TW201219770A - Test module incorporating spectrometer - Google Patents

Abstract

A test module having an outer casing having a receptacle for receiving a biological sample, an excitation source mounted in the outer casing for sequentially illuminating the biological sample with different wavelengths of light, and, a photosensor positioned to detect the different wavelengths of light transmitted through the biological sample, wherein during use, the photosensor output signal is used to generate a spectrogram for analysis of a characteristic of the biological sample.

Description

201219770 VI. Description of the Invention [Technical Field of the Invention] The present invention relates to a diagnostic apparatus using microsystem technology (MST). In particular, the present invention relates to the processing and analysis of microfluidics and biochemicals for molecular diagnostics. [Prior Art] Molecular diagnosis has been used to provide an area for early detection of disease before the onset of symptoms. Molecular diagnostic tests are used to detect: • genetic disorders • acquired diseases • infectious diseases • genes associated with health-related genetic factors due to high accuracy and rapid processing time, molecular diagnostic tests can reduce the incidence of ineffective health care, Improve patient outcomes, improve disease management, and individualized patient care. Many techniques for molecular diagnostics are based on the detection and identification of specific nucleic acids (both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) extracted and amplified from biological samples such as blood or saliva. The complementary nature of the nucleobase allows the short sequence (oligonucleotide) of the synthesized DNA to bind (hybridize) to a particular nucleic acid sequence for use in nucleic acid assays. If hybridization occurs, the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease that an individual will get in the future, to determine the type and pathogenicity of the infectious pathogen, or to determine the individual's response to the drug. 5 - 201219770 Nucleic acid-based molecular diagnostic test. Four separate steps: 1. Sample preparation 2. Nucleic acid extraction 3. Nucleic acid amplification (optional) 4. Detection of many sample types, such as blood, urine, sputum and tissue samples, for gene analysis. Diagnostic tests determine the type of sample required, as not all samples represent disease progression. These samples have various components, but usually only one of them is of interest. For example, in the blood, high concentrations of red blood cells can inhibit the detection of pathogenic microorganisms. Therefore, purification and/or concentration steps are typically required at the beginning of the nucleic acid assay. Blood is one of the most frequently requested sample types. It has three main components: white blood cells, red blood cells, and platelets. Platelets accelerate coagulation and maintain activity outside the body. To inhibit coagulation, the sample was mixed with a reagent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Red blood cells are typically removed from the sample to concentrate the target cells. In the human body, red blood cells account for about 99% of the cellular material, but they do not carry DNA because they do not have a nucleus. In addition, red blood cells contain components such as heme that may interfere with downstream nucleic acid amplification procedures (described below). Red blood cell removal can be accomplished by dissolving the solution differentially red blood cells, leaving the entire remaining cellular material, which can then be separated from the sample by centrifugation. This provides a concentrated target cell from which the nucleic acid is extracted. -6- 201219770 The exact procedure used to extract nucleic acids depends on the sample and the diagnostic analysis to be performed. For example, the protocol used to extract viral RNA is quite different from the protocol used to extract genomic DNA. However, extracting nucleic acids from a target cell typically involves a cell lysis step and subsequent nucleic acid purification. The cell lysis step ruptures the cell and nuclear membrane and releases the genetic material. This is often accomplished using a lyophilized detergent such as sodium lauryl sulfate, which also denatures a large amount of protein present in the cells. The nucleic acid is then purified by an alcohol precipitation step, which is usually carried out using ice ethanol or isopropanol, or via a solid phase purification step, which is usually carried out in a column of cerium oxide substrate, resin or high. The concentration is carried out on paramagnetic beads in the presence of a chaotropic salt, followed by washing of the nucleic acid, followed by elution with a buffer of low ionic strength. A selective step prior to precipitation of the nucleic acid is the addition of a protease which digests the protein to further purify the sample. Other lysis methods include mechanical lysis via ultrasonic vibration and heating of the sample to 94 °C to disrupt thermal lysis of the cell membrane. The target DNA or RNA can be present in the extracted material in very small amounts, especially if the target is from a pathogenic source. Nucleic acid amplification provides the ability to selectively amplify (i.e., replicate) a particular target in a low concentration to a detectable amount. The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). PCR is well known in the art, and a complete description of this type of reaction is provided by E. van Pelt-Verkuil et al. Principles and

Technical Aspects of PCR Amplification, Springer, 201219770 2008 ° PCR is a useful technique for amplifying target DNA sequences in the context of complex DNA. If RNA is to be amplified (by PCR), RNA must first be transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Subsequently, the obtained cDNA was amplified by PCR. PCR is an exponential method, as long as the conditions for maintaining the reaction are acceptable, the components of the PCR reaction are:

