A biochip and its preparation method and application

Abstract

The present invention relates to the technical field of biochip preparation, and in particular to a biochip and its preparation method and application. The biochip is a double-sided porous nano biochip with an anodic oxidation nanostructure on the surface. The surface of the chip can be chemically modified to provide the connection molecules required for the synthesis reaction of the polypeptide, thereby connecting the amino acid monomer to the surface of the biochip. The biochip provided by the present invention avoids complicated preparation and processing technology, and achieves the goal of high-throughput and flexible polypeptide synthesis at low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of biochip preparation, and in particular to a biochip and a preparation method and application thereof. Background Art

[0002] A polypeptide is a chain-like macromolecular substance formed by peptide bonds connected by the amino and carboxyl groups of one or more amino acid molecules through dehydration condensation. Polypeptides can be used to control human growth, development, immune regulation and metabolism. Their molecular structure is between that of amino acids and proteins, and they are naturally occurring active substances in organisms (Qu Peng et al., Modern Chinese Medicine, 2015(3), 285-289). With the development of proteomics, different types of active polypeptides have attracted more and more attention from scientists, and polypeptides have also been widely used in industries such as medicine, food, daily chemicals and medical treatment.

[0003] Peptide synthesis technology is mainly divided into biosynthesis, such as natural raw material extraction, enzymatic hydrolysis, fermentation, genetic recombination, etc., and chemical synthesis, among which chemical synthesis can be divided into liquid phase synthesis and solid phase synthesis (Zheng Long et al., Journal of Chemical Industry, 2021, 72 (7), 3538-3550). Merrifield first proposed the solid phase synthesis method of peptides (Solid Phase Peptide Synthesis, SPPS) in 1963. This method connects the carboxylic acid end of the amino acid to the resin particles through covalent bonds, introduces reactants and reaction reagents through the liquid phase, reacts chemically with the solid phase carrier, dehydrates and condenses the amino acids, and gradually extends the peptide chain (Merrifield, RB1963., 85 (14), 2149-2154). The SPPS synthesis method solves the problems of long time and cumbersome operation of the liquid phase synthesis method, and all purification steps can be completed by flushing and filtration, which greatly reduces the difficulty and cost of purification. Since then, solid phase synthesis has gradually become the mainstream technology for peptide chemical synthesis.

[0004] During the solid phase synthesis of polypeptides, the α-amino end of the peptide chain must be protected. Currently, the commonly used α-amino protection methods are the tert-butyloxycarbonyl (Boc) method and the 9-fluorenylmethyloxycarbonyl (Fmoc) method (Fu Youwei et al., Chemical Industry and Engineering, 2010, 27(4)). The solid phase carriers used in the synthesis process are all resins. These resins can only be combined with the initial amino acids after the introduction of reactive groups. According to the different reactive groups introduced into the resins, these resins and resin derivatives are divided into chloromethyl resins, carboxyl resins, amino resins or hydrazide resins. The Boc protection method often uses chloromethyl resins as solid phase carriers, such as Merrifield resins. However, during the synthesis process, it is necessary to use acid to repeatedly remove the Boc protecting group. This process will also remove part of the peptide chain from the carrier, affecting the final yield and synthesis effect. In the Fmoc method, hydroxy resins are often used as solid phase carriers, such as Wang resin, 2-Cl(Trt)-Cl resin, etc. The Fmoc synthesis method is favored by researchers because it can achieve deprotection in the final synthesis link and avoid the effect of acid deprotection on the peptide chain during the synthesis process. Since Fmoc is stable to acid, piperidine-CH2Cl2 or piperidine-DMF is often selected as a deprotection reagent. At the same time, the synthesis also requires excess reactants and reaction reagents, and the resin after synthesis needs a large amount of reagents to be cleaned before it can be recycled, which makes the overall safety and environmental friendliness of the synthesis low, and the synthesis cost is relatively high. When selecting a resin as a solid phase carrier, the amino acid connection sites that the resin can provide are also limited, which to a certain extent limits the synthetic capacity of the polypeptide per unit area of ​​the carrier. It is also difficult to achieve the goal of high-throughput synthesis using resin as a solid phase carrier for synthesis.

[0005] The current mainstream peptide synthesis method is the Fmoc method based on solid phase carrier synthesis. The synthesis process mainly involves first binding the first target amino acid to be synthesized to the solid phase carrier through a covalent bond, removing the amino group to provide a connection site for the coupling of the second amino acid, and then introducing the second target amino acid for condensation. It can be summarized into four major cycle steps: deprotection, cleaning, activation condensation, and cleaning. Repeat this cycle until the target peptide chain is obtained. By adding an acidic eluent, such as hydrofluoric acid, the target peptide chain can be cut off from the resin. The target product can be separated and purified by high performance liquid chromatography, capillary electrophoresis or any combination.

[0006] Traditional solid-phase synthesis methods are mostly based on resin synthesis. Due to the limited binding sites of the resin, it is still difficult to achieve the goal of high-load peptide synthesis per unit area. It is also difficult to track the peptides synthesized based on resin during the synthesis process. It is impossible to identify the information of the peptide library or a certain peptide fragment of the solid phase carrier, and it is difficult to directly select the desired target peptide. Since each cycle of peptide synthesis requires an excess of reaction reagents and each step of the cycle needs to be cleaned, the physical and chemical properties of the resin will decrease after repeated use, and the reusability is low, which also increases the cost of synthesis. At present, there are many peptide synthesizers based on different solid phase materials. According to the number of synthesizer channels, traditional peptide synthesis instruments can be divided into single-channel synthesizers and multi-channel synthesizers. According to the degree of automation of the synthesizer, it can be divided into fully automatic synthesizers and manual synthesizers. Major synthesizer manufacturers are also constantly innovating. The microwave peptide synthesizer launched by CEM, a US protein organic reaction equipment manufacturer, can increase the peptide synthesis rate by more than ten times compared with the first generation of peptide synthesizers, greatly improving the reaction speed (Jonathan McKinnon Collins. Microwave-assisted peptide synthesis. US7582728B2). The microarray spotter launched by Aurora Biotech in Canada realizes the solid phase synthesis of peptides based on biological microarray chips. It can synthesize peptides efficiently and with high throughput through the principle of microarray printing technology. Despite this, the number of spotting positions on a single chip of this type of microarray printing peptide synthesis device is only 96. If you want to increase the throughput, it will be limited by the conditions such as spotting density, machine model, disk surface, chip material, etc. It is difficult to meet the needs of convenient, fast, high-throughput, and multi-type peptide synthesis. In addition, the chip needs to be designed according to different throughput requirements, which further increases the cost and complexity of synthesis.

