A kind of mercaptonicotinic acid and gold nanoparticles modified graphene oxide material and its preparation method and application

Abstract

The present invention belongs to the technical field of analytical chemistry, and specifically relates to a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material and its preparation method and application. The present invention obtains a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material with higher selectivity for glycoprotein enrichment by improving the hydrophilicity, ionic interaction force and recognition ability of peptides of the material. The present invention uses graphene oxide as the initial material, and the surface of graphene oxide carries a large number of negatively charged oxygen-containing functional groups (epoxy groups and hydroxyl groups, etc.), so that it is easy to be modified by positively charged polymers through electrostatic interactions, and then the gold nanoparticles are modified by copolymerization, and the mercaptonicotinic acid is modified by gold-sulfur bond reaction. The prepared mercaptonicotinic acid and gold nanoparticle modified graphene oxide material has higher selectivity for glycoprotein enrichment and better hydrophilicity.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and specifically relates to a mercaptonicotinic acid and gold nanoparticle modified graphene material and a preparation method and application thereof. Background Art

[0002] Protein glycosylation is a common, important and complex post-translational modification of proteins in organisms. Glycoproteins are involved in many key biological processes. Therefore, the research and development of glycosylated proteins is not only of great significance for the in-depth study of the physiological mechanisms of organisms, but also of clinical value in disease diagnosis or biomarker discovery. However, glycoproteins have low abundance, low mass spectrometry detection signal response, and the coexistence of non-glycosylated proteins and glycosylated proteins in complex biological systems, which have serious ion suppression effects on glycoproteins. Therefore, the research and development of an efficient enrichment strategy is an essential step in the comprehensive analysis of glycoproteins.

[0003] Currently, the widely used enrichment methods include lectin affinity, hydrazine chemistry, boric acid affinity, and hydrophilic interaction chromatography. The existing enrichment methods have their own advantages and disadvantages. However, with the in-depth study of glycoprotein markers, researchers have gradually shifted their focus to the performance of actual sample enrichment, such as enrichment coverage, enrichment selectivity, operational stability, and mass spectrometry compatibility. In recent years, hydrophilic interaction chromatography has received increasing attention in the separation and enrichment of glycoproteins.

[0004] Hydrophilic interaction chromatography (HILIC) is a chromatographic mode that uses a polar stationary phase (such as silica gel, derivatized silica gel) and an organic solvent (such as acetonitrile)-water as a mobile phase. It has a mobile phase similar to reversed-phase liquid chromatography, and the elution order of the compounds is similar to normal-phase chromatography. Its mechanism of action relies on the distribution of the analyte between the organic phase and the water layer on the surface of the stationary phase to achieve separation. HILIC can effectively retain highly polar compounds that are not fully or not retained in reversed-phase chromatography, and has a good separation effect. It is a reliable means to solve the separation problems of various highly polar compounds and hydrophilic compounds. The mobile phase system used in HILIC is relatively simple, the separation conditions are mild, the application range is wide, the sample loss is small, and it can enrich glycopeptides of various structures. Therefore, it has developed rapidly in the fields of drug analysis and food science. However, existing glycoprotein enrichment methods such as HILIC generally have the problem of insufficient enrichment selectivity. Summary of the invention

[0005] The purpose of the present invention is to provide a mercaptonicotinic acid and gold nanoparticle modified graphene material and a preparation method and application thereof. The mercaptonicotinic acid and gold nanoparticle modified graphene material provided by the present invention has high selectivity for enriching glycoproteins.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material, comprising the following steps:

[0008] (1) mixing a hydrophilic polymer solution and a graphene oxide-methanol suspension to perform an electrostatic adsorption reaction to obtain a hydrophilic polymer-modified graphene oxide material;

[0009] (2) suspending the hydrophilic polymer modified graphene oxide material in methanol, and then mixing the obtained hydrophilic polymer modified graphene oxide material-methanol suspension with an aqueous solution of gold chloride for copolymerization to obtain an intermediate product; the intermediate product is a hydrophilic polymer and gold nanoparticle modified graphene oxide material;

[0010] (3) The intermediate product, 2-mercaptonicotinic acid and methanol are mixed to carry out a gold-sulfur bond reaction to obtain a mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material.

