Chip surface connector and its preparation method and application

Abstract

The present invention relates to the field of biochips and provides a chip surface connector, its preparation method, and application. The chip surface connector is prepared by reacting aromatic amine bonding molecules with the chip surface in the presence of acid and nitrite, applying a DC voltage, and forming bonding molecular groups attached to the chip surface. The functionalized molecular groups containing hydroxyl and ester groups are then modified with functionalized molecules. The chip surface connector obtained by the present invention can more stably bond to the chip surface, is stable in hot water and alkaline conditions, has good conductivity, electrical stability, and resistance to organic solvents required for nucleic acid synthesis, making it extremely beneficial for subsequent nucleic acid synthesis and other applications.

Description

Technical Field

[0001] The present invention relates to the technical field of biochip preparation, and in particular to a connector for electrically assisted chip synthesis of nucleic acids, and a preparation method and application thereof. Background Art

[0002] Stable linkers on metal or semiconductor chips are very important. On the one hand, they can be used for in situ surface synthesis or pre-preparation of large amounts of oligonucleotide polymers (oligo pools) and DNA probes, ensuring that nucleic acid molecules do not fall off during the synthesis process and ensuring the quality of synthesis. On the other hand, linkers can firmly connect nucleic acids to the chip surface to achieve different applications, such as on-chip in situ hybridization, chip screening, and chip diagnosis. By detecting and analyzing the hybridization signal of the sample, the specific biomarkers in the sample can be qualitatively or quantitatively analyzed, which can play a huge role in disease diagnosis, drug screening, new drug development, and agricultural and environmental research. However, the existing linkers used for chip synthesis of nucleic acids still have some problems, especially in linker stability and preparation cost. These problems are related to the methods used to prepare the linkers. The methods for preparing linkers in the existing technology mainly include small molecule adhesion method [1], metal-thiol reaction method [2], and polymer coating method [3,4]. Among them, the small molecule adhesion method uses the adhesion of high-concentration small molecules to adhere to the chip surface to form a linker. The disadvantage of this method is that the prepared linker has weak adhesion, especially during the nucleic acid synthesis process, it may fall off, resulting in an increase in synthesis errors. In addition, the linker prepared by this method is sensitive to water, especially during subsequent applications, it will fall off, and it is difficult to further meet the application needs of the majority of customers. The metal-thiol reaction method uses thiol groups to react with metals to form covalent bonds as linkers. The problem with this method is that when nucleic acids are synthesized under power, the thiol groups will be reduced and fall off from the metal surface, resulting in failure of nucleic acid synthesis and further difficulty in subsequent applications. The polymer coating method uses polymers or nanomaterials to coat the chip surface and then modify the corresponding molecules to form linkers. However, the stability of the coating material combined with the metal surface is limited, the thickness of each coating is difficult to control, and it is difficult to achieve a highly uniform surface. In addition, the stability under hot water or alkaline conditions is still insufficient and urgently needs to be solved. Summary of the Invention

[0003] In view of this, the present application provides a chip surface connector and its preparation method and application. The chip surface connector provided by the present application has good stability, is stable in hot water and alkaline conditions, has good conductivity, electrical stability and resistance to organic solvents required for nucleic acid synthesis, which is beneficial to applications such as nucleic acid synthesis.

[0004] The present invention provides a chip surface connector, which is prepared by the following steps: Step 1: in the presence of acid and nitrite, by applying a DC voltage, aromatic amine bonding molecules react with the chip surface to form bonding molecular groups connected to the chip surface; Step 2: using functionalized molecules for reaction modification to obtain a connector containing functionalized molecular groups, wherein the functionalized molecular groups include hydroxyl groups and ester groups.

[0005] In some embodiments, the bonding molecule is an aniline substance. In other embodiments, the bonding molecule is selected from p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine, preferably p-aminophenylacetic acid.

[0006] In some embodiments, the DC voltage is a constant DC voltage, and the applied DC voltage is selected from 0.5V to 5.0V, preferably 2.5V to 3.0V, and more preferably 2.5V. Other preferred constant DC voltages are 0.5V, 0.6V, 0.7V, 0.8V, 0.9V, 1.0V, 1.1V, 1.2V, 1.3V, 1.4V, 1.5V, 1.6V, 1.7V, 1.8V, 1.9V, 2.0V, 2.1V, 2.2V, 2.3V, 2.4V, 2.6V, 2.7V, 2.8V, 2.9V, 3.0V, 3.1V, 3.2V, 3.3V, 3.4V, 3.5V, 3.6V, 3.7V, 3.8V, 3.9V, 4.0V, 4.1V, 4.2V, 4.3V, 4.4V, 4.5V, 4.6V, 4.7V, 4.8V, 4.9V or 5.0V.

[0007] In some embodiments, the DC voltage is applied for 10 to 50 minutes, preferably for 10 to 30 minutes, more preferably for 20 minutes. Other preferred DC voltage applications are 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 minutes.

[0008] In some embodiments, the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite, preferably sodium nitrite.

[0009] In some embodiments, the acid is selected from hydrochloric acid, nitric acid, or sulfuric acid, preferably hydrochloric acid.

[0010] In some embodiments, the functionalized molecule is a hydroxyl substance comprising a long carbon chain and containing an ester group. In other embodiments, the functionalized molecule is selected from a base monomer modified by succinic anhydride, hydroxyethyl methacrylate, a base monomer modified by succinic acid or a base monomer modified by oxalic acid, preferably a base monomer modified by succinic anhydride. In some embodiments, the base monomer portion of the functionalized molecule is selected from one or more of adenine, guanine, cytosine, thymine and uracil. In a specific embodiment, the functionalized molecule is adenine, guanine, cytosine, thymine or uracil modified by succinic anhydride.