[0007] In order to realize the commercialization and automation of peptide solid phase chemical synthesis, reduce synthesis costs, and increase synthesis throughput, scientists have gradually set their sights on peptide synthesis technology based on microarray chips. Chip microarray synthesis is the second generation of synthesis technology after column synthesis. This technology usually selects micron-level biochips as synthesis carriers, achieving synthesis goals such as high integration, high throughput, and high sensitivity, and has an application prospect that cannot be ignored in peptide synthesis. The reactive area of ​​the biochip will directly affect the primer synthesis load. The larger the specific surface area of ​​the chip, the more amino acid reaction binding sites it can provide, and therefore the more peptide molecules it can carry. By constructing micro-nano structures on the surface of the biochip, the specific surface area of ​​the chip can be effectively increased, thereby achieving high-load synthesis. At present, the surface micro-nano structures of mainstream commercial biochips are mostly formed on their surfaces through micro-nano etching processes such as photolithography. The entire production process requires multiple thinning, cleaning, gluing, and etching. The process is complex, costly, and the processing accuracy still needs to be improved, which seriously limits the development of commercial peptide synthesis chips.

[0008] In general, although the current peptide synthesis technology is relatively mature and the synthesis speed has been greatly improved compared with the first generation of peptide synthesizers or liquid phase synthesis methods, it is still challenging to achieve the goals of high-throughput, traceable identification, low reagent consumption, low cost, and high-load peptide synthesis. How to use technological innovation to achieve low-cost, simple processing technology, high peptide synthesis capacity, reusable and recognizable biochips has become the focus of the development of synthetic peptide technology. Summary of the invention

[0009] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present invention provides a biochip and a preparation method and application thereof. The biochip is a double-sided porous nanobiochip with an anodized nanostructure on the surface. The surface of the chip can be chemically modified to provide the connecting molecules required for the synthesis reaction for the synthesis of polypeptides, thereby connecting the amino acid monomers to the surface of the biochip. The biochip provided by the present invention can be prepared by an anodization method with simple operation and low cost, which simplifies the preparation process and provides a higher load of synthetic products at a lower price.

[0010] To this end, the first aspect of the present invention provides a biochip, comprising:

[0011] Metal aluminum layer;

[0012] Anodized aluminum layers, the anodized aluminum layers are disposed on both sides of the metal aluminum layer and are connected to the metal aluminum layer through chemical bonds;

[0013] A chemical modification layer, the chemical modification layer is located on a surface of the anodized aluminum layer away from the metal aluminum layer, the chemical modification layer comprises a connecting molecule, and the connecting molecule is capable of connecting an amino acid monomer to a terminal of the connecting molecule;

[0014] The starting end of the linking molecule is a functional group that can react with an amino group, and the terminal end is a functional group that can react with a carboxyl group.

[0015] At present, the number of channels of multi-channel peptide synthesizers in peptide synthesis technology is only about ten, and the number of synthesis spots of a single single chip of a fully automatic microarray printing synthesizer is only 96. Improving the throughput will be limited by the machine model, chip structure and material, and it is difficult to meet the goal of high-throughput synthesis in a convenient and fast way. From the perspective of synthetic chips, in order to meet different throughput requirements, the material and structure of the peptide synthesis chip need to be redesigned and processed, and the process is cumbersome, and the synthesis cost will also increase. The chip-based synthesis method mainly adopts a planar structure with a small specific surface area, and the synthesis sites that can be provided are limited, so the synthesis load is not high. Even if it is processed to have a nanopore size and thus increase the specific surface area, this process has extremely high requirements on the structural design of the chip and the micro-nano processing accuracy, and the preparation process is also very complicated. In addition, the surface of the silicon-based chip is relatively easy to wear, which will affect its use effect to a certain extent. In summary, the synthesis cost of the existing peptide synthesis method is extremely high, and the processing accuracy of the biochip is high, which seriously limits the development of peptide synthesis chips.

[0016] The biochip provided by the present invention has an anodized aluminum layer distributed on both sides of the metal aluminum layer. The anodized aluminum layer can be prepared by an anodizing method with simple operation and low cost to obtain ordered microarray holes with a precision of 10-1000nm. The biochip can effectively avoid complicated preparation and processing technology, can adjust the pore size, cooperate with the synthesis of polypeptide chains of different lengths, and can adjust the specific surface area to achieve flexible requirements for synthetic loading, and achieve nmol-level polypeptide synthesis loading with lower reagent dosage and cost. The flux can be increased by 10-1000 times compared with traditional biochips, greatly improving the polypeptide synthesis flux, reducing the selection and processing costs of biochips, and providing a new model with great potential for polypeptide synthesis.

[0017] According to an embodiment of the present invention, the anodized aluminum layer is a porous nanostructure, and the inner diameter of the pores in the porous nanostructure is 100-1000 nm, thereby providing more active sites for subsequent connection of connecting molecules.

[0018] According to an embodiment of the present invention, the linking molecule includes at least one selected from Wang resin, 2-Cl(Trt)-Cl resin, and 4-hydroxymethylbenzoic acid.

[0019] According to an embodiment of the present invention, the surface of the anodized aluminum layer away from the metal aluminum layer is treated with a silanization agent to introduce amino groups on the surface. The amino groups can react with the starting groups of the connecting molecules, thereby connecting the connecting molecules to the biochip.

[0020] According to an embodiment of the present invention, one end of the silanization molecule in the silanization agent is an alkoxy silicon group for connecting to the anodized aluminum oxide layer; the other end of the silanization molecule is an amino group for connecting to the connecting molecule.

