WO2020119706A1 - 一种生物芯片及其制备方法与应用 - Google Patents

一种生物芯片及其制备方法与应用 Download PDF

Info

Publication number
WO2020119706A1
WO2020119706A1 PCT/CN2019/124473 CN2019124473W WO2020119706A1 WO 2020119706 A1 WO2020119706 A1 WO 2020119706A1 CN 2019124473 W CN2019124473 W CN 2019124473W WO 2020119706 A1 WO2020119706 A1 WO 2020119706A1
Authority
WO
WIPO (PCT)
Prior art keywords
chip
biochip
reaction
code
reagent
Prior art date
Application number
PCT/CN2019/124473
Other languages
English (en)
French (fr)
Inventor
汪建
徐讯
汪军
沈玥
倪鸣
章文蔚
王勇
江湘儿
黄小罗
Original Assignee
深圳华大生命科学研究院
深圳华大基因科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳华大生命科学研究院, 深圳华大基因科技有限公司 filed Critical 深圳华大生命科学研究院
Priority to US17/413,493 priority Critical patent/US20220395799A1/en
Priority to AU2019395588A priority patent/AU2019395588B2/en
Priority to JP2021533681A priority patent/JP7375998B2/ja
Priority to SG11202106282RA priority patent/SG11202106282RA/en
Priority to EP19896991.7A priority patent/EP3907293A4/en
Priority to CA3123119A priority patent/CA3123119A1/en
Priority to CN201980082590.8A priority patent/CN113166974B/zh
Publication of WO2020119706A1 publication Critical patent/WO2020119706A1/zh

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00529—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00547—Bar codes
    • B01J2219/00549—2-dimensional
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides

