TW201209406A - Test module with microfluidic device having LOC and dialysis device for separating pathogens from other constituents in a biological sample - Google Patents

Abstract

A test module for concentrating pathogens in a biological sample, the test module having an outer casing with a receptacle for receiving the sample, a dialysis device in fluid communication with the receptacle and configured to separate the pathogens from other constituents in the sample, and, a lab-on-a-chip (LOC) device being in fluid communication with the dialysis device and configured to analyze the pathogens.

Description

201209406 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a diagnostic apparatus using Microsystem Technology (MST). In more detail, the present invention relates to the processing and analysis of microfluidics and biosynthesis for molecular diagnostics. [Prior Art]

Molecular diagnostics has emerged as an area that guarantees effective early detection of disease before the onset of disease symptoms. Molecular diagnostic tests can be used to detect: • Hereditary diseases • Acquired diseases • Infectious diseases • Genetic quality of susceptible diseases that are highly susceptible to health and high turnover due to the high degree of accuracy and rapid turnover of molecular diagnostic tests Invalid health care services, improve medical outcomes, improve disease management and provide individualized patient care. Many techniques in molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleic acid base allows the synthetic DNA short sequence (oligonucleotide) to bind (hybrid) to a particular nucleic acid sequence for use in nucleic acid testing. If a hybrid occurs, the complementary sequence will appear in the sample. This will, for example, make it possible to predict a person's possible future illness, determine the identity and pathogenicity of an infectious pathogen, or -5 - 201209406 to determine that someone will respond to a drug. Nucleic Acid-Based Molecular Diagnostic Testing Nucleic acid-based testing has four distinct steps: 1. Sample preparation 2. Nucleic acid extraction 3 (optional) nucleic acid amplification 4. Detection

Many sample types are available for genetic analysis, such as blood, urine, sputum, and tissue samples. Since not all samples represent disease progression, this diagnostic test determines the type of sample required. These samples have a variety of different compositions, but usually only one composition is of interest. For example, high red blood cell concentrations in blood can inhibit the detection of pathogenic organisms. Therefore, purification and/or concentration steps are usually required at the beginning of the nucleic acid test.

Blood is a type of sample that is more commonly used. It has three main components: white blood cells (white blood cells), red blood cells (red blood cells), and blood clotting cells (platelets). Blood coagulation cells promote coagulation and remain active outside the cell. In order to inhibit blood agglutination, the sample is mixed with a chemical such as ethylenediaminetetraacetic acid (EDTA) before purification and concentration. Red blood cells are usually removed from the sample to concentrate the target cells. In humans, red blood cells account for about 99% of blood cell material, but because they do not have a nucleus, they do not carry DNA. Furthermore, red blood cells contain certain components such as heme that interfere with the downstream nucleic acid amplification process (described below). Removal of red blood cells can be achieved by distinguishing cytolytic red blood cells in a cytolytic solution to retain other cellular material integrity, and then -6-201209406 using centrifugation to separate intact cellular material from the sample. Thus, a concentrate of the target cells can be provided and nucleic acids can be extracted from the concentrates

The precise procedure for extracting nucleic acids will depend on the sample and the diagnostic test to be performed. For example, the procedure used to extract the viral RN A is significantly different from the protocol used to extract the genomic DNA DN A. However, the extraction of nucleic acids from a target cell typically involves a cell lysis step followed by nucleic acid purification. The cytolysis step of the cell disrupts the cells and cell membranes and releases the genetic material. This is usually done with a cytosolic detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cell. The nucleic acid can then be purified by an alcohol (usually ice-cold ethanol or isopropanol) precipitation step or by a solid phase purification step, typically based on a ruthenium oxide substrate on a column, resin or paramagnetic beads. The granules are carried out in the presence of a high concentration of chaotropic salts, followed by washing, followed by dissolution with a low ionic strength buffer. Any step prior to precipitation of the nucleic acid is the addition of a protease that digests the protein to further purify the sample. Other methods of cytolysis also include mechanical cytolysis by ultrasonic oscillations and heating of the sample to 94 °C to disrupt the thermal cytolysis of the cell membrane. The extracted material may also contain a very small amount of target DN A or RN A, especially when the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target at a low concentration to a detectable level. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). PCR has been known in the art and a detailed description of this type of reaction can be found in 201209406 E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008. PCR is a powerful technique for amplifying a target DNA sequence in a complex DNA background. If you want to amplify RNA (by PCR), you must first use an enzyme called reverse transcriptase to transcribe RNA into cDNA (complementary DNA). Then, the prepared cDNA was further amplified by PCR. PCR is an exponential process that continues as long as the conditions supporting the reaction permit. The ingredients of this reaction are:

1. Primer pair: a single strand of short DNA complementary to the flanking region of the target sequence and about 10-30 nucleotides long. 2. DNA polymerase: a thermostable enzyme capable of synthesizing DNA 3. Deoxyribonucleoside III Phosphoric acid (dNTPs): Nucleotide-buffered to provide newly synthesized DNA strands: The best chemical environment for DNA synthesis PCR typically involves the addition of such reactants to the vials containing the extracted nucleic acids (~ 10_50 microliters). Place the tube in a thermal cycler (a device that allows the reaction to react at different temperatures for varying lengths of time). The standard operating procedures for each thermal cycle involve the denaturation phase, the binding phase, and the extension phase. The extension period is sometimes referred to as the primer extension period. In addition to this three-step process, a two-step thermal process can be used, in which the combination period and extension period are combined. The denaturation phase typically involves raising the reaction temperature to 90-95 t to denature the DNA strand; during the binding phase, the temperature is lowered to about 50-60 °C to allow the primer to bind; then during the extension, the temperature is raised. 201209406 Up to 60-72 °C for DNA polymerase activity for primer extension. This process is repeated approximately 20-40 times, with the end result of producing a copy of the target sequence between millions of two primers. There are also a number of changes to the standard flow of the PCR, including, for example, multiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR, and reverse transcriptase PCR, which have been developed. For molecular diagnosis.

Multiplex PCR uses multiple primer sets in a single PCR mix to create amplicons of different lengths that are specific for different DNA sequences. By targeting multiple genes at the same time, more information can be obtained in a single test-operation, otherwise several experiments will be required. However, the optimization of multiplex PCR is more difficult and it is necessary to select primers with similar binding temperatures, and amplicon of similar length and base composition to ensure the amplification efficiency of each amplicon is equal to the leader-to-head PCR (also It is known to use conjugative linker PCR as a method for nucleic acid amplification of substantially all DN A sequences in a complex DNA mixture without the need for a target-specific primer. This method involves first digesting the target DN A population with an appropriate restriction enzyme (enzyme). A double-stranded oligonucleotide linker (also referred to as a linker) having a suitable overhanging end is then ligated to the end of the target DN A fragment with a ligase enzyme. Nucleic acid amplification is then carried out using oligonucleotide primers specific for the linker sequence. In this way, all DNA source fragments that are sandwiched by the linker oligonucleotide can be amplified. Direct PCR describes one that has not been subjected to any nucleic acid extraction or only some micronuclei 201209406

Acid extraction is performed on the sample directly on the PCR system. It has long been agreed that various components (e.g., heme components in blood) in unpurified biological samples inhibit the P C R reaction. Traditionally, P C R requires thorough purification of the target nucleic acid prior to preparation of the reaction mixture. However, by appropriately changing the chemical properties and sample concentration, PCR or direct PCR can be performed with minimal DNA purification. Direct PCR modulating PCR chemistry includes increasing buffer strength, using polymerases with high activity and continuous potency, and adding additives that chelate possible polymerase inhibitors.

Tandem PCR uses two independent fields of nucleic acid amplification reactions to increase the likelihood that the pair of amplicons will be amplified. One form of tandem PCR is nested PCR, which uses two pairs of PCR primers to amplify a single locus in each nucleic acid amplification field. The first pair of primers will hybridize to the nucleic acid sequence located in the region outside the target nucleic acid sequence. A second pair of primers (nested bed primers) for use in the second round of amplification will bind to the first PCR product and produce a second PCR product containing the target nucleic acid which will be shorter than the first PCR product. The logic behind this strategy is that if an incorrect locus is incorrectly amplified in the first round of nucleic acid amplification reactions, the probability that the locus will be amplified again by the second pair of primers will be extremely low. Come to ensure its specificity. Real-time PCR or quantitative PCR is used to measure the instantaneous amount of PCR product. The initial amount of nucleic acid in the sample can be quantified by using a probe containing a fluorophore or a fluorescent dye and a set of reaction standards. This is particularly useful for molecular diagnostics because the therapeutic regimen in molecular diagnostics will vary depending on the pathogen load within the sample. -10- 201209406 Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. Reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used for gene expression characterization to determine gene expression or to identify sequences of RNA transcripts, including transcription initiation and termination sites. It can also be used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus.

Isothermal amplification is another form of nucleic acid amplification whose amplification does not depend on the thermal denaturation of the target DN A, so no complicated mechanical means are required. Therefore, the isothermal nucleic acid amplification method can be carried out in situ or easily in an environment outside the laboratory. A variety of isothermal nucleic acid amplification methods have been described, including strand displacement amplification, transcription vector amplification, nucleic acid sequence amplification, recombinase polymerase amplification, rolling circle amplification, mesh Branch amplification, helicase-dependent isothermal DN A amplification and circular medium isothermal amplification. The isothermal nucleic acid amplification method does not rely on the continuous heat denaturation of the template DN A to produce a single molecule as a template for further amplification, but at a constant temperature by other methods such as using a specific restriction enzyme to the enzyme. Cut a gap in the DN A molecule or use an enzyme to separate the DN A strand. The strand displacement amplification method (SDA) relies on the ability of a specific restriction enzyme to cleave the unmodified gap in the semi-modified DNA and the extension of the 5'-3' exonuclease-deficient polymerase. The ability to replace the downstream strands. Exponential nucleic acid amplification is achieved by combining meaningful and antisense reactions, in which a meaningful reaction strand substitution (the resulting sequence) is used as a template for an antisense reaction. This reaction uses a nicking enzyme (which does not cut the DNA in a conventional manner, but instead cuts out a gap in the double-stranded DNA), such as N. -11 - 201209406

Alwl, N. BstNBl and Mlyl. SDA has been improved by the use of a combination of a thermostable restriction endonuclease (Jval) and a thermostable exonuclease-polymerase polymerase. This combination shows an amplification efficiency that increases the amplification of 1 〇 8 fold response to amplification 1 〇 1 (1 fold, so this technique can be used to amplify unique single copy molecules.

The transcription vector amplification method (TMA) and the nucleic acid sequence-based amplification method (NASBA) use RNA polymerase to replicate the RNA sequence 歹IJ instead of the corresponding genomic DNA. This technique uses two primers and two or three enzymes: RNA polymerase, reverse transcriptase and optionally RNase H (if the reverse transcriptase does not have RNase activity). A primer contains a promoter sequence of RNA polymerase. In the first step of nucleic acid amplification, the primer is hybridized to one of the target ribosomes RN A ( rRNA ). The reverse transcriptase can produce a DNA copy of the target rRNA by extending from the 3' end of the promoter. In the resulting RNA: DNA, the RNA is decomposed by the RNase activity possessed by the reverse transcriptase or the added RNase. Next, let the second primer be bound to the DN Α copy. A new DNA strand is synthesized from the end of the primer by reverse transcriptase to form a double stranded DNA molecule. RNA polymerase recognizes the promoter sequence within the DN A template and initiates transcription. Each newly synthesized RNA amplicon will re-enter this process and serve as a template for a new round of replication. In recombinant enzyme polymerase amplification (RPA), isothermal amplification of specific DNA fragments is achieved by binding the opposite oligonucleotide primer to template DN A and by extension of DN A polymerase. . There is no need to use heat to denature the double strand DNA (dsDNA) template. Alternatively, RPA uses a recombinase-introduction complex to scan dsDN A and promote share exchange of homologous sites.生-12- at . - 201209406 . • - · · V.· The structure is stabilized by the interaction between the single-stranded DNA-binding protein and the replaced template strand, thus preventing the primer from being evicted due to branch migration. . The dissociation of the degrading enzyme leaves the 3' end of the oligonucleotide of the parental replacement DNA polymerase (for example, a large fragment of Bacillus subtilis δα£^·//Μ·ϊ Pol I (Bsu)). Next the primer will extend. Exponential nucleic acid amplification can be accomplished by cyclically repeating this process.

The helicase-dependent amplification method (HDA) mimics the living system, using DN A helicase to generate a single-strand template for hybridization of the primer and then extending the primer with DN A polymerase. In the first step of the HAD reaction, the helicase moves along the target DN A, disintegrating the two hydrogen bonds, and the two separated molecules will bind to the single-stranded binding protein. The single-stranded target area exposed by the helicase allows primer binding. The DN A polymerase then extends from the 3' end of each primer using free deoxyribonucleoside triphosphates (dNTPs) to form two DN A replicators. These two replicated dsDN A shares will each enter the next HAD cycle, causing exponential nucleic acid amplification of the target sequence.

Other DN A-based isothermal techniques include rolling circle amplification (RCA), where the DNA polymerase allows the primer to extend continuously along the circular DNA template, resulting in a repeat containing many circular templates. Copy the strip of DN A product. At the end of the reaction, the polymerase produces a copy of thousands of circular templates that are ligated to the original target DNA. This allows for spatial resolution of the target and rapid nucleic acid amplification of the signal. Up to 101 2 template copies can be produced in one hour. The reticular branch is amplified as a change in RC A. It uses a locked circular probe (C_probe) or a padlock probe and a DNA polymerase with high continuous efficiency under isothermal conditions. - 201209406 Amplify the C-probe.

The circular medium isothermal amplification method (LAMP) is highly selective and employs DNA polymerase and a set of four primers that are specifically designed to recognize a total of six unique sequences on the target DNA. An intrinsic strand containing the target DN A and an intron within the antisense strand sequence can trigger LAMP. Subsequent replacement of DNA synthesis by a foreign-inducing strand releases a single strand of DNA. These single-stranded DN A can be used as a template for the synthesis of DN A, which is the lead of the second and second primers, which are heterozygous to the other end of the target sequence, which can form a stem-loop DNA structure. In the next LAMP cycle, an internal primer will hybridize to the loop of the product and initiate a replacement DN A synthesis reaction, producing the original stem-loop DNA and the new stem-loop DNA (the stem length is twice the original ). This cycle of reaction continues and less than an hour can accumulate 1 〇 9 copies of the target. The final product is a stem-loop DNA having a plurality of inverted copies of the target and a polycyclic broccoli structure formed by the incorporation of a replica of the target in the same strand.

After completion of the nucleic acid amplification, the amplification product must be analyzed to determine if the desired amplicon (amplification amount of the target nucleic acid) has been produced. The method of analyzing the product ranges from simply measuring the size of the amplicon by gel electrophoresis to identifying the nucleotide composition of the amplicon using DNA hybridization. Gel electrophoresis is the easiest way to check if a nucleic acid amplification reaction produces the desired amplicon. Gel electrophoresis applies an electric field to the gel matrix to separate the DNA fragments. Negatively charged DNA fragments move at different speeds (mainly by their size) on the substrate. When the electrophoresis is over, the fragments in the gel can be stained to make them visible to the naked eye. Ethidium bromide is a commonly used dye of -14-201209406, which emits fluorescence under UV light.

The size of the fragments is determined by alignment with a DNA size standard reference (DNA ladder containing DNA fragments of known size) which are placed next to the amplicon and run at the same time. Since the oligonucleotide primer binds to a specific site that encloses the target DNA, the size of the amplification product can be estimated and detected by a known size spectrum on the gel. In order to reliably identify the amplicon, or if multiple amplicons are simultaneously produced, the amplicon is usually hybridized with a DNA probe. DNA hybridization refers to the formation of double-stranded DNA by a pairing reaction of complementary bases. The DNA hybridization reaction used to positively identify a specific amplification product requires the use of a DNA probe of about 20 nucleotides in length. If the sequence of the probe is complementary to the amplicon (target) DN A sequence, the heterozygous reaction will occur under conditions of temperature, pH and ionic strength. If heterozygous occurs, the gene or DN A sequence indicating interest has appeared in the original sample. 0 Optical detection is the most common method for detecting heterozygosity. Amplicon or probe can be labeled by fluorescence or electrochemiluminescence to emit light. These methods differ in how the luminescent group is excited to form, but both can covalently label the nucleotide strand. In electrochemiluminescence (ECL), the light is generated after the luminescent group molecules or complexes are stimulated by a current, and is irradiated with excitation light that causes a radiation reaction. The fluorescent system uses an illumination source (which provides excitation light of a wavelength that can be absorbed by the fluorescent molecules) and a detection unit to detect. The detecting unit includes a photo sensor capable of detecting a radiation signal (for example, a photomultiplier tube or a charge coupled -15-201209406 device (CCD) array), and a mechanism for preventing excitation light from being included in the output of the photo sensor. (eg wavelength-select filter). The fluorescent molecules react with the excitation light to emit Stokes-shifted light, which is collected by the detecting unit. Stokes shifts the frequency difference or wavelength difference between the emitted light and the absorbed excitation light.

The ECL illumination is detected using a light sensor that is sensitive to the radiation wavelength of the ECL species being used. For example, a transition metal-ligand complex will emit light at visible wavelengths, so conventional photodiodes and CCDs can be used as photosensors. An advantage of ECL is that if the ambient light is excluded, the ECL illumination is the only light present in the detection system that enhances detection sensitivity.

Microarrays allow hundreds or thousands of DN A heterozygous experiments to be performed simultaneously. Microarrays are powerful tools in molecular diagnostics that can screen thousands of diseases or detect the presence of multiple infectious pathogens in a single test. The microarray consists of a number of different DN A probes that are fixed to the substrate and are spotted. The target DN A (amplicon) is first labeled with a fluorescent or luminescent molecule (either during or after nucleic acid amplification) and then applied to the probe array. The microarray is incubated in a controlled temperature, humid environment for hours or days, during which time the probe and the amplicon will hybridize. After incubation, the microarray must be rinsed with a series of buffers to remove unbound strands. Once the cleaning is complete, the surface of the microarray is blown dry with an air injection (usually a nitrogen injection). The stringency of hybridization and cleaning is severe. Insufficient stringency can result in a high degree of non-specific binding. Excessive rigor can result in inability to properly combine, resulting in reduced sensitivity. The heterozygous line is detected by the light emission of the labeled amplicons, which have formed a hybrid with the complementary probes of -16-201209406. The fluorescence emitted by the microarray is detected using a microarray scanner, typically a computer controlled inverted scanning fluorescent conjugated focus microscope, which typically uses a laser to excite the fluorescent dye. And using a light sensor (such as a photomultiplier tube or CCD) to detect the transmitted signal. The fluorescent molecules emit Stokes-shifted light (as described above), which are collected by the detection unit.

The emitted fluorescence must be collected, separated from the wavelength of the unabsorbed excitation light, and transmitted to the detector. Conjugate focal designs are often used in microarray scanners to remove defocus information by conjugated pinholes located on the image plane. This will only allow the light in the focus portion to be detected. Avoid light from above and below the object's focal plane into the detector, thereby increasing the ratio of signal to noise. The detected fluorescent photons are converted into electrical energy by the detector and then converted into a digital signal. This digital signal is translated into a number representing the intensity of the fluorescence of a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. The exact sequence and position of each probe on the microarray is known, so that the hybrid target sequences can be identified and analyzed simultaneously. More information on fluorescent probes can be found at: http : "www.premierbiosoft.com/tech_notes/FRET_probe, html and http : "www.invitrogen.com/site/us/en/home/

References/Molecular-Probes-The-Handbook/Technical-

Notes-and-Product-Highlights/Fluorescence- Resonance- 201209406

Energy-Transfer-FRET. html Molecular Diagnostics for Point-of-care

Although molecular diagnostic tests can provide these benefits, the growth of this type of test in clinical laboratories is slower than expected and only a small fraction of these tests are performed in laboratory medicine practice. This is mainly because the test using nucleic acid is more complicated and more expensive than other test methods that do not involve nucleic acid. In the clinical environment, the extensive use of molecular diagnostic tests is closely related to the development of equipped instruments. Significantly reduces costs, provides fast and automated verification from scratch (sample processing) to tail (results), and it does not require extensive human intervention. There are many benefits to using a point-and-care approach to a doctor's office, a hospital's clinical or even a user-focused home, including: • Quick results, immediate treatment and improved care quality.

• Ability to obtain laboratory-quality test results by testing very few samples. • Reduce clinical effort. • Reduce laboratory load and improve office performance by reducing administrative work. • Improve the cost of treatment for each patient by reducing the time spent on hospitalization, the time required to reach a conclusion for first-time medical outpatient consultation, and reducing the time spent on sample handling, storage, and transportation. • Accelerate clinical governance conclusions such as infection control and antibiotic use -18- 201209406 On-wafer laboratory (LOC)-based molecules, diagnostics

Molecular diagnostic systems based on microfluidics provide devices that automate and accelerate molecular diagnostic assays. The faster detection time is mainly based on the active small size of the body involved, the automated operation and the diagnostic process steps of the microfluidic device as a low overhead built-in cascade. The volume of nanoliters and microliters also reduces reagent wear and cost. The on-wafer laboratory (L 0 C) device is in the form of a common microfluidic device. The LOC unit has an MST structure (for fluid processing) integrated into the MST layer on a single struts (usually ruthenium). The use of the VLSI (very large scale integrated) etching technology of the semiconductor industry to manufacture can make the unit cost of the LOC device very low. However, large amounts of external piping and electronics are required to control the flow of fluid through the LOC device, to add reagents, to regulate reaction conditions, and the like. In fact, the connection of LOC devices to such external devices limits the use of LOC devices for molecular diagnostics in laboratory environments. The cost of such external devices and the complexity of their operation make LOC-based molecular diagnostics a practical solution for point-of-care devices. Based on the above points, there is a need for a molecular diagnostic system based on a LOC device that can be used for fixed-point care. SUMMARY OF THE INVENTION Various aspects of the invention are described in the following numbered paragraphs. -19- 201209406 GBS005.1 This aspect of the invention provides a microfluidic device for diagnostic analysis of a sample fluid, the microfluidic device comprising: a plurality of on-wafer laboratory (LOC) devices, each device having a support substrate and a microsystem technology (MST) layer for treating the sample fluid;

A top cover interconnecting the plurality of LOC devices, the top cover having a top cover channel for establishing fluid communication with the MST layers on the at least two LOC devices. GBS005.2 Preferably, the MST layer in each LOC device defines at least one microchannel for processing the sample fluid, and the cap is configured to be capable of one microchannel in one LOC device and at least another LOC A fluid connection is established between the microchannels of one of the devices. Preferably, the top cover is configured to draw fluid flow between the LOC devices by capillary action.

Preferably, the top cover has a reservoir in fluid communication with the top cover channel, the reservoir being configured to retain reagent by surface tension of the meniscus of the reagent. GBS005.5 Preferably, the plurality of LOC devices are a first LOC device and a second LOC device, the first LOC device having a dialysis zone capable of accepting a sample and having a composition greater than a threshold size and less than 闽値The components of the size are separated. Preferably, the dialysis zone has apertures corresponding to the threshold 値 size and components greater than the threshold 包括 include white blood cells and components less than the threshold 包括 include pathogens. -20- 201209406 GBS005.7 Preferably, the second LOC device has a hybrid region containing a probe nucleic acid sequence which can hybridize with a target nucleic acid sequence in the sample to form a probe-target A heterozygous region configured to detect the probe-target hybrid. GBS005.8 Preferably, the top cover has a layered structure and the top cover channels are formed in one of the layers and the reservoir is formed in the other adjacent layer.

