing a photodiode is discussed below: 1. Inhibition method

Figures 109, 110 and 111 show three possible configurations 77 8 , 7 8 0, 78 2 using KMshunt transistor 394. Mshunt transistor 394 has a very high turn-off ratio when the maximum 値 M = 5 V is enabled during excitation. As shown in FIG. 109, 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 184. The third option is to structure the Mshunt gate 3 84 within the photodiode 184, as shown in FIG. According to the third option, the effective area of the photodiode is less than 1 8 5 . These three configurations 77 8 , 7 80 and 7 82 reduce the average path length from all positions in the photodiode 184 to the Mshunt gate 3 84. In Fig. 109, Mshun, 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. However, any carrier remaining at the far end of the photodiode 184 takes a long time to diffuse through the MShunt gate 384. In FIG. 110, the Mshunt gate 384 surrounds the photodiode 184. This further reduces the average path length of the carriers in the photodiode 184 to the Mshunt gate 384. However, the Mshunt gate 3 84 is extended around the photodiode 184. -79- 201209405 The light-emitting diode active area 1 85 is greatly reduced. The configuration 782 in FIG. 1 1 1 positions the MShunt gate 384 in the 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 leakage path.

a. The flip-flop photodiode drives the shunt transistor with a fixed delay b. The flip-flop photodiode drives the shunt transistor with a programmable delay. The parallel transistor is driven by the LED drive pulse with a fixed delay d. The parallel transistor is driven as a 2c but with a programmable delay.

69 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 trigger photodiode 187. Since the intensity of the excitation light is high as compared with the fluorescence emission, the trigger light diode 187 having a small area is sufficient. The flip-flop photodiode 187 is sensitive to the excitation light 244. The flip-flop photodiode 187 shows that the excitation light 2 44 has extinguished and activates the photodiode 184 (see Fig. 2) after a short delay of Δί 300 . This delay causes the fluorescent photodiode 1 84 to detect the fluorescent emission from the FRET probe 186 when there is no excitation light 244. This enables detection and enhancement of signal to noise ratio.

S -80- 201209405

Under each of the hybridization chambers 180, both the photodiode 184 and the flip-flop photodiode 1S7 are located in the CMOS circuit 86. The photodiode array is combined with appropriate electronic components to form a photosensor 44 (see Figure 65). 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 of the phosphor to illuminate the active region 185 of the photodiode 184. The photodiode 184 has a field of view such that a fluorescent signal from the probe-target hybrid within the hybridization chamber 180 is 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 combinations with 2a and 2b above. Fluorescence Delay Detection The following derivation shows the fluorescence delay detection using the long life cycle fluorophore for the LED/fluorescent combination described above. The fluorescence intensity is derived as a function of time after an ideal pulse excitation of the fixed intensity Ie between times ί, and ~ shown in Figure 60. Let [S1] ( ί ) be equal to the intensity of the excited state at time t, then the number of excited states per unit volume per unit time during and after the excitation is described by the following differential equation: Back (0 + calendar 1 = 仏... (1) dt rF hve where C is the molar concentration of the fluorophore' ε is the molar extinction coefficient, Ve is the excitation: .) -81 - * t 201209405 frequency, and h = 6.62606896(1 0)·34 Js is the Planck constant . This differential equation has the general formula: ^ + p(x)y = q(x) αχ The solution is: fe^( )dxq(x)dx + k Λχ)=——...(2) Now use this to solve [幻](0

Leer t hve then at time + ke -// 7 ...(3) [S1](1))=0 is derived from equation (3)

k .εοτ. hve ...(4) Substituting (4) into (3): [zero)

IescTf IescTf __(<.,|)/Γ/ hve hve

At time h,: [Sl](t2) = ^^--l^Le~^~ll),Tf ...(5)

Ave hve at r 2 〇, the excited state decays exponentially and is described by equation (6): [51](〇= [51](ί2)£-(,-ω/Γ^ ...(6) Substituting (5) into (6) ): [s\m= hVe ...(7) The fluorescence intensity is obtained by the following equation dx

-82- S 201209405 where V/ is the fluorescence frequency, η is the quantum yield, and 1 is the optical path length. Then we can see from (7): = -^[1 - eHh-''), Tf ...(9) at hve Substituting (9) into (8):

If{t) = Ι^Ιη^-ΙΙ-β^-1'^ ...(10)

Because Γ/ 匕 Therefore, we can write the following approximation, which describes the attenuation of the fluorescence intensity after a sufficiently long excitation pulse (>> Tf): (7)=/e£c/77”^_( Bu ':) / r / for ί, 2 ... (11) When called, in the previous section, we summarize the situation for >> Tf,

If(t) = Ie€C^—e~°~, l)/T/ and then. From the above equation, we can derive the following expression: nf(t) = nefc/^e'(,_,I,/r/ ...(12) where «/(0 = -r— is per unit The number of fluorescent photons per unit time and i = 丄β is the number of excitation photons per unit area per unit time. Therefore, where ~ is the number of fluorescent photons per unit area and ^ is the photodiode turned on - 83- 201209405 Time point. Substituting (1 2 ) into (13 ): 00 nf =jne£clne~(",2)lT/dt (14) h At present, the light diode is reached per unit area per unit time. The number of fluorescent photons of the body, & (0, is obtained by: ^(0 = ^/(0^0 ...(15) where nine is the light collection efficiency of the optical system. Substituting (1 2 ) (1 5 ) We found