Definitions

  • This application relates to the field of biochemistry. Specifically, the present application relates to a biochip, its preparation method and application.
  • Biomacromolecules mainly include DNA, RNA, peptides, polysaccharides, etc. Due to the important use of these biomacromolecules in the fields of medicine, agriculture, food, materials, environment, etc., the in vitro synthesis of these biomacromolecules has also become a research and development hotspot and has huge market prospects. Since Todd in the 1950s, the Khorana group first reported on DNA synthesis (Michelwn, AM, Todd, ARJChem. Soc., 1955; Gilham, PT, Khorana, HG, JAmCliem. Soc., 1958) Since then, DNA synthesis methods have experienced long-term development.
  • the current classic synthesis methods include: the column synthesis method developed in the 1980s, and the high-throughput synthesis method based on microarrays developed in the 1990s. These methods are basically solid-phase synthesis methods, in which a single deoxyribonucleotide is used as the unit for synthesis, and the synthesis process mostly involves a four-step cycle based on phosphoramidite chemistry: deprotection, coupling, capping And oxidation steps. Due to the incompleteness of each step of the reaction, the accompanying side reactions (such as adenosine, etc.) and the concentration of reactants decrease with the progress of the reaction. With the extension of the DNA single strand, the error rate of DNA synthesis rises sharply, and the yield is sharp. decline.
  • the disadvantage of the column synthesis method is that the amount of reagents used is large and the flux is low, resulting in higher synthesis costs and time and effort.
  • the microarray-based synthesis method has high throughput and low reagent usage, its error rate is relatively high, its yield is low, and it is unstable.
  • Other biological macromolecules such as RNA, peptides, polyphosphoric acid, etc.
  • the synthesis method is similar to the solid phase synthesis of DNA, and they are repeated on a solid phase carrier for a single cycle of chemical reaction, and their synthesis characteristics are also similar.
  • the error rate is lower, but the reagent consumption is large and the flux is low, which is not conducive to cost savings.
  • Microarray synthesis has high flux and low material usage, but the error rate is relatively high and unstable. Therefore, it is particularly important to find a synthetic chip that can achieve high throughput, low cost, and low error rate.
  • Biochips also known as protein chips or gene chips, originated from the combination of DNA hybridization probe technology and semiconductor industry technology. This technique refers to the immobilization of a large number of probe molecules on a support and hybridization with fluorescently labeled DNA or other sample molecules (such as proteins, factors or small molecules), and then the detection of the intensity of the hybridization signal of each probe molecule to obtain Sample molecule number and sequence information.
  • Biochip technology originated from the hybridization of nucleic acid molecules.
  • the so-called biochip generally refers to a microarray hybrid chip (micro-arrays) of high-density biological information molecules (such as gene fragments, DNA fragments or peptides, proteins, sugar molecules, tissues, etc.) immobilized on mutually supporting media.
  • the sequence and position of each molecule are known, and it is a pre-set sequence lattice.
  • the biochemical analysis process is integrated on the surface of the chip, so that DNA, RNA, High-throughput rapid detection of peptides, proteins and other biological components.
  • the narrow sense of biochip refers to the fixation of biomolecules (oligonucleotides, cDNA, genomic DNA, peptides, antibodies, antigens, etc.) on silicon wafers, glass sheets (beads), plastic sheets (beads), gels by different methods , Nylon membrane and other solid molecular transmitters formed on the lattice.
  • biochip technology is also called microarray technology
  • the solid-phase matrix containing a large amount of biological information is called microarray, also known as biochip.
  • biochips have also developed microfluidics chips (microfluidics chips), also known as microelectronic chips (microelectronic chips), which are also microchip lab chips.
  • microfluidics chips also known as microelectronic chips (microelectronic chips)
  • microchips are integrated like electronic chips, but they are integrated biomaterials. Like laboratory tests, checking blood glucose, protein, enzyme activity, etc. on a biochip is based on the same biological reaction principle. So the biochip is a carrier platform. Since biochips have the potential advantages of high integration, high throughput, high sensitivity, and portability, they are widely used in a variety of scenarios, especially in biosynthesis and detection, and have great application prospects. .
  • representative commercial microarray synthesizers such as the CustomArray synthesizer, reduce the synthesis reaction to micron-level reaction holes, and there are tens of thousands of reaction holes on a chip, which increases the synthesis flux to a certain extent. It reduces the consumption of raw materials, but the output is low, the electrochemical reaction is not easy to control, and the error rate is high.
  • the chip also integrates temperature and humidity sensors, control circuits, etc., the manufacturing process is very complex, and the price is relatively expensive.
  • the Twist synthesizer uses high-speed micro inkjet printheads as a means of transporting reagents such as monomers, synthesizes Oligo on specially processed micron-level silicon-based chip through holes, and then uses a matching reactor to dock these micro holes. Realize in situ PCR and PCA, so as to directly obtain a large number of long fragments of DNA molecules.
  • the biochip uses a special silicon-based material with specific chemical modifications, a large flux, and requires physical isolation. In addition, its chip size is large (the size is comparable to a conventional 96-well plate), and it is not commercialized.
  • the synthesizer of Evonetix circuit control mode mainly uses the characteristics of semiconductor chips with large-scale parallel control to achieve independent control of the reaction of different sites.
  • the principle of controlling the synthesis process is as follows: at a large-scale addressable synthesis site with a special design Add a low-melting, re-heatable blocking material (for example, n-tetracosane) into the closed cavity of the spot, and the synthesizer can use the circuit signal to control whether each spot is energized or not, and then choose whether to heat the bit
  • the special material at the site can be adsorbed at the site to prevent subsequent reagents from reacting at the site. If subsequent synthesis is required at the site, the material can be washed away with a solvent , So that the site is exposed for the synthesis reaction, so that the individual control of the reaction of each site is realized.
  • the chip integration also integrates circuit control, and the manufacturing process is relatively complicated.
  • the invention proposes a biochip, its preparation method and application.
  • the biochip of the present invention can be applied to the synthesis and detection of biological macromolecules such as DNA, RNA, and peptides, antibody screening, antigen recognition, etc., and has great application prospects.
  • the present application provides a biochip that carries chemical entities and codes, where the codes have a unique correspondence with the chip.
  • the present application provides a method of preparing the biochip of the present invention, the method comprising the following steps:
  • the present application provides the application of the biochip of the present invention in the synthesis of biological macromolecules such as DNA, RNA, and polypeptides.
  • the present application provides the application of the biochip of the present invention in biological detection.
  • FIG. 1 schematically depicts an example of a recognizable two-dimensional code chip of the present invention.
  • FIG. 2 schematically depicts the flow of the chip of the present invention for synthesizing DNA.
  • FIG. 3 schematically depicts the flow of the chip of the present invention for synthesizing polypeptides.
  • FIG. 4A shows the HPLC spectrum of the T30 product synthesized in Experiment 1 in Example 6.
  • FIG. 4A shows the HPLC spectrum of the T30 product synthesized in Experiment 1 in Example 6.
  • FIG. 4B shows the HPLC spectrum of the T30 product synthesized in Experiment 2 in Example 6.
  • FIG. 4C shows the HPLC spectrum of the T30 product synthesized in Experiment 3 in Example 6.
  • Figure 5 shows a gel electrophoresis diagram of the product synthesized in Example 7, where Ctrl: standard synthetic primer control; lanes 1-3: Mix1-3.
  • the present application provides a biochip that carries chemical entities and codes, where the codes have a unique correspondence with the chip.
  • the substrate of the chip is prepared from any material suitable for use as a chip, which material does not adversely affect the activity of the reaction reagent or cause undesirable side reactions with the reaction reagent.
  • the chip is made of an inert material.
  • the chip is made of a material selected from the following: silicon wafer (silicon crystal), glass wafer (bead), ceramic, metal wafer, plastic wafer (bead), gel, nylon membrane , Or any combination thereof, is preferably a silicon wafer.
  • the chip is made of porous glass, and its particle size may be any one of 5 ⁇ m-2000 ⁇ m, and further, its particle size is any one of 25 ⁇ m, 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 500 ⁇ m;
  • Its aperture can be Any one of, furthermore, the aperture may be Any of them.
  • Such chips may have any desired shape.
  • the chip may be in the shape of a sheet, a rectangular parallelepiped, a cylinder, a sphere, or the like.
  • the chip is a square sheet.
  • the biochip is a micro DNA synthesis chip, and its size is 2 mm*2 mm, even 1 mm*1 mm, 0.5 mm*0.5 mm or less.
  • the chips of the present invention can be recycled and reused.
  • the chip of the present invention has identifiable characteristics.
  • the chip needs to be encoded, which provides a specific signal for the chip.
  • the specific signal on the chip may be a magnetic signal, an electrical signal, an identification code, etc.
  • the specific signal is a two-dimensional code.
  • the biochip of the present invention carries a code, and the code has a unique correspondence with the chip.
  • “encoding” here means any feature that can be used to distinguish and identify the identity of the chip. Such features include, but are not limited to, numbers, symbols, graphics, identification codes such as barcodes, two-dimensional codes, preferably two-dimensional codes.
  • the code carried on the chip is a two-dimensional code.
  • Two-dimensional bar code/two-dimensional code (2-dimensional bar code) is a certain geometric figure according to a certain rule distributed on a plane (two-dimensional direction) of black and white graphics to record data symbol information; in the code preparation ingeniously Use the concept of "0" and "1" bit streams that form the internal logic basis of the computer, use several geometric shapes corresponding to binary to represent text value information, and automatically read through image input equipment or photoelectric scanning equipment to achieve information Automatic processing. It has some common characteristics of bar code technology: each code system has its specific character set; each character occupies a certain width; it has a certain check function, etc.
  • the two-dimensional code has a built-in error correction function, which can recover data in the case where the code is damaged or smeared. It can use mathematical error correction (Reed-Solomon) to recover data.
  • the code carried on the chip is a combination of at least two or more features.
  • the code carried on the chip may be a combination of an identification code (such as a barcode or two-dimensional code) and an electrical signal, a combination of an identification code (such as a barcode or two-dimensional code) and an optical signal (such as fluorescence), and an identification code (such as a barcode or Two-dimensional code) and digital combination, identification code (such as bar code or two-dimensional code) and RFID tag combination, RFID tag and electrical signal combination, RFID tag and optical signal (such as fluorescence) combination, RFID tag and digital combination , A combination of numbers and optical signals (such as fluorescence), or a combination of numbers, graphics and identification codes (such as barcodes or two-dimensional codes), etc.
  • code has a unique correspondence with the chip
  • each chip corresponds to a unique code.
  • the codes carried by each chip are different from each other. Since each chip has a unique correspondence with the code it carries, the code can be identified to easily distinguish multiple chips, or to quickly and conveniently find and identify the chip of interest from multiple chips.
  • detectors are used (eg, by identifying numbers, symbols, graphics, identification codes (eg, barcodes, two-dimensional codes), RFID tags, optical signals (eg, fluorescence, chemiluminescence, Raman spectroscopy) , Quantum dots, magnetic signals, electrical signals, or any combination of detectors) to detect the code carried on the chip, and use the processor to analyze the signal detected by the detector to identify the identity (code) of the chip.
  • identification codes eg, barcodes, two-dimensional codes
  • RFID tags e.g, optical signals (eg, fluorescence, chemiluminescence, Raman spectroscopy)
  • Quantum dots e.g, magnetic signals, electrical signals, or any combination of detectors
  • optical signals Recognizer such as optical signals Recognizer, magnetic signal recognizer, electrical signal recognizer, image recognizer, or any combination thereof.
  • such an instrument may be a two-dimensional barcode detector, barcode detector, or RFID tag reader.
  • the surface of the chip is chemically modified to achieve the purpose that the chip can meet different functional requirements.
  • the specific type of molecules modified on the surface of the chip depends on the actual application of the chip. For example, a chip used to synthesize biological macromolecules is modified on the surface with molecules capable of initiating a synthesis reaction; a chip used to detect antigens is modified on the surface with specific antibodies; and a chip used for screening antibodies is modified on the surface with Specific antigen.
  • each chip needs its corresponding chemical modification of the chip surface; in addition, it is necessary to ensure that there are enough modified molecules on the chip surface, and at the same time Ensure uniform distribution of modified molecules on the chip surface and avoid uneven signal distribution.
  • the present application provides a biochip that carries a chemical entity and a code, wherein the chemical entity can react with a reaction reagent to connect the monomer in the reaction reagent to the At the end of the chemical entity, the code has a unique correspondence with the chip.
  • the biochip of the present invention is chemically modified, that is, the surface of the biochip is modified by connecting chemical entities.
  • the chemical entity is a linker molecule (linker molecule), which can react with a reaction reagent (coupling reaction or linking reaction), thereby linking the monomer in the reaction reagent to the end of the linker molecule .
  • the biochip used to synthesize nucleic acid is modified with linker molecules, wherein the linker molecule preferably has a functional group reactive with an amino group at the beginning and a hydroxyl group with an acid-labile protective group at the end.
  • the hydroxyl protecting group can be removed to expose the hydroxyl group, and the middle can be ester group, lipid group, thioester group, o-nitrobenzyl group, coumarin group, hydroxyl group, mercapto group, mercapto ether group, carboxyl group, aldehyde
  • the compound of any one or more functional groups in the group, amino group, amine group, amide group, alkenyl group, alkynyl group, the preferred starting functional group is a carboxyl group, the terminal hydroxyl protecting group is trityl, such as 4,4'-di Methoxytrityl (DMT) can be removed when treated with a solution of dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid in an organic solvent (such as dichloromethane, acetonitrile).
  • DMT 4,4'-di Methoxytrityl
  • the Linker molecule is a molecule that can be disconnected and easily removed during the aminolysis treatment after DNA synthesis is completed.
  • the preferred Linker molecule is Universal Linker, where Universal Linker refers to exo-nitrogen-phenyl-5 -(Succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide, exo-nitro-methyl-5-(succinyloxy Yl)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dimethylformimide or exo-nitro-methyl-5-(diethylene glycoloxy)-6 -Dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide.
  • the chemical structure of Universal Linker is as follows:
  • the biochip used for synthesizing the polypeptide is modified with a chemical entity, wherein the chemical entity is a polymer carrier (resin) for synthesizing the polypeptide by a solid phase method.
  • the chemical entity is a polymer carrier (resin) for synthesizing the polypeptide by a solid phase method.
  • These resins can only be directly connected to the first amino acid (initial amino acid) by introducing reactive groups.
  • These resins and resin derivatives are classified into chloromethyl resins, carboxyl resins, amino resins, or hydrazide type resins according to the introduced reactive groups.
  • BOC synthesis method usually chooses chloromethyl resin, such as Merrifield resin
  • FMOC synthesis method usually chooses carboxyl resin such as Wang resin, 2-Cl(Trt)-Clresin.
  • the fixation of the starting amino acid is mainly achieved by protecting the covalent bond formed between the carboxyl group of the amino acid and the reactive group of the resin.
  • chloromethyl resin usually the protected amino acid is prepared first Tetramethylammonium salt or sodium salt, potassium salt, cesium salt, and then at the appropriate temperature, directly react with the resin or in a suitable organic solvent such as dioxane, DMF or DMSO;
  • carboxyl resin usually add the appropriate A condensing agent such as DCC or carboxydiimidazole can form a co-ester between the protected amino acid and the resin to complete the fixation of the amino acid; an amino resin or hydrazide type resin is added after a suitable condensing agent such as DCC is passed between the protecting amino acid and the resin An amide bond is formed to complete the fixation of amino acids.
  • the chemical entity (linker molecule) used to modify the chip is a carboxyl resin, that is, the terminal contains a group that can react with carboxyl condensation, such as Wang resin, 2-Cl (Trt)-Clresin, etc., or a molecule with a similar chemical structure, preferably has a functional group that can react with an amino group at the beginning and a functional group that can react with a carboxyl coupling of an amino acid monomer at the end.
  • the middle can be ester group, lipid group, thioester group, o-nitrobenzyl group, coumarin group, hydroxyl group, mercapto group, mercapto ether group, carboxyl group, aldehyde group, amino group, amine group , Amide group, alkenyl group, alkynyl group of any one or more functional groups of compounds, such as 4-hydroxymethylbenzoic acid, 4-chloromethylbenzoic acid, or substituted 4-hydroxymethylbenzoic acid, 4- Chloromethyl benzoic acid, etc.
  • the preferred linker molecule is 4-hydroxymethyl benzoic acid.
  • the present application provides a biochip that carries a specific antigen/antibody and a code, and the code has a unique correspondence with the chip.
  • a chip used for detecting antigens has a surface modified with specific antibodies;
  • a chip used for screening antibodies has a surface modified with specific antigens.
  • the present invention provides a method for preparing a biochip, the method includes the following steps:
  • the chemical modification is detected or quantitatively analyzed.
  • encoding the chip means adding a unique identity tag to the chip. It should be understood that “encoding” here means any feature that can be used to distinguish and identify the identity of the chip. Such features include, but are not limited to, numbers, symbols, graphics, identification codes, such as barcodes, two-dimensional codes.
  • "encoding the chip” includes adding a two-dimensional code on the chip, the two-dimensional code uniquely corresponding to the chip. In other words, the two-dimensional codes added on different chips are different from each other.
  • the present invention uses laser printing to print a two-dimensional code on the chip as an identifiable signal.
  • it is necessary to design and produce chips with different polished and two-dimensional code types, that is, unpolished single-sided two-dimensional code, unpolished double-sided two-dimensional code, single-polished single-sided two-dimensional code, single-polished two-sided two-dimensional code Dimension code, double-polished single-sided two-dimensional code, double-polished double-sided two-dimensional code chip, you can also perform special treatment on the chip surface in special application scenarios, such as frosting or nano-point modification to increase the surface area to ensure the two The identification of the dimensional chip is fast, accurate and efficient.
  • the present invention adopts the following technology: the size of the chip for DNA synthesis proposed by the present invention can be adjusted according to the application, for example, it can be 2mm*2mm or even 1mm*1mm , 0.5mm * 0.5mm or less, the smallest can be sub-millimeter level (for example, see Figure 1). Due to the small size of the chip, in the selection of the coding mode, the dotted two-dimensional code mode is adopted. In order to ensure the cutting quality and no burrs, the two-dimensional code chip proposed by the present invention uses mechanical cutting. In order to ensure the depth of the code, the red laser is selected to ensure that the dots are deep and clear.
  • Chip identification that is, the process of taking pictures, magnifying and identifying the chip. In order to better identify the chip, make sure that the dots in the QR code are as large and deep as possible.
  • a plurality of two-dimensional codes different from each other may be printed on the entire silicon wafer at equal intervals, for example, the size of the two-dimensional code may be 1 mm*1 mm. Then cut the silicon wafer into 2mm*2mm chips, and ensure that the two-dimensional code is located in the center of each chip when cutting.
  • pre-treating the chip is to achieve the purpose of cleaning the surface of the chip, while exposing more hydroxyl groups to improve surface reactivity.
  • the pretreatment step includes acid treatment, alkali treatment, ultrasound, plasma cleaning, acetone cleaning, or a combination thereof, wherein the acid can be selected from sulfuric acid, hydrochloric acid, phosphoric acid, piranha, etc., and the alkali can be selected from sodium hydroxide and potassium hydroxide Wait.