GBS005.9 Preferably, the layered structure includes an interfacial layer for establishing fluid communication between the cap passage and the LOC devices. Preferably, the MST layer on the second LOC has an active valve that blocks the flow of the sample from the MST channel to the cap channel until the active valve is activated to allow the sample to reflow. GBS 005.il Preferably, the sample is blood and the reagent in the reservoir is an anticoagulant and the cap is configured to allow the anticoagulant to mix with the blood prior to entering the dialysis zone. GBS 00 5.12 Preferably, the microfluidic device further has a cytolytic zone in fluid communication with a lysing reagent reservoir containing a cytolytic reagent, the cytolysis zone being configured to lysate cells and release the same The genetic material inside. GBS 005.1 3 Preferably, the microfluidic device further has a nucleic acid amplification region for amplifying the nucleic acid sequence within the fluid. GBS 005.14 Preferably, the nucleic acid amplification region is a polymerase chain reaction (PCR) region and the cap has a pCR reagent reservoir containing dNTPs and primers to bind the PCR reagents before amplifying the nucleic acid sequence. The samples are mixed together. GBS00 5.1 5 Preferably, the cap has a poly-21 - 201209406 synthase reservoir containing a polymerase to mix the enzyme with the fluid prior to amplifying the nucleic acid sequence. Preferably, the The microfluidic device also has a CMOS circuit between the support substrate and the MST layer to operatively control the pcr region

Preferably, the microfluidic device further has a heterozygous region having a hybrid chamber containing a probe nucleic acid sequence capable of binding to a target nucleic acid sequence within the fluid. GBS005.18 Preferably, the probe nucleic acid sequence comprises an Electrochemical Luminescence Resonance Energy Transfer (ERET) probe. Preferably, each of the hybrid chambers has a photodiode capable of detecting the luminescence of the ERET probe, which reacts with the target nucleic acid sequence. GBS005.20 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the sample.

Multiple surface-micromachined chips are fluidly integrated to provide more adequate and progressive functionality. The microfluidic multi-wafer assembly provides greater modularity. The surface MEMS wafer within the assembly is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. The use of a less expensive process with the best function to fabricate the various surface-micro-electrode wafer components of the microfluidic multi-chip assembly, such as a only-bioMST dialysis wafer, can omit all CMOS process steps and can employ inexpensive glass wafers. GB S006.1 provides a test module according to this aspect of the invention, the package -22-201209406 includes: a housing for carrying by hand; a storage tank for accommodating the sample fluid; and a fluid connection with the inlet a microfluidic device having a plurality of on-wafer laboratory (LOC) devices and a top cover associated with each LOC device; wherein the cap is configured to establish a fluid connection between at least two LOC devices

through. GBS006.2 Preferably, each LOC device has a support substrate and a microsystem technology (MST) layer on the support substrate and a top cover overlying the MST layer. An MST channel and a plurality of fluidic connections in fluid communication with the cap and having a cap channel GBS006.3 in fluid communication with a fluid connection of each LOC device, preferably the sample fluid is The biological sample and each MST channel has a cross-sectional area of 1 square micrometer to 400 square micrometer to biochemically process the components in the biological sample, and each of the cap channels has a cross-sectional area larger than 400 square micrometers to receive the biological The sample and the cells in the biological sample are transported to a predetermined location within the MST channel. Preferably, the housing has a lancet for puncturing the patient's finger to obtain a blood sample that is applied to the sample inlet. GBS 006.5 Preferably, the lancet is switchable between a compressed and extended position, the housing having a biasing mechanism to bias the lancet to the extended position, and a user actuated catch To retract the lance-23-201209406 blood needle to the compression position until the user starts again. GBS006.6 Preferably, the cap is configured to drive fluid flow through the cap channels by capillary action. Preferably, the top cover has a reservoir in fluid communication with the top cover channel, the reservoir being configured to retain the reagent by the surface tension of the meniscus of the reagent.

GBS006.8 Preferably, the plurality of LOC devices are a first LOC device and a second LOC device, the first LOC device having a dialysis zone capable of accepting a sample and having a composition greater than a threshold size and a threshold less than The ingredients are separated. Preferably, the dialysis zone has apertures corresponding to the size of the crucible and components greater than the crucible include white blood cells and components smaller than the crucible include pathogens.

GBS006.1 0 Preferably, the second LOC device has a hybrid region comprising a probe nucleic acid sequence capable of hybridizing with a target nucleic acid sequence in the sample to form a probe-target hybrid The hybrid region is configured to detect the probe-target hybrid. GBS 00 6.1 1 Preferably, the top cover has a layered structure and the top cover passage is formed in one of the layers and the reservoir is formed in the other adjacent layer. GBS006.12 Preferably, the layered structure includes an interfacial layer for establishing fluid communication between the canopy channel and the LOC devices. Preferably, the MST layer on the second LOC has an active valve that blocks the flow of the sample from the MST channel to the cap channel until the active valve is activated to allow the sample to reflow. -24- 201209406 GBS006.14 preferably 'the sample is blood and the reagent in the reservoir is an anticoagulant and the cap is configured to mix the anticoagulant with blood before the blood enters the dialysis zone Together. Preferably, the microfluidic device further has a cytolytic zone in fluid communication with a cytolytic reagent reservoir containing a lysing reagent, the cytolysis zone being configured to lysate the cells and release the genes therein. substance.

GBS006.16 Preferably, the microfluidic device further has a nucleic acid amplification region for amplifying a nucleic acid sequence in the fluid. GBS006.1 7 Preferably, the nucleic acid amplification region is a polymerase chain reaction (PCR) region and the cap has a PCR reagent reservoir containing dNTPs and primers for amplifying the nucleic acid sequence before amplifying the nucleic acid sequence. The reagent is mixed with the sample. Preferably, the cap has a polymerase-containing polymerase reservoir to mix the enzyme with the fluid prior to amplifying the nucleic acid sequence. GBS006.19 Preferably, the microfluidic device further has a CMOS circuit between the support substrate and the MS T layer to operatively control the PCR zone GBS006.20. Preferably, the microfluidic device further has A hybrid region of a hybrid chamber containing a probe nucleic acid sequence capable of binding to a target nucleic acid sequence within the fluid. Multiple surface-microelectromechanical wafers are fluidly integrated to provide more adequate and progressive functionality. The microfluidic multi-wafer assembly provides greater modularity. The surface MEMS wafer within the assembly is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Using the most inexpensive process with the best function - 25, 201209406 to manufacture the various surface of the microfluidic multi-chip module - the components of the MEMS wafer, for example, the only -bioMST dialysis wafer can omit all CMOS process steps and can use inexpensive glass Substrate. GDI0 15.1 This aspect of the invention provides a microfluidic device for treating a sample fluid containing a target molecule, the microfluidic device comprising: a dialysis device for receiving a sample and concentrating a target molecule in a portion of the sample :

- a wafer-on-lab (L0C) for analyzing the target molecules; and a cap covering the LOC and the dialysis device to establish fluid communication between the LOC and the dialysis device. GDI0 15.2 Preferably, the fluid sample is a sample of biological material containing cells of different sizes, the dialysis zone has at least two channels and a plurality of small holes and the small holes are fluidly connected to the channels, a plurality of small The size of the pores corresponds to the size of the cells within the fluid sample.

GD10 1 5.3 Preferably, the at least two channels and the plurality of apertures are configured to allow the sample to flow through the channels and apertures by capillary action. Preferably, the GD 1015.4 includes at least two channels including a target channel and a waste channel, the target channel being coupled to the top cover for driving the flow to the LOC by capillary. GD 10 1 5.5 Preferably, the target molecules are target nucleic acid sequences within the cells of the sample fluid, and the LOC has a nucleic acid amplification region for amplifying the target nucleic acid sequence. Preferably, the target nucleic acid sequence is in a cell smaller than the established 闽値-26-201209406. Preferably, the LOC has a hybrid region with a hybrid probe array. The probe can hybridize to the target nucleic acid sequence to form a probe-target hybrid. GDI015.8 Preferably, the probe is designed to form a probe-target hybrid with the target nucleic acid sequence, the probe-target hybrid being designed to emit photons of light in response to an excitation current. .

Preferably, the LOC device has a CMOS circuit for operatively controlling the PCR zone, the CMOS circuit having a photosensor for sensing photons emitted by the probe-target hybrid. GDI0 15.10 Preferably, the hybrid region has an array of hybrid chambers containing probes that are capable of hybridizing to the target nucleic acid sequence. GDI0 1 5 · 1 1 Preferably, the photo sensor is an array of photodiodes, and the photodiodes are respectively adjacent to the hybrid chambers. Preferably, the CMOS circuit has a digital memory for storing data associated with the fluid processing, the data including the probe details and the position of each probe in the array of hybrid chambers. GDI015.13 Preferably, the CMOS circuit has at least one temperature sensor to sense the temperature of the hybrid chamber array. Preferably, the L0C device has a heater controlled by a CMOS circuit that utilizes feedback from a temperature sensor to maintain the probe and target nucleic acid sequence below the hybrid temperature. Preferably, the photodiode is less than 1600 microns from the corresponding hybrid chamber. -27- 201209406 GDI015.16 Preferably, the probe has an electrochemiluminescence (ECL) luminophore of photons in an excited state. Preferably, the hybrid chambers have electrodes for emitting the ECL luminophores. Preferably, each of the ECL probes has a quenching probe that emits a photon emitted by the illuminating group to quench the photon emitted by the illuminating group, and when the hybridization of a target nucleic acid sequence is mixed, the quenching material is moved far away. Photons are no longer quenched. GD1015.19 Preferably, the CMOS circuit has an interface, electrically connected to an external device, and configured to convert the output of the polar body into an indication signal indicating that the ECL probe has been associated with the target nucleic acid, and Signals are provided to the bond pads to transfer the device. Preferably, the top cover has at least - fluid communication between the passage means and the LOC, and a plurality of reservoirs for containing the test reagent. The easy-to-use, mass-produced and inexpensive microfluidic multi-energy accepting biological sample 'using dialysis wafers to functionally extract nucleic acid content wafers of different sizes and separately process the size-differentiated cells can be more extracted from the test Information and increase the sensitivity, signal-to-noise ratio and dynamic range. The microfluidic multi-wafer assembly provides greater modularity. The microelectromechanical wafer is much cheaper and disproportionately much smaller than a single unit that provides all of the functionality of the assembly. Alternatively, a large group emits a current stimulator and a sticker when the luminescent group (bond-mixed from the photodiode sequence to the outside and the dialysis sample)

Wafer component cell separation, . The dialysis system has a small wafer size but a 280-201209406 cost-effective dialysis wafer provides advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. Each of the surface-microelectromechanical wafer components of the microfluidic multi-chip assembly is fabricated using a less expensive process that is optimal in function, i.e., the only-bioMST dialysis wafer can omit all CMOS process steps and can employ inexpensive glass wafers. GDI 0 16.1 This aspect of the invention provides a dialysis device for dialysis of fluids containing different sized components, the dialysis device comprising:

a first material layer defining a first channel and a second channel, the first channel being configured to accept a fluid having components of different sizes; a second layer having a plurality of apertures and at least one fluid connector, a plurality of small holes are open to the first passage and the fluid connection member is connected to the second passage from the small holes to establish fluid communication between the first passage and the second passage: wherein the small holes are sized according to the predetermined The size of the crucible is such that the component flowing to the second channel is a smaller component smaller than the predetermined crucible size, and the component remaining in the first channel comprises a larger component GDI0 16.2 larger than the predetermined crucible size. The second layer is a layered structure having a top wall layer, and the at least one fluid connecting member is a series of adjacent passages closed by the top wall layer, the small holes being in the first wall layer at the first The formation of a channel and the adjacent channels. GDI0 16.3 Preferably, the first and second passages and the series of contiguous passages are configured to be filled with the sample by capillary action. GDI016.4 preferably, wherein the dialysis device has a further between the -29th and the 201209406

a bypass passage between a passage and a second passage, wherein each of the adjacent passages is configured to fix a fluid meniscus to block capillary flow between the first passage and the second passage; and the first passage And a bypass passage between the second passages, the bypass passages merging into the second passage upstream of the series of adjacent passages and configured to provide an unimpeded capillary drive flow from the first passage to the second passage; After the meniscus is formed in each of the adjoining passages, the liquid flow from the bypass passage sequentially removes each meniscus when it reaches the meniscus fixed to each of the adjoining passages, and the sample liquid flows through the same The adjoining channel and the bypass channel flow from the first channel to the second channel. GDI016.5 Preferably, the sample is a biological sample containing components of different sizes, and the adjacent channels and the bypass channels have small holes corresponding to a predetermined threshold size, such that the component flowing into the second channel is smaller than the predetermined size. The smaller component of the 値 size, while the component remaining in the first channel contains a larger component than the predetermined 闽値 size.

GDI 0 16.6 Preferably, the biological sample is blood and the larger component comprises white blood cells and the smaller components include red blood cells. GDI0 16.7 Preferably, the contiguous channel is a series of parallel adjacent channels and typically extends to the first channel and the second channel. GDI0 16.8 Preferably, the dialysis device further has a waste reservoir for containing downstream processing or for analyzing unwanted components. GDI 016.9 Preferably, the aperture is a hole having a diameter of less than 8 microns. Preferably, the smaller component includes the target, and therefore the processing of the smaller component includes detection of the target. GDI01 6.1 1 Preferably, the dialysis device further has a cytolysis zone attached to the target -30-201209406 channel to lyse the target cells to release the target nucleic acid sequence therein. GDI01 6.12 Preferably, the dialysis device further has a nucleic acid amplification region for amplifying the target nucleic acid sequences. GDI 0 16.13 Preferably, the dialysis device further has a heterozygous region of a hybrid probe array that is hybridized to the target nucleic acid sequence amplified by the nucleic acid amplification region.

Preferably, the probe is designed to form a probe-target hybrid with the target nucleic acid sequence, the probe-target hybrid capable of reacting with a current pulse to produce electrochemiluminescence. GDI016.15 preferably, the dialysis device further has a CMOS circuit for operatively controlling the nucleic acid amplification region and the hybrid region, the CMOS circuit further having a photo sensor to sense the electrochemical of the probe-target hybrid Luminous radiation. Preferably, the hybrid region has an array of hybrid chambers containing probes that are capable of hybridizing to the target nucleic acid sequences. GDI016.17 Preferably, the photo sensor is an array of photodiodes, and the photodiodes are respectively adjacent to the hybrid chambers. Preferably, the CMOS circuit has a digital memory for storing fluid processing related data, including detailed description of the probes and the position of each probe in the array of hybrid chambers. GDI016.19 preferably, the CMOS circuit has a bond pad for electronically connecting an external device, and is configured to convert an output from the photodiode into an indication signal indicating that the probe has been hybridized with the target nucleic acid sequence, and This signal is provided to the bond pads for transmission to an external device. Preferably, the dialysis device has a plurality of reservoirs for containing the liquid reagents added to the sample. The easy-to-use, mass-produced, and inexpensive microfluidic multi-wafer assembly is capable of accepting biological samples, separating the cells of different sizes using dialysis wafers, and separately processing the nucleic acid contents of the cells differentiated by size. The dialysis wafer functionally extracts more information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.

The microfluidic multi-wafer assembly provides greater modularity. The surface MEMS of this component is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Alternatively, a large but cost-effective dialysis wafer can provide advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. Each of the surface-microelectromechanical wafer components of the microfluidic multi-chip assembly is fabricated using a less expensive process that is optimal in function, i.e., the only-bioMST dialysis wafer can omit all CMOS process steps and can employ inexpensive glass wafers.

GDI0 17.1 This aspect of the invention provides a dialysis device having a capillary drive, flow channel structure for dialysis of fluids having different sized components, the dialysis device comprising: a first passage configured to be fluidly filled by capillary action a second passage configured to be fluidly filled by capillary action; a plurality of fluid connections connecting the first passage and the second passage, each connector being configured to fix a fluid meniscus to block the first Capillary flow between a channel and a second channel; a channel between the first channel and the second channel, the bypass channel is merged into the second channel upstream of the -32-201209406 plurality of fluid connectors and configured to be capable of Providing an unimpeded capillary drive flow from the first passage to the second passage; wherein during use, after the meniscus is formed in each of the fluid connections, the flow from the bypass passage is fixed to each of the fluid connections Each meniscus is sequentially removed during the meniscus, and the sample stream flows from the first channel to the second channel via the plurality of fluid connections and bypass channels.

GDI0 17.2 Preferably, the fluid is a biological sample containing components of different sizes, and the plurality of fluid connecting members and the bypass passage have small holes corresponding to a predetermined threshold size, such that the component flowing to the second channel is smaller than the predetermined The smaller component of the threshold 値 size, while the component remaining in the first channel contains a larger component greater than the predetermined threshold 値 size. GD 1017.3 Preferably, the biological sample is blood and the larger component comprises white blood cells and the smaller components include red blood cells. GDI 017.4 Preferably, the dialysis device further comprises: a first layer defining a first channel and a second channel; and a second layer defining a plurality of fluid connections and bypass channels. GDI0 17.5 Preferably, the second layer is a layered structure having a top wall layer, the plurality of fluid connecting members being a series of adjacent channels closed by the top wall layer, the small holes being in the top wall layer Formed between the first channel and the adjacent channels. GDI01 7.6 Preferably, the contiguous channel is a series of parallel adjacent channels and extends generally to the first channel and the second channel. GDI0 17.7 Preferably, the dialysis device also has a waste reservoir -33-201209406 for containing components that are not needed for downstream processing or analysis. GDI0 17.8 Preferably, the aperture is a hole having a diameter of less than 8 microns. GDI0 17.9 Preferably, the smaller component comprises a target, so processing of the smaller component includes detection of the target. GDI01 7.10 Preferably, the dialysis device further has a cytolysis zone attached to the target channel to lyse the target cell to release the target nucleic acid sequence therein.

GDI0 17.il Preferably, the dialysis device further has a nucleic acid amplification region for amplifying the target nucleic acid sequence. GDI01 7.12 Preferably, the dialysis device further has a hybrid region comprising a hybrid probe array that is hybridizable to the target nucleic acid sequence amplified by the nucleic acid amplification region. GDI0 17.13 Preferably, the probe is designed to form a probe-target hybrid with a target nucleic acid sequence that is capable of reacting to a current pulse to produce electrochemiluminescence.

GDI017.14 Preferably, the dialysis device further has a CMOS circuit for operatively controlling the nucleic acid amplification region and the hybrid region, the CMOS circuit further having a photo sensor for sensing the electrochemistry from the probe-target hybrid Learn to emit radiation. GDI 017.15 Preferably, the CMOS circuit is configured to provide electrical pulses to the electrodes. Preferably, the hybrid region has an array of hybrid chambers containing probes that hybridize to the target nucleic acid sequence. GDI0 17.17 Preferably, the photo sensor is a photodiode array, -34-201209406 and the photodiodes are respectively adjacent to the hybrid chambers respectively. GDI017.18 preferably, the CMOS circuit has a use The digital memory for storing fluid processing related data, including detailed description of the probe and the position of each probe in the array of hybrid chambers.

GDI017.19 preferably, the CMOS circuit has a bond pad for electronically connecting an external device, and is configured to convert an output from the photodiode into an indication signal indicating that the probe has been hybridized to the target nucleic acid sequence, and This signal is provided to the bond pads for transmission to an external device. Preferably, the dialysis device has a plurality of reservoirs for containing the liquid reagents added to the sample. The easy-to-use, mass-produced and inexpensive microfluidic multi-wafer assembly is capable of accepting biological samples, separating different sized cells using dialysis wafers, and separately processing nucleic acid contents of cells differentiated by size. More information from the sample is extracted and the sensitivity, signal-to-noise ratio and dynamic range of the assay system are increased. The microfluidic multi-wafer assembly provides greater modularity. The surface MEMS of this component is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Alternatively, a large but cost-effective dialysis wafer can provide advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. Each of the surface-microelectromechanical wafer components of the microfluidic multi-chip assembly is fabricated using a less expensive process that is optimal in function, i.e., the only-bioMST dialysis wafer can omit all CMOS process steps and can employ inexpensive glass wafers. This special flow channel structure initiates capillary motion of the dialysis wafer without trapping air bubbles. GDI0 1 9.1 This aspect of the invention provides a dialysis device for separating a pathogen from a biological sample, the dialysis device comprising: a first channel for receiving a biological sample; - a second channel: and a plurality of small holes ;among them

The second passage is fluidly coupled to the first passage via the apertures to enable pathogens to flow from the first passage to the second passage while a larger component of the biological sample remains in the first passage. GDI0 19.2 Preferably, the dialysis device further has a series of adjoining passages extending between the first passage and the second passage, wherein the small holes are located at the upstream end of the adjacent passages, and the adjacent passages are configured to be fixed a sample meniscus to block capillary flow between the first passage and the second passage: and

a bypass passage between the first passage and the second passage, the bypass passage sinking into the second passage upstream of the adjacent passages and configured to provide unimpeded capillary drive flow from the first passage To the second passage; wherein during use - after the meniscus is formed in each of the adjacent passages, the flow from the bypass passage sequentially removes each of the liquid flows when they reach the meniscus fixed to each of the adjacent passages In the meniscus, the sample stream flows from the first channel to the second channel via the adjacent channels and bypass channels. GDI019.3 Preferably, the dialysis device further comprises: a top cover layer, the first passage and the second passage are formed therein; and a support layer, the adjacent passages are formed therein, and the cover layer covers -36- 201209406 On the support layer. GD 1019.4 Preferably, the support layer is a layered structure comprising a top wall layer, the top wall layer defining the apertures. GDI 019.5 Preferably, the contiguous channels are parallel and generally extend to the first and second channels. GDI 019.6 Preferably, the biological sample is blood and the cap has a reagent reservoir containing an anticoagulant added to the blood.

GDI019.7 Preferably, the reagent reservoir has a surface tension valve having a meniscus anchor to fix the meniscus to leave the reagent in the reservoir, thus when the sample flow is from the meniscus The anticoagulant is loaded into the sample stream when the meniscus is removed from the face anchor. The easy-to-use, mass-produced, and inexpensive microfluidic multi-wafer assembly can accept biological samples, separate the pathogens in the sample using dialysis wafers, and separately process the nucleic acid contents of the isolated pathogens in the samples. The dialysis wafer functionally extracts more information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The multi-chip lab provides higher modularity. The surface MEMS of the assembly is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Alternatively, a large but cost-effective dialysis wafer can provide advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The best-functioning, inexpensive process is used to fabricate the microfluid. The various surface of the multi-chip assembly - the component of the MEMS wafer, the only-bioMST dialysis wafer, omits all CMOS process steps and can be used with inexpensive glass wafers. -37- 201209406 GD 102 3.1 This aspect of the invention provides a dialysis device for separating nucleated cells from a smaller component in a biological sample, the dialysis device comprising: a first channel for receiving a biological sample; a second channel; and a plurality of small holes; wherein

The second channel is fluidly connected to the first channel via the apertures. The apertures are smaller than the nucleated cells such that smaller components flow from the first channel to the second channel and within the biological sample Nucleated cells will remain in the first channel. Preferably, the dialysis device further has a series of adjacent passages extending between the first passage and the second passage, wherein the small holes are located at the upstream end of the adjacent passages, and the adjacent passages are configured to Capturing a sample meniscus to block capillary flow between the first passage and the second passage; and

a bypass passage between the first passage and the second passage, the bypass passage sinking into the second passage upstream of the adjoining passage and configured to provide an unimpeded capillary drive flow from the first passage to the second passage a channel; wherein, during use, after the meniscus is formed in each of the adjoining passages, the flow from the bypass passage sequentially removes each meniscus when reaching the meniscus fixed to each of the adjoining passages The sample flow flows from the first passage to the second passage via the adjacent passages and the bypass passage. GDI023.3 Preferably, the dialysis device further comprises: a cap layer, the first channel and the second channel are formed therein; and a support layer, wherein the adjoining channels are formed therein; the cap layer covers -38- 201209406 On the support layer. GDI 023.4 Preferably, the support layer is a layered structure comprising a top wall layer, the top wall layer defining the apertures. Preferably, the GDI 023.5 is parallel and generally extends to the first and second channels. Preferably, the biological sample is blood and the cap has a reagent reservoir containing an anticoagulant added to the blood.

