An ethanol biosensor electrode and its preparation method and application

Abstract

The present invention discloses an ethanol biosensor electrode and a preparation method and application thereof. In the present application, the preparation method of the ethanol biosensor electrode comprises the following steps: S1 preparation of a conductive substrate modified by a three-dimensional gold nanoarray; S2 preparation of a conductive substrate modified by a Prussian blue / three-dimensional gold nanoflower array; S4 preparation of an ethanol biosensor electrode. The ethanol biosensor electrode prepared in the present application can perform in-situ detection, and has good timeliness and strong anti-interference ability.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation process detection and relates to an ethanol biosensor electrode and a preparation method and application thereof. Background Art

[0002] Ethanol is an important industrial raw material, widely used in food, medicine and chemical production and other fields. At the same time, ethanol has been gradually developed as a clean fuel in recent years; fermentation is the main method for producing ethanol, but the domestic fermentation production of ethanol generally adopts an "extensive" production mode, the understanding of the fermentation process is in a "black box" state, and the production depends on the experience of the operator. In fact, the ethanol fermentation process is a typical product-inhibited fermentation reaction, so real-time and full-concentration analysis of ethanol in the reaction process can accurately control the metabolism of microorganisms. Therefore, ethanol concentration is an important process parameter in the fermentation process, which directly affects the control and optimization of the fermentation process and the quality and yield of the fermentation product. In addition, in the prior art, commercial ethanol detection technology, commonly used ethanol detection methods include gas chromatography, high performance liquid chromatography, refractometry, near infrared spectroscopy, ultraviolet-spectrophotometry, etc. When the above-mentioned ethanol detection method is implemented, the test liquid usually needs to be pre-treated, and the detection personnel also have high requirements for operation skills. In addition, the detection equipment usually used is also relatively expensive, and the detection process is also time-consuming, which cannot meet the direct detection needs of the fermentation liquid. In order to effectively optimize and control the fermentation process, it is urgently necessary to detect the ethanol concentration parameters in real time. The development of new electrochemical biosensors has also become one of the key technologies for online ethanol detection that researchers have focused on. However, since the detection system of biomass is often complex in composition and contains many interfering substances, it is easy to cause sensor failure or inaccuracy. Therefore, there are few biosensors suitable for detecting ethanol in biomass. Moreover, the core of the biosensor is the sensing electrode. Therefore, it is particularly important to develop a sensing electrode that can perform in-situ detection, has good timeliness and strong anti-interference ability. Summary of the invention

[0003] Aiming at the problems of poor timeliness, poor anti-interference ability, non-in-situ detection, etc. in the current diagnosis process of ethanol in biomass, the present invention proposes a novel biosensor for rapid ethanol detection, a preparation method thereof, and an application thereof.

[0004] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0005] A method for preparing an ethanol biosensor electrode comprises the following steps:

[0006] S1 Preparation of conductive substrate modified with three-dimensional gold nanoarrays:

[0007] 1-1) preparing an acidic electrodeposition solution from chloroauric acid, a complexing agent and a solvent;

[0008] 1-2) immersing the conductive substrate fixed on the three-electrode system in an electrodeposition solution to perform constant potential electrodeposition, and then taking out the conductive substrate and rinsing it, and drying it at a constant temperature to obtain a conductive substrate modified with a three-dimensional gold nanoarray;

[0009] Preparation of S2 Prussian blue / 3D gold nanoflower array modified conductive substrate:

[0010] 2-1) preparing a Prussian blue reaction solution from a transition metal salt solution, a surfactant and a transition metal potassium cyanide reaction solution;

[0011] 2-2) immersing the conductive substrate modified with the three-dimensional gold nanoflower array fixed on the three-electrode system in a Prussian blue reaction solution, and then performing cyclic voltammetry electrodeposition, and then taking out the conductive substrate, rinsing it, and drying it at a constant temperature to obtain a conductive substrate modified with Prussian blue / three-dimensional gold nanoflower array;

[0012] S3 Preparation of conductive substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modification:

[0013] The conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array is immersed in L-cysteine ​​solution, and then taken out and dried after a period of time to obtain a conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine;

[0014] Preparation of S4 ethanol biosensor electrode:

[0015] The ethanol oxidase solution is fixed on a conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​by cross-linking or embedding, and then dried at low temperature to obtain a conductive substrate with ethanol oxidase on the surface, namely, an ethanol biosensor electrode.

