A method for preparing a silver ion catalyzed protein imprinting optical and electrochemical sensor

Abstract

The present invention provides a method for preparing a silver ion-catalyzed protein imprinting optical and electrochemical sensor. First, a mixed aqueous solution of template protein, functional monomer, cross-linking agent and sodium alginate is coated on a bare carbon electrode into which amino groups are introduced. The electrode modified with protein molecular imprinting hydrogel is prepared by silver ion-catalyzed free radical polymerization reaction initiated by ammonium persulfate. Subsequently, the silver ions in the hydrogel are reduced to obtain silver nanoparticles uniformly dispersed in the hydrogel. The silver nanoparticles serve as an enhanced substrate to greatly improve the detection sensitivity of proteins. A Raman spectroscopy probe is combined with an electrode modified with protein molecular imprinting hydrogel to form an optical sensor that can quickly detect and identify proteins; the electrode modified with protein molecular imprinting hydrogel prepared by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection, and has good application prospects.

Description

Technical Field

[0001] The invention relates to a method for preparing a silver ion catalyzed protein 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, electrochemistry, etc. Specifically, it relates to a preparation method and application of a silver ion catalyzed protein 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 and microbial sensors. Electrochemical biosensors are selective and specific, and are ideal analytical tools for quantitative or semi-quantitative detection of substances. They have been widely used in many fields such as environmental monitoring, human health assessment, biomedical research, fermentation industry production, food safety, etc., which has greatly promoted the development and progress of these fields. Molecular imprinting electrochemical sensor is an advanced technology that combines molecular imprinting technology with analytical sensor technology. It combines the advantages of molecular imprinting, avoids the shortcomings of traditional sensors, improves the selectivity and sensitivity of electrochemical sensors, and shortens the response time. It is more popular in more and more fields because of its simple design, economical and practical advantages. The principle of molecular imprinting technology is similar to the specific binding between antibodies and antigens. Although molecular imprinting technology has developed rapidly, most templates are small molecules, and macromolecular templates (such as proteins) are relatively rare. The main reason is the complexity of protein structure, high flexibility of conformation and diversity of protein sequence. Zhang et al. prepared a new molecular imprinting membrane with specific recognition sites for the separation of paclitaxel. For the separation of chiral substances, the advantages of MIP are more obvious. Li et al. proposed a new method, namely, controllable surface imprinting of boronate affinity anchor epitopes, which can control the thickness of the imprinted layer by adjusting the imprinting time and produce good binding properties for specific recognition of proteins and peptides.

[0004] Hydrogels have many properties such as high water content, high elasticity, soft wettability, molecular diffusivity, etc., and have broad application prospects. Molecular imprinting polymers based on hydrogels are a kind of biomimetic materials formed by non-covalent or covalent bonds in the presence of template molecules, functional monomers, and cross-linking agents. They are widely used in biosensing, molecular recognition, medical diagnosis, environmental analysis and other fields because of their specific recognition function. Due to the limitations of the polymerization conditions of functional monomers, only a few examples involve macromolecular proteins with biological activity, most of which are the detection of microorganisms and small organic compounds in complex environments. In order to overcome the traditional method of initiating the polymerization of functional monomers through ultraviolet light or initiators at higher temperatures, which easily leads to protein denaturation and long polymerization time, the stability of the template protein structure is ensured.

[0005] Surface enhanced Raman scattering technology is widely used in chemical and biological analysis due to its advantages such as ultra-high sensitivity, narrow spectrum, rapidity and non-destructive detection. In recent years, the combination of molecular imprinting technology and surface enhanced Raman scattering technology has produced a series of advanced protein detection methods, showing unique advantages and receiving widespread attention.

[0006] The present invention provides a method for preparing a silver ion-catalyzed protein imprinting optical and electrochemical sensor. First, a mixed aqueous solution of template protein, functional monomer, cross-linking agent and sodium alginate is coated on a bare carbon electrode into which amino groups are introduced. The electrode modified with protein molecular imprinting hydrogel is prepared by silver ion-catalyzed free radical polymerization reaction initiated by ammonium persulfate. Subsequently, the silver ions in the hydrogel are reduced to obtain silver nanoparticles uniformly dispersed in the hydrogel. The silver nanoparticles serve as an enhanced substrate to greatly improve the detection sensitivity of proteins. A Raman spectroscopy probe is combined with an electrode modified with protein molecular imprinting hydrogel to form an optical sensor that can quickly detect and identify proteins; the electrode modified with protein molecular imprinting hydrogel prepared by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection, and has good application prospects. 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 technical solution of the present invention to solve 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 is to provide a preparation method of silver ion catalyzed protein imprinting optical and electrochemical sensor.