1. A short single strand DN A of a nucleotide having a complement of about 10 to 30 nucleotides complementary to the region of the flanking standard.  DNA polymerase-synthesis of DNA thermostable enzymes 3.  Deoxyribonucleoside triphosphate (dNTP) - provides nucleotides that are incorporated into newly synthesized DNA strands.  Buffer one provides the ideal chemical environment for DNA synthesis. PCR typically involves placing these reactants in a vial containing from the extracted nucleic acid (approximately 1 Torr to 50 microliters). The tube is placed in a thermal cycler; this reactor is an instrument that allows the reaction to take unequal time at a range of different temperatures. The standard procedures for each thermal cycle involve the variable phase, the annealed phase, and the extended phase. The extension phase is sometimes referred to as the primer extension phase. In addition to these three-step procedures, a two-step thermal procedure can also be used in which the annealing and extension phases are combined. The denatured phase generally involves raising the reaction temperature to 90 to 95 °C to denature the DNA strand; in the annealed phase, the temperature is lowered to 50 to 60 °C to bond the primer; then in the extended phase, the temperature is raised The optimal DNA polymerase activity temperature is 60 to 72 ° C for primer extension. This process is repeated about 20 to 40 times, and the end result is the production of -8 - 201219770 of millions of sets of target sequence copies between primers. Variants of many standard PCR protocols for molecular diagnostics have been developed, including, for example, multi-primer PCR, linker-primed PCR, direct PCR 'tandein PCR, real-time PCR, and reverse transcriptase PCR. Multiple primer set PCR uses multiple primer sets in a single PCR mix to generate different sizes of amplicons specific for different DNA sequences. Additional information can be obtained from a single trial by targeting (targeting) multiple genes at once (in other ways, several trials are required). Optimization of multi-initiator PCR is difficult because it requires the selection of primers with approximate binding temperatures and amplicon of approximate length and base composition to ensure equal amplification efficiency of each amplicon. Linker-primed PCR, also known as ligation adaptor PCR ' is a type of primer that does not require a target-specific primer to amplify nucleic acids of virtually all DNA sequences in a complex DNA mixture. The method. This method first digests the target DNA population with an appropriate restriction endonuclease (enzyme). A double stranded oligonucleotide linker (also referred to as a conjugate) having a suitable overhang is then ligated to the end of the target DNA fragment using a ligase. Nucleic acid amplification is next carried out using an oligonucleotide primer specific for the linker sequence. Thereby, all DNA-derived fragments adjacent to the linker oligonucleotide can be amplified. Direct PCR describes a system that performs PCR directly on a sample without requiring any (or minimal) nucleic acid extraction. It has long been believed that many components present in unpurified biological samples, such as pro-heme components in the blood, inhibit the PCR reaction. Therefore, it is customary to enhance the purification of the target nucleic acid prior to preparation of the PCR reaction mixture. However, with appropriate changes in chemical properties and sample concentrations, it is possible to perform minimal PCR or PCR by direct PCR. Modification of PCR chemistries for direct PCR involves increasing buffer strength, using highly active and processable polymerases and additives that chelate with potential polymerase inhibitors. Repeated sequence PCR utilizes two separate nucleic acid amplification cycles to increase the probability of amplifying the correct amplicon. One type of repetitive PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in different nucleic acid amplification cycles. The first pair of primers hybridize to a nucleic acid sequence located in a region other than the target nucleic acid sequence. A second pair of primers (nested primers) is used for the second amplification, the pair of primers being incorporated within the first PCR product to produce a second PCR product comprising the target nucleic acid, and the second product is short. The rationale used in this strategy is that if the wrong locus is amplified due to an error during the first nucleic acid amplification, the probability that the wrong locus is amplified again by the second pair of primers is very low, thus ensuring specificity. Real-time PCR or quantitative PCR was used to measure the amount of PCR products in real time. The initial amount of nucleic acid in the sample can be determined by using a probe containing a fluorophore or a fluorescent dye and a set of standards in the reaction. This is especially useful for molecular diagnostics where treatment options may vary depending on the amount of pathogen in the sample. Reverse transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. The reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used to express the expression -10- 201219770 expression pro filing to determine the expression of a gene or to recognize the sequence of an rna transcript, including transcription initiation and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus. The isothermal amplification is another form of nucleic acid amplification that does not rely on the thermal denaturation of the target DN A during the amplification reaction, thus eliminating the need for sophisticated instruments. Therefore, the thermostatic nucleic acid amplification method can be carried out in a field place or simply in an environment other than the laboratory. Some thermostatic nucleic acid amplification methods have been described, including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Expansion Recombinase Polymerase Amplification, Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent I so th e rm al DN AA mp 1 ificati ο n) and Loop-Mediated Isothermal Amplification. Constant-temperature nucleic acid amplification does not rely on continuous heating to denature template DNA to produce a single-stranded molecule as a template for continued amplification, but to generate single-stranded molecules using other methods at a constant temperature, such as DNA molecules by specific restriction of endonucleases Enzyme cleavage, or the use of enzymes to separate DNA double strands. The ability of strand-substituted amplification (SDA) to rely on specific restriction enzymes to cleave unmodified strands of hemi·modified DNA, and the ability of polymerases to extend and replace downstream stocks lacking 5'-3' exonuclease activity . An exponential nucleic acid -11 - 201219770 amplification is then achieved by coupling sense and antisense, wherein the stock from the sense reaction replaces the template for the antisense reaction. The nicking enzyme used in this reaction does not cut DNA in a conventional manner, but instead produces an incision in one of the strands of DNA, such as N.  Alwl, N.  BstNBl and M ly 1. SDA is improved by using a combination of a thermostable restriction enzyme (Aval) and a thermostable external polymerase (Bst polymerase). This combination has been shown to increase the amplification efficiency of the reaction from 1 〇 8 fold amplification to 10 1 倍 amplification, so it is possible to use this technique to amplify unique single copy molecules. Transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA) RNA polymerase is used to replicate RNA sequences rather than corresponding genomic DNA. This technique uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase, and selective RNase® (if reverse transcriptase does not have Rnase activity). One of the primers contains a promoter sequence of RN A polymerase. In the first step of nucleic acid amplification, the primer hybridizes to a target ribosomal RNA (rRNA) at a defined site. The reverse transcriptase then extends from the 3' end of the promoter primer to produce a DNA copy of the target rRNA. The RNA in the formed RNA:DNA dimer is decomposed by the RNase activity (if any) of the reverse transcriptase or additional RNase. In the next step, the second primer binds to the DNA copy. The new DNA strand is synthesized by the reverse transcriptase from the end of this primer to produce a double-stranded DNA molecule. RNA polymerase recognizes the promoter sequence in the DNΑ template and initiates transcription. Each newly synthesized RNA amplicon is re-entered as a template for a new replication cycle. In recombinase polymerase amplification (RPA), isothermal amplification of a specific DNA fragment is achieved by binding an oppositely directed oligonucleotide primer to the template DNA and then extending the primers by DNa polymerase. . -12- 201219770 The denaturation of the double-stranded DNA (dsDNA) template does not require heating. Instead, RPA uses a recombinase-primer complex to scan dsDNA to facilitate strand exchange at the homologous site. The resulting structure is stabilized by the interaction of a single strand of DNA binding protein with the substituted template strand, thereby preventing the primer from exiting via branch migration. The enzymatic dissociation of the pediatric group allows the stranded DNA polymerase (such as a large fragment of Bacillus subtilis Pol I (Bsu) to be accessed to the 3' end of the oligonucleotide, followed by primer extension. The amplification of the index nucleic acid is accomplished by repeated cycles of this process. Helicase-dependent amplification (HDA) mimics in vivo systems. Wherein DNA helicase is used to generate a single strand template for hybridization of the primer, followed by extension of the primer by DNA polymerase. In the first step of the HDA reaction, the de-enzyme is passed along the target DNA to interrupt the hydrogen bond between the two strands, which in turn binds to the single-stranded binding protein. The primers are bonded by exposing the single-strand target region by helicase. The DNA polymerase then uses the free deoxyribonucleoside triphosphate (dNTP) to extend the 3' end of each primer to make two DNA replicas. The two dsDNA replication strands each enter the next HDA cycle, resulting in amplification of the exponential nucleic acid of the target sequence. Other DNA-based thermostating techniques include rolling circle amplification (RCA), in which a DNA polymerase extends the primer around a circular DNA template to produce a long DNA product consisting of many repeating copies of the loop. Prior to the end of the reaction, the polymerase produced tens of thousands of copies of the circular template and the copies of the strands were tethered to the original target DNA. This approach allows for spatial dissociation of the target and rapid nucleic acid amplification of the signal. A copy of 1 to 12 templates can be generated in one hour. A variant of the branched-type amplification system, RCA, which enables the use of a closed circular probe (C-probe) or a padlock probe and a DNA polymerase with high processivity at a constant temperature for 13-201219770 The C_probe was amplified exponentially. Circular thermostat amplification (LAMP) provides high selectivity using DNA polymerase and a set of four specially designed primers that recognize a total of six different sequences on the target DN A. The primers within the sense and antisense strand sequences containing the target DNA initiate LAMP. The strands initiated by the exogenous primers then replace the DNA synthesis to release a single strand of DNA. The single-stranded DNA can serve as a template for DNA synthesis initiated by a second primer and an external primer, and the second primer and the foreign primer are hybridized to the other end of the target, and the DNA is synthesized to produce stem-loop DNA. structure. In the subsequent LAMP cycle, an internal primer hybridizes to the loop on the product and initiates the replacement DNA synthesis, producing the original stem-loop DNA and the new stem-loop DNA with twice as long stems. The cycle reaction continued to accumulate 109 copies of the target within one hour. The final product is a stem-loop DNA having a plurality of inverted repeats of the target and a cauliflower-like structure. The plurality of loops in the cauliflower-like structure are formed by mutual overlapping of the target repeats in the same strand. . After completion of nucleic acid amplification, the amplification product must be analyzed to determine whether or not the expected amplicon (amplification amount of the target nucleic acid) is produced. The method of analyzing the product can be carried out by simply measuring the size of the amplicon by gel electrophoresis to the use of DNA hybridization to identify the nucleotide composition of the amplicon. Gel electrophoresis is the easiest way to check whether a nucleic acid amplification method produces the desired amplicon. Gel electrophoresis utilizes an electric field applied to a gel matrix to separate DNA fragments. Negatively charged DNA fragments will move in the matrix at different speeds "14- 201219770, depending on the fragment size. After the electrophoresis is completed, the fragments in the gel are stained to visualize them. Ethyl bromide is commonly used as a dye which exhibits fluorescence under ultraviolet light. The size of the fragments is determined by comparison with DNA ladders, which contain DNA fragments of known size and The gel was incubated with the amplicons side by side. Since the oligonucleotide primer binds to a specific site adjacent to the target DNA, the size of the amplification product can be predicted and detected as a band of known size on the gel. In order to determine the correctness of the amplicon, or to generate a plurality of amplicons, a DNA probe that hybridizes to the amplicon is usually employed. DNA hybridization refers to the formation of double stranded DNA by complementary base pairing. DNA hybridization of about 20 nucleotides in length is required for DNA hybridization to clearly identify a particular amplification product. If the probe has a sequence complementary to the amplicon (target) DN A sequence, hybridization will occur at the appropriate temperature, pH and ion concentration. If hybridization occurs, the gene or DNA sequence of interest is present in the original sample. Optical detection systems are most commonly used to detect hybridization methods. One of the amplicon and probe is labeled with a fluorescent agent or an electrochemiluminescent agent to emit light. These methods differ in that they cause the photo-generating group to produce an excited state, but both can be used to covalently label nucleotide strands. In the case of electrochemiluminescence (ECL), the light system is generated by current stimulating luminescent group molecules or complexes. In the case of fluorescence, it is irradiated with excitation light to cause light to be emitted. The fluorescent system is detected by a light source and a detection unit that provides excitation light of a wavelength absorbed by the fluorescent molecule. The detection unit includes a -15-201219770 light sensor that detects a transmitted signal (such as a photomultiplier tube or a charge coupled device (CCD) array) and a device that prevents excitation light from being included in the output of the light sensor (such as Wavelength selection filter). The fluorescent molecules emit Stokes shifted light in response to the excitation light, which is collected by the detection unit. The Stokes shift is the difference in frequency or wavelength between the emitted light and the absorbed excitation light. The ECL emission system is detected using a light sensor that is sensitive to the emission wavelength of the ECL species used. For example, transition metal-ligand complexes emit light of a visible wavelength, so conventional photodiodes and CCDs can be used as photosensors. One of the advantages of ECL is that if the ambient light is shielded, the ECL's emitted light is the only light in the detection system, thus increasing sensitivity. Microarrays allow hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are powerful molecular diagnostic tools that screen thousands of genetic diseases or detect the presence of numerous infectious pathogens in a single experiment. The microarray consists of a number of different DNA probes that are fixed at the point of acceptance. The target DNA (amplicon) is first labeled with fluorescent or luminescent molecules (either during nucleic acid amplification or after nucleic acid amplification), followed by administration of the target DNA to the probe microarray. The microarray is cultured in a temperature controlled, humid environment for hours or days to allow hybridization between the probe and the amplicon. After incubation, the microarray must be washed through a series of buffers to remove unbound strands. After cleaning, the surface of the microarray is dried with a gas stream (usually nitrogen). The rigor of hybridization and cleaning is critical. Insufficient stringency may result in highly non-specific binding. Too high a rigor may result in an inability to properly combine -16-201219770, resulting in reduced sensitivity. Hybridization is identified by detecting the light emitted by the labeled amplicon that forms a hybrid with the complementary probe. Fluorescent systems from microarrays are detected using a microarray scanner, which is typically a computer-controlled inverted scanning fluorescent conjugated focus microscope that typically uses laser-excited fluorescent dyes and photosensors (such as The photomultiplier tube or CCD) detects the emission signal. The fluorophore emits a Stokes bit shift (as described above) which is collected by the detection unit. The emitted fluorescent light must be collected, separated from the unabsorbed excitation wavelength and transmitted to the detector. In a microarray scanner, a conjugate focal configuration of a conjugate focal hole aperture mounted on the image side is typically used to eliminate out-of-focus information. This device allows only the light of the focused portion to be detected. Light from above and below the focal plane of the target cannot enter the detector, thus increasing the signal to noise ratio. The detected fluorescent photons are converted into electrical energy by a detector and then converted into a digital signal. This digital signal is translated into a number that represents the intensity of the fluorescence from a given pixel. Each feature of the array consists of one or more of these pixels. The final result of the scan is an image of the surface of the array. Since the exact sequence and position of each probe on the microarray is known, the target sequence to which it is hybridized can be simultaneously identified and analyzed. For more information on fluorescent probes please see: http://www. Premierbiosoft. Com/tech_notes/FRET_probe. Html and http://www. Invitrogen. Com/site/us/en/home/ References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-T ran sfer -FRET. Html -17- 201219770 In-situ Care (POINT-OF-CARE) Molecular Diagnostics Although molecular diagnostic tests offer many benefits, the growth of such tests in clinical laboratories is still slower than expected and is not the mainstream of laboratory medical testing. This is mainly because nucleic acid detection leads to higher complexity and cost than detection without nucleic acid methods. The extensive use of molecular diagnostic testing in clinical settings is closely related to the development of instrumentation, which must significantly reduce costs, provide rapid (automatic) analysis from initial (sample processing) to final (resulting), and does not require The operation of substantial human intervention. In-situ care technology can provide care in the physician's office, on the hospital bed side or even in a consumer-oriented home environment. This technology offers many advantages including: - Quick results, immediate treatment and improved care quality - from very A small number of sample tests to obtain laboratory data - reduce clinical workload - reduce laboratory workload and reduce administrative work to improve office efficiency - by reducing the number of hospital stays, outpatients can be diagnosed at the initial diagnosis and reduced sample processing , storage and delivery to improve the cost per patient - useful for clinical management decisions such as infection control and antibiotic use. On-wafer laboratory (LOC)-based molecular diagnostics-18- 201219770 Molecular diagnostics based on microfluidics The system provides a means to automate and accelerate molecular diagnostic analysis. The shorter detection time is primarily due to the fact that the required sample volume is less active, automated, and low-cost built-in cascaded diagnostic method steps within the microfluidic device. The volume of nanoliters and microliters also reduces reagent consumption and cost. The on-wafer laboratory (LOC) device is a common form of microfluidic device. The LOC device has an MST structure within the MST layer for integrating fluid processing onto a single support substrate (usually helium). The VLSI (Ultra Large Integrated Circuit) lithography technology manufactured by the semiconductor industry makes the unit cost of each LOC device very low. However, controlling the flow of fluid through the LOC device, adding reagents, controlling reaction conditions, and the like requires a large external hydroelectric engineering device. The connection of the LOC devices to these external devices greatly limits the molecular diagnostic use of the LOC devices in a laboratory environment. The cost of external instruments and their operational complexity excludes LOC-based molecular diagnostics as an option in a local care environment. In view of this, there is a need for a molecular diagnostic system based on a LOC device that can be used for local care. SUMMARY OF THE INVENTION Various aspects of the invention are now described by the following numbered paragraphs. GAS070. 1 This aspect of the invention provides a test module comprising: a housing having a container for receiving a biological sample; an excitation source disposed in the housing to continuously illuminate the biological sample with light of different wavelengths; -19- 201219770 Photosensor, where the photosensor is located at a different wavelength of light that can pass through the biological sample: where, when used, the signal output by the photosensor is used to generate an analysis of the organism The spectrum of the characteristics of the sample. GAS070. Preferably, the excitation source is an array of light emitting diodes (LEDs) that emit light of different wavelengths, and the LEDs are configured for continuous activation. GAS070. Preferably, the housing is configured for hand movement. GAS 070. Preferably, the test module also has a data connection to transmit the output signal of the photo sensor to the external device. GAS070. Preferably, the data connection is connected to an electrical connection of an external device, the test module being configured to electrically draw power from an external device. GAS070. Preferably, the electrical connection is a USB connector for insertion into a universal serial bus (USB) port of an external device. GAS070. Preferably, the test module also has a wafer-on-lab (LOC) device in communication with the container fluid, the LOC device having an array of chambers configured to charge the biological sample from the container by capillary drive. GAS070. Preferably, the photo sensor is inserted into the LOC device and located adjacent to the array of chambers. GAS070. Preferably, the LOC device has a support substrate and a CMOS circuit on the support substrate, the CMOS circuit having a photo sensor and a series of pads connected to the USB connector. •20- 201219770 GAS070.  1 较佳 Preferably, the CMOS circuit has an LED driver GAS 070 which controls the activation of the LED array via pads. Il photodiode array GAS 070. 1 2 LED array light window G AS070. 1 3 GAS070. Preferably, the photosensor is aligned with the array of chambers. Preferably, each chamber has an exposed biological sample. Preferably, the biological sample is blood. Preferably, the analyzed characteristic blood glucose comprises GAS 070. Preferably, the test module also has a shunt transistor between each photodiode anode and a voltage source, the shunt cell system being configured to remove the excitation light in the photodiode The carrier generated by the photon. GAS 070. Preferably, the shunt cell system is configured to activate upon deactivation of each LED. GAS 070. Preferably, the CMOS circuit has memory storage identification data for the external device to identify the test module. GAS 070. Preferably, the photodiode array is less than 249 microns from the hybridization chamber array. The easy-to-use, mass-produced, inexpensive and lightweight microfluidic test module accepts samples, analyzes the sample using an integrated image sensor with multiple discrete spectrometers, and multiple LED sources to provide electronic results at the output. - 21 - 201219770 [Embodiment] This summary describes the main components of the molecular diagnostic system in which the embodiment of the present invention is embodied. The full details of the system structure and operation are discussed in the following description. Referring to Figures 1, 2, 3, 98 and 99, the system has the following most important components: Test Modules 〇 and 1 1 are the size of a conventional U S B flash drive, which can be produced very cheaply. Test modules 10 and 1 each comprise a microfluidic device, typically in the form of a lab-on-lab (LOC) device 30, which preloads reagents and typically more than one probe for molecular diagnostic analysis ( See Figures 1 and 9 8). The test module 10 illustrated in Figure 1 uses a fluorescent substrate detection technique to identify the target molecule, whereas the test module 11 of Figure 98 uses an electrochemiluminescent substrate detection technique. The LOC device 30 has an integrated photosensor 44 (described in detail below) for fluorescence or electrochemiluminescence detection. Test modules 1 and 1 1 use standard micro-USB connector 14 for power supply, data and control. Both test modules have a printed circuit board (PCB) 57 and an external power supply capacitor 3 2 and an inductor 15 . Test Modules 1 and 1 1 are for single use only, and are mass-produced and distributed for use in sterile packaging for immediate use. The outer casing 13 has a large container 24 that accepts a biological sample and a removable sterile sealing tape 22, which preferably has a low viscosity adhesive to cover the large container prior to use. Membrane Seal with Membrane Guard 410 -22- 201219770 Part of the housing 13 is formed to reduce the humidity reduction in the test module while providing a pressure relief when the pressure changes. Membrane guard 4 10 protective film seal 4 0 8 is free from damage. The test module reader 1 2 supplies power to the test module 1 〇 or 1 1 via the micro-USB 埠 16. The test module reader 12 can be in many different forms, the choice of which is described later. The reader 1 2 version shown in Figures 1, 3 and 98 is an implementation of the smart phone. The block diagram of this reader 12 is shown in Figure 3. The processor 42 executes the application software from the program storage 43. The processor 42 is also interfaced with a display screen 18 and a user interface (UI) touch screen 17 and a button 19, a cellular radio 2 1 , a wireless network connection 23, and a satellite navigation system 25. Honeycomb radio 2 1 and wireless network connection 2 3 are used for communication. The satellite navigation system 25 is used to update the epidemiological database with location information. The location data can optionally be manually entered via touch screen 17 or button 19. The data store 27 stores gene and diagnostic information, test results, patient information, analysis and probe data for identifying each probe and its array position. The data store 27 and the program store 43 can be shared by a common memory device. The application software installed in the Test Module Reader 1 2 provides results analysis and other test and diagnostic information. To perform a diagnostic test, insert Test Module 1 (or Test Module 1 1) into Micro-U S B埠1 6 on Test Module Reader 12. Ripped back. The sterile sealing tape 22 and the biological sample (in liquid form) are loaded into the sample large container 24. Press the start button 20 to start the test via the application software. The sample flows into the LOC device 30 and the device is subjected to on-board analysis to extract the 'culture, amplification and pre-synthesized hybrid-reactive oligonucleoside-23-201219770 acid probe with the sample nucleic acid (target) ) hybridization. In the case of a test module 1 ( (which uses detection of a fluorescent substrate), the probes are fluorescently labeled and provide the necessary excitation light by LEDs 26 mounted in the housing 13 to induce hybridized probes. Fluorescent emission (see Figures 1 and 2). In the case of test module 1 (which uses electrochemiluminescence (ECL) detection), LOC device 30 is loaded with an ECL probe (as described above) and LED 26 is not required to produce luminescent emissions. The excitation current is actually provided by electrodes 860 and 870 (see Figure 99). The emission (fluorescence or illumination) is detected by a photosensor 44 integrated into the CMOS circuitry of each LOC device. The detection signal is amplified and converted to a digital output for analysis by the test module reader. The reader then displays the results. This information can be stored locally and/or uploaded to a network server containing patient records. The test module 10 or 1 1 is removed from the test module reader 1 2 and processed appropriately. Figure 1 '3 and 9 8 show the test module reader designed as a mobile phone/smartphone 2 8 . Other forms of test module readers can be laptop/notebook 101, dedicated reader 103, e-book reader 1〇7 'tablet 109 or desktop computer 1〇5 for hospitals, private clinics Or laboratory (see Figure 100). The reader can interface with additional applications such as patient records, accounting, online databases and multi-user environments. It can also interface with some local or remote peripherals, such as printers and patient smart cards. Referring to Figure 1 0 1 'The data generated by the test module 1 可 can be used to update the epidemiological database and genetic data stored in the epidemiological data host system through the reader 1 2 and the network 1 2 5 The electronic system stored in the host system 丨 3 3 - 201219770 gene database, electronic health record (EHR) host system 1 1 5 stored electronic health record, electronic medical record (EMR) host system 121 stored electronic Medical records, as well as personal health records stored in the Personal Health Record (PHR) host system. Conversely, the epidemiological data stored in the epidemiological data host system 1 1 1 , the genetic data stored in the genetic data host system 1 13 , and stored in the electronic health record (EHR) host system 1 15 An electronic health record, an electronic medical record stored in an electronic medical record (EMR) host system 121, and a personal health record stored in a personal health record (PHR) host system 123 can be used to access the network 125 and The reader 12 updates the digits in the LOC 30 of the test module 1〇. Referring to Figures 1, 2, 9 8 and 99, the reader 1 2 in the mobile phone configuration uses battery power. The mobile phone reader contains all pre-loaded test and diagnostic information. Data can also be downloaded or uploaded via some wireless or contact interface to enable communication with peripheral devices, computers or online servers. The Micro USB 埠1 6 is used to connect a computer or a primary power supply for charging the battery. Figure 70 shows an embodiment of a test module 10 for a positive or negative test result requiring only a particular target, such as for testing whether a person is infected with, for example, influenza A virus H1N1. Only the USB power/indicator module 47 built for a particular purpose is required. No other readers or application software is required. The indicator 45 on the USB power/indicator module 47 alone shows no positive or negative results. This configuration is ideal for a large number of inspections. Other items that may be provided by the system may include a test tube containing reagents for pre-treating a particular sample and a spatula and lancet for sample collection. Figure -25- 201219770 7〇 shows the implementation of the test module with the spring-loaded telescopic lancet 3 90 and the lancet release button 3 92 for convenience. Satellite phones can be used in remote areas. Test Module Electronics Figures 2 and 99 are block diagrams of the electronic components in test modules 10 and 11, respectively. The CMOS circuit integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB compatible LED driver 29, a clock 3 3, a power conditioner 3 1 , a RAM 3 8 and a program and data flash memory 40. These components provide a photosensor 44, a temperature sensor 170, a liquid sensor 1 74, and various heaters 152, 154, 182, 234 and associated drivers 37 and 39 for the entire test module 10 or 11. And the control and memory of the registers 35 and 41. Only LED 26 (with test module 10 as an example), external power capacitor 32 and micro USB connector 14 are external to LOC device 30. The LOC device 30 includes pads for connection to these external components. RAM 3 8 and program and data flash memory 40 have application software and diagnostic and test information for more than 1 000 probes (flash/guarantee storage, for example via encryption). In the test module 1 1 designed for ECL detection, the test module 1 1 does not contain the LED 26 (see Figures 98 and 99). The data is encrypted by the LOC device 30 for secure storage and secure communication with external devices. . The LOC device 30 is loaded with electrochemiluminescent probes each having a pair of ECL excitation electrodes 860 and 870. Many types of test modules 10 are manufactured in several test formats for stock use. The difference between the different test formats is the reagents and probes for on-board analysis (on -26 - 201219770 board assay). Some examples of infectious diseases that can be quickly identified using this system include: • Influenza-influenza virus A, B, C, infectious salmon virus (Isavirus), Tohogotovirus (phogotovirus) • Pneumonia-respiratory fusion Virus (RSV), adenovirus, interstitial pneumonia virus, pneumococci, staphylococcus aureus, tuberculosis-mycobacterium tuberculosis, mycobacterium bovis, mycobacteria, mycobacteria, and mycobacteria • Plasmodium falciparum, Toxoplasma gondii and other parasitic protozoa • Typhoid- typhoid bacillus • Ebola virus • Human immunodeficiency virus (HIV) • Dengue fever – Yellow fever virus • Hepatitis (A to E) • Medical source Sexual infections - such as Clostridium pneumoniae, vancomycin-resistant enterococci and drug-resistant Staphylococcus aureus • Herpes simplex virus (HSV) • Cytomegalovirus (CMV) • Epstein-Barr virus (EBV) • Brain Inflammation - Japanese encephalitis virus, Zhangdipula virus • Pertussis - pertussis • Measles - paramyxovirus • Meningitis - Streptococcus pneumoniae and Neisseria meningitidis -27- 201219 770 • Anthracnose-Bacillus anthracis Examples of some genetic diseases that can be identified using this system include: • Cystic fibrosis • Hemophilia • Sickle cell anemia • Black Mongolian idiots • Hemochromatosis • Cerebral arterial disease • Cloning Disease • Polycystic kidney disease • Congenital heart disease • Leier's disease A few examples of cancers identified by this diagnostic system include: • Ovarian cancer • Colon cancer • Multiple endocrine neoplasms • Retinal blastoma • Turk disease (Turcot syndrome) The above list is incomplete and the diagnostic system can be configured to utilize nucleic acid and proteomic analysis to detect more diverse diseases and conditions. Detailed Structure of System Components LOC Device The L0C device 30 is the core of the diagnostic system. The device rapidly performs the four major steps of molecular diagnostic analysis of nucleic acid substrates on the Microfluid-28-201219770 platform, ie sample preparation, nucleic acid extraction, nucleic acid amplification and detection. The LO C device is also of selective use and will be described in more detail below. As discussed above, test modules 10 and 11 can take many different configurations to detect different targets. Similarly, LOC device 30 can also be used to create a variety of different embodiments for the target of interest. One form of LOC device 30 is used in a LOC device 3〇1 for fluorescent detection of a target nucleic acid sequence of a pathogen in a whole blood sample. For the purpose of the description, the structure and operation of the LOC device 301 will now be described in detail with reference to Figures 4 to 26 and 27 to 57. 4 is a representative diagram of the structure of the LOC device 301. For the sake of convenience, the processing stages shown in Fig. 4 are denoted by the component symbols corresponding to the functional portions of the LOC device 301 that implements the processing stage. The processing stages associated with each of the major steps in the molecular diagnostic analysis of the nucleic acid substrate are also shown: sample input and preparation 288, extraction 290, culture 291, amplification 292, and detection 294. The various reservoirs, chambers, valves and other components of the LOC device 301 will be described more closely below. Figure 5 is a perspective view of the LOC device 301. The device is fabricated using high volume CMOS and MST (microsystem technology) fabrication techniques. The layered structure of the LOC device 30 1 is illustrated in a schematic (not to scale) partial cross-sectional view of Figure 12. The LOC device 301 has a germanium substrate 84 that supports a COMS + MST wafer 48 that includes a CMOS circuit 86 and an MST layer 87 and has a cover 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 a sample treated with various reagents. Accordingly, these structures and components are configured to define a flow path having a feature size of -29-201219770 that will support capillary flow to drive the flow of liquid similar to the physical properties of the sample of the treatment section. In view of this, MST layers and components are typically fabricated using surface micromachining techniques and/or stereomachining techniques. However, other manufacturing methods can also produce structures and components that are sized for capillary drive flow and for handling very small samples. The particular embodiment described in the specification shows that the MST layer is a structure and active component supported by CMOS circuitry 86, but does not have the upper cover 46. However, those skilled in the art will appreciate that the MST layer does not require a lower CMOS or even a top cover to process the sample. The overall dimensions of the LOC device shown in the following figures are 1 760 micro X 5824 microns. Of course, LOC devices made for different applications can be of different sizes. 6 shows the features of the MST layer 87, which are superimposed on the features of the upper cover. The AA to AD, AG, and AH regions shown in FIG. 6 are enlarged at 13, 3, 4, 3, 5, 5, 5, and 6 3, respectively. The details of the various structures within the LOC device 301 are fully explained below. Figures 7 through 10 show the features of the cover 46 independently, while Figure 11 shows the structure of the CMOS + MST device 48 independently. Hierarchical Structure Figures 12 and 22 illustrate the hierarchical structure of the CMOS + MST device 48, the upper cover, and the fluid interaction therebetween. The drawings are not drawn to scale for purposes of illustration. Figure 12 is a schematic cross-sectional view through the sample inlet 68, and Figure 22 is a schematic cross-sectional view through the reservoir 54. The CMOS+ MST device 48 has a germanium substrate 84 supporting a CMOS circuit 86, the CMOS circuit 86 operating thereon. The CMOS+MST device 48 has a germanium substrate 84 supporting the CMOS circuit 86. Active components within the MST layer 87. The passivation layer 8 8 seals and protects the C Μ Ο S layer 8 6 from flowing into the liquid flowing through the MST layer 87. The liquid flows through both the upper cap channel 46 and the MST channel 90 in the upper cap layer 46 and the MST channel layer 100 (see, for example, Figures 7 and 16). Cell transport occurs in the larger channel 94 made in the upper cap 46, while biochemical treatment takes place in the smaller MST channel 90. The size of the cell delivery channel is designed to deliver cells in the sample to a predetermined location in the MST channel 90. Cells that transport greater than 20 microns (e.g., certain white blood cells) require channel sizes greater than 20 microns, so the cross-sectional area across the flow must be greater than 400 square microns. The position of the MST channel, particularly in L〇c, which does not require delivery of cells, can be significantly smaller. It will be understood that the 'top cover channel 94 and the MST channel 90 are generic, and the particular MST channel 90 may also be referred to as, for example, a heated microchannel or a dialysis MST channel due to its particular function. The MST channel 90 is formed by etching a MST channel layer 1 deposited on the passivation layer 88 and patterned by a photoresist. The MST channel 90 is closed by a top layer 66 that forms the top of the CMOS + MST device 48 (shown in the figure). Although sometimes shown in separate layers, the upper cover channel layer 8 and the reservoir layer 78 are formed from a single piece of material. Of course, the sheet material can also be non-unitary. Both sides of the sheet material are etched to form an upper cover channel layer 8 and a reservoir layer 78, and an upper cover channel 94 is etched in the upper cover channel layer 80, and reservoirs 54, 56, 58, 60 are etched in the reservoir layer 78. 62. Alternatively, the -31 - 201219770 reservoir and capping channel are formed by micromolding. Both etching and micromolding techniques are used to fabricate channels having a cross-sectional area of up to 20,000 square microns and a minimum of 8 square microns across the fluid. For various locations in the LOC device, a variety of suitable channel cross-sectional areas across the fluid can be selected. When a large number of samples or samples having a large composition are accommodated in the channel, a cross-sectional area of up to 20,000 square micrometers (e.g., a channel having a width of 200 micrometers in a layer having a thickness of 100 micrometers) is appropriate. When the channel contains a small amount of liquid or a mixture free of large cells, it is preferred that the cross-sectional area across the fluid is very small. The lower sealing layer 64 closes the upper cover passage 94, and the upper sealing layer 82 closes the reservoirs 54, 56, 58, 60 and 62. Five reservoirs 54, 56, 58, 60, and 62 preload reagents for specific assays. In the embodiments described herein, the reservoirs are preloaded with the following reagents, but can be easily replaced with other reagents: • Reservoir 54 : Anticoagulant, optional including red blood cell lysate • reservoir 5 6 : lysis reagent • reservoir 5 8 : restriction enzymes, ligase and linker (for linker initiation PCR (see Figure 69, excerpt from T.  Stachan et al. , Human Molecular Genetics 2, Garland Science, NY and London, 1999)) • Reservoir 60: amplification mixture (dNTPs, primers, buffers), and • reservoir 62: DNA polymerase. Upper cover 46 and CMOS + MST layer 48 are in fluid communication via corresponding openings in lower seal 64 and top layer 66. These openings are referred to as riser 96 and drop 92 depending on the flow regime from the -32-201219770 MST passage 90 to the upper cover passage 94 or vice versa. LOC Device Operation The operation of the LOC device 301 is described step by step as an example of the analysis of pathogenic DNA in a blood sample. Of course, other types of biological or non-biological fluids may also be analyzed using appropriate reagents, test protocols, groups of LOC variants and detection systems, or a combination thereof. Referring to Figure 4, the analysis of the biological sample is largely divided into five steps, including sample input and preparation 288, nucleic acid extraction 290, nucleic acid culture 291, nucleic acid amplification 292, and detection and analysis 294. The sample input and preparation steps 2 8 8 involve mixing the blood with the anticoagulant 116, followed by separation of the pathogen from the pathogen dialysis unit 70 with white blood cells and red blood cells. As best seen in Figures 7 and 12, the blood sample enters the device via sample inlet 68. Capillary action draws the blood sample along the upper cover channel 94 to the reservoir 54. When the sample blood flow opens the surface tension valve 1 18 of the reservoir 54, the anticoagulant is released from the reservoir 54 (see Figures 15 and 22). Anticoagulants prevent clot formation from obstructing flow. As best shown in Fig. 22, the anticoagulant 116 is aspirated from the reservoir 54 by capillary action and enters the MST channel 90 via the descending port 92. The drop port 92 has a capillary initiation feature (CIF) 1〇2 to control the geometry of the meniscus such that the meniscus is not fixed at the edge of the drop port 92. The venting opening 12 in the upper sealing layer 82 allows air to replace the anticoagulant that is aspirated from the reservoir 54. The MS T channel 90 shown in Fig. 22 is one of the surface tension valves 118 -33 - 201219770 minutes. The anticoagulant 116 fills the surface tension valve 118 and secures the meniscus 120 to the meniscus anchor 98 of the riser 96. The meniscus 120 remains fixed to the riser 96 prior to use so that the anticoagulant does not flow into the upper cover passage 94. As blood flows through the upper lid passage 94 to the ascending port 96, the meniscus 110 is removed and the anticoagulant is drawn into the flow. Figs. 15 to 21 show the AE area. The AE area is a part of the AA area which is not shown in Fig. 13. As shown in Figures 15, 16 and 17, surface tension valve 118 has three separate MST passages 90 extending between respective lower and upper risers 92, 96. The number of MST channels 90 in the surface tension valve can vary to change the flow rate of reagent into the sample mixture. When the sample mixture and reagents are mixed by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample stream. Thus, the surface tension valves of each reservoir are configured to meet the desired reagent concentration. The blood enters the pathogen dialysis section 70 (see Figures 4 and 15) where the target cells are concentrated from the sample using an array of apertures 1 64 sized according to a predetermined valve. Cells smaller than the valve plaque pass through the well, while large cells cannot pass through the well. The undesired cells are either larger cells that are blocked by the pore array I 64 or smaller cells that pass through the pores, which are transduced to the waste unit 76, whereas the target cells are still part of the analysis. In the pathogen dialysis section 70 described herein, the pathogen system from the whole blood sample is concentrated for microbial DNA analysis. The array of holes is formed by a plurality of 3 micron diameter holes 164 that fluidly communicate with the input flow in the upper cover channel 94 to the target channel 74. The 3 micron diameter aperture 164 and the dialysis riser aperture 168 in the target channel 74 are connected by a series of dialysis MST channels - 34 - 201219770 204 (best shown in Figures 15 and 21). The pathogen is small in volume and can therefore be filled through the 3 micron diameter aperture 164 and filled through the target channel 74 via the dialysis MST channel 204. Cells larger than 3 microns, such as red blood cells and white blood cells, remain in the waste channel 72 of the upper cover 46, which leads to the waste reservoir 76 (see Figure 7). Other pore shapes, sizes, and aspect ratios can be utilized to isolate specific pathogens or other target cells, such as white blood cells for human DNA analysis. Detailed instructions for the dialysis section and dialysis variants are provided below. Referring again to Figures 6 and 7, fluid is drawn through target channel 74 to surface tension valve 128 of lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 that extend between the lysis reagent reservoir 56 and the target channel 74. When the meniscus is removed by the sample stream, the flow rate from all seven MST channels 90 will be greater than the flow rate from the anticoagulant reservoir 54, where the surface tension valve 118 of the reservoir 54 has three MST channels 90 (hypothesis) The physical properties of the liquids are approximately equal). Thus the ratio of lytic reagent to the sample mixture is greater than the ratio of anticoagulant to the sample mixture. The lysis reagent and the target cells are mixed by diffusion in the target channel 74 in the chemical lysis unit 130. The boiling start valve 1 2 6 stops the flow until sufficient time for diffusion and lysis occurs to release the genetic material from the target cells (see Figures 6 and 7). The structure and operation of the boiling start valve are described in detail below with reference to Figs. 31 and 32. Other active valve types (as opposed to passive valves such as surface tension valve 118) have also been developed by the applicant, and other types of active valves can be used herein to replace the boiling start valve. These alternative valves -35- 201219770 are also described below. When the boiling start valve 126 is opened, the lysed cells flow into the mixing section 1 31 to perform restriction enzyme cleavage and linker ligation before amplification. Referring to Fig. 13, when the fluid removes the meniscus of the surface tension valve 133 at the beginning of the mixing portion 131, the restriction enzyme, linker and ligase are released from the reservoir 58. The mixture flows through the length of the mixing section 133 to diffusely mix. The end of the mixing portion 131 is a descending port 134 leading to the incubator inlet passage 133 of the culture portion 114 (see Fig. 13). The incubator inlet channel 133 feeds the mixture into a heated microchannel 210 in a crimped configuration that provides a culture chamber for holding the sample during restriction enzyme cleavage and linker ligation (see Figures 13 and 14). Figures 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 in the AB zone of Figure 6. The figures show the layers of continuous salt addition to form the structure of CMOS + MST layer 48 and upper cover 46. The AB region shows the end point of the fostering unit 1 14 and the starting point of the amplifying portion 112. As best seen in Figures 14 and 2, the fluid fills the microchannel 2 1 0 of the culture section 1 1 4 until it reaches the boiling start valve 1 〇 6, where the fluid stops to allow diffusion to occur. As discussed above, the microchannels 210 in the upstream of the boiling start valve 106 become the culture chamber containing the sample, restriction enzyme, ligase, and linker. The heater crucible 54 is then activated and maintains a stable temperature for a specific period of time to allow restriction enzyme cleavage and linker ligation. Those skilled in the art will appreciate that this culturing step 29 1 (see Figure 4) is optional and is only required for some types of nucleic acid amplification assays. Also in some instances, it may be desirable to provide a heating step at the end of the incubation period to allow the temperature to rise sharply -36-201219770 above the culture temperature. This temperature rise causes the restriction enzyme and the ligase to deactivate before entering the amplification section 112. Deactivation of restriction enzymes and ligases is particularly important when using isothermal nucleic acid amplification. After the incubation, the boiling start valve 106 is activated (opened) and the fluid continues to enter the amplifying portion 112. Referring to Figures 31 and 32, the mixture is filled with heated microchannels 158 in a curved configuration until reaching the boiling start valve 1 〇 8, which forms one or more amplification chambers. As is clear from the cross-sectional view of Fig. 30, the amplification mixture (dNTP 'primer' buffer) is released from the reservoir 60 and the polymerase is then released from the reservoir 62 into the junction culture section and the amplification section (1 Μ and 1 respectively) 12) The intermediate MST channel 212. Figures 35 through 51 show the layers of LOC device 301 in the AC region of Figure 6. The figures show the layers of successive additions to form the structure of CMOS+MST device 48 and upper cover 46. The AC region is the end point of the amplification unit 1 12 and the starting point of the hybridization and detection unit 52. The cultured sample, amplification mixture, and polymerase flow through the microchannel 158 to the boiling start valve 108. After sufficient time for diffusion mixing, the heater 154 in the microchannel 158 is activated for thermal cycling or isothermal amplification. The amplification mixture undergoes a predetermined number of thermal cycles or a predetermined amplification time to amplify sufficient target DNA. After the nucleic acid amplification procedure, the boiling start valve 108 is opened and the fluid continues to enter the hybrid and detection portion 52. The operation of the boiling start valve is described in detail below. As shown in Fig. 52, the hybridization and detection unit 52 has a hybridization chamber array 1 10 . Figures 5, 5, 3, 4 and 5 show the hybridization chamber array 110 and the single-hybridization chamber 180 in detail. A diffusion barrier 175 is provided at the entrance of the hybridization chamber 180 to prevent diffusion of the target nucleic acid, probe strands, and hybridized probes between the -37-201219770 hybridization chambers during hybridization to prevent erroneous hybridization. Test results. The diffusion barrier 175 represents a flow path of sufficient length to prevent the target sequence and probe from diffusing out of one chamber and contaminating another chamber during the time required for the probe and nucleic acid hybridization and signal to be detected, thus avoiding errors result. Another mechanism to prevent erroneous results is to include the same probe in multiple hybridization chambers. The CMOS circuit 86 derives a single result from the photodiode 1 8 4 corresponding to the hybridization chamber 180 containing the same probe. When deriving a single result, the anomalous results can be ignored or given different weights. The thermal energy required for hybridization is provided by a CMOS controlled heater 182 (described in more detail below). After the heater is activated, hybridization occurs between complementary target-probe sequences. The LED driver 29 in the CMOS circuit 86 transmits a message to the LED 26 located in the test module to illuminate it. These probes only fluoresce when hybridization occurs, so the cleaning and drying steps typically required to remove unbound strands are omitted. Hybrid forced FRET probe 186 has a stem-loop structure that allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as described in more detail below. Fluorescence is detected by photodiode 184 located in CMOS circuit 86 below each hybridization chamber U0 (see description of hybridization chamber below). The photodiode 184 and associated electronics for all of the hybridization chambers together form a photosensor 44 (see Figure 64). In other embodiments, the photosensor can be an array of charge coupled devices (CCD arrays). The detection signal from the photodiode 1 84 is amplified and converted to a digital output for analysis by the test module reader 12. Further details of the detection method are described below. -38- 201219770 Other Detailed Description of LOC Device The modular design LOC device 3〇1 has a number of functional parts (including reagent reservoirs 54, 56, 58, 60 and 62, dialysis unit 70, lysis unit 130, culture unit 114). And amplification unit 1 1 2), valve type, humidifier and humidity sensor. In other embodiments of the LOC device, these functional portions may be omitted, but additional functional portions may be added or the functional portions may be used for different purposes than those described above. For example, the culture portion 114 can be used as the first amplification portion 11 2 of the tandem amplification analysis system, and the chemical lysis reagent reservoir 56 is used to add the first amplification primer 'dNTP and buffer The mixture, and reagent reservoir 58 are used to add reverse transcriptase and/or polymerase. If the sample is to be chemically lysed, a chemical lysis reagent (along with the amplification mixture) may also be added to the reservoir 56' or alternatively, the sample may be heated for a predetermined period of time to cause thermal dissolution in the culture section. Cell. In some embodiments, if chemical lysis is desired and it is desired to separate the chemical lysis reagent from the mixture of primers, dNTPs, and buffer, an additional reservoir can be introduced upstream of the reservoir 58 immediately adjacent to the mixture. In some cases it may be desirable to omit the steps, such as the culturing step 291. In this case, the 〇C device may be specially fabricated to avoid the reagent reservoir 58 and the culture portion 114, or may simply not be loaded with the reagent, or If an active valve is present, the active valve is not activated to release the reagent into the sample stream. Thus, the culture portion becomes a passage for transferring only the sample from the lysis unit 30 to the amplification portion 112. The heater can be operated independently, so when the reaction depends on -39 - 201219770 heating - such as hot lysis reaction, setting the heater not to start during this step ensures that hot lysis does not occur in LOC devices that do not require hot lysis in. The dialysis section 70 can be located at the beginning of the fluid system within the microfluidic device as shown in Figure 4 or can be located at any other location within the microfluidic device. For example, in some cases, it may be advantageous to dialyze after the amplification phase 2 92 to remove cellular debris prior to the hybridization and detection step 294. Alternatively, two or more dialysis sections can be inserted at any location in the LOC device. Similarly, it is possible to inject additional amplifications 1 1 2 so that the multiplex targets can be amplified simultaneously or sequentially before being detected by the specific nucleic acid probes in the hybridization chamber array 1 1 。. It is not necessary to perform dialysis for analyzing a sample such as whole blood, so the dialysis portion 70 is simply omitted from the sample input and preparation portion 28 of the LOC design. In some cases, it is not necessary to omit the dialysis section 70 of the LOC device, even if the analysis does not require dialysis. If the presence of the dialysis section does not cause a geometrical impediment to the analysis, the sample input and preparation section can still be used to have the LOC of the dialysis section 70 without losing the desired function. In addition, the detection portion 294 can include a protein body array array that is identical to the hybrid chamber array but carries a probe that is designed to conjugate or hybridize to a sample target protein present in the non-amplified sample, rather than a design A nucleic acid probe for hybridization to a target nucleic acid sequence. It will be appreciated that the LOC devices manufactured for use with this diagnostic system are different combinations of functional components selected for a particular LOC application. The vast majority of functional units are the same for many LOC devices, and the design of additional LOC devices for new applications relates to the combination of appropriate functional components from the various functional options used in existing LOC devices. -40- 201219770 Only a few LOC devices are shown in this description. More LOC devices are illustrated in a diagram to illustrate the design flexibility of the L 0 C device manufactured for this system. Those skilled in the art will readily appreciate that the LOC devices described in this specification are not exhaustive, and that many additional LOC designs are related to integrating appropriate functional combinations. Sample Type LOC Variants can accept and analyze a variety of nucleic acid or protein contents in a liquid form, including but not limited to blood and blood products, saliva, cerebrospinal fluid 'urine, semen, amniotic fluid' umbilical cord , breast milk, sweat, pleural effusion, tears, pericardial fluid 'peritoneal fluid, environmental water samples and dip samples. Amplicon obtained from macroscopic nucleic acid amplification can also be analyzed using the LOC device; in this case, all reagent reservoirs will be empty or configured to not release their contents, and the dialysis, lysis The culture and amplification section will only be used to deliver the sample from the sample inlet 68 to the hybridization chamber 180 for nucleic acid detection as described above. Some sample types require a pre-treatment step prior to input into the LOC device. For example, semen may require liquefaction and mucus may require pre-treatment with enzyme to reduce viscosity. Sample input