[0021] According to an embodiment of the present invention, the silanization molecule in the silanization agent includes at least one selected from APTES (3-aminopropyltriethoxysilane), APTS (3-aminopropyltriethoxysilane), and MSTFA (N-methyl-N-trimethylsilane trifluoroacetamide).

[0022] According to an embodiment of the present invention, the biochip carries a code, and the code has a unique corresponding relationship with the biochip, thereby achieving the distinction and identification of the chip, and the synthetic information carried on the chip corresponding to the code can be obtained by reading the code.

[0023] According to an embodiment of the present invention, the code is provided on a surface of each anodized aluminum layer away from the metal aluminum layer.

[0024] According to an embodiment of the present invention, the code includes at least one of numbers, symbols, graphics, and identification codes.

[0025] According to an embodiment of the present invention, the code is a QR code.

[0026] According to an embodiment of the present invention, the chip is in the shape of a sheet, a cuboid or a cylinder.

[0027] According to an embodiment of the present invention, the chip is in the shape of a square sheet with a side length of 1-2 mm.

[0028] According to an embodiment of the present invention, the thickness of the metal aluminum layer is 0.3-0.5 mm.

[0029] According to an embodiment of the present invention, the thickness of the anodized aluminum layer is 5-1000 μm.

[0030] The second aspect of the present invention provides a method for the biochip of the first aspect, the method comprising the following steps:

[0031] (1) performing anodizing treatment on both sides of the metal aluminum layer to obtain an anodized aluminum chip;

[0032] (2) The anodized aluminum chip is subjected to silanization treatment and chemical modification in sequence to obtain the biochip.

[0033] The preparation method of the biochip provided by the present invention uses a metal aluminum sheet as a substrate, avoids the problems of low resin reuse rate and unstable physical and chemical properties, and solves the problems of large brittleness, easy wear and high processing difficulty of traditional silicon-based chips. The preparation of ordered microarray holes with an accuracy of 10-1000nm is achieved by an anodic oxidation method with simple operation and low cost. After that, the surface is modified to provide the connection molecules required for the synthesis reaction for the synthesis of the polypeptide, so that the amino acid monomer is connected to the surface of the anodized aluminum chip. The preparation method is easy to operate, simplifies the preparation process, and realizes the provision of a synthetic product with a higher load at a lower cost.

[0034] According to an embodiment of the present invention, step (1) further includes encoding the anodized aluminum chip. The encoding process enables the biochip to have an identification function, which can effectively trace the information of the polypeptide fragments carried on the chip surface, and is conducive to the practical application of synthetic polypeptides.

[0035] According to an embodiment of the present invention, the encoding process includes printing the code on the anodized aluminum chip by laser etching.

[0036] According to an embodiment of the present invention, the encoding process further includes printing the two-dimensional code on the anodized aluminum chip in a dotted two-dimensional code mode.

[0037] According to an embodiment of the present invention, the encoding process includes performing double-sided mirror coding on the anodized aluminum chip.

[0038] According to an embodiment of the present invention, the silanization treatment in step (2) comprises immersing the anodized aluminum chip in an organic solvent containing a silanization agent.

[0039] According to an embodiment of the present invention, the silanization molecule in the silanization agent includes at least one selected from APTES, APTS, and MSTFA.

[0040] According to an embodiment of the present invention, the organic solvent is acetone, isopropanol or toluene.

[0041] According to an embodiment of the present invention, the chemical modification in step (2) includes connecting the linking molecule on the surface of the anodized aluminum chip.

[0042] The third aspect of the present invention provides the use of the biochip described in the first aspect or the biochip prepared according to the method described in the second aspect in polypeptide synthesis.

[0043] The biochip described in the first aspect or the biochip prepared according to the method described in the second aspect has a simple manufacturing process, low price, high synthetic load and throughput, is reusable, has recognition function, and can be used in the synthesis of polypeptides.

[0044] A fourth aspect of the present invention provides a method for synthesizing a polypeptide, the method comprising the following steps:

[0045] S1: providing the biochip described in the first aspect or the biochip prepared according to the method described in the second aspect;

[0046] S2: providing a reaction pool for adding the required amino acid monomers;

[0047] S3: determining the target amino acid monomer to be added according to the amino acid sequence of the polypeptide to be synthesized, and sorting the biochip into the reaction pool corresponding to the target amino acid monomer;

[0048] S4: connecting the target amino acid monomer to the biochip in the reaction pool, and performing a deprotection treatment on the target amino acid monomer;

[0049] S5: Based on the amino acid sequence, determine the next amino acid monomer connected to the target amino acid monomer, repeat step S4 until the amino acid monomer consistent with the amino acid sequence is connected to the biochip to obtain a polypeptide chain.

[0050] The polypeptide synthesis method provided by the present invention is based on the biochip provided by the first aspect of the present invention or the biochip prepared by the method according to the second aspect of the present invention, and can achieve nmol-level polypeptide synthesis loading capacity, while the flux can be increased by 10-1000 times compared with traditional biochips, which greatly improves the polypeptide synthesis flux, reduces the selection and processing costs of biochips, and provides a new model with great potential for polypeptide synthesis.

[0051] According to an embodiment of the present invention, the biochip carries a code corresponding to the amino acid sequence of the polypeptide to be synthesized.

[0052] According to an embodiment of the present invention, step S3 further includes: identifying the code carried by the biochip, and determining the target amino acid monomer to be added according to the amino acid sequence corresponding to the code, and sorting the biochip into a reaction pool corresponding to the target amino acid monomer.

[0053] According to an embodiment of the present invention, the method further comprises the following steps:

[0054] S6: cutting the polypeptide chain from the biochip to obtain the target polypeptide.

[0055] According to an embodiment of the present invention, step S6 further includes cutting the polypeptide chain from the biochip using a cutting agent.

[0056] According to an embodiment of the present invention, the cutting agent includes at least one selected from hydrofluoric acid, trifluoroacetic acid, and tetrafluoroboric acid.