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

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

[0011] b) preparing a mixture aqueous solution A having a protein mass percentage concentration of 0.01%-5%, a sodium alginate mass percentage concentration of 0.1%-5%, an acrylamide mass percentage concentration of 0.5%-10%, an ammonium persulfate mass percentage concentration of 0.01%-1%, and a carboxylated graphene mass percentage concentration of 0.01%-1%, standing to defoam, and placing in a sterile container for standby use; preparing a mixture aqueous solution B having a N,N-methylenebisacrylamide mass percentage concentration of 0.01%-0.5% and a silver nitrate mass percentage concentration of 0.01%-0.5%; preparing a calcium nitrate aqueous solution having a calcium nitrate mass percentage concentration of 0.1%-5%;

[0012] c) quickly mixing the mixture aqueous solution A obtained in step b) with the mixture aqueous solution B, coating the mixed aqueous solution on the bare carbon electrode with amino groups introduced on the surface obtained in step a), then putting the coated bare carbon electrode into a plastic bag and passing nitrogen to exclude air, silver ions catalyzing free radical polymerization reaction, regulating the time of initiation of polymerization by regulating the concentration of silver ions, obtaining a chemically cross-linked hydrogel on the surface of the bare carbon electrode, immersing the bare carbon electrode with the chemically cross-linked hydrogel on the surface into the calcium nitrate aqueous solution obtained in step b), calcium ions cross-linking sodium alginate and carboxylated graphene, obtaining a chemically and ionically double cross-linked hydrogel on the surface of the bare carbon electrode; then reducing the silver ions in the hydrogel with sodium borohydride, and eluting the protein with an eluent, A molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying proteins is obtained; after the electrode is placed in a protein aqueous solution to adsorb the protein, the adsorbed protein is detected using a Raman spectroscopy probe; since the hydrogel contains silver nanoparticles as an enhanced substrate, the detection sensitivity of the protein is greatly improved; the Raman spectroscopy probe is combined with the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying proteins to obtain an optical sensor capable of rapidly detecting and identifying proteins; at the same time, the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying proteins is used as a working electrode, a silver electrode is used as a reference electrode, and a platinum sheet is used as an auxiliary electrode to assemble an electrochemical sensor capable of rapidly detecting and identifying proteins;

[0013] d) The silver ion catalyzed protein molecular imprinting optical and electrochemical sensor obtained by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection. The protein molecular imprinting optical and electrochemical sensor has good application prospects in the field of protein analysis and detection.

[0014] The protein described in the present invention is any one of bovine serum albumin, ovalbumin, adhesion protein, transforming growth factor, and lysozyme, or a mixture of two or more thereof; the enhancer described is any one of graphene oxide, carboxylated multi-walled carbon nanotubes, carboxymethyl chitosan, and carboxylated titanium dioxide, or a mixture of two or more thereof; and the eluent described is any one of phosphate buffer solution, Tris-HCl buffer solution, acetate buffer solution, and borate buffer solution. DETAILED DESCRIPTION

[0015] Specific embodiments of the present invention are described below, but the present invention is not limited to the embodiments.

[0016] Example 1.

[0017] a) immersing the cleaned bare carbon electrode in a 0.01% by mass 3-aminopropyltriethoxysilane aqueous solution to introduce amino groups on the surface of the bare carbon electrode;

[0018] b) preparing a mixture aqueous solution A 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 0.5% acrylamide, a mass percentage concentration of 0.01% ammonium persulfate, and a mass percentage concentration of 0.01% carboxylated graphene, standing and defoaming, and placing the mixture in a sterile container for standby use; preparing a mixture aqueous solution B with a mass percentage concentration of 0.01% N,N-methylenebisacrylamide and a mass percentage concentration of 0.01% silver nitrate; preparing a calcium nitrate aqueous solution with a mass percentage concentration of 0.1% calcium nitrate;