Referring to Figures 1 and 12, the sample is added to the large container 24 of the test module 1 . The large container 24 is a truncated cone that is fed by capillary action into the inlet 68 of the LOC unit 301. The sample is here flowed into a 64 μιη wide X-41 - 201219770 6 0 μηι deep cap channel 94 and is also attracted to the anticoagulant reservoir 54 by capillary action. Reagent Reservoirs A small amount of reagents required for the analysis system of a microfluidic device, such as LOC device 301, are used to allow the reagent reservoirs to contain all of the reagents required for biochemical treatment in each of the reagent reservoirs having a small volume. This volume must be less than · 1,000,000,000 cubic microns, mostly less than 3 inches, 〇〇〇, 〇〇〇 cubic microns, typically less than 70,000,000 cubic microns, and less than 20,000,000 cubic microns for the LOC device 301 shown in the figures. . Dialysis Section Referring to Figures 15 through 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate pathogen target cells from the sample. As previously described, a plurality of wells 1 64 having a 3 micron diameter opening in the top layer 66 filter the target cells from the sample body. As the sample flows through the 3 μm diameter hole 164, the microbial pathogen enters a series of dialysis MST channels 204 through the opening and flows back to the target channel 7 4 via the 16 μm dialysis rising hole 168 (see Figure 3). 3 and 3 4). The remaining sample (red blood cells, etc.) remains in the upper cover channel 94. Downstream of the pathogen dialysis section 70, the upper cover passage 94 becomes a waste passage 72 to the waste reservoir 76. For a biological sample type that produces a large amount of waste, the foam (f 0 am) insert or other porous element 49 in the outer casing 13 of the test module 10 is configured to be in fluid communication with the waste receptacle 76 (see Figure • 42- 201219770 The function of the pathogen dialysis section 70 is entirely dependent on the capillary action of the fluid sample. The 3 micron diameter hole 1 64 at the upstream end of the pathogen dialysis section 70 has a capillary initiation feature (CIF) 166 (see Figure 33) to allow fluid It is drawn downward into the dialysis MST channel 204 below it. The first rising hole 198 of the target channel 74 also has a CIF 02 (see Figure 15) to prevent fluid from forming a meniscus easily in the dialysis rising hole 1 68. The small component dialysis section 682 shown in Fig. 74 may have a structure similar to that of the pathogen dialysis section 70. The small component dialysis section separates the small target cells from the sample by the size (and shape if necessary) of the pores. Alternatively, the pores are adapted to allow small target cells or molecules to pass through the target channel and continue to be further analyzed. Large size cells or molecules are removed to waste reservoir 766. Thus, the LOC device 30 (see Figures 1 and 98) )and Limited to pathogens with a size less than 3 μm, can also be used to isolate cells or molecules of any desired size. The lysate is again referred to Figures 7, 11 and 13 and the genetic material in the sample is treated by chemical lysis. The cells are released. As described above, the lysing reagent from the lysate 56 is mixed with the sample stream in the target channel 74 downstream of the surface tension valve 126 of the lysis reservoir. However, some diagnostic assays It is more suitable to use hot lysis treatment, or even a combination of chemical and thermal lysis of target cells. The LOC device 301 mixes the heated microchannels of the culture portion 114. The sample stream is filled with the culture portion 1 1 4 And stop at the boiling start with the same 〇6. The culture microchannel 2 10 heats the sample to the temperature of the cell membrane rupture -43-201219770 degrees. In some hot lysis applications, the enzyme in the chemical lysis unit 130 is not required, The enzymatic reaction in the chemical lysis section 130 is completely replaced by hot lysis. Boiling Start Valve As described above, the LOC unit 301 has three boiling starts 126, 106 and 108. The positions of these valves are shown in Figure 6. Figure 31 shows the boiling start valve 1 separately An enlarged plan view of 08, located at the end of the heating microchannel 158 of the amplification 112. The sample stream 1 1 9 is attracted by capillary action through the heating micropass 1 58 until the boiling start valve 108 is reached. The former guiding fluid 120 is fixed to the meniscus anchor 98 of the valve inlet 146. The geometry of the meniscus 98 stops the advancing meniscus to prevent capillary flow. See Figures 31 and 32. As shown, the meniscus anchor 98 is a hole provided by the MST channel 90 to the raised opening of the upper cover channel 94. The surface tension of the meniscus 120 keeps the valve closed. The ring heater is located around the valve inlet 146. The ring heater 152 is controlled by C Μ Ο S via the boiling valve heater contact 1 5 3 . To open the valve, the CMOS circuit 86 delivers an electrical pulse to the heater contact 153. The annular heater crucible 52 is heated by electrical resistance until the liquid sample 119 is boiled. This boiling causes the meniscus 120 to be removed from the valve inlet 146 and begin to wet the upper cover passage 94. The capillary action is restored once the upper cover is opened 94'. The fluid sample 1 1 9 is filled with the upper cover 9 4 and flows through the valve lowering port 1 50 to the valve outlet 1 4 8 , and the capillary action counter valve at this point is the side surface fixing white 52 the opening body track drive - 44-201219770 The flow of the motion continues to advance to the hybridization and detection unit 52 along the expansion outlet channel 160. Liquid sensor 1 74 is placed before and after the valve for diagnostic purposes. It will be understood that once the boiling start valve is opened, it can no longer be closed. However, since the LOC device 3 and the test module 1 are used in a single use, it is not necessary to close the valve. Culture section and nucleic acid amplification section Figs. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50, and 51 show the culture section Π4 and the amplification section 112. The culture portion 114 has a single strip of heated culture microchannel 210' which is etched into the MST channel layer 100 and etched into a meandering pattern 134 starting at the lowering port 134 and finally boiling the activation valve 106 (see Figures 13 and 14). Controlling the temperature of the culture section 1 14 allows the enzyme reaction to occur with higher efficiency. Similarly, the amplifying portion 112 has a heated augmentation microchannel 158 (see Figs. 6 and 14) that begins in a curved structure with the boiling start valve 106 leading to the boiling start valve 108. When mixing, culture, and nucleic acid amplification occurs, the valves stop the flow to retain the target cells in the heated culture or amplification microchannel 210 or 158. The curved pattern of the microchannels also promotes (to some extent) the mixing of the target cells with the reagents. In the culture unit 1 14 and the amplification unit 1 1 2, the sample cells and reagents are heated by a heater 1 54 controlled by a pulse width modulation (PWM) CMOS circuit 86. Each curved corner of the heated culture microchannel 210 and the expanded microchannel 158 has three independently operable heaters 154 extending between the individual heater contacts 156 (see Figure-45-201219770 1 4)), which provides two-dimensional control of the input heat flux density. As best shown in Figure 51, the heater 154 is supported by the top layer 66 and embedded in the lower sealing layer 64. The material of the heater is TiAl, but many other conductive metals are also suitable. The elongated heater 154 is parallel to the longitudinal length of each of the channel portions forming the wide curve of the curved structure. In the amplification section 112, each wide curve can be controlled via a separate heater as a separate PCR chamber. The use of a small volume of amplicons required for the analysis system of a microfluidic device, such as LOC device 301, allows the use of a small volume of amplification mixture for amplification in the amplification section. This volume must be less than 400 nanoliters, mostly less than 170 nanoliters, typically less than 70 nanoliters, and the LOC device 301 is typically between 2 nanoliters and 30 nanoliters. Increasing heating rate and better diffusion mixing The small cross-sectional area of each channel portion increases the heating rate of the augmentation fluid mixture. The distance between all fluids and heaters 154 is quite short. Reducing the cross-sectional area of the channel (i.e., the cross-section of the augmented microchannel 158) to less than 100,000 square microns provides a significantly higher heating rate than a "large scale" device. The lithography manufacturing technique provides the amplifying microchannel 158 with a cross-sectional area across the flow path of less than 1 6,000 square microns which provides a substantially higher heating rate. Dimensional features of 1 micron size can be easily obtained using lithography manufacturing techniques. If only a very small amount of amplicons are required (in the case of LOC device 301), the cross-sectional area can be reduced to less than 2,500 square microns. For a diagnostic analysis performed on a LOC device with 1,000 to 2,000 probes and requiring "input samples, results" in 1 minute, between 400 and 46 - 201219770 square micrometers to 1 square micrometer The cross-sectional area of the traversing fluid is appropriate. The heater element in the amplification microchannel 158 heats the nucleic acid sequence at a rate of greater than 8 每秒 absolute temperature (K) per second, in most cases per The second is greater than the rate of 100 K. Typically the heater element heats the nucleic acid sequence at a rate greater than 1 〇〇〇K per second, and the heater element often heats the nucleic acid sequence at a rate of greater than 1 000 K per second. The system requires the heater element to be greater than 100,000 K per second, greater than 1.000 0.000 κ per second, greater than 10,000,000 K per second, greater than 20.000 000 K per second, greater than 40,000,000 κ per second, greater than 8 每秒 per second,核酸, 〇〇〇κ and the nucleic acid sequence at a rate greater than 160, 〇〇〇, 〇〇〇 K per second. The small cross-sectional channel is also beneficial for the diffusion mixing of any reagent with the sample fluid. prior to, The phenomenon that the liquid diffuses to another liquid is most pronounced at the interface between the two liquids. The concentration decreases as the distance from the interface increases. Two types of microchannels having a relatively small cross-sectional area across the fluid direction are used. The fluid flows close to the interface for faster diffusion mixing. Reducing the cross-sectional area of the channel to less than 100,000 square microns provides a significantly higher mixing rate than "large scale" equipment. The lithography manufacturing technique allows the traversing of the microchannel The cross-sectional area of the flow path is less than 1 600 square microns, which provides a significantly higher mixing rate. If only a very small volume is required (in the case of the LOC device 301), the cross-sectional area can be reduced to less than 2,500 square feet. Micron. Between 400 square microns and 1 square micron for diagnostic analysis on a LOC device with 1 to 2000 probes and requiring "input sample, result" in 1 minute Cross-47- 201219770 The cross-sectional area of the fluid is appropriate. The rapid thermal cycle time keeps the sample mixture close to the heater and uses a very small amount of fluid to allow for nucleic acid amplification. Rapid thermal cycling during the period. For each target sequence of up to 150 base pairs (bp) length, each thermal cycle (ie, denaturation, adhesion, and extension of the primer) is completed in less than 30 seconds. In the diagnostic analysis, the individual thermal cycle time is less than 11 seconds, and most of the system is less than 4 seconds. For the LOC device 30 for some of the most common diagnostic analyses, up to 150 base pairs (bp) of the target sequence The thermal cycle time is between 〇45 seconds and 1.5 seconds. The thermal cycling of this speed allows the test module to complete the nucleic acid amplification procedure in much less than 1 minute; usually less than 2 2 0 It can be done in seconds. For most analyses, the amplification produces enough amplicons within 80 seconds of the sample fluid entering the sample inlet. Many assays produce enough amplicons in 3 seconds. When a predetermined number of amplification cycles are completed, the amplicon is fed to the hybridization and detection portion 52 via the boiling start valve 108. Hybridization Chambers Figures 52, 53, 54, 56 and 57 show hybridization chambers 1 80 in the hybrid chamber array 11〇. The hybridization and detection unit 52 has a 24 x 45 array 1 10 hybridization chamber 180'. Each of the hybridization chambers has a hybrid-reactive FRET probe 186, a heater element 182, and an integrated photodiode 184. The photodiode 1 8 4 is introgressed to detect fluorescence produced by hybridization of the labeled nucleic acid sequence or protein to the -48-201219770 FRET probe 186. Each photodiode 184 is independently controlled by a CMOS circuit 86. Any substance between the FRET probe 186 and the photodiode 1 84 must be transparent to the emitted light. Therefore, the wall portion 97 between the probe 186 and the photodiode 184 can also be optically transmitted by the emitted light. In the LOC device 301, the wall portion 97 is a thin layer of ruthenium dioxide (about 5 μm). Direct incorporation of photodiode 184 beneath each hybridization chamber 180 allows for a detectable fluorescent signal to be produced by the extremely small probe-target hybrid volume (see Figure 5 4). This small amount allows the use of a small volume of hybridization chamber. The amount of detectable probe-target hybrid required for pre-hybridization probes must be less than 270 picograms (corresponding to 900,000 cubic micrometers), in most cases less than 60 picograms (corresponding to 200,000 cubic meters) Micron), typically less than 12 picograms (corresponding to 40,000 cubic micrometers), and is less than 2.7 picograms (corresponding to a chamber volume of 9,000 cubic micrometers), as exemplified by the LOC device 301 shown in the drawings. Of course, reducing the size of the hybridization chamber allows for a higher density chamber' thus allowing the LOC device to have more probes. In the LOC device 301, the hybrid portion has more than one, and one chamber (i.e., less than 2,250 square microns per chamber) in an area of 1,50 Å micrometers by 1,500 micrometers. Smaller volumes also reduce reaction time, so hybridization and detection can be faster. Another advantage of requiring a small number of probes in each chamber is that only a very small amount of probe solution needs to be spotted into each chamber during manufacture of the LOC device. Embodiments of the LOC device of the present invention can be spotted using a probe solution having a volume of 10-»2 or less. After nucleic acid amplification, the boiling start valve 108 is activated and the amplicon -49 - 201219770 flows along the flow path 176 and flows into each of the hybridization chambers 180 (see Figures 52 and 56). The endpoint liquid sensor 178 shows the point at which the hybridization chamber 180 is filled with the amplicon and the heater 182 can be activated. After a sufficient hybridization time, LED 26 (see Figure 2) is activated. The opening in each hybrid chamber 180 is provided with a light window 136 to expose the FRET probe 186 to the excitation radiation (see Figures 52, 54 and 56). The LED 26 emits light for a time long enough to induce a high intensity fluorescent signal from the probe. Photodiode 184 is shorted during excitation. After a preprogrammed delay 300 (see Figure 2), the photodiode 184 is enabled and detects fluorescent emissions in the absence of excitation light. The incident light on the active region 185 (see Fig. 54) of the photodiode 184 is converted into a photocurrent which can then be measured by the CMOS circuit 86. Hybridization chambers 1 80 each carry a probe for detecting a single target nucleic acid sequence. Each hybridization chamber 180 can carry probes that detect more than 1,000 different targets, if desired. Alternatively, many or all of the hybridization chambers may carry the same probe to repeatedly detect the same target nucleic acid. Copying the probes in the hybridization chamber array 110 in this manner results in increased confidence in the results obtained, if desired to provide a single result by combining the results of the photodiodes adjacent to the hybridization chambers. Those skilled in the art will appreciate that there may be from 1 to more than 1,000 different probes on the hybrid chamber array 110, depending on the analytical specifications. Humidifier and Humidity Sensor The AG zone of Figure 6 indicates the location of the humidifier 1 96. The humidifier prevents the reagents and probes from evaporating during operation of the L0C device 301. The best as shown in Figure 55-50-201219770 enlarged view, the water reservoir 1 8 8 series with three evaporators 1 90 fluid phase water storage device 1 8 8 contains molecular biological grade water and is best sealed during manufacturing As indicated by 55 and 67, water is drawn to the three lowerings 1 94 by capillary action and to the riser 1 93 of the evaporator 1 90 along the individual water supply passages 1 92. The meniscus is fixed to each of the risers 1 93 to retain the evaporator having a ring heater 191 which surrounds the riser 193. The heater 191 is connected to the metal layer 195 of the CMOS circuit 86 by a conductive post 376 (see Figure 37). At startup, the ring heater 191 adds water to evaporate and wet the surrounding devices. Figure 6 also shows the location of the humidity sensor 232. However, as shown in the enlarged view of the AH zone of the optimum 63, the humidity sensor has a capacitive structure. The lithographically etched first electrode 296 and the lithographically etched first pole 298 are opposite each other such that their teeth are interleaved. The opposing electrode capacitor has a capacitance that can be monitored by CMOS circuit 86. As the degree increases, the permittivity of the air gap between the electrodes increases, resulting in an increase in power. The humidity sensor 232 is adjacent to the hybridization chamber array 1 1 , and the humidity measurement is important to slow the evaporation of the feedback sensor temperature containing the exposed probe and the liquid sensor is inserted into the L Ο C device 3 0 1 feedback and diagnosis during the operation of the device. Referring to Fig. 35, nine thermostats 170 are distributed to the amplification unit 112. Similarly, the culture section 1 has nine temperature sensors 170. These sensors each use a 2x2 array of bipolar junction transistors (BJT) to monitor fluid temperature and provide reverse. seal. Three waters. The top of the ring is heated as shown in the figure. The heat is also formed to provide the humidity. The sense is also provided. -51 - 201219770 C Μ O S circuit 8 6. The c Μ 0 S circuit 8 6 utilizes this to accurately control thermal cycling during nuclear acid amplification as well as thermal lysis and any heating during incubation. In the hybridization chamber 180, the CMOS circuit 86 uses the hybridization heater 182 as a temperature sensor (see Figure 56). The resistance of the hybrid heater 182 is temperature dependent and the CMOS circuit 86 utilizes this to derive temperature readings for each of the hybrid chambers 180. The LOC device 301 also has a number of MST channel liquid sensors 174 and an upper cover channel liquid sensor 208. Figure 35 shows a row of MST channel liquid sensors 174 at one of the other corners of the heated microchannels 1 58. As best seen in Figure 37, the MST channel liquid sensor 174 is formed by a pair of electrodes formed by exposed regions of the top metal layer 195 in the CMOS structure 86. The liquid closes the current between the electrodes to indicate their presence at the sensor. Figure 25 shows an enlarged perspective view of the upper cover channel liquid sensor 208. The TiAl electrode pairs 218 and 220 are deposited on the top layer 66. Between electrodes 2 18 and 22 0 is a gap 222 for keeping the circuit open in the absence of liquid. The circuit is turned off when the liquid is present and the CMOS circuit 86 utilizes this feedback to monitor the flow. Non-gravity dependent test module 1 is non-directional dependent. These modules do not need to be fixed to a smooth surface to operate. The fluid flow driven by the capillary action and the external tubing that does not have to be connected to the auxiliary device make the module truly portable and can be easily inserted into a similar portable handheld reader, such as a mobile phone. -52- 201219770 Operation with non-gravity dependence means that these test modules also help to be independent of all practical applications. They can withstand shock and vibration, they can operate on moving vehicles, or when the mobile phone is moving. Nucleic acid amplification variant