[0011] Preferably, the hydrophilic polymer in the hydrophilic polymer solution includes one or more of poly-(2-ethyl-2-oxazoline), polyethylene diamine and chitosan.

[0012] Preferably, in the step (1), the mass ratio of the hydrophilic polymer in the hydrophilic polymer solution to the graphene oxide in the graphene oxide-methanol suspension is 20:1-3; the concentration of the hydrophilic polymer solution is 10-50 mg / mL; the concentration of the graphene oxide-methanol suspension is 10-50 mg / mL.

[0013] Preferably, the electrostatic adsorption reaction is carried out under stirring conditions, at a temperature of 20 to 30° C., and for a heat preservation time of 12 to 30 hours.

[0014] Preferably, in the step (2), the mass ratio of graphene oxide in the hydrophilic polymer modified graphene oxide material-methanol suspension to gold chloride in the gold chloride aqueous solution is 1-10:1-30; the concentration of the gold chloride aqueous solution is 2-10 mg / mL; the concentration of the hydrophilic polymer modified graphene oxide material-methanol suspension is 2-10 mg / mL.

[0015] Preferably, the copolymerization reaction is carried out under stirring conditions, the reaction temperature is 30 to 80° C., and the insulation time is 0 to 2 hours.

[0016] Preferably, the mass ratio of the intermediate product to 2-mercaptonicotinic acid is 1 to 5:1.

[0017] Preferably, the gold-sulfur bond reaction is carried out under stirring conditions, the reaction temperature is 10 to 30° C., and the insulation time is 12 to 36 hours.

[0018] The present invention also provides a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material obtained by the preparation method described in the above scheme, comprising graphene oxide, gold nanoparticles and hydrophilic polymers modified on the graphene oxide, and 2-mercaptonicotinic acid modified on the gold nanoparticles.

[0019] The present invention also provides the use of the mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material in the above scheme in glycoprotein enrichment.

[0020] The present invention provides a method for preparing a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material. The present invention obtains a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material with higher selectivity for glycoprotein enrichment by improving the hydrophilicity, ion interaction and peptide recognition ability of the material:

[0021] (1) Improving the hydrophilicity of the graphene oxide surface: The present invention adds a hydrophilic polymer to the graphene oxide surface through electrostatic adsorption to improve the hydrophilicity of the graphene oxide surface. For example, when the hydrophilic polymer is poly-(2-ethyl-2-oxazoline) (PEOz), an amide-type hydrophilic material, poly-(2-ethyl-2-oxazoline) modified graphene oxide GO / PEOz, can be formed. Since PEOz contains a large number of amide groups, the introduction of PEOz to the graphene oxide surface can increase the hydrophilicity of the graphene oxide surface. The hydrophilicity of the graphene oxide surface; when the hydrophilic polymer is polyethylenediamine, polyethylenediamine-modified graphene oxide GO / PEI can be formed. Since polyethylenediamine contains a large number of amine groups, PEI is introduced into the graphene oxide surface to increase the hydrophilicity of the graphene oxide surface; when the hydrophilic polymer is chitosan, chitosan-modified graphene oxide GO / CS can be formed. Since chitosan contains a large number of glycosyl and amine groups, chitosan is introduced into the graphene oxide surface to increase the hydrophilicity of the graphene oxide surface.

[0022] (2) Increasing the peptide recognition sensitivity of the material: Taking advantage of the in-situ growth characteristics of gold, nano-gold is introduced on the basis of hydrophilic polymer modified graphene oxide material, and further utilizing the reaction between Au-S bonds to obtain hydrophilic polymer and gold nanoparticle modified graphene oxide material. The gold nanoparticles have a specific recognition function for peptides, thereby increasing the peptide recognition sensitivity of the material;

[0023] (3) Increasing the ionic interaction force on the surface of the material: The present invention selects 2-mercaptonicotinic acid containing both cations and anions, combines the characteristics of graphene oxide containing sp2 hybridized carbon hexagonal ring structure, and the anion and cation of 2-mercaptonicotinic acid forming π-π conjugation, and adds 2-mercaptonicotinic acid to the hydrophilic polymer and gold nanoparticle modified graphene oxide material to obtain mercaptonicotinic acid and gold nanoparticle modified graphene oxide material GO / PEOz / Au / MA.

