Microporous polypeptide chip, preparation method and application thereof, and label-free affinity screening method of targeted polypeptide

Abstract

The invention belongs to the technical field of medical analysis and detection articles, and particularly relates to a microporous polypeptide chip, a preparation method and application thereof and a label-free affinity screening method of targeted polypeptide. According to the microporous carrier chip provided by the invention, the same or different polypeptides can be subjected to a bonding reaction with chemical groups on the inner surfaces of the micropores in the hollow micropores, and the obtained microporous polypeptide chip is a trace, efficient and controllable polypeptide chip; the polypeptide of the microporous polypeptide chip provided by the invention does not need to be subjected to signal labeling by using a fluorophore or other labeling technologies, and when the microporous polypeptide chip provided by the invention is used for carrying out a targeted affinity screening test, protein labeled by fluorescence or other labels is not needed, so that a polypeptide compound labeling or protein labeling process is omitted, and the cost is reduced. Meanwhile, the dosage of the target protein is small, the screening operation process is greatly simplified, the screening efficiency is improved, the screening cost is saved, and negative effects generated by signal labeling groups such as fluorescence are eliminated.

Description

Technical Field

[0001] The invention belongs to the technical field of medical analysis and detection products, and specifically relates to a microporous polypeptide chip and a preparation method and application thereof, as well as a label-free affinity screening method for targeted polypeptides. Background Art

[0002] As a high-throughput research tool, peptide array is a new type of biochip that uses automated instruments to place hundreds or even thousands of specially designed peptides at extremely high density on a specially treated carrier matrix (such as glass or filter paper, etc.) through in situ synthesis technology. Peptide arrays are widely used in drug screening, target confirmation, epitope localization, vaccine development, and structural function research. At the same time, due to the increasing interest in peptide drugs, this technology can also meet the growing demand for peptide drug research and development of a large number of pharmaceutical companies.

[0003] However, the current peptide chips are planar chips. When conducting targeted affinity screening tests, the amount of target protein solution used is very large, resulting in a huge waste of solution and high screening test costs. Summary of the invention

[0004] The purpose of the present invention is to provide a microporous polypeptide chip and a preparation method and application thereof, as well as a label-free affinity screening method for targeted polypeptides. The microporous polypeptide chip provided by the present invention is used for polypeptide affinity screening, and a small amount of target protein solution is used, and the protein or polypeptide can be used for polypeptide affinity screening without labeling. It has the characteristics of simple operation, high screening efficiency, and low screening cost. At the same time, the negative impact of signal labeling groups such as fluorescence is eliminated, and the detection sensitivity is high and the accuracy is good.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a microporous carrier chip, comprising a carrier substrate and a microporous array arranged on the carrier substrate, wherein the microporous array is formed by arranging a hollow microporous array; the hollow micropores are through holes, and the inner surfaces of the hollow micropores have chemical modification groups, and the chemical modification groups include at least one of an aldehyde group, an amino group, an epoxy group, a PEG carboxylic acid and a PEG amino group.

[0006] Preferably, the pore diameter of the hollow micropores is 200-500 μm, and the pore depth is 1-1.5 mm.

[0007] Preferably, the column spacing of the hollow micropores is 400-600 μm, and the row spacing of the hollow micropores is 400-600 μm.

[0008] Preferably, the carrier substrate comprises a glass substrate, a silicon substrate or a ceramic substrate.

[0009] The present invention provides a method for preparing the microporous carrier chip described in the above technical solution, comprising the following steps: Performing micro-hole drilling on the carrier substrate to obtain a semi-finished product; The semi-finished product is coated or immersed in a chemical modification group reaction solution to perform chemical group modification to obtain the microporous carrier chip.

[0010] Preferably, the micro-holes are opened by laser processing, and the conditions of the laser processing include: laser wavelength of 1065nm, frequency of 100-200kHz, positioning accuracy of ±20μm, repeatability of ±5μm, and hole processing accuracy of ±5μm.

[0011] The present invention provides a microporous polypeptide chip, comprising the microporous carrier chip described in the above technical solution or the microporous carrier chip prepared by the preparation method described in the above technical solution; and a polypeptide bonded to a chemical modification group on the inner surface of the hollow micropore.

[0012] The present invention provides a method for preparing the microporous polypeptide chip described in the above technical solution, comprising the steps of performing quality control detection on the microporous polypeptide chip; The step of performing quality control detection on the microporous polypeptide chip comprises: adding water into the hollow micropores of the microporous polypeptide chip; then irradiating the microporous polypeptide chip with ultraviolet light, and qualitatively and / or quantitatively detecting the bonding amount of the polypeptide in the microporous polypeptide chip by using the ultraviolet light absorption signal of the hollow micropores.

[0013] The present invention provides the use of the microporous polypeptide chip described in the above technical solution in label-free affinity detection and / or screening of polypeptide compounds.

[0014] The present invention provides a label-free affinity screening method for a targeting polypeptide, comprising the following steps: Adding unlabeled target protein into the hollow micropores of the microporous polypeptide chip described in the above technical solution and incubating the protein to obtain a protein-incubated chip; The chip after the protein incubation is irradiated with ultraviolet light, and the ultraviolet light absorption signal of the hollow micropores is used to qualitatively and / or quantitatively screen to obtain a polypeptide compound having the targeting affinity of the target protein.

[0015] Preferably, the wavelength of the ultraviolet light is 280 nm, the photomultiplier factor is 300-650, and the resolution is 10±5 nm.

[0016] The present invention provides a microporous carrier chip, comprising a carrier substrate and a microporous array arranged on the carrier substrate, wherein the microporous array is formed by arranging a hollow microporous array; the hollow micropores are through holes, and the inner surfaces of the hollow micropores have chemical modification groups, and the chemical modification groups include at least one of an aldehyde group, an amino group, an epoxy group, a PEG carboxylic acid, and a PEG amino group. First, compared with ordinary planar chips, the present invention constructs hollow micropores on the carrier substrate, and constructs a three-dimensional solid binding surface in the hollow micropores. Compared with a single point of an ordinary planar chip and a single hole of the hollow micropore of the present invention, the surface area of ​​a single hole in the microporous carrier chip provided by the present invention is larger. Compared with an ordinary planar chip, the area of ​​a point and a micropore of the same size differs by several times (S point = πr², S micropore = 2πrh, h≥r). Second, the chemical modification groups of ordinary planar chips such as Figure 1 As shown, the immobilization method of the polypeptide compound is bonding on a two-dimensional plane. When immobilized, the polypeptide is in a solution state. Since the solid carrier is extremely small (nL or pL level), the polypeptide compound has not yet fully bonded with the carrier surface, and the solution evaporates to interrupt the liquid phase bonding reaction. In comparison, the chemical modification groups on the inner surface of the micropores in the microporous carrier chip provided by the present invention are as follows Figure 2 As shown, the present invention constructs a three-dimensional immobilization binding surface in the micropores. After the polypeptide solution is added, the surface of the carrier is sealed with a film to avoid the volatilization of the polypeptide solution, greatly prolonging the bonding reaction time, thereby ensuring that the polypeptide compound is fully bonded to the chemical modification group in the hollow micropores. Third, the immobilization method of the ordinary planar chip is a two-dimensional planar immobilization. Since the polypeptide is in a solution state during immobilization, the size and position of each immobilization point will inevitably change and shift with the difference in the properties of the polypeptide solution, the change in mechanical precision, and the change in ambient temperature and humidity. The present invention uses hollow micropores of the same size, and the polypeptide is immobilized in the hollow micropores. When the polypeptide solution is loaded, the change in the properties of the solution and the change in temperature and humidity have no effect on the size and position of the hollow micropores (the size and position deviation of each immobilized micropore is only affected by the mechanical precision when the pre-carrier micropore is formed), and the signal point size and position of the carrier chip can be determined to be consistent. Thus, the microporous carrier chip provided by the present invention can obtain a microporous polypeptide chip with high sensitivity, strong signal, low background interference, uniform chip signal, and high polypeptide bonding efficiency.