Direct PCR Conventionally, PCR requires extensive purification of target DNA prior to preparation of the reaction mixture. However, with appropriate changes in chemical properties and sample concentrations, it is possible to perform nucleic acid amplification or direct amplification with minimal DNA purification. When the nucleic acid amplification program is PCR, this method is referred to as direct P C R. When the nucleic acid amplification is carried out in a controlled constant temperature LO C device, the method is directly thermostated. Direct nucleic acid amplification techniques have significant advantages when used in LOC devices, particularly with regard to simplifying the fluid design required. Adjustments to the amplification chemistry of direct PCR or direct isothermal amplification include boosting buffer strength, the use of reactive and processivity polymerases and additions to potential polymerase inhibitors. It is also important to dilute the inhibitor in the sample. In order to utilize direct nucleic acid amplification techniques, the LOC device design incorporates two additional features. The first feature is a reagent reservoir (e.g., reservoir 58 in Figure 8) that is suitably sized to supply a sufficient amount of amplification reaction mixture or diluent to cause the sample to interfere with amplification chemistry The final concentration of the ingredients is low enough to allow for successful nucleic acid amplification. The desired dilution factor of the non-cellular sample component is between 5 and 20 times. Different LOC structures are used where appropriate to confirm that a sufficiently high target nucleic acid sequence concentration -53 - 201219770 is maintained for amplification and detection, such as the pathogen dialysis section 70 of Figure 4. In this embodiment (further illustrated in Figure 6), a concentration effective to concentrate the pathogen that is small enough to enter the amplification portion 292 and discharge the larger cells to the waste container 76 is used upstream of the sample extraction portion 290. Dialysis department. In another embodiment, a dialysis section is used to selectively remove proteins and salts in plasma while retaining cells of interest. A second LOC structural feature that supports direct nucleic acid amplification is the channel aspect ratio design to adjust the mixing ratio between the sample and the amplified mixed components. For example, to ensure that the inhibitor associated with the sample is diluted to a preferred range of 5 to 20 times via a single mixing step, the length and cross-section of the sample and reagent channels are designed to be at the beginning of the mixing The upstream sample channel has a flow impedance that is 4 to 19 times higher than the flow impedance of the channel through which the reagent mixture flows. The flow impedance of the microchannel can be easily controlled by controlling the design geometry. In terms of a fixed cross-sectional area, the flow impedance of the microchannel increases linearly with the length of the channel. It is important for hybrid designs that the flow impedance in the microchannel is most dependent on the smallest cross-sectional area size. For example, when the aspect ratio is extremely non-uniform, the flow resistance of a microchannel having a square cross section is inversely proportional to the cube of the smallest vertical dimension. Reverse Transcriptase PCR (RT-PCR) When the sample nucleic acid species analyzed or extracted is RNA, such as from RNA virus or messenger RNA', the RNA must first be reverse transcribed into complementary DNA (cDNA) before PCR amplification. The reverse transcription reaction can be carried out in the same chamber as the PCR (one-step RT-PCR), or it can be an initial reaction (two-step RT-PCR) starting from -54 to 201219770. In the body described herein, a one-step RT-PCR can be carried out simply by adding a reagent reservoir 62 of reverse transcription j polymerase and staging the heater 1 54 to: perform a nucleic acid amplification step after the transcription step . The 2 2 PCR can also be carried out by using the reagent reservoir 58 for storing and dispensing sputum, primers, dNTPs, and reverse transcriptase, using the transcript step of the culture portion 1 14 J, followed by performing the 于 in the amplification portion 112 in an ordinary manner. Simply done. Thermostatic Nucleic Acid Amplification For some applications, thermostatic nucleic acid amplification is the preferred nuclear method, so that the reaction component does not need to be reloaded at different temperatures but maintains the amplification at typically about 37. (: to 41. C constant some constant temperature nucleic acid amplification methods have been described, including stock take (SDA), transcription-mediated amplification (TMA), nucleic acid sequence IJ-based (NASBA), recombinase polymerase amplification ( RPA), helicase-based temperature DNA amplification (HDA), rolling cycle amplification (RCA), branching (RAM), and circular media constant temperature amplification (LamP), among these methods or other constant temperature amplification methods Can be used in the LOC specific embodiments described herein. To perform isothermal nucleic acid amplification, the adjacent amplification sections of tests 60 and 62 will carry the appropriate reagents for a particular constant temperature method with a PCR amplification mixture. And the polymerase. For example, in the case of s 〇a, the reservoir 60 contains an amplification buffer, a primer, and a dNTP, and the reagent! LOC is changed to contain a buffer such as RT-such as an anti-increase to increase by i acid [loop, i. The amplification of the bottom amplification of the constant type amplification: the reagent reservoir of any device is not loaded with the reagent reservoir 62-55-201219770 contains the appropriate endonuclease and exo-DNA polymerase. For RPA, the reagent reservoir 60 contains amplification buffer, primer, dNTP, and recombinase protein. The reagent reservoir 62 contains a strand-substituted DNA polymerase, such as Bsu. Similarly, in the case of HDA, the reagent reservoir 60 contains an amplification buffer, an primer, and dNTP, and the reagent reservoir 62 contains a suitable DNA polymerase and The helicase removes the double stranded DNA instead of using heat. It will be appreciated by those skilled in the art that the necessary reagents can be dispensed into the two reagent reservoirs in any manner suitable for the nucleic acid amplification method. For viruses such as HIV or hepatitis C virus, NASBA or TMA is suitable because it does not need to first transcribe RNA into cDNA. In this example, the reagent reservoir 60 is filled with amplification buffer, Primer and dNTP, reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase and any RNase Η. Some thermostated nucleic acid amplification forms must have an initial denaturation cycle to separate the double-stranded DNA template before maintaining a suitable thermostated nucleic acid. The temperature of the amplification is carried out in a favorable reaction. This can be easily achieved in the embodiment of all LOC devices described herein, since the temperature of the mixing in the amplification section 112 can be obtained by the amplification microchannel 1 58 Heater 1 54 Fine control (see Figure 14). Thermostatic nucleic acid amplification is more resistant to potential inhibitors in the sample' and is therefore generally suitable for direct nucleic acid amplification from the sample. Therefore, thermostatic nucleic acid amplification sometimes It can be used for LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677, etc. shown in Figures 75, 76 and 77, respectively. Direct thermostatic amplification can also be compared with Figure 75-56-201219770 and 77. Illustrating one or more pre-amplification dialysis steps 70 '6 8 6 or 682 ' and/or combining pre-hybridization dialysis step 682 as shown in FIG. 76 to assist the target cells in the sample prior to nucleic acid amplification, respectively Partially concentrated, or remove unwanted cell debris before the sample enters the array of hybridization chambers. Those skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybridization dialysis can be used. Thermostatic nucleic acid amplification can also be performed in parallel amplification sections such as those shown in Figures 71, 72 and 73. 'Multiplexing and some methods of constant temperature nucleic acid amplification such as LAMP lines are compatible with the initial reverse transcription step to amplify rnA . Additional Details of Fluorescence Detection System Figures 58 and 59 show hybridization-reactive FRET probes 236. These probes, often referred to as molecular beacons, are stem-loop probes produced from single-stranded nucleic acids that fluoresce when hybridized to complementary nucleic acids. Figure 58 shows a single FRET probe 236 prior to hybridization to the target nucleic acid sequence 238. The probe has a ring 240, a stem 242, a fluorophore 246 at the V-end, and a quencher 248 at the 3 end. This loop 240 is composed of a sequence complementary to the target nucleic acid sequence 238. The complementary sequences flanking the probe sequence are joined together to form a stem 242 °. When the complementary target sequence is absent, the probe is closed as shown in Figure 58. The stem 242 keeps the fluorophore-quenching agent pairs close to each other such that significant resonance energy transfer can occur between them, substantially eliminating the ability of the fluorophore to emit fluorescence when illuminated by the excitation light 244. . Figure 59 shows FRET probe 236 in an open or hybridized configuration. When -57-201219770 hybridizes with the complementary target nucleic acid sequence 2 3 8 , the stem-loop structure is destroyed, and the fluorescent group and the quenching agent are spatially separated, thereby restoring the ability of the fluorescent group 246 to emit fluorescence. . The fluorescent emission 250 is optically detected as an indicator that the probe has hybridized. The probe hybridizes to the complementary target with very high specificity because the stem helix of the probe is designed to be more stable than the probe-target helix with a non-complementary single nucleotide. Since the double-stranded DNA is quite robust, the probe-target helix and the stem helix cannot coexist in the stereoscopic space. The probe-attached probe and primer-attached stem-loop probe and the primer-connected linear probe (also known as the scorpion-type probe) are alternative molecular beacons that can be used in LOC devices for immediate and quantitative Nucleic acid amplification. Immediate amplification can be performed directly in the hybridization chamber of the LOC device. An advantage of using a probe coupled to a primer is that the probe element is actually linked to the primer, so that only a single hybridization event is required during nucleic acid amplification without the need for separate primer hybridization and probe hybridization. This ensures an immediate and efficient response, resulting in a stronger signal, shorter reaction times and better recognition than when using separate primers and probes. The probes (and polymerase and amplification mixture) will be deposited in the hybrid chamber 180 during manufacture' without the need to provide separate amplifications on the LOC device. Alternatively, the amplification portion is not used or used for other reactions. Linear probes linked to primers Figures 78 and 79 show linear probes 692 linked to primers -58-201219770 during the first round of nucleic acid amplification, and primers linked to hybridization during subsequent nucleic acid amplification. Linear probe. Referring to Figure 7 8, the linear probe 692 coupled to the primer has a double-stranded stem segment 242. One of the probe sequences 696, which is homologous to the region 696 on the target nucleic acid, and which is linked to the primer, is labeled with a fluorophore 246 at the 5' end and amplified at the 3' end of the other. Blocker 694 is ligated to oligonucleotide primer 700. The other end of the stem 242 is labeled with a quencher group 248. Upon completion of the first round of nucleic acid amplification, the probe can be rolled up and hybridized to an extended strand having sequence 698 (now complementary). During the first round of nucleic acid amplification, the oligonucleotide primer 700 is affixed to the target DNA 23 8 (Fig. 78) and then extended to form a DNA strand containing both the probe sequence and the amplification product. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 696. Upon subsequent denaturation, the extended oligonucleotide primer 700/template hybridization system is separated, and the double-stranded stem 242 of the linear probe attached to the primer is also separated, thereby releasing the quencher 248. When the temperature is lowered for the bonding and extension step, the primer-linked probe sequence 696 of the linear probe linked to the primer is rolled up and hybridized with the amplified complementary sequence 69 8 on the extended strand, and can be detected. Fluorescence of the presence of the target DNA is displayed. A linear probe connected to the primer that is not extended retains the double stem and the fluorescence remains quenched. This test method is especially suitable for rapid detection systems because it requires only a single molecule of processing. Stem ring probes attached to the primers Figures 80A through 80F show the operation of the stem loop probe 704 attached to the primer. Referring to Fig. 80A, the stem loop probe 704 attached to the primer has a stem 242 complementary to the -59-201219770 double stranded DNA and a loop 24〇 having a probe sequence. The 5' end of one of the stems 708 is labeled with a fluorophore 246. The other 710 line is labeled with a 3'-end quencher 248 and carries both amplification blocker 694 and oligonucleotide primer 7 00. During the initial denaturing phase (see Figure 80B), the strands of the target nucleic acid 23 8 are separated, and the stem 242 of the stem loop probe 704 attached to the primer is also separated. When the temperature is cooled to effect the binding phase (see Figure 80C), the oligonucleotide primer 700 on the stem-loop probe 704 linked to the primer hybridizes to the target nucleic acid sequence 23 8 during extension (see Figure 80D), the complementary sequence 706 of the target nucleic acid sequence 23 is synthesized to form a DNA strand containing both the probe sequence 704 and the amplification product. The amplification blocker 694 prevents the polymerase from reading and replicating the probe region 7〇4. When the probe is bonded after denaturation, the probe sequence of the loop segment 240 of the stem-loop probe attached to the primer (see Figure 80F) is bonded to the complementary sequence 706 on the stretched strand. This configuration is such that the fluorophore 246 is far removed from the quencher 248, resulting in significantly enhanced fluorescene emission. Control Probes Hybridization chamber array 110 includes a number of hybridization chambers 180 with positive and negative control probes for analytical quality control. Figures 94 and 95 schematically illustrate negative control probes containing no fluorophores 796, and Figures 9.6 and 197 show positive control probes without quencher 798. The positive and negative control probes have a stem-loop structure as described above for the FRET probe. However, regardless of whether the probes hybridize to an open configuration or remain closed, the positive control probe 798 will emit a fluorescent signal 250' and the negative control probe 796 will never emit fluorescence-60-201219770 Signal 2 5 0. Referring to Figures 94 and 95, the negative control probe 796 does not have a fluorophore (which may or may not have a quencher 248). Thus, the reaction to excitation light 244 can be ignored regardless of whether the target nucleic acid sequence 2 3 8 hybridizes to the probe (see Figure 95) or the probe maintains its stem-loop configuration (see Figure 94). Alternatively, the negative control probe 796 can be designed such that it remains quenched forever. For example, by synthesizing a loop 24' of the probe molecule having a probe sequence that does not hybridize to any of the nucleic acid sequences in the study sample, the stem 242 of the probe molecule will self-hybridize, allowing the fluorophore and quenching The agents remain in close proximity without emitting visible fluorescent signals. This negative control signal corresponds to a small amount of emission from the hybridization chamber 180, wherein the probes are not hybridized but the quencher does not quench all of the emission from the reporter. Conversely, the positive control probe 798, which does not contain a quencher, was constructed as shown in Figures 96 and 97. Regardless of whether the positive control probe 798 is hybridized to the target nucleic acid sequence 238, no material will quench the fluorescent emission from the fluorophore 246 back to the luminescent 244. Figure 5 2 shows the possible distribution of positive and negative control probes (3 78 and 380, respectively) in the hybrid chamber array. The control probes 3 78 and 380 are placed in a hybridization chamber 180 located on a line that traverses the hybridization chamber array 110. However, the configuration of the control probes in the array is arbitrary (as in the configuration of the hybrid chamber array 1 1 0). The fluorophore design requires a fluorophore with a long fluorescence lifetime, which is to allow the excitation light to have -61 - 201219770 Sufficient time to decay to a low intensity compared to the intensity of the fluorescent emission when the photosensor 44 is enabled, thereby providing a sufficient signal to noise ratio. Moreover, a longer fluorescent lifetime represents a larger integrated fluorescence photon count. The fluorophore 246 (see Figure 59) has a fluorescence lifetime greater than 100 nanoseconds, often greater than 200 nanoseconds, more typically greater than 300 nanoseconds, and in most cases greater than 400 nanoseconds. Metal-coordination complexes based on transition metals or lanthanide metals have long lifetimes (from hundreds of nanoseconds to milliseconds), appropriate quantum yields, and high thermal, chemical, and photochemical stability. Both are advantageous for the needs of fluorescent detection systems. In particular, the metal-coordination complex based on the transition metal ion ruthenium (Ru (II)) is ginseng (2,2 bipyridyl) ruthenium (II) ([Ru(bpy)3]2 + ), the life of his is about Ιμβ. This complex is commercially available from Biosearch Technologies' under the trade name Pulsar 650. Table 1: Photophysical properties of Pulsar 650 (钌 合物 合物)