Λ〇Λ〇= Φ〇^1Ψ~°',2),Τ, (16) Similarly, the number of fluorescent photons per unit of fluorescent area reaching the photodiode will be as follows: 〇〇ns =[ns {t)dt ^ and substituting (1 6 ) and integrating:

Hs = φ^εοίητ fe~(,l',l)Ur Therefore,

Rts ^φ^ή^Ιητ (17) The ideal 値 of ί3 is when the electron rate generated by the photon in the photodiode 184 is equal to the electron rate generated by the photon in the photodiode 184. Because the excitation photon flux decays more quickly than the fluorescence photon flux decays. Since the fluorescence output per unit of fluorescence area of the sensor is: έ> (〇 = ^; (〇 where " is the quantum efficiency of the sensor at the fluorescent wavelength. Substitute (1 7) we get : ef (ί) = φ/ φΰ ne eel ηβ ~(,~h}'r/ (18) -84- s 201209405 Similarly, the sensor output electrons per unit of fluorescence area of the excited photons Rate: Production -..(19)

Where is the quantum efficiency of the sensor at the excitation wavelength and is the time constant relative to the "off" characteristic of the excited LED. After time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and 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: Therefore, from (1 8 ) and (1 9 ) we get: (f>f<^0nea:^e~U3~t2), Tf = and After the whole we get: \η(εοΙη^-) --1~~f~ -(20)

Tf Te From the above two paragraphs, we get the following two working equations: ns =<^0neFTfe^,Tf ...(21) Δ/ = -Α Λ ) ...(22) τί Te where F = and Δί = 33 - ί2, we also know 'actually, -[ » Γ,. The ideal time for fluorescence detection and the number of fluorescent photons detected using Philips LXK2-PR 14-R00 LED and Pulsar 6 5 0 dye are determined as follows. -85- 201209405 The ideal detection time is determined using equation (22): recall the concentration of the amplicon, and assuming that all amplicons are hybridized, then the fluorescence concentration of the fluorescing is: c = 2.89 (l 〇K6mol/L. The height of the chamber is the optical path length 1 = 8(10)-6 m. The fluorescent area has been regarded as equivalent to the photodiode area, but the actual fluorescent area is substantially larger than the photodiode area. Therefore, it can be assumed that Lu 0 = 0.5 is the light collection efficiency of the optical system. The characteristics of the photodiode, t = 1 〇 is the quantum efficiency of the photodiode at the fluorescence wavelength to the quantum efficiency of the photodiode at the excitation wavelength. The ratio is extremely conservative. With a typical LED attenuation life cycle factory = 〇 _ 5 nanoseconds and use

Pulsar650 specification, can be determined Δί: F = [1.48(10)6][2.89(10)-6][8(10)-6](1) =3.42(10)·5 ln([3.42(10)- 5](10)(0.5)) Δί = —ii− 1(10)-6 _ 0.5(10)-9 =4.34( 10)'9s The number of detected photons is determined using equation (2l). First, the number of excitation photons emitted per unit time is determined by the detection illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian radiation pattern, so: ή, = «, 〇cos(^) ---(23) where $ is the angle of the unit with the angle of the forward axis of the LED The number of photons emitted per unit time 'and the heart is 4 in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: -86- ... (24) 201209405 ή, =|^Ω = J)i; 0cos (Ning Ω Ω Now, △ Ω = 2; τ [1 — cos(0 + A0)] — 2;τ[1 — cos(0)] ΑΩ = 2;r[cos(0) - cos(0 + Δ0)] 4^sin(^) cos .(Αθλ 1 2 J l 2 J + 4; rcos(0)sin , (ΑΘ k

dQ - 2^sin(0)d0 Substituting (24): π Ί = 12ml0 cos(0)sin(0)ci9 o =^0 Rearrange, we get: n)〇=- -(26) π LED output The power is 0.515 watts and ve = 6.52 (10) 14 Hz, so hve .•彳27) __0515_ ~ [6.63(10)-34][6.52(10)14] = 1.19(10)18 photon 渺In (26) we get: ... 1.19(10)18 «/〇=- π =3.79(10)17 photon shape' glow surface with reference to Fig. 61, optical center 25 2 and lens 26 of LED 26 are shown as 201209405 The figure shows. The photodiode is 16 microns x 16 microns, and for the photodiode in the middle of the array, the solid angle (Ω) of the cone of light emitted from the LED 26 to the photodiode 184 is approximately: Ω = sensor area / r2 [16 (10)~6] [16(10)'6] ~[2_825(10)_3f~ =3.21(10)_5 Sphericality It will be understood that the central photodiode 184 of the photodiode array 44 is used for these calculations. use. The photosensor at the edge of the array receives only 2% of the photons for the Lambertian excitation source intensity distribution at the hybridization event. The number of excitation photons emitted per unit time: ne = η, Ω ..J28) = [3.79 ( 1 0) ι7][3.21(1〇Γ5] = 1.22(10)13 Photon 渺 Now refer to equation (29): = ^0^^Γ/β"Δ,/Γ/ ns = (0.5)^.22 (10)^3^.42(10)-^11(10)-6^-4340^9^00^ = 208 photons per sensor. Therefore, use Philips LXK2-PR 1 4-R00 LED and Pulsar 650 With the fluorophore, we can easily detect any hybridization events that cause the number of photons that are excited. The SET LED illumination geometry is shown in Figure 62. At ID = 20 mA, the LED has a minimum optical power output of Pl = 240 microwatts. The wavelength center is at λ6 = 340 nm (the absorption wavelength of the ruthenium chelate). The LED is driven linearly at Id = 200 mA to increase the output power to Pi = 2.4 mW. By placing the LED -88-201209405 Optical Center 2 52 placed at a distance of 17.5 mm from the hybrid chamber array 1 10, we concentrated the output flux to a diameter of 2 mm with a maximum diameter of 2 mm in the plane of the hybrid array plane The photon flux obtained by the equation (27).