[0016] Preferably, in step 1-1), the concentration of chloroauric acid in the electrodeposition solution is 5-20 mM; the concentration of the complexing agent is 50-500 mM; and the pH value of the electrodeposition solution is 1-5.

[0017] Preferably, in step 1-1), the complexing agent is one of ammonium chloride, perchloric acid and sulfuric acid.

[0018] Preferably, in step 1-2), the conductive substrate is any one of conductive glass, gold sheet and carbon cloth.

[0019] Preferably, in step 1-2), the conductive substrate is first placed in acetone, 10% sodium hydroxide, ethanol and distilled water in sequence for ultrasonic cleaning before being fixed on the three-electrode system.

[0020] Preferably, in step 1-2), the three-electrode system is a working electrode / reference Ag|AgCl electrode / counter electrode system; during the constant potential deposition process, the constant potential voltage is one of -0.3V, -0.1V, 0.1V and 0.3V, and the electrodeposition time is 2-15min.

[0021] Preferably, in step 1-2), the conductive substrate is rinsed with deionized water and dried at a temperature of 20-50°C.

[0022] Preferably, in step 2-1), the transition metal salt solution and the surfactant are first mixed evenly to obtain a mixed solution, and then the mixed solution and the transition metal potassium cyanide reaction solution are mixed evenly to obtain a Prussian blue reaction solution.

[0023] Preferably, in step 2-1), the transition metal salt is any one of FeCl3, MnCl2 and NiCl2; in the mixed solution, the molar concentration of the transition metal salt is 5-25 mM;

[0024] Preferably, in step 2-1), the surfactant is sodium citrate or pvp; in the mixed solution, the molar concentration of the surfactant is 1-30 mM.

[0025] Preferably, in step 2-1), the transition metal potassium cyanide reaction solution is prepared by uniformly mixing transition metal potassium cyanide and an inorganic acid.

[0026] Preferably, in step 2-1), the transition metal potassium cyanide is potassium ferrocyanide or potassium cobalt cyanide; the inorganic acid is any one of hydrochloric acid, sulfuric acid and nitric acid; in the transition metal potassium cyanide reaction solution, the molar concentration of the transition metal potassium cyanide is 5-25mM; the volume ratio of the mixed solution to the transition metal potassium cyanide reaction solution is 1:(1-3).

[0027] Preferably, the three-electrode system in step 2-2) is the same as the three-electrode system in step 1-2); during the cyclic voltammetry electrodeposition process, the electrodeposition potential range is 0-0.6V, and the deposition times are 20-30 times.

[0028] Preferably, in step 2-2), the conductive substrate is rinsed with deionized water and dried at a constant temperature of 20-50° C. for at least 0.5 h.

[0029] Preferably, in step S3, the molar concentration of the L-cysteine ​​solution is 0.1-0.4 M, and the conductive substrate modified with the Prussian blue / three-dimensional gold nanoflower array is immersed in the L-cysteine ​​solution for at least 0.5 h.

[0030] Preferably, the Prussian blue / three-dimensional gold nanoflower array modified conductive substrate prepared in step S3 is stored at a low temperature of -5-10°C.

[0031] Preferably, in step S4), the step of fixing the ethanol oxidase solution on the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​obtained in step S3 by cross-linking is as follows: using PBS buffer as a solvent to prepare an ethanol oxidase solution, and then adding glutaraldehyde aqueous solution to obtain a cross-linking solution; then drop-coating the cross-linking solution on the surface of the conductive glass modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine, and drying at low temperature to obtain a conductive substrate with ethanol oxidase on the surface, namely, an ethanol biosensor electrode.