[0057] According to an embodiment of the present invention, in step S6, the polypeptide chain is cut from the biochip at 20-120° C. to obtain the target polypeptide.

[0058] According to an embodiment of the present invention, the cutting time in step S6 is 0.5-48 hours.

[0059] According to an embodiment of the present invention, the method further comprises the following steps:

[0060] S7: separating and purifying the target polypeptide.

[0061] According to an embodiment of the present invention, the separation and purification method comprises any one selected from high performance liquid chromatography, affinity chromatography, and capillary electrophoresis.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) The biochip provided by the present invention adopts aluminum-based materials, which avoids the problems of low resin reuse rate and unstable physical and chemical properties, and solves the problems of high brittleness, easy wear and high processing difficulty of traditional silicon-based chips, thereby ensuring the reuse of chips and reducing production costs;

[0064] (2) The biochip provided by the present invention has an anodized aluminum layer on both sides, which is simple to prepare and low in cost. It realizes the preparation of ordered microarray holes with a precision of 10-1000nm, increases the reactive area of ​​the biochip, and increases the synthetic flux of peptides to 10,000. At the same time, the synthesis of peptides with different loadings and lengths can be achieved by adjusting the chip specific surface area and pore size, and the required peptide library can be quickly synthesized;

[0065] (3) The biochip provided by the present invention has identifiable features and can effectively trace the information of polypeptide fragments carried on the chip surface, which is conducive to the practical application of synthetic polypeptides.

[0066] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0068] Figure 1 The structure diagram of the biochip provided by the present invention is shown;

[0069] Figure 2The SEM image of the surface of the anodized aluminum layer in the biochip provided by the present invention is shown;

[0070] Figure 3 The cross-sectional SEM image of the anodized aluminum nanochannel in the biochip provided by the present invention is shown;

[0071] Figure 4 The schematic diagram of the biochip nanostructure provided by the present invention is shown;

[0072] Figure 5 A schematic diagram showing the HPLC detection results of the crude polypeptide provided by the present invention is shown;

[0073] Figure 6 A schematic diagram of HPLC detection results after the polypeptide is purified provided by the present invention is shown. DETAILED DESCRIPTION

[0074] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0075] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0076] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0077] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0078] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0079] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0080] According to an embodiment of the present invention, the present invention provides a biochip, the chip structure diagram is as follows Figure 1 As shown, including:

[0081] A metal aluminum layer 102;

[0082] Anodized aluminum layers 101 and 103, the anodized aluminum layers are disposed on both sides of the metal aluminum layer 102 and are connected to the metal aluminum layer 102 through chemical bonds;

[0083] A chemical modification layer, the chemical modification layer is located on the surface of the anodized aluminum layer away from the metal aluminum layer 102, the chemical modification layer includes a connecting molecule, and the connecting molecule can connect the amino acid monomer to the end of the connecting molecule;

[0084] The starting end of the linking molecule is a functional group that can react with an amino group, and the terminal end is a functional group that can react with a carboxyl group.

[0085] According to a specific embodiment of the present invention, the biochip can be processed into any shape and size, such as sheet, cuboid, cylinder, etc., according to actual production applications, in accordance with different load requirements and synthesizer requirements. Preferably, the chip is a square sheet with a side length of 1-2 mm. Preferably, the side length is 2 mm. Preferably, the thickness of the metal aluminum layer is 0.3-0.5 mm, and the thickness of the anodized aluminum layer is 5-1000 μm. The anodized aluminum layer is a porous nanostructure, and its SEM image is as follows: Figure 2 As shown, the anodized aluminum nanochannel (its cross-sectional SEM image is shown in Figure 3 The pore depth, pore diameter, pore angle, and pore density of the nanochannel can be adjusted according to actual production applications. Preferably, the inner diameter of the pores in the porous nanostructure is 100-1000nm, and the pore array is highly ordered and evenly distributed ( Figure 4 ), the pore surface and inner wall are relatively smooth and have hydroxyl active sites for subsequent connection of linker molecules, thereby solving the problem of low loading capacity of synthetic products in current biochips.

[0086] According to a specific embodiment of the present invention, the biochip carries a code, and the code has a unique corresponding relationship with the biochip. The code provides a specific signal for the chip, and achieves one code for one chip, so that the chip has an identifiable characteristic. By reading the code, the synthesis information carried on the chip corresponding to the code can be obtained, and it can be transported to the corresponding reaction pool for synthesis reaction. The specific signal can be an optical signal, a magnetic signal, an identification code, an RFID tag, etc., and there can also be such characteristic coding combinations for marking and identification. The code includes but is not limited to numbers, symbols, graphics, and identification codes. Preferably, the code is a two-dimensional code. Preferably, the front and back surfaces of the biochip carry the same and mirror-image two-dimensional code mark, which can be identified by a detector to distinguish different synthetic information of multiple chips, or quickly find the target chip from multiple chips for sorting. The code point marking range can be adjusted according to the chip size, and the marking range can be changed by adjusting the code point diameter and interval. Preferably, the entire biochip is cut into a 2mm×2mm square by laser cutting, and the code point marking range is adjusted to 1mm×1mm.

[0087] According to a specific embodiment of the present invention, after adding a unique code, the biochip can be silanized and chemically modified on the surface to meet the needs of polypeptide synthesis, so that the surface of the chip can be connected to an amino acid monomer reagent through a specific molecular structure, ensuring that there are enough reaction sites on the chip surface and that the sites are evenly distributed. Before silanization and chemical modification, the chip with the code added can be placed in an organic reagent, and the type of organic reagent can be acetone, isopropanol, or toluene with low water content. Preferably, acetone is selected as the organic reagent. Subsequently, a silanization reagent, such as APTES, APTS, or MSTFA, is added to react with the hydroxyl groups exposed on the chip surface and connect to the chip surface. This process can adjust the spacing of the reaction sites by adjusting the concentration of the silanization reagent, the time, temperature, and type of reagent for the silanization treatment, and optimize the density of the reaction sites on the chip surface. After the silanization process is completed, chemical modification can be performed, i.e., a connecting molecule is connected to the surface of the chip, and the connecting molecule is used to connect the amino acid monomer to the end of the connecting molecule. When performing peptide synthesis, the ends of the linker molecules carried on the biochip will be connected to the initial amino acids, and after each round of injection synthesis, a polypeptide molecule chain connected to the surface of the biochip through the linker molecules is formed. The starting end of the linker molecule is a functional group that can react with an amino group; the middle end can be a group with any one or more functional groups of ester, thioester, o-nitrobenzyl, coumarin, hydroxyl, sulfhydryl, sulfhydryl, carboxyl, aldehyde, amino, amine, amide, alkenyl, and alkynyl; the end is a functional group that can react with the carboxyl group in the amino acid monomer. The linker molecule can be dissociated with acid after the peptide synthesis is completed, so that the peptide product is separated from the biochip. Preferably, the linker molecule is a molecule with a similar chemical structure such as Wang resin, 2-Cl (Trt) -Cl resin or 4-hydroxymethylbenzoic acid.