[0017] The present invention provides a microporous polypeptide chip, comprising the microporous carrier chip described in the above technical solution or the microporous carrier chip prepared by the preparation method described in the above technical solution; and a polypeptide bonded to the chemical modification group on the inner surface of the hollow micropore. The microporous polypeptide chip provided by the present invention has the following beneficial effects: The present invention provides a microporous polypeptide chip whose microporous polypeptide bonding rate far exceeds that of an ordinary planar chip. The present invention uses hollow micropores to immobilize polypeptide compounds. The polypeptide immobilization efficiency is due to the three-dimensionality of the hollow micropores, the surface area is larger, the chemical groups on the inner surface of the hollow micropores are more, and the reaction time after the hollow micropores are coated is not affected by the volatilization of the polypeptide solution, the liquid phase bonding reaction time is extended, and the polypeptide bonding rate far exceeds that of an ordinary planar chip.

[0018] At present, peptide chips all fix compounds on the surface of carriers. When such planar peptide chips are used for various screening tests, the strength of the binding signal is affected by many factors, including the shape, size, and peptide bonding efficiency of the immobilization point. The present invention uses micropores to immobilize peptide compounds. Under the premise of uniform micropores, the shape and size of the immobilized micropores are not affected by the peptide solution. The peptide immobilization efficiency is due to the three-dimensionality of the micropores, the surface area is larger, and there are more chemical groups on the inner surface of the micropores, so the bonding possibility is much greater than that of ordinary planar chips.

[0019] The microporous polypeptide chip provided by the present invention is simple and fast to read. Since the polypeptide is in solution state when immobilized on the ordinary planar chip, the size and position of each immobilization point will inevitably change and deviate with the difference in the properties of the polypeptide solution, the change in mechanical precision, and the change in ambient temperature and humidity (such as Figure 4 As shown in the figure, it is inconvenient to determine the position of the signal point and the value of the signal size later. However, the present invention uses micropores of uniform size, and the position and size are uniform and consistent, which is convenient for rapid positioning and value acquisition (as shown in the figure). Figure 5 as shown).

[0020] The background interference of the microporous polypeptide chip provided by the present invention can be completely eliminated. When incubating the target protein, an ordinary planar chip may form non-specific binding in all areas of the carrier surface, forming a relatively strong background signal, and this background cannot be directly deducted, which greatly affects the interpretation of the signal value of the binding between the polypeptide and the ligand protein at the fixed point position. However, in the microporous polypeptide chip of the present invention, the non-microporous area will not bind to the target protein, and all non-specific binding can be directly ignored. The signal value in the micropore can be directly read or the signal value strength of the binding between the polypeptide and the target protein in each micropore can be compared.

[0021] The microporous polypeptide chip provided by the present invention greatly saves protein costs. When incubating target proteins, ordinary planar chips need to be completely immersed in protein solutions. However, the microporous polypeptide chip of the present invention directly points the target proteins into the micropores, and the quantitative determination is accurate. There is no need to incubate proteins in non-micropore areas, and the protein concentration and volume usage can be accurately calculated, which greatly saves the use of various types of expensive proteins and greatly saves screening costs.

[0022] The present invention provides a method for label-free affinity screening of targeted polypeptides, comprising the following steps: adding a label-free target protein into the hollow micropores of the microporous polypeptide chip described in the above technical solution and incubating the mixture to obtain a protein-incubated chip; irradiating the protein-incubated chip with ultraviolet light, and using ultraviolet light to qualitatively and / or quantitatively screen the ultraviolet light absorption signals of the hollow micropores to obtain polypeptide compounds with the target protein targeting affinity. The principle of the label-free affinity screening method for targeted polypeptides provided by the present invention is: a large number of different polypeptides are orderly fixed on the inner surface of the hollow micropores of the carrier microporous chip (i.e., the microporous polypeptide chip), and a specific ligand target protein may have a specific affinity with the polypeptide fixed in the hollow micropores, and the results of the possible specific reactions are identified and read. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of chemical functional group modification of ordinary planar chip; Figure 2 A schematic diagram of chemical functional group modification of a microporous carrier chip provided by the present invention; Figure 3 A schematic diagram of the structure of a microporous carrier chip provided by the present invention; Figure 4 This is the imaging picture of a common planar chip; Figure 5 An imaging diagram of the microporous carrier chip provided by the present invention; Figure 6 This is the quality inspection and partial magnification of the aldehyde-modified peptide chip prepared in Example 1; Figure 7 This is a diagram of the biochip screening of aldehyde-modified polypeptides prepared in Example 1 (partial magnification). DETAILED DESCRIPTION

[0024] The present invention provides a microporous carrier chip, comprising a carrier substrate and a microporous array arranged on the carrier substrate, wherein the microporous array is formed by arranging a hollow microporous array; the hollow micropores are through holes, and the inner surfaces of the hollow micropores have chemical modification groups, and the chemical modification groups include at least one of an aldehyde group, an amino group, an epoxy group, a PEG carboxylic acid and a PEG amino group.

[0025] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0026] Figure 3 The schematic diagram of the structure of the microporous carrier chip provided by the present invention is shown below. Figure 3 The microporous carrier chip provided by the present invention is described in detail.

[0027] The microporous carrier chip provided by the present invention comprises a carrier substrate. In the present invention, the carrier substrate preferably comprises a glass sheet, a silicon substrate or a ceramic sheet.

[0028] The microporous carrier chip provided by the present invention comprises a microporous array arranged on the carrier substrate, and the microporous array is formed by arranging a hollow microporous array; the hollow micropore is a through hole, and the inner surface of the hollow micropore has a chemical modification group, and the chemical modification group includes at least one of an aldehyde group, an amino group, an epoxy group, a PEG carboxylic acid and a PEG amino group. In the present invention, the pore size of the hollow micropore is preferably 200-500 μm, and in the embodiment, it can be 300 μm; the pore depth of the mesoporous micropore is preferably 1-1.5 mm, and in the embodiment, it can be 1 mm. The column spacing of the hollow micropores is preferably 400-600 μm, and in the embodiment, it can be 500 μm. The row spacing of the hollow micropores is preferably 400-600 μm, and in the embodiment, it can be 500 μm. In the present invention, the column spacing and the row spacing are both the spacing between the center points of the adjacent hollow micropores in the same column or row. In the present invention, the chemical modification group includes an aldehyde group, an amino group, an epoxy group, a PEG carboxylic acid or a PEG amino group. The chemical modification groups form a chemical group active modification layer on the inner surface of the hollow micropores. The chemical group active modification layer is used for bonding reaction with the polypeptide compound.

[0029] The present invention provides a method for preparing the microporous carrier chip described in the above technical solution, comprising the following steps: Performing micro-hole drilling on the carrier substrate to obtain a semi-finished product; The semi-finished product is coated or immersed in a chemical modification group reaction solution to perform chemical group modification to obtain the microporous carrier chip.

[0030] The present invention performs micro-hole drilling on a carrier substrate to obtain a semi-finished product. In the present invention, the micro-hole drilling is preferably performed by a laser processing method, and the conditions of the laser processing preferably include: the laser wavelength is preferably 1065nm, the frequency is preferably 100-200kHz, and in the embodiment it can be 100kHz, the positioning accuracy is preferably ±20μm, the repeatability is preferably ±5μm, and the hole processing accuracy is preferably ±5μm. During the laser processing, the present invention preferably sets an optical camera CCD to monitor the positioning.