_ 2.4(10)'3

~[6.63(10)-34][8.82(10)14] = 4.10(10)15 Photon 渺

Using equation (28), we get: ne = n, Q = 4.10(10)15^7, for 1(10)-3]2 = 3.34(10)" photon 渺 now, call the equation again (22 And using the previously listed Tb chelate properties,

Af ln[(6.94(10)-5 )(10)(0.5)] 1(10)-3 ~ 0.5(10)-9 =3.98(10)·9 seconds Now from the equation (2 1 ): ns = (0.5)[3.34(10)1I][6.94(10)-5][l(10)-3]e-3 98(I〇rS/1(,〇r3 = 11,600 photons per sensor by hybridization The event uses the SET LED and the photon theory number emitted by the ruthenium chelate system to easily detect and far exceed the minimum of 30 photons, which is used for the photosensors indicated above. Reliable detection is required.

-89 - 201209405 Maximum separation between probe and photodiode The on-wafer detection avoids the need for confocal microscopy (see background of the invention). Deviation from traditional detection techniques is an important factor in saving time and cost associated with the system. Traditional inspection requires the use of imaging optics for 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 surface of the hybridization chamber of the photodiode, which has a planar active surface region parallel to the surface of the chamber. The cone of light in which the light can be absorbed by the photodiode is defined as having a firing 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 X 1 9 · 7 5 μm, The angle is 1 7 3 °. A separation of 1 micron or less between the surface of the chamber and the active surface of the photodiode is easily achieved. 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 the photodiode and the probe is determined as shown in Figure 54 below. Using the ruthenium chelate fluorophore and the SET UVT0P335T039BL LED, we calculated 1,1 600 photons from individual hybridization chambers 180 to 16 micron x 16 micron photodiodes 184. When implementing this calculation, we assume hybridization chambers

The light collection area of the room 90 of 201209405 has the same product as the active area 185 of the photodiode, and half of the total number of hybrid photons reaches the photodiode 184. That is, the light collection efficiency of the system is 〇=〇.5. More precisely, we can write each = [(the bottom area of the light collection area of the hybrid chamber) / (photodiode area)] [Ω / 4π], which is the opposite of the light diode at the representative point of the hybrid Ω = solid angle. For the square pyramid geometry: Q = 4arcsin ( a2 / ( 4d 〇 2 + a2 )), where d 〇 = the distance between the chamber and the pole body, and α is the size of the light diode. Each hybridization chamber releases 2 3 200 photons, and the selected photodiode has a detection minimum of 値1 to 7 photons. Therefore, the minimum optical efficiency required is 9=17/23200=7.33x10 Light collection area of hybridization chamber 180 The bottom area is 29 microns x microns. Solving dG will result in a large limiting distance between the hybridization chamber and the photodiode 1 84 of cU = 249 microns. In this limitation, the set cone angle as defined above is only 0.8°. It should be noted that this analysis ignores the effects of refraction. LOC Variants The LOC device 301, described and illustrated in detail above, is only one of many LOC device designs. The flow chart (from sample to test) will now be used to illustrate and/or display the input of the most appropriate negligible line of the chamber with the non-bottom optical domain of the various functional sections described above. 91 - 201209405

Some possible combinations are illustrated in conjunction with LOC device variants. The flow is appropriately divided into a sample input and preparation stage 288, 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 detail configuration is shown. Also for the sake of 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 following L Ο C device designs for each of the appropriate position.

LOC variant X

Figures 92 through 108 show LOC variant X 728. Referring to the operational schematic shown in Figure 92, the LOC device extracts 290, cultures 291, expands 292, and detects both human and pathogen nucleic acids, as well as human and pathogen proteins. By combining white blood cells and pathogen dialysis units (32 8 and 70, respectively) to produce white blood cells, pathogens, and red blood cell output streams, the output flows through the lysis and separately directed to the protein body analysis chamber array 1 24.1 - 1 24.3 for protein Detection. In the case of leukocytes and pathogens, part of the output stream is also introduced into cultures 114.1 - 114.2 and amplifications 112.1 - 112.2 for nucleic acid amplification, and then to hybridization chambers 110.1 and 110.2 for nucleic acid detection. Each output is processed separately for higher sensitivity and parallel analysis. Initially, best seen in Figure 98, a biological sample, such as whole blood, is added to the sample inlet 68. The sample stream passes through the lid channel 94 to the anticoagulant sump surface tension valve 118. When the anticoagulant from the reservoir 54 flows to the upstream end of the leukocyte output dialysis section 3 2 8 , the anticoagulant from the reservoir 54 (or -92-)

S 201209405 Other reagents) mixed with the sample.