[0024] The present invention uses graphene oxide as the initial material. The surface of graphene oxide carries a large number of negatively charged oxygen-containing functional groups (such as epoxy groups and hydroxyl groups, etc.), which makes it easy to be modified by positively charged polymers through electrostatic interactions. Then, gold nanoparticles are modified by copolymerization, and mercaptonicotinic acid is modified by gold-sulfur bond reaction. The prepared mercaptonicotinic acid and gold nanoparticle modified graphene oxide material has higher selectivity for glycoprotein enrichment and better hydrophilicity.

[0025] The present invention also provides a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material obtained by the preparation method described in the above scheme, comprising graphene oxide, gold nanoparticles and hydrophilic polymers modified on the graphene oxide, and 2-mercaptonicotinic acid modified on the gold nanoparticles. The mercaptonicotinic acid and gold nanoparticle modified graphene oxide material provided by the present invention has higher selectivity for glycoprotein enrichment and better hydrophilicity.

[0026] The present invention also provides the use of the mercaptonicotinic acid and gold nanoparticle modified graphene oxide material in the above scheme in glycoprotein enrichment. The mercaptonicotinic acid and gold nanoparticle modified graphene oxide material provided by the present invention has the advantages of high enrichment selectivity, good hydrophilicity and high enrichment efficiency for glycoprotein enrichment, and provides a new choice for the comprehensive analysis of glycoprotein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 The SDS-PAGE gel electrophoresis diagram of the enrichment of the materials prepared in Examples 1 to 2 and Comparative Examples 1 to 2; wherein, Lane 1 is a protein marker, Lane 2 is a mixture of HRP and BSA in a 1:1 molar ratio, and Lanes 3, 4, 5 and 6 are analysis graphs of the eluate after enrichment of the materials prepared in Comparative Example 1, Comparative Example 2, Example 2 and Example 1, respectively. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material, comprising the following steps:

[0030] (1) mixing a hydrophilic polymer solution and a graphene oxide-methanol suspension to perform an electrostatic adsorption reaction to obtain a hydrophilic polymer-modified graphene oxide material;

[0031] (2) suspending the hydrophilic polymer modified graphene oxide material in methanol, and then mixing the obtained hydrophilic polymer modified graphene oxide material-methanol suspension with an aqueous solution of gold chloride for copolymerization to obtain an intermediate product; the intermediate product is a hydrophilic polymer and gold nanoparticle modified graphene oxide material;

[0032] (3) The intermediate product, 2-mercaptonicotinic acid and methanol are mixed to carry out a gold-sulfur bond reaction to obtain a mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material.

[0033] The present invention mixes a hydrophilic polymer solution and a graphene oxide-methanol suspension for electrostatic adsorption reaction to obtain a hydrophilic polymer modified graphene oxide material. In the present invention, the hydrophilic polymer in the hydrophilic polymer solution preferably includes one or more of poly-(2-ethyl-2-oxazoline), polyethylene diamine and chitosan; the solvent in the hydrophilic polymer solution preferably includes one or more of methanol, ethanol and water; the preparation method of the hydrophilic polymer solution is preferably: after the hydrophilic polymer and the solvent are mixed, ultrasonic (referred to as the first ultrasonic) is obtained to obtain a hydrophilic polymer solution; the mixing method is preferably mechanical stirring; the frequency of the first ultrasonic is preferably 20 to 60 Hz, more preferably 40 Hz, and the time is preferably 1 to 3 hours, more preferably 2 hours; the preparation method of the graphene oxide-methanol suspension is preferably: suspending graphene oxide in methanol and ultrasonic (referred to as the second ultrasonic); the frequency of the second ultrasonic is preferably 20 to 60 Hz, more preferably 40 Hz, and the time is preferably 1 to 3 hours, more preferably 2 hours.

[0034] In the present invention, the mass ratio of the hydrophilic polymer in the hydrophilic polymer solution to the graphene oxide in the graphene oxide-methanol suspension is preferably 20:1-3, more preferably 20:1.5-2.5, and further preferably 20:1.8-2.2; the concentration of the hydrophilic polymer solution is preferably 10-50 mg / mL, more preferably 20-40 mg / mL, and further preferably 25-35 mg / mL; the concentration of the graphene oxide-methanol suspension is preferably 10-50 mg / mL, more preferably 20-40 mg / mL, and further preferably 25-30 mg / mL; the electrostatic adsorption reaction is preferably carried out under stirring conditions, the temperature of the electrostatic adsorption reaction is preferably 20-30°C, more preferably 24-27°C, and the insulation time is preferably 12-30h, more preferably 18-25h; the stirring method is preferably magnetic stirring.