Parameter symbol 値αα — single 兀 absorption wavelength ^-abs 460 nm emission wavelength ^em 650 nm absorption coefficient E 14800 M'1 cm'1 叩 叩 Tf 1.0 ps quantum yield Η 1 (deoxygenated) N/A Μ The honey complex is a lanthanide metal-coordination complex that has been successfully demonstrated as a fluorescent reporter for the FRET probe system and has a long life of 1600 ps. -62 - 201219770 Table 2: 铽 Chelation Photophysical properties of matter Parameter symbol 値 Element absorption wavelength ^abs 330-350 nm Emission wavelength λετη 548 nm Absorption coefficient Ε 13800 (with hbs and ligand dependence, up to 30000 @ λβ = 340 nm) Fluorescence lifetime Tf 1600 (Hybridization Probe) μδ Quantum Yield Η 1 (Coordination Dependent) Ν/Α The fluorescence detection system used by the LOC device 301 does not use filters to remove unwanted background fluorescence. Therefore, if the quencher 248 has no natural emission in order to increase the signal to noise ratio, it is advantageous. Quenching agents without natural emission 248 will not contribute to background fluorescence. High quenching efficiency is also important, so there is no fluorescence before the hybridization event occurs. The black hole quencher (BHQ) from Biosearch Technologies, Inc. of Novato, Calif., does not have a natural emission and has high quenching efficiency, and is suitable for the quenching agent of the system. The maximum absorption enthalpy of BHQ-1 occurs at 534 nm and the quenching range is 480-580 nm' making it suitable for quenching Tb-chelating fluorophores. The maximum absorption enthalpy of BHQ-2 occurs at 579 nm and the quenching range is 560-670 nm, making it suitable for quenching the Pulsar 650. Iowa Black Quenchers (I〇wa Black FQ and RQ) from Intergrated DNA Technologies, Coralville, Iowa, are suitable alternative quenchers with little or no background emission. I〇wa -63- 201219770