[0019] c) quickly mixing the mixture aqueous solution A obtained in step b) with the mixture aqueous solution B, coating the mixed aqueous solution on the bare carbon electrode with amino groups introduced on the surface obtained in step a), then putting the coated bare carbon electrode into a plastic bag and passing nitrogen to exclude air, silver ions catalyzing free radical polymerization reaction, regulating the time of initiating polymerization by regulating the concentration of silver ions, obtaining a chemically cross-linked hydrogel on the surface of the bare carbon electrode, immersing the bare carbon electrode with the chemically cross-linked hydrogel on the surface into the calcium nitrate aqueous solution obtained in step b), calcium ions cross-linking sodium alginate and carboxylated graphene, obtaining a chemically and ionically double cross-linked hydrogel on the surface of the bare carbon electrode; subsequently reducing the silver ions in the hydrogel with sodium borohydride, eluting the bovine serum albumin with a phosphate buffer solution, and obtaining a silver-containing nanoparticle. The invention discloses a molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying bovine serum albumin; after placing the electrode in an aqueous solution of bovine serum albumin to adsorb the bovine serum albumin, taking it out and detecting the adsorbed bovine serum albumin with a Raman spectroscopy probe; since the hydrogel contains silver nanoparticles as an enhanced substrate, the detection sensitivity of the bovine serum albumin is greatly improved; the Raman spectroscopy probe is combined with the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying bovine serum albumin to obtain an optical sensor capable of rapidly detecting and identifying bovine serum albumin; at the same time, the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying bovine serum albumin is used as a working electrode, a silver electrode is used as a reference electrode, and a platinum sheet is used as an auxiliary electrode to assemble an electrochemical sensor capable of rapidly detecting and identifying bovine serum albumin;

[0020] d) The silver ion-catalyzed bovine serum albumin molecular imprinting optical and electrochemical sensor obtained by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection. The bovine serum albumin molecular imprinting optical and electrochemical sensor has good application prospects in the field of bovine serum albumin analysis and detection.

[0021] Example 2.

[0022] a) immersing the cleaned bare carbon electrode in a 2% by mass aqueous solution of 3-aminopropyltriethoxysilane to introduce amino groups on the surface of the bare carbon electrode;

[0023] b) preparing an aqueous solution A of a mixture of 5% egg white protein, 5% sodium alginate, 10% acrylamide, 1% ammonium persulfate, and 1% carboxylated graphene, standing to defoam, and placing in a sterile container for later use; preparing an aqueous solution B of a mixture of 0.5% N,N-methylenebisacrylamide and 0.5% silver nitrate; preparing an aqueous solution of calcium nitrate of 5% by mass;

[0024] c) quickly mixing the mixture aqueous solution A obtained in step b) with the mixture aqueous solution B, coating the mixed aqueous solution on the bare carbon electrode with amino groups introduced on the surface obtained in step a), then placing the coated bare carbon electrode in a plastic bag and passing nitrogen to remove air, silver ions catalyzing free radical polymerization, regulating the time of initiation of polymerization by regulating the concentration of silver ions, obtaining a chemically cross-linked hydrogel on the surface of the bare carbon electrode, immersing the bare carbon electrode with the chemically cross-linked hydrogel on the surface into the calcium nitrate aqueous solution obtained in step b), calcium ions cross-linking sodium alginate and carboxylated graphene, obtaining a chemically and ionically double cross-linked hydrogel on the surface of the bare carbon electrode; then reducing the silver ions in the hydrogel with sodium borohydride, and eluting the ovalbumin with a Tris-HCl buffer solution. , a molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles capable of identifying ovalbumin is obtained; after placing the electrode in an aqueous solution of ovalbumin to adsorb the ovalbumin, the electrode is taken out and the adsorbed ovalbumin is detected using a Raman spectroscopy probe; since the hydrogel contains silver nanoparticles as an enhanced substrate, the detection sensitivity of ovalbumin is greatly improved; the Raman spectroscopy probe is combined with the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles capable of identifying ovalbumin to obtain an optical sensor capable of rapidly detecting and identifying ovalbumin; at the same time, the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles capable of identifying ovalbumin is used as a working electrode, a silver electrode is used as a reference electrode, and a platinum sheet is used as an auxiliary electrode to assemble an electrochemical sensor capable of rapidly detecting and identifying ovalbumin;

[0025] d) The silver ion-catalyzed ovalbumin molecular imprinting optical and electrochemical sensor obtained by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection. The ovalbumin molecular imprinting optical and electrochemical sensor has good application prospects in the field of ovalbumin analysis and detection.

[0026] Example 3.