[0035] In the present invention, after the electrostatic adsorption reaction, the obtained product liquid is preferably centrifuged, washed and dried in sequence; the centrifugal speed is preferably 5000-18000r / min, more preferably 8000-15000r / min, and the time is preferably 1-20min, more preferably 10-15min; the washing reagent is preferably methanol; the number of washings is preferably 1-8 times, more preferably 3-5 times; the drying is preferably vacuum drying; the vacuum degree of the vacuum drying is preferably 100-150Pa, more preferably 130Pa, the temperature is preferably 10-30°C, more preferably 20°C, and the time is preferably 8-15h, more preferably 10-12h.

[0036] After obtaining the hydrophilic polymer modified graphene oxide material, the present invention suspends the hydrophilic polymer modified graphene oxide material in methanol, and then mixes the obtained hydrophilic polymer modified graphene oxide material-methanol suspension with a gold chloride aqueous solution for copolymerization to obtain an intermediate product; the intermediate product is a hydrophilic polymer and gold nanoparticle modified graphene oxide material. In the present invention, the gold chloride in the gold chloride aqueous solution preferably includes one or both of gold chloride trihydrate and gold chloride; the gold content of the gold chloride trihydrate is 48.0%; the water in the gold chloride aqueous solution is preferably deionized water; the mass ratio of the graphene oxide in the hydrophilic polymer modified graphene oxide material-methanol suspension to the gold chloride in the gold chloride aqueous solution is preferably 1-10:1-30, more preferably 1-10:1-20, and further preferably 1-10:1-25; the concentration of the gold chloride aqueous solution is preferably 2-10 mg / mL, more preferably 4-8 mg / mL; the concentration of the hydrophilic polymer modified graphene oxide material-methanol suspension is preferably 2-10 mg / mL, more preferably 4-8 mg / mL.

[0037] In the present invention, the mixing of the obtained hydrophilic polymer modified graphene oxide material-methanol suspension and the gold chloride aqueous solution is preferably: the hydrophilic polymer modified graphene oxide material-methanol suspension and the gold chloride aqueous solution are stirred and then ultrasonicated; the power of the ultrasonication is preferably 200-600W, more preferably 300-500W, the time is preferably 0-5h, more preferably 1-4h; the copolymerization reaction is preferably carried out under stirring conditions, the reaction temperature is preferably 30-80°C, more preferably 40-70°C, the insulation time is preferably 0-2h, more preferably 0.5-1.5h; the stirring method is preferably magnetic stirring; the rotation speed of the magnetic stirring is preferably 200-1800r / min, more preferably 500-1200r / min; the copolymerization reaction is preferably carried out under oil bath conditions.

[0038] In the present invention, after the copolymerization reaction, the obtained product liquid is preferably centrifuged, washed with water and dried in sequence; the water used for washing is preferably deionized water; the number of washings is preferably 3 to 5 times, more preferably 4 times; the drying is preferably vacuum drying; the vacuum degree of the vacuum drying is preferably 100 to 150 Pa, more preferably 130 Pa, the temperature is preferably 10 to 30°C, more preferably 20°C, and the time is preferably 3 to 12 h, more preferably 12 h.

[0039] After obtaining the intermediate product, the present invention mixes the intermediate product, 2-mercaptonicotinic acid and methanol for gold-sulfur bond reaction to obtain mercaptonicotinic acid and gold nanoparticle modified graphene oxide material. In the present invention, the mass ratio of the intermediate product and 2-mercaptonicotinic acid is preferably 1 to 5:1, more preferably 2 to 4:1, and further preferably 3:1; the mass volume ratio of the intermediate product and methanol is preferably 1g:2 to 10L, more preferably 1g:4 to 8L; the intermediate product, 2-mercaptonicotinic acid and methanol are preferably mixed by: dissolving the intermediate product and 2-mercaptonicotinic acid in methanol and then ultrasonicating; the power of the ultrasound is preferably 200 to 600W, more preferably 300 to 500W, and the time is preferably 5 to 30min, more preferably 10 to 25min; the gold-sulfur bond reaction is preferably carried out under stirring conditions, the reaction temperature is preferably 10 to 30°C, more preferably 15 to 25°C, and the insulation time is preferably 12 to 36h, more preferably 18 to 30h; the stirring is preferably magnetic stirring.

