A method for preparing protein molecular imprinting optical and electrochemical sensor

Abstract

The present invention provides a method for preparing a protein molecular imprinting optical and electrochemical sensor. First, a bare carbon electrode is surface treated with 3-aminopropyltriethoxysilane to introduce amino groups. The bare carbon electrode with the introduced amino groups is immersed in a mixed aqueous solution of template protein, sodium alginate and carboxylated graphene, and then the bare carbon electrode coated with the mixed aqueous solution is immersed in a calcium chloride aqueous solution for cross-linking, the template protein is eluted with a buffer solution, silver ions are introduced by adsorption and reduced to obtain a protein molecular imprinting optical and electrochemical sensor containing silver nano-ions on the surface. The molecular imprinting hybrid hydrogel modified electrode capable of recognizing proteins prepared by the present invention can be used for both optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy, thereby greatly improving the efficiency and reliability of detection, and has good application prospects in the field of protein analysis and detection.

Description

Technical Field

[0001] The invention relates to a method for preparing a protein molecular imprinting optical and electrochemical sensor, and belongs to the intersecting field of functional materials, analytical chemistry, sensors and biomedicine.

[0002] The present invention relates to the technical fields of hydrogel, molecular imprinting, protein detection, Raman spectroscopy and electrochemistry, and more particularly to a preparation method and application of a protein molecular imprinting optical and electrochemical sensor. Background Art

[0003] With the development of artificial intelligence and the increasingly high detection requirements, various sensors have emerged, such as enzyme biosensors, cell sensors, DNA biosensors, tissue sensors, and microbial sensors. Electrochemical sensors are selective and specific, and are ideal analytical tools for quantitative or semi-quantitative detection of substances. They have been widely used in food analysis, environmental monitoring, medicine, and military fields. However, general electrochemical sensors lack selectivity.

[0004] The combination of molecular imprinting polymers and electrochemical biosensors has attracted widespread attention. Molecular imprinting is a technology for preparing polymers with specific binding sites, which has been widely used in solid phase extraction, biomimetic sensors, chromatographic separation and drug controlled release. Although molecular imprinting technology has developed rapidly, most imprinting templates are small molecules, and macromolecular templates, such as protein molecular imprinting, are relatively rare. The main reason is the complexity of protein structure, high flexibility of conformation and diversity of protein sequence. Many researchers have overcome the shortcomings of protein molecular imprinting and are committed to the combination of molecular imprinting and sensors. Li et al. [ACS Sensors, 2018, 3, 378-385] studied the detection of bovine hemoglobin by molecular imprinting polymer nanoparticles, and the electrochemical signal showed good dynamic response.

[0005] Hydrogels have high water content and good biocompatibility, and can maintain protein conformation, and can be used for protein molecular imprinting. Although hydrogel MIPs have high selectivity for template proteins, proteins diffuse very slowly in hydrogels. Surface imprinting can solve the problem of protein diffusion [Polymers, 2018, 10(2): 170-183]. Zhao Kongyin et al. prepared BSA imprinted polyacrylamide hydrogel using bovine serum albumin (BSA) as the template molecule and acrylamide as the functional monomer. However, the heat or initiation conditions of hydrogel polymerization can lead to changes and denaturation of protein conformation. Peppas et al. used BSA as a template, sodium alginate (NaAlg) as a functional monomer, and CaCl2 as a cross-linking agent to prepare BSA-imprinted calcium alginate membrane [J.Biomater.Sci., Polym.Ed.2011, 22, 1523-1534] and microcapsules [Ind.Eng.Chem.Res.2010, 49, 9811-9814]. However, since the calcium alginate hydrogel membrane was too thick and the microcapsules were large (2-3 mm in diameter), the adsorption rate of MIP to BSA was slow and the adsorption capacity was low.