[0032] Preferably, in step S4), the mass percentage of the glutaraldehyde aqueous solution is 25%, the concentration of the ethanol oxidase solution is 0.2-4.0U / μL, the volume ratio of the ethanol oxidase solution to the glutaraldehyde aqueous solution is 100:(0.5-1), and the amount of the cross-linking solution drop-coated on the surface of the Prussian blue / three-dimensional gold nanoflower array / L-cysteine-modified conductive glass is 0.5-10 μL.

[0033] Preferably, the step of fixing the ethanol oxidase solution on the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​obtained in step S3 by cross-linking method is as follows: preparing a solution containing EDC·HCl and NHS, and then immersing the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​in the solution containing EDC·HCl and NHS, taking it out after a period of time to obtain a pretreated modified electrode, and storing it at low temperature; then using PBS buffer as a solvent to prepare an ethanol oxidase solution, and then taking the ethanol oxidase solution and dropping it on the surface of the pretreated modified electrode, and drying it at low temperature; then dropping a Nafion membrane solution, and drying it at low temperature to obtain a conductive substrate with ethanol oxidase on the surface, i.e., an ethanol biosensor electrode.

[0034] Preferably, in step S4), in the solution containing EDC·HCl and NHS, the molar concentration of EDC·HCl is 5-20mM, and the molar concentration of NHS is 10-30mM; the storage temperature and the low-temperature drying temperature are both -5-10°C; the concentration of ethanol oxidase in the ethanol oxidase solution is 0.2-4.0U / μL; the volume ratio concentration of the Nafion membrane solution is 5%; and the low-temperature drying time is 3-12h.

[0035] Preferably, in step S4), the amount of the Nafion membrane solution added dropwise to the surface of the Prussian blue / three-dimensional gold nanoflower array / L-cysteine-modified conductive glass is 2-6 μL.

[0036] Preferably, in step S4), the step of fixing the ethanol oxidase solution on the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​obtained in step S3 by an embedding method is as follows: dissolving chitosan and acetic acid in deionized water, mixing evenly, and obtaining a chitosan fixing solution; then using PBS buffer as a solvent to prepare an ethanol oxidase solution; then mixing the chitosan fixing solution and the ethanol oxidase solution in a certain volume ratio to obtain an enzyme fixing solution; and then drop-coating the enzyme fixing solution on the surface of the gold sheet substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine.

[0037] Preferably, the solid-liquid ratio of chitosan and acetic acid is 1:(1-3) g / mL, the volume ratio of acetic acid to deionized water is 1:(100-200), the molar concentration of the ethanol oxidase solution is 0.2-4.0U / μL, and the volume ratio of the chitosan immobilization solution to the ethanol oxidase solution is 1:1; 5-15 μL of the enzyme immobilization solution is drop-coated on the surface of a gold sheet substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modified.

[0038] The invention discloses an ethanol biosensor electrode prepared by utilizing the preparation method of the ethanol biosensor electrode.

[0039] The invention discloses an application of an ethanol biosensor electrode in ethanol biosensor.

[0040] The invention discloses an application of an ethanol biosensor in the rapid detection of ethanol in a fermentation process.

[0041] Compared with the prior art, the advantages and positive effects of the present invention are:

[0042] The ethanol biosensor electrode based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modification prepared by the present invention has a three-dimensional gold nanoflower array structure. Compared with ordinary electrodeposited gold particles, on the same substrate, the present application has a three-dimensional structure through electrodeposition, and the three-dimensional gold nanoflower array structure has a larger specific surface area. The larger specific surface area of ​​the three-dimensional gold nanoflower array structure can provide more enzyme loading sites, enzyme catalytic sites and more enzyme reaction areas. Since the enzyme has specificity, the larger specific surface area of ​​the three-dimensional gold nanoflower array structure is directly related to the anti-interference and detection speed of ethanol in situ detection; According to experimental tests, the ethanol biosensor prepared in the present application can complete the detection of ethanol in as fast as 13 seconds, and the ethanol biosensor prepared in the present application has relatively strong anti-interference ability. From the results, common components in the fermented biomass, such as sucrose, glucose, fructose, glutamic acid, pyruvic acid, acetaldehyde, etc., have no interference with the ethanol biosensor prepared in the present application. Since the ethanol biosensor prepared in the present application has strong anti-interference ability, its detection accuracy is also high; and since the ethanol biosensor prepared in the present application has strong anti-interference ability, it is suitable for in situ detection of biomass systems and has the advantage of good timeliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is an electron microscope image of the three-dimensional gold nanoarray modification on the gold sheet based on the three-dimensional gold nanoarray modification in Example 2;