  • the pretreatment method is to perform acid treatment first, followed by plasma cleaning; more preferably, the pretreatment method is to perform sodium hydroxide treatment first, followed by deionized cleaning and acetone cleaning.
  • silanizing the surface of the chip refers to performing amino surface modification on the chip surface.
  • the silanization process can adopt one or more treatments such as vapor deposition (CVD), solution immersion, simple negative pressure vapor deposition, etc., where the silylation reagent is an alkoxy silicon group at one end and an amino group at one end. It is selected from APTMS, APTES, etc.
  • APTMS is used as the silylating agent, and further preferably, 50% of the silylating agent is used.
  • a simple negative pressure vapor deposition method is adopted, which can ensure efficient silanization, at the same time, can ensure easy operation, and ensure uniformity of surface modification.
  • chemical modification of the chip surface means that chemical entities are attached to the chip surface, and the specific type of chemical entity is determined by the specific use of the chip.
  • the chemical entity is preferably a linker molecule with a functional group that can react with an amino group at the beginning and a hydroxyl group with an acid-labile protecting group at the end.
  • the hydroxyl protecting group is removed to expose the hydroxyl group, which can be ester group, lipid group, thioester group, o-nitrobenzyl group, coumarin group, hydroxyl group, mercapto group, mercapto ether group, carboxyl group, aldehyde group, amino group, Compounds with any one or more functional groups in the amine group, amide group, alkenyl group, and alkynyl group, the preferred starting functional group is a carboxyl group, and the terminal hydroxyl protecting group is a trityl group, such as 4,4'-dimethoxytri Benzyl (DMT) can be removed when treated with a solution of dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid in an organic solvent (such as dichloromethane, acetonitrile).
  • DMT 4,4'-dimethoxytri Benzyl
  • the linker molecule is preferably a carboxyl resin, that is, the terminal contains a group that can react with a carboxyl group condensation, such as Wang's resin, 2-Cl(Trt)-Clresin, or the like with a similar chemical structure
  • the molecule is preferably a linker molecule with a functional group that can react with an amino group at the beginning and a functional group that can be coupled with a carboxyl group of an amino acid monomer at the end. After the final peptide is synthesized, it can be dissociated when treated with an acid.
  • the middle can be an ester.
  • the sequence of steps in the preparation of the biochip is preferably coded first and then chemically modified on the surface, so as to better protect the modified surface of the biochip and facilitate the subsequent application of the biochip.
  • the chip surface is subjected to pretreatment and silanization, and finally the chip surface is chemically modified; if the order is reversed, after the chip is pretreated and silanized
  • the target compound when the target compound is DNA, the target compound can be cleaved from the chip through an aminolysis reaction.
  • the reagents used to perform the ammonolysis reaction may be selected from ammonia water, ammonia gas, methylamine, or any combination thereof.
  • the ammonolysis reaction can be performed at a temperature selected from room temperature to 120°C, for example, room temperature to 60°C, 60-90°C, 90-120°C.
  • the aminolysis reaction can be carried out for 0.5h-48h, for example 0.5-2h, 2-5h, 5-10h, 10-18h, 18-24h.
  • the target compound (DNA) is separated and purified, for example, MOP, PAGE, PAGE Plus, HPLC, or any combination thereof can be used to separate the target compound (DNA) And purification.
  • the target compound when the target compound is RNA, the target compound can be cleaved from the chip by an aminolysis reaction.
  • the reagents used to perform the ammonolysis reaction may be selected from ammonia water, ammonia gas, methylamine, or any combination thereof.
  • the ammonolysis reaction can be performed at a temperature selected from room temperature to 120°C, for example, room temperature to 60°C, 60-90°C, 90-120°C.
  • the aminolysis reaction can be carried out for 0.5h-48h, for example 0.5-2h, 2-5h, 5-10h, 10-18h, 18-24h.
  • the target compound (RNA) is separated and purified, for example, PAGE, PAGE Plus, HPLC, or any combination thereof can be used to separate and purify the target compound (RNA) .
  • the isolation and purification are performed in an RNase-free environment.
  • HPLC purification is used, and all reagents and equipment used for separation and purification are free of RNase (ie, RNase-free) to avoid RNase contamination.
  • the target compound when the target compound is a polypeptide, the target compound can be cleaved from the chip by adding an eluent.
  • the reagent used to perform the elution may be selected from hydrofluoric acid, trifluoroacetic acid, tetrafluoroboric acid, or any combination thereof.
  • the elution reaction can be carried out at a temperature selected from the following: room temperature to 120°C, for example, room temperature to 60°C, 60-90°C, 90-120°C.
  • the elution reaction can be carried out for 0.5h-48h, such as 0.5-2h, 2-5h, 5-10h, 10-18h, 18-24h.
  • the target compound (polypeptide) is separated and purified. For example, high performance liquid chromatography, affinity chromatography, capillary electrophoresis, or any combination thereof can be used to separate and purify the target compound (polypeptide).
  • the DMT-protected hydroxyl group at the end of the linker molecule can be deprotected by adding an acid, and the color of the collected solution can be qualitatively observed.
  • the solution is red to indicate that the Linker connection is successful, that is, the chip modification is successful.
  • the collected red solution is quantified, that is, the DMT quantification is performed by an ultraviolet-visible spectrophotometer, and the number of Linker molecules on the surface of the modified chip is determined to determine the molecular load on the chip surface.
  • the DMT-free chip is coupled with the 6-FAM fluorescent monomer.
  • the coupled chip is cleaned and placed under a fluorescence microscope to observe the fluorescence.
  • the presence, intensity, and uniformity of the fluorescence can be used to characterize the modification.
  • the quality of the chip that is, the high fluorescence intensity on the surface of the chip and the uniform fluorescence signal indicate that the modified chip is of good quality.
  • the chip of the present invention can be used to synthesize nucleic acid (DNA/RNA).
  • the general methods and principles for solid-phase synthesis of nucleic acids are well known to those skilled in the art.
  • the solid-phase synthesis of nucleic acids generally uses the solid-phase phosphoramidite triester method, which connects multiple nucleotides one by one and sequentially to the chip through multiple rounds of cyclic reactions to obtain the inclusion
  • the target product of multiple nucleotides, where each round of the cyclic reaction may involve 4 steps: namely, deprotection, activation/coupling, capping, and oxidation.
  • the cycle is continuously repeated until the synthesis is completed, and finally the chip can be collected collectively to obtain a DNA/RNA library or a separate sorting technology can be used to obtain individual DNA/RNA fragments (see Figure 2 for a schematic diagram of the process).
  • peptide synthesis according to the types of amino acid monomers required for peptide synthesis and the synthesis flux requirements, first select the chip coding method and formulate the identification and sorting plan to make different throughput biochips for peptide synthesis, and corresponding Identifier, sorter, and drive device that controls chip movement, reagent liquid introduction and extraction.
  • the biochip described in the present invention can be used for biological detection, such as antigen, antibody screening, disease diagnosis, etc., especially for simultaneous detection of multiple biological samples.
  • Antigen and antibody have specific binding ability, that is, an antigen can specifically bind with affinity to the corresponding antibody. Using this specific affinity, specific antigen-antibody screening can be performed.
  • Conventional biological detection chips can only detect one biological sample at the same time, that is, if antibody A is to be screened, the antibody library to be screened is passed through a chip modified with specific antigen A, and then eluted and desorbed to obtain target antibody A. Or modify multiple antigen sites on a single chip to achieve the goal of high-throughput screening, but this modification is obviously more complex and difficult to distinguish.
  • the biochip of the invention can realize simple and high-throughput antibody screening.
  • the following uses a modified chip that can screen 1000 antibodies as an example to describe specific technical application solutions:
  • each chip is modified with specific antigen, and then add the solution of antibody library Go to the mixed chip and let it pass, because the antigen and antibody have a specific binding effect, after contacting for a period of time, the specific antibody will bind to the corresponding antigen on the chip, and then remove the antibody library, and Add cleaning reagent to clean the chip to ensure that the remaining unbound antibody is removed.
  • 1000 chips are sorted into 1000 different reaction wells, and then the desorption reagent is added to each reaction well containing the chip to remove the antibody bound on the chip Separated from the desorption of the antigen on the chip, and repeated many times to ensure that the antibody is completely desorbed. Then transfer the solution in 1000 reaction wells separately to the new 1000 reaction wells, that is, the corresponding 1000 specific antibodies can be screened at the same time. Finally, the obtained antibodies are analyzed and characterized, and the antibodies with positive results are displayed. Purification to obtain high-purity antibodies. Through the above technical solutions, a large number of high-purity specific antibodies can be quickly and efficiently obtained, and high-throughput screening of antibodies can be completed.
  • the application of the antibody screening proposed by the present invention is due to the small size of the antigen-modified chip and the simple manufacture, which can be mass-produced. At the same time, it is combined with the proposed two-dimensional code on the chip for rapid and efficient identification and sorting
  • the throughput of this antibody screening technology application can reach tens of thousands or even millions.
  • the biochip of the invention can realize simple and high-throughput antigen detection.
  • the following uses a modified chip that can detect 1000 antigens as an example to describe specific technical application solutions:
  • each chip is modified with specific antibodies, and then add the solution of the antigen library Go to the mixed chip and let it pass, because the antigen and antibody have a specific binding effect, after a period of contact reaction, the specific antigen will bind to the corresponding antibody on the chip, and then remove the antigen library, and Add cleaning reagent to clean the chip to ensure that the remaining unbound antigen is removed.
  • 1000 chips are sorted into 1000 different reaction wells, and then filled to each Add desorption reagents to the reaction wells where the chip is placed to desorb and separate the antigen bound on the modified chip from the antibody on the chip, and repeat several times to ensure complete desorption of the antibody, and then transfer the solution in the 1000 reaction wells separately Into the new 1000 reaction wells, you can screen for the corresponding 1000 specific antigens at the same time, and finally analyze and confirm the obtained antigens.
  • the technical application proposed by the present invention is due to its small chip size, simple manufacture, and mass production. At the same time, it is combined with the rapid and efficient identification and sorting by printing a two-dimensional code on the chip proposed by the present invention.
  • the flux of technology applications can reach tens of thousands or even millions.
  • Synthetic solid phase carrier conventional chip, 100nm silicon oxide chip, 300nm silicon oxide chip, frosted quartz chip, transparent quartz chip, size: 2*2*0.45mm
  • Phosphoramidite monomers A, T, C, G Sigma Aldrich
  • Oxidizing agent 0.05M Oxidizing, Beijing Dinaxing Branch
  • TA cloning kit pMDTM19-T, TaKaRa
  • Example 1 Preparation of a chip for synthesizing nucleic acid (DNA/RNA)
  • the DMT-protected hydroxyl group at the end of the linker molecule was deprotected by adding acid, and the color of the collected solution was qualitatively observed.
  • the red color of the solution indicates that the Linker connection was successful, that is, the chip modification was successful.
  • the collected red solution is quantified, that is, the DMT quantification is performed by an ultraviolet-visible spectrophotometer, and the number of Linker molecules on the modified chip surface is determined to determine the molecular load on the chip surface.
  • the DMT chip is coupled with 6-FAM fluorescent monomer, the coupled chip is cleaned and placed under a fluorescence microscope to observe the fluorescence. The presence, intensity and uniformity of the fluorescence can be used to further characterize the The quality of the modified chip, that is, the high fluorescence intensity on the surface of the chip, and the uniform fluorescence signal indicate that the modified chip is of good quality.
  • modified two-dimensional code chips were taken out, and the DMT on the surface of the chip was eluted using TCA deblock, the concentration of DMT was measured using an ultraviolet-visible spectrophotometer, and the Linker graft density of the modified two-dimensional code chip was measured.
  • the synthesis effect of the actual target compound is not positively correlated, that is, the higher the graft density, the better the chip synthesis effect, because this involves the problem of molecular crowding.
  • the connected oligonucleotide chain will not be too crowded, so as to affect the final synthesis quality and length. Therefore, in this embodiment, when exploring the optimal ratio of silylation reagent, it is necessary to comprehensively consider factors such as Linker graft density, single-step synthesis efficiency, and product purity.
  • the chips are used to synthesize T5 DNA sequences (ie TTTTT (SEQ ID NO. 15)).
  • TTTTT SEQ ID NO. 15
  • Example 3 the final product purity is obtained. See Table 1 for the efficiency of single-step synthesis. From the results in Table 1, it can be seen that when 50% silylation reagent is used for modification, although Linker graft density is not as high as that of 100% silylation reagent, the purity and single-step synthesis efficiency of the resulting product are the highest.
  • the inventors used the DNA synthesis method shown in FIG. 2 to synthesize DNA molecules using four single-base reaction cells. Specific steps are as follows.
  • reaction cells first, second, third and fourth reaction cells
  • deoxyribonucleotides A, T, C and G deoxyribonucleotides A, T, C and G, and deprotection in each reaction cell, Capping, oxidation and washing; of which,
  • Adenosine 5'-hydroxyl protected by DMT and 3'-hydroxyl protected by phosphoramidite and tetrazole are added to the first reaction cell;
  • Example 1 Five different types of chips (conventional chip, 100 nm silicon oxide wafer, 300 nm silicon oxide wafer, frosted quartz chip, transparent quartz chip) modified with 50% silanating reagent in Example 1 were used.
  • the equipment includes,
  • -QR code recognizer which can recognize the QR code carried on the chip and generate a signal
  • -A central processor capable of receiving the signal generated by the two-dimensional code identifier to identify the two-dimensional code and issuing instructions to control the chip sorter to sort the chip and/or control the driving device to move the chip;
  • control program is also designed according to the target DNA sequence to be synthesized, and the control program can determine the sorting scheme of the chip according to the predetermined DNA sequence.
  • the central processor controls the chip sorter to sort the chips according to the identification signal of the identifier, and according to the preset control program, each Move the chip to the desired reaction tank, and then introduce the deprotection reagent into the reaction tank for deprotection by controlling the driver.
  • the deprotection reagent is drawn out, and the cleaning solution is introduced to clean the chip, and then according to the corresponding For the monomer to be coupled, the coupling reagent is introduced for coupling.
  • the coupling reagent is introduced, the cleaning solution is introduced to clean the chip, and finally, the capping reagent, the oxidizing reagent and the cleaning solution are introduced in turn to perform capping and oxidation respectively.
  • the cleaning ends, and the addition/coupling of one base is completed.
  • the specific operation steps, reagents and reaction time used are shown in Table 2.
  • a two-dimensional code can be used to identify all the chips, sort and collect the target chips, and then perform ammonolysis on the target chips, thereby separating and purifying the target DNA molecules.
  • Discharge the coupling reagent add 250 ⁇ L of acetonitrile and wash once.
  • After draining the reagent add the reagent and react again for one time. The two reactions take 40 seconds.
  • Discharge the acetonitrile and add 150 ⁇ L of oxidant react for 20 s. After removing the reagent, re-add the reagent and react again for a total of 40 seconds.
  • a transparent quartz chip was used to synthesize 14 oligonucleotides with a length of 59nt.
  • the sequence is shown in Table 4, and the small fragment gene assembly and gel running based on the one-step PCA/PCR reaction strategy were performed.
  • the gel map verifies the correctness of the target band.
  • it is recovered by excising the gel, transformed by TA cloning, and sent to Sanger sequencing. By analyzing and comparing the sequencing results, the best solid phase synthesis carrier is finally determined.
  • the inventors used an RNA synthesis method based on immersion-sorting to synthesize RNA molecules using four single-base reaction cells. Specific steps are as follows.
  • the chip modified with 50% silanating reagent in Example 1 was used.
  • the equipment includes,
  • -A central processor capable of receiving the signal generated by the two-dimensional code identifier to identify the two-dimensional code and issuing instructions to control the chip sorter to sort the chip and/or control the driving device to move the chip;
  • -A memory which is used to store the sequence of the RNA molecules to be synthesized, the correspondence between the chip and the two-dimensional code, the correspondence between the RNA molecules to be synthesized and the chip, and the reactions each chip has undergone.
  • a control program is also designed according to the target RNA sequence to be synthesized, and the control program can determine the sorting scheme and movement scheme of the chip according to the predetermined RNA sequence.
  • First mix 1000 chips recognize the two-dimensional code pre-printed on the chip through the identifier, the central processor controls the chip sorter to sort the chips according to the identification signal of the identifier, and according to the preset control program, each chip A chip is moved to the desired reaction cell, and then the deprotection reagent is introduced into the reaction cell for deprotection by controlling the driver. After the reaction is completed, the deprotection reagent is led out, and the cleaning solution is introduced to clean the chip, and then corresponding to the reaction cell For the monomer to be coupled, the coupling reagent is introduced for coupling.
  • the coupling reagent is introduced, the cleaning solution is introduced to clean the chip, and finally the capping reagent, the oxidizing reagent and the cleaning solution are introduced in turn, respectively. Oxidation and cleaning, at the end of this cycle, complete the addition/coupling of a base.
  • the specific operation steps, reagents used and reaction time are shown in Table 6.
  • RNA molecules are synthesized.
  • RNA synthesis After the RNA synthesis is completed, you can choose to collect 1000 chips together for ammonolysis, thereby isolating and purifying all the synthesized RNA molecules.
  • two-dimensional codes can be used to identify 1000 chips, sort and collect the target chips, and then perform ammonolysis on the target chips, thereby isolating and purifying the target RNA molecules.
  • the inventors used the polypeptide synthesis method based on soaking-sorting as shown in FIG. 3 to synthesize polypeptide molecules using 21 single amino acid synthesis pools. Specific steps are as follows.
  • each synthesis pool contains an ⁇ -amino group protected by Fmoc and side chains Amino acid monomers that protect (if there are side chains), as well as tetramethylurea hexafluorophosphate, N-methylmorpholine, and DMF;
  • the chip prepared in Example 2 was used.
  • the equipment includes,
  • -A central processor capable of receiving the signal generated by the two-dimensional code identifier to identify the two-dimensional code and issuing instructions to control the chip sorter to sort the chip and/or control the driving device to move the chip;
  • -A memory which is used to store the sequence of the polypeptide molecules to be synthesized, the correspondence between the chip and the two-dimensional code, the correspondence between the polypeptide molecules to be synthesized and the chip, and the reactions each chip has undergone.
  • control program is designed according to the target polypeptide sequence to be synthesized, and the control program can determine the sorting scheme and the movement scheme of the chip according to the predetermined polypeptide sequence.
  • a two-dimensional code identifier is used to identify the specific two-dimensional code printed on the chip in advance.
  • the central processor controls the chip sorter to sort the chips according to the identification signal of the two-dimensional code identifier, and moves each chip to a desired synthesis pool according to a preset control program to perform a coupling reaction. After the coupling reaction is completed, all chips are transferred to the washing tank for washing twice, thereby completing the addition/coupling of one amino acid.
  • the reagent liquid driving device needs to be used to introduce the corresponding reagent into the pool. After the reaction is completed, the reagent liquid driving device is used to lead the reagent out of the pool.
  • Table 7 The specific operation steps, reagents and reaction time used are shown in Table 7.
  • two-dimensional codes can be used to identify 1000 chips, sort and collect the target chips, and then dissociate the target chips, thereby separating and purifying the target polypeptide molecules.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种化学修饰的可识别的生物芯片，其制备方法和应用。