[0011] In some embodiments, a linker molecule group is further included between the bonding molecule group and the functionalized molecule group. Preferably, the linker molecule group is connected to the bonding molecule group via a linker molecule reaction, and the functionalized molecule group is connected to the linker molecule group via a functionalized molecule reaction.

[0012] In some embodiments, the linker molecule is a diamine or diol. In other embodiments, the linker molecule is selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexamethylenediol, decanediol, or 1,8-octanediamine, preferably 1,8-octanediamine. In some embodiments, the linker molecule is a diamine. In other embodiments, the linker molecule is selected from ethylenediamine, hexamethylenediamine, decanediamine, or 1,8-octanediamine, preferably 1,8-octanediamine.

[0013] In some embodiments, the chip is a metal chip, preferably a gold, platinum or aluminum chip, more preferably a metal platinum chip.

[0014] The present invention also provides a method for preparing a chip surface connector, comprising the following steps:

[0015] Step 1: mixing aromatic amine bonded molecules with acid and nitrite to obtain a mixed solution;

[0016] Step 2: The mixed solution of step 1 is brought into contact with the chip surface, and a DC voltage is applied to react to form a bonding molecular group connected to the chip surface;

[0017] Step 3: Modify the reaction with a functionalized molecule to obtain a linker comprising a functionalized molecule group, wherein the functionalized molecule group comprises a hydroxyl group and an ester group.

[0018] In some embodiments, step 3 includes contacting the chip surface after the reaction in step 2 with a linker molecule to connect the linker molecule group. The chip surface after the reaction is further contacted with the functionalized molecule in step 3 to connect the functionalized molecule group.

[0019] In some embodiments, the bonding molecule is an aniline substance. In other embodiments, the bonding molecule is p-aminophenylacetic acid, p-aminophenylethanol, or p-aminophenylenediamine, preferably p-aminophenylacetic acid.

[0020] In some embodiments, the functionalized molecule is a hydroxyl substance comprising a long carbon chain and an ester group. In other embodiments, the functionalized molecule is selected from a base monomer modified with succinic anhydride, hydroxyethyl methacrylate, a base monomer modified with succinic acid, or a base monomer modified with oxalic acid, more preferably a monomer modified with succinic anhydride. The base monomer portion of the functionalized molecule is selected from one or more of adenine, guanine, cytosine, thymine, and uracil.

[0021] In some embodiments, the DC voltage in step 2 is a constant DC voltage, and the applied DC voltage is selected from 0.5V to 5.0V, preferably 0.5V to 3.0V, and more preferably 2.5V.

[0022] In some embodiments, in step 2, the mixed solution obtained in step 1 is brought into contact with the chip surface and a constant DC voltage is applied for reaction at a temperature of 4°C to 40°C, preferably 20°C to 37°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, preferably room temperature; the reaction time is 10 minutes to 50 minutes, preferably 10 minutes to 30 minutes, for example, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes or 30 minutes, more preferably 20 minutes.

[0023] In some embodiments, the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite, preferably sodium nitrite.

[0024] In some embodiments, the acid is selected from hydrochloric acid, nitric acid, or sulfuric acid, preferably hydrochloric acid.

[0025] In some embodiments, the linker molecule is a diamine or diol substance selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexamethylenediol, decanediol or 1,8-octanediamine, preferably 1,8-octanediamine. In some embodiments, the linker molecule is a diamine substance selected from ethylenediamine, hexamethylenediamine, decanediamine or 1,8-octanediamine, preferably 1,8-octanediamine.

[0026] In some embodiments, the chip is a metal chip selected from gold, platinum and aluminum chips, preferably a metal platinum chip.

[0027] The present invention also provides the use of the chip surface connector as described above in nucleic acid synthesis or chip preparation kits. Detailed Description of the Invention

[0029] The present invention provides a chip surface linker. The linker is formed by applying a constant DC voltage to a chip surface through aromatic amine bonding molecules in the presence of acid and nitrite to form a bonding molecule group connected to the chip surface. Then, a linker molecule reacts with the bonding molecule group to form a linker molecule group. Then, a functionalized molecule reacts with the linker molecule group to obtain a linker containing the functionalized molecule group, wherein the functionalized molecule group includes a hydroxyl group and an ester group.

[0030] The bonding molecules described in the present invention are aromatic amine substances, such as aniline substances, which can be p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine. In some embodiments of the present invention, the bonding molecule is p-aminophenylacetic acid. The bonding molecule group described in the present invention is a group covalently connected to the chip surface after the bonding molecule undergoes an electro-promoted reaction with the chip surface. In some embodiments, the bonding molecule group is a group formed by the bonding molecule of the aromatic amine type reacting with the chip surface under the conditions of acid and nitrite and applying a constant DC voltage. In some embodiments, the bonding molecule can be p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine. The bonding molecule of the aromatic amine type in the present invention is diazotized under the conditions of acid and nitrite, and a constant DC voltage is applied to generate aromatic carbon radicals, which react with the chip-type electrode material to form covalent bonds and bind.