The Black FQ ranged from 420 to 620 nm with a maximum absorption 531 at 531 nm and is therefore suitable as a quencher for Tb-chelating fluorophores. The maximum absorption enthalpy of Iowa Black RQ occurs at 656 nm and the quenching range is from 500 to 700 nm, making it an ideal quencher for the Pulsar 650. In the embodiments described herein, the quencher 248 is a functional group that is attached to the probe initially, but in other embodiments, the quencher may be a separate molecule that is free in solution. Excitation source In the implementation of the fluorescence detection as the substrate described in this paper, the LED is selected as the excitation source instead of the laser diode, high power lamp or laser because of the low power consumption, low cost and small size of the LED. . Referring to Figure 81, LEDs 26 are placed directly over hybrid array array 110 of the outer surface of LOC device 301. Opposite to the hybridization chamber array 110 is a photosensor 44 consisting of an array of photodiodes 84 for detecting fluorescent signals from the chambers (see Figures 5 3, 5 4 and 6). 4). Figures 82, 83 and 84 schematically illustrate other embodiments for exposing the probe to excitation light. In the LOC device 30 shown in Fig. 82, the excitation light 244 generated by the excitation LED 26 is directed by the lens 254 over the hybrid cell array 110. The excitation LED 26 is pulsed and the photodetector 44 detects the fluorescence emission.

In the LOC device 30 shown in FIG. 83, the excitation light 244 generated by the excitation LED 26 is directed by the lens 2 54 , the first optical stop 712 and the second optical stop 714 over the hybridization chamber array 110. The excitation LED -64 - 201219770 26 is pulsed and the fluorescent sensor 44 detects the fluorescent emission. Similarly, in LOC device 30 as shown in FIG. 84, excitation light 244 generated by excitation LED 26 is directed by lens 254, first mirror 716, and second mirror 718 onto hybridization cell array 110. Furthermore, the excitation LED 26 is pulsed and the photodetector 44 detects the fluorescence emission. The excitation wavelength of the LED 26 is dependent on the choice of fluorescent dye. Philips LXK2-PR14-R00 is the appropriate source of excitation for Pulsar 650 dyes. SET UVT0P3 3 5 T039BL LED is the appropriate excitation source for the ruthenium chelate label. Table 3: Philips LXK2-PR14-ROO LED Specifications

Parameter Symbol 値 Element Wavelength λεχ 460 nm Transmit frequency Vem 6.52(10)14 Hz Output power Pi 0.515 (min) @ ΙΑ W Transmit mode Lambertian data plot N/A Table 4 : SET UVTOP334TQ39BL LED Specifications

Parameter Symbol 値 Single 兀 Wavelength 340 nm Transmit frequency Ve 8.82(10)14 Hz Power Pi 0.000240 (min) @ 20mA W Pulse forward current I 200 mA Emission mode Lambertian N/A UV excitation 矽 Absorbs a small amount of UV light. Therefore, the use of UV excitation light is beneficial -65-201219770. An LED excitation source can be used, but the broad spectrum of LED 26 reduces the effectiveness of this method. To solve this problem, filtered UV light L E D can be used. Optionally, an ultraviolet laser can be used as an excitation source unless the relatively high cost of the laser is not practical for a particular test module market. LED Driver The time required for the LED driver 29 to drive the LED 26 at a fixed current. The low-power USB 2.0-certified unit can supply up to 1 unit load (1 毫 mA) with a minimum operating voltage of 4·4 volts. Standard power conditioning circuits are used for this purpose. Photodiode Figure 54 shows a photodiode 184 that breaks into the CMOS circuit 86 of the LOC device 301. The photodiode 180 is part of a CMOS circuit 86 that is fabricated without additional masking or steps. This is a significant advantage of CMOS photodiodes over CCDs, an alternative sensing technique that can be integrated on the same wafer or fabricated on adjacent wafers using non-standard processing steps. On-wafer inspection is inexpensive and reduces the size of the array system. This shorter optical path length reduces noise from the surrounding environment to effectively collect fluorescent signals and reduce the need for conventional optical assemblies for lenses and filters. The quantum efficiency of the photodiode 184 is the fraction of photons that are effectively converted into photoelectrons in the photons that collide with their active regions 185. In terms of standard enthalpy treatment, the quantum efficiency of visible light ranges from 〇.3 to 0.5, depending on the processing parameters such as the amount of cover layer and the absorption characteristics of -66 - 201219770. The detection valve of the photodiode 1 84 determines the minimum intensity of the fluorescent signal that can be detected. The detection valve 値 also determines the size of the photodiode 184, and thus the number of hybridization chambers 180 in the hybridization and detection section 52 (see Fig. 52). The size and number of such chambers are limited by the size of the LOC device (in the case of LOC device 301, which is 1760 micro X 5 8 24 microns) and into other functional modules such as the pathogen dialysis unit 70 and The technical parameters of the size of the available space after the amplification unit 1 1 2 . In terms of standard 矽 processing, the photodiode 1 8 4 detects a minimum of 5 photons. However, to ensure reliable detection, the minimum chirp can be set to 10 photons. Thus, in the quantum efficiency range of 0.3 to 0.5 (as discussed above), the fluorescence emission from the probes should be a minimum of 17 photons, but 30 photons will contain an appropriate margin of error for reliable detection. Calibration Chamber The non-uniformity of the electrical characteristics of the photodiode 184, the auto-fluorescence, and the residual excitation photon flux that is not fully attenuated introduces background noise and offset into the output signal. This background is removed from each output signal using one or more calibration signals. The calibration signal is generated by exposing one or more of the calibrated photodiodes 1 84 in the array to individual calibration sources. The low calibration source is used to determine the negative result of the target 尙 not reacting with the probe. The high calibration source is indicative of a positive result from the #needle-target complex. In the actual state described herein, the low calibration source is provided by a calibration chamber 382 in the hybrid chamber array , 0, 'the calibration chamber 3 82 : -67- 201219770 does not contain any probes A probe containing no fluorescent reporter: or a probe having a reporter and a quencher configured to cause quenching to occur forever. The output signal from the calibration chamber 382 is very close to the noise and bias in the output signal from all of the hybrid chambers in the L〇c device. The output signal generated by the other hybridization chamber minus the calibration signal substantially removes the background, leaving the signal generated by the fluorescent emission (if any). Signals generated by ambient light in the area of the chamber array are also removed. It will be appreciated that negative control probes as described above with reference to Figures 94 to 9 can be used in the calibration chamber. However, as shown in Figures 86 and 87, which are enlarged views of the DG and DH regions of LOC variant X 72 8 as described in Figure 85, another option is to make the calibration chamber 382 fluid with the amplicons. isolation. The background noise and bias can be determined by maintaining the fluid isolation chamber clearance or by using a probe containing no reporter or indeed any "standard" probe having both a reporter and a quencher, as the hybrid Blocked by fluid isolation. The calibration chamber 382 provides a high calibration source to produce a high signal at the corresponding photodiode. This high signal corresponds to all probes that have been hybridized in the chamber. Spotting a probe with a reporter but no quencher, or a probe with only a reporter will consistently provide a signal that approximates that most of the probes in the hybridization chamber have been hybridized. It will also be understood that the 'calibration chamber 382 can be used in place of or in addition to the control probe. The number and arrangement of calibration chambers 382 in the array of hybridization chambers is arbitrary. However, if the photodiode 184 is calibrated from a relatively close calibration chamber 382-68-201219770, the calibration will be more accurate. Referring to Figure 56, the hybridization chamber has one calibration chamber 382 for every eight hybridization chambers 180. That is, the calibration chamber 382 is placed in a square of every three by three hybrid chambers. In this configuration, the hybrid chambers are aligned with the calibration chamber. Figure 93 shows a differential imager circuit 788 which subtracts the signal from the fluorescent signal of the surrounding hybridization chamber 180 corresponding to the photodiode 184 of the 308 due to the excitation light. Imager circuit 78 8 is from pixel 790 and "virtual" pixel 792 number. In one embodiment, the "virtual" pixel 792 is illuminated by illumination' so its output signal provides a dark reference. Alternatively, the "pixel 792 can be exposed to the excitation light "virtual" pixel 792 together with other portions of the array to be exposed to light, and the signal generated by ambient light in the chamber region is also subtracted. The signal is very weak (ie, close to the dark signal), and it will be difficult to distinguish between the background and the very weak signal without the dark signal. During use, the "read_column" 794 and "read_795" are used. Startup, and the M4 797 and MD4 801 transistors are turned off by switches 807 and 809 such that the outputs from the pixel 790 and "virtual 792 are stored in pixel capacitor 803 and virtual imager 805, respectively. After the pixel signal is stored, The switch 807 is deactivated and then the "read_row" switch 8 1 1 and the virtual "read-line" are turned off, and the switched capacitor amplifier 8 1 5 at the output amplifies the indication. 8 17° column 1 1 0 is Said, in the middle. 3 82 school calibration system with the calibration room, the differential sampling signal to prevent virtual. In the array of elements 790 quasi-column a d" start. Open" pixel pixel 8 09 ° off 8 1 3 difference signal -69- 201219770 photodiode suppression and enable the photodiode 1 8 4 Must be suppressed during LED 26 excitation and must be enabled during fluorescence. Fig. 6 is a circuit diagram of a single photodiode 〖8 4, and Fig. 66 is a timing diagram of a photodiode control signal. The circuit has a photodiode 184 and six MOS transistors Mshunt 394, Mtx 396, Mreset 3 98, Msf 400, Mread 402, and Mbias 404. At the beginning of the excitation cycle t1, the transistors Mshunt 394 and Mreset 3 98 are turned on by pulling up the Mshunt gate 384 and resetting the gate 3 88. During this period, the excitation photons generate a carrier in the photodiode 184. These carriers must be removed because the amount of carrier generated is sufficient to saturate the photodiode 184. During this cycle, Mshunt 3 94 directly removes the carrier generated in photodiode 184, while Mreset 3 9 resets any accumulation of nodes 'NS' due to transistor leakage or carrier diffusion due to excitation in the substrate. Carrier of 406. After excitation, the capture cycle begins at t4. In this cycle, the emission reaction from the fluorophore is captured and integrated into the circuitry on node 'NS' 406. This is achieved by pulling up the tx gate 386, which turns on the transistor Mtx 3 96 and transfers any carrier accumulated on the photodiode 184 to the node 'NS' 460. The length of the capture cycle can be as long as the fluorophore emission. The outputs from all of the photodiodes 184 in the hybrid cell array 110 are simultaneously captured. There is a delay between the end of the capture cycle t5 and the beginning of the read cycle t6. This delay is due to the need to separately read each photodiode 184 in the hybrid array 810 after the capture cycle (see Figure 52). The first photodiode 184 that is read -70-201219770 will have the shortest delay before the read cycle, while the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, transistor Mread 402 is turned on by pulling high on read gate 3 93. The voltage of the 'NS' node 406 is buffered and read using the source follower transistor Msf 400. Other optional methods of activating or suppressing the photodiode are discussed below: 1. Inhibition Methods Figures 90, 91 and 92 show three possible configurations 778, 780, 782 of Mshunt transistor 394. The Mshunt transistor 394 has a very high turn-off ratio at maximum | VGS | = 5 V, which is enabled during excitation. As shown in FIG. 90, the Mshunt gate 3 84 is configured at the edge of the photodiode 184. Optionally, as shown in FIG. 91, the Mshunt gate 3 84 is configured to surround the photodiode 1 84. The third option is to configure the Mshunt gate 384 within the photodiode 184 as shown in FIG. In the third option, the active region 185 of the photodiode will be smaller. These three configurations 778, 780, and 782 reduce the average path length from all locations in the photodiode 184 to the Mshunt gate 3 84. In Fig. 90, the Mshunt gate 384 is located on one side of the photodiode 184. This configuration is the easiest to manufacture' and does not occupy the active area 1 85 of the photodiode. However, any carrier remaining on the far-end side of the photodiode 1 84 takes a long time to pass through the Mshunt gate 3 84. In Figure 91, the Mshunt 384 is surrounded by the photodiode -71 - 201219770 184. This further reduces the average path length of the carrier in the photodiode 184 to the Mshunt gate 3 84. However, the Mshunt gate 384 extending around the photodiode 184 causes a significant reduction in the photodiode active region 185. Configuration 782 in Figure 92 positions Mshunt Gate 3 84 in active area 185. This provides the shortest average path length to Mshunt Gate 3 84, so the transition time is the shortest. However, the encroachment of the active zone is the largest. It also creates a wider leak path. 2. Enabling method a. Trigger the photodiode to drive the shunt transistor with a fixed delay. b. Trigger the photodiode to drive the shunt transistor with a programmable delay. c. The shunting transistor is driven by the LED drive pulse with a fixed delay. d. Drive the shunt transistor as a 2c but with a programmable delay. Figure 68 is a schematic cross-sectional view of the hybridization chamber 180 showing the photodiode 184 and the trigger photodiode 187 embedded in the CMOS circuit 86. The small area in the corner of the photodiode 184 is replaced by the trigger photodiode 187. Triggering the photodiode 187 with a small area is sufficient because the intensity of the excitation light will be higher than the fluorescence emission. The trigger photodiode 187 is sensitive to the excitation light 244. The trigger photodiode 187 temporarily stores the excitation light 244 to be extinguished, and activates the photodiode 184 after a short delay of Δt 3 00 (see Fig. 2). This delay allows the fluorescent photodiode 184 to detect fluorescent emissions from the 'FRET Detector-72-201219770 pin 186 when there is no excitation light 244. This enables detection and enhancement of the signal photodiode 184 and the CMOS circuit 86 under the hybridization chamber 180 of the photodiode 187. The optoelectronic combination, together with appropriate electronic components, forms the photosensor 44. The photodiode 184 is fabricated during the fabrication of the CMOS structure without the need for additional masking or steps. A dielectric layer (not shown) over the MS T diode 184 is arbitrarily thinned using a marking technique to allow more phosphor to illuminate the active region 185 of 184. The photodiode 184 has a photodetector surface from a probe-target hybrid within the hybridization chamber 180. This fluorescent light is converted into a photocurrent which is measured by a CMOS circuit 86. Alternatively, one or more of the hybridization chambers 180 can be configured with only the contact 187. These choices can be combined with the above 2a and 2b to detect the delay of the monthly fluorescence. The following derivation illustrates the use of long-lived fluorophores for the delayed detection of fluorescence by a combination of lumps. The fluorescence intensity is excited by the g ideal pulse between time t and t2, as shown in Fig. 60. Let [si](t) be equal to the intensity of the excited state at time t and after excitation, the unit time per unit volume is described by the following differential equation: Noise ratio Both are located in the array of diodes (see Figure 64). During the fabrication of the pn fabrication, the photoelectric [quasi MST etches the field of view of the photodiode to cause the incoming signal to be incident on the sense current and then the function of the LED/fluorescent intensity U. Derived and then excited the number of excited states -73- 201219770 c/[51] { [S\](Q _ Ieec dt tf hve where c is the molar concentration of the fluorophore, ε is the molar furnace, coefficient , ye is the excitation frequency, and h = 6.62606 896(1 0)·34 Js is the Planck constant. This differential equation has the general formula: word + p(x) less = 9(x) αχ There is a solution: ...( 2) fe^( )dXq(x)dx + k — Now use this to solve equation (1), (3) hve at time h, [Sl](ti) = 〇, and from (3): /e £Cr=~^r