[0031] After obtaining the semi-finished product, the present invention coats or impregnates the semi-finished product with a chemical modification group reaction solution to perform chemical group modification to obtain the microporous carrier chip. In the present invention, the chemical group modification includes at least one of aldehyde modification, amino modification, epoxy modification, PEG carboxylic acid modification and PEG amino modification. The present invention preferably pre-treats the semi-finished product, and the pre-treatment preferably includes: placing the semi-finished product in a pre-treatment solution for micro-boiling treatment, and then washing with anhydrous ethanol. The pre-treatment solution preferably includes concentrated sulfuric acid and hydrogen peroxide. The mass content of the concentrated sulfuric acid is preferably 95-98%. The mass content of the hydrogen peroxide is preferably 20-23%. The volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is preferably 4-5:1. The time for the micro-boiling treatment is preferably 60min±5min. The present invention preferably evaporates the semi-finished product after micro-boiling treatment and returns it to room temperature before washing with anhydrous ethanol. The anhydrous ethanol washing preferably includes soaking and ultrasonic washing in sequence. The soaking time is preferably 60min±5min. The ultrasonic washing time is preferably 30min. After the anhydrous ethanol washing, the present invention preferably evaporates the washed semi-finished product and then dries it. The drying temperature is preferably 45° C. and the drying time is preferably 20 min±5 min.

[0032] In the present invention, the amino modification preferably includes the following steps: coating or impregnating the semi-finished product with an amino reaction solution, performing amino modification, and obtaining the microporous carrier chip. The amino reaction solution is preferably an anhydrous ethanol solution of aminosilane. The CAS number of the aminosilane is 919-30-2. The volume of aminosilane and anhydrous ethanol in the anhydrous ethanol solution of aminosilane is preferably 1:49. The pH value of the amino reaction solution is preferably 3 to 4.5. The present invention preferably uses acetic acid to adjust the pH value of the amino reaction solution. The impregnation preferably includes ultrasonic impregnation and static impregnation in sequence, and the time of the ultrasonic impregnation is preferably 20min±5min. The time of the static impregnation is preferably 30min±5min. After the amino modification is completed, the present invention preferably washes and dries the amino-modified semi-finished product in sequence to obtain the microporous carrier chip. The water used for the water washing is preferably distilled water. The water washing is preferably ultrasonic water washing, and the number of ultrasonic water washings is preferably 2 times, and the time of each ultrasonic water washing is preferably 10min±2min. The drying is preferably vacuum drying, the temperature of the vacuum drying is preferably 50±5° C., the time is preferably 60 min±5 min, and the obtained microporous carrier chip is an amino-modified microporous carrier chip.

[0033] In the present invention, the aldehyde modification preferably comprises the following steps: Obtaining an amino-modified microporous carrier chip according to the amino-modification method described above; The amino-modified microporous carrier chip is coated or impregnated with an aldehyde reaction solution to perform aldehyde modification to obtain the microporous carrier chip. In the present invention, the aldehyde reaction solution is preferably an aqueous solution of glutaraldehyde. The volume ratio of glutaraldehyde to water in the aqueous solution of glutaraldehyde is preferably 1:4. The water is preferably distilled water. The impregnation preferably includes ultrasonic impregnation and static impregnation in sequence, and the time of the ultrasonic impregnation is preferably 20min±5min. The time of the static impregnation is preferably 40min±5min. After the aldehyde modification is completed, the present invention preferably washes and dries the semi-finished product after the aldehyde modification in sequence to obtain the microporous carrier chip. The water used for the washing is preferably distilled water. The washing is preferably ultrasonic washing, and the number of ultrasonic washings is preferably 2 times, and the time of each ultrasonic washing is preferably 10±2min. The drying is preferably vacuum drying, and the temperature of the vacuum drying is preferably 50°C, and the time is preferably 60min±5min. The obtained microporous carrier chip is an aldehyde-modified microporous carrier chip.

[0034] In the present invention, the epoxy modification preferably includes the following steps: coating or impregnating the semi-finished product with an epoxy reaction solution, performing epoxy modification, and obtaining the microporous carrier chip. The epoxy reaction solution is preferably an anhydrous ethanol solution of epoxyalkyltrimethoxysilane. The CAS number of the epoxyalkyltrimethoxysilane is 2530-83-8. The volume of epoxyalkyltrimethoxysilane and anhydrous ethanol in the anhydrous ethanol solution of epoxyalkyltrimethoxysilane is preferably 2:49. The impregnation preferably includes ultrasonic impregnation and static impregnation, the time of the ultrasonic impregnation is preferably 20min±5min, the temperature of the static impregnation is preferably 80±5℃, and the time of the static impregnation is preferably 8~16h. After the epoxy modification is completed, the present invention preferably washes and dries the semi-finished product after epoxy modification in turn to obtain the microporous carrier chip. The ethanol washing is preferably ultrasonic washing, the number of ethanol washings is preferably 2 times, and the time of each ethanol washing is preferably 10min±2min. The drying is preferably vacuum drying, the temperature of the vacuum drying is preferably 50° C., the time is preferably 60 min±5 min, and the obtained microporous carrier chip is an epoxy-modified microporous carrier chip.

[0035] In the present invention, the PEG carboxylic acid modification preferably comprises the following steps: Obtaining an amino-modified microporous carrier chip according to the amino-modification method described above; The amino-modified microporous carrier chip is coated or impregnated with a PEG carboxylic acid reaction solution to perform PEG carboxylic acid modification to obtain the microporous carrier chip. In the present invention, the PEG carboxylic acid reaction solution is preferably a PEG dicarboxylic acid mixed solution, and the PEG dicarboxylic acid mixed solution preferably includes PEG dicarboxylic acid, a condensing agent and an organic solvent. The PEG dicarboxylic acid is preferably PEG （2-5） Dicarboxylic acid (COOH-PEG (2-5) -COOH), the molar concentration of the PEG dicarboxylic acid is preferably 40 μmol / L. The condensing agent preferably includes one or more of TBTU, HATU and HBTU, and the molar concentration of the condensing agent is preferably 40 μmol / L. The organic solvent is preferably N,N-dimethylformamide (DMF). The impregnation preferably includes a first ultrasonic impregnation and a second ultrasonic impregnation in sequence, and the time of the first ultrasonic impregnation is preferably 20min±5min. During the second ultrasonic impregnation, the present invention preferably also includes adding diisopropylethylamine (DIEA) to the PEG carboxylic acid reaction solution, and the molar concentration of diisopropylethylamine in the solution formed after adding the diisopropylethylamine is preferably 80 μmol / L. The time of the second ultrasonic impregnation is preferably 20min±5min. After the PEG carboxylic acid modification is completed, the present invention preferably washes and dries the semi-finished product modified with the PEG carboxylic acid in sequence to obtain the microporous carrier chip. The reagents used for the washing are preferably DMF and water, and the water is preferably distilled water. The washing is preferably performed by alternating washing with DMF and water. Repeat the washing twice with each washing reagent. The drying is preferably vacuum drying, the temperature of the vacuum drying is preferably 50°C, the time is preferably 60min±5min, and the obtained microporous carrier chip is a PEG carboxylic acid modified microporous carrier chip. In the present invention, during the PEG carboxylic acid modification process, the PEG carboxylation reagent (i.e., PEG dicarboxylic acid) in the PEG carboxylic acid reaction solution dehydrates and condenses with the amino groups on the surface of the amino-modified microporous carrier chip to form a tight C=N bond.