[0006] The present invention provides a method for preparing a protein molecular imprinting optical and electrochemical sensor. First, a bare carbon electrode is surface treated with 3-aminopropyltriethoxysilane to introduce amino groups. The bare carbon electrode with the introduced amino groups is immersed in a mixed aqueous solution of template protein, sodium alginate and carboxylated graphene, and then the bare carbon electrode coated with the mixed aqueous solution is immersed in a calcium chloride aqueous solution for cross-linking, the template protein is washed off with a buffer solution, silver ions are adsorbed and introduced, and a protein molecular imprinting optical and electrochemical sensor containing silver nano-ions on the surface is obtained after reduction. The molecular imprinting hybrid hydrogel modified electrode capable of recognizing proteins prepared by the present invention can be used for both optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy, thereby greatly improving the efficiency and reliability of detection, and has good application prospects in the field of protein analysis and detection. Summary of the invention

[0007] In view of the shortcomings of the prior art, the technical problems that the present invention intends to solve are the poor selectivity and sensitivity of trace protein detection, long time consumption, easy denaturation of protein templates in the molecular imprinting process, and difficulty in elution.

[0008] The present invention solves the problems of poor selectivity and sensitivity of trace protein detection, long time consumption, easy denaturation of protein template in molecular imprinting process and difficulty in elution by providing a method for preparing protein molecular imprinting optical and electrochemical sensors.

[0009] The present invention provides a method for preparing a protein molecular imprinting optical and electrochemical sensor, which is characterized by comprising the following steps:

[0010] a) immersing the cleaned bare carbon electrode in a 0.01% to 2% by mass 3-aminopropyltriethoxysilane aqueous solution for 2 to 24 hours to introduce amino groups on the surface of the bare carbon electrode;

[0011] b) preparing a mixture aqueous solution with a protein mass percentage concentration of 0.01%-5%, a sodium alginate mass percentage concentration of 0.1%-5%, a sodium silicate mass percentage concentration of 0.01%-2%, and a carboxylated graphene mass percentage concentration of 0.01%-10%, standing to defoam and placing in a sterile container for standby use;

[0012] c) preparing a calcium chloride aqueous solution having a calcium chloride mass percentage concentration of 0.1% to 10%;

[0013] d) uniformly coating the aqueous mixture solution obtained in step b) on the bare carbon electrode with amino groups introduced on the surface obtained in step a), and combining the sodium alginate molecules and the carboxylated graphene on the surface of the bare carbon electrode by utilizing the interaction between the amino groups and the carboxyl groups on the sodium alginate and the carboxylated graphene; immediately immersing the bare carbon electrode coated with the aqueous mixture solution obtained in step b) in the calcium chloride aqueous solution obtained in step c) for 0.5-24 hours to obtain a protein-containing hybrid hydrogel modified electrode; calcium chloride cross-links sodium silicate to generate uniformly dispersed calcium silicate nanoparticles in the hybrid hydrogel;

[0014] e) washing the protein from the protein-containing hybrid hydrogel modified electrode obtained in step d) with an eluent, then immersing it in a silver nitrate aqueous solution to adsorb silver ions, and then reducing it with a sodium borohydride aqueous solution to obtain a molecularly imprinted hybrid hydrogel modified electrode that can recognize proteins and contains silver nanoparticles on its surface; assembling the modified electrode as a working electrode, a silver electrode as a reference electrode, and a platinum sheet as an auxiliary electrode to form an electrochemical sensor that can rapidly detect and recognize proteins; combining a probe of a Raman spectrometer with the molecularly imprinted hybrid hydrogel modified electrode that can recognize proteins to form an optical sensor that can rapidly detect and recognize proteins;

[0015] f) placing the protein-recognizable molecular imprinting hybrid hydrogel modified electrode obtained in step e) in a protein aqueous solution to adsorb the protein and then taking it out, and using a Raman spectrometer to detect the adsorbed protein. Since the silver nanoparticles in the hybrid hydrogel obtained in step e) can serve as active hot spots for enhancing the substrate, the protein detection sensitivity is greatly improved. The protein-recognizable molecular imprinting hybrid hydrogel modified electrode prepared by the present invention can be used for optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy at the same time, thereby greatly improving the detection limit, reliability and detection efficiency. The protein molecular imprinting optical and electrochemical sensors have good application prospects in the field of protein analysis and detection.