The anticoagulant surface tension valve 118 has two channels 596 and 598 in the interface layer 594 (see Figures 93 and 98). Below the sump side interface channel 596, the conduit 92 connects the sump outlet' while the sample side interface channel 598 is connected to the conduit 96 by the cover channel 94. The valve system is filled with anticoagulant from the sump 54 through the sump side interface channel 596 into the MST channel 90 to be secured to the meniscus of the upper tube 96. As the sample flows back through the lid channel 94 and toward the upstream end of the leukocyte dialysis portion 32 8 , the sample flowing along the lid channel 94 is forced into the sample side interface channel 598 to cause the sample and anticoagulant meniscus to act. To release anticoagulant to the sample. The sample enters the upstream end of the white blood cell output dialysis section 328 through the large component interface channel 73 0 . The large component interface channel opens the 7.5 micron filter apertures 165 on the array, each being a filter that is coupled to the lateral dialysis MST channel 204 of the upper conduit aperture 168, which in turn opens within the panel interface channel 73 2 (see Figure 102). The first lateral dialysis MST channel of the dialysis section is the bypass channel 6 00, which allows all other lateral dialysis MST channels to be filled without trapping the bubbles (see Figure 107). The lateral dialysis MST channel 204, except for all of the fixed meniscus of the bypass passage, has an upper conduit 168 and only releases the meniscus once it has reacted with the fluid in the panel interface channel 73 2 . The fluid in the panel interface channel 732 begins upstream of the conduit 168 above the bypass passage 600, and the bypass passage 600 includes a capillary initiation feature 202' which is a hole configured to cause a geometrical instability of the meniscus, such that The meniscus remains unraveled and the capillary drive flow is not blocked. The sample stream travels downstream from the capillary initiation feature to gradually unravel the meniscus of the upper conduit 168 (see Figure 1.7). -93- 201209405

The downstream end of the white blood cell output dialysis section 3 28 is shown in FIG. The large component interface channel 730 is fed into the large component cover channel 736 and the component interface channel 732 is fed into the group cover channel 73 4 . The large component cover channel 73 6 directs the white blood cells (and any other large components) through the dialysis surface tension valve 1 28. 1, wherein the dialysis reagent from the reservoir 5 6.1 is added to the leukocyte dialysis unit 1 3 0.1 (see Figure 96). The white blood cell lysis section 130.1 outlet has a filtered down conduit 738 (see Figure 98) that blocks the large component from blocking the MST channel or boiling initiation valve 206 prior to completion of lysis. After lysis for a sufficient period of time, the boiling initiation valve 206 opens the effluent portion 130.1 outlet, and the sample stream is split into two streams, first pass through the restriction enzyme, ligase and junction storage tank 58.1 surface tension valve 13 2.1 while the other stream The protein body analysis chamber array 124.1 (see Figure 98) is directly attracted to the lysed leukocyte bypass channel 742 to the hybridization and detection portion.

In the case of a flow through the lysed leukocyte bypass channel 742, the sample is loaded with a proteomic analysis chamber array 124.1 (see Figure 100) containing probes for hybridization to the target human protein. The probe-target hybridization system is detected by photosensor 44 (see Figure 92). In the case where the fluid passes through the restriction enzyme, ligase, and junction storage tank, the sample enters the white blood cell culture section 114.1 while continuously mixing with the restriction enzyme, ligase, and linker primer from the storage tank 58.1 (see Figure 98). ). After limiting restriction and linker engagement, the culture outlet valve 207 (which may also be a boiling initiation valve) bleeds fluid through the PCR mixing reservoir 60" surface tension valve 138.1, then the polymerase reservoir 62.1 surface tension valve 140.1 (see Figure 97 and 99), when the reagent is left to the white blood cell DNA amplification unit 112.1, each

The S-94-201209405 sump reagent is mixed with the sample. The boiling initiation valve 108 is opened to send the amplicon to the hybridization chamber array 1 1 1, 1. The thermal cycle is performed in the amplification unit 1 1 2 · 1 , and the hybridization chamber array contains the human DNA target Probe (see Figure 100). The probe-target hybrid is detected by photosensor 44 (see Figure 92).

Returning to the downstream end of the leukocyte output dialysis section (see Figure 102), the fraction cover channel 73 4 directs white blood cells and pathogens to the upstream side of the pathogen output dialysis section 70 (see Figures 96, 98 and 108). The pathogen output dialysis section 70 operates in the same manner as the leukocyte output dialysis section 3 28 except that the filtered tubing has a 3 micron aperture 164 instead of a 7.5 micrometer aperture. The white blood cells remain in the large component interface channel 73 0 while the pathogen is filtered into the panel interface channel 73 2 . At the downstream end of the pathogen output dialysis section 70, as illustrated in Figure 103, the red blood cells and pathogen streams each exit into the large component cover channel 736 and the component cover channel 734. Note that the naming of the dialysis section flow is based on the relative representation size such as "large component" and "team component", which is only relevant for the dialysis section considered. When the fraction output of the leukocyte output dialysis section 328 is fed into the large component interface channel 730 of the pathogen output dialysis section 70, the composition of the dialysis section 70 is uniform, and the size of the components between the two dialysis sections is irrelevant. When the red blood cell flow is directed through the lysis reagent reservoir surface tension valve 128.3 to fill the red blood cell lysis chamber 130.3, the red blood cell flow system in the large component cover channel 736 is mixed with the lysis reagent (see Figures 96 and 97). The red blood 201209405 ball lysis chamber 130.3 outlet contains a filtered down conduit 738 to avoid large components blocking the M ST channel or boiling initiation valve 206, as described above with respect to complete lysis of the leukocyte lysis chamber 130.1 outlet. Upon completion of lysis, the boiling initiation valve 206 releases fluid into the proteomic analysis chamber array 124.3 (see Figure 1A), which contains probes that are coupled or hybridized to the target human protein. The probe-target composite system is detected by photosensor 44 (see Figure 92).