GDI023.7 Preferably, the reagent reservoir has a surface tension valve having a meniscus anchor to secure the meniscus to retain the reagent in the reservoir, thus removing the sample stream from the meniscus anchor The meniscus will load the anticoagulant into the sample stream. GD 1023.8 Preferably, the nucleated cells are white blood cells. This multi-wafer laboratory design has the advantage of directly selecting the sample component from the sample containing the target. This multi-wafer laboratory design has the advantage of increasing the concentration of the target (the target is part of the sample and will be further processed by the multi-wafer laboratory). This multi-wafer laboratory design has the advantage of removing sample components that may inhibit subsequent analysis steps. This multi-wafer laboratory design has the advantage of removing undesirable components of the processing mixture that may interfere with later detection of the target. This multi-wafer laboratory design has the advantage of removing components of the reaction chamber or connections that may block the multi-wafer laboratory and reducing the quality of the operation. The multi-chip lab provides higher modularity. The surface MEMS of the assembly is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Alternatively, a large dialysis wafer with a cost of -39-201209406 can provide advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. Each of the surface-microelectromechanical wafer components of the microfluidic multi-chip assembly is fabricated using a less expensive process that is optimal in function, i.e., the only-bioMST dialysis wafer can omit all CMOS process steps and can employ inexpensive glass wafers. GDI 028.1 This aspect of the invention provides a test module for analyzing a sample fluid containing a target molecule, the test module comprising:

An outer casing having a sump for containing the sample fluid; and a microfluidic device having: a dialysis device in fluid communication with the sump and configured to separate target molecules from other components of the sample; A lab-on-lab (LOC) device for analyzing the target molecule: and a cap covering the L0C device and the analyzing device to establish fluid communication between the L0C device and the analyzing device.

GDI028.2 Preferably, the fluid sample is a sample of biological material containing cells of different sizes, and the dialysis zone has at least two channels connected to the channels by a plurality of small holes, the plurality of small holes being of comparable size The predetermined threshold size of the fluid sample cells. GDI 028.3 Preferably, the at least two channels and the plurality of apertures are configured to allow the sample to flow through the channels and apertures by capillary action. GDI0 28.4 Preferably, the at least two channels comprise a target channel and a waste channel, the target channel being connected to the top cover for flowing the capillary flow to the LOC device. -40 - 201209406 GDI0 28.5 Preferably, the target molecules are target nucleic acid sequences within the cells in the sample fluid, and the LOC device has a nucleic acid amplification region for amplifying the target nucleic acid sequence. GDI02 8.6 Preferably, the target nucleic acid sequence is within a cell that is less than a predetermined threshold.

GDI 028.7 Preferably, the LOC device has a heterozygous region containing a hybrid probe array that can hybridize to the target nucleic acid sequence to form a probe-target hybrid. GDI 02 8.8 Preferably, the probe is designed to form a probe-target hybrid with the target nucleic acid sequence, the probe-target hybrid being designed to emit photons of light in response to an excitation current. . GDI 028.9 Preferably, wherein the LOC device has a CMOS circuit for operative control of the PCR region, the CMOS circuit having a photosensor for sensing photons emitted by the probe-target hybrid. GDI02 8.1 0 Preferably, the hybrid region has an array of hybrid chambers containing probes capable of hybridizing with the target nucleic acid sequence. GDI02 8.il preferably, the photosensor is a light two A polar body array, and the photodiodes are adjacent to the respective hybrid chambers. GDI02 8.1 2 Preferably, the CMOS circuit has a digital memory for storing fluid processing related data, including detailed description of the probe and the position of each probe in the array of hybrid chambers. GDI 02 8.1 3 Preferably, the CMOS circuit has at least one temperature sensor to sense the temperature of the hybrid chamber array.

Preferably, the LOC device has a heater controlled by a CMOS 201209406 circuit. The CMOS circuit utilizes feedback from the temperature sensor to maintain the probe and target nucleic acid sequence below the hybrid temperature. GD 102 8.1 5 preferably 'the photodiode is less than 160 microns from the corresponding hybrid chamber. GD 1028.1 6 Preferably, the probe has an electrochemiluminescent (ECL) luminophore that emits photons in an excited state. GDI 02 8.1 7 preferably 'the hybrid chambers have electrodes to excite the ECL luminophores with current. Preferably, each of the ECL probes has a luminophore and a quenching material adjacent to the luminophore for quenching the photons emitted by the luminophore, and the probe moves when it is hybridized with a target nucleic acid sequence. The quencher is remote from the luminophore so that the photons are no longer quenched. GDI028.1 9 preferably, the CMOS circuit has a bond pad for electronically connecting an external device, and is configured to convert an output from the photodiode into an indication signal indicating that the ECL probe has been hybridized with the target nucleic acid sequence, And the signal is provided to the bond pads for transmission to an external device. GDI02 8.20 Preferably, the cap has at least one passage for fluid communication between the dialysis device and the LOC device, and a plurality of reservoirs for holding the liquid reagent to be added to the sample. The easy-to-use, mass-produced, and inexpensive microfluidic multi-wafer assembly is capable of accepting biological samples, separating the cells of different sizes using dialysis wafers, and separately processing the nucleic acid contents of the cells differentiated by size. The dialysis wafer functionally extracts more information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. -42- 201209406 The microfluidic multi-chip assembly provides higher modularity. The surface MEMS of this component is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Alternatively, a large but cost-effective dialysis wafer can provide advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.

Each of the surface-microelectromechanical wafer components of the microfluidic multi-chip assembly is fabricated using a less expensive process that is optimal in function, i.e., the only-bioMST dialysis wafer can omit all CMOS process steps and can employ inexpensive glass wafers. GDI0 3 0.1 This aspect of the invention provides a test module for concentrating a pathogen in a biological sample, the test module comprising: a housing having a reservoir for housing the sample fluid; and a dialysis device, The sump is in fluid communication and is configured to separate the pathogen from other components within the sample: and a on-wafer laboratory (LOC) device in fluid communication with the dialysis device and configured to analyze the pathogen. GDI030.2 preferably, the dialysis device has a first passage for receiving the biological sample, a second passage and a plurality of small holes, and the second passage is fluid with the first passage via the small holes Connected to allow pathogens to flow from the first channel to the second channel and the larger component of the biological sample remains in the first channel. GDI0 30.3 Preferably, the dialysis device further has a series of adjacent passages extending between the first passage and the second passage, wherein the small holes are located at the upstream end of the adjacent passages, and the adjacent passages are configured to be capable of Fixing a sample meniscus to block capillary flow between the first channel and the second channel; and -43- 201209406 a bypass channel between the first channel and the second channel, the bypass channel is imported a second passage upstream of the adjacent passageway and configured to provide an unimpeded capillary drive flow from the first passage to the second passage; wherein during use, after the meniscus is formed in each adjacent passage, from the bypass When the liquid flow of the passage reaches the meniscus fixed to each adjacent passage, the liquid flow sequentially removes each meniscus, and the sample liquid flows from the first passage to the first passage through the adjacent passage and the bypass passage. Two channels.

GDI03 0.4 Preferably, the second passage is available as a target passage and the first passage is available as a waste passage, the target passage being configured to drive the flow of fluid to the LOC unit. GDI 0 3 0.5 Preferably, the pathogen contains a target nucleic acid sequence and the LOC device contains a nucleic acid amplification region for amplifying the target nucleic acid sequence. GDI030.6 Preferably, the LOC device has a cytosolic region to lyse the pathogen to release the target nucleic acid sequence therein.

GDI030.7 Preferably, the LOC device also has a hybrid region having a hybrid probe array that hybridizes to the target nucleic acid sequence to form a probe-target hybrid. GDI0 30.8 Preferably, the probe is designed to form a probe-target hybrid with the target nucleic acid sequence, the probe-target hybrid being designed to emit photons of light in response to an excitation current. . GDI030.9 Preferably, the LOC device has a CMOS circuit for operatively controlling the PCR zone, the CMOS circuit having a photosensor to sense photons emitted by the probe-target hybrid. GDI030.1 0 Preferably, the hybrid region has an array of hybrid chambers, and the -44-201209406 hybrid chamber contains a probe that is hybridizable to the target nucleic acid sequence. GDI03 0.11 Preferably, the photo sensor is an array of photodiodes, and the photodiodes are respectively adjacent to the hybrid chambers. GDI03 0.1 2 preferably 'The CMOS circuit has a digital memory for storing fluid processing related data, including detailed description of the probes and the position of each probe in the array of hybrid chambers.

GDI03 0.1 3 Preferably, the CMOS circuit has at least one temperature sensor to sense the temperature of the hybrid chamber array. Preferably, the LOC device has a heater controlled by a CMOS circuit that utilizes feedback from the temperature sensor to maintain the probe and target nucleic acid sequence below the hybrid temperature. GDI03 0.1 5 Preferably, the photodiode is less than 1 600 microns from the corresponding hybrid chamber. GDI03 0.1 6 Preferably, the probe has an electrochemiluminescent (ECL) luminophore that emits photons in an excited state. GDI03 0.17 Preferably, the hybrid chambers have electrodes to excite the ECL luminophores with electrical current. Preferably, each ECL probe has a luminophore and a quenching substance adjacent to the luminophore for quenching photons emitted by the luminophore, and the probe moves when it is hybridized with a target nucleic acid sequence. The quencher is remote from the luminophore so that the photons are no longer quenched. GDI030.1 9 preferably, the CMOS circuit has a bond pad for electronically connecting an external device, and is configured to convert an output from the photodiode into an indication signal indicating that the ECL probe has been hybridized with the target nucleic acid sequence, And -45-201209406 provides the signal to the bond pads for transmission to an external device. Preferably, the LOC device and the dialysis device are fluidly connected through a top cover having at least one passageway for fluid communication between the dialysis zone and the LOC, and a plurality of dialysis zones for accommodating the sample. A reservoir of liquid reagents.

The easy-to-use, mass-produced, inexpensive, and portable diagnostic test module is capable of accepting biological samples, separating the pathogens in the sample using the dialysis wafer, and separately processing the nucleic acid contents of the pathogens isolated from the samples. . The dialysis wafer can functionally extract more information from the sample and increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The microfluidic multi-wafer assembly provides greater modularity. The surface MEMS of this component is much smaller and disproportionately much cheaper than a single wafer that provides all of the functionality of the assembly. Alternatively, a large but cost-effective dialysis wafer can provide advanced dialysis capacity to further increase the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.

Each of the surface-microelectromechanical wafer components of the microfluidic multi-chip assembly is fabricated using a less expensive process that is optimal in function, i.e., the only-bioMST dialysis wafer can omit all CMOS process steps and can employ inexpensive glass wafers. [Embodiment] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT This description defines a main component of a molecular diagnostic system embodying many specific examples of the present invention. A full detailed description of the overall structure and operation of the system is shown in this patent specification. Referring to Figures 1, 2, 3, 85 and 86, the system has the following top-level components:

Test modules 1 and 1 have the size of a typical U S B memory (memory key) and are very inexpensive to manufacture. Test modules 10 and 1 each contain a microfluidic device 'typically in the form of a wafer-on-lab (LOC) device 30 that has been pre-loaded with reagents for molecular diagnostic assays and typically more than 1 000 probes ( See Figure 1 and Figure 8) Test Module 1 As shown in Figure 1, the ground-based detection technique is used to identify the target molecules, while the test module 1 in Figure 85 For the detection technology based on electrochemiluminescence. The LOC device 30 has an integrated light sensor 44 (described in more detail below) for sensing fluorescence or electrochemiluminescence. Both test modules 1 and 1 1 use a standard Micro-USB plug 14 to transfer power, data, and control, both of which have a printed circuit board (PCB) 57, and both have external power supplies. Capacitor 3 2 and inductor 15 are supplied. The test modules 10 and 1 1 are intended for single use and can be mass produced and delivered in sterile packaging. The outer casing 13 has a macroreceptacle 24 for receiving a biological sample and a detachable sterile sealing tape 22, preferably having a low viscosity adhesive to cover the large sump prior to use. A film seal 40 8 having a film cover 410 forms part of the outer casing 13 to reduce dewetting in the test module while providing a pressure relief to prevent small fluctuations in air pressure. The film cover 4 10 protects the film seal 40 8 from damage. The test module reader 12 supplies the test module 10 or 11 power through the micro-USB port 16. The test module reader 12 can take many different forms -47- 201209406

These options will be detailed later. The reader 12 version shown in Figures 1, 3 and 85 is an example of a smart phone. The block diagram of the reader 12 is shown in FIG. 3. The processor 42 can execute the application software from the program storage 43. The processor 42 also interfaces the display screen 18 and the user interface (UI) touch screen 17 and the button 19' cellular radio station 21, the wireless network connection 23 and the satellite navigation system 25» the cellular radio station 21 and the wireless Network connection 23 is used for communication. The satellite navigation system 25 can update the epidemiological database with location information. Alternatively, the location data can be manually entered via the touch screen 17 or button 19. The data store 27 contains genetic and diagnostic information, test results, patient information, assays, and probe data for confirming the position of each probe and its array. The data storage 27 and the program storage 43 can share the same memory device. The application software installed in the test module reader 1 2 provides results analysis and other test and diagnostic information.

For diagnostic testing, the test module 10 (or test module 1 1 ) is inserted into the micro-USB port 16 of the test module reader 12. The sterile sealing tape 22 is torn open and a biological sample (in liquid form) is loaded into the sample sump 24. Press the start button 20 to start the test through the application software. The sample will flow into the LOC device 30 and the plate assay will begin to extract, incubate, amplify, and hybridize with the sample nucleic acid (target) using a pre-synthesized hybrid-reactive oligonucleotide probe. In the example of test module 10 (which uses fluorescence-based detection), the probe is fluorescently labeled and LED 26 mounted in housing 13 provides the desired illumination for the hybrid probe. Excitation light (see Figures 1 and 2). In test module 1 1 (which uses electrochemiluminescence (ECL) detection), the LOC device 30 is equipped with an ECL probe (as discussed above), -48-201209406 and does not require LED 26 to produce luminescent radiation . Alternatively, the excitation current is provided by electrodes 860 and 870 (see Figure 86). Radiation (fluorescence or luminescence) is detected using a photosensor 44 integrated in a CMOS circuit on each LOC device. The detection signal will be amplified and converted into a digital output' which can be analyzed by the test module reader 12. The reader will then display the result.

This information can be stored locally and/or uploaded to a web server containing patient records. The test module 10 or 11 is removed from the test module reader 12 and properly discarded. Figures 1, 3 and 85 show a test module reader 12 for making a mobile phone/smartphone 28. In other forms, the test module reader can be a laptop/notebook 101, a dedicated reader 103, an e-book reader 107, a tablet 109 or a table for use in a hospital, private clinic or laboratory. The upper computer 105 (see Figure 87). The reader can interface with a wide range of other applications such as patient records, billing, online databases and multi-user environments. It can also interface with a variety of regional or remote peripheral devices such as printers or patient 1C smart cards. Referring now to FIG. 88, the data generated by the test module 10 can be updated by the reader 12 and the network 125 to update the epidemiological database in the epidemiological data host system 111, and the gene database in the genetic data host system 113. An electronic health record in the electronic health record (EHR) host system, an electronic medical record in the electronic medical record (EMR) host system 121, and a personal health record in the personal health record (PHR) host system 123. Conversely, the epidemiological database within the epidemiological data host system 1 1 1 , the gene database in the gene-49-201209406 data host system 113, and the electronic health record in the electronic health record (Ehr) host system 115 The electronic medical record in the electronic medical record (EMR) host system 121 and the personal health record in the personal health record (PHR) host system 123 can also update the LOC device 30 of the test module 10 through the network 125 and the reader 12. Digital memory inside.

Referring now back to Figures 1, 2, 85 and 86, the reader 12 uses battery power in the mobile phone configuration. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be loaded or updated via a variety of wireless or touch interfaces to communicate with peripherals, computers or online servers. A micro-USB port 16 is provided to communicate with the computer or to maintain power supply while the battery is charging.

Figure 62 shows a specific example of the test module 10 whose test only needs to show a positive or negative reaction to a particular target, such as testing whether a person is infected with, for example, the H1N1 influenza A virus. A USB-only power/indicator module 47 built for a specific purpose is well suited. No other readers or application software is required. The indicator 45 on the USB power/indicator module 47 will display a positive or negative reaction result. This configuration is quite suitable for a large number of screenings. Other items provided by this system include test tubes containing specific sample pretreatment reagents, as well as tongue depressors and lancets for sample collection. The specific example of the test module shown in Fig. 62 conveniently contains a collapsible lancet 3 90 and a lancet release button 3 92 which are held by a spring. Satellite phone test module electronics can be used in remote areas -50- 201209406

Figures 2 and 86 are block diagrams of the electronic components of test modules 10 and 11. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36' - a controller 34, a USB-compatible LED driver 29, a clock 33, a power conditioner 31, a RAM 38, and a program and data flash memory 40. . These devices provide control and memory for the entire test module 1 or 11 and include a light sensor 44, a temperature sensor 170, a liquid sensor 1 74', and different heaters 152, 154, 182, 234, and accompanying drivers 37 and 39 and registers 35 and 41. Only the LED 26 (in the example of the test module 10), the external power supply capacitor 32, and the micro-USB plug 14 are external to the LOC device 30. The LOC device 30 includes bond-pads that are coupled to such external components. RAM 38 and program and data flash memory 40 have diagnostic software and test information for more than one probe (flash/secure storage, such as encryption). For the example of test module 1 using ECL detection, the module does not have LED 26 (see Figures 85 and 86). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. The LOC device 30 houses an electrochemiluminescent probe and a hybrid chamber having a pair of ECL excitation electrodes 860 and 870. Many test module types are manufactured in different test formats and are easy to use. The difference between these test formats is based on plate assays using reagents and probes. Some examples of infectious diseases that can be quickly identified by this system include: • Influenza: Influenza A 'B 'C type, infectious salmon poor -51 - 201209406 Blood virus (Isavirus), Tokavirus ( Thogotovirus) • Pneumonia: respiratory syncytial virus (RSV), adenovirus, interstitial pneumonia virus, Streptococcus pneumoniae, Staphylococcus aureus • tuberculosis: Mycobacterium tuberculosis (iMftercM/oi/· ?), bovine tuberculosis (Μ., M. africanum

(M. a_/Wci/«MW), M. caneiiz and M. voles (Λ/, wicr ο ί / ) • Malignant sore/"a/cz'/jarM /w), Toxoplasma gondii (Γοχορ/ύ^»ΐίϊ) and other protozoan parasites • Typhoid: Salmonella typhimurium (*5a/TW〇«e//a ·?βλ·〇να/* typhi ) • Ebola virus • Human immunodeficiency virus (HIV)

• Dengue fever: Flavivirus • Hepatitis (types A to E) • Hospital-acquired infections: for example, Clostridium difficile, vancomycin-resistant enterococci (five "ierococcw") and dimethylation resistant Benzomycin S. aureus • Herpes simplex virus (HSV) • Cytomegalovirus (CMV) • Epstein-Barrvirus (EBV) • Encephalitis: Japanese encephalitis virus, Zhang Dipu La Virus (chandiPura -52- 201209406 virus ) • Hundreds of coughs · Haemophilus pertussis heart • Measles: Paramyxovirus • Meningitis: pneumococcal and meningococcal (iVeiwerM meningitidis) • Gray mark: Bacillus charredi

Some examples of hereditary diseases that can be identified by this system include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black idiots • Hemochromatosis « Cerebral arterial disease • Crohn's disease • Multiple Cystic nephropathy • Congenital heart disease • Reuters syndrome A small group of cancers identified by this diagnostic system include: • Ovarian cancer • Colon cancer • Multiple endocrine tumors • Retinoblastoma -53- 201209406 • The above list of syndromes is not exhaustive and the diagnostic system can be configured to detect a wide variety of diseases and conditions using nucleic acid and proteomic analysis. Detailed structure of system components LOC device

The LOC device 30 is the center of this diagnostic system. By using a microfluidic platform, it is capable of rapidly performing four major steps in nucleic acid-based molecular diagnostic assays, namely sample preparation, nucleic acid extraction, nucleic acid amplification and detection. There are other uses for this LOC device, which will be described later in detail. As discussed above, test modules 10 and 1 1 can take many different configurations to detect different targets. Similarly, the LOC device 30 also has a number of specific examples for the design of the target. One form of LOC device 30 is a LOC device 301 that uses fluorescence to detect a target nucleic acid sequence of a pathogen in a whole blood sample. For purposes of illustration, the structure and operation of the LOC device 301 will now be described in detail with reference to Figures 4 through 26 and Figures 27 through 57. Fig. 4 is a schematic representation of the overall structure of the LOC device 301. For convenience, the process stage shown in Figure 4 is represented by the reference number of the functional area of the LOC device 301 performing the process stage. The process stages associated with each of the major steps of the nucleic acid-based molecular diagnostic assay have also been labeled: sample input and preparation 288, extraction 2 90, incubation 291, amplification 292, and detection 294. The different reservoirs, chambers, valves and other components of the LOC device 301 will be described in detail later. Fig. 5 is a perspective view of the LOC device 301. Its high volume -54- 201209406

Manufactured by CMOS and MST (Microsystem Technology) manufacturing technology. The layered structure of the LOC device 301 is shown in a schematic (not to scale) partial cross-sectional view of Fig. 12. The LOC device 301 has a germanium substrate 84 supporting a CMOS + MST wafer 48, a CMOS circuit 86 and an MST layer 87, and a cap 46 overlying the MST layer 87. For the purposes of this patent specification, the term "MST layer" refers to a collection of structures and layers of film that will be treated with different reagents. Accordingly, the structures and members are configured to define a flow path having a feature size that supports capillary drive flow of liquid having the same physical properties as the sample during sample processing. In this regard, the MST layers and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other manufacturing techniques can also produce such structures and components that are capable of producing capillary drive flow and capable of handling extremely small volumes. The specific embodiment described in this patent specification will show the MST layer and use the MST layer as the structure and active components supported on the CMOS circuit 86, excluding the features of the top cover 46. However, readers familiar with this art should be aware that the MST layer can handle samples without the need for a substrate CMOS or a virtually overlying cap. The overall size of the LOC device shown in the following figures is 1 760 μm χ 5824 μηι. Of course, LOC devices for different applications can have different sizes. Figure 6 shows the features of the MST layer 87 superimposed thereon by the features of the top cover. Figure 6 shows the illustrations AD to AD, AG and ΑΗ magnified in Figures 13, 14, 35, 56, 55 and 58, respectively, and is described in detail below with -55-201209406 to give a thorough understanding of the LOC device 301. Various structures within. Figures 7 through 10 show the features of the top cover 46 independently and Figure 11 shows the structure of the CMOS + MST device 48 independently. Layered structure

Figures 12 and 22 are sketches showing the layered structure of the CMOS + MST device 48, the top cover 46, and the fluid interaction therebetween. For the sake of explanation, these drawings are not shown in the actual scale. Fig. 12 is a schematic sectional view through the sample inlet 68 and Fig. 22 is a schematic sectional view through the reservoir 54. As best shown in Fig. 12, the CMOS + MST device 48 has a germanium substrate 84 that supports the CMOS circuit 86 which is capable of manipulating the active components within the upper MST layer 87. A passivation layer 88 seals and protects the CMOS layer 8.6 from contact with the flow of liquid through the MST layer 87.