[0031] The linker molecule described in the present invention is a diamine or diol substance, which can be ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexamethylenediol, decanediol or 1,8-octanediol, and in some embodiments of the present invention, it is 1,8-octanediamine. The linker molecule group described in the present invention is a group generated by the reaction of the linker molecule with the bonding molecule group and the functionalized molecule. In some embodiments, the linker molecule group can be selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexamethylenediol, decanediol or 1,8-octanediol. In the present invention, the chip connected to the bonding molecule group is immersed in the linker molecule solution to modify the long-chain molecules, increase the distance between the functional group and the chip, and reduce the steric hindrance in subsequent applications.

[0032] The functionalized molecules in the present invention are hydroxyl substances containing long carbon chains and ester groups, which can be base monomers modified by succinic anhydride, hydroxyethyl methacrylate, base monomers modified by succinic acid or base monomers modified by oxalic acid. In some embodiments of the present invention, a mixed solution of two or more of succinic anhydride-modified adenine, guanine, cytosine or thymine is used. In some embodiments of the present invention, a solution of succinic anhydride-modified adenine, succinic anhydride-modified guanine, succinic anhydride-modified cytosine or succinic anhydride-modified thymine is used alone. In some embodiments, the functionalized molecular group can be selected from succinic anhydride-modified adenine, guanine, cytosine, thymine or uracil, hydroxyethyl methacrylate, succinic acid-modified adenine, guanine, cytosine, thymine or uracil, and a group in which the carboxylic acid in oxalic acid-modified adenine, guanine, cytosine, thymine or uracil reacts with an amino group to remove the hydroxyl group. In the present invention, functional molecules are modified on the connecting arms, and the formed functionalized molecular groups contain hydroxyl groups and ester groups, which facilitate the subsequent electrocatalytic synthesis and cutting of DNA on the chip.

[0033] The chip surface connector of the present invention comprises: (1) a bonding molecular group, wherein the bonding molecular group is covalently connected to the chip surface; (2) a functionalized molecular group, wherein the functionalized molecular group comprises a reactive group of a hydroxyl group and an ester group; and (3) a connecting arm molecular group connecting the bonding molecular group and the functionalized molecular group. Figure 1 a~ Figure 1 d is a schematic diagram of the preparation of the chip connector and the synthesis of DNA in an embodiment of the present invention, wherein: Figure 1 In step d, a chip surface connector is sequentially connected to a bonding molecule, a linker molecule, and a functionalized molecule to form a chip surface connector. The chip surface connector comprises a bonding molecule group, a linker molecule group, and a functionalized molecule group. The chip surface connector described in the present invention sequentially connects a bonding molecule of p-aminophenylacetic acid, a linker molecule of 1,8-octanediamine, and a functionalized molecule of adenine, guanine, cytosine, or thymine modified with succinic anhydride.

[0034] The chip surface connector described in the present invention is stably bonded to the chip surface via covalent bonds and can be used in applications such as electrically assisted chip synthesis of nucleic acids. The metal is generally a sheet-like metal material, i.e., a metal chip. The metal component can be selected from gold, platinum, or aluminum. In some embodiments of the present invention, it is a platinum metal chip. In a specific embodiment of the present invention, the chip is a CustomArray chip. In the embodiment of the present invention, the metal sheet can be rinsed with water and alcohol in sequence, then soaked in an acid solution, heated to a certain temperature, such as 40-70°C, left for 5 to 30 minutes, and finally rinsed with water and dried to obtain a dry and clean metal surface. The water is generally distilled water, the alcohol is ethanol, and the acid solution is mainly a Prinaha solution (H2SO4:H2O2 = 3:1 by volume), which mainly removes pollutants such as other impurities, metal dust, inorganic particles, and small organic molecules.

[0035] The present invention also provides a method for preparing a chip surface connector, comprising the following steps: mixing a bonding molecule with a hydrochloric acid and sodium nitrite solution to obtain a mixed solution; then contacting the chip surface with the mixed solution and applying a constant DC voltage for reaction; sequentially contacting the chip surface after the reaction with a connector arm molecule solution and a functionalized molecule solution for reaction modification to obtain a connector, such as Figure 1 In the embodiment of the present invention, a clean metal chip is first provided, a mixed solution is prepared by mixing bonding molecules with hydrochloric acid and sodium nitrite solution, and finally the metal chip is contacted with the mixed solution for reaction.

[0036] In some embodiments of the present invention, 15mM hydrochloric acid is preferably used as a solvent, and the cold bonding molecule is mixed with the solvent, sodium nitrite is added again, and the mixture is shaken quickly to prepare a mixed solution. The bonding molecule is an aromatic amine substance, preferably an aniline substance, including but not limited to one or more of p-aminophenylacetic acid, p-aminophenylethanol, and p-aminophenylenediamine. In some embodiments of the present invention, it is p-aminophenylacetic acid. In one embodiment of the present invention, 0.10-0.15mM cold p-aminophenylacetic acid is mixed with 15mM hydrochloric acid, 0.07-0.10mM sodium nitrite is added again, and the mixture is shaken quickly to prepare a mixed solution. The cleaned metal chip is quickly immersed in the above mixed solution, that is, the metal surface is in contact with the mixed solution, and a fixed DC voltage is applied and allowed to stand for reaction. In some embodiments of the present invention, the applied fixed DC voltage is 0.5V to 3.0V, preferably 2.5V; the reaction temperature is 20°C to 37°C, preferably room temperature; and the reaction time is 10 minutes to 30 minutes, preferably 20 minutes. In some embodiments of the present invention, the constant DC voltage applied to the chip is 0.5V to 3.0V, and the electrocatalytic reaction is allowed to stand at room temperature for 10 to 30 minutes. In a specific embodiment of the present invention, the constant DC voltage applied to the chip is 2.5V, and the electrocatalytic reaction is allowed to stand at room temperature for 20 minutes. The principle of this step is that aromatic amine molecules are diazotized to generate aromatic carbon radicals after applying a constant DC voltage in the presence of hydrochloric acid and sodium nitrite. The aromatic carbon radicals react with the electrode material to form covalent bonds and combine. The specific reaction is as follows:

[0037]

[0038] After the reaction, the chip is taken out and rinsed with water, alcohol, acid solution, and finally water and blown dry.

