TWI378237B - Apparatus for identifying a single biomolecule, apparatus for identifying biomolecules and method for identifying a single biomolecule - Google Patents

Description

6. Description of the Invention: [Technical Field] The present invention relates to a bioanalytical system including a plurality of optical detecting devices and a bioanalytical system for detecting and analyzing biomolecules, and biomolecules such as nucleic acids . In one embodiment, the invention relates to a bioanalytical system comprising at least 10,000 optical detection devices for monitoring, in some embodiments, a plurality of fluorophore molecules for parallel detection Measuring and analyzing biomolecules. [Prior Art] The Human Genome Project (HGP) stimulates a large increase in the amount of sequencing processing and leads to a relatively low ordering cost. Compared to the cost of 13 years and nearly $3 billion per genomic body, the cost of sequencing has been significantly reduced. To be exact, two separate genomes have recently been completed (McGuire et al., 317:1687 (2007) ). The personal genome represents a paradigm shift in medical treatment for both patients and health care providers. By managing the genetic risk factors for disease, health care providers can implement preventive drugs and provide customized treatments more immediately. Due to the completion of the large genomic banks, drug design and drug administration can be more effective, pushing the initial field of pharmacogenetics forward. In order to popularize custom-made drugs for individuals, the National Institute of Human Genetics (NIH) National Institute of Human Genetics (NhgRI) has determined that the cost of sequencing each genome has dropped from a few million to nearly a thousand dollars. However, the general two-flux capillary electrophoresis and automated genomic sequencing techniques do not meet the increased demand for individual genome sequencing. In addition, the ordering method requires complex and error-prone images 撷2 and analysis steps. For example, in order to obtain multiple images, many current techniques are either arrays or detection systems to move. The resulting images can then be arranged, aligned and analyzed sequentially. Image Capture, Processing, and Analysis Steps White tends to be erroneous, takes extra time, and requires expensive equipment. Conversely, current systems that do not include mobile optics are often limited because of the large number of pre-test units. Finally, the current component does not place the molecule to be detected close to a corresponding detection unit, which substantially limits the strength of the detection signal. Therefore, there is a need for a device that reduces the cost of nucleic acid sequencing. In order to achieve the "$1000 genomic, paradigm, components need to be able to sequence multiple molecules in parallel, have simplified design and manufacturing processes, and avoid the need for scanning and image analysis of complex and error-prone trends in existing components and methods. The element needs to have the ability to sequence a single molecule to avoid expansion of the clustered sequencing method (eg, drift between sequences of ideal replica templates) and sequencing (step-by-step sequence reaction between sequencing templates) Dephasing of the stepwise sequencing reaction. The difficulty of the known different periods in the two steps. [Invention] The present invention provides a biological analysis system including a plurality of optical detecting devices and uses for nucleic acid. A method of detecting a biological analysis system, such as sequencing. The bioanalytical system provided in the examples has the capability of a large-scale parallel sequencing reaction, 1378237, ie, a large number of different nucleic acid templates are simultaneously performed. Sequencing. Each sequencing reaction makes - using a single molecule as a template (ie 'single molecule sequencing') The provided components and components also have a simplified design - eliminating the need for complex, expensive = error-prone scan and detection steps of the current components. The simplified design and system functionality provided by the present invention is based in part on To be directly associated with the linker site of the [detected nucleic acid (not directly, for example, by a polymerase molecule) with one or more detection units (eg. , the light detector), and based in part on the short distance between the link and the detection unit. In some embodiments, the large solid angle (s〇Hd φ angle) is displayed. The short distance between the nucleic acid and the detection unit. In one aspect, a biometric system for identifying a single biomolecule in a detection unit is provided. The bioanalysis system can include a plurality of optical detection devices. a substrate having a photodetector, the substrate being configured to detect light emitted from biomolecules. In some embodiments, the photodetector can include a charge-coupled device, a complementary metal oxygen One of a semiconductor (compoundary metal-oxide semiconductor) sensor or a photodiode. The optical detecting device can include a blind sheet on the substrate, and the masking sheet includes A pinhole, and a filter layer are provided between the masking sheet and the substrate and are provided below the pinhole, the filter layer being non-planar and arranged to filter light emitted from the biomolecule. In some embodiments, portions of the non-planar filter layer can be arcuate, spherical, elliptical or arcuate. The optical detection device also includes a "coupling position" which is provided close to the pinhole, and the bonding site is processed to place the biomolecule close to the pinhole. In another aspect, a bioanalytical system for identifying a single biota [s] 6 1378237 : sub is provided in a detection unit. The bioanalytical system can include a plurality of optical detection devices, each of which can include a substrate having a photodetector, the slab being configured to detect light emanating from the biomolecule. The optical detecting device can include a light emitting layer formed on the substrate, wherein the light emitting layer includes a recess, and a filter layer is provided between the recess and the substrate and under a pinhole. The layers are non-planar and are arranged to filter light that is emitted from biomolecules. In some embodiments, portions of the non-planar filter layer may be arcuate, spherical, elliptical or arcuate. The optical detection device can also include a nnker site, Φ being provided proximate to the recess and the pinhole, the bonding site being processed to place the biomolecules close to the pinhole. The optical detection device can further include an excitation light source to provide a light source for exciting a fluorophore attached to the biomolecule. In another aspect, another embodiment provides a link by attaching at least - a nucleic acid to an optical detection device of an embodiment (not directly, by binding to a nucleic acid polymerase that binds to a linker) And detecting a nucleic acid detecting nucleic acid on a corresponding photodetector. In some embodiments, the detection nucleic acid is hybridized with, for example, a labeled probe. In some embodiments, the nucleic acid is detected by performing nucleic acid sequencing on the optical detection device. In some embodiments, the nucleic acid sequencing method is selected from base-extension sequencing, terminally-labeled phosphate sequencing, and wobble sequencing. In other embodiments, the sequencing reaction is an experimental extension of the sequencing reaction. In other embodiments, the base extension sequencing reaction further includes the step of adding blocked and labeled nucleotides to the optical detection device. In other embodiments, the nucleotides are fluorescently labeled. 1378237 In another aspect, the embodiments also provide methods of detecting the same molecule. In some embodiments, the methods include the step of attaching the marked sample to a link on an optical detection device provided by the present invention and detecting a sample molecule on a corresponding photodetector. In some embodiments, the sample molecules are attached to a linking site by a linking molecule. In some embodiments, the linker molecule comprises 1) a capture molecule suitable for binding to the sample molecule and 2) a nucleic acid tag. In other embodiments, the sample molecules are provided with light that has been attached to the linking sites by the linking molecules provided by the examples.