[0088] According to an embodiment of the present invention, the present invention also provides a method for preparing the aforementioned biochip, the method comprising the following steps:

[0089] (1) performing anodizing treatment on both sides of the metal aluminum layer to obtain an anodized aluminum chip;

[0090] (2) The anodized aluminum chip is subjected to silanization treatment and chemical modification in sequence to obtain the biochip.

[0091] According to a specific embodiment of the present invention, step (1) further includes encoding the anodized aluminum chip. The encoding process can be performed by laser etching or dotted QR code mode to print the QR code on the anodized aluminum chip. As a unique identification signal, the QR code corresponds one-to-one to the chip to which it belongs, that is, each chip carries a different QR code. In order to ensure the recognition rate of the chip during use, the chip can be double-sided mirrored, that is, the surface of the anodized aluminum layer has the same and mirror-image QR code mark. The code point marking range can be adjusted according to the chip size, and the marking range is changed by adjusting the code point diameter and interval. Preferably, the marking range is 1mm×1mm.

[0092] According to a specific embodiment of the present invention, the anodized aluminum chip can be cut after step (1), that is, the size and geometry of the chip prepared by the anodizing method can be adjusted by processing. For example, by laser cutting, a square porous nano biochip with a side length of 1-2 mm is obtained. Preferably, the chip size is determined to be 2mm×2mm. In order to ensure the accuracy of cutting and improve the yield, while avoiding damage to the nanostructure of the anodized aluminum layer and the burr-free cutting edge of the coding site, the cut section should clearly show the nanopores of the upper and lower surface anodized aluminum layers and the intermediate metal aluminum layer.

[0093] According to a specific embodiment of the present invention, step (2) may also include pre-treating the anodized aluminum chip before silanization. The pre-treatment method includes a physical method or a chemical method, or a method combining physical and chemical methods. The physical method includes, but is not limited to, ultrasound and heat treatment. The chemical method includes cleaning with an organic reagent. The pre-treatment is, on the one hand, to clean the chip surface and remove impurities loaded on the chip surface during coding and cutting; on the other hand, to increase the surface activity of the chip, expose more hydroxyl groups, and increase silanization and subsequent reaction sites.

[0094] According to a specific embodiment of the present invention, the silanization treatment in step (2) includes immersing the pretreated anodized aluminum chip in an organic solvent containing a silanization agent, wherein the silanization agent may be APTES, APTS, or MSTFA, and the organic solvent may be acetone, isopropanol, or toluene with a low water content. The silanization treatment can uniformly introduce amino groups on the chip surface, and the spacing between synthesis sites can be controlled by APTES to adjust subsequent steric hindrance. Chemical modification includes connecting the connecting molecule to the surface of the anodized aluminum chip. The structure of the connecting molecule is as described above. When performing polypeptide synthesis, the connecting molecule carried on the chip will connect to the initial amino acid, connecting the amino acid monomer to the end of the connecting molecule, and after each round of injection synthesis, the chain will be extended to form a polypeptide molecule chain connected to the chip surface through the connecting molecule.

[0095] According to an embodiment of the present invention, the present invention also provides a method for peptide synthesis, which is based on the aforementioned biochip for peptide synthesis. Specifically, through multiple rounds of immersion cycle reactions, the target amino acid monomers are connected one by one to the connecting molecules on the surface of the biochip in the order of the target sequence to form a polypeptide chain. Each cycle mainly includes four major steps: deprotection, cleaning, condensation and secondary cleaning. In a specific embodiment, according to the type of amino acid monomers required for peptide synthesis and the requirements for synthetic flux, the chip encoding method is selected and the identification and sorting scheme is formulated to make peptide synthesis biochips with different fluxes. Specifically, 21 kinds of synthesis pools (corresponding to 21 conventional amino acid monomers) can be prepared, wherein each synthesis pool contains the following reagents: amino acid monomers with α-amino groups protected by Fmoc, coupling condensation reagents for peptide synthesis (preferably tetramethyluronium hexafluorophosphate), alkaline reagents (preferably N-methylmorpholine) and organic solvents (preferably N,N-dimethylformamide). Prepare corresponding identifiers, sorters, and drive devices for controlling chip movement, reagent liquid introduction and extraction. The amino acid monomers to be synthesized in this round are identified by the identifier and screened by the sorter. The chip is distributed to the corresponding amino acid reaction pool through the transport device. Through the control of the driver, the synthetic reagents required for each step are introduced according to the four major steps of each cycle. After the reaction, the reagents are drawn out until a cycle is completed. Through program settings, according to the required sequence, the cycle is repeated until the synthesis is completed, and finally the peptide library based on the biochip can be collected, or the target chip can be found by using the identification and sorting technology to obtain a single peptide fragment.

[0096] According to a specific embodiment of the present invention, the method further includes separating the chip from the synthetic product it carries. Specifically, the synthesized polypeptide chain can be cut from the chip using an acidic reagent. The acidic reagent used for elution and cutting can be hydrofluoric acid, trifluoroacetic acid, tetrafluoroboric acid, or any combination thereof. The cutting reaction can be carried out at a temperature from room temperature to 120°C, for example, 20-60°C, 60-90°C, 90-120°C. The reaction time can be 0.5h-48h, for example, 0.5-2h, 2-5h, 5-10h, 10-18h, 18-24h. After the elution and cutting are completed, the polypeptide product needs to be separated and purified, and high performance liquid chromatography, affinity chromatography, capillary electrophoresis, or any combination thereof can be used for separation and purification to finally obtain a high-purity polypeptide product.