[0039] After the reaction, the chip is modified with linker molecules through a cross-linking acylation reaction between carboxyl and amino groups. This increases the distance between the functional groups and the chip, reducing steric hindrance in subsequent applications. In some embodiments of the present invention, the linker molecules are diamines, including but not limited to one or more of ethylenediamine, hexamethylenediamine, decanediamine, and 1,8-octanediaminemethanol. In some embodiments, 1,8-octanediamine is used. The chip is immersed in a solution of 1,8-octanediaminemethanol and allowed to react for 8 to 12 hours, preferably 8 hours. It is then rinsed with water and air-dried.

[0040] The chip after the reaction is then modified with functionalized molecules. The functionalized molecules are hydroxyl substances containing long carbon chains and ester groups, including but not limited to base monomers modified with succinic anhydride, hydroxyethyl methacrylate, base monomers modified with succinic acid, or base monomers modified with oxalic acid. In some embodiments of the present invention, solutions of adenine, guanine, cytosine, thymine, or uracil modified with succinic anhydride are used alone. In other embodiments of the present invention, mixed solutions of two or more of adenine, guanine, cytosine, thymine, and uracil modified with succinic anhydride are used. The functionalized molecules contain ester groups and hydroxyl groups, which can be used for DNA synthesis and cleavage. The ester group is a cleavage site, in order to cut off the DNA synthesized on the chip to form an oligo pool in a free solution state. The hydroxyl groups contained in the functionalized molecules are the starting sites for DNA synthesis. In some embodiments of the present invention, the hydroxyl groups provided by the base monomer portion are used for DNA synthesis.

[0041] In summary, the DC-induced grafting of aniline compounds (e.g., p-aminophenylacetic acid, p-phenylenediamine, etc.) onto the surface of a metal chip (for ease of industrial production) creates a surface-stabilized covalent bond. This is then followed by modification of linkers and functional molecules, thereby forming a stable linker on the chip surface. The formation principle involves the following steps: ① Aromatic amine molecules are diazotized in the presence of hydrochloric acid and sodium nitrite. A fixed DC voltage is applied to generate aromatic carbon radicals, which react with the electrode material to form covalent bonds. ② Utilizing a carboxylamino cross-linking reaction, the chip is immersed in a solution of long-chain linker molecules to modify the long-chain molecules, increasing the distance between the functional groups and the chip and reducing steric hindrance for subsequent applications. ③ The functional molecules are then modified onto the linkers, which contain hydroxyl and ester groups, facilitating subsequent electro-induced DNA synthesis and cleavage on the chip.

[0042] The present invention also provides the use of the aforementioned chip surface connector in DNA synthesis or chip kit preparation. The chip surface connector of the present invention includes the following applications: on-chip DNA synthesis; chip detection of disease biomarkers; development of chip kits for point-of-care testing (POCT); and high-throughput chip screening kits.

[0043] The chip surface connector of the present invention can be used to synthesize DNA after being treated with hot water; the synthesized DNA can also be used to hybridize with oligonucleotides (such as DNA primers) after being hybridized with oligonucleotides (such as DNA primers) and then treated with alkali and hot TE. The DNA synthesized by the chip surface connector of the present invention can still maintain good stability after multiple nucleic acid hybridization, elution, and hybridization cycles. The chip surface connector prepared by the embodiment of the present invention has good conductivity and is stable when electrically applied. It can be used for electrically assisted synthesis of nucleic acid molecules such as DNA. If the connector does not have this property, it will affect DNA synthesis. The chip surface connector of the present invention is resistant to organic solvents required for DNA synthesis, otherwise DNA synthesis cannot be carried out. In the application of chip connectors for chip hybridization detection, the present invention can solve the problems of existing connectors being sensitive to water and unstable to heat, so the chip can be reused.

[0044] The term "chip" refers to a solid support made of an inorganic substance such as a semiconductor or a metal such as gold, silver, or platinum, which has a microarray of specific sites on its surface. These sites are usually arranged in rows and columns, where each site can be used for a certain type of chemical or biochemical analysis, synthesis, or method. These sites on the microarray are usually less than 100 microns. In the present invention, the chip is a metal platinum chip.

[0045] The term "linker" refers to a molecule that is or can be attached to a solid surface (e.g., a metal chip) at one end and has a reactive group at the other end that is or can be attached to a related chemical substance, such as a small molecule, oligomer, or polymer. A linker can be bound to a solid surface and / or its reactive groups have attached to the related chemical substance. The reactive groups of the linker can be attached to protecting groups, wherein the protecting groups can be removed chemically or electrochemically. A linker can contain multiple molecular groups, wherein the molecules are covalently attached to each other.