When the group cover channel 734 pathogen flow is directed through the lysis reagent reservoir 5 6.2 surface tension valve 1 2 8 · 2 to fill the lysis chamber 1 3 0.2, the group cover channel 734 pathogen flow system is mixed with the lysis reagent (See Figures 96 and 97). Upon completion of the lysis, the boiling initiation valve 206 opens the effluent chamber 130.2 outlet and the sample flow is split into two streams, with the flow passing through the restriction enzyme, ligase and junction reservoir 58.2 surface tension valve 132.2 while the other flow along The lysed pathogen bypass channel 744 is directly aspirated into the proteomic analysis chamber array 124.2 in the hybridization and detection portion 294 (see Figures 96, 97, 99 and 100).

In the case of flow through the lysing pathogen bypass channel 744, the sample is filled with a proteomic analysis chamber array 124.2 (Fig. 1A) containing probes coupled or hybridized to the target pathogen protein. The probe-target composite system is detected by photosensor 44 (see Figure 92). With flow through the restriction enzyme, ligase, and junction reservoir 58.2, the sample enters the pathogen culture section 1 1 4 · 2 while continuously mixing with the restriction enzyme, ligase, and linker primer reservoir 58.2 (see Figure 99). . After restriction enzyme digestion and junctional engagement, the culture vessel outlet valve 2〇7 (also a boiling initiation valve) releases fluid to continuously pass the PCR mixing tank 60.2 surface tension valve U8.2, followed by the polymerase storage tank 62.2 surface tension valve 140.2 (

S-96-201209405 See Figures 97 and 99), when the respective sump reagent stream enters the pathogen DNA amplification section 112.2, it is mixed with the sample (see Figure 99).

The thermal cycle is performed in the amplification section 112.2 before the boiling initiation valve 108 is opened to deliver the amplicon to the hybridization chamber array UO. 2 containing the probe for the pathogen DNA target (see Figure 1). Once the desired hybridization chamber has been full, the hybridization heater 1 82 is activated after a delay in time associated with the flow rate sensor 740 in the pathogen culture section 114.2 (see Figure 106). The probe-hybridization system is detected by photosensor 44 (see Figure 92). Within the hybridization chamber array 180 and the proteomic analysis chamber array 124, a calibration chamber (see Figures 104 and 105) is inserted to reduce the output noise of the photo sensor 44. Conclusion The devices, systems, and methods described herein promote 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 in 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 view of the electronic group -97-201209405 in the test module configured for fluorescence detection. 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 an LOC device having a lid 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 of the cover showing the top channel configuration. Figure 11 is a plan view of the LOC device showing the structure of the CMOS+ MST device independently. Figure 12 is a schematic cross-sectional view of the LOC device at the sample inlet.

Figure 13 is an enlarged view of the inset 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.

S - 98 - 201209405 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 illustrating the layered configuration of the LOC device inside the inset AE. Figure 22 is a schematic cross-sectional view showing the lysis reagent reservoir 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 inset AB. 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. Figure 32 is a schematic cross-sectional view of the boiling initiation valve of the line 3 2- 3 2 of Figure 31, -99-201209405. ® 33 is an enlarged view of the illustration AF shown in Fig. 15. Figure 34 is a cross-sectional view of the boiling initiation valve shown in Figure 34 of the line 34-34. Figure 35 is an enlarged view of the illustration AC shown in Figure 6. Fig. 13 6焉 shows a further enlarged view of the inside of the illustration AC of the amplification section. Fig. 37 shows a further enlarged view of the inside of the illustration AC of the amplification section. Fig. 38 shows a further enlarged view of the inside of the illustration AC of the amplification section. Figure 39 is a further enlarged view of the inside of the illustration AK shown in Figure 38. Figure 40A shows a further enlarged view of the inside of the illustration AC of the amplification chamber. Fig. 41 is a further enlarged view showing the inside of the illustration AC of the amplification section. Figure 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 shown in Figure 42. Fig. 44 is a further enlarged view showing the inside of the illustration AC of the amplification section. Figure 45 is a further 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. Figure 49 is a further enlarged view showing the inside of the illustration AC of the amplification chamber. Fig. 50 is a further enlarged view showing the inside of the illustration AC of the amplification section. Figure 51 is a schematic cross-sectional view of 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. -100- 201209405 Figure 55 is an enlarged view of the humidifier shown in the illustration 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 the FRET probe in a closed configuration. Figure 59 is a diagram of the FRET probe in an open and hybrid configuration. Figure 60 is a graph of excitation light density as a function of time.

Figure 61 is a graph of the excitation 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 of the humidity sensor shown in Figure AH of Figure 6. Figure 64 is a schematic cross-sectional view of the white blood cell target dialysis section. Figure 65 is a schematic view showing a portion of the photodiode array of the photo sensor. Figure 66 is a circuit diagram of a single photodiode. Figure 67 is a timing diagram of the photodiode control signal. Figure 68 is an enlarged view of the evaporator of the illustration AP shown in Figure 55. Figure 69 is a schematic cross-sectional view showing the detection of the photodiode and the trigger photodiode through the hybridization chamber. Figure 70 is a diagram of the linker-priming PCR. Figure 7 is a schematic view showing a test module having a lancet. Figure 72 is a graphical representation of the structure of LOC Variant VII. Figure 73 is a plan view of a LOC variant VIII having features and structures from all layers overlapping each other. -101 - 201209405 Figure 74 is an enlarged view of the inset CA shown in Figure 73.