Substituting (4) into (3): hvc hve at time t2: When t 2 t2, the excited state is exponentially decayed and described by equation (6): [5Ί](/) = [51](/2Κ(, ^)/γ/ (6) -74- 201219770 Substituting (5) into (6): [51] (0 = _ e-^Vrf hve The fluorescence intensity is obtained by the following equation h^ = -^~-hvfnl (8) where vf is the fluorescence frequency and η is the length of the diameter. Then from (7): dt hve substituting (9) into (8): /〆,)=/e£C/7 k[ l - el<1)/r,]e-(,_,2)/r, · When 匕^->00, b(〇4/e£c/77'e, 2)/r/ r / s Therefore, we can write the following near-intensity in a sufficiently long excitation pulse (t2-n >> when Q t2 If(t) = Ie£C!}]ke-(lh), T/ In the section, we target t2-t! >> 1 when = . ^ e From the above equation, we can derive rif{t) = «e£c/7e"(,",i)/r/ . (12) where...(7) sub-yield, and 1 is the optical path • •(9) ·0〇), which describes the attenuation after the fluorescent rf): ".(11) :f Summary 'column: -75- 201219770 ...If(t) = the number of fluorescent photons per unit area per unit time and 乂=7^- for each order Area per unit time of the excitation photon number. Therefore, «〇[rif(t)dt (13) where seven is the number of fluorescent photons per unit area and t3 is the time point at which the photodiode is turned on. Substituting (1 2 ) into (1 3 ): 00

Hf = \fieec^e'{,~,lVrr dt (14) At present, the number of photons t(7) per unit area per unit area of the photodiode is obtained by the following equation: ^(0 = «&gt ;W0 ...(15) where nine is the light collection efficiency of the optical system. Substituting (12) into (15) we find Κ(0 = Φ〇η^ηε~{,"ι)η/ ...( 16) Similarly, the number of fluorescent photons per unit of fluorescent area reaching the photodiode will be as follows: 〇〇屺 = (1) 代 and substituted (1 6) and integrated: h hs = < P〇nesc ^ Tfe '{,i~'l)lT/ Therefore, ns = φ^εοίητ^'^ (17) The ideal 値 of t3 is in the photodiode 1 8 4 because of the fluorescent photon-76-201219770 The resulting electron rate becomes equal to the electron rate produced by the excited photons in the photodiode 184 because the excitation photon flux decays more rapidly than the fluorescent photon flux decays. The electron rate per unit of fluorescent area of the sensor due to fluorescence is: where (Pf is the quantum efficiency of the sensor at the wavelength of the fluorescence. Substituting (17) we get: e'f (Ο = Φί Φ^εεΙ ηε~°~'ι),τ/ .. . (18) Similarly, the electron ratio of the sensor per unit of fluorescence area due to excitation of photons is: where is the sensor at the excitation The quantum efficiency at the wavelength is the time constant corresponding to the "off" characteristic of the LED. After time t2, the attenuated photon flux of the LED increases the intensity of the fluorescent signal and extends the decay time of the fluorescent signal, but We assume that this effect on If(t) can be ignored, so we take a conservative approach. Currently, as mentioned earlier, the ideal t3 is: ef(h) = ee(h) From (18) and (19) we get: and after the reformation we get: -77- 201219770 Ιη(εο1η^-) —I~~f— -(2〇)

Tf Te From the above two paragraphs, we get the following two expressions: ns = Φ〇ΚΡτίβ'^'Τί ...(21) ψΜ,

Ar= \--χ-' -(22) xf where corpse = «?/;7 and -f2. We also know that ί2 -ί, »Ζ7. The ideal time for fluorescence detection and the number of fluorescent photons detected using Philips LXK2-PR 14-ROO LED and Pulsar 650 dye are as follows. The ideal detection time is determined using equation (2 2): recalling the concentration of the amplicon, and assuming that all amplicons are hybridized, the concentration of the fluorescent fluorophore is: c = 2.89 (10) _ 6 mol / L. The height of the chamber is the optical path length 1 = 8 (1 0)_6 m. The fluorescent region has been regarded as equivalent to the photodiode region, but the actual fluorescent region is substantially larger than the photodiode region; therefore, it is assumed that nine = 0.5 is the light collecting efficiency of the optical system. From the characteristics of the photodiode, i = 1Q is extremely conservative, and the quantum efficiency of the photodiode at the fluorescence wavelength is proportional to the quantum efficiency at the excitation wavelength. With a typical LED decay life of 0.5 nanoseconds and the use of pulsar 6 50 specifications, Δί : -78- 201219770 F = [1.48(10)6 ] [2.89(10)'6 ] [8(10)' 6 ](1) = 3.42(10)-5 1η([3·42(1〇Γ5](10)(0.5))

At~ 1 1 1(10)-6 ~ 0.5(10)-9 = 4.34(10)'9 s The number of photons detected is determined by equation (2 1). First, the number of excitation photons emitted per unit time is determined by examining the illumination geometry.

The Philips LXK2-PR14-R00 LED has a Lambertian emission mode, so: n, = nl0 cos(0) ...(23) where) ϊ, the angle to the direction of the forward axis of the LED is 每 per unit solid angle per The number of photons emitted per unit time, and 6. For Κ, in the direction of the forward axis. The total number of photons emitted by the LED per unit time is: /?, = |η; ί/Ω Ω a ... (24) Now, ΔΩ = 2π[1 - cos(0 + Δ0)] - 2π[1 - Cos(9)] ΔΩ = 2^[cos(^) - cos(^ + Δ ^)] :4;rsin(0)cos •^X'^J + ^cos^sin2 άΩ. = 2nsm{6)d0 Substitute this (24): 79- 201219770 ή, = J2^i;ocos(0)sin(0)i/^ 0 =%0 Rearrange, we get:

...(26) The output power of the LED is 0.515 watts and ve = 6 _52(10)14 Hz, therefore: h-Pj_ ni~ , ...(27) =_0.515_ "[6.63(10)- 34][6.52(10>14]=1.19(10)18 Photon 値 Bring this 入 into (26) We get: ..._ 1.19(10)18 n,0 = — =3.79(10)17 Photon shape Referring to Fig. 6, the optical center of the LED 26 is shown in Fig. 6. The photodiode of the photodiode system is 16 micrometers x 16 microarrays, and the solid angle of the light cone emitted from the LED 26 is (Ω). Approx.: Ω = sensor area / r2 and lens 2 5 4 shows meters, and for the array of photodiodes 184 [16 (10) ^ 1116 (10) -6] 2.825 (10) - 3] 2 = 3.21(10)·5 Sphericality It will be understood that the central light of the photodiode array 44 is used for these calculations. The edge of the array is located in the diode 1 8 4 by the sensor at -80 - 201219770

The Lambertian excitation source intensity distribution of the hybridization event only receives photons. Number of excitation photons emitted per unit time: ne = ri, Q. ...(28) = [3.79(1 0),7][3.21(10)-s] =1.22(10)13 Photon reference equation now (29): ns = (0.5)[1.22(10)13][3.42(10)-s][l(10)-6]e-434(1〇)',/,(,0^ =208 photons /Sensor Thus, using the Philips LXK2-PR14-R00 LED and 650 Fluorescent Cluster, we can easily detect any hybridization events that cause these numbers to be emitted. The SET LED illumination geometry is shown in Figure 62. = ampere, the LED has a minimum optical power output Pl = 240 wavelength center at = 340 nm (absorption wavelength of ruthenium chelate). J 2 00 mA drive the LED will linearly increase the output power to Pl = watt. By placing the optical center 25 2 of the LED at approximately 1 7.5 mm from the hybridization array, we approximate this output flux to a dot size of 2 mm in diameter. In the 2 mm diameter point of the hybrid array plane Photon flux 27 is obtained. At 2%