[0040] In the present invention, after the gold-sulfur bond reaction, the obtained product liquid is preferably centrifuged, washed and dried in sequence; the centrifugal speed is preferably 5000-18000 r / min, more preferably 9000-15000 r / min, and the time is preferably 1-10 min, more preferably 5-8 min; the washing reagent is preferably methanol; the number of washings is preferably 3-5 times, more preferably 4 times; the drying is preferably vacuum drying; the vacuum degree of the vacuum drying is preferably 100-200 Pa, more preferably 140-180 Pa, the temperature is preferably 10-30°C, more preferably 15-25°C, and the time is preferably 5-12 h, more preferably 12 h.

[0041] The present invention also provides a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material obtained by the preparation method described in the above scheme, comprising graphene oxide, gold nanoparticles and hydrophilic polymers modified on the graphene oxide, and 2-mercaptonicotinic acid modified on the gold nanoparticles.

[0042] In the present invention, in the mercaptonicotinic acid and gold nanoparticle modified graphene oxide material, the mass ratio of the graphene oxide to the gold nanoparticles is preferably 1-10:1-30, more preferably 1-10:1-20; the mass ratio of the graphene oxide to the hydrophilic polymer is preferably 1-30:1-70, more preferably 1-40:1-60; the mass ratio of the graphene oxide to 2-mercaptonicotinic acid is preferably 1:1-6, more preferably 1:1-2.

[0043] The present invention also provides the use of the mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material in the above scheme in glycoprotein enrichment.

[0044] In the present invention, the application of the mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material in glycoprotein enrichment preferably includes the following steps: mixing mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material with an acetonitrile aqueous solution and then centrifuging for the first time, then vortexing and incubating the obtained solid product, a loading buffer and a protein mixture of horseradish peroxidase and bovine serum albumin, then centrifuging the obtained incubation product for the second time, discarding the supernatant, and then eluting, centrifuging for the third time to obtain the supernatant and freeze-drying in sequence to obtain enriched glycoprotein.

[0045] In the present invention, the concentration of the suspension solution obtained by mixing the mercaptonicotinic acid with the gold nanoparticle-modified graphene oxide material and the acetonitrile aqueous solution is preferably 0.5-3.0 μg / μL, more preferably 1.6 μg / μL; the volume of the suspension solution is preferably 50 μL; the mass concentration of the acetonitrile aqueous solution is preferably 70%-90%, more preferably 78%.

[0046] In the present invention, the loading buffer is preferably 78 wt % acetonitrile (ACN) and 1 wt % trifluoroacetic acid (TFA); the volume of the loading buffer is preferably 300 μL.

[0047] In the present invention, the molar ratio of the horseradish peroxidase (standard glycoprotein HRP) to bovine serum albumin (standard non-glycoprotein BSA) is preferably 1-5:2-8, more preferably 1:1; the concentration of the protein mixture is preferably 0.001-0.003 nmol / μL, more preferably 0.00125 nmol / μL; the loading amount of the horseradish peroxidase is preferably 0.01-0.5 nmol, more preferably 0.1 nmol.

[0048] In the present invention, the temperature of the vortex is preferably room temperature, and the time is preferably 2 to 10 minutes, more preferably 5 minutes.

[0049] In the present invention, the incubation temperature is preferably room temperature, and the incubation time is preferably 10 to 120 minutes, more preferably 60 minutes.

[0050] In the present invention, the rotation speed of the second centrifugation is preferably 5000-15000 rpm, more preferably 10000 rpm, and the time is preferably 1-10 min, more preferably 3 min.

[0051] In the present invention, the eluent used for elution is preferably 50wt% acetonitrile / 1wt% FA solution, the volume of the eluent is preferably 300 μL, the temperature is preferably room temperature, and the vortex time is preferably 10 min.