Figure 75 is a partial perspective view showing the layered structure of the LOC variant VIII shown in the inset CA in Figure 73. Figure 76 is an enlarged view of the inset CA shown in Figure 74. Figure 77 is a graphical representation of the structure of the LOC variant VIII. Figure 78 is a diagram showing the structure of the LOC variant XIV. Figure 79 is a diagram showing the structure of the LOC variant XLI. Figure 80 is a diagram showing the structure of the LOC variant XLII. Figure 81 is a diagram showing the structure of the LOC variant XLIII. Figure 82 is a diagram showing the structure of the LOC variant XLIV. Figure 83 is a diagram showing the structure of the LOC variant XLVII. Figure 8 is a diagram of the linear fluorescent probe linked by the primer during the initial amplification. Figure 85 is a diagram of a linear fluorescent probe coupled to a primer during a subsequent amplification cycle. Figures 86A through 86F illustrate the thermal cycling of the primer-linked fluorescent stem-and-loop probe. Figure 87 is a schematic illustration of an excitation LED for a hybrid chamber array and a photodiode. Figure 88 is a schematic illustration of the excitation LED and optical lens that direct light into the hybridization chamber array of the LOC device. Figure 89 is a schematic illustration of an excitation LED, optical lens, and optical cartridge for directing light into the hybrid chamber array of the LOC device. Figure 90 is a schematic illustration of the LED, optical lens and mirror arrangement of the hybridization chamber array for directing light into the LOC device. Figure 91 is a diagram of an alternative dialysis section. Figure 92 is a graphical representation of the LOC variant X structure. Figure 93 is a perspective view of the LOC variant X. Figure 94 is a plan view showing the LOC variant X of a separate CMOS + MST device structure. Figure 95 is a perspective view of the bottom surface of the lid having the reagent reservoir shown in phantom. Figure 96 is a plan view showing only the features of the separate lid. Figure 97 is a plan view showing all the features overlapping each other and showing the positions of the insets D A to DK. Figure 98 is an enlarged view of the inset DA shown in Figure 97. Figure 99 is an enlarged view of the illustration DB shown in Figure 97. Figure 100 is an enlarged view of the inset DC shown in Figure 97. Figure 101 is an enlarged view of the inset DD shown in Figure 97.

Figure 102 is an enlarged view of the inset DE shown in Figure 97. Figure 103 is an enlarged view of the illustration DF shown in Figure 97. Figure 104 is an enlarged view of the illustration DG shown in Figure 97. Figure 105 is an enlarged view of the inset DH shown in Figure 97. Figure 106 is an enlarged view of the inset DJ shown in Figure 97. Figure 107 is an enlarged view of the illustration DK shown in Figure 97. Figure 108 is an enlarged view of the inset DL shown in Figure 97. Figure 109 shows a specific embodiment of a parallel transistor for an optical diode. -103- 201209405 Figure no shows a specific embodiment of a parallel transistor for a photodiode. Figure 111 shows a specific embodiment of a parallel transistor for an optical diode. Figure 112 is a circuit diagram of the differential imager. Figure 1 13 illustrates a negative control fluorescent probe in a stem-and-loop configuration. Figure η 4 illustrates the negative control fluorometer of Figure 13 in an open configuration. Figure 115 schematically illustrates the positive control fluorescent probe in the stem-and-loop configuration. Figure 1 1 6 shows a positive control fluorescent probe of Figure 115 in an open configuration. Figure 1 17 illustrates a CMOS controlled flow rate sensor. Figure 1 18 shows the test module and test module reader used with configuration and ECL testing. Figure 1 1 9 is a pictorial summary of the electronic components in the test module that are configured for use with ECL testing. Figure 1 20 shows the test module and the alternative test module reader. Figure 1 2 1 shows the test module and test module reader and the host system that houses the various data banks. Figures 122A and 122B are diagrams illustrating the attachment of an aptamer to a protein to make a detectable signal. Figures 123A and 123B are diagrams illustrating the attachment of two aptamers to a protein to make a detectable signal. Figures 1 24A and 1 24B are diagrams illustrating the incorporation of two antibodies to a protein to produce a -104-201209405 detectable signal. Figure 125 is a plan view showing the LOC variant L showing all the features overlapping each other and the positions of the insets G A to GL. Figure 126 is an enlarged view of the inset GD shown in Figure 125. [Main component symbol description]

1 〇: Test module 1 1 : Test module 1 2 : Test module reader 1 3 : Case 14 : Micro-USB connector 1 5 : Sensor 1 6 : Micro-USB 埠 17 : Touch screen 18 : Display screen 19: Button 20: Start button 2 1 : Honeycomb radio 22: Aseptic sealing tape 2 3: Wireless network connection 24: Large container 25: Satellite navigation system 26: Light-emitting diode 2 7 : Data storage - 105 - 201209405 2 8 : Phone 29 : LED driver 30 : LOC device 3 1 : Power conditioner 32 : Capacitor 3 3 : Timer 3 4 : 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 42 : Processor

43 : Program Memory 44 : Light Sensor 45 : Indicator 46 : Cover 47 : Module 48 : CMOS + MST Device 49 : Porous Element 52 : Hybridization and Detection 54 : Anticoagulant Storage Tank