Pulsar light: 20 nanowatts, 2 ID = 2.4 millicolons 1 1 0 has the largest equation -81 - 201219770 Ρι hye 2.4(10)-3 ~ [6.63(10)-34][8.82(10) m] = 4.10(10)15 Photon 渺 Using Equation 28 we get: ne = η, Ω 4.10(10)15 [16(10)-6]2 r[l(l〇)·3]2 3.34(10 11 Photon shape Now, return to Equation 22 and use the previously listed Tb chelate properties, "ln[(6.94(10)-5)(10)(0.5)] Δ/ =-Γ~ 1 ' 1 ( 10) '3 ~ 0.5(10)-9 = 3·98(10)·9 seconds Now from the equation 21: ns = (0.5)[3.34(10)n][6.94(10)-5][l( 10)-3]e-398(,O),/,(,o>'5 = 11,600 photons/sensors are theoretical numbers of photons emitted by hybrid events using SET LED and M chelate systems It can be easily detected and far exceeds the low limit of 30 photons required for reliable detection of the photosensor as described above. The maximum spacing between the probe and the photodiode does not need to be detected on the wafer. The yoke microscope (see prior art) performs the test. This difference from the traditional detection technology represents an important factor in saving time and cost for this system. Traditional testing needs to be made -82 - 20121977 0 Imaging optics with lenses and curved mirrors. By using non-imaging optics, the diagnostic system avoids the need for complex and cumbersome strings of optical components. The placement of photodiodes in close proximity to the probes has the advantage of extremely high collection efficiency: When the material thickness between the probe and the photodiode is 1 micron, the collection angle of the emitted light is as high as 173°. This angle is considered by the surface center of the hybridization chamber closest to the photodiode. Calculated by the light emitted by the probe, the photodiode has a planar active surface area parallel to the surface of the chamber. Light within the cone of light emission can be absorbed by the photodiode, which is defined at its apex And a sensor probe at the corner of the sensor has a transmitting probe. Taking a 16 μm X 16 μm sensor as an example, the apex angle of the pyramid is 17 〇 °; the photodiode is expanded In a limiting case where the area is such that the area of the hybridization chamber of the 29 μm X 19.75 μm is satisfied, the apex angle is 173·. It is easy to achieve an interval of 1 μm or less between the surface of the chamber and the active surface of the photodiode. Should The non-imaging optical method does require that the photodiode 184 be in close proximity to the hybridization chamber to collect a sufficient amount of fluorescent emission photons. The maximum spacing between the photodiode and the probe is determined as described below with reference to Figure 54. For the fluorophore and SET UVT0P335T039BL LED, we calculated that 1 1 600 photons were passed from individual hybridization chambers 180 to a 16 micron to 16 micron photodiode 184. In carrying out this calculation, we assume that the light collection region of the hybridization chamber 180 has the same bottom area as the photodiode active region 185, and that half of the total number of hybrid photons reaches the photodiode 184. That is to say, the light collection efficiency of the optical system is ???=0.5. -83· 201219770 More precisely, we can write nine = [(the bottom area of the light collection area of the hybridization chamber) / (photodiode area)] [Ω / 4τγ], where Ω = the base of the hybridization chamber The solid angle of the photoelectric diode of the representative point on the opposite side. In terms of the geometry of the regular square pyramid: 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 . Each of the hybrid chambers released 23,200 photons. The selected photodiode has a detection low limit of 17 photons, so the minimum optical efficiency required is: φ0 = 1 7/23 200 = 7.3 3xlO'4 The light collection area of the hybridization chamber 180 The bottom area is 29 microns χ 1 9.75 microns. Decomposing dQ will result in a maximum limiting distance between the hybridization chamber and the photodiode 1 84 of d 〇 = 249 μm. Under this limitation, the collecting cone angle as defined above is only 0.8°. It should be noted that this analysis ignores the negligible effect of refraction. And a selective system for the LOC device. Figure 8 is an embodiment of a test module 10 configured to measure blood glucose levels. The LED spectrometer 772 is comprised of a set of LEDs 26.1, 26.2, 26.3, each of which transmits a different wavelength to the hybridization chamber 180 via a light window 136. The photodiode 184 detects light of different wavelengths that penetrate the untreated blood sample 770 and produces a spectrum. It should be noted that the LOC device 30 1 (see Figure 5) will be suitable for use with this type of test module. -84- 201219770 Alternative sources of excitation that do not require mobility or low cost. Alternative sources of excitation include lasers and gas flashes (such as helium). Figure 89 shows the configuration using laser 774 and directed to hybrid cell array 1 1 via optical element string 776. The laser 774 is activated to emit short pulses, and the photo sensor 44 (which includes an array of photodiodes 184) obtains a fluorescent emission. Combinations of different types of excitation sources can be used in the same system to excite fluorophores having different excitation spectra coupled to multiple probes in the hybrid chamber array. Fluorescent data for different probes can be obtained by continuously activating the different excitation sources, which in effect utilizes multiple analysis of each hybridization chamber. Conclusion The devices, systems, and methods described herein facilitate rapid, inexpensive, and suitable molecular diagnostic testing for local care. The above-described system and its components are merely illustrative, and many variations and modifications will be readily apparent to those skilled in the art without departing from the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings in which: FIG. 1 shows a test module and test module configured for fluorescence detection. Figure 2 is a schematic diagram of the electronic components in the test module configured for fluorescence detection; -85- 201219770 Figure 3 is a schematic diagram of the electronic components in the test module reader; Figure 4 is a schematic view of the structure of the LOC device; Figure 5 is a perspective view of the L Ο C device; Figure 6 is a plan view of the LOC device, the features and structures of all layers are overlapped with each other: Figure 7 is a plan view of the LOC device, It shows the upper cover's sag separately. Fig. 8 is a top view of the upper cover, showing the internal passage and the receptacle by dashed lines; Figure 9 is an exploded top view of the upper cover, showing the internal passage and the receptacle by dashed lines; Figure 10 is the upper cover The lower view, which shows the configuration of the upper channel; Figure 11 is a plan view of the LOC device, which shows the structure of the CMOS + MST device independently; Figure 12 is a schematic cross-sectional view of the sample inlet of the LOC device; Figure 13 is the AA area shown in Figure 6. Enlarged view; Figure 14 is shown in Figure 6. Figure 15 is an enlarged view of the AE area shown in Figure 13; a partial perspective view of Figure 16 illustrates the layered structure of the LOC unit in the AE area; a partial perspective view of Figure 17 illustrates the AE area The layered structure of the LOC device; a partial perspective view of Figure 18 illustrates the layered structure of the LOC device in the AE zone; a partial perspective view of Figure 119 illustrates the layered junction of the L Ο C device in the AE zone -86-201219770 Figure 20 is a partial perspective view showing the layered structure of the LOC device in the AE area; Figure 21 is a partial perspective view showing the layered structure of the LOC device in the AE area; Figure 22 is a lysing reagent reservoir shown in Figure 21. FIG. 23 is a partial perspective view illustrating the layered structure of the LOC device in the AB region; FIG. 24 is a partial perspective view illustrating the layered structure of the LOC device in the AB region; FIG. 25 is a partial perspective view illustrating the AI region Layered structure of the inner LOC device; part of the perspective view of Fig. 26 illustrates the layered structure of the LOC device in the AB zone; and a partial perspective view of Fig. 27 illustrates the layered relationship of the LOC device in the AB zone, part of Fig. 28. The perspective view illustrates the layered structure of the LOC device in zone AB; a partial perspective view of Figure 29 illustrates the layering of the LOC device in zone AB Figure 3 is a schematic cross-sectional view of the expansion mixing reservoir and the polymerase reservoir; Figure 31 shows the characteristics of the boiling start valve independently; Figure 32 is a schematic cross-sectional view of the boiling start valve, which is shown in Figure 31 Line 3 3 - 3 3 is obtained; -87- 201219770 Figure 33 is an enlarged view of the AF area shown in Figure 15; Figure 3 is a schematic cross-sectional view of the upstream end of the dialysis section, which is shown in Figure 33 Figure 35 is an enlarged view of the AC region shown in Figure 6; Figure 36 is a re-enlarged view of the AC region, which shows an amplification portion; Figure 37 is a re-enlarged view of the AC region, Figure 38 is an enlarged view of the AC region, which shows an amplification portion; Figure 39 is a re-enlarged view of the AK region shown in Figure 38; Figure 40 is a re-enlarged view of the AC region, Figure 41 is a re-enlarged view of the AC region, showing the amplification portion; Figure 42 is a re-enlarged view of the AC region, which shows the amplification chamber; Figure 43 is the AL region shown in Figure 42. FIG. 44 is a re-enlarged view of the AC area, and FIG. 45 is an enlarged view of the AM area shown in FIG. 44; FIG. 46 is a re-enlarged view of the AC area, which shows增室: Fig. 47 is a re-enlarged view of the AN region shown in Fig. 46; Fig. 48 is a re-enlarged view of the AC region, which shows an amplification chamber; and Fig. 49 is a re-enlarged view of the AC region, which shows amplification Figure 50 is a re-enlarged view of the AC region, which shows the amplification portion; Figure 5 is a schematic cross-sectional view of the amplification portion; Figure 52 is an enlarged plan view of the hybridization portion; Figure 53 is a re-enlargement of two independent hybridization chambers. Figure 54 is a schematic cross-sectional view of a single hybridization chamber; Figure 55 is an enlarged view of the humidifier in the AG region shown in Figure 6; -88 - 201219770 Figure 56 is an enlarged view of the AD region shown in Figure 52; An exploded perspective view of the LOC device of the 57-series AD region; Figure 58 is a diagram of a FRET probe in a closed configuration; Figure 59 is a diagram of a FRET probe in an open and hybrid configuration; Figure 60 is an excitation light Figure 6 is a diagram of the excitation illumination geometry of the hybrid chamber array; Figure 62 is a diagram of the sensor electronics technology LED illumination geometry; Figure 63 is the humidity sensor shown in the AH zone of Figure 6. An enlarged plan view; Fig. 64 is a schematic view showing a portion of the photodiode array of the photo sensor; Fig. 65 is a single photodiode Figure 66 is a timing diagram of the photodiode control signal; Figure 67 is an enlarged view of the evaporator shown in the AP area of Figure 55. Figure 68 shows the detection of the photodiode and the trigger photodiode. Schematic diagram of the cross section of the hybridization chamber, Fig. 69 shows the pCR induced by the linker; Fig. 7 is a representative diagram of the test module with the lancet; Fig. 71 is a representative diagram of the structure of the LOC variant VII; Fig. 72 is the LOC change Figure 73 is a representative diagram of the structure of the LOC variant XIV; Figure 74 is a representative diagram of the structure of the LOC variant XLI; Figure 75 is a representative diagram of the structure of the LOC variant XLIII; 76 is a representative diagram of the structure of the LOC variant XLIV; FIG. 77 is a representative diagram of the structure of the LOC variant XLVII; -89 - 201219770 FIG. 78 is a diagram of a linear fluorescent probe connected to the primer during the first round of amplification; Figure 7 is a diagram of a linear fluorescent probe coupled to a primer during a subsequent amplification cycle; Figures 80A through 80F illustrate the heating cycle of the fluorescent stem-loop probe coupled to the primer; Figure 81 is an excitation LED relative to Schematic diagram of the hybrid chamber array and photodiode; Figure 82 is used to guide the illumination to the LOC device Schematic diagram of the excitation LED and optical lens of the chamber array; Figure 83 is a schematic diagram of the excitation LED, optical lens and optical 用于 for guiding the illumination to the array of hybridization chambers of the LOC device; Figure 84 is for guiding illumination to the LOC device Schematic diagram of the array of excitation LEDs, optical lenses and mirrors of the hybrid chamber array; Figure 8 shows a plan view showing that all features overlap each other and indicates the position of the DA to DK region; Figure 86 is an enlargement of the DG region shown in Figure 85. Figure 87 is an enlarged view of the DH region shown in Figure 85; Figure 8 is a schematic diagram of an LED spectrometer that illuminates a hybridization chamber to measure blood glucose; Figure 89 is a laser excitation source that emits light through an optical element string to a hybridization chamber. Without intending, FIG. 90 shows an embodiment of a shunt transistor of a photodiode; -90 - 201219770 FIG. 91 shows an embodiment of a shunt transistor of a photodiode; FIG. 92 shows a photodiode One embodiment of the shunt transistor; Fig. 93 is a circuit diagram of the differential imager; Fig. 94 is a schematic illustration of the stem loop configuration of the negative control fluorescent probe; Fig. 95 is a schematic illustration of the negative control of the fluorescent probe of Fig. 94 Figure 96 is a schematic illustration of the stem loop configuration of the fluorescent probe being controlled; Figure 97 is a schematic illustration of the open configuration of the positive control fluorescent probe of Figure 96; Figure 98 shows the assay configured for ECL detection. Modules and test module readers; Figure 99 is a schematic diagram of electronic components in a test module configured for ECL detection; Figure 00 shows a test module and a selective test module reader; Figure 101 shows the test module and test module reader and the host system that stores various databases. [Main component symbol description] 10 : Test module 11 : Test module 12 : Test module reader 13 _ · Case 1 4 : Micro us B connector 15 : Inductor -91 - 201219770 1 6 : Micro USB port 17 : Touch Screen 〗 8: Display Screen 19: Button 2 0: Start Button 2 1 : Honeycomb Radio 22: Aseptic Sealing Tape 2 3: Wireless Network Connection 24: Large Container 2 5: Satellite Navigation System 26: Light Emitting Diode 2 7 : Data storage 28 : Mobile phone / smart phone 29 : USB compatible LED driver 30 : Laboratory chip (LOC) device 3 1 : Power conditioner 32 : Capacitor 33 : Clock 34 : Controller 35 : Temporary Memory 36: USB device driver 3 7 : driver

38 : RAM 39 : ECL excitation driver -92 201219770 40 : Program and data flash memory 41 : register 42 : processor 43 : program memory 44 : light sensor 45 : indicator 46 : upper cover 47 : only USB Power/Indicator Module 48: COMS + MST Wafer 49: Porous Element 52: Hybridization and Detection Section 54: Reservoir 5 6 : Reservoir 57: Printed Circuit Board (PCB) 5 8 : Reservoir 60: Reservoir 62 : Reservoir 6 4 : Lower sealing layer 66 : Top layer 6 8 : Sample inlet 7 0 : Pathogen dialysis section 72 : Waste channel 74 : Target channel 76 : Waste unit (reservoir) -93 201219770 7 8 : Reservoir layer 80: upper cover channel layer 8 2 : upper sealing layer 84 : 矽 substrate 86 : CMOS circuit 87 : MST layer 8 8 : passivation layer 90 : MST channel 92 : lowering port 94 : upper cover channel 96 : rising port 9 7 : wall portion 98 : Meniscus Anchor 100: MST Channel Layer 101: Laptop/Note 102: Capillary Start Feature (CIF) 103: Dedicated Reader 105: Desktop PC 106: Boiling Start Valve 107: E-book Reader 108: Boiling Start Valve 109: Tablet PC 1 1 〇: Hybrid Chamber Array 1 1 1 : Epidemiological Data Host System -94 201219770 1 1 2 : Increasing part 1 1 3 : Gene data host system 1 1 4 : Culture part 1 15 : Electronic health record (EHR) host system 1 1 6 : Anticoagulant 1 1 8 : Surface tension valve 1 2 0 : Meniscus 121: Electronic Medical Recording (EMR) host system 122: vent 123: personal health record (PHR) host system 125: network 126: boiling start valve 128: surface tension valve 1 3 0: chemical lysis unit 1 3 1 : Mixing section 1 3 2 : Surface tension valve 1 3 3 : Incubator inlet passage 1 3 4 : Lowering port 1 36 : Light window 1 4 6 : Valve inlet 1 48 : Valve outlet 1 5 0 : Lowering port 1 5 2 : Heater 1 5 3 : Valve heater contact -95- 201219770 1 5 4 : Heater 1 5 6 : Heater contact 1 5 8 : Amplification microchannel 160: Amplification section outlet channel 164: Hole array 166: Capillary start feature 1 6 8 : Dialysis riser hole 170 : Temperature sensor 174 : Liquid sensor 175 : Diffusion barrier 1 7 6 : Flow path 1 7 8 : End point liquid sensor 1 80 : Hybridization chamber 1 8 2 : Heater 1 84 : Photodiode 1 85 : Active area 186 : FRET Probe 187 : Trigger photodiode 1 8 8 : Water reservoir 190 : Evaporator 1 9 1 : Heater 192 : Water supply channel 1 9 3 :on Port 1 94 : Drop port - 96 201219770 1 9 5 : Top metal layer 196 : Humidifier 1 9 8 : First riser hole 202 : Capillary start feature 204 : Dialysis MST channel 208 : Liquid sensor 2 1 0 : Culture microchannel 212: intermediate MST channel 218: TiAl electrode 220: TiAl electrode 222: gap 23 2 : humidity sensor 2 3 4 : heater 23 6 : FRET probe 23 8 : target nucleic acid sequence 240: ring 242: Stem 244 : Excitation light 2 46 : Fluorescent group 2 4 8 : Quencher 2 5 0 : Fluorescence emission 2 5 2 : Optical center 2 5 4 : Lens 2 8 8 : Sample input and preparation 201219770 290 : Extraction 2 9 1 : Culture 2 9 2 : Amplification 294 : Detection 2 9 6 : First electrode 29 8 : Second electrode 300 : Preprogrammed delay 301 : Laboratory wafer (LOC) device 3 7 6 : Conductive column 3 82 : Calibration chamber 3 8 4 : Noisy 3 8 6 : Gate 3 8 8 : Gate 3 9 0 : Lancet 3 92 : Lancet release button 3 9 3 : No. 394: Mshunt transistor 3 9 6 : Μ Ο S transistor 398: MOS transistor 400: MOS transistor 4 0 2 : Μ Ο S transistor 404: MOS transistor 4 0 6 : node 408: film seal - 98 201219770 4 1 0 : membrane guard 682: group Division dialysis department 692: with reference Sub-ligated linear probe 694: amplification blocker 696: probe sequence 6 9 8 : sequence 7〇〇: oligonucleotide primer 704: stem loop probe 706 linked to the primer: complementary sequence 708: stem 712: first optical 稜鏡 714: second optical 稜鏡 7 1 6 : first mirror 7 1 8 : second mirror

728 : LOC variant X 766 : waste receptacle 7 7 0 : untreated blood sample 772 : LED spectrometer 7 7 4 : laser 776 : optical component string 778 : configuration 7 8 0 : configuration 7 8 2 : Configuration 7 9 0 : Pixel-99 201219770 792 : Virtual Pixel 7 94 : Read_Column 79 5 : Read - Column _d 796 : Negative Control Probe 797 : Μ 4 79 8 : Positive Control Probe 801 : MD4 8 0 3 : Pixel capacitor 805: virtual pixel capacitor 8 0 7 : switch 8 0 9 : switch 811: "read_row" switch 813: virtual "read_row" open 8 1 5: switched capacitor amplifier 8 1 7 :Differential signal 860: ECL excitation electrode 8 70 : ECL excitation electrode

Claims (1)

201219770 VII. Patent application scope 1. A test module comprising: a casing having a container for receiving a biological sample; an excitation source disposed in the casing to continuously illuminate the organism with light of different wavelengths a sample; and a light sensor positioned to detect light of different wavelengths that penetrate the biological sample; wherein when used, the signal output by the light sensor is used to generate the biological sample for analysis The spectrum of the characteristics of the sample. 2. The test module of claim 1, wherein the excitation source is an array of light emitting diodes (LEDs) that emit light of different wavelengths, and the LEDs are configured for continuous activation. The test module of claim 2, wherein the outer casing is configured for hand movement. 4. The test module of claim 3, further comprising a data connection for transmitting an output signal of the photo sensor to an external device. 5. The test module of claim 4, wherein the data connection is an electrical connection to an external device, the test module being configured to draw power from the external device via the electrical connection. 6. The test module of claim 5, wherein the electrical connection is a USB connector for insertion into a universal serial bus (USB) port of the external device. 7. The test module of claim 3, further comprising a wafer-on-lab (LOC) device in fluid communication with the container, the LOC device -101 · 201219770 having a configuration to drive from the container by capillary The chamber is filled with an array of chambers of the biological sample. 8. The test module of claim 7, wherein the light sensor is inserted into the LOC device and located adjacent to the array of chambers. 9. The test module of claim 8, wherein the LOC device has a support substrate and a CMOS circuit on the support substrate, the CMOS circuit having the photo sensor and a series of soldering to the USB connector 10. The test module of claim 9, wherein the CMOS circuit has an LED driver via the pad for controlling activation of the LED array. 1 1. The test module of claim 7, wherein the photosensor registers a photodiode array of the array of the chamber. 1 2 . The test module of claim 11, wherein each chamber has a light window for exposing the biological sample to the LED array. 1 3 . The test module of claim 1, wherein the biological sample is blood. 1 4. The test module of claim 13 of the patent application, wherein the analyzed characteristic is blood sugar content. 1 5 . The test module of claim 11, wherein the CMOS circuit further comprises a shunt transistor between each photodiode anode and a voltage source, the shunt cell system configured to remove A carrier generated by the photon of the excitation light in the photodiode. 16. The test module of claim 15 wherein the sub-102-201219770 galvanic crystal system is configured to be activated upon deactivation of each of the LEDs. 17. The test module of claim 12, wherein the CMOS circuit has memory storage identification data for the external device to identify the test module. 1 8 . The test module of claim 16 wherein the photodiode array is less than 249 microns from the hybridization chamber array. -103-