[0052] In the present invention, the rotation speed of the third centrifugation is preferably 5000-15000 rpm, more preferably 12000 rpm, and the time is preferably 1-10 min, more preferably 5 min.

[0053] In the present invention, the vacuum degree of freeze-drying is preferably 0 to 15 Pa, more preferably 3 to 8 Pa, the temperature is preferably -40 to -10°C, more preferably -40 to -20°C, and the insulation time is preferably 5 to 18 h, more preferably 10 to 12 h.

[0054] The mercaptonicotinic acid and gold nanoparticle modified graphene oxide material provided by the present invention has the advantages of high enrichment selectivity, good hydrophilicity and high enrichment efficiency for glycoprotein enrichment, and provides a new choice for the comprehensive analysis of glycoprotein.

[0055] In order to further illustrate the present invention, the scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0056] Example 1

[0057] (1) Weighing 100 mg of poly-(2-ethyl-2-oxazoline) and dissolving it in 4 mL of methanol for 2 h to obtain a poly-(2-ethyl-2-oxazoline) solution; Weighing 2 mg of graphene oxide and suspending it in 2 mL of methanol for 2 h to obtain a graphene oxide-methanol suspension; Transferring the poly-(2-ethyl-2-oxazoline) solution and the graphene oxide-methanol suspension to a microreactor, stirring them magnetically at 20-30° C. for 30 h, centrifuging and washing them with methanol for 5 times, and vacuum drying them for 15 h to obtain an amide-type hydrophilic material GO / PEOz;

[0058] (2) Weigh 40 mg of gold chloride trihydrate (48.0% Au) and dissolve it in 2 mL of deionized water, then add the 2 mg of GO / PEOz and stir evenly. After ultrasonication for 5 h, transfer the resulting solution into a microreactor, stir it magnetically for 2 h in an oil bath at 80 °C, centrifuge and wash it with deionized water for 5 times, and vacuum dry it for 12 h to obtain the intermediate product GO / PEOz / Au.

[0059] (3) Weigh 3.5 mg of 2-mercaptonicotinic acid and 1 mg of the intermediate product GO / PEOz / Au and dissolve them in 6 mL of methanol solution. After ultrasonication for 30 min, transfer them into a microreactor and stir them magnetically at room temperature for 36 h. After centrifugation, wash them with methanol 5 times and vacuum dry them for 12 h to obtain mercaptonicotinic acid and gold nanoparticles modified graphene oxide material GO / PEOz / Au / MA.

[0060] Example 2

[0061] (1) Weighing 200 mg of poly-(2-ethyl-2-oxazoline) and dissolving it in 4 mL of methanol for 2 h to obtain a poly-(2-ethyl-2-oxazoline) solution; Weighing 2 mg of graphene oxide and suspending it in 2 mL of methanol for 2 h to obtain a graphene oxide-methanol suspension; Transferring the poly-(2-ethyl-2-oxazoline) solution and the graphene oxide-methanol suspension to a microreactor, stirring them magnetically at 20-30° C. for 30 h, centrifuging and washing them with methanol for 5 times, and vacuum drying them for 15 h to obtain an amide-type hydrophilic material GO / PEOz;

[0062] (2) Weigh 60 mg of gold chloride trihydrate (48.0% Au) and dissolve it in 2 mL of deionized water, then add the 2 mg of GO / PEOz and stir evenly. After ultrasonication for 5 h, transfer the resulting solution into a microreactor, stir it magnetically for 2 h in an oil bath at 80 °C, centrifuge and wash it with deionized water for 5 times, and vacuum dry it for 12 h to obtain the intermediate product GO / PEOz / Au.

[0063] (3) Weigh 6 mg of 2-mercaptonicotinic acid and 1 mg of the intermediate product GO / PEOz / Au and dissolve them in 6 mL of methanol solution. After ultrasonication for 30 min, transfer them into a microreactor and stir them magnetically at room temperature for 36 h. After centrifugation, wash them with methanol 5 times and vacuum dry them for 12 h to obtain mercaptonicotinic acid and gold nanoparticles modified graphene oxide material GO / PEOz / Au / MA.