Description

一种生物芯片及其制备方法与应用 技术领域
本申请涉及生物化学领域。具体地，本申请涉及一种生物芯片，其制备方法和应用。
背景技术
生物大分子主要包括DNA、RNA、多肽、多糖等。由于这些生物大分子在医药、农业、食品、材料、环境等领域的重要用途，这些生物大分子的体外合成也成为研发的热点，并且具有巨大的市场前景。自二十世纪五十年代Todd，Khorana课题组第一次报道了DNA合成(Michelwn,A.M.,Todd,A.R.J.Chem.Soc.,1955；Gilham,P.T.,Khorana,H.G.,J.A.m.Cliem.Soc.,1958)以来，DNA的合成方法经历了长期的发展。目前经典的合成方法包括：八十年代发展起来的柱式合成法，以及九十年代发展起来的基于微阵列的高通量合成法。这些方法基本上为固相合成法，其中，以单个脱氧核糖核苷酸为单元进行合成，并且其合成过程大多涉及基于亚磷酰胺化学法的四步骤循环：即脱保护、偶联、加帽和氧化步骤。由于每一步反应的不完全性、伴随可能发生的副反应(如脱腺苷等)以及反应物浓度随反应进展的降低，随着DNA单链的延长，DNA合成的错误率急剧上升，产量急剧下降。此外，柱式合成法的缺点还在于试剂使用量大且通量低，导致合成成本较高，费时费力。而基于微阵列的合成方法虽然通量较高且试剂使用量少，但是其错误率相对较高，产量低，且不稳定。其他生物大分子如RNA、多肽、多聚磷酸等，其合成方法与DNA的固相合成类似，都是于固相载体上重复进行单循环的化学反应，其合成特点也类似，柱式合成有较低错误率，但是试剂消耗量大且通量低，不利于节省成本，而微阵列合成有通量高、物料使用量少，但是错误率相对较高且不稳定。所以寻找一种能实现高通量、低成本、低错误率的合成芯片尤为重要。
生物芯片，又称蛋白芯片或基因芯片，它们起源于DNA杂交探针技术与半导体工业技术相结合的结晶。该技术系指将大量探针分子固定于支持物上后与带荧光标记的DNA或其他样品分子(例如蛋白，因子或小分子)进行杂交，通过检测每个探针分子的杂交信号强度进而获取样品分子的数量和序列信息。生物芯片技术起源于核酸分子杂交。所谓生物芯片一般指高密度固定在互相支持介质上的生物信息分子(如基 因片段、DNA片段或多肽、蛋白质、糖分子、组织等)的微阵列杂交型芯片(micro-arrays)，阵列中每个分子的序列及位置都是已知的，并且是预先设定好的序列点阵，是根据生物分子间特异相互作用的原理，将生化分析过程集成于芯片表面，从而实现对DNA、RNA、多肽、蛋白质以及其他生物成分的高通量快速检测。狭义的生物芯片概念是指通过不同方法将生物分子(寡核苷酸、cDNA、genomic DNA、多肽、抗体、抗原等)固着于硅片、玻璃片(珠)、塑料片(珠)、凝胶、尼龙膜等固相递质上形成的生物分子点阵。因此生物芯片技术又称微陈列(microarray)技术，含有大量生物信息的固相基质称为微阵列，又称生物芯片。生物芯片在此类芯片的基础上又发展出微流体芯片(microfluidics chip)，亦称微电子芯片(microelectronic chip)，也就是缩微实验室芯片。生物芯片同电子芯片一样，也是集成的，不过是生物材料的集成。像实验室检测一样，在生物芯片上检查血糖、蛋白、酶活性等，是基于同样的生物反应原理。所以生物芯片就是一个载体平台。由于生物芯片具有集成度高、通量大、灵敏度高、携带便携等潜在优点，因此其被广泛应用于多种场景，特别是在生物合成和检测方向，应用前景巨大。。
目前代表性的商业化微阵列合成仪，例如CustomArray合成仪，是将合成反应缩小到微米级别的反应孔内，一张芯片上有上万个反应孔，这样虽然提高了合成通量也一定程度上减少了原料的消耗，然而产量低，电化学反应不易控制，且错误率较高。另外，该芯片上还集成了温湿度传感器、控制电路等，制作工艺非常复杂，价格相对较为昂贵。Twist合成仪是利用高速的微量喷墨打印头作为单体等试剂的输送方式，在特殊处理的微米级的硅基芯片通孔上合成Oligo，然后再利用匹配的反应器与这些微孔对接，实现原位的PCR和PCA，从而直接得到大量的长片段的DNA分子。该生物芯片采用特殊硅基材料，上有特定化学修饰，通量较大，需要物理隔离，此外其芯片尺寸较大(尺寸与常规96孔板相当)，且未进行商业化。Evonetix电路控制方式的合成仪，主要利用半导体芯片具有大规模并行控制的特性，实现了对于不同位点反应的独立控制。然而在实际应用中，一个重要的问题在于如何避免不同位点反应的相互影响和控制产物的输出。2018年初，英国Evonetix公司公布了一项基于半导体芯片控制的合成技术。Evonetix技术的重点在于理论上接近十亿个位点的Oligo合成和可实时监测的高保真DNA纠错组装技术，其控制合成过程的原理如下：在具有特殊设计的大规模可寻址的合成位点的封闭腔室内，加入低熔点的可反复加热的阻断材料(例如，正二十四碳烷)，合成仪可以利用电路信号控制每个位点的通电与否，进而 选择是否加热该位点，在加热情况下，该位点上的特殊材料可吸附在该位点阻止后续通入的试剂在该位点上反应，如果后续需要在该位点进行合成，可用溶剂将该材料清洗掉，使得该位点暴露出来以进行合成反应，这样就实现了每个位点反应的单独控制。但该芯片集成除了特殊化学修饰外，还集成了电路控制，制作工艺较为复杂。
因此，本领域亟须新的技术突破，尤其是制作工艺简单，价格低廉，多检测通量，可重复利用，有识别功能的生物芯片。
发明概述
本发明提出了一种生物芯片，其制备方法和应用。本发明的生物芯片能够应用于DNA、RNA、多肽等生物大分子合成与检测，抗体筛选，抗原识别等，具有很大的应用前景。
在一个方面，本申请提供了一种生物芯片，所述芯片上携带化学实体和编码，其中所述编码与所述芯片具有唯一对应关系。
在另一个方面，本申请提供了制备本发明的生物芯片的方法，所述方法包括以下步骤：
1)对芯片进行编码；
2)对芯片进行预处理；
3)对芯片表面进行硅烷化处理；
4)对芯片表面进行化学修饰；和
5)任选地，对所述化学修饰进行检测分析或定量。
在另一个方面，本申请提供了本发明的生物芯片在DNA、RNA、多肽等生物大分子的合成中的应用。
在另一个方面，本申请提供了本发明的生物芯片在生物检测中的应用。
附图概述
图1示意性描述了本发明的可识别的二维码芯片的实例。
图2示意性描述了本发明的芯片用于合成DNA的流程。
图3示意性描述了本发明的芯片用于合成多肽的流程。
图4A显示了实施例6中实验1合成的T30产物的HPLC谱图。
图4B显示了实施例6中实验2合成的T30产物的HPLC谱图。
图4C显示了实施例6中实验3合成的T30产物的HPLC谱图。
图4D显示了实施例6中实验4合成的T30产物的HPLC谱图。
图4E显示了实施例6中实验5合成的T30产物的HPLC谱图。
图4F显示了实施例6中T30标样的HPLC谱图。
图5显示了实施例7中合成的产物的凝胶电泳图，其中Ctrl：标准合成引物对照；泳道1-3：Mix1-3。
发明详述
在本申请中，除非另有定义，否则本文所用的全部技术和科学术语都具有本申请所属领域普通技术人员所通常理解的含义。在本申请的实施方案中，可以使用与本文所述的那些方法和材料类似或等同的方法和材料，下文仅仅是描述了例示性的适合的方法和材料。将所有公开出版物、专利申请、专利和其它参考文献并入本文作为参考。此外，所述材料、方法和实施例仅仅是示范性的而非限制性的。同时，为了更好地理解本发明，本申请中也提供了相关术语的定义和解释。当本申请中提供的术语定义和解释与本申请所属领域普通技术人员所通常理解的意思相冲突时，以本申请提供的术语定义和解释为准。
生物芯片
在一个方面，本申请提供了一种生物芯片，所述芯片上携带化学实体和编码，其中所述编码与所述芯片具有唯一对应关系。
如本领域技术人员已知，芯片的基片由适合用作芯片的任何材料制备而成，所述材料不会不利地影响反应试剂的活性或者与反应试剂发生不期望的副反应。优选地，芯片由惰性材料制成。在某些优选的实施方案中，所述芯片由选自下列的材料制成：硅片(硅晶)、玻璃片(珠)、陶瓷、金属片、塑料片(珠)、凝胶、尼龙膜，或其任何组合，优选为硅片。在一个实施方案中，芯片由多孔玻璃制成，其粒径可为5μm-2000μm中的任意一种，更进一步地，其粒径为25μm，50μm，100μm，200μm，500μm中的任意一种；其孔径可为
Figure PCTCN2019124473-appb-000001
中的任意一种，更进一步地，其孔径可为
Figure PCTCN2019124473-appb-000002
中的任意一种。此类芯片可以具有任何期望的形状。例如，芯片可以是片状、长方体、圆柱体、球体等形状，优选的，芯片为正方形的片状。在一个具体实施方式中，生物芯片是微型DNA合成用芯片，其尺 寸为2mm*2mm，甚至是1mm*1mm、0.5mm*0.5mm或更小。在某些优选的实施方案中，本发明的芯片可回收重复利用。
编码
本发明的芯片具备可识别特性。为此，需要对芯片进行编码，其为芯片提供特异性信号。芯片上的特异性信号可以为磁信号、电信号、识别码等，优选的，特异性信号为二维码。
因此，在具体实施方案中，本发明的生物芯片携带编码，所述编码与所述芯片具有唯一对应关系。应当理解，此处的“编码”意指，任何可用于区分和鉴别芯片身份的特征。此类特征包括但不限于，数字，符号，图形，识别码例如条形码，二维码，优选为二维码。
在优选的实施方案中，芯片上携带的编码为二维码。维条码/二维码(2-dimensional bar code)是用某种特定的几何图形按一定规律在平面(二维方向上)分布的黑白相间的图形记录数据符号信息的；在代码编制上巧妙地利用构成计算机内部逻辑基础的“0”、“1”比特流的概念，使用若干个与二进制相对应的几何形体来表示文字数值信息，通过图象输入设备或光电扫描设备自动识读以实现信息自动处理。它具有条码技术的一些共性：每种码制有其特定的字符集；每个字符占有一定的宽度；具有一定的校验功能等。同时还具有对不同行的信息自动识别功能、及处理图形旋转变化点。二维码具备内置的纠错功能，能够在代码损坏或被涂抹的情况下恢复数据。它可以采用数学形式的纠错(Reed-Solomon)来恢复数据。
在某些优选的实施方案中，芯片上携带的编码为至少两个或更多个特征的组合。例如，芯片上携带的编码可以为识别码(例如条形码或二维码)与电信号的组合，识别码(例如条形码或二维码)和光信号(例如荧光)的组合，识别码(例如条形码或二维码)与数字的组合，识别码(例如条形码或二维码)与RFID标签的组合，RFID标签与电信号的组合，RFID标签和光信号(例如荧光)的组合，RFID标签与数字的组合，数字和光信号(例如荧光)的组合，或数字、图形和识别码(例如条形码或二维码)的组合等等。
编码的一个重要功能是用于区分和鉴别芯片。表述“编码与芯片具有唯一对应关系”意指，每一个芯片对应于一个独特的编码。换言之，各个芯片所携带的编码是彼此不同的。由于每一个芯片与其所携带的编码具有唯一对应关系，因此，可通过识别 所述编码，方便地区分多个芯片，或者从多个芯片中快速、方便地寻找和鉴别感兴趣的芯片。
在本发明的方法中，可使用各种方式来识别芯片上携带的编码。在某些优选的实施方案中，使用检测器(例如，通过识别数字，符号，图形，识别码(例如条形码，二维码)，RFID标签，光信号(例如荧光，化学发光，拉曼光谱)，量子点，磁信号，电信号，或其任何组合的检测器)来检测芯片上携带的编码，并使用处理器来分析所述检测器检测到的信号，从而识别芯片的身份(编码)。用于检测/识别数字，符号，图形，条形码，二维码，RFID标签，荧光，发光，量子点，拉曼光谱等各种方法和仪器是本领域技术人员熟知的，包括但不限于光信号识别器、磁信号识别器、电信号识别器、图像识别器、或其任何组合。例如，此类仪器可以为，二维条码检测仪、条形码检测仪、或RFID标签阅读器等。
芯片表面化学修饰
在芯片上添加了编码之后，对芯片的表面进行化学修饰，以达到该芯片能满足不同功能需求的目的。芯片表面修饰的分子的具体类型取决于芯片的实际应用。例如，用于合成生物大分子的芯片，其表面修饰有能够起始合成反应的分子；用于检测抗原的芯片，其表面修饰有特异性的抗体；用于筛选抗体的芯片，其表面修饰有特异性的抗原。
A.用于生物大分子合成
要在芯片表面合成不同种类的生物大分子，如DNA、RNA、多肽等，则每一种芯片需要其对应的芯片表面化学修饰；此外，还需要保证芯片表面有足够多的修饰分子，同时需要保证芯片表面修饰分子分布均匀，避免信号分布不均一。
为此，在一个具体方面，本申请提供了一种生物芯片，所述芯片上携带化学实体和编码，其中所述化学实体能够与反应试剂发生反应从而将反应试剂中的单体连接至所述化学实体的末端，所述编码与所述芯片具有唯一对应关系。
如本文所使用的，本发明的生物芯片是化学修饰的，即生物芯片的表面通过连接化学实体而进行了修饰。在具体实施方案中，所述化学实体为linker分子(连接分子)，其能够与反应试剂发生反应(偶联反应或连接反应)，从而将反应试剂中的单体连接至所述linker分子的末端。
用于合成核酸(DNA/RNA)的生物芯片以linker分子进行修饰，其中所述linker 分子优选为始端为带有可与氨基反应的官能团，末端为带有酸不稳定性保护基的羟基，用酸处理时可将羟基保护基去除以暴露出羟基，中间可以是酯基、脂基、硫酯基、邻硝基苄基、香豆素基团、羟基、巯基、巯醚基、羧基、醛基、氨基、胺基、酰胺基、烯基、炔基中任意一种或多种官能团的化合物，优选的始端官能团为羧基，末端羟基保护基是三苯甲基，例如4,4’-二甲氧基三苯甲基(DMT)，可以用二氯乙酸、三氯乙酸或三氟乙酸在有机溶剂(如二氯甲烷，乙腈)中的溶液处理时将其去除。在优选实施方案中，Linker分子为：DNA合成完毕后，氨解处理时能断开、易脱掉的分子，优选的Linker分子为Universal Linker，其中Universal Linker是指外-氮-苯基-5-(琥珀酰氧基)-6-二甲基-7-氧杂双环[2.2.1]庚烷-2-3-二甲酰亚胺、外-氮-甲基-5-(琥珀酰氧基)-6-二甲基-7-氧杂双环[2.2.1]庚烷-2-3-二甲酰亚胺或外-氮-甲基-5-(二甘醇氧基)-6-二甲基-7-氧杂双环[2.2.1]庚烷-2-3-二甲酰亚胺。Universal Linker的化学结构式如下：
(1)外-氮-苯基-5-(琥珀酰氧基)-6-二甲基-7-氧杂双环[2.2.1]庚烷-2-3-二甲酰亚胺
Figure PCTCN2019124473-appb-000003
(2)外-氮-甲基-5-(琥珀酰氧基)-6-二甲基-7-氧杂双环[2.2.1]庚烷-2-3-二甲酰亚胺
Figure PCTCN2019124473-appb-000004
(3)外-氮-甲基-5-(二甘醇氧基)-6-二甲基-7-氧杂双环[2.2.1]庚烷-2-3-二甲酰亚胺
Figure PCTCN2019124473-appb-000005
用于合成多肽的生物芯片以化学实体进行修饰，其中所述化学实体为固相法合成多肽的高分子载体(树脂)。这些树脂只有导入反应基团，才能直接连上第一个氨基 酸(初始氨基酸)。根据所导入反应基团的不同，把这些树脂及树脂衍生物分为氯甲基树脂、羧基树脂、氨基树脂或酰肼型树脂。BOC合成法通常选择氯甲基树脂，如Merrifield树脂；FMOC合成法通常选择羧基树脂如王氏树脂、2-Cl(Trt)-Clresin。起始氨基酸的固定主要是通过保护氨基酸的羧基同树脂的反应基团之间形成的共价键来实现的，形成共价键的方法有多种：氯甲基树脂，通常先制得保护氨基酸的四甲铵盐或钠盐、钾盐、铯盐，然后在适当温度下，直接同树脂反应或在合适的有机溶剂如二氧六环、DMF或DMSO中反应；羧基树脂，则通常加入适当的缩合剂如DCC或羧基二咪唑，使被保护氨基酸与树脂形成共酯以完成氨基酸的固定；氨基树脂或酰肼型树脂，则是加入适当的缩合剂如DCC后，通过保护氨基酸与树脂之间形成的酰胺键来完成氨基酸的固定。
在优选的用于合成多肽的生物芯片的技术方案中，用于修饰芯片的化学实体(linker分子)为羧基树脂，即末端含有可与羧基缩合反应的基团，如王氏树脂，2-Cl(Trt)-Clresin等，或类似化学结构的分子，优选为始端带有可与氨基反应的官能团、末端带有可与氨基酸单体羧基偶联反应的官能团，最终多肽合成完毕后，用酸处理时能解离的linker分子，中间可以是酯基、脂基、硫酯基、邻硝基苄基、香豆素基团、羟基、巯基、巯醚基、羧基、醛基、氨基、胺基、酰胺基、烯基、炔基中任意一种或多种官能团的化合物，如4-羟甲基苯甲酸，4-氯甲基苯甲酸，或取代的4-羟甲基苯甲酸，4-氯甲基苯甲酸等，优选的linker分子为4-羟甲基苯甲酸。
B.用于生物检测
在另一个具体方面，本申请提供了一种生物芯片，所述芯片上携带特异性抗原/抗体和编码，所述编码与所述芯片具有唯一对应关系。例如，用于检测抗原的芯片，其表面修饰有特异性的抗体；用于筛选抗体的芯片，其表面修饰有特异性的抗原。
生物芯片的制备
在另一个方面，本发明提供了一种生物芯片的制备方法，所述方法包括以下步骤：
1)对芯片进行编码；
2)对芯片进行预处理；
3)对芯片的表面进行硅烷化处理；
4)对芯片的表面进行化学修饰；
5)任选地，对所述化学修饰进行检测或定量分析。