S -106 - 201209405 56,56.1,56.2,56.3: Storage tank 5 7 : Printed circuit board 5 8 , 5 8 · 1 , 5 8.2 : Storage tank 60, 60.1-60.12, 60.X: Storage tank 62, 62.1, 62.2, 62.3 , 62_4, 62.X: sump 64: lower seal 66: top layer

6 8 : sample inlet 70 : dialysis section 72 : waste channel 74 : target channel 76 : waste storage tank 78 : sump layer 80 : cover channel layer 8 2 : upper sealing layer 84 : 矽 substrate 8 6 : C Μ Ο S circuit 87 : MST layer 8 8 : Passivation layer 90 : MST channel 92 : Lower pipe 94 : Cover channel 96 : Upper pipe 9 7 : Wall - 107 - 201209405 98 : Meniscus holder 1 00 : MST channel layer 101 : Lap Computer/notebook 102: Capillary action starting feature 103: Dedicated reader 1 0 5: Desktop computer 106: Boiling trigger valve

107: e-book reader 108: boiling initiation valve 109: tablet 110, 110.1-110.12, 110.X: hybridization chamber array 1 1: epidemiological data 112, 112.1-112.12, 112.X: amplification department 1 1 3 : Genetic data 114, , 1 1 4.1 - 1 1 4.2 : Culture department

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 1 2 3 : Personal Health Record 1 2 4.1 - 1 24.3: Protein Body Analysis Chamber Array

S -108- 201209405 125 : Network 126 : Boiling Initiating Valve 128, 128.2, 128.3: Surface Tension Valve 130, 130.1-130.3: Lysis Department 1 3 1 : Mixing Department

132, 132.1, 132.3: Surface tension valve 1 3 3 : Incubator inlet channel 1 3 4 : Lower pipe 136 : Optical window 138.1, 138.2 : Surface tension valve 1 40.1 : Surface tension valve 141 : Aptamer 142 : Target protein 143 : Precursor 144 : Receptor 145 : Antibody 1 4 6 : Valve inlet 147 : Complementary oligonucleotide 1 4 8 : Valve outlet 1 4 9 : Junction 150 : Valve under the tube 152 : Ring heater 1 5 3 : Valve Heater contact 1 5 4 : Heater-109- 201209405 1 5 6 : Heater contact 158 : Micro channel 1 60 : Outlet channel 1 6 4 : 孑L port 1 6 5 : Hole

166: Capillary action initiation feature 168: Upper pipe opening 170: Temperature sensor 174: Liquid sensor 175: Diffusion barrier 1 7 6 : Flow path

1 7 8 : Liquid sensor 1 80 : hybridization chamber 1 8 2 : heater 1 84 : photodiode 1 85 : effective area 1 8 6 : probe 1 87 : photodiode 1 8 8 : water storage tank 190 : evaporator 1 9 1 : ring heater 192 : water supply channel 193 : upper pipe 194 : lower pipe

S -110- 201209405 1 9 5 : Top metal layer 196 : Humidifier 1 9 8 : Suction hole 202 : Capillary action initiation feature 204 : MST channel 206 : Boiling initiation valve 207 : Culture outlet valve

208: liquid sensor 2 1 0 : microchannel 2 1 2 : MST channel 2 1 8 : electrode 220 : electrode 222 : gap 23 2 : humidity sensor 2 3 4 : heater 23 6 : FRET probe 2 3 8: Target nucleic acid sequence 2 40: Ring 242: Stem 244: Excitation light 246: Fluorescent group 248: Quencher 250: Fluorescent signal 2 5 2: Optical center

-111 - 201209405 2 5 4 : Lens 28 8 : Sample input and preparation 290 : Extraction stage 291 : Culture stage 292 ′·Amplification stage 293 : Pre-hybridization stage

294: Detection phase 296: First electrode 298: Second electrode 3 00: Programmable delay 301: LOC device 328: White blood cell dialysis section 3 7 6 : Thermally conductive column

3 7 8 : Positive control probe 3 8 0 : Negative control probe 3 8 2 : Calibration chamber 3 8 4 : Noisy 3 8 6 : Wide pole 3 8 8 : Gate 3 90 : Retractable lancet 3 92: lancet release button 3 9 3 : smell pole 3 9 4 : Μ Ο S transistor 396: MOS transistor

S -112- 201209405 398 : MOS transistor 400: MOS transistor 402: MOS transistor 404: MOS transistor 406 : node 408 : film seal 4 1 0 : film guard

5 1 8 : LOC variant VIII 5 94 : interface layer 596 : interface channel 598 : interface channel 600 : bypass channel 602 : interface target channel 604 : interface waste channel 647 : AlexaFluor 673 : LOC variant 674 : LOC variant 677 : LOC variant 6 8 2 : dialysis section 6 8 6 : dialysis step 692 : primer-linked linear probe 694 : amplification blocker 696 : probe region 698 : complementary sequence - 113 201209405 700 : oligonucleoside Acid primer 704: stem-and-loop probe 706: complementary sequence 708: stem 710: strand

7 1 2 : first aperture 714 : second aperture 7 1 6 : first mirror 7 1 8 : second mirror 7 2 2 : dialysis section 724 : boiling initiation valve

728 : LOC variant X

729 : LOC variant L

73 0 : Large component interface channel 73 2 : Panel interface channel 73 4 : Panel cover channel 73 6 : Large component cover channel 7 3 8 : Filtered down conduit 742 : White blood cell bypass channel 740 : Flow sensor 766 : Waste storage tank 7 6 8 : Blind terminal 7 7 8 : Configuration 7 8 0 : Configuration