[0064] Comparative Example 1

[0065] (1) Weighing 100 mg of polyethylene glycol and dissolving it in 3 mL of methanol for 2 h to obtain a polyethylene glycol solution; weighing 2 mg of graphene oxide and suspending it in 2 mL of methanol for 2 h to obtain a graphene oxide-methanol suspension; transferring the polyethylene glycol solution and the graphene oxide-methanol suspension to a microreactor, stirring them magnetically at 20° C. for 30 h, centrifuging and washing them with methanol for 3 times, and vacuum drying them for 8 h to obtain an amide-type hydrophilic material GO / PEI;

[0066] (2) Weigh 40 mg of gold chloride trihydrate (48.0% Au) and dissolve it in 2 mL of deionized water, then add 2 mg of GO / PEI material and stir evenly. After ultrasonic treatment for 2 h, transfer the solution into a microreactor, stir it magnetically for 1 h in an oil bath at 30 °C, centrifuge and wash it with deionized water for 3 times, and vacuum dry it for 12 h to obtain the intermediate product GO / PEI / Au.

[0067] (3) Weigh 3.5 mg of 2-mercaptonicotinic acid and 1 mg of the intermediate product GO / PEI / Au and dissolve them in 6 mL of methanol solution. After ultrasonication for 30 min, transfer them into a microreactor and stir them magnetically at room temperature for 12 h. After centrifugation, wash them with methanol 4 times and vacuum dry them for 12 h to obtain mercaptonicotinic acid and gold nanoparticles modified graphene oxide material GO / PEI / Au / MA.

[0068] Comparative Example 2

[0069] (1) Weigh 0.4 g of chitosan and add it to 20 mL of acetic acid solution (2%, V / V), and ultrasonicate it at room temperature for 2 h to obtain a chitosan solution; weigh 0.3 g of graphene oxide and add it to 20 mL of deionized water, and ultrasonicate it at room temperature for 3 h to obtain a graphene oxide suspension; under mechanical stirring, mix the chitosan solution and the graphene oxide suspension, slowly stir and add 4 mL of glutaraldehyde solution (5%, V / V), place it in a 50°C water bath and continue stirring to react for 2 h; adjust the pH value to 9-10 with 0.1 mol / L NaOH solution, place it in a 80°C water bath and continue to react for 1 h; after the reaction system is cooled to room temperature, centrifuge it at 12000 r / min for 5 min, wash the black product with ethanol and deionized water in turn to a pH value of 7, and dry it at 37°C overnight to obtain a single-layer polymer graphene material GO / CS;

[0070] (2) Weigh 40 mg of gold chloride trihydrate (48.0% Au) and dissolve it in 2 mL of deionized water, then add 2 mg of material GO / CS and stir evenly. After ultrasonic treatment for 5 h, transfer the solution into a microreactor, stir it magnetically under 50 °C oil bath conditions for 2 h, centrifuge and wash it with deionized water for 3 times, and vacuum dry it for 12 h to obtain the intermediate product GO / CS / Au.

[0071] (3) Weigh 3.5 mg of 2-mercaptonicotinic acid and 1 mg of the intermediate product GO / CS / Au and dissolve them in 6 mL of methanol solution. After ultrasonication for 30 min, transfer them into a microreactor and stir them magnetically at room temperature for 15 h. After centrifugation, wash them with methanol 4 times and vacuum dry them for 12 h to obtain mercaptonicotinic acid and gold nanoparticles modified graphene oxide material GO / CS / Au / MA.

[0072] Application Example 1

[0073] A glycoprotein selective enrichment experiment was carried out. 3 mg of the GO / PEOz / Au / MA material prepared in Example 1 was weighed and prepared with 1000 μL of 80% ACN solution to obtain a 3 μg / μL GO / PEOz / Au / MA material suspension; 30 μL of the GO / PEOz / Au / MA material suspension was centrifuged, and 50 μL of loading buffer 78% ACN / 1% TFA, 0.1 nmol of standard glycoprotein HRP and standard non-glycoprotein BSA were added, vortexed and incubated at room temperature for 0.5 h, centrifuged at 12000 r / min for 5 min, the supernatant was discarded, 50 μL of elution buffer 50% ACN / 1% FA was added, vortexed at room temperature for 0.5 h, centrifuged at 12000 r / min for 5 min, and the supernatant was freeze-dried and analyzed by SDS-PAGE gel electrophoresis.