[0036] In the present invention, the PEG amino modification preferably comprises the following steps: Obtaining an amino-modified microporous carrier chip according to the amino-modification method described above; The amino-modified microporous carrier chip is coated or impregnated with a PEG amino reaction solution to perform PEG amino modification to obtain the microporous carrier chip. In the present invention, the PEG amino reaction solution is preferably a PEG amination reagent mixed solution, and the PEG amination reagent mixed solution preferably includes a PEG amination reagent, a condensing agent and an organic solvent. The PEG amination reagent is preferably amino-polyethylene glycol (2-5)-propionic acid (Amino-PEG (2-5) -Propionic Acid, NH2-PEG(2-5) -COOH), the molar concentration of the PEG amination reagent is preferably 20 μmol / L. The condensing agent preferably includes one or more of TBTU, HATU and HBTU, and the molar concentration of the condensing agent is preferably 40 μmol / L. The organic solvent is preferably N,N-dimethylformamide (DMF). The impregnation preferably includes a first ultrasonic impregnation and a second ultrasonic impregnation in sequence, and the time of the first ultrasonic impregnation is preferably 20 min ± 5 min. During the second ultrasonic impregnation, the present invention preferably also includes adding diisopropylethylamine (DIEA) to the PEG amino reaction solution, and the molar concentration of diisopropylethylamine in the solution formed after adding the diisopropylethylamine is preferably 80 μmol / L. The time of the second ultrasonic impregnation is preferably 20 min ± 5 min. After the PEG amino modification is completed, the present invention preferably washes and dries the semi-finished product after the PEG amino modification in sequence to obtain the microporous carrier chip. The reagents used for the washing are preferably DMF and water, and the water is preferably distilled water. The washing is preferably washed alternately with DMF and water. Repeat the washing twice with each washing reagent. The drying is preferably vacuum drying, the temperature of the vacuum drying is preferably 50°C, the time is preferably 60min±5min, and the obtained microporous carrier chip is a PEG amino-modified microporous carrier chip. In the present invention, during the PEG amino modification process, the carboxylic acid end of the PEG linker of the PEG amination reagent in the PEG amino reaction solution and the amino group on the surface of the amino-modified microporous carrier chip condense to form a stable amide bond.

[0037] The present invention provides a microporous polypeptide chip, comprising the microporous carrier chip described in the above technical solution or the microporous carrier chip prepared by the preparation method described in the above technical solution; and a polypeptide bonded to the chemical modification group on the inner surface of the hollow micropore. In the present invention, the polypeptide on the inner surface of the hollow micropore is a polypeptide of the same or different sequence, and the polypeptide and the chemical modification group in the hollow micropore form a bond between the microporous carrier chip and the polypeptide through a chemical reaction, thereby fixing the polypeptide on the inner surface of the hollow micropore of the microporous carrier chip.

[0038] In the present invention, the microporous polypeptide chip preferably has at least 4 groups of 5 hollow micropores each without polypeptide bonded, of which 1 group of 5 is blank hollow micropores, and the other 3 groups of 15 hollow micropores are respectively bonded with polypeptide standards, and the polypeptide standard is preferably nonapeptide-1, the CAS number of nonapeptide-1 is 158563-45-2, and the purity of nonapeptide-1 is 98.2%. The amount of polypeptide standards bonded in every 3 hollow micropores in the 15 hollow micropores is the same, and the amount of polypeptide standards bonded in the 15 hollow micropores is 5 levels from small to large.

[0039] The present invention provides a method for preparing the microporous polypeptide chip described in the above technical solution, preferably comprising the following steps: A microarray spotting device is used to spot the polypeptide solution into the hollow micropores of the microporous carrier chip, and the microporous polypeptide chip is obtained by bonding the polypeptide compounds in the polypeptide solution with the chemical modification groups.

[0040] In the present invention, the microarray spotting device is preferably a microarray chip spotter. The polypeptide solution is a polypeptide solution with a large amount of different sequence structure information. In the present invention, the microporous carrier chip preferably has at least 4 groups of 5 micropores each without the use of a microarrayer to spot polypeptides, of which 1 group of 5 is a blank well, and the other 3 groups of 15 micropores are respectively spotted with solutions of polypeptide standards, and the polypeptide standard is preferably nonapeptide-1, the CAS number of nonapeptide-1 is 158563-45-2, and the purity of nonapeptide-1 is 98.2%. The solvent in the solution of the polypeptide standard is preferably distilled water. The molar concentration of the polypeptide standard in the solution of the polypeptide standard is preferably: 0.02μmol / mL, 0.1μmol / mL, 0.2μmol / mL, 0.5μmol / mL and 1.0μmol / mL. The solution of each molar concentration of the polypeptide standard is spotted into 3 hollow micropores.

[0041] In the present invention, before the polypeptide solution is dripped into the microporous carrier chip, the present invention preferably coats the bottom surface of the microporous carrier chip with a film, and after dripping, the present invention preferably coats the top surface of the microporous carrier chip with a film before bonding. The bonding is performed in a constant temperature oscillator, the bonding temperature is preferably 25±2°C, and the bonding time is preferably 8-12h.

[0042] After the bonding reaction is completed, the present invention preferably removes the coating on the bottom and top surfaces of the bonded microporous carrier chip, and then washes and dries. The cleaning is ultrasonically cleaned with distilled water, and the cleaning is to remove the unbonded excess polypeptides; the drying is preferably vacuum drying, and the temperature of the vacuum drying is preferably 30±5°C, and the time is preferably 2~4h; after drying, the bottom surface of the cleaned bonded chip is coated again, and after the bottom surface is coated again, the unreacted chemical modification groups in the hollow micropores of the cleaned bonded chip are passivated using a blocking reagent. The blocking reagent is preferably an aqueous solution of sodium borohydride. The mass concentration of the aqueous solution of sodium borohydride is 0.1g / mL. The passivation treatment preferably includes: placing the cleaned bonded chip in a blocking reagent, removing it after ultrasonic treatment, and immediately coating the top surface after taking it out and performing a passivation reaction. The passivation reaction is carried out in a constant temperature oscillator, and the temperature of the passivation reaction is preferably 30±5°C, and the time is preferably 1~2h. After the passivation treatment, it also includes cleaning and drying the passivated chip. The cleaning and drying methods are the same as those after bonding.

[0043] The present invention provides a method for preparing the microporous polypeptide chip described in the above technical solution, further comprising a step of performing quality control detection on the microporous polypeptide chip; The step of performing quality control detection on the microporous polypeptide chip comprises: adding water into the hollow micropores of the microporous polypeptide chip; then irradiating the microporous polypeptide chip with ultraviolet light, and qualitatively and / or quantitatively detecting the bonding amount of the polypeptide in the microporous polypeptide chip by using the ultraviolet light absorption signal of the hollow micropores.

[0044] In the present invention, the binding concentration of the polypeptide in each micropore of the microporous polypeptide chip is preferably 0.013 μmol / mL to 0.019 μmol / mL.

[0045] In the present invention, the water is preferably distilled water. The wavelength of ultraviolet light used in the quality control detection method of the microporous polypeptide chip is preferably 220nm, the photomultiplier coefficient is preferably 400-650, and the resolution is preferably 10nm. In the present invention, the ultraviolet light is emitted from one end of the hollow micropore of the microporous polypeptide chip, and is received at the other end of the microporous polypeptide chip after passing through the hollow micropore. The ultraviolet absorption values ​​of the blank micropores and the known polypeptide standard concentration gradient micropores of the microporous polypeptide chip are recorded and a standard curve is drawn. The bonded polypeptides in the hollow micropores form a difference in ultraviolet light absorption according to the amount of bonding. Since the pore size of the hollow micropores is uniform, the volume of the solution in the hollow micropores is basically the same (the light range is consistent), and the difference in ultraviolet light absorption is only related to the amount of bonded polypeptides in the hole. At the same time, according to the ultraviolet absorption of the blank micropores and the ultraviolet absorption standard curve of the polypeptide standard, the qualitative and quantitative detection of the bonding amount of the polypeptide in the micropores can be achieved.