[0027] a) immersing the cleaned bare carbon electrode in a 0.5% by mass 3-aminopropyltriethoxysilane aqueous solution to introduce amino groups on the surface of the bare carbon electrode;

[0028] b) preparing a mixture aqueous solution A with a concentration of 1.5% by mass percentage of adhesion protein, 1% by mass percentage of sodium alginate, 2% by mass percentage of acrylamide, 0.2% by mass percentage of ammonium persulfate, and 0.1% by mass percentage of carboxylated graphene, standing to defoam and placing in a sterile container for later use; preparing a mixture aqueous solution B with a concentration of 0.1% by mass percentage of N,N-methylenebisacrylamide and 0.15% by mass percentage of silver nitrate; preparing a calcium nitrate aqueous solution with a concentration of 0.3% by mass percentage of calcium nitrate;

[0029] c) quickly mixing the mixture aqueous solution A obtained in step b) with the mixture aqueous solution B, coating the mixed aqueous solution on the bare carbon electrode with amino groups introduced on the surface obtained in step a), then placing the coated bare carbon electrode in a plastic bag and passing nitrogen to exclude air, silver ions catalyzing free radical polymerization, regulating the time of initiating polymerization by regulating the concentration of silver ions, obtaining a chemically cross-linked hydrogel on the surface of the bare carbon electrode, immersing the bare carbon electrode with the chemically cross-linked hydrogel on the surface into the calcium nitrate aqueous solution obtained in step b), calcium ions cross-linking sodium alginate and carboxylated graphene, obtaining a chemically and ionically double cross-linked hydrogel on the surface of the bare carbon electrode; then reducing the silver ions in the hydrogel with sodium borohydride, and eluting the adhesion protein with an acetate buffer solution to obtain A molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles that can identify adhesion protein; after placing the electrode in an aqueous solution of adhesion protein to adsorb adhesion protein, taking it out and detecting the adsorbed adhesion protein with a Raman spectroscopy probe; since the hydrogel contains silver nanoparticles as an enhanced substrate, the detection sensitivity of adhesion protein is greatly improved; the Raman spectroscopy probe is combined with the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles that can identify adhesion protein to obtain an optical sensor that can quickly detect and identify adhesion protein; at the same time, the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles that can identify adhesion protein is used as a working electrode, a silver electrode is used as a reference electrode, and a platinum sheet is used as an auxiliary electrode to assemble an electrochemical sensor that can quickly detect and identify adhesion protein;

[0030] d) The silver ion-catalyzed adhesion protein molecular imprinting optical and electrochemical sensor obtained by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection. The adhesion protein molecular imprinting optical and electrochemical sensor has good application prospects in the field of adhesion protein analysis and detection.

[0031] Example 4.

[0032] a) immersing the cleaned bare carbon electrode in a 1.5% by mass 3-aminopropyltriethoxysilane aqueous solution to introduce amino groups on the surface of the bare carbon electrode;

[0033] b) preparing a mixture aqueous solution A with a mass percentage concentration of 4.5% transforming growth factor, a mass percentage concentration of 3.5% sodium alginate, a mass percentage concentration of 7% acrylamide, a mass percentage concentration of 0.75% ammonium persulfate, and a mass percentage concentration of 0.6% carboxylated graphene, standing to defoam and placing in a sterile container for standby use; preparing a mixture aqueous solution B with a mass percentage concentration of 0.35% N,N-methylenebisacrylamide and a mass percentage concentration of 0.25% silver nitrate; preparing a calcium nitrate aqueous solution with a mass percentage concentration of 3.5% calcium nitrate;