Fluid flows through the top cover channel 94 of the cap layer 46 and the MST channel 90 located at the MST channel layer 100 (see, for example, Figures 7 and 16). Cell transport occurs in the larger channel 94' built into the top cover 46 and biochemical treatment takes place in the smaller MST channel 90. The size of the cell transport channel is capable of transporting cells within the sample to a predetermined location within the MST channel 90. Transport of cells larger than 20 microns (e.g., specific white blood cells) would require channels with a diameter greater than 20 microns, and thus the cross-sectional area perpendicular to the flow would be greater than 400 square microns. The MST channel, especially in the LOC where there is no need to transport cells, is significantly smaller. It should be understood that the top cover channel 94 and the MST channel 90 are both generic terms. The special M S T channel 90 can be referred to as a heated micro-56-201209406 channel or a dialysis MST channel depending on its particular function. The MST channel 90 is formed by etching a MST channel layer 100 deposited over the passivation layer 88 and patterning with a photoresist. The MST channel 90 is surrounded by a top wall layer 66 which forms the top end of the CMOS + MST device 48 (relative to the orientation of the drawing)

Although sometimes represented by individual layers, the top channel layer 80 and the reservoir layer 78 are formed on a single piece of material. Of course, this piece of material may not be a single whole. The sheet material is etched from both sides to form a cap channel layer 80 etched with a cap channel 94 and a reservoir layer 78 etched with reservoirs 54, 56, 58, 60 and 62. Alternatively, the reservoir and cap channel can be formed by microforming. Both etching and microforming techniques can be used to create channels that can be as large as 20,000 square microns or as small as 8 square microns perpendicular to the flow. In the LOC device, the channel at different locations has a wide selection of cross-sectional areas perpendicular to the flow. When the channel contains a large number of samples or when the sample contains a large structural component, the cross-sectional area of the channel can be as high as 2 〇, 〇〇〇 square micron (for example, 200 μm wide in a 100 μm thick film layer) Channel). When the channel contains only a small amount of liquid or the mixture contains no large cells, the cross-sectional area of the channel perpendicular to the flow is preferably very small. The lower seal 64 encloses the top cover channel 94 and the upper sealing layer 82 encloses the reservoir 54. , 56, 58, 603⁄4 62 ° Five reservoirs 54, 56, 58, 60 and 62 have been previously loaded with assay-specific reagents. In the specific examples described herein, the following reagents are pre-filled with -57-201209406, but are easily replaced with other reagents: • Reservoir 54: anticoagulant, optionally containing red blood cells Dissolving buffer•Storage 5 6 : lysing reagent

• Reservoir 5 8: restriction enzymes, ligase and linker sequences (for linker-to-head PCR (see Figure 61 'selected from T. Stachan et al, Human Molecular Genetics 2, Garland Science, NY And London, 1999)) • Reservoir 60: amplification mixture (dNTPs, primers, buffer) and • reservoir 62: DNA polymerase. The top cover 46 and the CMOS + MST layer 48 are in fluid communication through the corresponding openings in the lower seal 64 and the top wall layer 66. These openings are referred to as uptakes 96 or downtakes 92 from the MST channel 90 to the cap channel 94 or to the reverse flow.

LOC Device Operation The operation of the LOC device 301 will be described in a stepwise manner with reference to the analysis of pathogenic DNA in a blood sample. Of course, other types of biological or abiotic fluids can also be analyzed using a suitable set of reagents, test procedures, L Ο C variants and detection systems, or a combination thereof. Referring back to Figure 4, there are five main steps involved in the analysis of biological samples, including sample placement and preparation 288, nucleic acid extraction 290, nucleic acid incubation 291, nucleic acid amplification 292, and detection and analysis 294. -58- 201209406 Sample Placement and Preparation Step 288 involves mixing the blood with the anticoagulant 116 and then separating the pathogen from the white blood cells and red blood cells in the pathogen dialysis zone 70. As best shown in Figures 7 and 12, the blood sample enters the device via sample inlet 68. The capillary action draws the blood sample into the reservoir 54 along the top cover channel 94. The anticoagulant is released from the reservoir 54 when the sample blood flow opens the surface tension valve 118 (see Figures 15 and 22). The anticoagulant prevents clot formation and the clot blocks the flow.

As best shown in Fig. 22, the anticoagulant 116 is drawn from the reservoir 54 by capillary action and enters the MST channel 90 via the downcomer 92. The downcomer 92 has a capillary initiation feature (C IF ) 102 to shape the geometry of the meniscus so that it does not rest at the edge of the downcomer 92. The venting opening 122 in the upper seal 82 can replace the anticoagulant with air as the anticoagulant 166 is drawn out of the reservoir 54. The MST channel 90 shown in Fig. 22 is part of the surface tension valve 118. The anticoagulant 116 will fill the surface tension valve 118 and secure a meniscus 120 to the meniscus anchor 98 of the upper conduit 96. Prior to use, the meniscus 120 remains attached to the upper catheter 96 so that the anticoagulant does not flow into the canopy channel 94. As blood flows through the canopy passage 94 to the upper conduit 96, the meniscus 120 is removed and the anticoagulant is introduced into the flow. Figures 15 through 21 show an illustration AE, which is part of the illustration AA shown in Figure 13. As shown in Figures 15, 16 and 17, the surface tension valve 118 has three separate MST channels 90 extending between the respective downcomers 92 and the upper conduits 96 between -59 and 201209406. The number of MST channels 90 within the surface tension valve can vary to vary the flow rate of reagent flow into the sample mixture. When the sample mixture and the reagent are mixed together by diffusion, the flow rate of the reagent out of the reservoir determines the concentration of the reagent in the sample stream. Therefore, the surface tension valve of each reservoir will be configured to meet the desired reagent concentration.

The blood then flows into the pathogen dialysis zone 70 (see Figures 4 and 15) where the target cells are concentrated from the sample by using an array of small wells 1 64 having a pore size of a given size. Cells smaller than the sputum pass through the small holes and larger cells cannot pass through the small holes. Useless cells, which can be cells stuck in the array of small holes 1 64 or cells that pass through the small holes, are redirected to the waste unit 76 and the target cells remain in the assay as part of the assay. Share.

In the pathogen dialysis zone 70 described herein, pathogens from whole blood samples are concentrated for microbial DN A analysis. The aperture array is formed by a plurality of 3 micron diameter holes 164 and is capable of fluidly coupling the input stream within the header channel 94 to the target channel 74. The 3 micron diameter aperture 164 and the dialysis upper pilot aperture 168 leading to the target channel 74 are connected by a series of dialysis MST channels 2 〇 4 (best shown in Figures 15 and 21). The pathogen will be small enough to pass through the aperture 164 of diameter 3 microns and fill the target channel 74 through the dialysis MST channel 206. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 in the top cover 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-60-201209406 DNA analysis. More details of this dialysis zone and dialysis variants will be provided later.

Referring again to Figures 6 and 7, the flow is drawn through target passage 74 to surface tension valve 128 of lytic reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending between the lysate reservoir 56 and the target channel 74. When the meniscus is collapsed by the sample stream, the flow rate of the lysing reagent reservoir 56 (from a total of seven MST channels 90) will be greater than the clotting reservoir 54 (the surface tension valve 118 has three MST channels 90) The flow rate is faster (assuming the physical properties of the streams are approximately the same). Therefore, the proportion of the lysing reagent in the sample mixture is higher than the ratio of the anticoagulant. The cytolytic reagent and target cells are mixed by diffusion in the target channel 74 in the chemical cytolytic zone 130. A boiling-initiated valve 126 can stop the flow for a sufficient period of time for diffusion and cytolysis to release hereditary material from the target cells (see Figures 6 and 7). The structure and operation of the boiling-starting valve will be described in more detail below with reference to Figures 31 and 32. The applicant has also developed other active valve types (as opposed to passive valves such as surface tension valve 118) that can be used in place of the boiling-start valve. These additional valve designs are also described later. When the boiling-start valve 126 is opened, the lysed cells will flow into the mixing zone 133 for pre-amplification restriction endonuclease digestion and linker ligation. Referring now to Figure 13, when the liquid phase collapses the meniscus of the surface tension valve 132 at the beginning of the mixing zone 133, the restriction enzymes, linkers and ligase are released from the reservoir 58. . The mixture flows through the length of the mixing zone 133 over -61 - 201209406 for diffusion mixing. The end of the mixing zone 131 is a downcomer 134 that leads to the incubator inlet channel 133 of the incubation zone 114 (see Figure 13). The incubator inlet channel 133 feeds the mixture to the heated microchannel 210 of the crucible configuration, which provides a chamber for holding the sample for restriction digestion and linker ligation (see paragraph 13 and 14 picture).

Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the inset AB of Figure 6. Each of the figures shows the structure of the CMOS + MST layer 48 and the top cover 46 in a sequential process of displaying the layers. The illustration AB is the end point of the incubation zone 114 and the starting point of the amplification zone 112. As best shown in Figures 14 and 23, the flow will continue to flow into the microchannel 2 1 0 of the incubation zone until it reaches the boiling-start valve 106 where it will stop and diffuse. As discussed above, the microchannel 210 upstream of the boiling-start valve 106 becomes a chamber containing a sample, restriction enzyme, ligase, and linker. The heater 154 is then activated and maintains a constant temperature for a limited period of time for limiting digestion and linker bonding.

Workers familiar with this technique should be aware that this incubation step 291 (see Figure 4) is optional and will only be required for certain nucleic acid amplification assay types. Again, in some instances, a heating step may be required at the end of the incubation period to raise the temperature above the incubation temperature. The temperature increase causes the restriction endonuclease and ligase to be deactivated before entering the amplification zone 112. The inactivation of the restriction enzymes and ligases is particularly important when isothermal nucleic acid amplification is employed. After incubation, the boiling-start valve 106 is activated (opened) and the flow re-enters the expansion zone 112. Referring to Figures 31 and 32, the mixture will continuously flow from -62 to 201209406 into the heated microchannel 158 of the crucible configuration (which will constitute one or more amplification chambers) until the liquid stream reaches the boiling-start valve 1〇8. . As best shown in the schematic cross-sectional view of Figure 30, amplification mixtures (dNTPs, primers, buffers) are released from reservoir 60 and then polymerase is released from reservoir 62 into the junction incubation zone and expanded. The intermediate MST channel 212 of the zones (114 and 112, respectively).

Figures 35 through 51 show the layers of the LOC device 301 in the inset AC of Figure 6. Each of the figures shows a sequential addition process of the layers to form the structure of the CMOS + MST device 48 and the top cover 46. The illustration AC is the endpoint of the amplification zone 112 and the beginning of the hybrid and detection zone 52. The incubated sample, amplification mixture, and polymerase will flow through the microchannel 158 to the boiling-start valve 108. After diffusion mixing for a sufficient period of time, the heater 154 within the microchannel 158 is activated to initiate thermal cycling or isothermal amplification. The amplification mixture is subjected to a predetermined number of thermal cycles or an existing amplification time to amplify sufficient target DNA. After the nucleic acid amplification process, the boiling-start valve 108 opens and the flow re-flows into the hybrid and detection zone 52. The operation of the boiling-starting valve will be described in more detail after the tip. As shown in Fig. 52, the hybrid and detection zone 52 has a hybrid chamber array 110»52, 53, 54, and 56 showing the hybrid chamber array 110 and the detailed individual hybrid chambers 180. At the entrance of the hybrid chamber 180 is a diffusion barrier 175 which prevents target nucleic acid, probe strands and hybrid probes from diffusing between the hybrid chambers 180 during hybridization to avoid erroneous hybrid detection results. The diffusion barrier 175 is as long as the flow path, and is long enough to prevent the target sequence and the probe from diffusing out of the chamber during the probe and nucleic acid hybridization and signal detection, and contaminating the other chamber -63-201209406, such a To avoid erroneous results·> Another mechanism for preventing erroneous reading is to use the same probe in several hybrid chambers. The CMOS circuit 86 will obtain a single result from the photodiode 184 corresponding to the hybrid chamber 180 containing the same probe. When a single result is derived, the unreasonable results are eliminated or weighted differently.

The thermal energy required for hybridization is provided by a CMOS-controlled heater 182 (described in more detail below). Hybridization occurs between complementary target-probe sequences after the heater is activated. The LED driver 29 in the CMOS circuit 86 signals the LED 26 located within the test module 10 to illuminate. These probes fluoresce when heterozygous occurs, thus eliminating the need for cleaning and drying steps typically required to remove unbound strands. As will be explained in more detail later, hybridization forces the stem-loop structure of the FRET probe 186 to open, and the open structure allows the fluorophore to react to the LED excitation light to emit fluorescent energy. Fluorescence can be detected by photodiode 184 lining the CMOS circuit 86 at the bottom of each of the hybrid chambers 180 (see the description of the hybrid chamber below). The photodiode 184 of all hybrid chambers and associated electronic components collectively form a photosensor 44 (see Figure 59). In other embodiments, the photosensor can be an array of charge coupled devices (CCD arrays). The detection signal from photodiode 184 can be amplified and converted to a digital output, which can be analyzed by test module reader 12. More details of this detection method will be explained later. More detailed design of the LOC device is modularized. The LOC device 301 has a number of functional areas including reagent reservoirs 54-64-201209406, 56, 58, 60 and 62, dialysis zone 70, cytolysis zone 130, incubation zone 114. And the amplification zone 1 1 2, a variety of valve types, humidifiers and humidity sensors. In other specific examples of the LOC device, such functional areas may be omitted, other functional areas may be added or other functional areas may be used for other purposes as described above.

For example, the incubation zone 4 can be used as the first amplification zone 112 of the tandem amplification assay system, and the chemical lysate reagent reservoir 56 can be used to add primers, dNTPs, and a first amplification mixture of buffer. Reagent reservoir 58 can be used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, the chemical lysing reagent can be added to the reservoir 56 along with the amplification mixture; alternatively, the sample can be heated in the incubation zone by heating the sample for a predetermined period of time. Hot cell solution. In some embodiments, if chemical cytolysis is required and the mixture of primers, dNTPs, and buffer is separated from the chemical lysing reagent, it can be upstream of the reservoir 58 of the primer, dNTPs, and buffer mixture. Add more storage. In some cases it may be necessary to omit a step, such as incubation step 291. In this case, a LOC device may be specially fabricated to omit the reagent reservoir 58 and the incubation zone 114; or simply do not load the reagent in the reservoir; or if there is an active valve, the active valve may not be activated to dispense the reagent to The sample is flowed so that the incubation zone simply becomes a passage for transporting the sample from the cytolytic zone 130 to the amplification zone 112. The heater operates independently, and the heater can be activated when the reaction is dependent on heat (eg, hot cytolysis); however, in a LOC device that does not require thermolysis, the heater can be programmed to ensure that the heater is not during this step. Will be activated and thermolysis will not occur. The dialysis zone 70 can be located at the beginning of the microfluidic system within the microfluidic device as shown in Figure 4, -65-201209406

Or it can be located anywhere else within the microfluidic device. For example, in some instances the dialysis system is performed after the amplification period 292 to remove the cell debris prior to the hybridization and detection step 294, which may be advantageous. Alternatively, two or more dialysis zones can be combined anywhere in the entire LOC device. Similarly, more amplification regions 1 1 2 can also be combined, such that multiple targets can be detected prior to detection with a specific nucleic acid probe for hybrid chamber assay 110. Amplify simultaneously or continuously. In order to analyze a sample that does not require dialysis, such as a whole blood sample, the dialysis zone 70 can be simply omitted from the sample placement and preparation zone 28 of the LOC design. In some instances, the dialysis zone 70 is not removed from the LOC device, even if dialysis is not required for analysis. If the presence of the dialysis zone does not cause geometrical obstruction, even the LOC containing the dialysis zone 70 in the sample placement and preparation zone can be used without loss of the desired functionality.

Further, the detection zone 294 may contain a proteomic chamber assay that is identical to the hybrid chamber assay but is loaded into a probe designed to engage or hybridize with the target protein of the sample in the unamplified sample, rather than Nucleic acid probes designed to hybridize to a target nucleic acid sequence. The L 0 C device that is known to be used in this diagnostic system is a different combination of functional regions selected for a particular LOC application. For most LOC devices, the vast majority The functional area is very common, and the additional LOC device design with novel applications is simply to completely eliminate the functional areas used in the existing LOC device and edit the appropriate combination of functional areas. Only a few LOC devices are shown in this description, and more devices are shown to show the design flexibility of the LOC device used in this system. Those skilled in the art -66 - 201209406 can easily understand that the L0C devices shown in these descriptions are not fully enumerated and many other L 0 C designs are related to how to edit the appropriate combination of functional regions. Sample type

The LOC variant accepts and analyzes nucleic acid or protein contents of a variety of liquid form sample types including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine 'semen, amniotic fluid, cord blood , milk, sweat, pleural effusion, tears, pericardial, ascites, environmental water samples and beverage samples. Amplicon obtained from macronucleic acid amplification can be analyzed using the LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, the dialysis, lysis The incubation and expansion zones are only used to transport the sample from the sample inlet 68 to the hybrid chamber 180 for nucleic acid detection, as described above. Some sample types may require a pretreatment step. For example, the semen may need to be liquefied prior to placing the semen and mucus into the LOC device. The mucus may need to be pretreated with an enzyme to reduce viscosity. Sample Placement Referring to Figures 1 and 12, the sample is applied to a large sump 24 of the test module 1 。. The large sump 24 is a truncated cone and the sample can flow to the inlet 68 of the LOC unit 301 via capillary action. Here, the sample will flow into the top cover channel 94 of the 64 μηι 宽 60 μηι deep, where the sample is also pulled toward the anticoagulant reservoir 54 by capillary action. -67- 201209406 Reagent storage

The volume of reagent required to use a microfluidic device assay system (e.g., LOC device 301) is such that the reagent reservoir contains all of the reagents required for biochemical processing, even if only a small volume. This volume can easily be less than 1,000,000,000 cubic microns, in most cases less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns and the LOC device 301 shown in the figures is less than 20,000,000 cubic microns. . Dialysis area

Referring to Figures 15 through 21, 33 and 34, the pathogen dialysis zone 70 is designed to concentrate pathogenic target cells from the sample. As previously described, a plurality of apertures in the form of a 3 micron diameter hole 164 in the top wall layer 66 filter the target cells from the sample body. As the sample flows through the 3 micron diameter orifice 164, microbial pathogens pass through the pores into a series of dialysis MST channels 204 and flow back to the target channel 74 via the 16 μιη dialysis vias 168 (see Figures 33 and 34). ). The remainder of the sample (red blood cells, etc.) will remain in the top cover channel 94. Downstream of the pathogen dialysis zone 70, the canopy channel 94 becomes a waste channel 72 that directs waste to the waste reservoir 76. Foam illustrations or other apertured elements 49 in the outer casing 13 of the test module 10 can be configured to be in fluid communication with the waste reservoir 76 for a type of biological sample that will produce substantial amounts of waste (see paragraph 1). Figure). The pathogen dialysis zone 70 functions entirely by the capillary action of the fluid sample. The 3 micron diameter orifice 164 at the upstream end of the pathogen dialysis zone 70 has a -68-201209406 capillary activation feature (CIFs) 1 66 (see Figure 33) so that fluid can be drawn to the underlying dialysis MST channel 204. The first upper pilot hole 198 of the target channel 74 also has a CIF 202 (see Figure 15) to prevent fluid flow from easily securing the meniscus across the orifice of the dialysis upper pilot hole 168.

The small component dialysis zone 682, shown schematically at Figure 70, can have a configuration similar to the pathogen dialysis zone 70. The small component dialysis zone can separate all small target cells or molecules from the sample by means of small pores of different sizes (if desired, by different shapes) to allow small target cells or molecules to pass through and into the target. Channel and continue for further analysis. Larger cells or molecules are moved to waste reservoir 766. Thus, the LOC device 30 (see Figures 1 and 85) is not limited to isolation of pathogens having a size of less than 3 microns, and can be used to isolate cells or molecules of any desired size. Cytolysis zone Referring back to Figures 7, 11, and 13, the genetic material in the sample can be released from the cell by chemical cytolysis. As discussed above, the lysing reagent from the lysis reservoir 56 will mix with the sample stream within the target channel 74 downstream of the surface tension valve 128 of the lysis reservoir 56. However, some diagnostic tests are more suitable for thermocytolysis, even for chemically cytolysis and thermocytolysis of target cells. The LOC device 301 can accommodate this problem by the heated microchannel 210 of the incubation zone Π4. The sample stream flows into the culture zone 114 and stops at the boiling-start valve 106. Incubating the microchannel 210 heats the sample to the temperature at which the cell membrane collapses. In some thermocytolysis applications, the enzyme cytolysis zone 130 does not require the enzyme -69-201209406 reaction and the thermocytosine completely replaces the enzyme reaction in the chemical cytolysis zone 130. Boiling Start Valve As discussed above, the LOC unit 310 has three boiling start valves 126, 106 and 108. The location of these valves is shown in Figure 6. Figure 31 shows an enlarged plan view of the boiling starter valve 108 at the end of the heated microchannel 158 of the amplification zone 112.

The sample stream 1 19 is drawn to the boiling start valve 108 along the heated microchannel 158 by capillary action. The flow guiding meniscus 120 is fixed to the meniscus anchor 98 of the valve inlet 146 before the sample. The geometry of the meniscus anchor 98 stops the advancing meniscus to stop capillary flow. As shown in Figures 31 and 3, the meniscus anchor 98 is a small aperture from the MST channel 90 to the opening of the conduit above the canopy channel 94. The surface tension of the meniscus 1 120 causes the valve to close. A ring heater 152 surrounds the valve inlet 146, which is CMOS-controlled through the boiling start valve heater contact 153.

To open the valve, the CMOS circuit 86 sends a current pulse to the valve heater contact 153. The annular heater 1 52 is continuously heated until the liquid sample 119 is boiled. Boiling disintegrates the meniscus 120 of the valve inlet 146 and causes the cap passage 94 to wet. Once the top cover passage 94 begins to wet, the capillary flow resumes. The fluid sample 119 fills the cap passage 94 and flows through the valve downcomer 150 to the valve outlet 148 where the capillary drive flow continues along the expansion zone outlet passage 160 into the hybrid and detection zone 52. The liquid sensor 174 is placed before or after the valve for diagnosis. It should be understood that once the boiling start valve is opened, it cannot be closed. However, -70-201209406, since the LOC device 301 and the test module 10 are used and lost, it is not necessary to close the valves.

Figures 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubation zone 114 and the amplification zone 112. The incubation zone 114 is coupled from the downcomer opening 134 to the MST channel layer 1 of the boiling starter valve 106 and has a single heated incubation microchannel 210 etched into a 蜿蜒 pattern (see Figures 13 and 14). Temperature control of the incubation zone 114 allows the enzyme reaction to proceed with better performance. Similarly, the amplification zone 11 2 has a heated amplifying microchannel 158 that is connected from the boiling start valve 106 to the boiling start valve 108 and in a 蜿蜒 configuration (see Figures 6 and 14). These valves are capable of stopping flow, leaving the target cells in heat-increasing or amplifying microchannels 210 or 158 for mixing, incubation, and nucleic acid amplification. The ruthenium pattern of the microchannel also accelerates (to some extent) the mixing of target cells with reagents. In the incubation zone 1 14 and the amplification zone 1 12, the sample cells and reagents are heated by a heater 154 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each of the chirp configurations of the heated incubation microchannel 210 and the amplification microchannel 158 has three independently operable heaters 154 extending between the individual heater contacts 156 (see Figure 14). The contacts provide two-dimensional regulation of the input heat flux density. As best shown in Fig. 51, the heater 154 is supported by the top wall layer 66 and embedded in the lower seal 64. The heater material is TiAl, but other conductive metals can also be used. The elongated heaters 154 are parallel to the longitudinal portions of the channel regions which form the wide labyrinth of the dome shape. -71 - 201209406 In the amplification zone 112, each of the wide labyrinths can be manipulated as a separate PCR chamber operation by individual heaters. The size of the amplicon required for an assay system employing a microfluidic device, such as LOC device 301, is so small that the volume of the amplified mixture that amplifies the amplified region 112 is also small. This volume can easily be less than 400 nanoliters, and in most cases it is less than 1 70 nanoliters, typically less than 70 nanoliters, and in the case of L Ο C device 3 0 1 in 2 Rise to between 30 nanoliters.