[0046] In the method for preparing the microporous polypeptide chip described in the above technical solution provided by the present invention, the step of performing quality control detection on the microporous polypeptide chip is a necessary step for the normal use of the microporous polypeptide chip.

[0047] The present invention provides the use of the microporous polypeptide chip described in the above technical solution in label-free affinity detection and / or screening of polypeptide compounds.

[0048] The present invention provides a label-free affinity screening method for a targeting polypeptide, comprising the following steps: Adding unlabeled target protein into the hollow micropores of the microporous polypeptide chip described in the above technical solution and incubating the protein to obtain a protein-incubated chip; The chip after the protein incubation is irradiated with ultraviolet light, and the ultraviolet light absorption signal of the hollow micropores is used to qualitatively and / or quantitatively screen to obtain a polypeptide compound having the targeting affinity of the target protein.

[0049] The present invention adds unlabeled target protein into the hollow micropores of the microporous polypeptide chip described in the above technical solution and incubates the micropores to obtain a chip after incubating the protein. In the present invention, the target protein is preferably spotted into the hollow micropores by a microarray spotting device in the form of a target protein diluent. The mass concentration of the target protein in the target protein diluent is preferably 1±0.2μg / mL. The spotting volume of the target protein diluent is preferably 5~10nL. The temperature of the target protein diluent spotting is preferably 25~30℃, and the relative humidity is 40~50%. After the addition is completed, the present invention preferably incubates the microporous polypeptide chip after coating. The coating can prevent the target protein diluent from volatilizing. The incubation is preferably first oscillated on an oscillator, and then a constant temperature reaction is performed. The oscillation time during the incubation is preferably 30±5min, the constant temperature reaction is performed in a constant temperature box, the temperature of the constant temperature reaction is preferably 30±5℃, and the time is preferably 1~2h.

[0050] After the incubation is completed, the present invention preferably removes the chip coating, and then washes, dries, and pre-scans the chip after incubation of the protein in sequence. The washing is preferably carried out in a bath. The washing is preferably performed in a PBS (pH 6.5) buffer solution. The pre-scanning treatment preferably includes: coating the bottom surface of the washed incubated protein chip and placing it in a microarray spotter, and spotting the PBS (pH 6.5) buffer solution into the micropores of the microporous polypeptide chip, with 3 to 5 nL being spotted into each well, wherein a group of 5 blank wells are spotted, and 5 target protein solutions with a concentration gradient are spotted into the control wells, and the mass concentration of the target protein solution is preferably 0.01 μg / mL, 0.05 μg / mL, 0.12 μg / mL, 0.2 μg / mL, and 0.3 μg / mL, and 3 micropores are spotted for each concentration, and 3 to 5 nL are spotted into each well. After the spotting is completed, the front of the chip is immediately coated to prevent the solution in the micropores of the chip from volatilizing, and at the same time, be careful to handle it gently to prevent the liquid in the micropores from seeping out.

[0051] After obtaining the chip after incubation of protein, the present invention uses ultraviolet light to irradiate the chip after incubation of protein, and uses ultraviolet light to qualitatively and / or quantitatively screen the ultraviolet light absorption signal of the hollow micropores to obtain a polypeptide compound with the target protein targeting affinity. In the present invention, the wavelength of the ultraviolet light is 280nm, the photomultiplier coefficient is 300-650, the energy is 50-100, and the resolution is 10nm. In the present invention, after the protein is incubated and washed on the microporous polypeptide chip, the ultraviolet light is emitted from one end of the hollow micropore by using the ultraviolet light absorption principle, and is received at the other end of the chip after passing through the micropores. The polypeptide chip washed after incubation of the protein forms a light absorption difference based on whether the protein can bind to the bonded polypeptide in each micropore and the amount of binding. The ultraviolet absorption values ​​of the blank micropores and the micropores with known protein standard concentration gradient are recorded and a standard curve is drawn. Since the pore size is uniform and the volume of the solution in the micropores is basically the same (the light range is consistent), the difference in ultraviolet light absorption is only related to the amount of protein in the pore. At the same time, based on the ultraviolet absorption of the blank micropores and the ultraviolet absorption standard curve of the protein standard solution, the qualitative and quantitative detection of the binding of the protein and the polypeptide can be achieved. The absorption value determines whether the polypeptide in the micropore has affinity with the protein and the affinity, thereby qualitatively and quantitatively screening to obtain polypeptide compounds with the protein targeting affinity.

[0052] In the present invention, the nouns are annotated as follows: DIEA: N,N-diisopropylethylamine (basic catalyst, CAS No.: 7087-68-5); TBTU: O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (condensation agent, CAS No.: 125700-67-6); HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (condensation agent, CAS: 148893-10-1); HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (condensation agent, CAS No.: 94790-37-1); DMF: N,N-dimethylformamide (solvent, CAS number: 68-12-2); PEG: polyethylene glycol; DMSO: dimethyl sulfoxide (solvent, CAS number: 67-68-5); Chip material: refers to ceramic, silicon-based, glass, plastic or similar products; Microporous substrate: refers to the use of laser burning or etching to produce hollow micropores according to parameters of chip materials of ceramic, silicon, glass, plastic or similar products; (Aldehyde) microporous substrate: refers to a microporous substrate modified with aldehyde chemical groups; (Epoxy) microporous substrate: refers to a microporous substrate modified with epoxy chemical groups; (Amino) microporous substrate: refers to a microporous substrate modified with amino chemical groups; (PEG carboxylic acid) microporous substrate: refers to a microporous substrate modified with PEG carboxylic acid chemical groups; (PEG amino) microporous substrate: refers to a microporous substrate modified with PEG amino chemical groups; Microporous peptide chip: refers to a microporous substrate with chemical group modifications (including aldehyde, amino, epoxy, and PEGylation) bonded to the peptide.

[0053] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] The peptides used in the following examples are derived from the sub-peptide library in the Novotide® peptide entity library independently constructed by Hangzhou Hetai Jianyu Biotechnology Co., Ltd., and 3840 continuous peptides are selected. The characteristics of the peptide library used in the following examples are shown in Table 1.

[0055] Table 1 Characteristics of peptide libraries used in the examples

[0056] Example 1 This embodiment provides a microporous carrier chip, including a carrier substrate and a microporous array disposed on the carrier substrate, wherein the microporous array is formed by arranging a hollow microporous array; the hollow microporous is a through hole, and the inner surface of the hollow microporous has a chemical modification group, and the chemical modification group is an aldehyde group. The carrier substrate is a ceramic sheet.

[0057] The method for preparing the microporous carrier chip provided in this embodiment includes: A laser processing system is used to make micro-holes on a ceramic sheet. The laser wavelength is set to 1065nm, the frequency is 100kHz, the positioning accuracy is ±20μm, the repeatability is ±5μm, the hole processing accuracy is ±5μm, the hole diameter is 300μm, the hole depth is 1mm (through hole), the row and column spacing (center point spacing) between holes is 500μm, and an optical camera CCD is set to monitor positioning to obtain a semi-finished product; The semi-finished product is immersed in an aldehyde reaction solution for aldehyde modification to obtain a microporous carrier chip; the specific method of aldehyde modification includes: 1) Use 95wt% concentrated sulfuric acid and 20wt% hydrogen peroxide (volume ratio 4:1) to slightly boil the semi-finished product for 60min±5min; 2) After taking it out, evaporate it and return it to room temperature, then soak it in anhydrous ethanol for 60min±5min, ultrasonic wash it for 30min, take it out and evaporate it, and put it in a 45℃ oven to dry for 20min±5min; 3) Prepare aminosilane (CAS: 919-30-2) solution (10 mL aminosilane and 490 mL anhydrous ethanol), adjust the pH value to between 3.0 and 4.5 with acetic acid, place the semi-finished product obtained in step 2) in the aminosilane solution, soak in ultrasound for 20 min ± 5 min, let stand for 30 min ± 5 min, replace with distilled water and ultrasonically wash for 10 min ± 2 min (repeat 2 times), and dry in a vacuum oven at 50 ° C for 60 min ± 5 min. 4) Place the amino chip obtained in step 3) into the aldehyde reaction solution (20% glutaraldehyde 100mL + distilled water 400mL) and ultrasonicate for 20min±5min, let it stand for 40 minutes, replace the distilled water and ultrasonicate for 10min (repeat 2 times), and dry in a vacuum oven at 50℃ for 60min±5min to obtain a microporous carrier chip.