[0097] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0098] Example 1 Preparation of biochip and synthesis of polypeptides

[0099] 1. Preparation of biochip

[0100] (1) Anodizing the upper and lower surfaces of the metal aluminum sheet to obtain an anodized aluminum chip;

[0101] (2) Coding and cutting the anodized aluminum chip substrate. Using laser printing technology, a 1mm×1mm QR code is printed in order on the anodized aluminum chip substrate material; using positioning cutting technology, the coded substrate material is cut into 2mm×2mm×0.45mm microchips. Each chip carries a specific QR code with the QR code point centered. Use acetone to pre-treat the coded and cut microchips, followed by plasma cleaning;

[0102] (3) Silanization treatment and chemical modification

[0103] Silanization treatment: prepare 100 mL of 50% silanization reagent and divide it into two 100 mL beakers. Put the cleaned QR code chip in each beaker, sonicate for 30 minutes in an ultrasonic instrument, clean it with acetone 5 times, and place it in a drying oven at 80°C for 10 minutes before taking it out.

[0104] The silanization agent is a 1 vol.% (APTES: PTES = 1:1) anhydrous ethanol solution. Taking a total volume of 100 mL as an example, the components are 500 μL APTES, 500 μL PTES and 99 mL anhydrous ethanol;

[0105] Chemical modification: 1000 mg of 4-hydroxymethylbenzoic acid, 800 mg of HATU, and 2000 μL of DIPEA were added to 100 mL of acetonitrile, shaken and divided into two 50 mL centrifuge tubes. 2000 silanized QR code chips were placed in each centrifuge tube and stirred overnight in a vertical stirrer. After the reaction was completed, the chips were collected, washed three times with acetonitrile and acetone respectively, and collected in centrifuge tubes after drying.

[0106] 2. Synthesis of peptides

[0107] Step 1: Prepare synthetic monomers, reaction reagents and reaction pool

[0108] 23 pools available, including:

[0109] (1) A synthesis pool of 21 conventional amino acid monomers, each used to add a different amino acid monomer. Each synthesis pool contains the following reagents: an amino acid monomer with an α-amino group protected by Fmoc, tetramethyluronium hexafluorophosphate, N-methylmorpholine, and N,N-dimethylformamide;

[0110] (2) Deprotection pool, which contains a 20% piperidine solution in dimethylformamide (DMF) as a deprotection reagent;

[0111] (3) Washing tank. It contains DMF / DCM as cleaning liquid;

[0112] Step 2: Prepare the chip

[0113] Take 1000 biochips that have been chemically modified and cleaned in Example 1 for use;

[0114] Step 3: Prepare your equipment and software program

[0115] The device comprises:

[0116] (1) A two-dimensional code reader capable of recognizing the two-dimensional code information carried by the biochip and generating a signal;

[0117] (2) Chip sorter, which can sort the chips;

[0118] (3) a driving device, which can drive the chip to move;

[0119] (4) a reagent driving device capable of introducing and removing reaction reagents from various reaction cells;

[0120] (5) a central processing unit capable of receiving the signal generated by the two-dimensional code reader and issuing instructions to control the chip sorter to sort the chips or to control the driving device to move the chips;

[0121] (6) A memory, used to store the sequence of the polypeptide molecules to be synthesized, the correspondence between the chip and the QR code, the correspondence between the polypeptide molecules to be synthesized and the chip, and the reactions completed by each chip.

[0122] In addition, a control program is designed according to the target polypeptide sequence to be synthesized, and the control program can determine the sorting scheme and movement scheme of the chip according to the predetermined polypeptide sequence;

[0123] Step 4: Peptide synthesis

[0124] Place the biochip in step 2 in the deprotection tank for deprotection for 15 seconds. After the reaction is completed, transfer it to the washing tank for washing twice, soaking for 60 seconds each time. After the specific QR code pre-printed on the chip is recognized by the QR code reader, the central processing unit receives the recognition signal of the identifier, controls the chip sorter to sort the chip, and moves each chip to the target synthesis tank according to the pre-set control program for coupling reaction. After the reaction is completed, transfer all chips to the washing tank for washing twice to complete the coupling reaction of one amino acid. According to the target polypeptide sequence information, the above reaction steps are repeated until 1000 polypeptide molecules are synthesized.

[0125] In each step of the reaction, the required reagents need to be introduced into the reaction pool by using a reagent driving device, and after the reaction is completed, the reaction reagents are drawn out by the reagent driving device;

[0126] Step 5: Isolation and purification of peptides

[0127] After the peptide synthesis is completed, you can choose to dissociate all 1,000 chips in a unified manner to separate and purify all synthesized peptide molecules; you can also choose to use an identifier to identify the specific QR code, sort and collect the target chips, dissociate the target chips, and separate and purify the target peptide molecules.

[0128] The peptide molecules synthesized on each chip were separated and purified, and then sequenced. The results showed that the synthesized peptide molecules all had the expected target sequence.

[0129] Example 2 Preparation of biochip and synthesis of polypeptides

[0130] In this embodiment, 21 kinds of amino acid reaction pools are used to synthesize peptides based on the soaking-sorting synthesis method. The specific equipment materials and steps are as follows.

[0131] 1. Equipment

[0132] High-throughput parallel synthesis equipment (BGI).

[0133] 2. Materials and reagents

[0134] Materials: Porous nanobiochip;

[0135] Reagents: APTES (98%) and PTES (97%) were purchased from J&K. Linker reagent (≥97%) was purchased from Wuhu Huaren Technology Co., Ltd. HATU (99%) was purchased from Aladdin. DIPEA (99%) was purchased from Adamas-beta. Acetonitrile (ACN), acetone, DEAPA, activator HOBT, coupling agent HBTU, organic base DIEA, TFA, DMF, DCM, methanol, ethanol, chromatographic acetonitrile, and ammonia were all purchased from Anaiji Chemical. Fmoc-protected amino acids ACDEFGHIKLMNPQRSTVWY were all purchased from Jier Biochemical.