[0016] No toxic or harmful reagents are introduced in the preparation process of the present invention, and there is no change in protein conformation caused by polymerization reaction heat. The process of eluting proteins is gentle, and the obtained molecular imprinting hydrogel modified electrode capable of recognizing proteins has good biocompatibility and high recognition performance. The protein described in the present invention is any one of bovine serum albumin, ovalbumin, catalase, and lysozyme, or a mixture of two or more thereof. The eluent described in the present invention is any one of phosphate buffer solution, Tris-HCl buffer solution, and acetate buffer solution. DETAILED DESCRIPTION

[0017] Specific embodiments of the present invention are described below, but the present invention is not limited to the embodiments.

[0018] Example 1.

[0019] a) immersing the cleaned bare carbon electrode in a 0.01% by mass 3-aminopropyltriethoxysilane aqueous solution for 2 hours to introduce amino groups on the surface of the bare carbon electrode;

[0020] b) preparing a mixture aqueous solution with a mass percentage concentration of 0.01% bovine serum protein, a mass percentage concentration of 0.1% sodium alginate, a mass percentage concentration of 0.01% sodium silicate, and a mass percentage concentration of 0.01% carboxylated graphene, standing and defoaming, and placing the mixture in a sterile container for standby use;

[0021] c) preparing a calcium chloride aqueous solution having a calcium chloride mass percentage concentration of 0.1%;

[0022] d) uniformly coating the aqueous mixture solution obtained in step b) on the bare carbon electrode with amino groups introduced on the surface obtained in step a), and utilizing the interaction between the amino groups and the carboxyl groups on sodium alginate and carboxylated graphene to combine the sodium alginate molecules and the carboxylated graphene on the surface of the bare carbon electrode; immediately immersing the bare carbon electrode coated with the aqueous mixture solution obtained in step b) in the calcium chloride aqueous solution obtained in step c) for 0.5 hours to obtain a hybrid hydrogel modified electrode containing bovine serum albumin; calcium chloride cross-links sodium silicate to generate uniformly dispersed calcium silicate nanoparticles in the hybrid hydrogel;

[0023] e) washing the hybrid hydrogel modified electrode containing bovine serum albumin obtained in step d) with a phosphate buffer solution to remove the bovine serum albumin, then immersing it in a silver nitrate aqueous solution to adsorb silver ions, and then reducing it with a sodium borohydride aqueous solution to obtain a molecularly imprinted hybrid hydrogel modified electrode that can recognize bovine serum albumin and contains silver nanoparticles on the surface; assembling the modified electrode as a working electrode, a silver electrode as a reference electrode, and a platinum sheet as an auxiliary electrode to form an electrochemical sensor that can quickly detect and identify bovine serum albumin; combining a probe of a Raman spectrometer with the molecularly imprinted hybrid hydrogel modified electrode that can recognize bovine serum albumin to form an optical sensor that can quickly detect and identify bovine serum albumin;

[0024] f) placing the molecular imprinted hybrid hydrogel modified electrode capable of identifying bovine serum albumin obtained in step e) in an aqueous solution of bovine serum albumin to adsorb the bovine serum albumin and then taking it out, and detecting the adsorbed bovine serum albumin using a Raman spectrometer. Since the silver nanoparticles in the hybrid hydrogel obtained in step e) can serve as active hot spots of the enhanced substrate, the detection sensitivity of the bovine serum albumin is greatly improved. The molecular imprinted hybrid hydrogel modified electrode capable of identifying bovine serum albumin prepared by the present invention can be used for both optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy, thereby greatly improving the detection limit, reliability and detection efficiency. The bovine serum albumin molecular imprinted optical and electrochemical sensors have good application prospects in the field of bovine serum albumin analysis and detection.

[0025] Example 2.