[0058] Three ceramic microporous carrier chips were prepared in this example.

[0059] This embodiment provides a microporous polypeptide chip, and the preparation method thereof comprises the following steps: 1) Take 3 prepared ceramic microporous carrier chips, numbered HT-C-Am-24030041, HT-C-Am-24030042, and HT-C-Am-24030043, cover the bottom surface of the ceramic microporous carrier chip with a film (to prevent liquid from seeping out. Although in actual experiments, due to the small micropores and factors such as liquid surface tension, almost no liquid seepage was observed, but in order to ensure the consistency of reaction volume and conditions, this embodiment performs the operation of covering the bottom surface before spotting and covering the top surface after spotting), place them on a microarray chip spotter, and load the peptide solution ( 10 384-peptide well plates (derived from the sub-peptide library in the Novotide® peptide entity library independently constructed by Hangzhou Hetai Jianyu Biotechnology Co., Ltd.), with a total of 3840 different peptides, were used. A microarray spotter (SmartArrayer™136) was used to spot different peptides into the micropores of the aldehyde-modified ceramic microporous carrier chip. The spotting needle had a caliber of 75μm, the spotting volume per well was 3~5nL, and the multiple spotting was 2 (i.e., every 2 wells were the same peptide). Three microporous peptide chips were spotted at the same time. After each chip was spotted, the top surface was immediately covered with a film (to prevent liquid evaporation) and placed in a constant temperature oscillator at 25℃±2℃ for 8h (overnight) for reaction.

[0060] 2) Take out the microporous polypeptide chip that has been fully reacted in step 1), remove the coating on the top and bottom surfaces, place the entire microporous polypeptide chip in a distilled water bath, and ultrasonicate for 10 minutes to wash away the unbonded free polypeptides, replace the distilled water, and repeat the washing for 3 times, evaporate the water and place it in a 30°C vacuum drying oven for 4 hours, and then coat the bottom surface for use (the purpose of the bottom coating here is: after washing away the unbonded polypeptides, the bottom surface needs to be coated again to prepare for the blocking of the unbound groups in the micropores, to prevent the subsequent non-specific binding of proteins to the substrate surface groups, and to ensure that the subsequent target protein can only bind to the bonded polypeptide or not).

[0061] 3) Place the microporous polypeptide chip after cleaning in step 2) in a bath, add a blocking reagent sodium borohydride aqueous solution (2 g of sodium borohydride dissolved in 20 mL of water), immerse the chip, and ultrasonicate for 10 minutes. After taking it out, immediately cover it with a film and place it in a constant temperature oscillator at 30°C for 2 hours (the unstable C=N double bond formed by the aldehyde group on the substrate and the N-terminal amino group on the polypeptide will be reduced to a stable CN bond, forming a more stable polypeptide-chip bond).

[0062] 4) Remove the film from the polypeptide chip obtained in step 3) above, place the entire microporous polypeptide chip in a distilled water bath, and ultrasonicate for 10 minutes to wash away excess sodium borohydride, replace the distilled water, and repeat the washing 3 times. After evaporating the water, place it in a vacuum drying oven at 30°C for 4 hours, and then number and coat it for use.

[0063] Example 2 This embodiment provides a microporous carrier chip, including a carrier substrate and a microporous array disposed on the carrier substrate, wherein the microporous array is formed by arranging a hollow microporous array; the hollow microporous is a through hole, and the inner surface of the hollow microporous has a chemical modification group, and the chemical modification group is an aldehyde group. The carrier substrate is a glass sheet.

[0064] The method for preparing the microporous carrier chip provided in this embodiment includes: A laser processing system is used to make micro-holes on a glass sheet. The laser wavelength is set to 1065nm, the frequency is 100kHz, the positioning accuracy is ±20μm, the repeatability is ±5μm, the hole processing accuracy is ±5μm, the hole diameter is 300μm, the hole depth is 1mm (through hole), the row and column spacing (center point spacing) between holes is 500μm, and an optical camera CCD is set to monitor positioning to obtain a semi-finished product; The semi-finished product is immersed in an amino reaction solution for amino modification to obtain a microporous carrier chip; the specific method of amino modification includes: 1) Use 95% concentrated sulfuric acid and 20% hydrogen peroxide (volume ratio 4:1) to slightly boil the semi-finished product for 60min±5min; 2) After taking it out, evaporate it and return it to room temperature, then soak it in anhydrous ethanol for 60min±5min, ultrasonic wash it for 30min, take it out and evaporate it, and put it in a 45℃ oven to dry for 20min±5min; 3) Prepare aminosilane (CAS: 919-30-2) solution (10 mL aminosilane and 490 mL anhydrous ethanol), adjust the pH value to between 3.0 and 4.5 with acetic acid, place the semi-finished product in the above step 2) in the aminosilane solution, soak in ultrasound for 20 min ± 5 min, let stand for 30 min ± 5 min, replace with distilled water and ultrasonically wash for 10 min ± 2 min (repeat 2 times), and dry in a vacuum oven at 50°C for 60 min ± 5 min to obtain a microporous carrier chip.

[0065] Three glass microporous carrier chips were prepared in this example.

[0066] This embodiment provides a microporous polypeptide chip, and the preparation method thereof comprises the following steps: 1) Take 3 prepared glass (Amino) microporous carrier chips, numbered HT-G-Am-24030011, HT-G-Am-24030012, HT-G-Am-24030013, cover the bottom of the glass microporous carrier chip with a film, place it on the microarray chip spotter, and load the configured peptide solution (derived from the Novotide® peptide entity independently constructed by Hangzhou Hetai Jianyu Biotechnology Co., Ltd.) dissolved and diluted to a concentration of 2 μmol / mL using dimethyl sulfoxide (DMSO) The sub-peptide library in the library) was used to prepare 10 384-peptide well plates, with a total of 3840 different peptides. The different peptides were spotted into the micropores of the amino-modified microporous carrier chip using a microarray spotter (SmartArrayer™136). The spotting needle had a caliber of 75μm, the spotting volume per well was 3~5nL, and the multiple spotting was 2 (i.e., the same peptide was spotted in every 2 wells). During the spotting, 3 chips (same type of peptide, same arrangement, etc.) were processed simultaneously. After the spotting of each chip was completed, the top surface was immediately coated and left to react in a constant temperature box at 25℃±2℃ for no less than 8h (overnight). During the reaction, the peptide solution fully reacted with the amino groups on the substrate, so that the amino groups in the micropores condensed with the carboxylic acids on the peptides to form stable amide bonds, thereby fixing the peptides on the inner surface of the micropores of the chip.

[0067] 2) Take out the fully reacted peptide chip, remove the coating on the top and bottom surfaces, place the entire microporous peptide chip in a distilled water bath, and ultrasonicate for 10 minutes to wash away the unbonded free peptides. Replace the distilled water and repeat the washing 3 times. After evaporating the water, place it in a 30℃ vacuum drying oven and dry it for 4 hours. Then cover the bottom surface with a film for later use.