[0136] 3. Experimental steps

[0137] 3.1 Chip processing: First, the porous nano biochip (AAO biochip) is coded and cut. Using laser printing technology, the 1mm×1mm QR code is printed on the wafer in order. Through positioning and cutting technology, the wafer is processed into a 2mm×2mm×0.45mm microchip with the QR code point position as the center. Each chip center carries a specific QR code. The coded and cut chips are pre-treated with piranha liquid, followed by plasma cleaning.

[0138] 3.2 Chemical modification

[0139] (1) Silanization treatment: Prepare 100mL of 50% silanization reagent and divide it into two 100mL beakers. Place the cleaned QR code chip in step 1 in each beaker, sonicate for 30 minutes in an ultrasonic instrument, clean it with acetone 5 times, and place it in an 80℃ drying oven for 10 minutes before taking it out. The silanization reagent is: 1vol.% (APTES: PTES=1:1) anhydrous ethanol solution. Taking the total volume of 100mL as an example, the components are 500μL APTES, 500μL PTES and 99mL anhydrous ethanol;

[0140] (2) Linker modification: 1000 mg of linker, 800 mg of HATU, and 2000 μL of DIPEA were added to 100 mL of acetonitrile, shaken and divided into two 50 mL centrifuge tubes. 2000 silanized QR code chips were placed in each centrifuge tube and stirred overnight in a vertical stirrer. After the reaction was completed, the chips were collected and washed three times with acetonitrile and acetone respectively, and then collected in centrifuge tubes after drying;

[0141] (3) Take the AAO biochip that has been chemically modified in (1) and (2) and set it aside.

[0142] 3.3 Prepare synthetic monomers, reagents and corresponding reaction pools

[0143] Provide 23 kinds of synthesis reaction cells, including:

[0144] (1) Adding a different amino acid monomer to each of the 21 synthesis pools, wherein each synthesis pool contains an Fmoc-protected amino acid monomer and a coupling reagent HBTU, an organic base DIEA, and an organic solvent DMF, as well as the reaction reagents required for each round of reaction (deprotection, capping, oxidation, and washing);

[0145] (2) In the deprotection cell, a dimethylformamide (DMF) solution containing 20% ​​piperidine was added as a deprotection reagent;

[0146] (3) In the washing tank, add DMF / DCM mixed reagent as a cleaning reagent.

[0147] 3.4 Preparing equipment and software programs

[0148] Using a high-throughput synthesis apparatus, wherein the apparatus comprises,

[0149] (1) A two-dimensional code reader capable of recognizing the two-dimensional code information carried by the chip and generating a signal;

[0150] (2) a chip sorter, which can sort the chips;

[0151] (3) a driving device for driving the chip to move;

[0152] (4) a reagent liquid driving device capable of introducing and removing the reagent liquid from the synthesis reaction cell;

[0153] (5) a central processing unit, which can receive the signal generated by the two-dimensional code recognizer when recognizing the two-dimensional code, and issue instructions to control the chip sorter to sort the chips, or control the driving device to move the chips;

[0154] (6) A memory for storing the sequence of the polypeptide molecules to be synthesized, the correspondence between the chip and the QR code, the correspondence between the polypeptide molecules to be synthesized and the chip, and the reactions that each solid phase carrier has undergone.

[0155] In addition, a control program is set according to the target polypeptide sequence to be synthesized, and the control program can determine the sorting and movement scheme of the chip according to the predetermined polypeptide sequence.

[0156] 3.5 Peptide synthesis

[0157] First, all chips are mixed and placed in a deprotection tank for deprotection. After the reaction is completed, they are transferred to a washing tank to introduce a cleaning reagent and washed twice. The QR code pre-printed on the chip is recognized by the identifier. The central processor controls the chip sorter to sort the chips according to the recognition signal of the identifier, and moves each chip to the corresponding target reaction tank according to the preset control program for coupling reaction (90s). After the reaction is completed, the coupling reagent is introduced; all chips are transferred to a washing tank, and a cleaning solution is introduced to clean the chip (60s), thereby completing the coupling of one amino acid. According to the predetermined peptide synthesis sequence information, the above steps are repeated cyclically until all peptide molecules are synthesized.

[0158] 3.6 Isolation and purification of peptides

[0159] After the peptide synthesis is completed, all the synthesized chips can be collected together and dissociated to separate and purify all the synthesized peptide molecules. Alternatively, all the chips can be identified by QR codes, and the target chips can be sorted and collected, and then the target chips can be dissociated to separate and purify the target peptide molecules.

[0160] In this example, the AAO biochip was used to synthesize a polypeptide sequence (PGYTVYPGI, SEQ ID NO: 1) with a length of 9. The purity of the synthesized polypeptide crude product was 75.88% ( Figure 5 ). The HPLC high performance liquid chromatography method is used for purification, and the purity after purification can reach 99.15% ( Figure 6 ).

[0161] The results of mass spectrometry show that the porous AAO nanobiochip can be applied in the field of peptide synthesis.

[0162] Example 3 Comparative Example

[0163] A 100nm oxidized silicon wafer (silicon wafer) was selected as the control group. The specific peptide synthesis steps are as follows:

[0164] (1) Using laser printing technology, a 1mm×1mm two-dimensional code is printed in order on the substrate material of the silicon oxide chip; using positioning and cutting technology, the material is processed into a 2mm×2mm×0.45mm microchip with the two-dimensional code point position as the center. After the silicon wafer is coded and cut, the chip is pre-treated with acetone and then plasma cleaned;

[0165] (2) Preparation of biochip:

[0166] The processed and cleaned silicon wafer is subjected to silanization treatment and chemical modification, and the specific operations are the same as those in Example 1.