[0026] a) immersing the cleaned bare carbon electrode in a 2% by mass 3-aminopropyltriethoxysilane aqueous solution for 24 hours to introduce amino groups on the surface of the bare carbon electrode;

[0027] b) preparing an aqueous solution of a mixture having a mass percentage concentration of 5% egg white protein, a mass percentage concentration of 5% sodium alginate, a mass percentage concentration of 2% sodium silicate, and a mass percentage concentration of 10% carboxylated graphene, standing the mixture to defoam, and placing the mixture in a sterile container for later use;

[0028] c) preparing a calcium chloride aqueous solution having a calcium chloride mass percentage concentration of 10%;

[0029] d) uniformly coating the aqueous mixture solution obtained in step b) on the bare carbon electrode with amino groups introduced on the surface obtained in step a), and combining the sodium alginate molecules and the carboxylated graphene on the surface of the bare carbon electrode by utilizing the interaction between the amino groups and the carboxyl groups on the sodium alginate and the carboxylated graphene; immediately immersing the bare carbon electrode coated with the aqueous mixture solution obtained in step b) in the calcium chloride aqueous solution obtained in step c) for 24 hours to obtain a hybrid hydrogel modified electrode containing ovalbumin; calcium chloride cross-links sodium silicate to generate uniformly dispersed calcium silicate nanoparticles in the hybrid hydrogel;

[0030] e) washing the hybrid hydrogel modified electrode containing ovalbumin obtained in step d) with a Tris-HCl buffer solution to remove the ovalbumin, then immersing it in a silver nitrate aqueous solution to adsorb silver ions, and then reducing it with a sodium borohydride aqueous solution to obtain a molecularly imprinted hybrid hydrogel modified electrode that can recognize ovalbumin and contains silver nanoparticles on its surface; assembling the modified electrode as a working electrode, a silver electrode as a reference electrode, and a platinum sheet as an auxiliary electrode to form an electrochemical sensor that can rapidly detect and recognize ovalbumin; combining a probe of a Raman spectrometer with the molecularly imprinted hybrid hydrogel modified electrode that can recognize ovalbumin to form an optical sensor that can rapidly detect and recognize ovalbumin;

[0031] f) placing the molecular imprinting hybrid hydrogel modified electrode capable of identifying ovalbumin obtained in step e) in an aqueous solution of ovalbumin to adsorb the ovalbumin and then taking it out, and detecting the adsorbed ovalbumin using a Raman spectrometer. Since the silver nanoparticles in the hybrid hydrogel obtained in step e) can serve as active hot spots for enhancing the substrate, the detection sensitivity of ovalbumin is greatly improved. The molecular imprinting hybrid hydrogel modified electrode capable of identifying ovalbumin prepared by the present invention can be used for both optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy, thereby greatly improving the detection limit, reliability and detection efficiency. The ovalbumin molecular imprinting optical and electrochemical sensors have good application prospects in the field of ovalbumin analysis and detection.

[0032] Example 3.

[0033] a) immersing the cleaned bare carbon electrode in a 0.5% by mass 3-aminopropyltriethoxysilane aqueous solution for 10 hours to introduce amino groups on the surface of the bare carbon electrode;

[0034] b) preparing a mixture aqueous solution with a mass percentage concentration of 1.5% catalase, a mass percentage concentration of 0.5% sodium alginate, a mass percentage concentration of 0.2% sodium silicate, and a mass percentage concentration of 1% carboxylated graphene, standing and defoaming, and placing the mixture in a sterile container for standby use;

[0035] c) preparing a calcium chloride aqueous solution having a calcium chloride mass percentage concentration of 1%;

[0036] d) uniformly coating the aqueous mixture solution obtained in step b) on the bare carbon electrode with amino groups introduced on the surface obtained in step a), and combining the sodium alginate molecules and the carboxylated graphene on the surface of the bare carbon electrode by utilizing the interaction between the amino groups and the carboxyl groups on the sodium alginate and the carboxylated graphene; immediately immersing the bare carbon electrode coated with the aqueous mixture solution obtained in step b) in the calcium chloride aqueous solution obtained in step c) for 4 hours to obtain a hybrid hydrogel modified electrode containing catalase; calcium chloride cross-links sodium silicate to generate uniformly dispersed calcium silicate nanoparticles in the hybrid hydrogel;