[0044] Figure 2 This is a photo of a gold sheet modified based on a three-dimensional gold nanoarray in Example 2;

[0045] Figure 3 This is a linear range test diagram of the ethanol biosensor electrode prepared in Example 1;

[0046] FIG4(A) is a test diagram of the linear range of the ethanol biosensor electrode prepared in Example 2; FIG4(B) is a test diagram of the anti-interference ability of the ethanol biosensor electrode prepared in Example 2;

[0047] Figure 5 This is a linear range test chart of the ethanol biosensor electrode prepared in Example 2. DETAILED DESCRIPTION

[0048] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0049] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0050] Example 1

[0051] This embodiment provides a method for preparing an ethanol biosensor electrode, comprising the following steps:

[0052] S1 Preparation of conductive substrate modified with three-dimensional gold nanoarrays:

[0053] 1-1) 0.068 g of chloroauric acid and 1.07 g of ammonium chloride were added to 40 mL of deionized water, and the mixture was uniformly mixed to obtain an acidic electrodeposition solution, wherein the pH value of the electrodeposition solution was 2.0; wherein the concentration of chloroauric acid in the electrodeposition solution was 5 mM, and the concentration of ammonium chloride was 0.5 M;

[0054] 1-2) The cleaned conductive glass is fixed on the three-electrode system, and then the conductive glass fixed on the three-electrode system is immersed in an electrodeposition solution, and then constant potential deposition is performed, the constant potential voltage is -0.1V, after 10 minutes of electrodeposition, the conductive glass is taken out and rinsed with deionized water, and then dried in an oven at a constant temperature of 30°C for 0.5h to obtain a conductive glass modified with a three-dimensional gold nanoarray; wherein the cleaned conductive glass refers to a conductive glass obtained by placing the conductive glass in acetone, 10% sodium hydroxide, ethanol and distilled water in sequence for ultrasonic cleaning;

[0055] S2) Preparation of Prussian blue / 3D gold nanoflower array modified conductive substrate:

[0056] 2-1) 20 mL of a FeCl3 solution having a density of 0.000812 g / mL was mixed with 0.038 g of sodium citrate to obtain a mixed solution, wherein the molar concentration of FeCl3 in the mixed solution was 5 mM, and the molar concentration of sodium citrate was 7.5 mM, and then the mixed solution was mixed with 20 mL of a transition metal potassium cyanide reaction solution to obtain a Prussian blue reaction solution; wherein the transition metal potassium cyanide reaction solution was prepared in the following manner;

[0057] 0.036 g of potassium ferrocyanide was dissolved in 19.8 ml of deionized water, and then 0.2 ml of 1 mol / L hydrochloric acid solution was added and mixed evenly to obtain a transition metal cyanide potassium solution. The molar concentration of potassium ferrocyanide in the transition metal cyanide potassium solution was 5 mM, and the molar concentration of HCL was 10 mM.

[0058] 2-2) The three-dimensional gold nanoarray-modified conductive glass obtained in step 1-2) fixed on the three-electrode system is immersed in a Prussian blue reaction solution, and then electrodeposited by cyclic voltammetry, the potential range of the electrodeposition is 0-0.6V, the number of electrodepositions is 20 times, and then the conductive glass is taken out and rinsed with deionized water for more than 10s, and then dried in an oven at a constant temperature of 30°C for 1h to obtain a conductive glass modified based on Prussian blue / three-dimensional gold nanoflower arrays;

[0059] S3 Preparation of conductive substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modification:

[0060] The conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array is immersed in a L-cysteine ​​solution with a volume molar concentration of 0.2M, and taken out after immersion for 1 hour, and naturally dried at room temperature; the conductive glass modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​is obtained; the conductive glass modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​is stored in a refrigerator at 4°C;

[0061] Preparation of S4 ethanol biosensor electrode:

[0062] Take 50U of ethanol oxidase (ethanol oxidase was purchased from Sigma Biotechnology Co., Ltd.) and use 100μL of PBS buffer as a solvent to prepare an ethanol oxidase solution with a concentration of 0.5U / μL, and then add 1μL of 25% mass percentage glutaraldehyde aqueous solution to obtain a cross-linking solution; then, take 2μL of cross-linking solution I and drop it on the surface of conductive glass modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine, and dry it at -5°C for 12h to obtain an ethanol biosensor electrode.