Learn _ device. In other instances, the sample molecule is allowed to bind to a linker molecule and the bond complex is then provided to the optical device and allowed to adhere to the link site. In other embodiments t, the sample molecule is a biomolecule such as a multi-peptide, nucleic acid, lipid, polysaccharide or metabolite. The above and other objects, features, and advantages of the present invention will become more apparent from the description of the preferred embodiments illustrated herein The analytical system can be used to monitor a large number (e.g., in some embodiments, greater than 1G, _) of single-molecules in parallel. The bioanalysis system can include a plurality of optical (four) devices. Each optical device can detect the presence of camp light _u(10)Ph〇re) on a single molecule, and emit photons of self-operated light groups by _. With parallel-aged optics, the bioanalytical system can be identified by high-throughput 1378237 ,, such as a sequence of genes or a profile of genes expressed in a tissue sample. Referring to Figure 1, a bioanalysis system 1 is shown. The bioanalysis system 1 may include a bioanalysis substrate 10 and a plurality of optical detecting devices 20 formed on the substrate 10. Each optical detection device 20 can be independently operated to detect and identify a single biomolecule attached to it. For example, the sequence of a single strand of DNA can be confirmed by continuously performing a test. • Extension and detection of light emitted from a fluorescent group bound to the extended base by the optical detecting device 20. By integrating a large number of optical detecting devices 20 on the substrate 10, a large number of single biomolecules can be detected and confirmed in parallel. Depending on the design choice, the bioanalysis system 1 may include at least, for example, 10,000 (1, 〇〇〇), 250,000 (250,000), 2 million (2,000,000), or even 10 million (10,000,000) or more. An optical detecting device 20 is formed on the substrate 10. The biometric system 1 can further include a detection and recording system 2 coupled to the substrate 10 for controlling the operation of the optical detection device 20 and for recording data retrieved from the optical detection device 20. In addition, the bioanalysis system 1 can include an excitation source (not shown). The excitation source produces excitation light to induce the fluorophore to emit fluorescence. In one embodiment, the excitation source can be separately disposed from the optical detection device 20 or the bioanalytical substrate 10. In an alternate embodiment, the excitation source can be integrated with optical detection device 20 or bioanalytical substrate 10. In this embodiment, as shown in FIG. 1, the optical detecting device 20 may have a circular shape when viewed from above. It will be appreciated that the optical detection device 20 can have other geometric shapes, such as a square shape, a polygonal shape, an oval shape, and the like. Further, reference numeral 1378237 1 shows that a plurality of optical detecting devices 20 are arranged in a one-frame pattern. It can be appreciated that the optical detection device 20 can be configured in other patterns, such as a triangular lattice pattern, a honeycomb pattern, and the like. Since the plurality of optical detecting devices 20 of the bioanalytical system 1 are independently operable, one of the optical detecting devices 20 to be used will be described below in accordance with various embodiments. Although an optical detecting device will be described, it will be understood that the different optical detecting devices 2 in the biological analysis system 1 are not necessarily the same. Depending on the design choice, different forms of optical detection device 20 can be constructed in accordance with various embodiments. Referring to Fig. 2', a cross-sectional view of the optical detecting device 20 in accordance with an embodiment is shown along line a_a of Fig. 1. As shown in FIG. 2, the optical detecting device 20 includes a photodetector 21 formed on the substrate 1 and a connecting portion 220 formed on the photodetector 21A. In addition, the optical detecting device 20 may further include a control circuit 215 formed on the substrate 1Q for controlling the operation of the photo sensor 210. Control circuitry 215 can be coupled to detection and recording system 2 for accepting control indications from detection and recording system 2 and transmitting the detected signals to detection and recording system 2. In some embodiments, the substrate 10 can be a glass substrate, a semiconductor substrate (e.g., germanium), or a plastic substrate. In some embodiments, one or more control circuits 215 can be appropriate for each photodetector 210. In some embodiments, 'photodetector 210 can include a single photoconductive photon detector or a set of photoconductive photodetectors' such as one or more electrically coupled components (charged-coupled) Device) or a complementary metal-oxide semiconductor (CMOS) sensor. In an alternate embodiment, photodetector m 1378237 .-210 may include a single photon photon detector or a set of photovoltage photon detectors. In an alternate embodiment, photodetector 210 can include a single avalanche photodiode or a set of discrete photodiodes. In an alternate embodiment, photodetector 210 can comprise a single phototransistor or a group of optoelectronic crystals. In an embodiment, the optical detecting device 20 further includes a blind sheet 230 on the photodetector 210. The masking sheet 230 can include a pinhole 235. In an embodiment, the pinhole 235 can have a circular shape and can have a diameter D1 that is less than or equal to one of 10,000, 1,000, 500, 300, 200, 150, or 100 nm. It will be appreciated that the pinhole 235 can have other shapes, such as an oval shape, a square shape, and the like. In an embodiment, the masking sheet 230 can include an opaque material to block unwanted light from reaching the photodetector 210. Therefore, the desired light can reach the photodetector 210 via the pinhole 235. A linker site 220 can be formed proximate to the pinhole 235. In the embodiment shown in this figure, the joint position 220 is formed within the pinhole 235. • In one embodiment, the junction 220 formed to be close to the pinhole 235 can be separated from the photodetector 210 by a distance Η1 less than or equal to 100 μm. In an alternate embodiment, the distance Η1 can be less than or equal to 75, 50, 25, 15, 10, 5, or 3 μπι. The optical detecting device 20 may further include a filter layer 240 (optical) and a microlens 250 (optical) interposed between the photodetector 210 and the shielding sheet 230. Although FIG. 2 shows that the filter layer 240 is formed on the microlens 250, it is understood that the 'filter layer 240 may be formed under the microlens 25'. In some embodiments, the filter layer 240 can comprise a single transparent layer or a plurality of transparent layers (4) of a different refractive index (4). As shown in FIG. 3a, in some embodiments, the plurality of sub-layers may be non-planar non-planar may include, but is not limited to, hemispherical, elliptical, arcuate or otherwise H-shaped shapes and/or surfaces. . When the 24 〇 includes a plurality of arcuate sublayers, the filter layer 24G can be formed by continuously depositing the sublayer on the substrate. In the operation of the towel, the plurality of arc-sublayers may have optical transmissions that are - highly reflective of a particular wavelength or range of wavelengths and are inversely related to a different wavelength or range of wavelengths. For example, a plurality of arcuate filters in an embodiment can reflect light having a wavelength of 633 nm and emit light having a wavelength of 680 nm. In some embodiments, a sub-layer having a higher refractive index can be lost between two layers having a lower refractive index. Alternatively, a sublayer having a lower refractive index may be sandwiched between two layers having a higher refractive index. In some embodiments, filter layer 240 can include a layer having a single region or a layer having a plurality of sub-regions, while the plurality of sub-regions have different transparency for different wavelength ranges. m Returning to Figure 2, the link 22 can be treated to attach the biomolecule 30 thereto. In one embodiment, biomolecule 30 can include a single strand of DNA molecule 32 and an end primer 34 in combination with a single strand of DNA molecule 32. The biomolecule 3 can be attached to the linking site 220 by the terminal linking primer 34. Further, a fluorescent cluster 36 can be used to mark the DNA molecule 32. When excited by the excitation light of a first wavelength λ , , the fluorophore 36 can emit fluorescence of a second wavelength λ 2 . In some embodiments, the first wavelength λ 短 is shorter than the second wavelength λ 2 . In some embodiments, the first wavelength χι is longer than the second wavelength λ2, e.g., in a multi-photon excitation (mUiti_ph〇ton). The photodetector 210 then detects the fluorescence emitted from the phosphor group 36 to identify the base form to which the fluorophore 12 1378237 36 is attached' thereby thereby continuously determining the sequence of the DNA molecule 32. Referring to Figure 3b, a cross-sectional view of optical detection device 20 in accordance with an embodiment is shown. As shown in Fig. 3b, the masking sheet 230 is formed on the photometric detector 210 and is vertically spaced from the photosensor 210 by a distance H1. The masking sheet 23 having a thickness T includes a pinhole 235 having a radius (i.e., one half of the diameter D1). In this embodiment, a bonding site 220 can be formed in the pinhole 235 to bond with a biomolecule (not shown). When the luminescent group 36 is disposed at one of the first positions 36A at the joint position 220 and separated from the joint position 220 by a distance H2, the ray detector 21 having the radius R2 may be at a first solid angle ( In the 仙 ang ang ) ) 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集When the luminescent group 36 is disposed at a second position 36B and is almost in contact with the bonding position 22 (ie, the distance H2 is near zero or less than 1 μm), the photodetector 210 can be collected in a second solid angle Θ2. Fluorescence emitted from the fluorescent group 36. The second solid angle θ2 is greater than the first solid angle Θ1 and provides a substantially stronger signal. In order to expose the photodetector 210 to the fluorescent light emitted from the fluorescent group 36 via the pinhole 235, the radius R2 of the photodetector 21 must be greater than or equal to that projected on the upper surface of the photodetector 21 The radius of the second solid angle θ2. By bringing the masking sheet 230 (or the joining position 220) closer to the photodetector 210 (ie, by reducing the distance Η1), the photodetector can then collect more concentrated light from a solid angle (ie, Strong light signal). In one embodiment, the masking sheet 230 (or the joint location 220) is separated from the photodetector 210 by a '! distance Η1' such that the second solid angle θ2 is at least 〇8SI sphere [S} 13 1378237 Steradian. Referring to Fig. 4, there is shown a cross-sectional view of optical detecting device 20 in accordance with another embodiment along line A-A of Fig. 1. In this embodiment, an excitation source 40 is integrated with the optical detection device 20. As shown in Fig. 4, the excitation light source 40 is formed on the masking sheet 230 of the optical detecting device 20. In one embodiment, the excitation source 40 can include a p-type and an n-type semiconductor layer (410 and 430), and a light-emitting layer 420 is interposed between the junction regions between the layer 410 and the layer 430. Layer 410 and layer 430 can be connected to a power source. Depending on the physical and atomic structure of the materials and/or materials used for layers 410, 420, and 430, excitation light source 40 can be a light emitting diode (LED), a light emitting laser (light emitting laser). Diode, LD), an organic light emitting diode (OLED) or a polymer light emitting diode (PLED). Inorganic materials 'for example, arsenic arsenide, indium phosphide, gallium antimonide and gallium nitride, or organic materials' such as a conjugated polymer having a poly-para-pheneylene-vinylene skeleton, They are all examples of semiconductor materials that can be used to fabricate junction junction diodes. In other embodiments, the excitation source 40 can form a masking sheet 230 or can be formed within the masking sheet 230. In some embodiments, the excitation source 4A integrated with the optical detection device 20 can emit light in a wavelength band or a plurality of wavelength bands. The excitation light source 40 can emit light intermittently or continuously. The excitation light source 40 can emit light of one wavelength band at a time or simultaneously emit light of several wavelength bands. Referring again to Figure 4, the excitation source 4A can include a recess 450 in its central portion to expose the pinhole 235. In this embodiment, the joint position 220 " may not be formed in the pinhole 235. Of course, the joint location 220 can be formed in the recess 450 and proximate to the pinhole 235. In an embodiment, wherein the excitation source 40 forms a masking sheet 230 or is formed within the masking sheet 230, the recess 450 forms a pinhole 235 or is formed within the pinhole 235. In some embodiments, the pinholes 235 can be formed in a central portion of both the layer 410 and the masking sheet 230 by etching the layer 410 and the masking sheet 230, for example, using a suitable process. In addition, an excitation source 40 can be coupled to a power source 440 via a metal contact 415 formed on the lower layer 410 and a metal contact 435 formed on the upper layer 430. The power source 440 can be separately provided and controlled by the detection and recording system 2, or can be integrated with the debt measurement and recording system 2. The light emitting layer 420 of the excitation light source 4 emits excitation light into the concave portion 45 沿着 in a horizontal direction, and the horizontal side is as indicated in Fig. 4 on the light emitting layer. In this embodiment, the excitation light is emitted in the direction of the upper surface of the shield plate 230. Therefore, the excitation: can not interfere with the light reaching the light detecting H 21G. Optical detection devices are more accurate in identifying biomolecules than can be used. 2. Nucleic Acid Detection A bioanalytical system (including, for example, not a single optical detection, measurement: a plurality of such devices) can be used as part of the system for molecular inversion. In the method or process of knife detection, molecular detection, such as nucleic acid sequencing, and the methods and processes utilizing the same are useful for, for example, splitting. These applications can be for personal, public, [ 15 1378237 commercial or industrial. In some embodiments the 'biological analysis system is suitable for a large number of parallel sequencing of nucleic acids. In part, due to the direct connection of the link to the photodetector of the bioanalytical system and/or the close proximity of the link to the photodetector (shown as a large solid angle in some embodiments), embodiments may be used A bioanalytical system is provided to sequence nucleic acids without the need for expensive, complicated, and error-prone knowledge, such as a m〇ving scanning lens or a moving component (9) (10) ton Device stage) and subsequent image analysis 'thus reducing errors and costs. Bioanalytical systems detect signals with substantially improved signal intensities that make single molecule analysis possible. Bioanalytical systems can be used in a widely varying sequence, and the bioanalytical system is suitable for sequencing a single molecule. In addition, compared to current bio-two-piece 7L devices, the optical detection element has a simplified design, assembly, and, for example, randomization of the nucleic acid to be sequenced onto the array to avoid deposition or synthesis at predetermined locations. The time consumption of nucleic acids and the use of the machine. The tissue can be used in bioassay systems for both biomolecule detection methods and portions of the program t system, including nucleic acid hybridization or for, for example, whole gene sequence sequencing, transcriptional profiling, competitive transcriptional expression, or genetic identification. . Biomolecular detection can also include detection and/or measurement of binding to each other as a protein such as protein/protein, antibody/antigen, receptor/ligand, and nucleic acid/egg. These applications are useful for analysis and diagnostic procedures and methods.