[0046] The term "bonding molecule group" refers to a chemical molecule located at the end of a linker, one end of the group being covalently attached to a solid surface (e.g., a metal chip) and the other end having a reactive group, wherein the reactive group is or can be attached to a related chemical substance, such as a small molecule, oligomer, or polymer, and in the present invention, can be attached to a linker molecule or a functionalized molecule. The bonding molecule group is already bound to a solid surface and / or its reactive group is already attached to a related chemical substance (e.g., a linker molecule group or a functionalized molecule group). The reactive group of the bonding molecule group may be attached to a protecting group, wherein the protecting group can be removed chemically or electrochemically.

[0047] The term "linker group" refers to a chemical molecule located in the middle portion of the linker, one end of the group being capable of being connected to a bonding group, and the other end having a reactive group, wherein the reactive group is or can be connected to a related chemical substance, such as a small molecule, oligomer, or polymer, and in the present invention, can be connected to a functionalized molecule. The linker group can be combined with the bonding group and / or its reactive group is already connected to a related chemical substance (such as a bonding group or a functionalized group). The reactive group of the linker group can be connected to a protecting group, wherein the protecting group can be removed chemically or electrochemically. The linker group can be formed in situ on the bonding group. The linker group can be formed first and then connected to the bonding group already connected to the solid surface. The linker group can be synthesized externally on the related chemical substance and then connected to the bonding group already connected to the solid surface. The related chemical substance can be connected to the linker group connected to the bonding group, and then the entire structure can be connected to the reaction site on the solid surface. The purpose of the linker group is to extend the distance between the related molecule and the solid surface of the chip to reduce steric hindrance during subsequent applications. In the present invention, the linker molecular group may be an ethylenediamine group, a hexamethylenediamine group, a decanediamine group, a 1,8-octanediamine group, an ethylene glycol group, a hexamethylenediol group, a decanediol group, a 1,8-octanediol group, or the like.

[0048] The term "functionalized molecular group" refers to a chemical molecule located at the terminal portion of a connector, one end of which can be connected to a linker molecular group or a bonding molecular group, and the other end of which has a reactive group, wherein the reactive group is or can be connected to a related chemical substance, such as a small molecule, oligomer or polymer, and in the present invention, can be connected to a deoxyribonucleotide molecule. The functionalized molecular group can be connected to the linker molecular group and / or the reactive group to which the related chemical substance is connected. The reactive group of the functionalized molecular group can be connected to a protecting group, wherein the protecting group can be removed by chemical or electrochemical methods. In the present invention, the functionalized molecule can be adenine, guanine, cytosine, thymine or uracil modified with succinic anhydride, hydroxyethyl methacrylate, adenine, guanine, cytosine, thymine or uracil modified with succinic anhydride, adenine, guanine, cytosine, thymine and uracil modified with oxalic acid, etc.

[0049] The term "base monomer" refers to a molecule that can undergo polymerization to form a unit of a macromolecule, such as an oligomer, co-oligomer, polymer, or copolymer. Examples of monomers include A, C, T, G, adenylic acid, guanylic acid, cytidylic acid, uridylic acid, amino acids, and other compounds.

[0050] The term "aryl" refers to an aromatic carbocyclic group having a monovalent ring and approximately 4-20 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. One or more hydrogen atoms of a substituted aryl group may be replaced with other groups. Although aryl is defined as monovalent, as used herein, aryl includes groups having multiple valencies, thereby meeting the substitution requirement. An aryl group can be part of a fused ring structure, for example, N-hydroxysuccinimide combined with phenyl (benzene) to form N-hydroxyphthalimide.

[0051] The term "aromatic amine" refers to an amine with an aromatic substituent—i.e., -NH2, -NH-, or a nitrogen-containing group—attached to an aromatic ring. Aromatic hydrocarbons typically contain one or more benzene rings. Aniline is the simplest example of this type of compound. In the present invention, the aromatic amine can be p-aminophenylacetic acid, p-aminophenylethanol, or p-aminophenylenediamine.

[0052] The term "oligomer" refers to a molecule of intermediate relative molecular mass whose structure essentially comprises a small number of units derived, in fact or in concept, from molecules of lower relative molecular mass. If the properties of a molecule are indeed significantly different after removing one or more units, the molecule may be considered to have an intermediate relative molecular mass. If part or all of the molecule has an intermediate relative molecular mass and essentially comprises a small number of units derived, in fact or in concept, from molecules of lower relative molecular mass, it may be described as oligomeric, or described using the adjective "oligomer". Oligomers are typically composed of monomers.

[0053] Beneficial effects

[0054] Compared to existing technologies, the present invention utilizes aniline substances under constant DC voltage conditions to effectively bind to the electrode surface via carbon free radicals, thereby forming a metal-carbon covalent bond, stably binding the molecule to the chip surface. The molecule is then modified with linker molecules and functionalized molecules to form a stable connector on the chip surface. The present invention produces a highly adhesive chip surface connector that can stably bind to the chip surface. The chip surface connector of the present invention is stable in hot water and alkaline conditions, exhibits good conductivity, is electrically stable, and is resistant to organic solvents required for nucleic acid synthesis, making it extremely advantageous for subsequent nucleic acid synthesis and other applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the preparation of a chip surface connector and its use for DNA synthesis in Example 1 of the present invention, wherein ① is the metal chip, ② is the linker prepared by this method, and ③ is the DNA synthesized on this linker.