[0063] The linear range of the ethanol biosensor electrode prepared in Example 1 was tested using the differential pulse technology of the Shanghai Chenhua CHI660E electrochemical workstation. The voltage variation range was -0.1-0.6 V with an increment of 0.005 V. The ethanol biosensor electrode (working electrode), platinum wire (counter electrode) and Ag / AgCl (reference electrode) prepared in Example 1 were connected to the corresponding electrode wires on the electrochemical workstation.

[0064] The linear range test results of the ethanol biosensor electrode prepared in Example 1 are as follows: Figure 3 As shown. Figure 3 It can be seen that the sensitivity of the ethanol biosensor electrode prepared in Example 1 of the present application to ethanol is 7.31 μA·mM -1 cm -2When the ethanol concentration is greater than or equal to 0.01mM, the ethanol biosensor electrode prepared in Example 1 of the present application will have a more obvious current response, the linear range is 0.05mM-5mM, and the detection time is only 13s. In addition, after completing the standard curve test, the ethanol biosensor electrode prepared in Example 1 of the present application was stored in a PBS buffer solution at 4°C for 30 days, and its response signal was 90% of the initial signal, indicating that the ethanol biosensor electrode has excellent stability.

[0065] Example 2

[0066] The conditions not specifically described in this embodiment and subsequent embodiments are consistent with those in embodiment 1. This embodiment provides a method for preparing an ethanol biosensor electrode, comprising the following steps.

[0067] S1 Preparation of conductive substrate modified with three-dimensional gold nanoarrays:

[0068] 1-1) 0.136 g of chloroauric acid and 0.267 g of 72% perchloric acid were added to 40 mL of deionized water, and the mixture was uniformly mixed to obtain an acidic electrodeposition solution, wherein the pH value of the electrodeposition solution was 2.5; wherein the concentration of the chloroauric acid in the electrodeposition solution was 10 mM, and the concentration of the perchloric acid was 0.05 M;

[0069] 1-2) The cleaned gold substrate is placed on the three-electrode system, and then the gold substrate fixed on the three-electrode system is immersed in the electrodeposition solution, and then constant potential deposition is performed, and the constant potential voltage is -0.3V. After 5 minutes of electrodeposition, the gold substrate is taken out and rinsed with deionized water, and dried in an oven at a constant temperature of 20°C for 0.5 hours to obtain a gold substrate modified with a three-dimensional gold nanoarray. The photo of the gold substrate modified with a three-dimensional gold nanoarray is shown in FIG. Figure 2 As shown, Figure 2 The black position in the figure is the position covered by the three-dimensional gold nanoarray. Figure 2 It can be seen that the area of ​​the gold substrate covered by the three-dimensional gold nanoarray accounts for about 80% of the area of ​​the entire gold substrate; In addition, in the second embodiment, the cleaned gold substrate refers to the gold substrate obtained by placing the gold substrate in acetone, 10% sodium hydroxide, ethanol and distilled water for ultrasonic cleaning in sequence;

[0070] S2) Preparation of Prussian blue / 3D gold nanoflower array modified conductive substrate:

[0071] 2-1) 20 mL of a MnSO4 solution having a density of 0.003 g / mL was mixed with 1.35 g of PVP to obtain a mixed solution, wherein the molar concentration of MnSO4 in the mixed solution was 20 mM, and the molar concentration of PVP was 30 mM, and then the mixed solution was mixed with 20 mL of a transition metal potassium cyanide reaction solution to obtain a Prussian blue reaction solution; wherein the transition metal potassium cyanide reaction solution was prepared in the following manner;

[0072] 0.166 g of potassium cobalt cyanide was dissolved in 19.8 ml of deionized water, and then 0.2 ml of 1 mol / L sulfuric acid solution was added and mixed evenly to obtain a transition metal cyanide potassium cyanide reaction solution. The molar concentration of potassium cobalt cyanide in the transition metal cyanide potassium cyanide reaction solution was 25 mM, and the molar concentration of H2SO4 was 10 mM.