Suitable for detecting nucleic acids on the system provided by the present invention, in some real iS

In the embodiment, in order to link the octa 2 A interaction molecule, :=:, which will be suitable for analysis of the binding phase or small molecule attached protein shell, other subtraction, carbohydrate moiety. Link position. In one example, in her case, the "linking molecule may include - the capture molecule, which binds to the molecule to be broken by the binding of the acid. The determination of the nucleus in the -linked molecule: direct sequencing or heterozygous as the capture of the linked molecule. The method provided by the method includes attaching the molecule to be detected to the element provided by the actual example. A step of an address array. In some embodiments, the 'address array' can include a plate 230 having a plurality of pinholes 235, and can be formed in or around the pinholes 235. See, for example, Figures 1 and 2. Therefore the bioanalysis system. Take 5 million nucleic acid fragments at 5 °. If each fragment is, for example, 1000 bases long, a single-piece can obtain billions of bits of sequence data, and it is possible to sequence and resequend the entire genome. 2.1 The molecule to be detected is suitable for detecting the nucleic acid by the method provided in the examples, which may include any such as DNA, RNA or PNA (peptide nucleic acid) and may contain Any sequence - known and unknown includes self-sequences. Nucleic acids can be obtained naturally, recombinantly, or chemically synthesized. Nucleic acids may include naturally occurring (tetra) acids, nucleoside analogs that are not present, or modified nucleotides. The length of the nucleic acid being detected can be varied based on the actual application. In some embodiments, the nucleic acid comprises at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000 ί S 1 17 1378237 sterility or more. In some embodiments, the nucleic acid can be from 10 to 20, from 10 to 50, from 10 to 100, from 50 to 100, from 50 to 500, from 50 to 1000, from 50 to 5000, from 500 to 2000, from 500 to 5000, or from 1000 to 5000 bases. The nucleic acid used for detection can be a single strand. The single-stranded nucleic acid template can be derived from a duplex nucleic acid template by methods known in the art, such as heat or alkali or other chemical treatment. A single-stranded nucleic acid template can also be produced by, for example, chemical-^, (10) synthesis. In some embodiments, it is to be phased (4) with its 5, or 3, terminal attachment-linking sites. In the case of _, _ may further include - or a plurality of terminal linking primers which are bonded to the 5' end, the 3' end or the 5' end and the 3' end (4) of the nucleic acid. In other embodiments, the '-end linkage scorpion is attached to the 3' end of the nucleic acid. The gentleman's terminal can be used to attach the nucleic acid to be detected to the element = = Γ for - or a complementary sequence of multiple detected primers, such as a sequence of primers. 2.1.1 End-linking 5|Sub-end: Junction 2: Short nucleic acid molecule, usually from less than one nuclear two-two i5, two 〇, t-applications, the length of the end-linking primer is to ^1' 30 ' 50, 75, 90 nucleotides or more. In the examples, the length of the terminal is & 20, from 10 to 30, or from 1 to 30, the end-linking primer is undivided and can be branched. In other embodiments, end-linking primers can be used to attach the nucleic acid to be tested to the junction on the array of bits 18 1378237. In some embodiments, an 'end-linking primer can link a nucleic acid directly to the surface of the array, such as by covalent attachment (eg, ester or thiol linkage) or non-covalent linkage, eg, antigen/antibody Biotin/avidin binding. See, for example, Figure 6, Figure 7, and Figure 8. In some embodiments, the 'terminally linked tibial can bind the nucleic acid non-directly to the surface of the array, e.g., by binding to an intermediate molecule, such as a polymerase. See, for example, Figure 8. Thus, the end-linking primer may comprise a modified φ nucleic acid or otherwise modified to assist in attachment to a linker, by methods known in the art, for example, disulfide, thioester, guanamine, phosphodiester or ester. Linking; or by, for example, antibody/antigen, or biotin/avidin binding, for example, a terminal linker comprises - a nucleotide comprising an antigenic region or a biotinylated nucleotide. In other embodiments, a modified nucleotide is located on the 3' end of the terminating primer. In some embodiments, one end of the primer link 5, the end comprising a modified core acid. The ® end-linking primer can also be used as a complement to detect one or more of the nucleic acids, for example, a sequencing sequence. In some embodiments, the primer is used by hybridization to detect nucleic acids, for example, the primer comprises a detectable marker, such as a fluorescent or radioisotope marker. In some embodiments, the 5' end of the 'end joining primer includes a sequence that is complementary to a certain sequence of primers. In some embodiments, the terminal junction scorpion line complementary to the sequencing primer is positioned such that the 3' end of the sequencing primer is contiguous with the first nucleotide in the nucleic acid to be sequenced. For example, Fig. 6 is a diagram showing the sequence to be sequenced attached to an optical detecting device [S 1 19 1378237 20]. A single-stranded nucleic acid 32, an end-linked and an annealing sequence primer 346 is attached to the linker 220 having a reactive functional group, and the linker 220 is coupled to a modified nucleoside at the terminal linker 34. Acid 344 binds. In some embodiments, the nucleic acid 32 can be attached via its 5' end to the linker position 220, and the end linker 34 can be attached to the 3' end of the nucleic acid 32 to serve as a complement to the sequence leader 346. In some cases, an end-linking primer is added to the end of the nucleic acid to be detected by a ligase, such as a DNA ligase. In some of the examples, before the ligati〇n, the end-linking primer and the nucleic acid to be detected are both single-stranded. In other embodiments, both are double stranded. In still other embodiments, one is a single share and the other is a double share. Connections are well known in the art. For example, in the polymerization community sequencing method (p〇l〇ny sequencing method) t, Shendure et al. (Science, 309: 1728-1732 (2005)) quickly connected to the New England Biolab (NEB) The Quick Ligation kit links a T30 end linker (32 bp) to the same DNA fragment. Among them, the ligation reaction solution included 0.26 pMole of DNA, 0.8 pMole of T30 end ligated primer, and 4.0 μM of T4 DNA ligase in lx Quick Ligation buffer. After mixing, the reaction solution was incubated at room temperature for about 10 minutes and then placed on ice. By heating the sample to 65. 〇, 1 〇 minutes to stop the ligation reaction. In other embodiments, an end-linking primer can be synthesized on the nucleic acid to be detected. For example, the 'end-linking primer' may be a homopolymer (h〇mopolymer) which is intrinsically entangled by, for example, a terminal transferase. Example [S] 20 1378237 As in 'Harne et al. (5We«ce, 320: 106·109 (2008)), a poly A tail is added to a DNA template as a viral gene. The complement of one of the polythymidine (poly T) sequencing primers in the single molecule sequence of the body. 2.1.2 Sequencing primers A pre-sequence primer is a quinone oligo-nucleotide complementary to a fragment of a nucleic acid to be detected or a ligated end-linking primer thereof. In some embodiments, the sequencing primers are at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or more in length. In a particular embodiment, the sequencing primers can be from 8 to 25, from 10 to 20, from 10 to 30, or from 10 to 50 nucleotides. The sequencing primer can be formed from any form of nucleotide acid, including naturally occurring nucleotides, nucleotide analogs that are not found in nature, or modified nucleotides. In an embodiment, after a certain sequence of primers is hybridized with a nucleic acid to be sequenced comprising one or more terminal linker molecules, the 5th end of the sequence can be modified to facilitate its alignment with the positional array. A joint is combined. In some embodiments, the sequence primer comprises a modified nucleotide, 'eg, locked nucleic acid (LNA) (modified ribonucleotide) that provides enhanced assays in a polynucleic acid Base stacking interaction). For example, the effect of the locked nucleic acid solution -t Levin et al. (Nucleic Acid Research 34 (20): 142 (2006)) shows that a primer containing a locked nucleic acid has improved specificity and a stronger binding relative to the corresponding Unlocked primer. Three variations of the mcp 1 primer (5'-cttaaattttcttgaat-3') were made to contain three locked nucleic acids (in the cap) at different positions in the primer: [S ] 21 1378237 MCP1 -LAN-3 '(5 '-cttaaattttCtTgaAt-3 ') ; MCP1 -L AN-5 ' (5,-CtTaAattttcttgaat-3,) ; and MCPl-LAN-even (5'-ctTaaatTttctTgaat-3'). All primers substituted with locked nucleic acids have an elevated melting temperature (Tm), while MCP1-LNA-5' primers show a particularly improved sequence accuracy (Phred Q30 counts). Thus, in other embodiments, the sequencing primer may comprise at least one locked nucleotide in its 5' end region, i.e., one-half, one-third or one-quarter of the 5' end of the 'sequencing primer. Prior to being provided to an optical detection element, the sequencing primer can be hybridized to a single sample nucleic acid (i.e., a nucleic acid to be detected comprising at least one end-linking primer). The sequencing primer can be hybridized to the sample nucleic acid by mixing the sample nucleic acid with a molar excess of the sequencing primer in a saline solution, such as 5xSSC (or 5xSSPE), 0.1% Tween20 ( Or 0.1% SDS) with 0.1% BSA buffer solution. The mixture can be heated to 65 ° C for at least 5 minutes and slowly cooled to room temperature to allow the primer/template to bond. Residual primers can be removed by suitable methods, such as molecular sieves. S{S 1 can be designed by suitable methods, including visual review of sequences or computer-assisted primer design, including end-linking and Both are introduced. Many software kits are available to assist with primer design, including DNAStarTM (DNAstar, Inc., Madison, WI), OLIGO 4.0 (National Biosciences, Inc.), Vectoe NTI® (Invitrogen), Primer Premier 5 (Premierbiosoft), and Prime3 ( Whitehead Institute for Biomedical Research, Cambridge, MA). Primer design, taking into account, for example, the molecularity to be sequenced, specificity, length, required melting temperature, 22 1378237 \ secondary structure, primer dimer, GC content, pH and ionic strength of the buffer solution and used Enzyme (ie, polymerase or ligase). See, for example, k〇 Joseph Sambrook and David Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition (2001). 2.1.3 Binding to the surface of the array After binding the sequencing primer to the nucleic acid to be sequenced, including one or more end-linking primers, the complex is prepared in a suitable buffer solution and supplied to the substrate. - Position the surface of the array and allow it to bond. In some embodiments, the sample nucleic acid (the nucleic acid to be detected and one or more end-linking primers) is attached to the conjugation site, and then sequencing or detection primers are provided. In other embodiments, the composite is first hybridized prior to being provided to a component. Only one binding site of a nucleic acid sample is known to be a valid position. In an embodiment, the complex is provided to the optical detection element and the sample nucleic acid is attached to a random linkage at the position array. In other embodiments, the sample nucleic acid can be provided to a predetermined junction on the array of locations by a suitable 'method, such as by a machine or liquid handling system. Suitable methods for attaching nucleic acids to a solid support are well known in the art. In some embodiments, the sample nucleic acid can be attached directly to a linker by covalent attachment, such as disulfide, thioester, guanamine, phosphonium phosphate or ester linkage; or by non-covalent linkage, for example Antibody/antigen, or biotin/biotin protein binding. In some embodiments, the sample nucleic acid can be attached to a conjugation site by an intermediate molecule. In some embodiments, the intermediate molecule of W can be a polymerase, such as a DNA polymerase. [S 1 23 1378237] As an illustrative example of direct, covalent nucleic acid attachment, Adeesietal (Nucleic Acid Research, 28:87 (2000)) modifies the 5' end of an primer to include an SH g group. According to the method of Adeesi et al., a nucleic acid can be prepared in 50 μM phosphate buffered saline (PBS) (NaPi: 0.1 M NaH2P04 pH 6.5, 0.1 M NaCl). The primer solution can then be supplied to a salted glass slide and cultured in a humidity control box at room temperature of about 5 % to allow the primer to bind to the wafer surface. After the binding reaction is completed, shake the pBS solution twice at room temperature for 5 minutes each time to remove unbound DNA. After 'month/leather, add 1 mM β-thiol to one. The PBS solution was used to rinse the position array at room temperature to deactivate the thiol groups of the unbound DNA. Again, the array surface is cleaned, for example once with 5xSSC, 0.1% Tween and once with 5xSSC buffer solution. Thus, in some embodiments, the method used by Adeesi et al can be used in the methods provided herein to attach a sample nucleic acid complex, for example, via a sequence primer or a 5' end of a sample nucleic acid to a linker. . In an alternate embodiment, the sample nucleic acid can comprise, for example, a biotinylated nucleotide' and can bind to an avidin on the surface of the linker. In another embodiment, the sample nucleic acid can comprise an antigenic moiety, such as BrdU or digoxigenin, which is bound to the binding site by an antibody (or a fragment thereof). By "antibody," it will be appreciated that this phrase includes fragments of immunoglobulin molecules, including, for example, one or more CDR regions; or variable heavy or variable light fragments. Antibodies may occur naturally, Recombinant or synthetic. Antibodies may also include, for example, multiple strains (P〇lyCl〇ne) and monoclone variants. In some embodiments, the antibodies are at least 1〇6, 1〇7, 8 9 10, 10 Μ. Or higher binding constant (ass〇ciati〇n constant) binds to 24 1378237 • its antigen. The structure, function and production of antibodies are well known in the art. See, for example, Gary Howard and Matthew Kasser,