[0074] Application Example 2

[0075] The GO / PEOz / Au / MA material prepared in Example 2 was subjected to glycoprotein separation and enrichment according to the method of Application Example 1.

[0076] Comparative application example 1

[0077] The GO / PEI / Au / MA material prepared in Comparative Example 1 was subjected to glycoprotein separation and enrichment according to the method of Application Example 1.

[0078] Comparative Application Example 2

[0079] The GO / CS / Au / MA material prepared in Comparative Example 2 was subjected to glycoprotein separation and enrichment according to the method of Application Example 1.

[0080] The materials prepared in Examples 1 to 2 and Comparative Examples 1 to 2 were subjected to a glycoprotein selective enrichment experiment. The results are as follows: Figure 1 As shown. Figure 1It can be seen that the three materials have strong enrichment effects and selectivity for various types of glycoproteins, and the GO / PEOz / Au / MA prepared in Example 1 of the present invention has the best enrichment effect.

[0081] It can be seen from the above examples that the mercaptonicotinic acid and gold nanoparticle modified graphene oxide material provided by the present invention has the advantages of high enrichment selectivity, good hydrophilicity and high enrichment efficiency for glycoprotein enrichment, and provides a new choice for the comprehensive analysis of glycoproteins.

[0082] 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 method for preparing a mercaptonicotinic acid and gold nanoparticle modified graphene oxide material, comprising the following steps: (1) mixing a hydrophilic polymer solution and a graphene oxide-methanol suspension to perform an electrostatic adsorption reaction to obtain a hydrophilic polymer-modified graphene oxide material; (2) suspending the hydrophilic polymer modified graphene oxide material in methanol, and then mixing the obtained hydrophilic polymer modified graphene oxide material-methanol suspension with an aqueous solution of gold chloride for copolymerization to obtain an intermediate product; the intermediate product is a hydrophilic polymer and gold nanoparticle modified graphene oxide material; (3) mixing the intermediate product, 2-mercaptonicotinic acid and methanol to perform a gold-sulfur bond reaction to obtain a mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material; In the step (1), the mass ratio of the hydrophilic polymer in the hydrophilic polymer solution to the graphene oxide in the graphene oxide-methanol suspension is 20:1-3; The hydrophilic polymer in the hydrophilic polymer solution includes one or more of poly-(2-ethyl-2-oxazoline), polyethylene diamine and chitosan.

2. The preparation method according to claim 1, characterized in that: In the step (1), the concentration of the hydrophilic polymer solution is 10 to 50 mg / mL; the concentration of the graphene oxide-methanol suspension is 10 to 50 mg / mL.

3. The preparation method according to claim 1 or 2, characterized in that: The electrostatic adsorption reaction is carried out under stirring conditions at a temperature of 20 to 30° C. and a heat preservation time of 12 to 30 hours.

4. The preparation method according to claim 1, characterized in that: In the step (2), the mass ratio of the hydrophilic polymer modified graphene oxide material in the hydrophilic polymer modified graphene oxide material-methanol suspension to the gold chloride in the gold chloride aqueous solution is 1-10:1-30; the concentration of the gold chloride aqueous solution is 2-10 mg / mL; and the concentration of the hydrophilic polymer modified graphene oxide material-methanol suspension is 2-10 mg / mL.

5. The preparation method according to claim 1 or 4, characterized in that: The copolymerization reaction is carried out under stirring conditions, the reaction temperature is 30-80° C., and the insulation time is 0-2 hours.

6. The preparation method according to claim 1, characterized in that: The mass ratio of the intermediate product to 2-mercaptonicotinic acid is 1 to 5:

1.

7. The preparation method according to claim 1 or 6, characterized in that: The gold-sulfur bond reaction is carried out under stirring conditions, the reaction temperature is 10-30° C., and the insulation time is 12-36 hours.

8. The mercaptonicotinic acid and gold nanoparticle modified graphene oxide material obtained by the preparation method according to any one of claims 1 to 7 comprises graphene oxide, gold nanoparticles and hydrophilic polymers modified on the graphene oxide, and 2-mercaptonicotinic acid modified on the gold nanoparticles.

9. Use of the mercaptonicotinic acid and gold nanoparticle-modified graphene oxide material according to claim 8 in glycoprotein enrichment.

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