Higher heating rate and better diffusion mixing

The small cross-section of each channel zone increases the heating rate of the augmentation fluid mixture. All fluids maintain a relatively short distance from the heater 154. Shrinking the channel section (this is the section of the augmented microchannel 158) to less than 100,000 square microns has a higher heating rate than a more "large size" device. The etch fabrication technique allows the ablation microchannels 158 to have a cross-sectional area perpendicular to the flow that is less than 1 6,000 square microns, which provides a substantially higher heating rate. Characteristic components of about 1 micron in size can be easily provided by etching techniques. If the table requires a very small amplicon (as in the case of the L O C device 310), then the cross-sectional area can be reduced to less than 2,500 square microns. For a diagnostic test that contains 1, 〇〇〇 to 2,0 0 probes on the LOC device and from "sample placement" to "result output" must be less than 1 minute, preferably The vertical cross-sectional area of the liquid flow should be between 400 square microns and 1 square micrometer. The heating elements in the amplifying microchannel 158 can be used at a speed of more than 8 每秒 per second (Kelvin (K)). The nucleic acid sequence is heated at a rate of more than 1 sec per second. Typically, the heating element can heat the nucleic acid sequence at a rate of from -72 to 201209406 per 1,000 K per second, in many cases per second. The nucleic acid sequence is heated at a rate of more than 10,000 Torr. Typically, the heater can exceed 100,000 sec per second, more than 1,000,000 sec per second, more than 10,000,000 sec per second, more than 20.000 per second, based on the requirements of the assay system.核酸, more than 40,000,000 每秒 per second, more than 80.000. 000 sec per second and more than 160,000,000 每秒 per second to heat the nucleic acid sequence.

The passage of the small cross-sectional area is also advantageous when diffusing and mixing any of the reagents with the sample fluid. Before the diffusion mixing is completed, the diffusion of one liquid to another is the fastest at the junction of the two. The concentration decreases as the distance from the interface increases. By using a microchannel with a smaller cross-section perpendicular to the direction of flow, both fluids can flow close to the interface and diffuse and mix more quickly. Shrinking the cross-section of the channel to less than 100,000 square microns has a better mixing rate than devices with more "large size". Etch fabrication techniques produce microchannels having a cross-sectional area perpendicular to the flow path of less than 16,000 square microns, which substantially provides a higher mixing rate. If a small volume is required (as in the case of LOC device 301), the cross-sectional area can be reduced to less than 2,500 square microns. For a diagnostic test located at 1^0 (the device contains 1,0() 0 to 2,000 probes and must be less than 1 minute from "sample-load" to "result-read" A cross-sectional area perpendicular to the flow is suitably between 400 and 1 square micron. The short thermal cycle keeps the sample mixture close to the heater and uses a very small fluid volume -73 - 201209406

It allows for rapid thermal cycling during nucleic acid amplification. For a target sequence of up to 150 base pairs (bp) long, each regenerative cycle (i.e., denaturation, binding, and primer extension) is completed in less than 30 seconds. In most diagnostic tests, individual thermal cycling times are less than 11 seconds, and most will be less than 4 seconds. The LOC device 30 with some of the most common diagnostic tests has a thermal cycle time of between 0.45 seconds and 1.5 seconds for a target sequence of up to 150 bp. This rate of thermal cycling allows the test module to complete the nucleic acid amplification process in less than 10 minutes, usually in less than 260 seconds. For most assays, the amplification zone can generate sufficient amplicons from the sample fluid entering the sample inlet in less than 80 seconds. For a very large number of assays, sufficient amplicons can be generated in 30 seconds. After completing the current number of amplification cycles, the amplicon can be fed to the hybrid and detection zone 52 via the boiling start valve 1 〇 8. Hybrid room

Figures 52, 53, 54, 56 and 57 show the hybrid chamber 180 of the hybrid chamber assay 110. The hybrid and detection zone 52 has an array 110 of 24 x 45 hybrid chambers 180, each having a hybrid reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. Photodiode 184 is conjugated to detect fluorescence of the target nucleic acid sequence or protein produced by hybridization with FRET probe 186. Each of the photodiodes 184 independently control the material between the FRET probe 186 and the photodiode 184 via the CMOS circuit 86 must be transparent to the radiation. Accordingly, the wall region 97 between the probe 186 and the photodiode 184 is also optically transparent to the emitted light. In the LOC device -74- 201209406 301, the wall region 97 is a thin layer of cerium oxide (about 0.5 micron).

Combining the photodiode 184 directly under each of the hybrid chambers 180 allows the probe-target hybrid to be small in size while still producing a detectable fluorescent signal (see Figure 54). The small amount allows the hybrid chamber to have a small volume. The detectable amount of probe-target hybrid requires a probe before the hybrid, and the amount is easily less than 270 pg (equivalent to 900,000 cubic microns), which is less in most cases. At 60 picograms (equivalent to 200,000 cubic micrometers), typically less than 12 picograms (equivalent to 40,000 cubic micrometers), and the LOC device 301 in the following figures is less than 2.7 picograms (equivalent to 9,0) 00 cubic micrometers). Of course, reducing the size of the hybrid chamber allows for a higher density of hybrid chambers and thus more probes on the LOC device. In the LOC unit 301, the hybrid region has more than 1 000 chambers in the region of 1500 microns by 1500 microns (i.e., less than 2250 square microns per chamber area). 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 only a small amount of probe solution needs to be dropped into each chamber during manufacture of the LOC device. In the specific example of the LOC device of the present invention, a probe solution of 1 picolitre or less may be added. After nucleic acid amplification, the boiling start valve 108 is activated and the amplicon flows along the flow path 176 into each of the hybrid chambers 180 (see Figures 52 and 56). When the hybrid chamber 180 is full of amplicons, an endpoint liquid sensor ι 78 will display this phenomenon and the heater 182 will be activated. After ample mixing time, the LED 26 (see Figure 2) will be activated. The opening of each of the hybrid chambers 180 provides a window 136 for exposing the FRET probe 186 to excitation radiation (see Figures 52' 54 and 56). Let the -75-201209406 LED 26 be illuminated for a sufficient period of time to induce the probe. The high intensity fluorescent signal of the needle. During the excitation, the photodiode 1 84 is shortened. After a stylized pre-delay of 300 (see Figure 2), the photodiode 184 is activated and detects fluorescence emission when there is no excitation light. The incident light of the active region 185 of the photodiode 184 (see Fig. 54) is converted into a photocurrent, which is then measured by a CMOS circuit 86.

Each of the hybrid chambers 180 is equipped with a probe for detecting a single target nucleic acid sequence. If desired, each of the hybrid chambers 180 can be equipped with probes capable of detecting more than 1,000 different targets. Alternatively, the same probe can be loaded into many or all of the hybrid chambers to repeatedly detect the same target nucleic acid. Reloading such probes in this manner throughout the array of hybrid chambers 110 can increase the confidence of the results obtained; if desired, the results can be combined with light diodes adjacent to such hybrid chambers. Provide a single result. Those skilled in the art will appreciate that there may be from 1 to over 1,000 different probes in the hybrid chamber array 1 10 depending on the specifications of the inspection.

Hybrid Chambers for Electrochemiluminescence Detection Figures 78, 90, 115 and 116 show the hybrid chamber 180 used in the ECL variant of the LOC device - LOC variant L 729. In a specific example of the L0C device, the 24x45 array 110 of the hybrid chamber 180 (each of which has a hybrid reactive ECL probe 23 7 ) is disposed with an array of corresponding photodiodes 184 integrated in the CMOS. . In a manner similar to a LOC device employing fluorescence detection, each photodiode 1 84 is combined to detect the ECL produced by hybridization of the target nucleic acid sequence or protein to the ECL probe 237. Each of the photodiodes 184-76-201209406 is independently controlled by the CMOS circuit 86. Again, the transparent wall region 97 between the probe t86 and the photodiode 184 is transparent to the radiation.

The photodiode 184 adjacent to each of the hybrid chambers 180 produces a detectable ECL signal even when the probe-target hybrid is small (see Figure 78). The small amount of the mixture allows the hybrid chamber to have a small volume. The detectable amount of probe-target hybrid requires a probe before the hybrid, which is easily less than 270 pg (equivalent to a volume of 900,000 cubic microns), for the most part. Medium is less than 60 pg (equivalent to 200,000 cubic microns), typically less than 12 pg (equivalent to 40,000 cubic microns), and less than 2.7 pg in the LOC device shown in the following figure (equivalent At room volume of 9,000 cubic microns). Of course, reducing the size of the hybrid chamber allows for a higher density of hybrid chambers and thus more probes on the LOC device. In the L0C device shown, the hybrid region can have more than 1000 chambers in an area of 1500 microns by 1500 microns (i.e., less than 2250 square microns per chamber area). Smaller volumes also reduce reaction time, making hybridization and detection faster. Another advantage of the small amount of probe required for each chamber is that only a small amount of probe solution needs to be applied to each chamber during the manufacture of the LOC device. In the example of the LOC device shown in the figures, the desired amount of probe can be added in a solution volume of 1 picoliter or less. After nucleic acid amplification, the boiling start valve 108 is activated and the amplicon flows along the flow path 176 into each of the hybrid chambers 180 (see Figures 52 and 116). When the hybrid chamber 180 is full of amplicons, the endpoint liquid sensor 178 will indicate that the heater 182 can be activated. After a sufficient amount of hybrid time, the photodiode 184 can be activated by -77-201209406 and collect the ECL signal. The ECL excitation driver 39 (see Fig. 86) will activate the ECL electrodes 860 and 870 for a predetermined length of time. After the ECL excitation current is stopped, the photodiode 184 continues to be activated for a short period of time to maximize the signal-to-noise ratio. For example, if the photodiode 184 remains active for five times the lifetime of the luminescence decay, then the signal attenuation will be less than 1% of the initial enthalpy. The incident light of the photodiode 1 84 is converted into a photocurrent, which can then be measured by a CMOS circuit 86.

Humidifier and humidity sensor

The illustration AG of Fig. 6 indicates the position of the humidifier 196. The humidifier prevents reagents and probes from volatilizing during operation of the LOC device 301. As best shown in the enlarged view of Fig. 55, a reservoir 188 is fluidly coupled to three evaporators 190. The reservoir 188 is loaded with molecular biology-grade water and sealed during manufacture. As best shown in Figures 55 and 60, water is drawn into the three lower conduits 194 and flows by capillary action along the individual water supply passages 192 into 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 an annular heater 191 that encloses the upper conduit 193. The ring heater 191 is connected to the CMOS circuit 86 by a conductive string 376 connected to the metal top layer 195 (see Figure 37). When the ring heater 191 is activated, the heater heats the water to cause evaporation and humidifies the surrounding portion of the device. The position of the humidity sensor 2 3 2 is also shown in Fig. 6. However, as best shown in the enlarged view of the illustration AH of Fig. 5, the humidity sensor has a capacitive comb structure. A first electrode 296 etched by an etching technique and a second electrode 298 etched by an etch-78-201209406 technique face each other and are interdigitated. The opposite electrode forms a capacitor having a capacitance that can be monitored by CMOS circuitry 86. As the humidity increases, the dielectric constant of the air gap between the electrodes increases, so the capacitance also increases. The humidity sensor 232 is adjacent to the hybrid chamber array 1 10 where humidity measurement is of the utmost importance to slow the evaporation of the solution containing the exposed probe.

The feedback sensor combines the temperature and liquid sensors into the entire LOC device 301 to provide feedback and diagnostics during operation of the device. Referring to Fig. 35, there are nine temperature sensors 170 dispersed throughout the amplification zone 112. Similarly, the incubation zone 114 also has nine temperature sensors 170. These sensors use a 2x2 array of bipolar junction transistors (BJTs) to monitor fluid temperature and provide feedback to CMOS circuitry 86. The CMOS circuit 86 uses this feedback to precisely control the cycling of temperature during nucleic acid amplification and the thermal lysis and heating during incubation. In the hybrid chamber 180, the CMOS circuit 86 uses a hybrid heater 182 as a temperature sensor (see Fig. 56). The resistance of the hybrid heater 182 is temperature dependent and the CMOS circuit 86 utilizes this characteristic to obtain temperature readings for each of the hybrid chambers 180. The LOC device 301 also has a plurality of MST channel liquid sensors 174 and a cap channel liquid sensor 208. Figure 35 shows a row of MST channel liquid sensors 174 at the end of each of the spaced labyrinths in the heated microchannel 158. As best shown in FIG. 37, the MST channel liquid sensor 174 is formed as a counter electrode by the exposed regions of the metal top layer 195 of the CMOS structure 86. The liquid will turn off -79- 201209406 to close the circuit between the electrodes and point out the presence of liquid at the location of the sensor. Figure 25 shows an enlarged perspective view of the top cover channel liquid sensor 208. Opposite electrical TiAl electrode pairs 218 and 22 are deposited on top wall layer 66. Between the electrodes 218 and 220 is a gap 222 that maintains circuit communication when there is no liquid. The presence of liquid shuts down the circuit and CMOS circuit 86 uses this feedback to monitor the flow.

Gravity irrelevance

The test module 10 is independent of orientation. They do not need to be fixed to operate on a smooth surface. The capillary drive flow is flowed and no external tubing is connected to the auxiliary device, making the module truly portable and simply plugged into a similar portable handheld reader such as a mobile phone. Operating operability that is not related to gravity means that the test module is also unrelated to acceleration at all utility levels. They are resistant to violent shocks and swings and can be operated on mobile vehicles or when the mobile phone is carried around. Dialysis Variations The dialysis zone with flow channels to prevent trapped air bubbles The following is a specific example of a LOC device known as L0C variant VIII 518 and is shown in Figures 64, 65, 66 and 67. The LOC device has a dialysis zone filled with a fluid sample and having no trapped bubbles in the channel. The L0C variant VIII 518 has an additional layer of material known as the interface layer 5 94. The interface layer 594 is located between the top channel layer -80 - 201209406 80 of the CMOS + MST device 48 and the MST channel layer 100. The interfacial layer 594 allows for more complex fluid interconnections between the reagent reservoir and the M S T layer 87 without increasing the size of the tantalum substrate.

Referring to Fig. 65, the bypass passage 600 is designed to introduce a time delay when the fluid sample stream flows from the interface waste passage 604 to the interface target passage 602. This time delay causes the fluid sample to flow through the dialysis MST channel 204 to the dialysis upper conduit 168 where a meniscus is secured. By passing from the bypass passage 600 to the capillary priming feature (CIF) 202 of the conduit above the interface target passage 206, the sample fluid will fill the interface from one point upstream of all dialysis upcomers 168 from the dialysis MST channel 204. When the target channel 6 02 does not have the bypass channel 600, the interface target channel 602 will still be charged from the upstream end, but the forward-moving meniscus will eventually reach and pass over the unfilled MS channel. The upper tube is trapped and the air is trapped at this point. The trapped air reduces the flow of sample through the white blood cell dialysis zone 328

Nucleic Acid Amplification Variants Parallel PCR Several variants of the LOC device have multiple amplification regions operating in parallel. For example, Figure 63 shows that LOC variant VII 492 has a parallel amplification region of 112.1 to 112.4 that allows multiple nucleic acid amplification assays to be performed simultaneously. The LOC variant XI 746 shown in Figure 74 also has parallel amplification zones 112.1 to 112.4, but there are also parallel incubation zones 114.1 to 114.4, -81 - 201209406 so the samples can be treated differently before amplification. Other LOC variants, such as LOC XIV 641, shown schematically in Figure 69, show that the number of parallel amplification zones can be an "X" number, and X is only limited by the size of the LOC device. Larger LOC devices can be fabricated to accommodate a greater number of parallel amplification zones. The separate amplification zones can be configured to be treated at different cycle times and/or temperatures for a particular target size or particular amplification mixture component. When several amplification zones are run in parallel, the LOC device can perform a multiple nucleic acid amplification process or a single (unipl ex) amplification process in each zone. In the multiplex nucleic acid amplification method, more than one target sequence is amplified by using one or more primers. A parallel nucleic acid amplification system having "m" chambers can perform an equivalent of n-reamplification, where n = n(l) + n(2) +... + n(i) +...+ n(m And η ( i ) is the number of different primer pairs used in the multiplex amplification method performed in chamber "i", it should be borne in mind that the SNR (signal-to-noise ratio) is higher in the parallel amplification system than in the single chamber system Performed η-reamplification method. In the special case of n ( i ) = 1, the amplification method of the chamber "i" becomes a single amplification method.

Direct PCR Traditionally, PCR requires thorough purification of the target DNA prior to preparation of the reaction mixture. However, by appropriate chemical properties and sample concentration, nucleic acid amplification or direct nucleic acid amplification can be performed after purification of some micro-DN A. When the nucleic acid amplification process is PCR, this method is called direct PCR. When the nucleic acid amplification is carried out at a controlled constant temperature in a LOC device, this method is a direct isothermal amplification method. Direct nucleic acid amplification techniques have considerable advantages when used in LOC devices, especially with regard to the simplified aspect of 201209406 for the desired fluid design. Modification of amplification chemistry by direct PCR or direct isothermal amplification involves increasing buffer strength, using a polymerase with high activity and continuous potency, and an additive that can sequester possible polymerase inhibitors. The dilution of the inhibitor in the sample is also important.

In order to utilize direct nucleic acid amplification techniques, the design of the LOC device incorporates two additional features. The first characteristic member is a reagent reservoir (e.g., reservoir 58 of Figure 8) that is sized to supply a sufficient amount of amplification mixture or diluent and thus may interfere with the final concentration of the amplification chemistry sample component. It will be low enough to successfully perform nucleic acid amplification. The dilution required for the non-cellular sample components is in the range of 5X to 20X. When it is properly determined that the concentration of the target nucleic acid sequence is maintained at a high level sufficient for amplification and detection, a different LOC structure such as the pathogen dialysis zone 70 of Figure 4 can be employed. In this specific example (further shown in Figure 6), there is a dialysis zone upstream of the sample extraction zone 290 which is effective to concentrate pathogens small enough to enter the amplification zone 292 and to exclude larger cells. To the waste storage tank 76. In another embodiment, a dialysis zone is used to selectively remove proteins and salts from the plasma while retaining the cells to be studied. The second LOC structural feature that supports direct nucleic acid amplification is the channel aspect ratio design to adjust the mixing ratio of the sample and the amplification mixture components. For example, to ensure that the single mixing step can dilute the accompanying inhibitor in the sample to a preferred range of 5X to 20X, the length and cross-section of the sample and reagent channels are designed to begin mixing. The flow path impedance at the upstream sample channel is 4X to 19X higher than the flow impedance of the reagent mixture flow channel. The control of the flow impedance in the microchannel can be easily achieved by designing the control. When the cross-sectional size is constant, the flow impedance of the microchannel increases linearly with the length of the channel. Important for hybrid designs, the flow impedance of the microchannel is more primarily dependent on the smallest cross-sectional dimension. For example, when the aspect ratio is very inconsistent, the flow impedance of the microchannel having a rectangular cross section is inversely proportional to the cube of the smallest orthogonal dimension. Reverse transcriptase PCR (RT-PCR)

When the sample nucleic acid species to be analyzed or extracted is RN A, such as RNA from RN A virus or messenger RNA, the RNA must be reverse transcribed into complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction can be carried out in the same chamber in which PCR is carried out (single-step RT-PCR) or an initial reaction which can be separated (two-step RT-PCR). In the LOC variants described herein, single-step RT-PCR can be performed by simply adding the reverse transcriptase and polymerase to the reagent reservoir 62 and programmatically controlling the heater 1 54, so that the temperature cycle is appropriate. The nucleic acid amplification step is carried out after performing the reverse recording step. The two-step RT-PCR can also be carried out by using the reagent reservoir 58 to store and dispense buffers, primers, dNTPs and reverse transcriptase and performing a reverse transcription step in the incubation zone 14 followed by normal amplification in the amplification zone 112. Amplification is achieved. Isothermal Nucleic Acid Amplification For some applications, isothermal nucleic acid amplification is a preferred method of nucleic acid amplification that maintains the amplified region at a constant temperature (typically about 37 ° C to 41 ° C) It is avoided to cyclically heat the reaction to a temperature of -84-201209406 at different temperature cycles. Several isothermal nucleic acid amplification methods have been described, including stock exchange amplification (SDA), transcription vector amplification (TMA), nucleic acid sequence amplification (NASBA), and recombinase polymerase amplification ( RPA), helicase-dependent isothermal DNA amplification (HDA), rolling circle amplification (RCA), reticulated branch amplification (RAM), and circular medium isothermal amplification (LAMP), Any of these isothermal amplification methods or other isothermal amplification methods can be used in the detailed examples of the LOC devices described herein.

For isothermal nucleic acid amplification, the PCR amplification mixture and polymerase are reloaded into the middle of reagent reservoirs 60 and 62 adjacent to the amplification zone instead of the appropriate reagents for the particular isothermal method. For example, in the case of the SDA method, the reagent reservoir 60 contains amplification buffers, primers, and dNTPs, while the reagent reservoir 62 contains appropriate nicking enzymes and Exo-DNA polymerase. In the case of the RPA method, the reagent reservoir 60 contains amplification buffers, primers, and dNTPs, while the reagent reservoir 62 contains a stock-replacement DNA polymerase such as Bsu. Similarly, for the 'HDA method, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, while reagent reservoir 62 contains the appropriate DNA polymerase and helicase to displace the double strand DNA strand in place of heat. Those skilled in the art will appreciate that the reagents required can be placed in two reagent reservoirs in any manner suitable for the nucleic acid amplification method. For amplification of viral nucleic acids from RN A viruses such as HIV or hepatitis C virus, NASBA or TMA methods are well suited because they do not require prior transcription of RNA into cDNA. In this example, reagent reservoir 60 contains amplification buffer, primers, and dNTPs, while reagent reservoir 62 contains RNA polymerase, reverse transcriptase, and optionally RNase H. -85- 201209406 For some forms of isothermal nucleic acid amplification, it may be necessary to have a preliminary denaturation cycle to separate the double-stranded DNA template before maintaining the temperature for isothermal nucleic acid amplification. This object can be easily achieved in the specific examples of all of the LOC devices described herein because the temperature of the mixture in the amplification zone 小心2 can be carefully controlled by the heaters 1 54 in the amplification microchannels 158 (see Figure 14)).

Isothermal nucleic acid amplification is more tolerant to possible inhibitors in the sample, so such methods are generally preferred when direct nucleic acid amplification from the sample is required. Therefore, isothermal nucleic acid amplification is sometimes used in LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677, as shown in Figures 71, 72 and 73, respectively. Direct isothermal amplification may also be combined with one or more pre-amplification dialysis steps 70, 686 or 6 82 (as shown in Figures 71 and 73) and/or prehybridization dialysis steps. 682 (as shown in Figure 72), respectively, helps to partially concentrate the target cells in the sample prior to nucleic acid amplification or to remove unwanted cell debris before the sample enters the hybrid chamber array 110. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybrid dialysis can be employed.