如本发明所使用的，“对芯片进行编码”，意指为芯片添加一个独特的身份标签。应当理解，此处的“编码”意指，任何可用于区分和鉴别芯片身份的特征。此类特征包括但不限于，数字，符号，图形，识别码，例如条形码，二维码。在优选的实施方案中，“对芯片进行编码”包括在芯片上添加二维码，该二维码与所述芯片是唯一对应的。换言之，不同的芯片上添加的二维码是彼此各不相同的。
在具体实施方式中，本发明采用激光打印的方式，将二维码打印在芯片上，作为可识别信号。为了适应不同的应用场景，需要设计制作不同抛光及二维码类型的芯片，即不抛光单面二维码、不抛光双面二维码、单抛单面二维码、单抛双面二维码、双抛单面二维码、双抛双面二维码芯片，还可以在特殊需要的应用场景对芯片表面进行特殊处理，如磨砂或纳米级点位修饰以增加表面积，以确保二维码芯片的识别快速、准确、高效。
以DNA合成用二维码芯片制作及识别为例，本发明采取了如下技术：本发明提出的用于DNA合成的芯片尺寸可根据应用情况调整，例如可以是2mm*2mm，甚至是1mm*1mm、0.5mm*0.5mm或更小，最小可到亚毫米级别(实例请见附图1)。由于芯片尺寸微小，在打码模式的选择上，采用了打点二维码模式，为了确保切割质量，无毛刺，本发明提出的二维码芯片采用机械切割。为了保证打码深度，选择红光的激光，保证点位深且清楚，即便在后续长时间使用过程中有些许磨损，仍然不会影响二维码的识别。为了解决大量芯片使用过程中不可避免的黏连问题，采用震荡反应容器，在保证使用过程中高效率的同时，不影响芯片表面芯片反应的均一性。芯片的识别，即芯片拍照、放大、识别过程。为了更好地进行芯片识别，在打码时要保证二维码中的点尽量大、尽量深。
在具体的实施方式中，可首先将多个彼此不同的二维码等间距打印在整张硅片上，例如二维码大小可以是1mm*1mm。然后将硅片切割为2mm*2mm的芯片，切割时确保二维码位于每张芯片的中央。
如本文所使用的，“对芯片进行预处理”是为了达到清洁芯片表面的目的，同时暴露出更多羟基，提高表面反应活性。所述预处理步骤包括酸处理、碱处理、超声、等离子清洗、丙酮清洗或其组合，其中酸可选自硫酸、盐酸、磷酸、食人鱼液等，碱可选自氢氧化钠、氢氧化钾等。在优选的实施方案中，预处理方法为先进行酸处理，然后进行等离子清洗；更优选地，预处理方法为先进行氢氧化钠处理，然后进行去离子 清洗和丙酮清洗。
如本文所使用的，“对芯片表面进行硅烷化处理”是指进行芯片表面氨基修饰。硅烷化过程可采用气相沉积法(CVD)、溶液浸泡、简易负压气相沉积法等一种或多种处理，其中硅烷化试剂为一端为烷氧基硅基团，一端为氨基的试剂，可选自APTMS、APTES等，优选的，采用APTMS为硅烷化试剂，进一步优选地，采用50％的硅烷化试剂。在优选的实施方案中，采用简易负压气相沉积法，在能保证高效硅烷化的同时，又能保证操作的简便，保证表面修饰的均一性。
如本文所使用的，“对芯片表面进行化学修饰”意指在芯片表面连接化学实体，化学实体的具体种类由芯片的具体用途决定。
当生物芯片用于合成核酸(DNA/RNA)时，所述化学实体优选为始端带有可与氨基反应的官能团、末端带有酸不稳定性保护基的羟基的linker分子，用酸处理时可将羟基保护基去除以暴露出羟基，中间可以是酯基、脂基、硫酯基、邻硝基苄基、香豆素基团、羟基、巯基、巯醚基、羧基、醛基、氨基、胺基、酰胺基、烯基、炔基中任意一种或多种官能团的化合物，优选的始端官能团为羧基，末端羟基保护基是三苯甲基，例如4,4’-二甲氧基三苯甲基(DMT)，可以用二氯乙酸、三氯乙酸或三氟乙酸在有机溶剂(如二氯甲烷，乙腈)中的溶液处理时将其去除。
当生物芯片用于合成多肽时，所述linker分子为优选羧基树脂，即末端含有可与羧基缩合反应的基团，如王氏树脂，2-Cl(Trt)-Clresin等，或类似化学结构的分子，优选为始端带有可与氨基反应的官能团、末端带有可与氨基酸单体羧基偶联反应的官能团，最终多肽合成完毕后，用酸处理时能解离的linker分子，中间可以是酯基、脂基、硫酯基、邻硝基苄基、香豆素基团、羟基、巯基、巯醚基、羧基、醛基、氨基、胺基、酰胺基、烯基、炔基中任意一种或多种官能团的化合物，如4-羟甲基苯甲酸，4-氯甲基苯甲酸，或取代的4-羟甲基苯甲酸，4-氯甲基苯甲酸等，优选的link分子为4-羟甲基苯甲酸。
需要说明的是，生物芯片制备的步骤顺序，优选为先进行编码，后进行表面化学修饰，以便更好地保护修饰后的生物芯片表面，有利于生物芯片的后续应用。例如，当采用激光打印的方式将二维码打印在芯片上之后，经过预处理与硅烷化处理，最后再进行芯片表面化学修饰；如果将顺序颠倒，在对芯片进行预处理与硅烷化处理之后，进行芯片表面化学修饰，最后采用激光打印的方式将二维码打印在芯片上，则容易在激光打印过程中对芯片表面修饰的化学分子造成破坏，影响芯片后续的使用。
易于理解的是，在进行目标化合物合成时，芯片上携带的化学实体(linker分子)每一次接触反应试剂，都将使得反应试剂中的单体连接至所述linker分子的末端，使其延长。重复进行接触和反应，即可不断延长合成的化合物的长度，直至获得目标化合物。
在某些优选的实施方案中，在完成芯片上的合成反应后需要能够方便地将合成产物(目标化合物)从芯片上切割/分离。
例如，当目标化合物为DNA时，可通过氨解反应将目标化合物从芯片上切割下来。在某些优选的实施方案中，用于进行氨解反应的试剂可以选自氨水，氨气，甲胺，或其任何组合。在某些优选的实施方案中，氨解反应可以在选自下列的温度下进行：室温到120℃，例如，室温到60℃，60-90℃，90-120℃。在某些优选的实施方案中，氨解反应可以进行0.5h-48h，例如0.5-2h，2-5h，5-10h，10-18h，18-24h。在某些优选的实施方案中，在氨解反应后，对目标化合物(DNA)进行分离和纯化，例如，可使用MOP，PAGE，PAGE Plus，HPLC或其任何组合对目标化合物(DNA)进行分离和纯化。
例如，当目标化合物为RNA时，可通过氨解反应将目标化合物从芯片上切割下来。在某些优选的实施方案中，用于进行氨解反应的试剂可以选自氨水，氨气，甲胺，或其任何组合。在某些优选的实施方案中，氨解反应可以在选自下列的温度下进行：室温到120℃，例如，室温到60℃，60-90℃，90-120℃。在某些优选的实施方案中，氨解反应可以进行0.5h-48h，例如0.5-2h，2-5h，5-10h，10-18h，18-24h。在某些优选的实施方案中，在氨解反应后，对目标化合物(RNA)进行分离和纯化，例如，可使用PAGE，PAGE Plus，HPLC或其任何组合对目标化合物(RNA)进行分离和纯化。在某些优选的实施方案中，分离和纯化在无RNase的环境中进行。例如，在某些优选的实施方案中，采用HPLC纯化，并且用于分离和纯化的所有试剂和设备都不含RNase(即RNase-free)，避免RNase污染。
例如，当目标化合物为多肽时，可通过加入洗脱剂将目标化合物从芯片上切割下来。在某些优选的实施方案中，用于进行洗脱的试剂(洗脱剂)可以选自氢氟酸、三氟乙酸，四氟硼酸，或其任何组合。在某些优选的实施方案中，可以在选自下列的温度下进行洗脱反应：室温到120℃，例如，室温到60℃，60-90℃，90-120℃。在某些优选的实施方案中，洗脱反应可以进行0.5h-48h，例如0.5-2h，2-5h，5-10h，10-18h，18-24h。在某些优选的实施方案中，在洗脱反应后，对目标化合物(多肽)进行分离 和纯化。例如，可使用高效液相色谱、亲和层析、毛细管电泳，或其任何组合对目标化合物(多肽)进行分离和纯化。
任选地，在修饰步骤之后，为了表征芯片的修饰质量，可采取多种技术手段对所述化学修饰进行检测或定量分析。例如，可首先通过加入酸对linker分子末端的带有DMT保护的羟基进行脱保护，对收集溶液的颜色进行定性观测，溶液为红色表明Linker连接成功，即芯片修饰成功。进一步的，对收集的红色溶液进行定量，即通过紫外可见分光光度计进行DMT定量，确定修饰完毕的芯片表面Linker的分子数，以确定芯片表面分子载量。最后将脱掉DMT的芯片与6-FAM的荧光单体偶联，将偶联完毕的芯片清洗干净后置于荧光显微镜下观测荧光，通过荧光的有无、强度及均一性，可定性该修饰芯片的质量，即芯片表面荧光强度高，同时荧光信号均一，表明该修饰后的芯片质量好。
生物芯片用于核酸合成
本发明的芯片可用于合成核酸(DNA/RNA)。用于固相合成核酸的一般方法和原理是本领域技术人员所熟知的。通常而言，核酸的固相合成一般都采用固相亚磷酰胺三酯法，其通过多个轮次的循环反应，将多个核苷酸逐个、按顺序地连接至芯片上，从而获得包含多个核苷酸的目标产物，其中，每个轮次的循环反应可涉及4个步骤：即，脱保护，活化/偶联，盖帽，和氧化。
在DNA/RNA合成的具体实施技术方案中，根据合成所需单体的种类及合成通量要求，首先选择芯片编码方式和制定识别分选方案，制作不同通量的DNA/RNA合成用生物芯片，和相应的识别器、分选器、及控制芯片移动、试剂液体引入和引出的驱动装置。通过识别器识别芯片上的特异性信号，将其分选入含有相应的偶联单体的“反应池”中，然后通过控制驱动器将脱保护试剂引入反应池中进行脱保护，待反应完毕后引出脱保护试剂，然后再根据反应池对应的待偶联的单体，引入偶联试剂进行偶联，引出偶联试剂，最后再依次引入盖帽试剂与氧化试剂，分别进行盖帽与氧化，至此一个循环结束。通过程序设定，不断重复该循环直至合成完毕，最终可集中收集芯片获得DNA/RNA库或利用识别分选技术以获得单独DNA/RNA片段(流程示意图请见附图2)。
在多肽合成的具体实施方案中，根据多肽合成所需氨基酸单体的种类及合成通量 要求，首先选择芯片编码方式和制定识别分选方案，制作不同通量的多肽合成用生物芯片，和相应的识别器、分选器、及控制芯片移动、试剂液体引入和引出的驱动装置。通过识别器识别芯片上的特异性信号，将其分选入含有相应的偶联氨基酸单体的“反应池”中，然后通过控制驱动器将脱保护试剂引入反应池中进行脱保护，待反应完毕后引出脱保护试剂，然后再根据反应池对应的待偶联的氨基酸单体，引入偶联试剂进行偶联，引出偶联试剂，至此一个循环结束。通过程序设定，不断循环直至合成完毕，最终可集中收集芯片获得多肽库或利用识别分选技术以获得单独多肽片段(流程示意图请见附图3)。
生物芯片用于生物检测
本发明所述的生物芯片，可用于生物检测，如抗原、抗体筛选、疾病诊断等，尤其是可同时进行多生物样本检测。抗原抗体有特异性结合力，即一种抗原能特异性的与对应的抗体进行亲和性结合，利用这种特异性亲和力，即可进行特异性的抗原抗体筛选。常规的生物检测芯片，只能同时检测一种生物样本，即如果要筛选抗体A，将待筛选的抗体库通过有特异性抗原A修饰的芯片，然后洗脱、解吸附来获得目标抗体A。或在单一芯片上修饰多种抗原位点，以达到高通量筛选的目的，但是此种修饰显然较为复杂，且不易区分。
A抗体筛选
本发明的生物芯片，可实现简单高通量的抗体筛选。下面以可筛选1000种抗体的修饰芯片为例，描述具体的技术应用方案：
将1000片芯片(芯片尺寸为2mm*2mm，甚至是1mm*1mm、0.5mm*0.5mm或更小)混合在一起，每一张芯片上修饰有特异性的抗原，然后将抗体库的溶液加到混合后的芯片上，并使其没过，由于抗原和抗体有特异性结合作用，接触反应一段时间后，特异性的抗体会与其相应的芯片上的抗原结合，然后移除抗体库，并加入清洗试剂清洗芯片，以确保残留的未结合的抗体移除干净。随后根据芯片上标识的二维码，将1000片芯片分选到1000个不同的位点反应孔中，然后向每个盛放有芯片的反应孔中加入解吸附试剂，将芯片上结合的抗体与芯片上的抗原解吸分开，并反复多次以确保抗体完全解吸附。然后将1000个反应孔中的溶液再单独转移到新的1000个反应孔中，即可以同时筛选得到相应的1000种特异性抗体，最后对得到的抗体进行分析表征，对有阳性结果显示的抗体进行纯化以得到高纯度的抗体。通过以上技术方案，即 可快速高效的得到大量高纯度的特异性抗体，完成抗体的高通量筛选。
本发明提出的抗体筛选方面的应用，由于经抗原修饰的芯片体积微小，并且制作简单，可大批量生产，同时配合本发明提出的通过芯片上标记二维码进行快速、高效的识别和分选，该抗体筛选的技术应用的通量可达上万甚至百万级别。
B抗原检测
本发明的生物芯片，可实现简单高通量的抗原检测。下面以可检测1000种抗原的修饰芯片为例，描述具体的技术应用方案：
将1000片芯片(芯片尺寸为2mm*2mm，甚至是1mm*1mm、0.5mm*0.5mm或更小)混合在一起，每一张芯片上修饰有特异性的抗体，然后将抗原库的溶液加到混合后的芯片上，并使其没过，由于抗原和抗体有特异性结合作用，接触反应一段时间后，特异性的抗原会与其相应的芯片上的抗体结合，然后移除抗原库，并加入清洗试剂清洗芯片，以确保残留的未结合的抗原移除干净，随后根据芯片上标识的二维码，将1000片芯片分选到1000个不同的位点反应孔中，然后向每个盛放有芯片的反应孔中加入解吸附试剂，将修饰芯片上结合的抗原与芯片上的抗体解吸分开，并反复多次以确保抗体完全解吸附，然后将1000个反应孔中的溶液再单独转移到新的1000个反应孔中，即可以同时筛选得到相应的1000种特异性抗原，最后对得到的抗原进行分析表征确认。通过以上技术方案，即可快速高效的完成抗原的高通量检测。
本发明提出的技术应用，由于其芯片体积微小，并且制作简单，可大批量生产，同时配合本发明提出的通过在芯片上打印二维码进行快速、高效的识别和分选，该抗原检测的技术应用的通量可达上万甚至百万级别。
本发明通过以下多个实施例进行详细展开说明，其中实施例中使用到的主要试剂与耗材如下：
合成固相载体：常规芯片、100nm氧化硅片、300nm氧化硅片、磨砂石英芯片、透明石英芯片，尺寸：2*2*0.45mm
ACN(乙腈)：北京迪纳兴科
去保护试剂：3％TCA Deblock，北京迪纳兴科
活化剂：0.25M Activator，北京迪纳兴科
亚磷酰胺单体A、T、C、G:Sigma Aldrich
氧化剂：0.05M Oxidizing，北京迪纳兴科
CAP A：乙酸酐/吡啶/四氢呋喃1/1/8，北京迪纳兴科
CAP B：17.6％w/v氮-甲基咪唑/乙腈，北京迪纳兴科
氨水：国药
T30标样：深圳国家基因库
TA克隆试剂盒：pMDTM19-T，TaKaRa
实施例1.用于合成核酸(DNA/RNA)的芯片的制备
首先，委托广瑞特电子进行定制化的二维码芯片打印与切割。采用红光激光打印，二维码大小为1mm*1mm，将打印有二维码的裸硅片切割为2mm*2mm*0.45mm的微型芯片，每个微型芯片居中打印有特异性的二维码，使用食人鱼液对已经进行二维码打印、切割的芯片进行预处理，然后进行等离子清洗。之后，采用APTMS通过简易负压气相沉积法对预处理后的芯片进行硅烷化处理。最后，采用购自芜湖华仁科技的Universal Linker偶联于芯片上。
之后，通过加入酸对linker分子末端的带有DMT保护的羟基进行脱保护，对收集溶液的颜色进行定性观测，溶液为红色表明Linker连接成功，即芯片修饰成功。然后，对收集的红色溶液进行定量，即通过紫外可见分光光度计进行DMT定量，确定修饰完毕的芯片表面Linker的分子数，以确定芯片表面分子载量。或将脱掉DMT的芯片与6-FAM的荧光单体偶联，将偶联完毕的芯片清洗干净后置于荧光显微镜下观测荧光，通过荧光的有无、强度及均一性，可进一步定性该修饰芯片的质量，即芯片表面荧光强度高，同时荧光信号均一，表明该修饰后的芯片质量好。
具体修饰步骤如下：
1、配制100mL 0.1M NaOH溶液，分装于两个100mL烧杯中，每个烧杯放入2000片二维码常规芯片，手摇震荡清洗3min，首先使用去离子水清洗三次，丙酮清洗三次，晾干，使芯片表面洁净以便于后续进行硅烷化修饰。
2、配制100mL 50％的硅烷化试剂,分装于两个100mL烧杯中,每个烧杯放入2000片清洗过的二维码芯片，于超声仪超声30min，丙酮清洗5次，晾干。
其中，50％硅烷化试剂为：1％(APTES：PTES＝1:1)的丙酮溶液，以总体积100mL为例，各组分为APTES 500μL、PTES 500μL和丙酮99mL。
3、于干燥箱80℃干燥10min，取出待后续实验。
4、取1000mg Linker、800mg HATU、2000μL DIPEA加入100mL乙腈中，摇匀后分装于两个50mL离心管中,每个离心管放入2000片硅烷化的二维码芯片，于垂直搅拌仪搅拌过夜，反应完成后，收集芯片，分别用乙腈、丙酮清洗三次，晾干。
之后，取出30片修饰的二维码芯片，使用TCA deblock洗脱芯片表面DMT，使用紫外可见分光光度计测定DMT的浓度，测定修饰二维码芯片的Linker接枝密度。
5、将修饰完毕，干燥后的芯片收集至50mL离心管中，待用。
与此同时，比较了1％、100％硅烷化试剂对芯片修饰的影响，步骤同上，只是分别使用1％、100％硅烷化试剂替代50％硅烷化试剂，其中1％硅烷化试剂为：1％(APTES：PTES＝1:99)的丙酮溶液，以总体积100mL为例，各组分为APTES 10μL、PTES 990μL和丙酮99mL，100％硅烷化试剂为：1％(APTES：PTES＝1:0)的丙酮溶液，以总体积100mL为例，各组分为APTES 1000μL、PTES 0μL和丙酮99mL。最终获得三种不同硅烷化配比的修饰芯片的Linker接枝密度，结果如表1所示。