[0034] c) quickly mixing the mixture aqueous solution A obtained in step b) with the mixture aqueous solution B, coating the mixed aqueous solution on the bare carbon electrode with amino groups introduced on the surface obtained in step a), then placing the coated bare carbon electrode in a plastic bag and passing nitrogen to remove air, silver ions catalyzing free radical polymerization, regulating the time of initiating polymerization by regulating the concentration of silver ions, obtaining a chemically cross-linked hydrogel on the surface of the bare carbon electrode, immersing the bare carbon electrode with the chemically cross-linked hydrogel on the surface into the calcium nitrate aqueous solution obtained in step b), calcium ions cross-linking sodium alginate and carboxylated graphene, obtaining a chemically and ionically double cross-linked hydrogel on the surface of the bare carbon electrode; subsequently reducing the silver ions in the hydrogel with sodium borohydride, and eluting the transforming growth factor with a borate buffer solution to obtain silver-containing nanoparticles. A molecular imprinting hybrid hydrogel modified electrode capable of identifying transforming growth factor; placing the electrode in an aqueous solution of transforming growth factor to adsorb transforming growth factor, taking it out and detecting the adsorbed transforming growth factor with a Raman spectroscopy probe; since the hydrogel contains silver nanoparticles as an enhanced substrate, the detection sensitivity of the protein is greatly improved; the Raman spectroscopy probe is combined with the molecular imprinting hybrid hydrogel modified electrode capable of identifying transforming growth factor containing silver nanoparticles to obtain an optical sensor capable of rapidly detecting and identifying transforming growth factor; at the same time, the molecular imprinting hybrid hydrogel modified electrode capable of identifying transforming growth factor containing silver nanoparticles is used as a working electrode, the silver electrode is used as a reference electrode, and the platinum sheet is used as an auxiliary electrode to assemble an electrochemical sensor capable of rapidly detecting and identifying transforming growth factor;

[0035] d) The silver ion-catalyzed transforming growth factor molecular imprinting optical and electrochemical sensor obtained by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection. The transforming growth factor molecular imprinting optical and electrochemical sensor has good application prospects in the field of transforming growth factor analysis and detection.

Claims

1. A method for preparing a silver ion catalyzed protein imprinting optical and electrochemical sensor, characterized in that The following steps are involved: a) treating the cleaned bare carbon electrode with a 0.01%-2% by mass 3-aminopropyltriethoxysilane aqueous solution to introduce amino groups on the surface of the bare carbon electrode; b) preparing a mixture aqueous solution A having a protein mass percentage concentration of 0.01%-5%, a sodium alginate mass percentage concentration of 0.1%-5%, an acrylamide mass percentage concentration of 0.5%-10%, an ammonium persulfate mass percentage concentration of 0.01%-1%, and a carboxylated graphene mass percentage concentration of 0.01%-1%, standing to defoam, and placing in a sterile container for standby use; preparing a mixture aqueous solution B having a N,N-methylenebisacrylamide mass percentage concentration of 0.01%-0.5% and a silver nitrate mass percentage concentration of 0.01%-0.5%; preparing a calcium nitrate aqueous solution having a calcium nitrate mass percentage concentration of 0.1%-5%; c) quickly mixing the mixture aqueous solution A obtained in step b) with the mixture aqueous solution B, coating the mixed aqueous solution on the bare carbon electrode with amino groups introduced on the surface obtained in step a), then putting the coated bare carbon electrode into a plastic bag and passing nitrogen to exclude air, silver ions catalyzing free radical polymerization reaction, regulating the time of initiation of polymerization by regulating the concentration of silver ions, obtaining a chemically cross-linked hydrogel on the surface of the bare carbon electrode, immersing the bare carbon electrode with the chemically cross-linked hydrogel on the surface into the calcium nitrate aqueous solution obtained in step b), calcium ions cross-linking sodium alginate and carboxylated graphene, obtaining a chemically and ionically double cross-linked hydrogel on the surface of the bare carbon electrode; then reducing the silver ions in the hydrogel with sodium borohydride, and eluting the protein with an eluent, A molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying proteins is obtained; after the electrode is placed in a protein aqueous solution to adsorb the protein, the adsorbed protein is detected using a Raman spectroscopy probe; since the hydrogel contains silver nanoparticles as an enhanced substrate, the detection sensitivity of the protein is greatly improved; the Raman spectroscopy probe is combined with the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying proteins to obtain an optical sensor capable of rapidly detecting and identifying proteins; at the same time, the molecular imprinting hybrid hydrogel modified electrode containing silver nanoparticles and capable of identifying proteins is used as a working electrode, a silver electrode is used as a reference electrode, and a platinum sheet is used as an auxiliary electrode to assemble an electrochemical sensor capable of rapidly detecting and identifying proteins; d) The silver ion catalyzed protein molecular imprinting optical and electrochemical sensor obtained by the present invention can be used for both surface enhanced Raman spectroscopy optical sensors and electrochemical sensors, thereby greatly improving the efficiency and reliability of detection. The protein molecular imprinting optical and electrochemical sensor has good application prospects in the field of protein analysis and detection.

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

3. The method for preparing a silver ion catalyzed protein 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.