Isothermal nucleic acid amplification can also be performed in parallel amplification regions as shown schematically in Figures 63, 68 and 69. Multiple isothermal nucleic acid amplification methods and certain isothermal nucleic acid amplification methods such as LAMP can be used with the original The reverse transcription step is compatible to amplify the RNA photodiode. Figure 54 shows the light-86-201209406 diode 184 integrated into the CMOS circuit 86 of the LOC device 301. The photodiode 184 is fabricated as part of the 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 on adjacent wafers using non-standard processing steps. The cost of detection on the wafer is low and the size of the assay system can be reduced. The shorter optical path reduces noise in the surrounding environment to efficiently collect fluorescent signals and eliminate the need for optical components for conventional lenses and filters.

The quantum efficiency of the photodiode 184 is the fraction of photons impinging on the active region 185, which are effectively converted into photo-electrons. For the standard tantalum process, the quantum efficiency is in the range of 0.3 to 0.5 under visible light, depending on the method parameters such as the amount of the cover layer and the absorption properties. The detection threshold of the photodiode 184 determines the minimum intensity of the detected fluorescent signal. The detection threshold 値 also determines the size of the photodiode 184, thus also determining the number of hybrid chambers 180 in the hybrid and detection zone 52 (see Figure 52). The size and number of chambers are technical parameters that are limited by the size of the LOC device (in the case of the LOC device 301, which is 1 760 μιη X 5824 μηι), and the actual estimates are in other functional modules such as the pathogen dialysis zone 70. And the amplification region (one or more) is not known until it is invaded. For standard 矽 process, photodiode 184 can detect a minimum of a minimum of 5 photons. However, to ensure reliable detection, the minimum amount can be set to 10 photons. Therefore, when the quantum efficiency is in the range of 0.3 to 0.5 (as discussed above), the fluorescent emission of the probe should be a minimum of 17 photons, but in the case of a reliable detection, it should be regarded as 30 photons. Enter the appropriate error side

-87- 201209406 Electrochemiluminescence as another detection method Electrochemiluminescence (ECL) involves the generation of chemical species on the surface of an electrode, which is then subjected to an electron-transfer reaction to form an excited state that emits light. Electrochemiluminescence differs from common chemiluminescence in that the formation of stimulated species depends on the oxidation or reduction of the luminophore or co-reactant on the electrode. In this context, the co-reactant is an additional reagent added to the ECL solution that enhances ECL emission performance. In common chemiluminescence, stimulated species are formed purely by mixing a suitable reagent. Emissive atoms or complexes are traditionally referred to as luminophores. Briefly, the ECL system relies on the generation of the excited state of the luminophore, which emits photons in the excited state. When either of these processes is employed, the excitation state may also take another path to produce the desired light emission (i.e., quenching). A specific example of a test module that uses ECL instead of fluorescent detection does not require an excitation LED. The electrode system is placed in the hybrid chamber to provide electrical pulses to generate the ECL and the photo sensor 44 is used to detect photons. The duration and voltage of the electrical pulse are controlled; in some specific cases, the current is controlled to replace the control voltage. The luminophore and quenching compound [Ru(bpy) 3]2+, as described above, is used as a fluorescent reporter in the probe and can also be used as a glow in a hybrid indoor ECL reaction. And TPrA (tri-n-propylamine (CH3CH2-CH2) 3N) as a co-reactant. An advantage of the co-reactant ECL is that the luminescent group and -88-201209406 are not consumed after the photon emission and the reagent can be repeatedly carried out by this method. Still further, the [Ru(bpy)3]2 + /TPrA ECL system provides a good signal level under physiologically relevant pH conditions in aqueous solution. Another co-reactant which can produce the same or better results as the TPr A and hydrazine complexes is N-butyldiethanolamine and 2-(dibutylamino)ethanol.

Figure 76 shows the reaction occurring in the ECL method with [Ru(bpy)3]2+ as the luminophore 864 and TPrA as the co-reactant 866. The ECL emission of the [Ru(bpy)3]2 + /TPrA ECL system 862 is followed by oxidation of both [Ru(bpy) 3]2 + and TPrA at the cathode 8 60 . The reaction is as follows:

(2) (3) (4)

Ru(bpy)32+ -e' Ru(bpy)33+ TPrA -e* -> [TPrA*]+ -> TPrA* + H+ Ru(bpy)33+ + TPrA· 4 Ru(bpy)3' The 2+ + product Ru(bpy)3*2+ Ru(bpy)32+ +ho emits light 862 at a wavelength of about 620 nm and the cathode potential is about 1.1 V with respect to the Ag/AgCl reference electrode. For the [Ru ( bpy ) 3] 2 + /TPrA ECL system, either the Black Hole Quencher 'BHQ 2 or the Iowa Black RQ is a suitable quenching. Things. In the specific examples described herein, the quencher is a functional group attached to the probe from the beginning, but in other specific examples, the quencher may be an individual molecule that is free in solution. Hybrid probe for ECL detection-89- 201209406 反.m - from the combination of miscellaneous and indicating the indications of the picture 4 h 9 points and 93 said that the first is the general production of the acid E, the nuclear nucleus The needle probe explores it. The stem of the ring 37 luminesces when hybridized to a complementary nucleic acid. Figure 93 shows a single ECL probe 237 prior to hybridization to the target nucleic acid sequence 23 8 . The probe has a ring 240, a stem 242, a luminophore 864 at the 5' end, and a quencher 248 at the 3' end. Loop 240 is composed of a sequence complementary to the target nucleic acid sequence 238. The complementary sequences on both sides of the probe sequence are joined to each other to form a stem 242. In the absence of a complementary target sequence, the probe remains closed as shown in Figure 93. The stems 242 allow the lumino-quenching pairs to be in close proximity to each other, so that significant resonance energy transfer can occur between the two, substantially eliminating the ability of the luminescent group to emit light after electrochemical excitation. Figure 94 shows the ECL probe 23 7 in an open or hybrid configuration. After hybridization to the complementary target nucleic acid sequence 23 8 , the stem-loop structure collapses, and the fluorophore 864 and the quencher 248 are spatially separated, so that the luminophore 864 regains the ability to emit light. The ECL launch 862 can be optically detected as an indicator that the probe has been hybridized. Since the stem helix of the probe is designed to be more stable than the probe-target helix of a non-complementary single nucleotide, these probes will hybridize to the complementary target with very high specificity. Since the double-stranded DNA is relatively rigid, it is spatially impossible to coexist with the probe-target helix and the stem helix. Primer-Linked ECL Probe Primer-Linked Stem Loop Probe and Lead-Linked Linear Probe (Additional -90 - 201209406

The 蝎 probe is known as another molecular indicator and can be used for both real-time and quantitative nucleic acid amplification of LOC devices. The immediate amplification is performed directly in the hybrid chamber of the L0C device. The advantage of using a primer to link the probe is that the probe element is physically linked to the primer, so that only a single heterozygous event occurs during nucleic acid amplification without the need to heterozygous the primer and probe. This ensures that the reaction occurs more efficiently and with a stronger signal, shorter reaction times, and better discrimination than using separate primers and probes. The probe (and polymerase and amplification mixture) is deposited in the hybrid chamber 180 during manufacture so that no amplification region is required on the LOC device. Alternatively, the amplification region can be left unused or used in other reactions. Primer-Linked Linear ECL Probes Figures 95 and 96 show the primer-ligated linear ECL probe 693 during the first round of nucleic acid amplification and the heterozygous ECL probe during subsequent nucleic acid amplification, respectively. Referring to Figure 95, the primer-linked linear ECL probe 693 has a double-stranded stem segment 242. One of them will break into a probe sequence 696 that has been ligated to the primer, which is homologous to a region of the target nucleic acid 696 and is labeled with a luminophore 864 at its 51-end, and is ligated to the amplification blocker 694 at the 31-end. An oligonucleotide primer 7〇〇. The other strand of the stem 242 is labeled with a quencher molecule 248 at the 3' end. After the first round of nucleic acid amplification is completed, the probe will be looped again and now hybridized to the extended strand by complementary sequence 698. During the first round of nucleic acid amplification, the oligonucleotide primer 7 结合 binds to the target DN A 23 8 (see Figure 95) and then extends to form a DN A strand that contains both the probe sequence and the amplified product. . Amplification blocker 694 prevents polymerase from reading and copying probe -91 - 201209406

Area 696. During subsequent denaturation, the extended oligonucleotide primer 700/template hybrid will detach, and likewise the double-strand 242 of the primer-linked linear probe will also detach to release the quencher. . Once the temperature is lowered for the binding and extension step, the primer-ligated probe sequence 696 of the primer-ligated linear ECL probe will curl up and hybridize to the amplified complementary sequence 69 8 on the stretched strand and the light emission is detected. It is indicated that the target DNA is present. Linear ECL probes with unextended primers retain their bistrand and the light emission remains quenched. This detection method is especially suitable for fast detection systems because it relies on a single-molecule process. Lead-chained ECL probe

Figures 97A through 97F show the operation of the lead-coupled stem-loop ECL probe 705. Referring to Figure 97A, the primer-ligated stem-loop ECL probe 705 has a stem portion 242 of complementary double-stranded DNA and a loop portion 240 of the conjugate probe sequence. One of the stem strands 708 is marked with a luminophore 864 at the 5' end. The other 71 0 is labeled with quencher 248 at the 3' end and carries amplification blocker 694 and oligonucleotide primer 700. During the initial degeneration period (see 97B), the double strands of the target nucleic acid 23 8 are separated from each other, as is the stem portion 242 of the primer-ligated stem-loop ECL probe 705. When the temperature is cooled during the binding phase (see Figure 97C), the oligonucleotide primer 700 on the primer-ligated stem-loop ECL probe 705 will hybridize to the target nucleic acid sequence 23 8 . During extension (see Figure 97D), the complement 706 of the target nucleic acid sequence 298 is synthesized and forms a DNA strand containing the probe sequence 705 and the amplification product. The amplification blocker 694 prevents the polymerase from reading and copying the probe region 705. When the probe binds after denaturation (see Figure 97E, Figure 92-201209406), the probe sequence of the linker 240 of the primer-ligated stem-loop ECL probe 705 (see Figure 97F) is bound to the extended strand. Complementary sequence 706. This configuration leaves the luminophore 864 relatively far from the quencher 248, resulting in a significant increase in light emission. ECL control probe

Hybrid chamber array 110 includes a number of hybrid chambers 180 with forward and reverse ECL control probes for assay quality control. Figures 98 and 99 schematically show reverse control ECL probes 786 without luminophores, and panels 100 and 101 are thumbnails of forward control ECL probes 787 without quenching. The forward and reverse control ECL probes have a stem-loop structure similar to that described above for ECL probes. However, regardless of whether the probes are synthesized into an open configuration or remain latched, an ECL signal 862 (see Figure 94) is always emitted by the forward control ECL probe 787 and the reverse control ECL probe 786 does not. The ECL signal 862 is transmitted. Referring to Figures 98 and 99, the reverse control ECL probe 786 has no luminophores (and may or may not have quencher 248). Therefore, regardless of whether the target nucleic acid sequence 23 8 is heterozygous with the probe shown in FIG. 99, or the probe remains in the configuration of the stem portion 242 and the loop portion 240 as shown in FIG. 98, the ECL signal can be ignore. Alternatively, the reverse control ECL can be designed to always remain quenched. For example, the probe can be provided with an artificial probe (loop) sequence 240 that does not hybridize to any of the nucleic acid sequences in the sample, such that the stem portion 242 of the probe molecule is hybridized with itself and luminophore and quenched. The extinguisher is kept close without an appreciable ECL signal being detected. This reverse control can account for any low-volume emissions that occur when quenching is incomplete. In contrast, the forward control ECL probe 78 7 is constructed to be free of quenching as shown in Figures 100 and 101. Regardless of whether the forward control probe 787 is heterozygous to the target nucleic acid sequence 2 3 8 , nothing will quench the ECL emission 862 from the luminophore 864.

Figures 91 and 92 show another possibility for constructing a forward control room. In this case, the ECL luminophore solution can be loaded into the calibration chamber 382 that is sealed to avoid contact with the amplicon (or any liquid stream containing the target molecule) so that a positive signal is always detected at the electrode. Similarly, the control room can be a reverse control chamber that prevents any target from contacting the probe due to the lack of access to the control chamber, so that the ECL signal is never detected.

Figure 52 shows the possible distribution of the forward and reverse control probes (3, 7 and 380, respectively) throughout the hybrid chamber array 110. For ECL, the forward and reverse control ECL probes 786 and 78 7 can be replaced with control fluorescent probes 378 and 380, respectively. The control probes can be disposed in the hybrid chamber 180 along diagonally diagonal lines of the hybrid chamber array 1 10. However, the configuration of the control probes within the array is arbitrary (as is the configuration of the hybrid chamber array Π 0). The inconsistency of the electrical characteristics of the photodiode 1 84 (which reacts to any ambient light present in the sensor array and from other locations in the array) in the calibration chamber of the ECL detection introduces background noise and shifts to Inside the output signal. This background is removed from each output signal by a calibration chamber 382 in the hybrid chamber array 110. The array may be free of any probes, probes containing no ECL luminophores, or -94-201209406 containing luminophores and Quenching but designed as a probe that is always quenched. The number and configuration of calibration chambers 382 throughout the array of hybrid chambers are optional. However, if the photodiode 184 is calibrated by a closer calibration chamber 382, the calibration will be correct. Referring to Fig. 116, the hybrid chamber array 110 has a calibration chamber 382 in every eight hybrid chambers 180. That is, the calibration chamber 382 is located in the center of the hybrid chamber 180 arranged in a 3x3 square shape. In this configuration, the hybrid chamber 18 is calibrated by the calibration chamber 382 immediately adjacent.

Fig. 75 shows a differential imager circuit 788 which subtracts the signal generated by the photodiode 184 corresponding to the calibration chamber 382 from the ECL signal of the surrounding hybrid chamber 180 after the application of the electrical pulse. The differential imager circuit 78 8 samples the signal from the pixel 790 and the "virtual" pixel 792. Signals generated by ambient light from the array area of the chamber are also subtracted. The signal from pixel 790 is very weak (i.e., close to a dark signal) and it is difficult to distinguish between a background signal or a very weak signal when there is no darkness reference. During use, "read column (read_row)" 794 and "read column d (read_row_d)" 795 are activated and M4 797 and MD4 801 transistors are activated" switches 807 and 809 are turned off, such that from pixel 790 and "virtual The outputs of the pixels 792 are stored in the pixel capacitor 803 and the virtual pixel capacitor 850, respectively. After the pixel signal is stored, switches 8 07 and 08 09 are deactivated. The "read_col" switch 811 and the virtual "read row" switch 813 are then turned off and the output switched capacitor amplifier 815 amplifies the differential signal 8 1 7 . -95- 201209406 ECL Amount and Signal Efficiency The normal efficiency measure of ECL is the number of photons obtained for each "Faraday" electron (ie, each electron participating in the electron). The ECL efficiency is ♦ ECL is not: Φεα = [ΐάτ

Where I is the photon intensity per second 'i is the current amperage, F is the Faraday constant and NA is the Yafox constant. Co-reactant ECL efficiency

An annihilation ECL in a deoxygenated, aprotic solution (such as a 'nitrogen flushed acetonitrile solution) is simply sufficient for efficiency measurements, and φΕ(:Ι^—to permit a 値 of about 5%. However, it is generally considered The reactant system does not have a meaningful direct measurement efficiency. Alternatively, the emission intensity is measured by a standard solution that is easily prepared, for example, a Ru(bpy) 32+ alignment measured in the same format. (See, for example, JK Lei and And MJ Powell, J. El ectrochem. Soc., 137, 3 1 2 7 (1 990 ), and R. Pyati and Μ. M. Richter > Annu. Rep. Prog. Chem. C, 103, 12-78 (2007)) pointed out (without accelerators such as surfactants): the efficiency peak of the RU (bpy) 32 + ECL and TPrA co-reactant is equivalent to 5% of the degree of ec湮 ECL seen in acetonitrile (eg 2% efficiency; see I. Rubinstein & A. J. Bard, J. Am. Chem. Soc., 1 03 5 1 2 - 5 1 6 (1 9 8 1 )) -96- 201209406 ECL potential

The voltage at the working electrode of the Ru(bpy) 32 + /TPrA system is about +1.1 V (generally measured in the literature relative to the Ag/AgCl reference electrode). This high voltage will shorten the electrode life, but this is not a problem for a LOC device used in a diagnostic device such as the disposable device.

The ideal voltage between the cathode and the anode depends on the combination of the solution composition and the electrode material. Choosing the right voltage requires compromise between the highest semaphore, reagent and electrode stability, and poorly activated side reactions such as electrolysis of water in the room. In the test of buffered aqueous Ru(bpy)32+/co-reactant solution and platinum electrode, the ECL emission is at most 2.1-2.2 V (depending on the choice of co-reactant). When the voltage is below 1.9V and above 2.6V, the emission intensity will drop to <75% peak. When the voltage is below 1.7V and above 2.8V, the emission intensity will drop to <50% peak. Therefore, the preferred cathode-anode voltage difference for ECL operation in such systems is 1.7-2.8 yen, which is particularly good in the range of 1.9-2.6. This maximizes the emission intensity as a function of voltage and avoids voltages that cause significant gas loss at the electrodes. ECL Emission Wavelength The wavelength of the ECL emission light 862 has an intensity peak of about 620 nm (measured in air or vacuum) and the emission covers a relatively wide range of wavelengths. Significant emission systems are at wavelengths from about 5 50 nm to 700 nm. Furthermore, the peak emission wavelength may vary by about 1% due to the different chemical environments surrounding the active species. The specific example of the LOC device described herein, which does not include a wavelength-specific filter, has two advantages for capturing signals of such wide and varying spectra from -97 to 201209406. The first advantage is sensitivity: any wavelength filter (even within its passband) reduces light penetration, so efficiency is improved without filters. The second advantage is flexibility: there is no need to adjust the filter passband after making some reagent changes, and the signal comparison does not depend on the slight difference of the non-target components of the input sample.

The volume of solution involved in ECL relies on the availability of luminescent groups (and co-reactants) in solution. However, as shown in Figure 78, the stimulated species 868 are only produced in solution 872 near electrodes 860 and 870. The depth of the boundary layer of the parameter in the module shown herein is the depth of the layer 872 of the solution around the electrode 860 that produces the stimulated species 868. This is a simplified result because the kinetics of the solution can lead to an increase or decrease in the available concentration:

Increased availability: diffusion and electrophoretic effects allow for interchange with more solutions. Reduced availability: The reagent is adsorbed on the electrode and becomes unusable in the ECL process. When the boundary layer depth 値 is 0.5 μηι, the following phenomenon can be observed: ECL can be observed in an experiment in which a magnetic bead having a diameter of at most 4.5 μπ is used in combination to attract the luminophore 864 to the cathode 860. In the electrode array of the fingers, it can be found that the Ru ( bpy ) 32 + /TPrA ECL emission 862 system which is a function of the electrode spacing is the largest at the electrode spacing of -98 - 201209406 0.8 μηι. When the electrode spacing is about 2 μηι, the need for the aqueous solution 8 72 to have a co-reactant 866 is increased. This phenomenon indicates that the stimulated species 868 will diffuse by a few microns, suggesting that the stimulated species are diffusely exchanged at a scale similar to the ground state. Steady state and pulsed operation

During pulsed activation of electrodes 860 and 870, the intensity of the ECL emission 862 (see Figure 94) will generally be higher than the intensity of the emission 862 when the electrode is activated in a steady state. Accordingly, the activation signals of the electrodes 860 and 870 are pulse-width modulated (PWM) by the CMOS circuit 86. Reagent Recycling and Species Lifetime The Ru complex is not consumed in the Ru ( bpy ) 32 + /TPrA ECL system, so the intensity of the emitted 8 62 does not decrease with subsequent reaction cycles. The rate limiting step has a lifetime of about 0.2 milliseconds and can provide a total reaction cycle time of about 1 millisecond. Electrophoretic effects and other limitations Given the complexity of the solution in the hybrid chamber, multiple phenomena can occur when the ECL voltage is activated. The capacitive effects of macromolecular electrophoresis, ohmic conduction, and small ion migration occur simultaneously. Electrophoresis of oligonucleotides (probes and amplicons) complicates the detection of probe-target hybrids because of DNA. It has a strong negative charge and is adsorbed on the cathode 860. The time scale for such activities is typically very short (-99-201209406 on a millisecond scale). Even if the voltage intensity is only moderately strong (about 1 v), the electrophoretic effect is still strong because the separation distance between the cathode 860 and the anode 870 is small. Electrophoresis enhances ECL emission 862 and degrades other emissions in certain instances of certain LOC devices. This phenomenon is manifested in the fact that the electrophoretic effect increases or decreases concomitantly when the electrode spacing is increased or decreased. The finger-like intersection of the cathode 860 and the anode 8 70 above the photodiode 184 represents an extreme example of such a minimum spacing. These configurations can produce ECL 〇 ohmic heating (DC current) even if there is no co-reactant 8 66 between carbon electrodes 860 and 870. The current required to maintain the ECL voltage at about 2.2 V can be referred to the ECL battery shown schematically in Fig. 79. 874 to decide. The DC current flowing through the chamber is determined by two impedances: the interfacial resistance Ri between the electrodes 860 and 870 and the body of the solution, and the solution resistance Rs derived from the conductivity of the host solution and the geometry of the conduction path. For solutions in which the ionic strength is related to the conditions of the LOC device, the chamber resistance is dominated by the interfacial resistance of electrodes 8 60 and 870 and Rs can be ignored. The effect of interface resistance is estimated by measuring the amount of giant current in a similar solution under the electrode geometry of the LOC device. A giant spectroscopy method using a uranium electrode and passing a current density of a similar solution is used. Consistent with the worst case (high current) approach, the overall ionic strength and ECL reactant concentration in the test solution is higher than that of the LOC device. The cathode area is smaller than the anode area and is surrounded by an anode having a substantial area of the ring geometry -100 - 201209406. For a cathode consisting of a 2 mm diameter circle, the measured current is 1 · 1 mA, which produces a current density of 305 A/m2. In the heating mode, the electrode area is the square annular geometry shown schematically in Figure 79. The cathode is a ring having a width of 1 μηι and a thickness of 1 μηι. The surface area is 196 square microns and thus the current is calculated as 1 = 69 ιιΑ.

Heating (power = V2/R) is designed with the worst case model in which all of the heat is used to increase the temperature of the water in the room. If the LOC device body is not allowed to remove heat, this phenomenon heats the chamber contents at a rate of 5.8 °C/s and has a voltage difference of 2.2 V. Heating the chambers at about 20 °C denatures most of the hybrid probes. For highly specific probes for mutation detection, it is preferable to further limit the heating to 4 ° C or lower. With this degree of temperature stability, single base mismatching-sensitivity hybridization can be achieved using appropriate design sequences. This allows for the detection of allelic differences in mutations and single nucleotide polymorphism levels. Therefore, the DC current is applied to the electrodes 8 60 and 870 for 0.69 seconds to limit the heating to 4 °C. A current of about 69 ιιη through the chamber is difficult to accommodate as a Faraday current of the EML species of micromolar concentration. Therefore, the low power-cycle pulsing of the electrodes 860 and 870 can further reduce the heating (down to 1 ° C or less) while maintaining sufficient ECL emission 862, and will not introduce Disorders related to reagent depletion. In other embodiments, the current will drop to 〇 1 nA, which removes the need for the electrode to be pulse activated. Even if the current is as low as 0.1 nA, the ECL emission 8 62 still has a luminophore-restricted. -101 - 201209406 Chamber and Electrode Geometry Optimum optical coupling between ECL luminescence and photosensor The most direct chemical precursor for ECL luminescence is produced within a few nanometers of the working electrode. Referring again to Figure 78, light emission (excited species 868) typically occurs within a few microns or less of the location. Therefore, the volume adjacent to the working electrode (cathode 860) can be felt by the photodiode 184 corresponding to the photo sensor 44. Accordingly, electrodes 860 and 870 are directly adjacent to the active surface region 185 of the corresponding photodiode 184 within photosensor 44. Further, the cathode 860 is shaped to increase the shape of the length of the side "sensed" by the photodiode 184. The purpose is to maximize the volume of the excited species 86 8 that can be detected by the photodiode 184 of the substrate. Fig. 77 schematically shows three specific examples of the cathode 860. The cathode 878 of the comb structure has the advantage that the parallel fingers 880 can be interdigitated with the fingers of the anode 870. The configuration of the finger joint is shown in Fig. 84, and the 90th and 92th views are partial views of the LOC layout. The configuration of the finger intersection provides a narrow (1 to 2 micron) uniform dielectric gap 8 76 (see Figure 78) and the finger-like comb structure is relatively simple for etching processes . As discussed above, the relatively narrow dielectric gap 876 between electrodes 860 and 870 can eliminate the need for co-reactants of certain solutions 876 because the excited species 864 will diffuse between the cathode and the anode. Eliminating the need for co-reactants can remove the possible chemical impact of the co-reactants on different assay chemistries and provide a wider range of possible assays. Referring again to Fig. 77, some specific examples of the cathode 860 have a 蜿蜒 group -102 - 201209406 state 8 82. In order to maintain the ability to withstand manufacturing errors when the side grows up, it is easy to make a wide rectangular path 8 84.