[0068] 3) Place the microporous polypeptide chip after cleaning in step 2) in a bath, add a blocking reagent sodium borohydride aqueous solution (2 g of sodium borohydride dissolved in 20 mL of water), immerse the chip, and ultrasonicate for 10 minutes. After taking it out, immediately cover it with a film and place it in a constant temperature oscillator at 30°C for 2 hours (the unstable C=N double bond formed by the aldehyde group on the substrate and the N-terminal amino group on the polypeptide will be reduced to a stable CN bond, forming a more stable polypeptide-chip bond).

[0069] 4) Remove the film from the polypeptide chip obtained in step 3) above, place the entire microporous polypeptide chip in a distilled water bath, and ultrasonicate for 10 minutes to wash away excess sodium borohydride, replace the distilled water, and repeat the washing 3 times. After evaporating the water, place it in a vacuum drying oven at 30°C for 4 hours, and then number and coat it for use.

[0070] Example 3 Characteristic Quality Inspection of Microporous Peptide Chip In this example, a ceramic (aldehyde-based Aldehyde) microporous polypeptide chip (the aldehyde-modified ceramic microporous polypeptide chip prepared in Example 1) is used as the object to be tested. The following steps are also applicable to the quality control of other amino-, epoxy-, PEG-carboxylic acid-, and PEG-amino-modified polypeptide chips made of glass or silicon in the present invention.

[0071] The characteristic quality inspection method of the microporous polypeptide chip provided in this embodiment specifically includes: Remove the film from any side of the microporous polypeptide chip and place it in a microarray spotter. Spot distilled water into each micropore of the microporous polypeptide chip (including blank wells). 3~5nL should be spotted into each well. After spotting, cover the chip immediately to avoid evaporation of the solution in the micropores of the chip. At the same time, be careful to handle it with care to avoid leakage of the liquid in the micropores. Place it at 25℃±2℃ and let it stand for 20min±5min.

[0072] Note 1: The microporous polypeptide chip provided by the present invention has at least 4 groups of 5 micropores each modified with the same surface chemical group, but no polypeptide is spotted using a microarrayer, of which 1 group of 5 are blank wells, and the other 3 groups of 15 micropores are spotted with 3-5 nL of polypeptide standard (nonapeptide-1, CAS: 158563-45-2, purity 98.2%) solution (dissolved in distilled water), with concentration gradients of 0.02 μmol / mL, 0.1 μmol / mL, 0.2 μmol / mL, 0.5 μmol / mL, and 1.0 μmol / mL as standard control wells, and 3 micropores are spotted at each concentration.

[0073] Note 2: For non-sampling microporous peptide chips, it is not necessary to spot the standard control wells and they can be left empty.

[0074] Scanning: Place the above-coated polypeptide microporous carrier chip into a preheated chip scanner for scanning (photomultiplier factor 500, wavelength 220nm, resolution 10nm). Ultraviolet light is emitted from one end through the coated micropores into the signal receiving end. There is only distilled water in the blank well, and there is no signal absorption at 220nm. The standard (polypeptide standards have more unsaturated bonds and usually have better ultraviolet absorption at 220nm) control wells obtain different ultraviolet absorption values ​​according to the concentration gradient difference. Since the light intensity and light range (solution volume) are consistent, the light absorption difference is only related to the amount of polypeptide in the micropore. A standard curve of the relationship between the amount of polypeptide and the absorption value is drawn (the average of the absorption values ​​of 3 micropores at each standard concentration). The polypeptides bonded to the inner surface chemical modification groups in other micropores show light absorption differences at 220nm according to the amount of bonding. The bonding effect and bonding amount are determined by comparing with the blank and standard wells.

[0075] The peptide standard control data in this example are shown in Table 2.

[0076] Table 2 Peptide standard control data obtained in Example 3

[0077] Figure 6 1 is a quality inspection and local magnification diagram of the microporous polypeptide chip prepared in Example 1. Table 3 is the light absorption reading of the local magnified area of ​​the microporous polypeptide chip prepared in Example 1.

[0078] Table 3 shows the local data of microporous polypeptide chip (HT-C-Al-RI-24030003)

[0079] Note: In the coordinates in Table 3, the X-axis is the horizontal direction and the Y-axis is the vertical direction. The positioning principle is to start from the first hole in the upper left corner of the microporous polypeptide chip, and count from left to right on the X-axis and from top to bottom on the Y-axis.

[0080] Through the actual test of this example, the linear correlation coefficient of the peptide standard reference curve is R 2 =0.994, the standard reference curve has good linear correlation, and the linear equation is y=5.3326x-0.0189. Taking the local amplified area as an example, the bonded concentration of the peptide in this area is converted to be between 0.013μmol / mL and 0.019μmol / mL. The bonding efficiency is in line with expectations, and the bonding uniformity is good. This batch of peptide chips can be used for subsequent affinity screening experiments with target proteins.

[0081] Example 4 Incubation and data processing of microporous polypeptide chip 1. Protein dilution: Dilute W1 protein (recombinant human prostate-specific membrane antigen (Recombinant Human PSMA) / Folate hydrolase 1 (FOLH1) protein, extracellular domain (ECD), histidine tag (His Tag, manufacturer: Sino Biological, catalog number: 15877-H07H) to 1 μg / mL with 1× PBS (pH 6.5) buffer solution.

[0082] 2. Incubation of microporous polypeptide chip: Take out one (aldehyde) microporous polypeptide chip prepared in Example 1 (with 3860 micropores (5 blank + 15 control + 3840 sample wells), containing 1920 different polypeptides, of which 20 micropores are blank micropores, divided into 4 groups, 1 group of 5 continuous micropores, located at the left and right edges of the micropore array, when the polypeptide is dotted for bonding, these blank micropores are not dotted with polypeptides, and serve as blank and control wells) and remove the single-sided film. The above-mentioned protein dilution solution is dotted into the micropores of the polypeptide chip using a microarray spotter (SmartArrayer™ 136), 5-10 nL is dotted into each well, and the temperature is controlled at 25°C-30°C and the relative humidity is 40%-50% during the spotting. After the protein spotting is completed, the chip surface is immediately covered with a film to prevent the solution from volatilizing, and then placed on an oscillator, gently shaken for 30 minutes, and then placed in a constant temperature box at 30°C±5°C for reaction for 1 hour.

[0083] 3. Cleaning: After incubation, remove the chip film, place the chip in a bath, add PBS (pH 6.5) buffer solution to the bath, soak the chip, ultrasonically clean it for 5 minutes, replace the PBS (pH 6.5) buffer solution, repeat the cleaning twice, and blow dry the solution for later use.

[0084] 4. Pre-scanning treatment: After coating the bottom surface of the above-mentioned polypeptide microporous carrier chip, place it in a microarray spotter, and spot PBS (pH 6.5) buffer solution into the micropores of the microporous polypeptide chip, with 3~5nL spotted into each well. Among them, 5 blank wells are spotted into 1 group, and 5 control wells are spotted into 5 concentration gradients of W1 protein (recombinant human prostate-specific membrane antigen (Recombinant HumanPSMA) / folate hydrolase 1 (FOLH1) protein), extracellular domain (ECD), histidine tag (His Tag). 3 micropores are spotted for each concentration, and 3~5nL is spotted into each well. After the spotting is completed, the front side of the chip is immediately coated to prevent the solution in the micropores of the chip from evaporating. At the same time, be careful to handle it with care to prevent the liquid in the micropores from seeping out.