[0056] Figure 2Schematic diagram showing the comparison of the stability of a chip surface interconnect prepared by the method of Example 1 of the present invention and a traditional interconnect in hot water;

[0057] Figure 3 The stability of the chip surface connector prepared by the method of Example 1 of the present invention in nucleic acid synthesis applications;

[0058] Figure 4 Schematic diagram of hybridization experiment of the chip surface connector prepared by the method of Example 1 of the present invention after being used for DNA synthesis;

[0059] Figure 5 Schematic diagram of the cleavability of DNA synthesized by the chip surface linker prepared by the method of Example 1 of the present invention;

[0060] Figure 6 Schematic diagram of DNA synthesized by the chip surface connector prepared by the method of Example 1 of the present invention;

[0061] Figure 7 This is an electrophoresis gel image of DNA synthesized by the chip surface linker prepared by the method of Example 1 of the present invention. The sample is DNA (120 nt) synthesized by the chip surface linker prepared in Example 1. The reference is DNA (120 nt) synthesized by the original linker chip disclosed in Example 1 of US20060105355A1. Ladder represents a standard (after electrophoresis, the nucleic acid positions of 50 nt, 75 nt, 150 nt, 200 nt, and 300 nt can be displayed). DETAILED DESCRIPTION

[0062] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0063] To further understand the present application, the chip surface connector, its preparation method, and application provided herein are described in detail below with reference to the following examples. However, it should be understood that these examples are based on the technical solutions of the present invention and provide detailed implementation methods and specific operating procedures. These examples are intended only to further illustrate the features and advantages of the present invention and are not intended to limit the claims of the present invention. The scope of protection of the present invention is not limited to the following examples.

[0064] Example 1: Chip linker preparation and application in DNA synthesis

[0065] Specific experimental steps:

[0066] 1. Chip cleaning: Metal platinum chips such as Figure 1 As shown in a, the chip was rinsed with distilled water 5 times, ethanol 5 times, and distilled water 3 times, and then immersed in Prinaha solution (H2SO4:H2O2=3:1, volume ratio) and placed in a 65°C oven for 30 minutes. The chip was rinsed with distilled water 5 times and dried with argon gas;

[0067] 2. Chip modification: Mix 18.15 mg of cold p-aminophenylacetic acid with 15 mM hydrochloric acid (1820 μL H2O, 180 μL 0.5 M HCl), then add 6.21 mg of sodium nitrite and shake quickly to mix.

[0068] 3. Quickly add this mixture to the chip surface, apply a 2.5V DC voltage to the chip, and let the electrochemical reaction stand at room temperature for 20 minutes. The chip modification reaction is as follows:

[0069]

[0070] 4. Remove the chip, rinse it with distilled water 5 times, ethanol 5 times, 15mM hydrochloric acid 5 times, distilled water 3 times, and blow dry with argon gas;

[0071] 5. Immerse the chip in 8.7 mg of 1,8-octanediamine in methanol (1 mL of methanol, 1.53 mg of NHS and 7.64 mg of EDC dissolved in 100 μL of water, where NHS is N-hydroxysuccinimide and EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). Allow to react for 8 hours, rinse with distilled water five times, and blow dry with nitrogen.

[0072] 6. Immerse the chip in a mixture of succinic anhydride-modified base monomers (10.8 mg of adenine, thymine, guanine, or cytosine, 1.53 mg of NHS, and 7.64 mg of EDC dissolved in 100 μL of water) and allow to react for 8 hours.

[0073] 7. Rinse the chip with ethanol, acetone, ethanol, and distilled water five times in sequence, and blow dry with nitrogen to obtain a stable linker. Figure 1 As shown in b;

[0074] 8. Place on CustomArray chip synthesizer and synthesize DNA, such as Figure 1 As shown in c.

[0075] The schematic diagram of the linker prepared by the above operation is as follows Figure 1As shown in Figure d, the chip is sequentially connected to the bonding molecule, the linker molecule and the functionalized molecule to obtain a chip connector. The connector includes a bonding molecule group, a linker molecule group and a functionalized molecule group. The obtained connector is then subjected to DNA synthesis (using the CustomArray chip synthesizer). The schematic diagram of the obtained effect is shown in Figure d. Figure 1 As shown in c, perfect DNA can be synthesized.

[0076] Example 2: Stability comparison of new and old linkers in hot water

[0077] After linker modification on the chip, the chip was placed in distilled water at 80°C for 2 days before DNA synthesis.

[0078] The original linker is to deposit a polyhydroxylated small molecule on the chip (see Example 1 in US20060105355A1), rinse with distilled water, dry, and then place in distilled water at 80°C for 2 days. After drying with argon, a 33nt DNA is synthesized on the chip using a CustomArray chip synthesizer. The chip is then scanned on a chip scanner (CustomArray, GenePix4000B). The results are as follows: Figure 2 As shown in a, most areas are unable to synthesize DNA.

[0079] The new linker was prepared according to the method of Example 1 of the present invention. It was placed in distilled water at 80°C for 2 days, dried with argon, and then synthesized on the chip using a CustomArray chip synthesizer. The chip was scanned on a chip scanner (CustomArray, GenePix4000B). The results are as follows: Figure 2 As shown in b, the DNA on the chip surface can still exist stably, indicating that this new linker is sufficiently stable in hot water, which is of great value for subsequent high-temperature hybridization or other detection applications on the chip surface.

[0080] Example 3: Stability of the new linker in nucleic acid synthesis applications

[0081] After being treated with hot water and synthesizing DNA, it is treated with hot TE and alkali solution and then hybridized for application.