The cathode can have a more complex configuration 8 8 6 if needed or desired. For example, it may have a fine scalloped area 888, a branched structure 890, or a combination of the two. A partial view of the LOC design with branch structure 890 is shown in Figures 1 15 and 11 6 . More complex configurations such as the 886 provide long sides because it is more difficult to pattern the opposite phase of the dense phase, so this configuration is best suited for solution chemistry using co-reactants. Electrode Thickness In general, an ECL battery involves a working electrode that appears flat from the outside. Similarly, conventional metal layer microfabrication techniques tend to form flat structures having a metal thickness of about 1 micron. As indicated earlier and schematically shown in Figures 77, 80 and 81, increasing the length of the sides enhances the coupling between the ECL emission and the photodiode 184. A second strategy to further increase the collection efficiency of the photodiode 184 to the emitted light 8 62 (see Figure 94) is to increase the thickness of the cathode 860. This is shown schematically in Figure 78. The portion of the participating volume 892 adjacent the working electrode wall is the region most efficiently coupled to the photodiode 184. Thus, for a working electrode 860 of a given width, the overall collection efficiency of the emitted light 862 can be increased by increasing the thickness of the electrodes. Moreover, since high current carrying capacitance is not required, the width of the working electrode 860 can be reduced as long as it is practical. The thickness of the electrodes 860 and 870 may not be increased without limitation. It should be noted that the separation dimension of the feature member and the electrode is about -10 - 201209406, and the liquid charge is disadvantageous for the gap having a larger width than the depth, and the optimum practical thickness of the electrode is 0.25 micrometer to 2 micrometer. Electrode Spacing The distance between the electrodes 860 and 870 is important for the quality of the signal in the LOC device (especially the specific example where the electrodes are finger-to-finger). The specific structure of the cathode 860 as a branched structure, for example, as shown in Figs. 7 and 8 1 is also important. Both the ECL emission efficiency and the collection efficiency of the emitted light should be maximized. The level of electrode spacing at 1 micron or less is most beneficial for the occurrence of ECL emissions. Smaller spacing is particularly advantageous when the ECL system is carried out in the absence of a co-reactant. The fact that this spacing corresponds to the wavelength of the emitted light 862 is of limited importance. Therefore, in many specific examples (the measurement of the radiation 8 62 (see Fig. 94) is where no light is transmitted between the electrodes 860 and 870), the target is usually to make the electrode spacing within practical limits. Try to be as small as possible. However, in the specific case where the radiation 862 must be transmitted between the electrodes 8 60 and 8 70, in addition to considering the ECL emission process, the pulsation property of the light is also considered. The emitted light 862 from the ECL of Ru(bpy) 32+ has a wavelength of about 620 nm and is therefore 460 nm (0.46 microns) in water. In the case where the photodiode 184 and the ECL excited species 868 are located on different sides of the electrode structure and the electrode structure is metallic, the emitted light 862 must pass through the gap between the elements of the metal structure. If the gap is equivalent to the wavelength of the light, the diffraction typically reduces the intensity of the -104 - 201209406 of the propagating light reaching the photodiode 1 84. However, when the emitted light 862 is incident on the gap at a large angle, an evanescent mode couPling can be used to improve the intensity of the collected signal. The LOC device employs two measures to enhance the coupling efficiency between the photodiode 184 and the emitted light 862. First, the spacing between the metal components is not less than the wavelength of the emitted light in water, i.e., about 0.4 microns. When combined with other observations associated with the small spacing between the finger-like electrodes, the optimum range of electrode spacing is 〇·4

To 2 microns. Secondly, the gap distance between the components and the photodiodes 1 8 4 is minimized. In the specific example of the LOC device described herein, this requirement indicates that the total thickness of the film between the electrodes 860 and 870 and the photodiode 184 is preferably 1 μm or less. Arranging its thickness to a quarter-wavelength or three-quarters of a wavelength in a specific example in which a plurality of layers are formed between the electrodes and the photodiode has an additional advantage of suppressing the reflection of the emitted light 862. Electrode Mode Figure 78 is a schematic cross-sectional view of a schematic portion of electrodes 860 and 870 in a hybrid chamber. The volume around the side of the cathode 860 that is filled with the excited species 868 is sometimes referred to as the participating volume of 8 92. The occlusion region 8 94 above the cathode 860 can be ignored because its optical coupling to the photodiode 184 is negligible. A technique for determining whether a particular electrode configuration can provide ECL emission 862 to the photodiode 184 of the substrate will be described below with reference to Figures 79, 80 and 81. Figure 79 is a toroidal geometry in which the cathode 860 surrounds the periphery of the light -105 - 201209406 diode 184. In Fig. 80, cathode 860 is located within the edge of light 184. Figure 81 shows a more complex configuration in which 860 has a series of parallel fingers 8 8 〇 to increase the length of its side edges. For all of the above configurations, the mode is calculated as follows. For the volume of the solution 892 VECL, the emitter has a N em of · Nem = N|um.Tp / TECL = VECLCLNA-tp / TECL (6) where the number of participants of the luminophore Nlum = VeclClNa, τ Ε EC L process The lifetime, CL is the concentration of the illuminant, τρ is the pulse duration and Να is the Yafot number. The number of isotropically emitted photons ^^^^ is: Nphot = <l> ECL Nem (7) where <)) ecl is the ECL efficiency, which is defined as the average number of photons that should be emitted by a single luminophore. Then, the electronic signal count S from the photodiode is S_Nph〇t. <|) 〇 <|)q, (8) where is the optical coupling efficiency (light absorbed by the photodiode 184) and Φ ς is the photodiode Quantum efficiency. Therefore, the signal is: S = VECLCLNA^EMq (9) TECL For the electrode configuration of Figures 79 and 80, φ. For: (|). = ( 2 5% : photon amount of guided photodiode 184) X (10%: unreflected photon amount) That is, for the configuration shown in Figures 79 and 80, Φ. = 2.5% diode The number of cathode effects CL is the time, the number of ECL counters -106- 201209406 For the electrode configuration of Fig. 81, 50% of the photons are emitted towards the direction of the photodiode 184, but at an angle The absorption efficiency of the function is constant, so φ. = ( 50% : photon amount of the guided photodiode) X (10%: unreflected photon amount) That is, for the configuration of Fig. 81, φ. = 5%.

The participating volume 8 92 depends on the electrode configuration and its detailed description is found in the corresponding chapter. The input parameters for the calculations are listed below: Table 5: Input Parameters

Number of parameters 値 Comments Luminous concentration CL 2.89 μΜ Previously calculated probe concentration ECL cycle (life) τΕα 1 ms Luminous reaction steps of the luminescent group boundary depth D 0.5 μιη Effective volume of solution involved in ECL (including diffusion And electrophoresis) Current application duration τρ 0.69 s Select to limit ohmic heating to 4 °C 洳 previously described) Chamber X size 28 μιη Room size 28 μπι Room height Ζ 8 μιη Light diode X size 16 μιη Light diode Υ Size 16 μη electrode thickness (ie, exposed edge height) 1 μηι Electrode layer minimum width and gap 1 μπι Process critical dimension electrode interface current density 350 A/m2 0 in ohmic heating solution volume resistance 0.5 Ω. m for ohmic heating application Voltage Difference (Working Electrode - Counter Electrode) 2.2 V -107 - 201209406 Ring Geometry Around the Light Diode Referring to Figure 79, the cathode 860 is a ring around the photodiode 184. In this configuration, the participating volume 892 is: VECL = 4χ [(film layer next to the electrode wall) + (quarter cylinder above the electrode wall)] Calculation result: Number of photons generated from the 0.5 μιη boundary layer: 3.1Μ05 Electronic Diode Electron Count: 2.3χ103 This signal can be easily detected by the substrate photodiode 184 of the LOC device photosensor 44. Additional Fingers to Increase the Length of the Circumference Referring to Figure 81, the parallel protrusions 880 are added throughout the cathode 8 60. » Only the horizontal edges in the figure provide the participating volume 892 to avoid repeating the counting of the vertical edges. Thus the participating volume 892 is: VECL=(8x2) X [(membrane layer beside the electrode wall) + (quarter cylinder above the electrode wall)] Figure 8 Configuration result calculated: From 0.5 μιη boundary layer Number of photons generated: 1.1Μ06 Electron count of photodiode 184: 8.〇χ1〇3 This signal can be easily measured with photodiode 1 84. -108- 201209406 Full coverage

The configurations shown in Figures 82 and 83 can be considered as extreme examples of maximum surface area coupling. In practice, a 90% or better coupling between the electrode surface area and the active surface area 185 of the photodiode 184 has achieved near-optimal results, even though the photodiode active surface area 185 is only 50% coupled to the electrode surface area. It offers most of the advantages of a full coverage configuration. The complete coverage can be achieved by two specific examples: the first specific example, as schematically shown in Fig. 82, uses a transparent cathode 860 which is placed on a plane parallel to the plane of the photodiode 184 and has an area. The region is matched to the area of the photodiode 184, and the cathode is disposed adjacent to the photodiode 184 such that the emitted light 862 can pass through the cathode to the photodiode. In a second embodiment schematically shown in Fig. 83, again, the cathode 860 is parallel to the photodiode region and the cathode is registered with the photodiode region, but the solution 872 is full. The cavity between the cathode 860 and the photodiode 1 84. In order to establish a signal pattern that is fully covered, the cathode is assumed to be a complete layer above the photodiode 184, with half-photons being directed to the photodiode 184 (absorption efficiency is still 10%). The amount of photons generated from the 0.5 μιη boundary layer: 7.7 The electron count of the MO5 photodiode: 1.2Χ104 The above mode can be improved by using a surfactant and fixing the probe to the cathode to improve the signal and verification. Maximum spacing between ECL probes and photodiodes -109- 201209406

Hybrid detection on the wafer avoids the need for detection via confocal microscopy (see background of the invention). This is an important factor in saving time and cost for the system. Traditional detection requires the use of a lens or curved mirror for optical imaging. By using non-optical imaging, the diagnostic system avoids the need for complex and bulky optical components. The close placement of the photodiode near the probe provides the advantage of providing extremely high collection efficiency: when the material thickness between the probe and the photodiode is at the 1 micron level, the emitted light can be collected at an angle of up to 174°. This angle is calculated by considering the light emitted from the probe at the centroid of the surface of the hybrid chamber closest to the photodiode having a flat active surface parallel to the surface of the hybrid chamber. The emission angle cone that the light can be absorbed by the photodiode is defined as the perimeter of the emission probe and the sensor angle falls to the perimeter of its flat surface. For a 16 micron χ 16 micron sensor, the apex angle of the cone is 170°; in the extreme case (where the photodiode is expanded to match the 28 micron χ26·5 micron hybrid chamber) The area angle, which is 174°», can easily have a spacing of 1 micron or less between the surface of the chamber and the active surface of the photodiode. The use of a non-optical imaging scheme requires that the photodiode 184 be in close proximity to the hybrid chamber to collect sufficient photons of the fluorescent emission. The maximum spacing between the photodiode and the probe can be determined in the following manner. Using a chelating luminescence group and the electrode configuration of Figure 81, we calculated that a 16 micron X16 micron sensor in a single hybrid chamber absorbed 27,000 photons, assuming a sensor quantum efficiency of 30%. 8,000 electronic. In doing this calculation, we assume that the collection area of the hybrid chamber has the same bottom area as the sensing area, and that one quarter of all the hybrid photons have an angular orientation of -110-201209406. The ratio of photons scattered by the detector-dielectric interface is conservatively estimated to be 1 〇%. That is, the light collection efficiency of the optical system is 0.025. More precisely we can write also = [(the bottom area of the hybrid chamber light collection area) / (photodetector area)] [Ω / 4π] [10% absorbed amount], where Ω = hybrid chamber substrate Represents the solid angle of the light detector on the point. For the regular pyramid geometry:

Ω = 4arcsin ( a2/ ( 4d 〇 2 + a2)), where d 〇 = the distance between the chamber and the photodiode, and a is the size of the photodiode. 1. The hybrid chamber can release 1. 1 X 1 〇 6 photons. The detection threshold of the selected photodetector is 17 photons. When dQ値 is greater than 10 times the size of the sensor (ie, substantially normal incidence), the ratio of photons that are not reflected by the sensor surface can be 10% increased to 90%. Therefore, the minimum optical efficiency required is: φ0 - 17/ (1.1χ106χ〇.9) - 1.72xl0's The bottom area of the luminescent region of the hybrid chamber 180 is 29 microns χ 19.75 microns

Solving d〇, we get the maximum distance dQ = 1 600 μm between the bottom of the hybrid chamber and the photodetector. Within this limit, the collection cone angle defined above is only 0.8°. It should be noted that this analysis ignores the slight effects of refraction. LOC Variability The LOC device 301, which is fully described herein and above, is one of many possible LOC device designs. LOC device changes using different combinations of the different functional zones described above will be described by placing the sample into the detection path -111 - 201209406 and/or displaying it in a schematic flow chart to show some possible combinations. Where appropriate, the flow chart will be divided into sample placement and preparation stage 28 8 , extraction stage 290, incubation stage 291, amplification stage 292, pre-hybrid stage 293, and detection stage 294. All of the LOC variations described above or simply in schematic form are 'simplified and straightforward' and do not show all of the accompanying complete layout. Also for the sake of clarity' does not show smaller functional units such as liquid sensors and temperature sensors' but it should be understood that such units have been incorporated into the appropriate locations in each of the following LOC device designs. Microfluidic device with dialysis device, LOC device and interconnected cap - Microfluidic device 78 3 with dialysis device 784, LOC device 785 and interconnected cap 51 can provide better modularity and higher Sensitivity. Separating the dialysis function from the LOC device relative to the LOC design that combines all functions (e.g., LOC Variant 729 in Figure 89) allows for the development of different specialized dialysis devices 7 84 to select different targets. These tailored dialysis devices 784 are combined with a LOC device 785 and an interconnecting cap 51 to form a complete assay system. Furthermore, different LOC devices 78 5 that are best suited for different verification strategies can be developed and cooperated with different dialysis devices 784, thus providing an extremely efficient and flexible method to develop the system. It is also possible to develop a LOC device without a dialysis device for a particular application, or to combine multiple LOC devices 785. The various surface-microelectromechanical wafer configurations of the microfluidic device 783 can be prepared using the best and most cost effective manufacturing method. For example, the dialysis zone -112-201209406 7 84 does not require CMOS circuitry and can be fabricated with less expensive materials and fewer process steps. Further, the larger and optimized dialysis device 7 84 provides better sensitivity, signal to noise ratio, and dynamic range of the assay system.

As shown in Figure 102, the microfluidic device 783 can utilize 12 individual amplification chambers (Ϊ 12.1 to 112.12) to prepare sample 288, then extract 290, incubate 291, amplify 292, and detect 294 pathogens. Sex DNA. The module uses a plurality of amplification chambers to increase the sensitivity of the assay and improve the signal-to-noise ratio. This device uses the hybrid chamber array 110.1 to 110.12 ugly (:1^ to detect the probe-target impurity The system is modularized to allow different dialysis devices 7 84 to be used to detect other targets in the liquid sample, such as white blood cells, red blood cells, pathogens or molecules like free proteins or DNA, although the current description describes pathogenicity. DN A detection, but those skilled in the art will appreciate that the microfluidic device 783 is not limited to detecting only one target. Figure 103 shows the microdialysis device 784, the LOC device 785, and the interconnective top cover 51. Fluidic device 78 3. The interconnective cap 51 is comprised of a reservoir layer 78, a cap channel layer 80, and an interface layer 594. The interfacial layer 594 is located in the cap channel layer 80 and MST of the CMOS + MST device 48. Between the channel layers 100. The interface layer 5 94 may have a more complex fluid interconnection between the reagent reservoir and the MST layer 87 without increasing the size of the ruthenium substrate 84. Figure 104 overlays the reservoir, the upper channel, and the interface channel to display The interface layer 5 94 is caused by Complex piping system. Referring to Figures 1 and 4, a sample (e.g., blood) enters the sample inlet 68 and capillary action pulls blood along the top channel 94 to the anticoagulant surface tension valve 118. As best shown in Fig. 105, the anticoagulant surface sheet-113-201209406 force valve 118 has two interface channels 596 and 598 at the interface layer 594. The reservoir-side interface channel 5 96 is connected by a downcomer 92. To the reservoir outlet, the sample-side interface channel 598 is connected to the upper conduit 96 by a cap channel 94. The anticoagulant from the reservoir 54 flows through the reservoir-side interface channel 5 96 through the MST channel 90 until the bend The liquid level is fixed to the upper conduit 96. The sample flow flowing along the top cover passage 94 wets the sample-side interface passage 5 9 8 to remove the meniscus, so that the anticoagulant continues to flow in the blood sample. The pathogen dialysis zone is combined with the blood sample at 70. Referring to Figures 104 and 105, in this particular example, the pathogen dialysis zone contains an interface target channel 602 and an interface waste channel 604, which pass through a plurality of predetermined enthalpies. The size of the hole is fluidly coupled. Located in the dialysis area 70 The most upstream hole is different from the hole in the downstream; the downstream hole is a hole having a diameter of less than 8.0 μm, which is selected to allow the target to pass through and enter the interface target channel 602. In the present specific example, the hole is A hole 164 having a diameter of 3.0 microns is selected to allow passage of the pathogen into the interface target channel 602. The pathogen dialysis zone 7 is configured to enable the sample to flow through the channels and apertures under capillary action. In Figures 104 and 105, the blood sample flows through the top cover channel 94 to the upstream end of the interface waste cell channel 604. The interface waste cell channel 60 4 is connected to a 3.0 micron diameter aperture 1 64 leading to the dialysis MST channel 204. Each dialysis MST channel 204 connects a small aperture 1 64 having a diameter of 3.0 microns to a respective dialysis upper pilot 168. The dialysis upper pilot hole 168 leads to the interface target passage 602. However, the upper conduit is configured to secure the meniscus rather than allowing the capillary drive flow to continue to flow. -114- 201209406

The pathogen dialysis zone 7 has a bypass passage 600 to fill the flow channel structure without trapping air bubbles. The bypass passage 600 at the most upstream end of the pathogen dialysis zone 70 has a CIF (Capillary Start Feature) 202 to cause capillary drive flow to flow from the bypass passage 600 to the interface target passage 602 (see Figures 104 and 105). The bypass channel also has a wide labyrinth to extend the flow path of the interface waste cell channel 604 to the interface target channel 602. The longer flow path will retard the sample flow so that the sample flow can fill the interface target channel 602 when the meniscus is formed at the most upstream dialysis MS T channel 204. The sample stream begins at the upstream end and disintegrates the meniscus fixed to each of the dialysis upper pilot holes 168 as the liquid stream moves downstream along the interface waste channel 206. This ensures that the dialysis bottom channel fills the sample stream when the dialysis area is filled. Some dialysis MST channels 204 may not be filled if there are no bypass channels 600, or dialysis upper conduits 168 that are not configured to hold the meniscus. Similarly, bubbles may form within the interface target channel 602. In either case, the flow through the dialysis zone is substantially blocked. Interface waste channel 604 will flow to waste channel 72 and interface target channel 602 will flow to target channel 74 (see Figures 104 and 105). The five dialysis zones are connected together in series through the cap channels 72 and 74 to increase the efficiency of the dialysis process. At the exit of the fifth dialysis zone, the sample stream containing the target is drawn by capillary action from the dialysis device 784 along the target channel 74 in the cap channel layer to the LOC device 78 5 » the waste channel 72 It will flow into the waste reservoir 76 (see Figure 103). The LOC device 785 can also be used as a stand-alone microfluidic device with another top cover suitable for a single device, and any anti-115-201209406 coagulant reservoir 55 can be used in this configuration to supply an anticoagulant. . An example of this stand-alone LOC device 785 is its use for whole blood analysis. Referring to Figures 1 and 6 and 107, the target will flow along the top cover passage 74 into the LOC unit 78 5 . It flows through any of the anticoagulant surface tension valve 1 17 (which is used to add the contents of the optional anticoagulant reservoir 55)' to continue until it reaches the cytosolic surface tension valve 128. In the configuration described herein, any reservoir and surface tension valve are not used and do not affect sample flow or operation of the LOC device. In the case of the anticoagulant surface tension valve 118 described above, the cytolytic surface tension valve 128 has a cytosolic reservoir-side interface channel 606 and a cytolysis-sample side interface channel 60 8 (see 107 picture). The lysing reagent flows from the reservoir 56 through the cap passage 94 to the cytolysis reservoir-side interface channel 606. The reagent will flow into the downcomer 92 and through the MST channel 90 to the conduit 96 above the fluid level of the reagent (see Figure 107). The sample stream from target channel 74 will fill the cytolytic sample-side interface channel 608. The sample stream removes the meniscus at the upper conduit 96 and the cytosol is mixed with the sample as it flows into the chemical cytolytic zone. In the chemical cell compartment region 130, the cytolytic reagent is diffused into the liquid stream to lysate the target cells and release the genetic material therein. The sample stream is stopped at the mixing zone outlet valve 206. The mixing zone outlet valve is a boiling starter valve 206. The liquid sensor 174 upstream of the valve provides feedback as the sample stream is about to reach the valve 206. If the CMOS circuit 86 is programmed to delay the start to ensure that the target cells are completely lysed, then the feedback from the liquid sensor will cause the delay period. After any lag period has elapsed, the boiling starter valve 206 will be activated -116-201209406 and the downstream liquid sensor 174 will note that the flow has begun to flow again along the MS 通道 channel 90.

The lysate sample stream continues to flow to the restriction enzyme, ligase, and linker surface tension valve 132. The operation of the surface tension valve 132 is the same as described earlier for the anti-coagulant surface tension valve 186. Referring to Fig. 107, when the lysed sample stream reaches the meniscus fixed at the surface tension valve 132, the restriction enzyme, ligase and linker primer are released from the reservoir 58 and the sample is sampled. The streams are mixed together. The sample then flows through the MST channel 90 to the heated microchannels of the incubation zone 114. Referring to Figures 107 and 108, the incubation zone 114 is comprised of a microchannel 210 heated by a heater 154. Referring to Fig. 108, the sample stream will be stopped at the incubator outlet valve 207 for a sufficient period of time. The incubator outlet valve 207 is a boiling start valve similar to the mixing zone outlet valve 206. The liquid sensor 174 at the beginning of the incubation zone, coupled with the flow sensor 740 (see Figure 112) and the CMOS circuit 86, can cause incubation time delays. After sufficient incubation, the incubator outlet valve 207 will be activated and the flow will again flow along the MST incubation outlet channel 63 0 to the polymerase surface tension valve 140 (see Figure 109). The polymerase from reservoir 62 is mixed into the sample stream as it flows through the enthalpy path of amplification input channel 63 2 . Referring to Figures 108 and 109, the amplification input channel 63 2 directs the sample stream through 12 amplification mixing surface tension valves 138. The amplification mixture (see Figure 109) of each of the amplification mixing reservoirs 61.3-60.1 is passed through each of the top cap passages 94 to fix the meniscus at the amplifying mixing surface tension valve 138. The sample stream will alternately open the various surface tension valves, and the amplification mixture from each of the amplification mixtures stored in the sample stream is loaded into the sample stream and flows to 12 amplification chambers 112.1- 112.12 rooms. The LOC device has a CMOS circuit that operatively controls the amplification region through a temperature sensor and a heater.