[0037] e) washing the catalase-containing hybrid hydrogel modified electrode obtained in step d) with an acetic acid buffer solution to remove the catalase, then immersing it in a silver nitrate aqueous solution to adsorb silver ions, and then reducing it with a sodium borohydride aqueous solution to obtain a molecularly imprinted hybrid hydrogel modified electrode with silver nanoparticles on the surface that can recognize catalase; assembling the modified electrode as a working electrode, a silver electrode as a reference electrode, and a platinum sheet as an auxiliary electrode to form an electrochemical sensor that can rapidly detect and recognize catalase; combining a probe of a Raman spectrometer with the molecularly imprinted hybrid hydrogel modified electrode that can recognize catalase to form an optical sensor that can rapidly detect and recognize catalase;

[0038] f) placing the molecular imprinted hybrid hydrogel modified electrode capable of identifying catalase obtained in step e) in an aqueous solution of catalase to adsorb catalase and then taking it out, and detecting the adsorbed catalase using a Raman spectrometer. Since the silver nanoparticles in the hybrid hydrogel obtained in step e) can serve as active hot spots of the enhanced substrate, the detection sensitivity of catalase is greatly improved. The molecular imprinted hybrid hydrogel modified electrode capable of identifying catalase prepared by the present invention can be used for optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy at the same time, thereby greatly improving the detection limit, reliability and detection efficiency. The catalase molecular imprinted optical and electrochemical sensors have good application prospects in the field of catalase analysis and detection.

[0039] Example 4.

[0040] a) immersing the cleaned bare carbon electrode in a 1.5% by mass 3-aminopropyltriethoxysilane aqueous solution for 20 hours to introduce amino groups on the surface of the bare carbon electrode;

[0041] b) preparing a mixture aqueous solution with a lysozyme mass percentage concentration of 3.5%, a sodium alginate mass percentage concentration of 2.5%, a sodium silicate mass percentage concentration of 1.5%, and a carboxylated graphene mass percentage concentration of 8.5%, standing and defoaming, and placing in a sterile container for standby use;

[0042] c) preparing a calcium chloride aqueous solution having a calcium chloride mass percentage concentration of 6%;

[0043] d) uniformly coating the aqueous mixture solution obtained in step b) on the bare carbon electrode with amino groups introduced on the surface obtained in step a), and combining the sodium alginate molecules and the carboxylated graphene on the surface of the bare carbon electrode by utilizing the interaction between the amino groups and the carboxyl groups on the sodium alginate and the carboxylated graphene; immediately immersing the bare carbon electrode coated with the aqueous mixture solution obtained in step b) in the calcium chloride aqueous solution obtained in step c) for 16 hours to obtain a hybrid hydrogel modified electrode containing lysozyme; calcium chloride cross-links sodium silicate to generate uniformly dispersed calcium silicate nanoparticles in the hybrid hydrogel;

[0044] e) washing the lysozyme from the hybrid hydrogel modified electrode containing lysozyme obtained in step d) with a phosphate buffer solution, then immersing it in a silver nitrate aqueous solution to adsorb silver ions, and then reducing it with a sodium borohydride aqueous solution to obtain a molecularly imprinted hybrid hydrogel modified electrode that can recognize lysozyme and contains silver nanoparticles on its surface; assembling the modified electrode as a working electrode, a silver electrode as a reference electrode, and a platinum sheet as an auxiliary electrode to form an electrochemical sensor that can rapidly detect and recognize lysozyme; combining a probe of a Raman spectrometer with the molecularly imprinted hybrid hydrogel modified electrode that can recognize lysozyme to form an optical sensor that can rapidly detect and recognize lysozyme;

[0045] f) placing the molecular imprinting hybrid hydrogel modified electrode capable of identifying lysozyme obtained in step e) in an aqueous solution of lysozyme to adsorb the lysozyme and then taking it out, and detecting the adsorbed lysozyme using a Raman spectrometer. Since the silver nanoparticles in the hybrid hydrogel obtained in step e) can serve as active hot spots of the enhanced substrate, the detection sensitivity of lysozyme is greatly improved. The molecular imprinting hybrid hydrogel modified electrode capable of identifying lysozyme prepared by the present invention can be used for optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy at the same time, thereby greatly improving the detection limit, reliability and detection efficiency. The lysozyme molecular imprinting optical and electrochemical sensors have good application prospects in the field of lysozyme analysis and detection.