S -114- 201209405 7 8 2 : Configuration 788 : Differential Imager Circuit 790 : Pixel 792 : Virtual Pixel 7 94 : Read - Column 795 : Read _ Column

796: Negative control probe 7 9 7 : (Transistor) 7 9 8 : Positive control probe 801 : (Transistor) 8 03 : Pixel capacitor 8 05 : Virtual pixel capacitor 8 〇 7 : Switch 8 〇 9 : Switch 8 1 1 : switch 8 1 3 : switch 8 1 4 : heating element 815 : capacitor amplifier 8 1 7 : differential signal 860 : ECL excitation electrode 870 : ECL excitation electrode

-115-

Claims (1)

201209405 VII. Patent application scope: 1. A microfluidic device for processing a liquid sample, the microfluidic device comprising '· a first channel configured to be filled with the sample by capillary action; a second channel configured To fill the sample by capillary action; a plurality of fluid communication between the first passage and the second passage - one of the fluid communication having an active valve that blocks the flow of the sample into the second passage, and the remaining of the fluid communication are configured Fixing a meniscus of the sample that blocks capillary flow between the first passage and the second passage, the fluid communication with the active valve being upstream of the remaining fluid communication; wherein, during use, Activation of the active valve initiates the flow of the sample into the second passage' such that the flow of sample in the second passage progressively removes the meniscus at each meniscus holder. 2. A microfluidic device as claimed in claim 1 wherein one of the remaining fluid connections has a liquid sensor for triggering activation of the active valve. 3. The microfluidic device of claim 1, wherein the sample is a biological sample containing components of different sizes 'and the plurality of fluid communication ports having a threshold according to a predetermined size threshold such that flow into the second channel The components are smaller than the smaller component of the predetermined size threshold 且 and the components remaining in the first channel include a larger component greater than the predetermined size threshold 値. 4. The microfluidic device of claim 1, wherein the active-116-S 201209405 valve-type boiling initiation valve has a meniscus holder and a heater. The meniscus holder is used to fix the flow resistance. a meniscus of the liquid of the fluid, the heater being disposed adjacent to the meniscus holder, such that the heater activates to boil the liquid of the meniscus holder to move the meniscus and reflow The fluid. 5. The microfluidic device of claim 1, wherein the active valve has a meniscus holder and a bending actuator for fixing a meniscus of a liquid that blocks the fluid, The bending actuator is configured to flex at startup and to re-flow the fluid from the meniscus retainer. 6. The microfluidic device of claim 3, wherein the first channel, the second channel, and the plurality of fluid connections are portions of the dialysis portion of the microfluidic device for separately processing Smaller components and/or such larger components. 7. The microfluidic device of claim 6, wherein the biological φ sample is blood, and the larger components comprise white blood cells, and the smaller components comprise red blood cells. 8. The microfluidic device of claim 7, further comprising a microsystem technology (MST) layer for analyzing genetic material in white blood cells, wherein the MST layer has a cell for lysing the white blood cells to release therein The lytic portion of the target nucleic acid sequence. 9. The microfluidic device of claim 8, wherein the MST layer has a nucleic acid amplification portion for amplifying the target nucleic acid. 1. The microfluidic device of claim 9, further comprising a hybridization portion of -117-201209405, the hybrid having an array of probes for hybridizing to the target nucleic acid sequence, wherein the target nucleic acid sequence is The nucleic acid amplification unit is amplified. 1 1 . The microfluidic device of claim 10, wherein the probes are configured to form a probe-target hybrid with the target nucleic acid sequences, the probe-target hybridization system is responsive to excitation Fluorescent light. 1 2. The microfluidic device of claim 11, wherein the method further comprises a complementary metal oxide semiconductor (CMOS) circuit for operatively controlling a polymerase chain reaction (PCR) portion, the CMOS circuit having A light sensor from the fluorescent emission of the probe-target hybrid is sensed. 13. The microfluidic device of claim 12, wherein the hybrid has an array of hybridization chambers comprising the probes for hybridization to the target nucleic acid sequences. The microfluidic device of claim 1, wherein the micro-fluid device is a wafer-on-a-chip (LOC) device, wherein the photosensor is a photodiode array, and the photodiodes are respectively disposed adjacent to the photodiode Each of the hybrid chambers. 15. The microfluidic device of claim 14 which is a LOC device wherein the photodiode system is less than 249 microns from the corresponding hybridization chamber. 16. The microfluidic device of claim 15 which is an L〇c device wherein the probes are fluorescent resonance energy transfer (FRET) probes. 17. The microfluidic device of claim 16 which is an L〇C device wherein the hybridization chambers have optical windows disposed to expose the FRET probes to the excitation light. -118- 201209405 18. The microfluidic device of claim 1, wherein the FRET probes each have a fluorophore and a quencher, when the FRET probe has When the probe-target hybrid is formed, the fluorophore is configured to emit a fluorescent signal to the photodiode in response to the excitation light, the CMOS circuit being configured to energize the predetermined delay after the extinction of the excitation light The iso-dipole, the digital memory includes the predetermined delay. 19. The microfluidic device of claim 18, which is a LOC device, wherein the CMOS circuit has a bond pad for electrically connecting to an external device, and is configured to convert an output from the photodiodes A signal indicative of the FRET probes that hybridize to the target nucleic acid sequences and provide the signal to the bond pad for delivery to the external device. 20. The microfluidic device of claim 13 further comprising a plurality of reservoirs for retaining liquid reagent for addition to the sample. -119-