Referring to Fig. 110, each of the 12 expansion chambers 112.1-112.12 has an expansion outlet valve 207. The expansion outlet valve 207 is a boiling start valve similar to the cultivation outlet valve 207. The sample stream will stop at each of the amplification outlet valves 207. After amplification, the amplification outlet valve 207 opens to allow the amplicon to flow into the hybrid chamber array 110.1-110.12 containing the probes, which are designed to interact with the target nucleic acid sequence (here the pathogen) DNA) forms a probe-target hybrid. The sample flows along flow path 176 through respective individual arrays 110.1-110.12 and through respective diffusion barrier inlets 175 into respective hybrid chambers 180 (see Figures 90 and 1 1 1).

Referring to Figures 91 and 111, when the sample stream reaches the end liquid sensor 178, the hybrid heater 182 is activated after a period of delay to promote probe-target hybrids. The flow rate sensor 740 (see Figure 112) is included in the pathogen incubation zone 114 to determine the length of time delay. After a suitable delay for hybridization, the excitation current applied to the ECL electrodes 860 and 870 (see Figures 111 and 114) causes the probe-target hybrid to emit light photons, which can be used in a substrate CMOS circuit. The light sensor 44 in 86 is detected. The photosensor consists of an array of photodiodes 184 in close proximity to each of the hybrid chambers.

Figures 113 and 114 show the calibration chamber 382. As explained elsewhere in this patent specification, the calibration chamber is used to calibrate the photodiode 184 to adjust system noise and background artifacts. Similarly, the forward ECL control probe 7 87 and the reverse ECL-118-201209406 control probe 786 are placed in some of the hybrid chambers 180 for verification quality control. A variation of the calibration chamber 382 having finger-crossing ECL electrodes is shown in Fig. 92. The humidifier 196 and the humidity sensor 2 32 are used to control evaporation in the LOC device 785 (especially the hybrid chamber array 11). And condensation. Fig. 110 shows the main components of the humidifier 196, the water reservoir 188, and the evaporator 190.

Referring to Figure 109, an evaporation-based telltale 189 can indicate whether the packaging of the device has been damaged during storage and whether the integrity and reliability of the microfluidic device are insufficient. During manufacture, a small drop of liquid is added to the liquid sensor 174 in the center of the evaporative alarm 189. If the package seal is damaged during storage, the drop will evaporate. The presence or absence of the liquid can be detected by the liquid sensor 1 74 and thereby indicating the integrity of the microfluidic device seal. Conclusion The devices, systems, and methods described herein enable people to perform molecular diagnostic tests at a low cost and at a low cost. The systems and components thereof described above are purely display and can be easily understood by those skilled in the art. Numerous variations and modifications of the spirit and scope of the broad inventive concept of the present invention. [Brief Description of the Drawings] Preferred embodiments of the present invention will now be described by way of example with reference to the following drawings, in which: -119- 201209406 Figure 1 shows the test module and test module reader using the fluorescence detection: Figure 2 is a schematic view of the electronic components in the test module using the fluorescence detection; Figure 3 is the test module. Schematic view of the electronic components in the reader » Figure 4 is a schematic representation of the overall structure of the LOC device;

Figure 5 is a perspective view of the LOC device; Figure 6 is a plan view of the LOC device and the features and structures of all the layers laminated to each other; Figure 7 is a plan view of the LOC device showing the top cover structure separately; The top view of the top cover is a top view and the internal passage and the reservoir are indicated by broken lines; FIG. 9 is an exploded top perspective view of the top cover and the internal passage and the reservoir are indicated by broken lines;

Figure 1 is a bottom perspective view showing the channel configuration above the top cover: Figure 11 is a plan view showing the LOC device showing the structure of the CMOS + MST device separately; Figure 12 is a schematic view of the LOC device at the sample inlet. Fig. 13 is an enlarged view of the illustration AA shown in Fig. 6; Fig. 14 is an enlarged view of the illustration AB shown in Fig. 6; Fig. 15 is an enlarged view of the illustration AE shown in Fig. Figure 16 is a partial perspective view showing the layered structure of the LOC device in the illustration AE; -120-2012〇94〇6

To display the LOC device in the layered structure in the inset AE, the LOC device is shown in the portion of the layered structure in the inset AE of the LOC device, and the LOC device is displayed in the layered structure in the inset AE. A schematic cross-sectional view of the cytosol reservoir shown in Fig. 21 of the layered structure of the LOC device in Fig. AE is shown in Fig. AE; 箄23 tei H _舄 shows that the L〇C device is part of the layered structure in the illustration AB from the fins: ^ 2 4 fei ~ 松丄_舄 shows the part of the layered structure of the L0C device in the illustration AB

The first geese _ ; the first three-dimensional _ ; the second _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The 26th taog of the Ministry... _舄 shows that the L0C device is in the layered structure in the illustration AB, the 27th-like part + _ is the part of the layered structure showing the LOC device in the illustration AB from the fins; 2 隐 隐 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Figure 30 is a schematic cross-sectional view of the amplification mixture reservoir and polymerase reservoir; Figure 31 shows the features of a boiling starter valve separately; Figure 32 is the line 33 of the boiling start valve shown from Figure 31. 3 is a schematic sectional view cut at 3: Fig. 33 is an enlarged view of the illustration AF shown in Fig. 15;

Figure 34 is a schematic cross-sectional view of the upstream end of the dialysis zone cut from line 35-3 5 shown in Figure 33; Figure 35 is an enlarged view of the illustration AC shown in Figure 6: Figure 36 is an illustration AC Further enlarged view of the amplification region: Figure 37 is a further enlarged view of the amplification region in the illustration AC; Figure 38 is a further enlarged view of the amplification region in the illustration AC; Figure 39 is the 38th image. Illustration of AK inside further enlarged view

Figure 40 is a further enlarged view of the amplification chamber in the illustration AC; Figure 41 is a further enlarged view of the amplification region in the illustration AC: Figure 42 is a further enlarged view of the amplification chamber in the illustration AC; The figure is a further enlarged view of the inside of the illustration AL shown in Fig. 42; Fig. 44 is a further enlarged view of the enlarged area in the illustration AC: Fig. 45 is a further enlarged view of the inside of the illustration AM shown in Fig. 44. A further enlarged view of the amplification chamber in the illustration AC; Fig. 47 is a further enlarged view of the inside of the illustration AN shown in Fig. 46 - 122 - 201209406 Fig. 48 is a further enlarged view of the amplification chamber in the illustration AC; Figure 49 is a further enlarged view of the amplification chamber in the illustration AC; Figure 50 is a further enlarged view of the amplification region in the illustration AC; Figure 51 is a schematic cross-sectional view of the amplification region; Figure 52 is the hybrid Magnified plan view of the joint area; Figure 53 is a plan view of the further enlargement of the two hybrid chambers.

Figure 54 is a schematic cross-sectional view of a single hybrid chamber; Figure 55 is an enlarged view of the humidifier in the illustration AG shown in Figure 6; Figure 56 is an enlarged view of the illustration AD shown in Figure 52; The figure is an exploded perspective view of the LOC device illustration AD; Fig. 58 is an enlarged view of the humidity sensor shown in the illustration AH of Fig. 6, and Fig. 59 is a photodiode array showing the photosensor Schematic diagram of the components, Fig. 60 is an enlarged view of the evaporator shown in the illustration AP of Fig. 55; Fig. 61 is a diagram of the linker-headed PCRClinker-primed PCR. Fig. 62 is a lancet A schematic representation of the test module; Figure 63 is a graphical representation of the overall structure of the LOC variant VII; Figure 64 is a plan view of the LOC variant VIII and the features and structures of all of the layers laminated to each other; The figure is an enlarged view of the illustration CA shown in Fig. 64; Fig. 66 is a partial perspective view of the layer -123-201209406-like structure in the LOC variant VIII inset CA shown in Fig. 64; Fig. 67 is shown in the figure Figure 65 is a magnified view of the illustration CE; Figure 68 is a graphical representation of the overall structure of the LOC variant VIII Figure 69 is a schematic diagram of the overall structure of the LOC variant XIV; Figure 70 is a schematic diagram of the overall structure of the LOC variant XLI; Figure 71 is a schematic diagram of the overall structure of the LOC variant XLIII: Figure 72 is a LOC variant XLIV Schematic diagram of the overall structure; Figure 73 is a schematic diagram of the overall structure of the LOC variant XLVII; Figure 74 is a graphical representation of the overall structure of the LOC variant XI; Figure 75 is a circuit diagram of the differential imager

I Figure 76 shows the reactions that occur during electrochemiluminescence (ECL); Figure 7 shows schematically three different cathode configurations; Figure 78 shows a partial cross-sectional view of the cathode and anode of the hybrid chamber; Figure 79 Schematically showing the annular cathode near the edge of the photodiode; Fig. 80 schematically showing the annular cathode in the edge of the photodiode; Fig. 8 is a schematic view showing the cathode having a series of fingers to increase the length of the side edge; Figure 82 is a schematic illustration of the use of a transparent cathode to optimize surface area coupling and ECL signal detection: Figure 83 schematically shows the surface area coupling and ECL signal detection optimization fixed to the cathode of the top of the hybrid chamber. The use of Fig. 84 schematically shows the cathode which is in contact with the anode finger; Fig. 85 shows a test module and test module using ECL detection - 124-201209406; and Fig. 86 shows the detection using ECL A schematic diagram of the electronic components in the test module; Figure 8 shows a test module and another test module reader; Figure 8 shows a test module and test module reader and storage Host system of different databases System

Figure 89 is a plan view showing all of the feature members stacked on each other and the LOC variation type L showing the positions of the insets GA to GL; Fig. 90 is an enlarged view of the illustration GD shown in Fig. 89; An enlarged view of the illustrated illustration GG; Fig. 92 is an enlarged view of the illustration GH shown in Fig. 89, and Fig. 93 is an electrochemiluminescence resonance energy transfer probe in a closed configuration

Figure 94 is a diagram of an electrochemiluminescence resonance energy transfer probe in an open and hybrid configuration;

Figure 95 is a diagram of the primer-linked luminescence linear probe during initial re-amplification; Figure 96 is a diagram of the primer-linked luminescence linear probe during the subsequent amplification cycle; Figure 97A to 97F diagram A thermal cycle is shown in which a light-emitting primer is coupled to the stem-loop probe; Figure 98 is a schematic representation of a light-emitting probe in the reverse control of the stem-loop configuration; Figure 99 is a schematic representation of Figure 98 in an open configuration. Reverse control sends a -125-201209406 optical probe; Figure 100 shows schematically a positively controlled illuminating probe in a stem-ring configuration; Figure 101 shows schematically the positive 00th aspect of the open configuration To control the illuminating probe;

Figure 102 is a graphical representation of the overall structure of a microfluidic device having a dialysis device, a LOC device, an interconnected cap, and an electrochemiluminescence (ECL) detection; Figure 103 is a diagram showing a dialysis device, a LOC device, and FIG. 104 is a plan view showing a characteristic member of the dialysis device of the microfluidic device and a JA position of the illustration; FIG. 105 is an enlarged view of the illustration JA shown in FIG. Figure 106 is a plan view showing the features of the LOC device of the microfluidic device and the positions of the display illustrations JB to JJ;

Figure 107 is an enlarged view of the illustration JB shown in Figure 106; Figure 108 is an enlarged view of the illustration JC shown in Figure 106; Figure 109 is an enlarged view of the illustration JD shown in Figure 106; The figure is an enlarged view of the illustration JE shown in Fig. 106: Fig. 111 is an enlarged view of the illustration JF shown in Fig. 106; Fig. 112 is an enlarged view of the illustration JG shown in Fig. 1; The figure is an enlarged view of the illustration shown in Figure 1-6; Figure 114 is an enlarged view of the illustration shown in Figure 1-6; Figure 115 is an enlarged view of the hybrid chamber of the LOC variant L; -126 - 201209406 Figure 16 is a magnified view of the LOC variant L hybrid chamber array and showing the distribution of the calibration chamber; [Key component symbol description]

1 〇: Test Module 1 1 : Test Module 1 2 : Test Module Reader 1 3 : Case 14 : Micro-USB Plug 15 : Inductor 1 6 : Micro-USB 埠 17: User Interface (UI) Touch Screen 1 8 : Display Screen 1 9 : Button 20 : Start Button 21 : Honeycomb Radio 22 : Aseptic Sealing Tape 2 3 : Wireless Network Connection 24 : Large Storage Tank 25 : Satellite Navigation System

26 : LED 27 : Data Memory 28 : Mobile Phone / Smartphone 29 : USB-compatible LED Driver -127- 201209406 30 : LOC Device 3 1 : Power Conditioner 3 2 : Power Supply Capacitor 33 : Clock 3 4 : Control 35: register 36: USB device driver 3 7 : driver

38 : RAM 39 : ( ECL activated ) drive 40 : program and data flash memory 41 : register 42 : processor 4 3 : program memory 44 : light sensor 4 5 : indicator 46 : top cover 47 : USB power only / indicator module 48 : CMOS + MST chip (device) 49 : Foam illustration or other perforated element 5 1 : Interconnected top cover 52 : Intermediate MST channel 5 4 : (anticoagulant Reservoir 5 6 : (cytolytic reagent) reservoir -128 - 201209406 57 : printed circuit board (PCB) 5 8 : reservoir 60 : reservoir 62 : reservoir 64 : lower seal 66 : top wall layer 6 8 : Sample inlet

7 〇: dialysis zone 72: waste channel 74: target channel 76: waste unit (waste storage, waste storage tank) 7 8 : reservoir layer 80: roof channel layer 8 2 : upper seal layer 84:矽 Substrate 86: CMOS circuit 87: MS, 8 8 '·passivation layer 90: MST channel 92: downcomer 94: top cover channel 96: upper conduit 97: wall region 98: meniscus anchor-129- 201209406 1 00 : MST channel layer 106: Boiling-start valve 107: e-book reader 108: boiling-start valve 109: tablet

1 1 〇 : Hybrid chamber array 1 1 I : Epidemiological data 1 1 2 : Amplification area 1 1 3 : Genetic data 1 1 4 : Cultivation area

115: Electronic Health Record (HER) 1 1 6 : Anticoagulant 1 1 7 : Surface tension valve 1 1 8 : Surface tension valve 1 1 9 : Sample flow 1 2 0 : Meniscus 1 2 1 : Electronic medical record (EMR) 122: vent hole 123: personal health record (PHR) 125: network 126: boiling-start valve 1 28: surface tension valve 1 3 0 : chemical cytolysis zone 1 3 1 : mixing zone -130- 201209406 132: Surface tension valve 1 3 3 : incubator inlet passage 134: downcomer (opening)

1 3 6 : Window 140 : Surface tension valve 146 : Valve inlet 1 4 8 : Valve outlet 1 5 0 : Valve down conduit 152 : Heater 153 : Boiling - Start valve heater contact 154 : Heater 1 5 6 : Heating Connector 158: microchannel 160: amplification zone outlet channel 164: aperture 168: dialysis upper catheter aperture 170: temperature sensor 174: liquid sensor 175: diffusion barrier 176: flow path 178: liquid sensor 180 : Hybrid chamber 1 8 2 : Heater 184 : Light diode -131 - 201209406 1 8 5 : Activation zone 186: FRET probe 1 8 8 : Water reservoir 189 : Alarm device

190: Evaporator 1 9 1 : Heater 192 : Water supply channel 1 93 : Upper pipe 1 94 : Down pipe 1 9 5 : Metal top layer 196 : Humidifier

1 9 8 : First upper conduit hole 202 : CIF 204 : Dialysis MST channel 206 : Mixing zone outlet valve

207: incubation zone outlet valve 208: top cover channel liquid sensor 210: microchannel 212: intermediate MST channel 2 1 8 : T i A1 electrode 2 2 0 : T i A1 electrode 2 2 2 : gap 2 3 2 : humidity Sensor 2 3 4 : Heater-132-201209406 237: ECL probe 23 8 : Target nucleic acid sequence 240: Ring 242: Stem 2 4 8 : Quenching 288: Sample placement and preparation 290: Nucleic acid extraction

291: Nucleic Acid Breeding 292: Nucleic Acid Amplification 294: Detection and Analysis 2 9 6 : First Electrode 298: Second Electrode 3 00: Delay before Stylization 301: LOC Device 3 2 8 : White Blood Cell Dialysis Zone 3 76 : Conductive Tube Column 3 7 8 : Forward control probe 3 8 0 : Reverse control probe 3 82 : Calibration chamber 3 90 : Lancet 3 9 2 : Lancet release button 408 : Film seal 410 : Mask cover

492 : LOC variant VII 201209406 518 : LOC variant VIII 5 9 4 : interface layer 5 96 : interface channel 5 98 : interface channel 600 : bypass channel 602 : interface target channel 604 : interface waste (abandoned cells) 606 : Interface channel 608: Interface channel 630: Cultivate exit channel 63 2 : Amplification input channel

641 : LOC XIV

673 : LOC variant XLIII 674 : LOC variant CLIV 677 : LOC variant XLVn 6 8 2 : dialysis zone 6 86 : pre-amplification dialysis step 693 : linear ECL probe 694 : amplification 6 96 : primer-linking probe 6 9 8 : Amplified complementary sequence 700 : Oligonucleotide primer 7 05 : ECL probe 706 : Complementary sequence channel

-134- 201209406 708 : Stem strand 710 : Another strand 729 : LOC variant 740 : Flow sensor 766 : Waste reservoir 783 : Microfluidic device 784 : Dialysis unit

785 : LOC device 786 : reverse control ECL probe 7 87 : forward control ECL probe 788 : differential imager circuit 7 9 0 : pixel 792 : "virtual" pixel 7 9 4 : "read column" 795 : " Read column d" 797: M4 transistor 801: MD4 transistor 803: pixel capacitor 805: virtual pixel capacitor 8 07: switch 8 0 9 : switch 811: "read row" switch 813: virtual "read row" switch 815: Capacitor Amplifier - 135 201209406 8 1 7 : Differential Signal 846 : Luminous Cluster

860: ECL excitation electrode (cathode) 8 62 : ECL emission (ECL signal) 864 : Luminous group 8 66 : Co-reactant 8 68 : Excited species 8 70 : ECL excitation electrode (anode) 8 7 2 : Solution 8 74 : ECL battery 8 7 6 : Dielectric gap 878: comb structure cathode 8 8 0 : parallel finger 8 82 : 蜿蜒 configuration 8 8 6 : more complex configuration 8 8 8 : fine scalloped area 8 9 0 : branch Structure 8 92: Participation volume 8 9 4: Interrupted area -136-

Claims (1)

201209406 VII. Patent application scope: 1. A test module for concentrating a pathogen in a biological sample, the test module comprising: an outer casing having a storage tank for accommodating the sample; and a fluid connection with the storage tank (fluid Communication) and configured as a dialysis device capable of separating the pathogen from other components in the sample; A on-wafer laboratory (LOC) device in fluid communication with the dialysis device and configured to analyze the pathogens. 2. The test module of claim 1, wherein the dialysis device has a first passage, a second passage, and a plurality of small holes for receiving the biological sample, the second passage passing through the small passage The aperture is fluidly coupled to the first passageway such that the pathogen can flow from the first passage to the second passage and the larger component of the biological sample remains in the first passage. 3. The test module of claim 2, wherein the dialysis device further has a series of adjacent channels extending between the first channel and the second channel, wherein the small holes are located at an upstream end of the adjacent channels Each adjacent channel is configured to fix a sample meniscus to block capillary flow between the first channel and the second channel; and a bypass channel between the first channel and the second channel, the side The passageway merges into a second passage upstream of the adjoining passageway and is configured to provide an uninterrupted capillary drive flow from the first passage to the second passage; wherein during use, after the meniscus is formed, from the bypass passage When the liquid flow reaches the meniscus fixed to each of the adjacent passages, the liquid flow sequentially removes each meniscus, and the sample flow passes through the adjacent passages and the -137-201209406 bypass passage from the first One channel flows to the second channel. 4. The test module of claim 3, wherein the second channel is for a target channel and the first channel is for a waste channel, the target channel being configured to drive the flow with capillary Flow to the L Ο C device. 5. The test module of claim 4, wherein the pathogen contains a target nucleic acid sequence, and the LOC device has a nucleic acid amplification region to amplify the target nucleic acid sequences. 6) The test module of claim 5, wherein the l 0 C device has a cytosolic region to lyse the pathogens to release the target nucleic acid sequence therein. 7. Test as in claim 5 A module wherein the LOC device has a hybrid region with a hybrid probe array that hybridizes to a target nucleic acid sequence to form a probe-target hybrid. 8 _ As in the test module of claim 7, wherein the probe is designed to form a probe-target hybrid with the target nucleic acid sequence, the probe-target hybrid is designed to be capable of exciting current A photon that produces a reaction that emits light. 9. The test module of claim 8 wherein the LOC device has a complementary metal oxide semiconductor (CMOS) circuit for operative control of a polymerase chain reaction (PCR) region, the CMOS circuit having a photosensor To sense the photon emitted by the probe-target hybrid. The test module of claim 9, wherein the hybrid region has an array of hybrid chambers containing probes that are hybridizable to the target nucleic acid sequence. -138- 201209406 11. The test module of claim 1, wherein the photosensor is an array of photodiodes, and the photodiodes are respectively adjacent to the hybrid chambers. 12. The test module of claim 11, wherein the CMOS circuit has a digital body for storing fluid processing related information, the probe includes a detailed description of the probe and each probe is in the hybrid chamber. Array position 13. The test module of claim 12, wherein the CMOS circuit has at least one temperature sensor to sense the temperature of the hybrid chamber array. 14. The test module of claim 13, wherein the LOC device has a CMOS circuit controlled heater that uses feedback from a temperature sensor to maintain the probe and the target nucleic acid sequence in a hybrid Below the temperature. 15. The test module of claim 14 wherein the photodiodes are less than 1 600 microns from the corresponding hybrid chamber. 16. The test module of claim 15 wherein the probe has an electrochemiluminescent (ECL) illuminant that emits photons in an excited state. 17. The test module of claim 16, wherein the hybrid chambers have electrodes to excite the ECL luminophores with electrical current. . 1 8 . The test module of claim 17 , wherein each ECL probe has a luminophore and a quenching substance adjacent to the luminophore for quenching photons emitted by the luminophore, when the probe Hybridization with a target nucleic acid sequence moves the quencher away from the luminophore such that the photons are no longer quenched. -139- 201209406 1 9 · As in the test module of claim 18, the circuit has bond-pads for electronic connection, which is configured to convert the output from the photodiode into a target The nucleic acid sequence is hybridized with an indicator signal and the bond pads are transported to an external device. 20. The test module of claim 13 wherein the dialysis device is fluidly connected through a cap, and the channel is in fluid communication with the dialysis device and the L0C device to accommodate the storage of the liquid reagent added to the sample. Device. Wherein the CMOS external device is provided with the ECL probe signal provided to the LOC top cover having at least a plurality of -140-