Claims

1. A method for preparing a protein molecular imprinting optical and electrochemical sensor, characterized in that The following steps are involved: a) immersing the cleaned bare carbon electrode in a 0.01%-2% by mass 3-aminopropyltriethoxysilane aqueous solution for 2-24 hours to introduce amino groups on the surface of the bare carbon electrode; b) preparing a mixture aqueous solution with a protein mass percentage concentration of 0.01%-5%, a sodium alginate mass percentage concentration of 0.1%-5%, a sodium silicate mass percentage concentration of 0.01%-2%, and a carboxylated graphene mass percentage concentration of 0.01%-10%, standing to defoam and placing in a sterile container for standby use; c) preparing a calcium chloride aqueous solution having a calcium chloride mass percentage concentration of 0.1% to 10%; d) uniformly coating the aqueous mixture solution obtained in step b) on the bare carbon electrode with amino groups introduced on the surface obtained in step a), and combining the sodium alginate molecules and the carboxylated graphene on the surface of the bare carbon electrode by utilizing the interaction between the amino groups and the carboxyl groups on the sodium alginate and the carboxylated graphene; immediately immersing the bare carbon electrode coated with the aqueous mixture solution obtained in step b) in the calcium chloride aqueous solution obtained in step c) for 0.5-24 hours to obtain a protein-containing hybrid hydrogel modified electrode; calcium chloride cross-links sodium silicate to generate uniformly dispersed calcium silicate nanoparticles in the hybrid hydrogel; e) washing the protein from the protein-containing hybrid hydrogel modified electrode obtained in step d) with an eluent, then immersing it in a silver nitrate aqueous solution to adsorb silver ions, and then reducing it with a sodium borohydride aqueous solution to obtain a molecularly imprinted hybrid hydrogel modified electrode containing silver nanoparticles on the surface that can recognize proteins; assembling the modified electrode as a working electrode, a silver electrode as a reference electrode, and a platinum sheet as an auxiliary electrode to form an electrochemical sensor that can quickly detect and recognize proteins; The probe of the Raman spectrometer is combined with a molecularly imprinted hybrid hydrogel modified electrode that can recognize proteins to form an optical sensor that can quickly detect and recognize proteins; f) placing the protein-recognizable molecular imprinting hybrid hydrogel modified electrode obtained in step e) in a protein aqueous solution to adsorb the protein and then taking it out, and detecting the adsorbed protein using a Raman spectrometer. Since the silver nanoparticles in the hybrid hydrogel obtained in step e) can serve as active hot spots for enhancing the substrate, the protein detection sensitivity is greatly improved. The prepared protein-recognizable molecular imprinting hybrid hydrogel modified electrode can be used for both optical sensors and electrochemical sensors of surface enhanced Raman spectroscopy, thereby greatly improving the detection limit, reliability and detection efficiency. The protein molecular imprinting optical and electrochemical sensors have good application prospects in the field of protein analysis and detection. g) No toxic or harmful reagents are introduced during the preparation process, there is no polymerization reaction heat to cause changes in protein conformation, the protein elution process is gentle, and the obtained protein-recognizable molecular imprinting hydrogel modified electrode has good biocompatibility and high recognition performance.

2. A method for preparing a protein molecular imprinting optical and electrochemical sensor as claimed in claim 1, characterized in that The protein is any one of bovine serum albumin, ovalbumin, catalase and lysozyme or a mixture of two or more thereof.

3. The method for preparing a protein molecular imprinting optical and electrochemical sensor as claimed in claim 1, characterized in that The eluent is any one of a phosphate buffer solution, a Tris-HCl buffer solution, and an acetate buffer solution.