Using Antibodies: A Practical Handbook CRC Press; 1st edition (2006). In yet another embodiment, the sample nucleic acid can be attached to the conjugation site by polymerization of a polymerase such as DN A. Those skilled in the art will appreciate that in order to preserve the function of the enzyme, information such as one, two and three levels of enzymes should be considered. For example, the structure of Taq and Phi29 polymerase is known in the art, see: Kim et al., 376:612-616 (1995) and Kamtekar et al., Μ〇/· Ce//, 16:609 -618 (2004). A method of immobilizing a polymerase to a surface while maintaining activity is known in the art and is described, for example, in U.S. Patent Publication No. 2,8/199932, filed on Aug. 21, 2008, and K 〇rlachetal WAS 1G5: 1176-1181 (2GG8). Figure 8 is a graphical representation of an embodiment in which sample nucleic acids (i.e., nucleic acid 32 to be sequenced, end-linking primer 34, and leader primer 346) are bound by a polymerase 38 to a linker 220 and polymerized. The enzyme 38 has been bound to the linker 22 by method 384, such as direct non-covalent adsorption, an antibody, biotin or chemical linkage, such as a guanamine linkage. In some examples, the aidehyde-modified surface of a linker is treated with an acetaldehyde-containing silane (Silane) reagent. Acetaldehyde quickly reacts with a primary amine on the protein to form a Sehiff,s base linkage. Since many proteins exhibit lytic acid on their surface in addition to the more reactive alpha-amines generally at the terminal end, they can be attached to the slide in various directions to allow the different sides of the protein to be in the solution. Protein or small. L ^ 1 25 1378237 Molecular reaction. In another embodiment, by photoactivation of a uv light, a photo NHS (photoNHS) (N-hydroxy succimido carboxylate is linked by a carbon chain) A linker, attached to azidonitrobenzene, is attached to an amine-modified surface on the element. In these examples, by eliminating nitrogen, the UV light excites the monoazide nitrobenzene moiety to produce a highly reactive nitrene. The nitrene immediately reacts with NH2 on the surface of the element to form a hydrazine bond. The other end of the linker is an NHS carboxylate which reacts with the amine acid on the surface of the polymerase to produce a guanamine covalent bond. In another embodiment, the NHS carboxylate moiety reacts with a primary amine on the surface of the component in a buffered environment. The UV light is used to activate the monoazide nitrobenzene moiety and form a highly reactive nitrone which is an electron deficient group and reacts immediately with the primary amine of the amino acid residue on the surface of the polymerase. These methods are described in more detail in Example 4 below.

2.2 Sequencing Forms The bioanalytical system provided by the examples can be used to detect and sequence nucleic acids by methods known in the art, as reviewed, for example, in U.S. Patent No. 6,946,249 and Shendure et al., #扣· ~ν· Gend. 5:335-44 (2004). In some embodiments, the sequencing method relies on the specificity of not being a DNA polymerase or DNA ligase, and includes, for example, base extension sequencing (single base stepwise extension), By synthetic multi-sequence sequencing (including, for example, sequencing with terminally labeled nucleotides) and wobble 26 1378237 sequencing, which is a pertinent linkage. All methods typically require a single strand of sample nucleic acid comprising at least one end-linking primer to be attached to a linker (not directly or indirectly). The sequencing then begins with a certain sequence of primers (the junction sequence is generally referred to as an anch〇r primer, which provides for analog use for sequencing primers). For all sequencing forms, the examples provide a single molecule that can be reordered. For example, after the sequencing reads are completed, the sequencing primers and the extended nucleotides can be deleted from the sample nucleic acid, the components washed, and the sequence repeated. In various embodiments, reordering can be re-executed by the same or different methods. By reordering the same molecules, it is expected that the sequencing error will fall to the power of the number of sequential reads. For example, if the error of reading each base in a single time is 1〇·3, then after two readings, it drops to (1(Γ3)2, ,1().6. This is a single molecule sequencing. A particular advantage, since the modified geranic acid used for sequencing can lose its "δ;!" or hinder the generation of groups, such as false deletions. 2.2.1 Base Extension Sequence: Progressively in some implementations In an example, the photodetecting device provided by the embodiments can be used to perform base extension sequencing, as disclosed in, for example, U.S. Patent No. 5,3,2,5,9. In some embodiments, Included is a single-stranded nucleic acid 32 to be sequenced, a terminally linked primer 34 to be ligated to the single-stranded nucleic acid 32 to be sequenced, and a partial double-replicating nucleic acid attached to a certain sequence of primers 346 bonded thereto. Up to a link 220 to initiate the base extension sequence as shown in Figure 6. In some embodiments, 'polymerase 38 and modified nucleotide acid are then provided to the photodetecting element for appropriate buffering. Solution In some embodiments, the county subtractive complex is attached to the t S 1 27 1378237 junction by a polymerase at the conjugation site. In some embodiments, the nucleotide comprises a covalently linked detectable marker, such as a fluorescent marker, and a barrier group to avoid any secondary extension. Thus, a single nucleotide is added to the sequencing primer 346. After the 3' end, the sequencing is interrupted. Figure 7 is a graphical representation of the first step of an embodiment of a one-base extension reaction. The nucleotide 362 having the fluorescent blocking group 364 is passed by DNA polymerase 38. Add to the 3' end of the sequencing primer 346. In some embodiments, the fluorescent labels act as blocking groups. In other embodiments, they are separate portions. A single nucleotide is incorporated into the sequencing primer 346-3 'End, and identified by its corresponding photodetector 210. The fluorescent marker and the blocking group are then removed, for example, by chemical or enzymatic decomposition to allow for additional cycles of base extension. In embodiments, the marker and the blocking group can be removed simultaneously or sequentially and in any order. The sequence of the sample nucleic acid is deduced one column at a time in the 3' to 5' direction by editing the sequence of addition. Figure 9 shows some samples in parallel A cycle of nucleic acid extension, detection, and deblocking/de-marking. In general, there are two ways to recognize the added nucleotide during the stepwise extension. In a first example, the four nucleotides have the same identity. The detectable marker is added one at a time in a predetermined order. The identification of the extended nucleotide is confirmed by the order of the nucleotides added in the extension reaction. The integrated base is recognized upon extension. In the second mode, four different nucleotides are added simultaneously and each is combined with a distinguishable detectable marker. In various embodiments, the excitation or emission spectrum and/or intensity of the marker may be different. The intensity and/or wavelength of the marker (i.e., excitation or emission spectrum) is detected to confirm the strength of the nucleotide added to the extension. The implementation of these two methodologies is shown in Example 5. 2.2.2 Sequence by synthesis: multi-step extension

In some embodiments, the sequencing by synthesis can be performed by multiple uninterrupted extensions, e.g., without the use of blocking groups. In these examples, the polymerization was monitored by detecting the release of pyrophosphate after hydrolysis of the nucleoside triphosphates, i.e., the release of the beta and gamma phosphate complexes. The complex can be detected directly, for example by combining the fluorescent moiety on the complex, or indirectly detecting the complex, by combining pyrophosphate with a chemical or biological luminescence detection system. In some embodiments, the nucleic acid sample is sequenced by using a terminal-phosphate-labeled nucleotide. Examples of end-phosphate-labeled nucleotides and methods of use thereof are described in, for example, U.S. Patent No. 7,361,466 and U.S. Patent Publication No. 2007/0141598, issued Jun. 21, 2007. Briefly, the nucleotides are provided to the device provided in the Examples. When hydrolyzed in the polymerization, the labeled phosphate is detected by a corresponding photodetector. In some embodiments, all four of the nucleotides include a distinctive signature' and can be added simultaneously. In some embodiments, the nucleotides include indistinguishable, i.e., the same, and are continuously added in a predetermined order. Continuous, cyclic addition of nucleotides with indistinguishable markers still allows multiple, uninterrupted polymerization steps, such as in homopolymer sequences.

29 1378237 2·2·3 Ligase-Based Sequencing

In other embodiments, the same nucleic acid is sequenced on an optical detection device provided by the examples by ligase sequencing. The ligase sequencing method is described, for example, in U.S. Patent No. 5,750,341, PCT Publication WO

06/073504 and Shendure et al. iSc/ewce, 309: 1728-1732 (2005). In the method of Shendure et al., for example, an unknown single-strand DNA sample can be located on the side of the two end-linking primers and immobilized on a solid support. At a specific position in an unknown sequence (ie, an nth base close to a specific end-linking primer), a so-called anchoring primer (which is an analog of the ~-sequence primer) can be bonded to the end-linking primer, and The combination of the four degraded 9-mers is then provided to the mixture for review. The four nine-mers are white, and the difference is that the cursor is far away, and except for the question position (query p0siti〇n), it is degraded at all positions, and the 9-mer is a distinguishable base at the question position. Ask A, C, G or T. The sample is cleaned, scanned, and the base of the question is identified. Thereafter, the anchoring primer is removed from the sample nucleic acid, and the 9-mer, the cleaning element, and the steps are repeated to challenge a different position. Advantageously, the method is non-progressive, i.e., the test base need not be challenged in turn. Therefore, the error will not accumulate. In addition, this method can be used to question nucleotides from not 5, that is, 3, that is, without standard 5, ~3, Ε) ΝΑ synthesis. By this method, a sample nucleic acid of about 13 bases in total can be usually sequenced. 2·3 application