[0082] The new linker was made according to the method of Example 1 of the present invention, placed in distilled water at 80°C for 2 days, dried with argon, and then synthesized on the chip using the CustomArray chip synthesizer. The chip was scanned on the chip scanner. The results are as follows. Figure 3 As shown in a and b; two fluorescent DNA primers (100 pM each) were hybridized with the DNA synthesized on the chip (room temperature, hybridization reaction for 2 hours), washed with PBS buffer and scanned on the chip scanner. The results are shown in Figure 3As shown in Figure c, different fluorescent spots are displayed, indicating that the two DNA primers can hybridize with the DNA synthesized on the chip. The chip is then rinsed with 1M NaOH solution to remove the hybridization primers. The chip is then placed in 80°C TE buffer (a mixture of 5mL pH 8.0 1M Tris-HCl Buffer and 1mL pH 8.0 0.5M EDTA) for 2 days. After drying with argon gas, the chip is scanned on a chip scanner. The results are shown in Figure 4. Figure 3 d; then two fluorescent DNA primers (100 pM each) were hybridized with the DNA synthesized on the chip (room temperature, hybridization reaction for 2 hours), washed with PBS buffer and scanned on the chip scanner. The results are shown in Figure 3 As shown in Figure e, different fluorescent spots are displayed, indicating that the two DNA primers can still hybridize with the DNA synthesized on the chip. This shows that this new linker is sufficiently stable in hot water and is resistant to alkali and heat, which is of great value for subsequent high-temperature hybridization or other detection applications on chip surfaces.

[0083] Example 4: Stability of the new linker during multiple hybridization-elution-hybridization applications

[0084] The new linker is made according to the method of embodiment 1 of the present invention, such as Figure 4 As shown, after drying with argon, 33nt DNA was synthesized on the chip using the CustomArray chip synthesizer and scanned on the chip scanner. The results are shown in Figure 4 As shown in b; two fluorescent DNA primers (100 pM each) were hybridized with the DNA synthesized on the chip (room temperature, hybridization reaction for 2 hours), washed with PBS buffer and scanned on the chip scanner. The results are shown in Figure 4 c, showing different fluorescent spots; then rinse the chip with 100mM NaOH solution, remove the hybridization primers, blow dry with argon gas, and scan the chip on a scanner. The results are shown on the right. Figure 4 d; two fluorescent DNA primers (100 pM each) were hybridized with the DNA synthesized on the chip (room temperature, hybridization reaction for 2 hours), washed with PBS buffer and scanned on the chip scanner. The results are shown on the right. Figure 4 As shown in Figure e, after multiple cycles, the linker can still be used for hybridization applications with obvious fluorescence signals, indicating that the linker is stable enough to withstand multiple hybridizations and alkaline elutions.

[0085] Example 5: Cleavage of DNA from Chip After Synthesis

[0086] The linker ( Figure 5 a), and then synthesize 120nt DNA on the chip using CustomArray chip synthesizer (such as Figure 5b) The formed linker contains a cleavage group (ester group), which can be used to cleave the oligo from the chip under alkaline (ammonia) heating conditions (65°C) (16 hours) to form free oligo. After washing the chip, scan it with a chip scanner ( Figure 5 c), indicating that almost all the DNA has been cut off.

[0087] Example 6: DNA quality characterization using the new linker for DNA synthesis

[0088] The linker was prepared by the method of Example 1 of the present invention, and then a 120 nt DNA (such as Figure 6 ), the formed linker contains a cleavage group (ester group), which can cut the oligonucleotide (oligo) from the chip under alkaline (ammonia) heating conditions (65℃) (16h) to form a free oligonucleotide pool (oligo pool), and the oligonucleotide pool was prepared by Thermo Scientific TM NanoDrop TM The concentrations of the two chips measured by One Microvolume UV-Vis Spectrophotometers were 21.4 ng / uL and 19.4 ng / uL, which met the requirements. PCR amplification was then performed and the resulting products were analyzed by electrophoresis gel analysis. Figure 7 As shown, the product is similar in size to the standard product and is located at the same position, indicating that the product synthesized using this linker is correct.

[0089] References:

[0090] 1.Sharma R.Small-molecule surfactant adsorption,polymer surfactant adsorption,and surface solubilization:An overview[M].1995.

[0091] 2.Kokkin DL, Zhang R, Steimle TC, et al. Au–S bonding revealed from the characterization of diatomic gold sulfide, AuS [J]. The Journal of Physical Chemistry A, 2015, 119(48): 11659-11667.

[0092] 3.Toma M,Tawa K.Polydopamine thin films as protein linker layer forsensitive detection ofinterleukin-6by surface plasmon enhanced fluorescencespectroscopy[J].ACS applied materials&interfaces,2016,8(34):22032-22038.

[0093] 4.Saaem I,Ma K S,Marchi A N,et al.In situ synthesis of DNA microarrayon functionalized cyclic olefin copolymer substrate[J].ACS applied materials&interfaces,2010,2(2):491-497