考虑到芯片表面的Linker接枝密度，与实际目标化合物的合成效果并不是正相关关系，即不是接枝密度越高，芯片合成效果越好，因为这其中涉及到分子拥挤的问题。为了能够保证芯片表面分子数足够多的同时，又需要同时权衡芯片在实际合成使用过程中，连接上的寡核苷酸链不会太拥挤，以至于影响最终的合成质量及长度。因此，本实施例在摸索最佳硅烷化试剂配比时，需要综合考虑Linker接枝密度、合成单步效率以及产物纯度等因素。本实施例中，在获得上述不同硅烷化试剂配比修饰的芯片之后，将芯片用于合成T5DNA序列(即TTTTT(SEQ ID NO.15))，具体方法参见实施例3，最终获得的产物纯度与单步合成效率见表1。从表1的结果可知，当使用50％硅烷化试剂进行修饰时，Linker接枝密度虽然没有100％硅烷化试剂配比的高，但是其所得产物的纯度及单步合成效率最高。
表1
Figure PCTCN2019124473-appb-000006
实施例2.用于合成多肽的芯片的制备
首先，委托广瑞特电子进行定制化的二维码芯片打印与切割。采用红光激光打印，二维码大小为1mm*1mm，将打印有二维码的裸硅片切割为2mm*2mm*0.45mm的微 型芯片，每个微型芯片居中打印有特异性的二维码，使用食人鱼液对已经进行二维码打印、切割的芯片进行预处理，然后进行等离子清洗。之后，采用APTMS通过简易负压气相沉积法对预处理后的芯片进行硅烷化处理。最后，将采购自安耐吉的4-羟甲基苯甲酸作为Universal linker偶联于芯片上。
实施例3.DNA的合成
在本实施例中，发明人使用如图2所示的DNA合成法，利用4种单碱基反应池进行DNA分子合成。具体步骤如下。
S1.准备合成单体、反应试剂及反应池
提供4种反应池(第一、第二、第三、第四反应池)，其分别用于添加脱氧核糖核苷酸A、T、C和G，以及在每个反应池中进行脱保护、盖帽、氧化和洗涤；其中，
-在偶联时，
第一反应池加入5’-羟基被DMT保护、3’-羟基被亚磷酰胺保护的腺苷酸以及四唑；
第二反应池加入5’-羟基被DMT保护、3’-羟基被亚磷酰胺保护的鸟苷酸以及四唑；
第三反应池加入5’-羟基被DMT保护、3’-羟基被亚磷酰胺保护的胞苷酸以及四唑；
第四反应池加入5’-羟基被DMT保护、3’-羟基被亚磷酰胺保护的胸苷酸以及四唑；
-在脱保护时，4个反应池均加入三氯乙酸的二氯甲烷溶液作为脱保护试剂；
-在盖帽时，4个反应池均加入乙酸酐/吡啶/四氢呋喃和N-甲基咪唑的乙腈溶液作为盖帽试剂；
-在氧化时，4个反应池均加入0.01M碘液作为氧化剂；
-在洗涤时，4个反应池均加入乙腈作为清洗液。
S2.准备芯片
使用实施例1中50％硅烷化试剂修饰后的5种不同类型的芯片(常规芯片、100nm氧化硅片、300nm氧化硅片、磨砂石英芯片、透明石英芯片)。
S3.准备设备和软件程序
提供设备，其中，所述设备包括，
-二维码识别器，其能够识别芯片上携带的二维码并产生信号；
-芯片分选器，其能够对芯片进行分选；
-驱动芯片移动的驱动装置；
-试剂液体驱动装置，其能够将各种“池”中的试剂液体引入和引出；
-中央处理器，其能够接收二维码识别器识别二维码所产生的信号，并发出指令控制芯片分选器分选芯片和/或控制驱动装置移动芯片；和
-存储器，其用于存储待合成的DNA分子的序列，芯片与二维码的对应关系，待合成的DNA分子的序列与芯片的对应关系，以及，每一个固相基质已经历的反应。
另外，还根据待合成的目标DNA序列设计控制程序，所述控制程序能够根据预先确定的DNA序列，确定芯片的分选方案。
S4.DNA合成
首先混合所有芯片，通过识别器识别预先印制在芯片上的二维码，中央处理器根据识别器的识别信号，控制芯片分选器分选芯片，并按照预设的控制程序，将每一张芯片移动至期望的反应池中，然后通过控制驱动器将脱保护试剂引入反应池中进行脱保护，待反应完毕后引出脱保护试剂，引入清洗液对芯片进行清洗，然后再根据反应池对应的待偶联的单体，引入偶联试剂进行偶联，待反应完毕后引出偶联试剂，引入清洗液对芯片进行清洗，最后再依次引入盖帽试剂、氧化试剂和清洗液，分别进行盖帽、氧化与清洗，至此一个循环结束，完成一个碱基的添加/偶联。具体的操作步骤、所使用的试剂及反应时间，如表2所示。
在一个碱基偶联后，根据预定的DNA合成序列信息，循环重复以上步骤，直至DNA分子合成完毕。
表2.
Figure PCTCN2019124473-appb-000007
Figure PCTCN2019124473-appb-000008
S5.DNA的分离和纯化
待DNA合成完毕后，可以选择将所有芯片统一收集在一起，进行氨解，由此分离和纯化所有合成的DNA分子。或者，可以利用二维码对所有芯片进行识别，分选和收集目的芯片，然后对目的芯片进行氨解，由此分离和纯化目的DNA分子。
本实施例中共选取了5种不同类型的芯片进行T30DNA(序列为：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(SEQ ID NO.16))合成，其中这5种芯片为：常规芯片、100nm氧化芯片、300nm氧化芯片、磨砂石英芯片、透明石英芯片。具体步骤如下：
(1)实验1-T30
取40片常规芯片(规格：2*2*0.45mm，双面具有二维码)，将其混合，根据合成的目标序列确定本次循环需要添加的单体，在芯片分选器分选之后，待合成相同单体的芯片被分选至同一个反应池，加入由75μL Cap A和75μL Cap B组成的盖帽试剂，将芯片浸泡在反应池中反应1次，把试剂排出之后再加入1次试剂，将芯片再次浸泡反应1次，两次共反应40s，然后排出盖帽试剂，加入乙腈清洗3次。排出乙腈加入去保护试剂TCA 150μL，反应15s，排出试剂后重新加入试剂再反应1次，15s。完成去保护步骤后，排出去保护试剂，加入250μL乙腈清洗3次。排出乙腈加入40μL亚磷酰胺单体T和60μL活化剂ACT，进行偶联反应60s，排掉试剂后重新加入试剂再反应1次，两次反应共120s，完成偶联步骤。排出偶联试剂，加入250μL乙腈清洗1次。排出乙腈加入由75μL Cap A和75μL Cap B组成的盖帽试剂，反应1次，排掉试剂后重新加入试剂再反应1次，两次反应共40s。排出盖帽试剂，加入250μL乙腈清洗1次。排出乙腈加入150μL氧化剂，反应20s，排掉试剂后重新加入试剂再反应1次，两次反应共40s，排出氧化试剂，加入250μL乙腈清洗3次，至此一个循环完成。将上述合成步骤循环30次，最后去保护，氨水氨解，处理后得到T30产物，进行Nanodrop定量测定单片合成量，并通过HPLC分析测定HPLC纯度，计算单步合成效率(单步合成效率由HPLC纯度分析获得，例如：合成T10的HPLC纯度为 80％，则单步合成效率为
Figure PCTCN2019124473-appb-000009
)，HPLC检测结果见图4A。
并且需要注意，在首次循环的脱保护之前，先进行盖帽处理，以封闭芯片表面端部基团不完整的区域，使其无法偶联构建单元，从而提高合成效率。从第二次循环开始，不需要再次进行盖帽，而是从识别分选开始，历经“脱保护-偶联-盖帽-氧化”步骤，如此循环直至合成完毕。
(2)实验2-T30
取40片100nm氧化芯片进行T30DNA合成，具体步骤同实验1，HPLC检测结果见图4B。
(3)实验3-T30
取40片300nm氧化芯片进行T30DNA合成，具体步骤同实验1，HPLC检测结果见图4C。
(4)实验4-T30
取40片磨砂石英芯片进行T30DNA合成，具体步骤同实验1，HPLC检测结果见图4D。(5)实验5-T30
取40片透明石英芯片进行T30DNA合成，具体步骤同实验1，HPLC检测结果见图4E。
将上述5个实验的DNA合成结果进行汇总，结果如表3所示。从表3可知，使用透明石英芯片进行DNA合成，所得产物纯度和单步合成效率最高。相对于单步合成效率最低的100nm氧化芯片，单步合成效率提高了2.5％，即每一循环的合成效率由96.8％提高到99.3％，而整个合成过程需要30个循环，那么在经历整个合成过程后所得产物的纯度显著提高，由37.9％提高至81.0％。当合成的序列越长，反应循环数越多，产物纯度的提高就越显著。
表3
Figure PCTCN2019124473-appb-000010
Figure PCTCN2019124473-appb-000011
(6)实验6基于透明石英芯片的59nt寡核苷酸合成及基因组装测试、测序结果分析
为了进一步确认最佳合成载体，使用透明石英芯片进行14条59nt长度的寡核苷酸的合成，序列如表4所示，并进行基于一步法PCA/PCR反应策略的小片段基因组装、跑胶胶图验证目标条带正确性，最后通过切胶回收、TA克隆转化、送Sanger测序，通过分析比对测序结果，最终确定最佳固相合成载体。
表4
Figure PCTCN2019124473-appb-000012
Figure PCTCN2019124473-appb-000013
具体步骤如下：
取42片透明石英芯片按照实施例3中的实验1的方法步骤进行寡核苷酸的合成，最后去保护，分别合成14条上表序列寡核苷酸，每条序列使用平行的3张芯片进行合成(即每条序列合成使用3组平行试验)，总共合成42条。将42条引物3*(59nt-1-59nt-14)，分成3组(14片/组)氨解，处理后得到目标3组59nt Mix产物各50μL。取10μL样品，分别加入4μL dNTPs、5μL Buffer、4μL首尾引物、0.5μL DNA聚合酶，用水将其体积补至50μL混匀，进行一步法PCA/PCR反应。使用降落PCR扩增35个循环后，产物于12℃保存。取2μL PCR产物点样至胶孔中，调节电压为180V，电泳时间为30min，进行跑胶检测。结果如图5所示。从图5中可看出，基于透明石英芯片合成的59nt引物，组装成的560bp基因条带，与标准合成引物对比清晰、正确。
另将条带正确的PCR产物切胶回收后，利用TaKaRa公司的TA克隆试剂盒(pMDTM19-T)进行克隆转化实验，菌落PCR验证条带正确的TA克隆转化子送Sanger测序。Sanger测试结果如表5所示，3组平行实验的平均错误率为0.34％。
表5
Figure PCTCN2019124473-appb-000014
Figure PCTCN2019124473-appb-000015
经过对不同类型的二维码芯片进行化学修饰，以及基于修饰芯片进行基于浸泡-识别-分拣的一系列59nt寡核苷酸合成、基因合成测试，并对多种芯片合成HPLC分析结果进行对比，表明：经过化学修饰的二维码芯片，可以进行寡核苷酸的合成，且透明石英芯片的合成效果最好，相应的T30引物的单片合成量26.8pmol，HPLC纯度81.0％，单步合成效率达到99.3％，最终通过14条59nt引物合成、小片段组装、Sanger测序进一步验证了透明石英芯片效果更佳，且Mix组测序结果错误率可低至0.3％。
以上实施例及实验结果均表明本发明提出的生物芯片可用于DNA合成，且最终测试对比的透明石英芯片合成效果好，与目前商业化主流合成效果有可比性，进一步表明本发明提出的化学修饰的可识别的生物芯片的可行性高，且有很大的应用前景。
实施例4.RNA的合成
在本实施例中，发明人使用基于浸泡-分选的RNA合成法，利用4种单碱基反应池进行RNA分子合成。具体步骤如下。
S1.准备合成单体、反应试剂及各种“池”
提供4种池(第一、第二、第三、第四反应池)，其分别用于添加核糖核苷酸A、U、C和G，以及在每个反应池中进行脱保护、盖帽、氧化和洗涤；其中，
-在偶联时，
第一反应池加入5’-羟基被DMT保护、2'-羟基被TBDMS保护、3’-羟基被亚磷酰胺保护的腺苷酸以及四唑；
第二反应池加入5’-羟基被DMT保护、2'-羟基被TBDMS保护、3’-羟基被亚磷酰胺保护的鸟苷酸以及四唑；
第三反应池加入5’-羟基被DMT保护、2'-羟基被TBDMS保护、3’-羟基被亚磷酰胺保护的胞苷酸以及四唑；
第四反应池加入5’-羟基被DMT保护、2'-羟基被TBDMS保护、3’-羟基被亚磷酰胺保护的尿苷酸以及四唑；
-在脱保护时，4个反应池均加入三氯乙酸的二氯甲烷溶液作为脱保护试剂；
-在盖帽时，4个反应池均加入乙酸酐/吡啶/四氢呋喃和N-甲基咪唑的乙腈溶液作为盖帽试剂；
-在氧化时，4个反应池均加入0.01M碘液作为氧化剂；
-在洗涤时，4个反应池均加入乙腈作为清洗液。
S2.准备芯片
使用实施例1中50％硅烷化试剂修饰后的的芯片。
S3.准备设备和软件程序
提供设备，其中，所述设备包括，
-二维码识别器，其能够识别芯片携带的二维码并产生信号；
-芯片分选器，其能够对芯片进行分选；
-驱动芯片移动的驱动装置；
-试剂液体驱动装置，其能够将各种“池”中的试剂液体引入和引出；
-中央处理器，其能够接收二维码识别器识别二维码所产生的信号，并发出指令控制芯片分选器分选芯片和/或控制驱动装置移动芯片；和
-存储器，其用于存储待合成的RNA分子的序列，芯片与二维码的对应关系，待合成的RNA分子与芯片的对应关系，以及，每一个芯片已经历的反应。
另外，还根据待合成的目标RNA序列设计控制程序，所述控制程序能够根据预先确定的RNA序列，确定芯片的分选方案和移动方案。
S4.RNA合成
首先混合1000张芯片，通过识别器识别预先印制在芯片上的二维码，中央处理器根据识别器的识别信号，控制芯片分选器分选芯片，并按照预设的控制程序，将每一张芯片移动至期望的反应池中，然后通过控制驱动器将脱保护试剂引入反应池中进行脱保护，待反应完毕后引出脱保护试剂，引入清洗液对芯片进行清洗，然后再根据反应池对应的待偶联的单体，引入偶联试剂进行偶联，待反应完毕后引出偶联试剂，引入清洗液对芯片进行清洗，最后再依次引入盖帽试剂、氧化试剂和清洗液，分别进行盖帽、氧化与清洗，至此一个循环结束，完成一个碱基的添加/偶联。具体的操作步骤、所使用的试剂及反应时间，如表6所示。
在一个碱基偶联后，根据预定的RNA合成序列信息，循环重复以上步骤，直至1000条RNA分子合成完毕。
表6.
Figure PCTCN2019124473-appb-000016
S5.RNA的分离和纯化
待RNA合成完毕后，可以选择将1000张芯片统一收集在一起，进行氨解，由此分离和纯化所有合成的RNA分子。或者，可以利用二维码对1000张芯片进行识别，分选和收集目的芯片，然后对目的芯片进行氨解，由此分离和纯化目的RNA分子。
分离和纯化每一张芯片所合成的RNA分子，并进行测序。测序结果显示，所合成的每一种RNA分子均具有预期的目标序列。
实施例5.多肽的合成
在本实施例中，发明人使用如图3所示的基于浸泡-分选的多肽合成法，利用21种单氨基酸合成池进行多肽分子合成。具体步骤如下。
S1.准备合成单体、反应试剂及各种“池”
提供23种池，其中，
-21种合成池(对应于21种常规的氨基酸单体)，其各自用于添加一种不同的氨基酸单体；其中，每一种合成池包含一种α-氨基被Fmoc保护、侧链被保护(如果有侧链的话)的氨基酸单体，以及四甲基脲六氟磷酸盐、N-甲基吗啉和DMF；
-脱保护池，其包含20％哌啶的二甲基甲酰胺(DMF)溶液作为脱保护试剂；
-洗涤池，其包含DMF/DCM作为清洗液。
S2.准备芯片
使用实施例2中制备好的芯片。
S3.准备设备和软件程序
提供设备，其中，所述设备包括，
-二维码识别器，其能够识别芯片携带的二维码并产生信号；
-芯片分选器，其能够对芯片进行分选；
-驱动芯片移动的驱动装置；
-试剂液体驱动装置，其能够将各种“池”中的试剂液体引入和引出；
-中央处理器，其能够接收二维码识别器识别二维码所产生的信号，并发出指令控制芯片分选器分选芯片和/或控制驱动装置移动芯片；和
-存储器，其用于存储待合成的多肽分子的序列，芯片与二维码的对应关系，待合成的多肽分子与芯片的对应关系，以及，每一个芯片已经历的反应。
另外，还根据待合成的目标多肽序列设计控制程序，所述控制程序能够根据预先确定的多肽序列，确定芯片的分选方案和移动方案。
S4.多肽合成
首先将1000张芯片置于脱保护池中进行脱保护，待反应完毕后，转移至洗涤池中进行清洗两次。然后，使用二维码识别器识别预先印制在芯片上的特异性二维码。中央处理器根据二维码识别器的识别信号，控制芯片分选器分选芯片，并按照预设的控制程序，将每一张芯片移动至期望的合成池中，进行偶联反应。待偶联反应完毕后，将所有芯片转移至洗涤池中进行清洗两次，由此完成一个氨基酸的添加/偶联。其中，每一步反应时，都需要先利用试剂液体驱动装置将对应的试剂引入池中，待反应结束之后，再利用试剂液体驱动装置将试剂从池中引出。具体的操作步骤、所使用的试剂及反应时间，如表7所示。
在一个氨基酸偶联后，根据预定的多肽序列信息，循环重复以上步骤，直至1000条多肽分子合成完毕。
表7
Figure PCTCN2019124473-appb-000017
Figure PCTCN2019124473-appb-000018
S5.多肽的分离和纯化
待多肽合成完毕后，可以选择将1000张芯片统一收集在一起，进行解离，由此分离和纯化所有合成的多肽分子。或者，可以利用二维码对1000张芯片进行识别，分选和收集目的芯片，然后对目的芯片进行解离，由此分离和纯化目的多肽分子。
分离和纯化每一张芯片所合成的多肽分子，并进行测序。测序结果显示，所合成的每一种多肽分子均具有预期的目标序列。