The IS Bioanalytical System detects millions of nucleic acid fragments simultaneously. If each piece of sheep is again, for example, 1 验 base length, a single component can obtain more than 1,378,237 billion base sequences at a time. Additional applications and methods for the components provided herein will be discussed below. 2.3.1 Whole Genome Sequencing Bioanalytical systems can be used to perform whole or partial genotyping of viruses, bacteria, fungi, eukaryotes or vertebrates, such as mammals, such as humans. For sequencing, the genomic DNA can be cleaved into fragments of at least 2, 5, 1, 2, 200, 300, 500, 800, 1200, 1500 nucleotides or longer. In some embodiments, the 'cutted genomic DNA can be from 2 to 50, from 20 to 100, from 20 to 500, from 20 to 1 , from 500 to 12, or from 500 to 1500. The acid is long and bitter. In some embodiments, for sequencing as described above, the nucleic acid' to be sequenced with the bound end-linking primers is made into a single strand, bonded to a sequencing primer and provided to the elements provided by the examples. 2.3.2 Gene Expression Profiling 'In other shells' cases] For gene expression studies, bioanalytical systems can be used to sequence cDN A. For example, the degree of mRNA can be quantified by measuring the relative frequency of a particular sequence that is speculated on the element. Millions of cDNA molecules can be sequenced in parallel to one of the elements provided in the examples. If a cell contains an average of 350, mRNA mRNA molecules, in one million sequencing reactions, it is expected that a transcript that replicates exactly one cell will be sequenced three times. Thus, the elements provided by the examples are suitable for single molecule sequencing with a single number of sensitivities.

t SI 31 1378237

cDNA synthesis is well known in the art and generally includes total RNA extraction and mRNA enrichment as desired. By steps including, for example: reverse transcription for the first synthesis; RNAse treatment to remove residual RNA; first random hexamer priming and second synthesis by DNA polymerase, To generate cDNA from mRNA. The synthesized cDNA is suitable for sequencing on the elements provided in the examples. Methods for isolating and preparing both DNA and RNA are well known in the art. See, joseph Sambrook and David Russell, Molecular Cloning: A

Cold Spring Harbor Laboratory Press, 3rd edition (2001). In some embodiments, the cDNA can be sequenced by the methods described in U.S. Patent Nos. 6,812,005 and 7,361,488. Briefly, the cDNA is conjugated to an adapter nucleic acid' to treat the conjugate by a specific restriction enzyme' and the final treated nucleic acid is conjugated to a complementary oligonucleotide attached to the junction of the elements provided in the Examples. In an embodiment, the conjugate molecule is an end-linking primer. In some embodiments, the mRNA polyp tail can be used as a suitable end-linking primer that is complementary to a poly-thymidine (p〇ly τ tail) sequencing primer. 2.3.3 Detection and/or Measurement of Binding Interactions In other embodiments, a bioanalytical system can be used to detect various binding interactions, including, for example, DNA/DNA, RNA/RNA or DNA/RNA base pairing, nucleic acids. /protein interactions, antigen/antibody, receptor/ligand binding combined with enzyme/substrate. In general, the same molecule is attached to a linker comprising 32 1378237 - an identifying nucleic acid tag (ID). In some embodiments, the linker molecule further comprises a capture molecule that binds to the sample molecule. Linking molecules also include means for binding to the linking sites, such as a moiety that promotes covalent chemical bonding, for example, a disulfide, thioester, guanamine, phosphodiester or ester linkage; or by non-covalent linkage, For example antibody/antigen or biotin/avidin binding. In some embodiments, the linker molecules are attached to the array by an ID tag. The same molecule is provided to a component, and the linker molecule thereof attaches Φ to a random link, for example by binding to a capture molecule located on the linker molecule. In some embodiments, the sample molecules are mixed with the linking molecules, allowed to bind, and then provided to one of the elements provided by the embodiments. In some embodiments, the linker molecule is first provided to the element, allowing it to attach to a linker, and then providing the sample molecule. Again, the ID is detected by identifying the combined sample molecules (e. g., by hybridization or sequencing). A plurality of sample molecular species can be attached to the same array and distinguished by their logo, and the unique interaction ID of the capture molecule combined with them can be characterized by the binding interaction of B. Thus, in some embodiments, the method of detecting a labeled sample molecule comprises linking the nucleic acid of the same molecule to a link of the element by a linker comprising a nucleic acid tag (ID) The step of sequencing and detecting the labeled sample molecules. In an embodiment, nucleic acid sequencing is base extension sequencing. In some embodiments, the nucleic acid sequence is selected from ligase sequencing or nucleotide sequencing via a terminal-phosphate label. By using nucleotide "bits", up to 4n distinct capture molecules can be attached to and identified on the bioanalytical system, where n is a natural number showing the length of the ID being sequenced. For example, 5 nucleotides can provide [33 1378237 more than a thousand unique IDs, while 12 nucleotides provide more than 16 million combinations. For example, a linker molecule is attached to a component and its position is confirmed by detecting its corresponding ID tag. The molecule is then linked as a probe to, for example, investigate the binding interaction with one or more labeled sample molecules. That is, an element having one or more linking molecules attached thereto can serve as a probe array. In an embodiment, the labeled sample molecule is a fluorescent marker. When combined with the capture molecule of the linker molecule, the labeled sample molecule is detected by a photodetector corresponding to the linker to which the linker is attached. Thus, in some embodiments, the method can further include the steps of providing a labeled sample molecule to a component and detecting the labeled sample molecule. In an embodiment, the element has a linker molecule attached to the linker and the linker molecule comprises a nucleic acid tag (ID) attached to the linker. Multiple-marker sample molecules can be provided simultaneously to a probe array and can be distinguished by their signature, such as by the intensity and/or wavelength of their fluorescent signature. At a concentration given by the marker molecule, based on kinetics (eg, docking/undocking) and statistics (eg, at any given time, portions of the sample molecule are bound or The state of binding) both can interfere with the separation constant between the binding interaction between the sample molecule and the labeled interrogating molecule. In some embodiments, the ID of a linker molecule is at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 90, 100, 150, 200 or more nucleotides in length. In some embodiments, the ID is from 5 to 10, 20, 40, 80 or 160; or from 10 to 20 or 50; or from 20 to 35 nucleotides in length. The ID contains a unique nucleic acid sequence, ie, a nucleic acid to be detected. In embodiments, the unique nucleic acid sequence can be at least 1, 2, 4, 6, 8, 10, 34 1378237 12, 14, 16, 20, 24, 30 or more nucleotides in length. In some embodiments, the unique nucleic acid sequence is from 4 to 1 , 12, 15 or 20, or from 10 to 2 nucleotides in length. The ID includes at least one end-linking primer, ie, the ID comprises a sequence that is complementary to a sequence of primers, which in some embodiments is modified to attach to a linker', for example by including a biotinylated Nucleotide. In some embodiments, the end joining primer portion of the ID is from the 3' end to a unique nucleic acid sequence. In some embodiments, it is 5, end to a unique nucleic acid sequence. In yet another embodiment, the 'end link primer appears

In the unique nucleic acid column 3, and 5, both. In embodiments, the sample molecules and capture molecules include portions selected from the group consisting of - carbohydrates, lipids, proteins, peptides, antigens, nucleic acids, hormones, small knife (e.g., pharmaceutically) or a vitamin moiety, or a combination thereof. No part 8. For example, h 77 may be naturally occurring (for example, biochemically purified) or synthesized (for example, chemical synthesis or recombinant production). In addition, these matrices may contain no, some or all of the non-natural ingredients (e.g., unnatural amino acids, hindered or white matter groups, etc.). In the examples, the present molecule or capture molecule is an egg, such as a growth factor, a peptide antigen, an antibody or a receptor. A method in which a domain binds a nucleic acid to a linker molecule or a linker is in the art of <33>'' as reviewed in ', for example, U.S. Patent Publication No. 2004/0038331. The above disclosure discloses the formation of a protein-raised nucleotide conjugate in a solid support. U.S. Patent No. 4,74M11 provides a binding protein to a nucleic acid of 3, a specific example. Wherein the 'endotransferase is first used to add a ribose residue to the 3' portion. Oxidation of molybdenum (perate (jate oxidation) reaction a, no 3' acetaldehyde group on a nucleic acid, 3, acetaldehyde group and covalent bond with protein temporary + amine group When the protein binds to the 3' [S 1 35 1378237 end of the ID, it is attached to the junction via the 5' end of the ID. In some embodiments, a capture molecule, such as a protein, is linked to an ID 5 'End. In these examples, the ''3', the 5' end of the end or a certain sequence of primers is used to attach the capture molecule to a linker. U.S. Patent No. 6,013,434 ', for example, discloses oligo-picoic acid-polymerization The linker is made by the 5' end of the nutrient acid. U.S. Patent No. 6,197,513 discloses that both PNA and DN A bind to a molecule having a slow acid moiety, such as a protein, via a 5' end of the nucleic acid. PNA and The DNA molecule comprises an aryiamine or an aminooxyacetyl moiety. U.S. Patent No. 6,153,737 discloses a nucleus acid comprising at least one of the nucleus of the nucleus, which is suitable for binding to a plurality of molecules. 4 additional detection methods