[0085] 5. Scanning: Place the above chip in a preheated chip scanner for scanning (recommended parameters: Power = 10, photomultiplier factor 400, resolution 5nm, wavelength 280nm). The more the ligand target protein binds to the bonded polypeptide in the microwell, the stronger the absorption of the microwell. Since the light range (volume of microwell solution) is consistent, the final absorbance intensity is only related to the concentration of W1 protein in each microwell, which means that it is related to the amount of W1 protein in each microwell that binds to the bonded polypeptide in the microwell.

[0086] 6. Data reading: Import the data reading template file into the scanner, make each locking circle fit with the microwell array in the chip, click to obtain the absorption signal data, that is, obtain the affinity binding data of the peptide chip and the W1 protein. At the same time, combine each peptide with 2 duplicate wells to examine the uniformity of the absorption signal. The blank well only has PBS solution without protein, and the absorption at 280nm is used as the solution blank. The control wells obtain different ultraviolet absorption values ​​according to the concentration gradient difference (take the average of the absorption values ​​of 3 microwells for each standard concentration). Due to the consistency of light intensity and light range (solution volume), the difference in absorbance value is only related to the amount of W1 protein in the microwell. Draw a standard curve of the relationship between the amount of protein and the absorption value. The proteins bound to the peptide in other microwells show different absorption values ​​at 280nm according to the amount of binding. The protein concentration of each well is determined by comparing with the blank and standard curves.

[0087] The priority of affinity with W1 protein is screened out according to the absorption signal value, and the peptide compounds with specific affinity targeting W1 protein are efficiently obtained qualitatively and quantitatively, which are used for the next step of screening of potential candidate drug lead compounds or screening research of other targeted functional active peptide molecules.

[0088] The W1 protein standard control curve data in this example are shown in Table 4.

[0089] Table 4 W1 protein standard control curve data

[0090] Figure 7 This is a screening image of the aldehyde polypeptide biochip prepared in Example 1 (partial enlargement).

[0091] The screening experiment results of this embodiment show that the affinity screening experiment results of 3840 different polypeptides and W1 protein using the microporous polypeptide chip and the proposed test method provided by the present invention show that only 6 groups of different polypeptide compounds are lit up, which are located at the coordinate positions of the chip (X3Y34, X4Y34), (X7Y31, X7Y31), (X7Y35, X8Y35), (X15Y35, X16Y35), (X17Y31, X18Y31), (X17Y35, X18Y35), where the X axis in the coordinate position of the chip is the horizontal direction and the Y axis is the vertical direction. It indicates that these 6 polypeptides may have targeting affinity with W1 protein. Except for 3 groups of 15 W1 standard concentration microwells and these 6 groups of binding signals, no binding signals were detected in the remaining microwells with bonded polypeptides, indicating that the bonded polypeptides at the remaining microwell positions failed to bind to the W1 target protein, the incubated W1 target protein was eluted, and no absorption signals of the bound protein were detected at the remaining microwell positions.

[0092] The screening data of the aldehyde polypeptide biochip prepared in Example 1 is shown in Table 5.

[0093] Table 5 Screening data of aldehyde peptide biochip prepared in Example 1

[0094] Note: In the coordinates in Table 5, the X-axis is the horizontal direction and the Y-axis is the vertical direction. The positioning principle is to start from the first hole in the upper left corner of the microporous polypeptide chip, and count from left to right on the X-axis and from top to bottom on the Y-axis.

[0095] The correlation coefficient of the protein standard reference product is R 2 =0.9977, the standard control curve has a good linear correlation, and the linear equation is y=11.457x-0.1335. The test binding data show that the protein concentration in the binding area is approximately between 0.017μg / mL and 0.049μg / mL, which is between the linear concentrations of the protein standard concentration curve, indicating that the W1 target protein has good specific binding ability with the six groups of different polypeptides, and this specific binding strength is positively correlated with the polypeptide content. Therefore, the polypeptide compounds obtained by screening these six groups of data can be used for further in vitro affinity screening studies. Furthermore, other traditional methods such as ELISA and SPR in vitro efficacy tests can be used to confirm whether these six groups of polypeptide compounds have excellent W1 protein targeting binding properties and whether they can be used for further development of related pharmaceutical purposes.

[0096] From the above embodiments, it can be seen that the present invention provides a microporous carrier chip, so that the same or different polypeptides can bond and react with the chemical groups on the inner surface of the micropores in the hollow micropores, and the obtained microporous polypeptide chip is a trace, efficient and controllable polypeptide chip. The polypeptides of the microporous polypeptide chip provided by the present invention do not need to be signal-labeled using fluorescent groups or other labeling technologies. When the microporous polypeptide chip provided by the present invention is used for targeted affinity screening tests, no fluorescent labeling or other labeled proteins are required, which eliminates the polypeptide compound labeling or protein labeling process, greatly simplifies the screening operation process, improves screening efficiency, saves screening costs, and eliminates the negative effects of signal labeling groups such as fluorescence.

[0097] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A microporous carrier chip, characterized in that: It includes a carrier substrate and a micropore array arranged on the carrier substrate, wherein the micropore array is formed by arranging a hollow micropore array; the hollow micropores are through holes, and the inner surfaces of the hollow micropores have chemical modification groups, and the chemical modification groups include at least one of an aldehyde group, an amino group, an epoxy group, a PEG carboxylic acid and a PEG amino group.

2. The microporous carrier chip according to claim 1, characterized in that: The pore diameter of the hollow micropores is 200-500 μm, and the pore depth is 1-1.5 mm.

3. The microporous carrier chip according to claim 1 or 2, characterized in that: The column spacing of the hollow micropores is 400-600 μm, and the row spacing of the hollow micropores is 400-600 μm.

4. The microporous carrier chip according to claim 1, characterized in that: The carrier substrate includes a glass substrate, a silicon substrate or a ceramic substrate.

5. The method for preparing a microporous carrier chip according to any one of claims 1 to 4, characterized in that: The following steps are involved: Performing micro-hole drilling on the carrier substrate to obtain a semi-finished product; The semi-finished product is coated with or immersed in a chemical modification group reaction solution to perform chemical group modification to obtain the microporous carrier chip.

6. The preparation method according to claim 5, characterized in that: The micro-holes are opened by laser processing, and the conditions of the laser processing include: laser wavelength of 1065nm, frequency of 100-200kHz, positioning accuracy of ±20μm, repeatability of ±5μm, and hole processing accuracy of ±5μm.

7. A microporous polypeptide chip, characterized in that: The invention comprises the microporous carrier chip according to any one of claims 1 to 4 or the microporous carrier chip prepared by the preparation method according to claim 5 or 6; and a polypeptide bonded to the chemical modification group on the inner surface of the hollow micropore.

8. The method for preparing the microporous polypeptide chip according to claim 7, characterized in that: The method comprises the steps of performing quality control detection on the microporous polypeptide chip; The step of performing quality control detection on the microporous polypeptide chip comprises: adding water into the hollow micropores of the microporous polypeptide chip; then irradiating the microporous polypeptide chip with ultraviolet light, and qualitatively and / or quantitatively detecting the bonding amount of the polypeptide in the microporous polypeptide chip by using the ultraviolet light absorption signal of the hollow micropores.

9. Use of the microporous polypeptide chip according to claim 7 in label-free affinity detection and / or screening of polypeptide compounds.

10. A label-free affinity screening method for a targeting polypeptide, characterized in that: The following steps are involved: Adding unlabeled target protein into the hollow micropores of the microporous polypeptide chip according to claim 7 and incubating the micropores to obtain a protein-incubated chip; The chip after the protein incubation is irradiated with ultraviolet light, and the ultraviolet light absorption signal of the hollow micropores is used to qualitatively and / or quantitatively screen to obtain a polypeptide compound having the targeting affinity of the target protein.

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