Claims (23)

  1. 一种生物芯片，所述芯片上携带化学实体和编码，其中所述编码与所述芯片具有唯一对应关系。
  2. 根据权利要求1所述的生物芯片，其中所述化学实体能够与反应试剂发生反应从而将反应试剂中的单体连接至所述化学实体的末端。
  3. 根据权利要求2所述的生物芯片，其中所述化学实体为能够起始DNA/RNA合成反应的linker分子，优选为始端带有可与氨基反应的官能团、末端带有酸不稳定性保护基的羟基的linker分子，更优选为Universal Linker。
  4. 根据权利要求2所述的生物芯片，其中所述化学实体为能够起始多肽合成反应的linker分子，优选为始端带有可与氨基反应的官能团、末端带有可与氨基酸单体羧基偶联反应的官能团，最终多肽合成完毕后，用酸处理时能解离的linker分子，更优选为4-羟甲基苯甲酸。
  5. 根据权利要求1所述的生物芯片，其中所述芯片上携带特异性抗原，用于筛选抗体；或者，所述芯片上携带特异性抗体，用于检测抗原。
  6. 根据权利要求1-5任一项所述的生物芯片，其中所述编码为所述芯片身份的特异性表征，其包括数字，符号，图形，和/或识别码；优选地，所述编码为二维码。
  7. 根据权利要求1-6任一项所述的生物芯片，其中所述芯片具有下列一项或多项特征：
    (1)所述芯片选自不抛光单面二维码、不抛光双面二维码、单抛单面二维码、单抛双面二维码、双抛单面二维码、双抛双面二维码芯片；
    (2)所述芯片尺寸小于2mm*2mm，优选为0.5mm*0.5mm；
    (3)所述芯片由选自下列的材料制成：硅片(硅晶)、玻璃片(珠)、陶瓷、金属片、塑料片(珠)、凝胶、尼龙膜，或其任何组合，优选为硅片；
    (4)所述芯片由多孔玻璃制成，所述多孔玻璃粒径为5μm-2000μm；所述多孔玻璃孔径为
    Figure PCTCN2019124473-appb-100001
    (5)所述芯片形状为片状、长方体、圆柱体、或球体，优选地，所述芯片为正方形的片状；
    (6)所述芯片选自100nm氧化硅片、300nm氧化硅片、磨砂石英芯片或透明石英芯片；优选地，所述芯片为透明石英芯片；
    (7)所述芯片的表面经过硅烷化试剂处理，优选地，所述硅烷化试剂选自APTMS或APTES，更优选地，所述硅烷化试剂为50％的硅烷化试剂。
  8. 制备根据权利要求1-7任一项所述的生物芯片的方法，所述方法包括以下步骤：
    1)对所述芯片进行编码；
    2)对所述芯片进行预处理；
    3)对所述芯片的表面进行硅烷化处理；
    4)对所述芯片的表面进行化学修饰；
    5)任选地，对所述化学修饰进行检测或定量分析。
  9. 根据权利要求8所述的方法，其中所述预处理包括酸处理、碱处理、超声、等离子清洗、丙酮清洗或其组合，其中酸选自硫酸、盐酸、磷酸、食人鱼液，碱选自氢氧化钠、氢氧化钾；优选地，预处理方法为先进行酸处理，然后进行等离子清洗；更优选地，预处理方法为先进行氢氧化钠处理，然后进行去离子清洗和丙酮清洗。
  10. 根据权利要求8所述的方法，其中通过在所述芯片表面连接化学实体，从而实现对所述芯片的表面进行化学修饰。
  11. 根据权利要求8所述的方法，其中采用激光打印的方式将二维码打印在所述芯片上，优选地，采用红光的激光通过打点二维码模式进行打码。
  12. 根据权利要求8所述的方法，其中通过将所述芯片浸泡在硅烷化试剂中并进行超声处理来对所述芯片的表面进行硅烷化处理，优选地，所述硅烷化试剂包括APTES和PTES。
  13. 根据权利要求12所述的方法，其中所述硅烷化试剂为50％的硅烷化试剂。
  14. 根据权利要求1-7任一项所述的生物芯片或根据权利要求8-13任一项所述的方法制备的生物芯片在生物大分子合成中的应用。
  15. 根据权利要求14所述的应用，其中所述生物大分子为核酸或多肽。
  16. 权利要求1-7任一项所述的生物芯片或根据权利要求8-13任一项所述的方法制备的生物芯片在抗原或抗体检测中的应用。
  17. 一种核酸合成方法，所述方法包括以下步骤：
    (1)提供根据权利要求1-7任一项所述的生物芯片或根据权利要求8-13任一项所述的方法制备的生物芯片，其中，所述生物芯片上携带的编码对应于待合成的核酸的序列；
    (2)提供4种反应池，其分别用于添加脱氧核糖核苷酸A、T、C、G或核糖核 苷酸A、U、C、G，以及在每个所述反应池中进行脱保护、盖帽、氧化和洗涤；
    (3)识别所述生物芯片携带的编码，并根据编码所对应的序列，确定待添加的脱氧核糖核苷酸或核糖核苷酸，将待添加相同脱氧核糖核苷酸或核糖核苷酸的芯片分选至同一反应池；
    (4)在所述反应池中加入脱保护试剂，将所述生物芯片浸泡在所述反应池中进行脱保护，反应结束后排出所述脱保护试剂；
    (5)在所述反应池中加入相应的脱氧核糖核苷酸或核糖核苷酸的偶联试剂，将所述生物芯片浸泡在所述反应池中进行偶联反应，反应结束后排出所述偶联试剂；
    (6)在所述反应池中加入盖帽试剂，将所述生物芯片浸泡在所述反应池中进行盖帽反应，反应结束后排出所述盖帽试剂；
    (7)在所述反应池中加入氧化试剂，将所述生物芯片浸泡在所述反应池中进行氧化反应，反应结束后排出所述氧化试剂，由此完成一个核苷酸的添加；
    (8)根据编码所对应的待合成核酸序列，重复步骤(3)至(7)一次或多次，从而在所述生物芯片上产生具有预先确定序列的核酸；
    任选地，所述方法还包括下述步骤：
    (9)将所述核酸从所述生物芯片上切割下来，从而获得所述核酸。
  18. 根据权利要求17所述的方法，在进行所述核酸合成之前，将所述生物芯片分选至所述反应池后，在所述反应池中加入盖帽试剂，将所述生物芯片浸泡在所述反应池中进行盖帽反应。
  19. 根据权利要求17或18所述的方法，其中使用多个芯片同时进行合成反应。
  20. 一种高通量筛选抗体的方法，所述方法包括以下步骤：
    (1)将多个芯片混合，其中所述芯片为权利要求1-7任一项所述的生物芯片或根据权利要求8-13任一项所述的方法制备的生物芯片，并且每一个芯片上修饰有特异性的抗原并带有唯一编码；
    (2)将所述多个芯片浸入待检测样品溶液中并充分反应；
    (3)移除样品溶液并清洗所述芯片；
    (4)识别每个所述芯片上携带的编码，并根据编码将每个所述芯片彼此分开；和
    (5)将所述芯片上结合的抗体与芯片上的抗原解吸附，并鉴定得到的抗体。
  21. 一种高通量检测抗原的方法，所述方法包括以下步骤：
    (1)将多个芯片混合，其中所述芯片为权利要求1-7任一项所述的生物芯片或根据权利要求8-13任一项所述的方法制备的生物芯片，并且每一个芯片上修饰有特异性的抗体并带有唯一编码；
    (2)将所述芯片浸入待检测样品溶液中并充分反应；
    (3)移除所述样品溶液并清洗所述芯片；
    (4)识别每个所述芯片上携带的编码，并根据编码将每个所述芯片彼此分开；和
    (5)将所述芯片上结合的抗原与芯片上的抗体解吸附，并鉴定得到的抗原。
  22. 根据权利要求20或21所述的方法，其中在步骤(4)中，通过识别每个生物芯片上携带的编码对芯片进行分选，并通过驱动芯片移动的驱动装置将每个芯片移动到不同的反应容器中。
  23. 根据权利要求22所述的方法，其中通过向装有分选后的芯片的反应容器中加入解吸附试剂而使抗原和抗体解吸附。
PCT/CN2019/124473 2018-12-12 2019-12-11 一种生物芯片及其制备方法与应用 WO2020119706A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US17/413,493 US20220395799A1 (en) 2018-12-12 2019-12-11 Biochip, method of preparation and use thereof
AU2019395588A AU2019395588B2 (en) 2018-12-12 2019-12-11 Biochip and manufacturing method and application thereof
JP2021533681A JP7375998B2 (ja) 2018-12-12 2019-12-11 バイオチップ、その調製方法及び使用
SG11202106282RA SG11202106282RA (en) 2018-12-12 2019-12-11 Biochip, method of preparation and use thereof
EP19896991.7A EP3907293A4 (en) 2018-12-12 2019-12-11 BIOCHIP AND MANUFACTURING PROCESS AND USE THEREOF
CA3123119A CA3123119A1 (en) 2018-12-12 2019-12-11 Biochip, method of preparation and use thereof
CN201980082590.8A CN113166974B (zh) 2018-12-12 2019-12-11 一种生物芯片及其制备方法与应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811514682.2 2018-12-12
CN201811514682 2018-12-12