2.3.4.1 FRET In some embodiments, detecting a knife 'F〇rster resonance energy transfer (FRET)' is sometimes also known as fluorescence on a photodetecting device provided by the present invention. Fluorescence resonance energy transfer (FRET). As is known in the art, FRET occurs when an excited donor molecule non-radioactively transfers energy to a recipient molecule, which emits energy, typically light. FRET can help reduce the background signal by, for example, providing a large spectral separation between the effective excitation and emission wavelengths of the molecule to be detected. The eFRET sag is used to detect the proximity of the molecular interaction due to its efficacy decay. The sixth energy between the distance between the provider and the recipient. For example, Zhang 36 1378237: et a1· (#如', 施, 4:826-31 (2005)) detects nuclear heterozygous by FRET. Wherein, a biotinylated nucleic acid target is combined with a quantum dot provider covering the avidin, which is followed by a Cy5-bound DNA probe. In some embodiments of the invention, a labeled capture molecule and a labeled sample molecule can form a pair of providers/recipients (or vice versa) for detection by FRET. In some embodiments of the nucleic acid sequencing provided by the embodiments, for a chromophore attached to a polymerase or ligase provider, • Lloyd's logo nucleotide-receptor with chromophore . Thus, in these embodiments, a provider located on a polymerase or ligase chromophore excites a recipient with a chromophore, and the nucleic acid is polymerized or attached to the sample nucleic acid. Due to the rapid decline in FRET efficacy, the nucleotide acid that is not close to the polymerase is not excited. In some embodiments, the donor molecule is, for example, an additional-fluorescent group, such as a quantum dot. Quantum dots, for example, semiconductor quantum dots are known in the art and are described, for example, in International Publication No. WO _ 〇 3/〇03015. Methods for binding quantum dots to, for example, biomolecules are known to the art as reviewed, for example, Mednitz et al (Gaga Her (10) Oil, 4·235 46 (2005)) and U.S. Patent Publication No. 4 (6) 6盥2〇 _087843 was published on March 20, 2006 and April, April 17, respectively. In some embodiments, the quantum dots are combined with a -carrier polymerase molecule, which is further described in the following. As discussed earlier, in order to be, Su and even! Alpha-Bound Combinations A person skilled in the art will undoubtedly understand that when fluorophores are bound to, for example, a DNa polymerase or ligase, care must be taken to reduce the binding of the fluorophore to the enzyme-level, Any effect on the tertiary structure of the plate to maintain the enzyme function. [S3 37 1378237 2.3.4.2 Multiphoton Excitation In some embodiments, a band of chromons is excited by two or more photons. For example, in some embodiments, in FRET, it is not the provider that the recipient's chromophore excitation system passes two or more photons. Two photons and multiphoton excitation are further described, for example, in U.S. Patent Nos. 6,344,653 and 5,034,613. 2·3·4·3 Time Resolved Detection In some embodiments, the light source and photodetector provided by the present invention can be adjusted to have a characteristic phase shift. The use of methods known in the art, for example, as disclosed in U.S. Patent Publication No. 2008/0037008, issued Feb. 14, 2008, discloses the disclosure of the light of the detected molecules on the elements provided in the examples, It can be measured by a corresponding optical detector without interference from the excitation source. 3. Biomolecular Analysis Service Using a Bioanalytical System The embodiment also provides a method for providing biomolecule analysis services by using the bioanalytical system according to the present invention. In some embodiments, the method = provided by the service requester - includes - eight batches of donors of the biomolecule to be analyzed 'and the service requester accepts from the service provider" wherein the invention is provided by using the invention Component production 22: Co-consider to implement the method, service requester and (4) provide k, is ] 38 1378237 • Shipping. In some embodiments, the service provider or vendor may be geographically located outside of the United States, such as in another country. Unless otherwise stated, all numerical expressions used in the specification, including the ingredients, reaction conditions, etc. in the scope of the patent application, are to be understood. In all cases, by the wording "about," are modified. The method states that the wording 'at least' of a series of elements can be understood to mean each element of the series. [Embodiment] Example 1: Construction of a high-throughput bioanalytical system by reference to the first -4 Figure 'The method of manufacturing a bioanalysis system 1 will be described in detail below. First, a substrate 10 is formed and formed by a commercially available 0.25 μm semiconductor fabrication process for general logic and optical components. a plurality of photodetectors 21 on the upper surface of the substrate 1. The photodetector 210 is a photodiode photon detector each having an exposed area of 24 μm and a thickness of 452 μm. The photodetectors are arranged adjacent to each other such that an array of 512 rows and 512 columns of the photodetector 210 is formed on the substrate 10. "The plurality of control circuits 215 are formed on the upper surface of the substrate 10 without forming light. Detecting the position of state 210. In this embodiment, a control circuit 215 corresponds to an optical debt detector 210' to control the operation and control of the corresponding photodetector 210 between the optical detection device 210 and the recording device. 1378237 In this embodiment, a filter layer 240 is formed on the upper surface of the photodetector 210 and the control circuit 215. Before the filter layer 240 is formed, a global planarization process is performed on the upper surface of the photodetector 210 and the control circuit 215. Filter layer 240 includes a plurality of sublayers. In this embodiment, the germanium layer 240 can include a plurality of sublayers formed on a base substrate material that can be formed, placing, or deposited with a substrate material, such as SiO 2 on the substrate. 1〇 is available. In one embodiment, a semiconductor fabrication or deposition process can be implemented. For example, deposition methods may include, but are not limited to, physical deposition, chemical deposition, molecular beam epitaxy (MBE), and at mic layer deposition (ALD). In some embodiments, the substrate material can include one or more depressions formed on a surface. In some cases, the depressions may be non-planar, such as hemispherical or arcuate with a defined radius. For example, referring to Figure 3a, the radius of the sub-layer closest to the filter layer 240 of the substrate 10 in one embodiment may be about 6.1 μηη and may vary from 1 μπι to 100 μηη. The size of the recess in a system can vary depending on the design and application of the system and the size of the corresponding pinhole. In one embodiment, the radius of the system can be varied, for example, based on the application, design, characteristics, photodetector, and/or source of the system and the characteristics of the biomolecule to be detected. In some embodiments, the recess can be formed as part of the substrate formation process or by removing portions of the substrate material using conventional semiconductor process removal techniques. For example, the removal method may include wet etching, dry etching, or a method thereof. In other embodiments, a depression may be formed using pressure or pressure when the substrate material is formed. IS 3 Once the substrate material of filter layer 240 is formed as described above, a series of sublayers 40 1378237 ' can be formed on top of the substrate material to produce a filter layer ^ 240 as shown in Figure 3a. In some examples, a sub-layer can be formed by depositing a replacement layer of a material having different dielectric properties. For example, the filter layer 240 can be formed by first depositing a sub-layer having a higher refractive index on the surface of the planarized photodetector 21 and the control circuit 215 on the substrate material. Thereafter, a sublayer having a lower refractive index is deposited on the formed sublayer having a higher refractive index. In some embodiments, the sub-layer having a higher refractive index may comprise Nb 205, and the sub-layer having a lower refractive index may have Si 〇 2 . The tantalum layer 240 is formed by successively depositing a lower layer of higher refractive index and lower refractive index until a large number of sublayers have been deposited on the substrate material. In this embodiment, filter layer 240 includes 69 sublayers. The number of sublayers, their refractive indices, their materials, and/or other parameters can be varied based on various considerations, such as system applications, designs, features, photodetectors, and/or light sources. Referring to Figure 5, a table summarizing the construction of one of the filters, layer 240, is shown. In Fig. 5, a lower numbered sublayer provides a sublayer closer to the bottom surface of the filter layer 240, and a higher numbered sublayer provides a sublayer closer to the upper surface of the filter layer 240. As shown in Fig. 5, in this embodiment, the odd-numbered layers of the filter layer 240 are made of, for example, bismuth pentoxide (Nb205), which has a higher refractive index. The even sub-layer is made of, for example, SiO 2 (Si 〇 2), which has a lower refractive index. The sublayer can be formed by using a sputtering system, and examples of the sputtering system include Model No. RAS 110 of Radical Assisted Sputtering Series, by Shincron Co" 1^(1.(8110^8&\¥&-1 〇1, 丁〇1^〇, 八八)^). The thickness of each layer in this embodiment is also provided in the table of Figure 5. Synthetic filter layer 240 for the fluorescent group Cy5 It is highly transparent, while the light emitted from the [S] 41 1378237 (Helium-Neon) laser has a low penetration at a wavelength of about 633 nm, and the laser is used as an external light source to excite the fluorophore Cy5. Referring again to Figures 2 and 4, a masking sheet 230 having pinholes 235 is formed in the filter layer 240. In one embodiment, the pinholes 235 can be about 0.2 μm in size and can vary from 〇1 to 1 pm. The size of the system based design and application and the corresponding layer can vary depending on the pinhole size in the system. In one embodiment, based on many considerations, such as system applications, designs, features, photodetectors, and/or The light source and the characteristics of the biomolecule to be detected can change the radius. A masking sheet having a pinhole 235 is fabricated. A process of 230 on the filter layer 240 or the substrate 10 will be described in detail below. First, a photoresist layer is formed on the filter layer 240 by, for example, spin coating a photoresist layer (if filtering) The layer 24 is formed on the upper surface of the photodetector 210 and the control circuit 215 as needed or on the surface of the planarized photodetector 210 and the control circuit 215 (if the filter layer 240 is not formed). Thereafter, the photoresist is developed to form a photoresist pattern in the pinhole region. The pinhole region is covered by using a photomask, and the photoresist is exposed such that only the region covered by the photomask is maintained in the filter layer 240 or planarized. A photoresist pattern is formed on the upper surface of the photodetector 21 and the control circuit 215. The 'metal layer is then deposited on the filter layer 24 that has formed the photoresist pattern. In this embodiment, the metal layer may include chromium (Cr). By performing a magnetron sputtering process, it is deposited on the upper surface of the filter layer 24 or the planarized photodetector 210 and the control circuit 215. Then 'removed in the pinhole The portion of the metal layer on the region and the light 11 in the pinhole region and the pattern 'by forming a masking sheet 230 having a pinhole 235. Or 'by first depositing a metal layer (e.g., 'Cr) on the filter layer 240 ij 1 42 1378237 •, then forming a mask on the metal layer, thereby forming the metal layer A portion of the upper surface is exposed to form a masking sheet 230. The % portion of the exposed metal layer is then etched until the filter layer 240 is exposed, thereby forming pinholes on the metal layer. Thereafter, the mask is removed from the metal layer and a masking sheet 230 having pinholes 235 is formed on the filter layer 240. In some embodiments, the hemisphere formed by the space between the masking sheet 230 and the sub-layer closest to the masking sheet 230 is filled with SiO2, and the hemisphere is polished to a flat surface before the masking sheet 230 is formed. The metal layer has a pinhole 235 formed in the center of the half Φ ball of the filter layer 240. Referring again to Figures 2 and 4, in this embodiment, the joint location 220 is formed by filling a support material in the pinhole 235 or recess 450. The support material may be a polymer or inorganic material that is transparent to the fluorescent light emitted from the fluorescent group 36. Referring again to Figure 1, only 12 optical detection devices 20 are shown. It can be understood that at least 10,000 optical detecting devices 20 can be formed on the substrate 10. For example, in this embodiment, each of the optical detecting devices 20 has a circular shape having a radius of about 5 μm or less, which can occupy an area of about 100 μm 2 . For a substrate 10 having an area of 1 square inch (i.e., 2.54 cm by 2.54 cm), it is possible to construct more than six million optical detecting devices 20 on the substrate 10. By simultaneously operating those six million optical detection devices 20, biomolecules can be detected with high throughput. Example 2: Attachment and detection of biomolecules in high-throughput bioanalytical systems [S ] 43 1378237

Once the fluorescent dye Cy5 is used, the nucleic acid is used to test the detection system. Cy5 and biotin are attached to the 3, and 5, respectively, of a 60-mer (mer) oligonucleotide. Dissociation of the labeled and biotinylated DNA into TrisMg (1〇 mM