Claims

1. A chip surface connector, characterized in that: The linker is prepared by the following steps: Step 1: In the presence of acid and nitrite, a DC voltage is applied to allow aromatic amine bonding molecules to react with the chip surface to form bonding molecular groups connected to the chip surface; Step 2: Modify with a functionalized molecule to obtain a linker comprising a functionalized molecule group, wherein the functionalized molecule group comprises a hydroxyl group and an ester group, Wherein, the bonding molecule is selected from p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine. 2 . The chip surface connector according to claim 1 , wherein the bonding molecule is p-aminophenylacetic acid. 3 . The chip surface interconnector according to claim 1 , wherein the DC voltage is a constant DC voltage, and the applied DC voltage is 0.5 V to 5.0 V. 4 . The chip surface interconnector according to claim 3 , wherein the applied DC voltage is 0.5 to 3.0 V. The chip surface interconnector according to claim 1 , wherein the DC voltage is applied for 10 to 50 minutes. 6 . The chip surface interconnector according to claim 5 , wherein the DC voltage is applied for 10 to 30 minutes. The chip surface interconnector according to claim 1 , wherein the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite. The chip surface interconnector according to claim 7 , wherein the nitrite is sodium nitrite. 9 . The chip surface interconnector according to claim 1 , wherein the acid is selected from hydrochloric acid, nitric acid or sulfuric acid. 10 . The chip surface interconnector according to claim 9 , wherein the acid is hydrochloric acid. The chip surface connector according to any one of claims 1 to 10, wherein the functionalized molecule is a hydroxyl substance comprising a long carbon chain and an ester group. 12 . The chip surface connector according to claim 11 , wherein the functionalized molecule is selected from a base monomer modified with succinic anhydride, hydroxyethyl methacrylate, a base monomer modified with succinic acid, or a base monomer modified with oxalic acid. The chip surface connector according to claim 12 , wherein the functionalized molecule is a base monomer modified with succinic anhydride. The chip surface connector according to claim 12 , wherein the base monomer portion in the functionalized molecule is selected from one or more of adenine, guanine, cytosine, thymine and uracil. 15 . The chip surface connector according to claim 1 , further comprising a linker molecular group between the bonding molecular group and the functionalized molecular group. 16 . The chip surface connector according to claim 15 , wherein the linker molecule group is connected by reacting the linker molecule with the bonding molecule group, and the functionalized molecule group is connected by reacting the functionalized molecule with the linker molecule group. The chip surface connector according to claim 16 , wherein the linker molecule is a diamine or diol substance. 18 . The chip surface connector according to claim 16 , wherein the linker molecule is selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexamethylenediol, decanediol or 1,8-octanediol. The chip surface connector according to claim 18 , wherein the linker molecule is 1,8-octanediamine. 20 . The chip surface connector according to claim 1 , wherein the chip is a metal chip. The chip surface connector according to claim 20 , wherein the chip is a gold, platinum or aluminum chip. The chip surface interconnector according to claim 20 , wherein the chip is a metal platinum chip.

23. A method for preparing a chip surface connector, characterized in that The following steps are involved: Step 1: mixing aromatic amine bonded molecules with acid and nitrite to obtain a mixed solution; Step 2: The mixed solution of step 1 is brought into contact with the chip surface, and a DC voltage is applied to react to form a bonding molecular group connected to the chip surface; Step 3: reacting the chip surface after the reaction with functionalized molecules to obtain a linker containing functionalized molecular groups, wherein the functionalized molecular groups include hydroxyl groups and ester groups. Wherein, the bonding molecule is selected from p-aminophenylacetic acid, p-aminophenylethanol or p-aminophenylenediamine.

24. The preparation method according to claim 23, wherein before step 3, the chip surface after the reaction in step 2 is contacted with the linker molecule to react and connect the linker molecule group. The preparation method according to claim 23 , wherein the bonding molecule is p-aminophenylacetic acid. The preparation method according to claim 23 , wherein the DC voltage is a constant DC voltage, and the applied DC voltage is 0.5 V to 5.0 V. The preparation method according to claim 26 , wherein the applied DC voltage is 0.5 V to 3.0 V. The preparation method according to claim 23 , wherein the DC voltage is applied for 10 to 50 minutes. The preparation method according to claim 28 , wherein the DC voltage is applied for 10 to 30 minutes.

30. The preparation method according to any one of claims 23-29, wherein the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite. The preparation method according to claim 30 , wherein the nitrite is sodium nitrite.

32. The preparation method according to any one of claims 23 to 29, wherein the acid is selected from hydrochloric acid, nitric acid or sulfuric acid. The preparation method according to claim 32 , wherein the acid is hydrochloric acid.

34. The preparation method according to any one of claims 23 to 29, wherein the functionalized molecule is selected from a base monomer modified with succinic anhydride, hydroxyethyl methacrylate, a base monomer modified with succinic acid, or a base monomer modified with oxalic acid. The preparation method according to claim 34 , wherein the functionalized molecule is a base monomer modified with succinic anhydride.

36. The preparation method according to claim 34, wherein the base monomer portion of the functionalized molecule is selected from one or more of adenine, guanine, cytosine, thymine and uracil.

37. The preparation method according to any one of claims 23 to 29, wherein the linker molecule is a diamine or diol substance.

38. The preparation method according to claim 37, wherein the linker molecule is selected from ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexamethylenediol, decanediol or 1,8-octanediol. The preparation method according to claim 38 , wherein the linker molecule is 1,8-octanediamine.

40. The preparation method according to any one of claims 23 to 29, wherein the temperature during the step 2 of applying a DC voltage for reaction is 4°C to 40°C, and the reaction time is 10 minutes to 50 minutes. The preparation method according to claim 40, wherein the temperature during the DC voltage application in step 2 is 20°C to 37°C, and the reaction time is 10 minutes to 30 minutes.

42. The preparation method according to any one of claims 23-29, wherein the chip is a metal chip.

43. The preparation method according to claim 42, wherein the chip is a gold, platinum or aluminum chip.

44. Use of the chip surface connector according to any one of claims 1 to 22 in a nucleic acid synthesis or chip preparation kit.

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