Publications (1)

Publication Number Publication Date
WO2020119706A1 true WO2020119706A1 (zh) 2020-06-18

Family

ID=71075896

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/124473 WO2020119706A1 (zh) 2018-12-12 2019-12-11 一种生物芯片及其制备方法与应用

Country Status (8)

Country Link
US (1) US20220395799A1 (zh)
EP (1) EP3907293A4 (zh)
JP (1) JP7375998B2 (zh)
CN (1) CN113166974B (zh)
AU (1) AU2019395588B2 (zh)
CA (1) CA3123119A1 (zh)
SG (1) SG11202106282RA (zh)
WO (1) WO2020119706A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112986556A (zh) * 2021-05-12 2021-06-18 佛山微奥云生物技术有限公司 一种用于快速检测的芯片及其加工方法
CN113096507A (zh) * 2021-03-19 2021-07-09 北京龙迈达斯科技开发有限公司 一种教学芯片及其制备方法
CN113092218A (zh) * 2021-03-19 2021-07-09 北京龙迈达斯科技开发有限公司 一种教学芯片及其制备方法
CN114682309A (zh) * 2020-12-29 2022-07-01 深圳华大生命科学研究院 芯片、制备芯片的方法及芯片的用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024065585A1 (zh) * 2022-09-30 2024-04-04 深圳华大生命科学研究院 生物芯片及其使用和制备方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130196880A1 (en) * 2012-01-27 2013-08-01 Ventana Medical Systems, Inc. Patterned devices and methods for detecting analytes
CN103645308A (zh) * 2013-12-09 2014-03-19 东南大学 一种微载体二维编码方法
US20160265032A1 (en) * 2015-03-11 2016-09-15 Hexna Corp. Portable dna analysis machine
CN106311365A (zh) * 2016-08-02 2017-01-11 清华大学 微液滴反应编码检测方法及其系统
CN106872562A (zh) * 2017-03-01 2017-06-20 北京毅新博创生物科技有限公司 质谱基片及制备方法与用途
WO2017127570A1 (en) * 2016-01-20 2017-07-27 Triv Tech, Llc Point-of-care nucleic acid amplification and detection
WO2017169196A1 (ja) * 2016-03-28 2017-10-05 富士フイルム株式会社 細胞のスクリーニング方法
CN107475071A (zh) * 2017-08-23 2017-12-15 清华大学 用于药敏实验的抗生素编码微芯片及其制备和检测方法

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2801904B1 (fr) * 1999-12-07 2002-02-08 Pasteur Institut Produits comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn
US6587579B1 (en) * 2000-01-26 2003-07-01 Agilent Technologies Inc. Feature quality in array fabrication
WO2001083826A2 (en) * 2000-05-03 2001-11-08 Massachusetts Institute Of Technology Methods and reagents for assembling molecules on solid supports
GB0113358D0 (en) * 2001-06-01 2001-07-25 Scient Generics Ltd Enhanced target capture
FR2835829B1 (fr) * 2002-02-13 2007-09-14 Centre Nat Rech Scient Nouveau procede de preparation de biopuces a adn ou a proteines et leurs applications
US7094537B2 (en) * 2002-04-30 2006-08-22 Agilent Technologies, Inc. Micro arrays with structured and unstructured probes
JP2003322652A (ja) 2002-05-07 2003-11-14 Sigma Koki Kk マイクロ検査素子
TWI308214B (en) * 2002-09-10 2009-04-01 Univ Nat Central Method of forming a biochip and application thereof
US7709047B2 (en) * 2003-01-24 2010-05-04 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target activated microtransfer
US20040152081A1 (en) * 2003-01-31 2004-08-05 Leproust Eric M. Viscosity control during polynucleotide synthesis
JP2007046933A (ja) 2005-08-08 2007-02-22 Tokyo Institute Of Technology 蛍光画像検出方法、蛍光画像検出基板、蛍光画像検出器、プログラムおよび記録媒体
US20080200342A1 (en) * 2007-02-15 2008-08-21 Rao Rupa S Device, Array, And Methods For Disease Detection And Analysis
EP2344687A4 (en) 2008-11-06 2013-11-13 Agency Science Tech & Res DEVICE FOR THE SYNTHESIS OF BIOPOLYMERS
CN102230003A (zh) * 2011-06-03 2011-11-02 中国人民解放军南京军区福州总医院 一种临床检测用适配子信标-液态芯片
JP2016204316A (ja) * 2015-04-24 2016-12-08 日東電工株式会社 核酸固相合成用リンカー及び担体

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130196880A1 (en) * 2012-01-27 2013-08-01 Ventana Medical Systems, Inc. Patterned devices and methods for detecting analytes
CN103645308A (zh) * 2013-12-09 2014-03-19 东南大学 一种微载体二维编码方法
US20160265032A1 (en) * 2015-03-11 2016-09-15 Hexna Corp. Portable dna analysis machine
WO2017127570A1 (en) * 2016-01-20 2017-07-27 Triv Tech, Llc Point-of-care nucleic acid amplification and detection
WO2017169196A1 (ja) * 2016-03-28 2017-10-05 富士フイルム株式会社 細胞のスクリーニング方法
CN106311365A (zh) * 2016-08-02 2017-01-11 清华大学 微液滴反应编码检测方法及其系统
CN106872562A (zh) * 2017-03-01 2017-06-20 北京毅新博创生物科技有限公司 质谱基片及制备方法与用途
CN107475071A (zh) * 2017-08-23 2017-12-15 清华大学 用于药敏实验的抗生素编码微芯片及其制备和检测方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GILHAM, P. T.KHORANA, H. G., J. A. M. CLIEM. SOC., 1958
MICHELWN, A. M.TODD, A. R., J. CHEM. SOC., 1955

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114682309A (zh) * 2020-12-29 2022-07-01 深圳华大生命科学研究院 芯片、制备芯片的方法及芯片的用途
CN114682309B (zh) * 2020-12-29 2024-04-09 深圳华大生命科学研究院 芯片、制备芯片的方法及芯片的用途
CN113096507A (zh) * 2021-03-19 2021-07-09 北京龙迈达斯科技开发有限公司 一种教学芯片及其制备方法
CN113092218A (zh) * 2021-03-19 2021-07-09 北京龙迈达斯科技开发有限公司 一种教学芯片及其制备方法
CN112986556A (zh) * 2021-05-12 2021-06-18 佛山微奥云生物技术有限公司 一种用于快速检测的芯片及其加工方法
CN112986556B (zh) * 2021-05-12 2022-02-18 佛山微奥云生物技术有限公司 一种用于快速检测的芯片及其加工方法

Also Published As

Publication number Publication date
AU2019395588B2 (en) 2023-11-09
CA3123119A1 (en) 2020-06-18
SG11202106282RA (en) 2021-07-29
JP7375998B2 (ja) 2023-11-08
CN113166974B (zh) 2024-04-05
CN113166974A (zh) 2021-07-23
EP3907293A1 (en) 2021-11-10
JP2022514227A (ja) 2022-02-10
EP3907293A4 (en) 2022-04-06
AU2019395588A1 (en) 2021-07-22
US20220395799A1 (en) 2022-12-15

Similar Documents

Publication Publication Date Title
WO2020119706A1 (zh) 一种生物芯片及其制备方法与应用
JP4294740B2 (ja) 分析物の系列的プロセシングのためのシステムおよび装置
US20040208800A1 (en) Apparatus and method for simultaneously conducting multiple chemical reactions
Cho et al. Microbead‐based affinity chromatography chip using RNA aptamer modified with photocleavable linker
US20220401957A1 (en) Information storage using enzymatic dna synthesis and digital microfluidics
US20060078927A1 (en) Methods for in situ generation of nucleic acid molecules
EP1307743B1 (en) Colloid compositions for solid phase biomolecular analytical systems
CN101221167A (zh) 一种毛细管微流控芯片
WO1999060170A1 (en) Linear arrays of immobilized compounds and methods of using same
EP1774033A2 (en) Multiplexing assays for analyte detection
US20070004027A1 (en) Method for manufacturing a biosensor element
Furka Forty years of combinatorial technology
Tozzi et al. Affinity chromatography techniques based on the immobilisation of peptides exhibiting specific binding activity
WO2019178033A1 (en) Ultrahigh throughput protein discovery
Nand et al. Emerging technology of in situ cell free expression protein microarrays
EP2090888B1 (en) Method for producing a biomolecule assay chip
CN103882532B (zh) 一种先导化合物的合成及筛选方法与试剂盒
WO2011150675A1 (zh) 包含多根微通道的生物芯片
CN103882531B (zh) 一种先导化合物的合成及筛选方法与试剂盒
US20100112558A1 (en) Probe Bead Synthesis and Use
US20110212842A1 (en) Method for screening ligand
US20060105383A1 (en) Biopolymer detecting method and biochip
US20050095626A1 (en) Method for separation and purification method of nucleic acid
Seneci Encoding techniques for pool libraries of small organic molecules
JP5294963B2 (ja) 標的物質の処理方法及び標的物質の総量を求める方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19896991

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021533681

Country of ref document: JP

Kind code of ref document: A

Ref document number: 3123119

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019395588

Country of ref document: AU

Date of ref document: 20191211

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019896991

Country of ref document: EP

Effective date: 20210712