Tris, 10 mM NaCl, 100 mM MgCl2, pH 8) was deposited in a buffer solution on a positional array and cultured in a humid chamber. After about 30 minutes, the unbound DNA was washed away with a Tris buffer solution. Excitation light is provided via a 635 nm light emitting diode, and a 635 nm light emitting diode can be formed on the masking sheet. In order to read the signal from each pixel 'the excitation light is turned on for about 1-5 seconds', the signal from each pixel is recorded, and this cycle is repeated for one hundred rounds. Then, the representative average signal of each pixel and the corresponding standard deviation are calculated accordingly. The signal before and after the deposition of the DNA sample is compared, and the pixel having an average signal difference greater than 3 times the standard deviation is regarded as a positive pixel, that is, (Avg after -Avg before)> 3x (after STD - Before STD). Example 3: Bonding Quantum Dots to a Polymerase The following are two methods for binding a functionalized quantum dot to a primary amine on a polymerase molecule, the first using an amine-activated dot, the second A carboxyl-activated dot is used. 3.1 Amine EVITAGtm binding to DNA polymerase via amine EVITAGTM (eg Evident Technologies, cat# E2-C11-AM2-0620; EVITAGTM kit QD products are also available [5 1378237 • eBioscience, Inc., San Diego, CA • sold under the eFluorTM mark) • Functionalized quantum dots (QD) by BS3 (Bis (sulfosuccinimidyl suberate)), homogenous bifunctional (homobifunctional) water Activated by a dissolved crosslinker comprising an amine reactive N-hydroxysuccinimide carboxylate (NHS) ester at each end of an 8 carbon spacer arm. At pH 7-9, the NHS ester reacts with the primary amine on the surface of the QD to form a stable guanamine bond and releases the NHS leaving group. Taq DNA polymerase or Phi29 DNA polymerase has a number of primary amines (eg, an amino acid (K) residue and the N-terminus of each multi-peptide), which is the target for the cross-linking of available NHS esters. 3.1.1 Surface activation of quantum dots with 25 μΐ 10 mM BS3 (Bis(sulfosuccinimidyl) suberate), ?161^,? &11#2158〇) with 25 011〇><?68 (phosphate buffer salt, ?117.4) 2.0 nmol of EVITAGTM was activated in a final volume of 250 μM with dH20. After 30 minutes of incubation, the solution was desalted using a P10 column (Amersham Biosciences, product number 17-0851-01) and eluted with lxPBS. The colored protein contains activated QD. 3.1.2 DNA polymerase binding Polymerase was bound by adding 100% DNA polymerase in 0.1 Μ sodium carbonate buffer solution (pH 9.2) to the mixture. After mixing well, the culture was carried out at 4 ° C and tilted for 2 hours. 3.1.3 Purification of QD-bound polymerase [S] 45 1378237 The conjugate was concentrated to a total volume of ~200 μΐ by centrifugation at 6000 i*pm for 5-10 minutes with a 30K Microspin filter (Pall Corporation, part # OD100C33). The conjugate was washed twice with dH20 by a 30K Microspin filter. Further, the conjugate was purified by size exclusion using Suprdex 30/75 Resin (GE Healthcare, part # 17-0902-10 or 17-1044-10 for small proteins and peptides). The column was pre-equilibrated with dH20 before loading the concentrated binding mixture onto the column and allowing it to enter the column bed. These samples were eluted with dH20 under black light excitation and the fluorescent fragments were collected. The glory fragments were added together and concentrated by centrifugation at 6000 rpm for 5-10 minutes to a total volume of ~1 〇〇 μΐ with a 100K Microspin filter. The purified and concentrated combination can be stored at 4 °C. 3.2 Binding of Carboxy DNA Polymerase QD functionalized with a thiol EVITAGtm (e.g., Evident Technologies, cat# E2-C11-CB2-0620) is activated via an ECD-centered sulfur-NHS ester coupling reaction. The amine-reactive sulfur-NHS ester is reacted with, for example, a primary amine in the side chain of the lysine (K) on Taq DNA polymerase or Phi29 DNA polymerase. 3.2.1 Surface activation of quantum dots 1 tsi Equivalent 2.0 nmol of EVITAGtm was diluted in 25 mM MES pH 5.0 buffer solution. EDC (1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide 46 1378237 hydrochloride) was dissolved in cold before use immediately. A 50 mg/ml solution of sulfur-NHS (Pierce, part #24525) in 25 mM MES pH 5.0 was prepared similarly in MES pH 5.0 at a concentration of 5 〇 mg/m. Thereafter, 50 μL of EDC solution and 50 μL of sulfur-NHS solution were added to the EVITAGTM solution. The mixture was mixed well and spun at room temperature for 30 minutes at a slow tilt. The solution was desalted using a ρι〇 column (Amersham Bi〇sciences, product number 17-0851-01) and eluted with a 4.8 μM sodium carbonate buffer (pH 9.2). The colored portion containing the activated qd is collected. 3.2.2 DNA polymerase binding DNA polymerase 100 of 0.1 Μ sodium carbonate buffer solution (pH 9.2) was added to the mixture. After mixing well, the samples were incubated at 4 ° C and tilted for 2 hours. 3.2.3 Purification of polymerase bound to QD by centrifugation at 6000 rpm for 5-10 minutes with a 30K Microspin filter (Pall Corporation, part # OD100C33), the conjugate is concentrated to a total volume of ~200 μb by 30K Microspin filter The conjugate was washed twice with dH20. Again, the conjugate was purified by size exclusion with Suprdex 30/75 Resin. Briefly, the column is pre-equilibrated with dH20. The concentrated binding mixture is then loaded onto the column and allowed to enter the column bed. The column was eluted with dH20 under black light excitation, and the fluorescent fragments were collected. The fluorophores were added together and concentrated to a total volume of ~1 〇〇 μ1 by centrifugation at 6000 rpm for 5-10 minutes with a 100K Microspin filter. The purified and concentrated [S ] 47 1378237 conjugate can be stored at 4 °C. Example 4: Linking a polymerase to a component using a light-NHS (photoNHS) (N-hydroxy succimido carboxylate is bonded to a stack of nitrogen wall groups by a carbon chain) Azidonitrobenzene is described in two ways to attach an enzyme, such as a polymerase, to a component. 4.1 UV Surface Activation By activation with UV light, a photoNHS is used to attach a polymerase to an amine-modified surface on a device. The UV light excites the monoazide nitrobenzene moiety by excluding nitrogen to produce a highly reactive nitrene group. The nitrone reacts with NH2 on the surface of the element to form a hydrazine bond. The other end of the linker is an NHS carboxylate which reacts with an amino acid residue on the polymerase to produce a guanamine covalent bond. 4.1.1 Surface preparation A solution of 1 mM light NHS (Sigma, Art No. A3407 ' molecule 390.35) was prepared in 95% ethanol. The amine modified surface was washed with a carbonate buffer solution followed by 95% ethanol. The photo NHS solution is then provided to the amine modified surface. 254-365 nm UV light was applied to the surface for 3 minutes and then washed three times with 95% ethanol.

4·1·2 amine end cap (en(j_cap) [S 48 1378237] The solution of l〇mM BK-N-acet〇XySuccinimide; prepared in carbonate buffer solution (pH 9.3) Provided to the surface to cover any unreacted amine at the end. Incubate the device for 2 hours at room temperature with gentle shaking. Then wash the surface three times, each time with carbonate buffer solution and steam deionized water. .3 DNA polymerase binds to β, and 1 mM polymerase # in carbonate buffer solution (ΡΗ9.3) is supplied to the surface and incubated for 2 hours at room temperature under gentle shaking. Then, the surface is washed twice a month. Each time with a carbonate buffer solution and pH 7 4 pBS (phosphate buffer salt). The polymer-bound surface can be stored at 4. 4.2 4.2 Buffered surface activation In another embodiment, in a buffered environment, The photoNHS can be activated and bound to the surface. UV light is then used in the presence of the polymerase to activate the azide φ nitro stupid moiety. Again, the highly reactive nitrene forms an electron deficient group under UV light (electron deficient) Group) and immediately with the polymerase surface - one level Amine reaction to form a covalent bond on the surface. 4.2.1 Surface preparation A solution of 1 m]V [photo NHS (Sigma, Art No. A3407, molecular weight 390.35) was prepared in a carbonate buffer solution. The surface was rinsed with a carbonate buffer solution (pH 9.3). The light NHS solution was supplied to the amine-modified surface and incubated for 2 hours with gentle shaking. The surface was washed three times with a carbonate buffer solution. [S] 49 1378237 4.2 .2 End cap of amine The 10 mM acetic acid-N-plyimide solution prepared in a carbonate buffer solution (pH 9.3) was supplied to the surface to cover any unreacted amine groups at the end. The solution was incubated for 2 hours with gentle shaking and then rinsed three times with PBS buffer (pH 7.4). 4.2.3 DNA polymerase binding

A solution of 1 mM DNA polymerase was prepared in a Pbs buffer solution (pH 7.4) and supplied to the end capped (611 (|_(;叩06(1) surface. 1^ light)

(254-365 nm) was irradiated on the surface for 3 minutes. The surface was then rinsed three times with a Pbs buffer solution (pH 7.4). The surface bound by the polymerase can then be stored at 4 °C. Example 5: Base Extension Sequencing Forms As discussed above, there are two general methods for identifying nucleotides added during the stepwise extension: consecutive addition of four nucleotides of the same identity or simultaneous addition of four differences The nucleotide of the sex marker. Examples of each of these forms are provided below. 5.1 Continuous addition of 4 kinds of nuclear carboxylic acid with the same identity

5.1.1 Adenine (A) Molecular Extension: Add a hindered and labeled adenine 呤 with a suitable polymerase in a sequence reaction buffer (eg, 4 mM