[0167] (3) Peptide synthesis:

[0168] The silicon wafer chemically modified in step (2) was used to carry out a peptide synthesis experiment. The specific experimental steps and operation methods were the same as those in Example 2.

[0169] After summarizing the experimental results, it was found that the chemically modified porous nano biochip can be used for the synthesis of peptides, and the synthesis quality is better than that of silicon wafers, and the synthetic load is an order of magnitude higher than that of silicon wafers. The above embodiments and experimental results all show that the biochip proposed by the present invention can be used for peptide synthesis, and the synthesis flux is more advantageous than the current commercial mainstream peptide synthesis flux. It further shows that the chemically modified identifiable porous nano biochip proposed by the present invention is highly feasible and has broad application prospects.

[0170] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0171] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A biochip, characterized in that: include: Metal aluminum layer; Anodized aluminum layers, the anodized aluminum layers are disposed on both sides of the metal aluminum layer and are connected to the metal aluminum layer through chemical bonds; A chemical modification layer, the chemical modification layer is located on a surface of the anodized aluminum layer away from the metal aluminum layer, the chemical modification layer comprises a connecting molecule, and the connecting molecule is capable of connecting an amino acid monomer to a terminal of the connecting molecule; The starting end of the linking molecule is a functional group that can react with an amino group, and the terminal end is a functional group that can react with a carboxyl group.

2. The biochip according to claim 1, characterized in that: The anodized aluminum layer is a porous nanostructure, and the inner diameter of the pores in the porous nanostructure is 100-1000 nm.

3. The biochip according to claim 1, characterized in that: The linking molecule comprises at least one selected from Wang resin, 2-Cl(Trt)-Cl resin, and 4-hydroxymethylbenzoic acid.

4. The biochip according to claim 1, characterized in that: The surface of the anodized aluminum layer away from the metal aluminum layer is treated with a silanization agent to introduce amino groups on the surface; Optionally, one end of the silanized molecule in the silanization agent is an alkoxysilyl group, which is used to connect to the anodized aluminum oxide layer; the other end of the silanized molecule is an amino group, which is used to connect to the connecting molecule; Optionally, the silanization molecule in the silanization agent includes at least one selected from APTES, APTS, and MSTFA.

5. The biochip according to claim 1, characterized in that: The biochip carries a code, and the code has a unique corresponding relationship with the biochip; Optionally, the code is disposed on a surface of each anodized aluminum layer away from the metal aluminum layer; Optionally, the code includes at least one of a number, a symbol, a graphic, and an identification code; Optionally, the code is a QR code.

6. The biochip according to claim 1, characterized in that: The chip is in the shape of a sheet, a cuboid or a cylinder; Optionally, the chip is in the shape of a square sheet with a side length of 1-2 mm; Optionally, the thickness of the metal aluminum layer is 0.3-0.5 mm; Optionally, the thickness of the anodized aluminum layer is 5-1000 μm.

7. A method for preparing the biochip according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) performing anodizing treatment on both sides of the metal aluminum layer to obtain an anodized aluminum chip; (2) The anodized aluminum chip is subjected to silanization treatment and chemical modification in sequence to obtain the biochip.

8. The method according to claim 7, characterized in that Step (1) further includes encoding the anodized aluminum chip; Optionally, the coding process includes printing the code on the anodized aluminum chip by laser etching; Optionally, the encoding process further comprises printing a two-dimensional code on the anodized aluminum chip in a dotted two-dimensional code mode; Optionally, the encoding process includes double-sided mirror coding on the anodized aluminum chip.

9. The method according to claim 7, characterized in that: The silanization treatment in step (2) comprises immersing the anodized aluminum chip in an organic solvent containing a silanization agent; Optionally, the silanization molecule in the silanization agent includes at least one selected from APTES, APTS, and MSTFA; Optionally, the organic solvent is acetone, isopropanol or toluene.

10. The method according to claim 7, characterized in that The chemical modification in step (2) includes connecting the linking molecule to the surface of the anodized aluminum chip.

11. Use of the biochip according to any one of claims 1 to 6 or the biochip prepared according to the method according to any one of claims 7 to 10 in polypeptide synthesis.

12. A method for synthesizing a polypeptide, characterized in that: The method comprises the following steps: S1: providing a biochip according to any one of claims 1 to 6 or a biochip prepared according to the method according to any one of claims 7 to 10; S2: providing a reaction pool for adding the required amino acid monomers; S3: determining the target amino acid monomer to be added according to the amino acid sequence of the polypeptide to be synthesized, and sorting the biochip into the reaction pool corresponding to the target amino acid monomer; S4: connecting the target amino acid monomer to the biochip in the reaction pool, and performing a deprotection treatment on the target amino acid monomer; S5: Based on the amino acid sequence, determine the next amino acid monomer connected to the target amino acid monomer, repeat step S4 until the amino acid monomer consistent with the amino acid sequence is connected to the biochip to obtain a polypeptide chain.

13. The method for synthesizing a polypeptide according to claim 12, characterized in that: The biochip carries a code corresponding to the amino acid sequence of the polypeptide to be synthesized; Optionally, step S3 further comprises: identifying the code carried by the biochip, and determining the target amino acid monomer to be added according to the amino acid sequence corresponding to the code, and sorting the biochip into a reaction pool corresponding to the target amino acid monomer.

14. The method for synthesizing a polypeptide according to claim 12, characterized in that: The method further comprises the following steps: S6: cutting the polypeptide chain from the biochip to obtain a target polypeptide; Optionally, step S6 further comprises cleaving the polypeptide chain from the biochip by a cleavage reagent; Optionally, the cleavage reagent comprises at least one selected from hydrofluoric acid, trifluoroacetic acid, and tetrafluoroboric acid; Optionally, step S6 is to cut the polypeptide chain from the biochip at 20-120° C. to obtain the target polypeptide; Optionally, the cutting time in step S6 is 0.5-48h.

15. The method for synthesizing a polypeptide according to claim 14, characterized in that: The method further comprises the following steps: S7: separating and purifying the target polypeptide; Optionally, the separation and purification method comprises any one selected from high performance liquid chromatography, affinity chromatography, and capillary electrophoresis.

Images