Tris-HCl pH 9.1, 1 mM MgCl2). Adenine is added to the 3' end of the sequencing primer only when the thymus squeezing (τ) is the nucleotide adjacent to the 5' end of the terminal linker in the 50 1378237 nucleic acid. If the nucleotide of the nucleic acid is closest to the 3' end of the primer, it is guanine (G), cytosine (C) or adenine (A), and no extension will occur thereafter. 5.1.2 Extension reaction cleaning step: After the extension reaction is completed, the array wafer is cleaned using 5xSSC and 0.1% SDS at a time, and 5xSSC is used at a time to remove adenine and unreacted solution. 5.1.3 Fluorescence Detection and Recording: Fluorescence signals are read at the junction to confirm whether adenine has been extended, indicating a corresponding thymine in the sequenced nucleic acid. 5.1.4 Removal of Protection and Fluorescent Groups: After detection, the protective and fluorescent groups are removed by chemical or enzymatic cleavage. 5.1.5 Cleaning Step: Clean the array wafer using 5xSSC and 0.1% SDS at a time, and use 5xSSC at a time to remove the cleavage protection and labeling groups. 5.1.6 Proofreading and Recording: Confirm that the protective and fluorescent groups were successfully removed from the previous extension step. If there is residual protection and fluorophore detection, for example, the analysis software on the detection and recording system 2 will record the location. The recording of the sequencing reaction can only continue if the protection and fluorophore removal are confirmed in the next cleaning step. 51 1378237 5.1.7 Repeat 5.1.1-5.1.6, this time use guanine for extension reaction. 5.1.8 Repeat 5.1.1-5.1.6, this time using cytosine for extension reactions. 5.1.9 Repeat 5.1.1-5.1.6, this time using thymine for the extension reaction. 5.1.10 Use four extension reactions of A, G, C, and T as one cycle. The sequence of a nucleic acid is confirmed in a stepwise manner by repeating the cycle. 5.2 Simultaneous addition of 4 different nucleotides with distinctive markers 5.2.1 Base extension reaction: Add 4 kinds of blocked and differentiated DNA nucleotides (A, G, C, T) and nucleic acid polymerase Up to a sequencing buffer solution on the array. The extension reaction can occur only at the 3' end of the sequencing primer. A nucleotide complementary to the nucleotide of the sequenced nucleic acid closest to the 5' end of the linker can be added to the 3' end of the sequencing primer, see, for example, Figure 6. 5.2.2 Extension reaction cleaning step: After completion of the extension reaction, the wafer was washed with 5xSSC and 0.1% SDS once, and once with 5xSSC to remove residual material in the reaction solution. [S ] 5.2.3 Fluorescence Detection and Recording: Detect each distinct firefly at each link 52 1378237 Optical signal to confirm the added nucleotide. 5.2.4 Removal of Protected and Fluorescent Groups: After detection, the protective and fluorescent groups are removed by chemical or enzymatic cleavage. 5.2.5 Cleaning Step: Clean the wafer using 5xSSC and 0.1% SDS at a time, and use VSSC at a time to remove the cleavage of the protective and fluorescent groups. 5·2·ό Proofreading and Recording: Confirmation that the protection and fluorescent groups were successfully removed from the previous extension step. If there is residual protection and fluorophore detection, for example, the analysis software on the detection and recording system 2 will record the location. The recording of the sequencing reaction can only continue if the protection and fluorophore removal are confirmed in the next cleaning step. 5.2·7 repeats 5.2.1-5.2.6. The reaction cycle is repeated to confirm the sequence of the nucleic acid. Example 6: Sequencing of a nucleic acid. A chemically synthesized 60-member oligonucleotide having a known sequence: biotin-55-tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tc ACACGGAGGTTCTA-3', as a certain Sequence template. This sequencing template was combined with a 14-member oligonucleotide sequencing primer (5'-TAGAACCTCCGTGT-3,). The 5' end of the template is modified to include a biotin molecule. The template is attached to a reactor surface containing streptavidin. The sequencing reaction allowed [s] 53 1378237 to use /DNA polymerase to perform a one-base extension reaction in a 1 χ sequencing buffer solution with 15 mM * DTT. Each extension reaction step only adds a nucleotide of the formula, which has a secondary extension protection (obstruction) group and a fluorescent marker (e.g., 'Cy5). If the nucleotide of the DNA template adjacent to the 3rd end of the sequencing primer is complementary to the added nucleotide, then the fluorescent marker base is added. The fluorescent signal is detected after the unreacted test substrate material is washed away. If the test base is not complementary, then a fluorescent signal will be detected. After detection, the fluorophore is chemically removed and the array is further cleaned using a saline solution (e.g., 5xSSC; 0.1% SDS) and detected again to confirm the removal of the fluorescent marker. After the removal and cleaning steps, it is considered that the fluorescent marker has been removed for the location where the fluorescent signal is not detected. The camping signal obtained in the next reaction cycle is then considered as the next extended sequence signal. After removal and cleaning, the software used is recorded as having an incomplete removal reaction for the position where the fluorescent signal is still maintained. From the location message: Sustainable to be recorded, as long as the removal step in the next cycle is confirmed. Based on this method, four types of reaction alkali-based materials can be continuously added, and the reaction is carried out cyclically. Therefore, the sequence of the template is confirmed. The present invention has been disclosed in the above preferred embodiments, and it is not intended to limit the invention to any skilled person, and may make some modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims. Is 3 54 13/8237 [Simple Description of the Drawings] Fig. 1 is a plan view showing a bioanalysis system including an array of optical detecting devices. Fig. 2 is a cross-sectional view taken along line A_A of Fig. 1 showing an optical detecting device of an embodiment. Figure 3a is a cross-sectional view showing details of an optical detection device in accordance with an embodiment. Figure 3b is a cross-sectional view showing details of an optical detecting device in accordance with another embodiment. Fig. 4 is a cross-sectional view along line A_a of Fig. 1 showing an optical detecting device of another embodiment. Figure 5 is a table showing the construction of a filter layer of one embodiment. Figure 6 shows a nucleic acid linked to the junction of an element. Fig. 7 shows a nucleic acid linked to a junction of a member after a cycle in which the base of the nucleotide which is hindered from the marker is stretched. Figure 8 shows an alternative embodiment of the base extension sequence reaction shown in Figure 7. Figure 9 shows a cycle in which a number of nucleic acids are sequenced in parallel by base extension sequencing. [Main component symbol description] 1~Bioanalysis system; 2~Detection and recording system,

55 1378237 1 〇 ~ biological analysis substrate; 20 ~ optical detection device; 210 ~ light detector; 220 ~ link position; 215 ~ control circuit; 230 ~ masking sheet; 235 ~ pinhole; Η 1 ~ distance; Η 2 ~ distance ; 240 ~ filter, layer; 250 ~ microlens; 30 ~ biomolecule; 32 ~ single strand of DNA molecule; 344 ~ modified nucleotide; 346 ~ adhesive sequence primer; 34 ~ end primer; 36 ~ fluorophore ; 3 62 ~ nucleotide acid; 364 ~ fluorescent blocking group; 3 6 Α ~ first position; 36 Β ~ second position; 38 ~ polymerase; 384 ~ method; Θ 1 ~ first solid angle; Θ 2 ~ second three Angle; 56 1378237 Θ 2 ~ second solid angle; 40 ~ excitation light source; 410 ~ p type semiconductor layer; 420 ~ light emitting layer; 430 ~ n type semiconductor layer; 450 ~ recess; 415, 435 ~ metal contact; power supply.

57

Claims (1)

1378237 No. 98135549 VII. Patent application scope: Date of amendment: 101.8.17 A multi-original device 1. A device for identifying a single biomolecule, comprising: a substrate having a photodetector, the substrate being configured to detect light emitted from the biomolecule; and a blind sheet on the substrate The masking sheet includes a pinhole having a diameter; a filter layer is provided between the masking sheet and the substrate and is provided under the pinhole. The filter layer is non-planar and Light is provided to filter light emitted from the biomolecule; and a linker site 'which is provided proximate to the pinhole, the joint is processed to place the biomolecule close to the pinhole. 2_ The device for identifying a single biomolecule as described in claim 1, wherein the photodetector is selected from a charge-coupled device, a complementary metal-oxide semiconductor (CMOS) One of a sensor or a photodiode. 3. The device for identifying a single biomolecule as recited in claim 1, wherein the non-planar surface comprises one of an arc, a sphere, an ellipse or an arc. 4. The device of claim 1, wherein the pinhole has a diameter less than or equal to about 1, 〇〇〇 nm. 5. The device of claim 1, wherein the pinhole has a diameter less than or equal to about 200 nm. 6. A device for identifying a single biomolecule, comprising: 58 1378237 No. 98135549. Amendment date: 101.8.17 Revision * A substrate having a photodetector, the substrate being arranged to detect emissions. a light of a molecule; a light emitting layer is formed on the substrate, wherein the light emitting layer comprises a recess; a filter layer is provided between the recess and the substrate and under a pinhole, the filter The layer is non-planar and is configured to filter light that emits the biomolecule; and a link that is provided proximate to the recess and the pinhole, the link being processed to place the biomolecule close to the needle hole. 7. The device for identifying a single biomolecule according to claim 6, wherein the light emitting layer passes through the recess and emits light from an excitation source in a horizontal direction parallel to a surface of the photodetector. Light to the link. 8. The device for identifying a single biomolecule of claim 6, further comprising a masking sheet on the substrate, the masking sheet comprising the pinhole, the pinhole having a diameter. * 9. The device for identifying a single biomolecule as described in claim 6 wherein the non-planar comprises one of an arc, a sphere, an ellipse or an arc. 10. The device for identifying a single biomolecule of claim 6, wherein the pinhole has a diameter less than or equal to about 1, 〇〇〇nm. 11. The device for identifying a single biomolecule of claim 6, wherein the pinhole has a diameter of less than or equal to about 200 nm. 12. The device for identifying a single biomolecule according to claim 6, wherein the excitation light source is selected from a light emitting diode (light emitting 59 1378237, No. 98135549, date of revision: 101.8.17, revision of the diode, LED ), an organic light emitting diode (OLED), a polymer light emitting diode (PLED) and a laser diode Qaser di〇de, LD). 13. An optical detection system' comprising at least one device, such as the device of claim 1 of the scope of the patent application. 14. An optical detection system' comprising at least 25 turns, such as the device of claim 1 of the patent application. 15 - An optical detection system 'includes at least 2, 〇〇〇, a device as described in claim 1 of the patent application. The 16_-type optical detection system 'includes at least 1 〇, 〇〇〇, 装置 a device as described in claim 1 of the patent application. 17. An apparatus for identifying biomolecules, comprising: a substrate having a photodetector; a masking sheet on the substrate, the masking sheet comprising an array of pinholes; a filter layer formed in the pinhole Under the array, a filter layer provided under at least some of the pinholes is non-planar; and a joint is formed adjacent to each pinhole, the joint being processed to attach the biomolecule The link. 18. The device for identifying a biomolecule according to claim 17, wherein the photodetector is selected from one of a charge coupled device, a complementary metal oxide semiconductor sensor, or a photodiode. 19. The device for identifying biomolecules according to claim 17, wherein the non-planar comprises an arc, a sphere, an ellipse or an arch, wherein 1378237 • No. 98135549 is amended: ΗΠ.8.Ι7 Amendment. 20 The device for identifying biomolecules according to claim 17, wherein each pinhole has a diameter of less than or equal to about 1,000 nm. The apparatus for identifying biomolecules of claim 17, wherein each pinhole has a diameter of less than or equal to about 200 nm. 22. An optical detection system comprising at least one device, such as the device of claim 6 of the scope of the patent application. 23. An optical detection system comprising at least 250,000 devices as described in claim 6 of the application. Ι φ 24. An optical detection system comprising at least 2,000,000 devices as described in claim 6 of the scope of the patent application. 25. An optical detection system comprising at least 10, 〇〇〇, 000 devices as described in claim 6 of the scope of the patent application. 26. The device for identifying a single biomolecule of claim 1, wherein the light passes through the filter layer at an orthogonal angle. 27. The device for identifying a single biomolecule of claim 6, wherein the light passes through the filter layer at an orthogonal angle. • 28. A method of identifying a single molecule, comprising: attaching a biomolecule to a bonding site, the bonding site being provided adjacent to a pinhole formed in a masking sheet, the pinhole having a size; A filter layer is used to filter a light emitted from the biomolecule, the filter layer; (i) is provided between the shielding sheet and a substrate, the substrate has a photodetector, .... Ii) being provided under the pinhole, and (iii) being non-planar; and using the photodetector to detect light passing through the filter layer. 61