[0073] 2-2) The gold sheet substrate modified with the three-dimensional gold nanoarray obtained in step 1-2) fixed on the three-electrode system is immersed in a Prussian blue reaction solution, and then subjected to cyclic voltammetry electrodeposition with an electrodeposition potential range of 0-0.6 V and a deposition number of 20 times. The gold sheet substrate is then taken out and rinsed with deionized water for more than 10 s, and then dried in an oven at a constant temperature of 30° C. for 1 h to obtain a gold sheet substrate modified with Prussian blue / three-dimensional gold nanoflower array.

[0074] S3 Preparation of conductive substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modification:

[0075] The gold sheet substrate modified with Prussian blue / three-dimensional gold nanoflower array prepared in step 2-2) is immersed in a 0.2M L-cysteine ​​solution, and taken out after immersion for 1 hour, and dried naturally at room temperature to obtain a gold sheet substrate modified with Prussian blue / three-dimensional gold nanoflower array / L-cysteine; the obtained gold sheet substrate modified with Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​is stored in a refrigerator at 4°C;

[0076] Preparation of S4 ethanol biosensor electrode:

[0077] 0.25 g of chitosan and 0.5 mL of 98% pure acetic acid were added to 50 mL of deionized water and mixed evenly to obtain a chitosan fixation solution; 50 U of ethanol oxidase (purchased from Sigma Biotechnology Co., Ltd.) was added to 100 μL of PBS buffer to prepare an ethanol oxidase solution with a concentration of 0.5 U / μL; then 5 μL of the chitosan fixation solution and 5 μL of the ethanol oxidase solution were mixed in a volume ratio of 1:1 to obtain an enzyme fixation solution; then 10 μL of the enzyme fixation solution was drop-coated on the surface of a gold sheet substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modified, and dried at 4°C for 12 hours to obtain an ethanol biosensor electrode.

[0078] The linear range of the ethanol biosensor electrode prepared in Example 2 was tested using the differential pulse technology of the Shanghai Chenhua CHI660E electrochemical workstation. The voltage variation range was -0.1-0.6V with an increase of 0.005V. The corresponding electrode wires on the ethanol biosensor electrical workstation prepared in Example 2 were connected.

[0079] The linear range test results of the ethanol biosensor electrode prepared in Example 2 are shown in FIG4(A). As can be seen from FIG4(A), the sensitivity of the ethanol biosensor prepared in Example 2 of the present application to ethanol is 6.79 μA·mM -1 cm -2 When the ethanol concentration is greater than or equal to 0.05mM, the ethanol biosensor electrode prepared in Example 2 of the present application will have a more obvious current response, the linear range is 0.1mM-11mM, and the detection time is only 15s. In addition, after completing the standard curve test, the ethanol biosensor electrode prepared in Example 2 of the present application was stored in a PBS buffer solution at 4°C for 30 days, and its response signal was 92% of the initial signal, indicating that the ethanol biosensor electrode has excellent stability.

[0080] Example 3

[0081] A method for preparing an ethanol biosensor electrode comprises the following steps:

[0082] S1 Preparation of conductive substrate modified with three-dimensional gold nanoarrays:

[0083] 1-1) 0.272 g of chloroauric acid and 0.988 g of 75% sulfuric acid were added to 40 mL of deionized water and mixed evenly to obtain an acidic electrodeposition solution, wherein the pH value of the electrodeposition solution was 1.5; wherein the concentration of chloroauric acid in the electrodeposition solution was 20 mM, and the concentration of H2SO4 was 0.25 M;

[0084] 1-2) The cleaned carbon cloth substrate is fixed on the three-electrode system, and then the carbon cloth substrate fixed on the three-electrode system is immersed in an electrodeposition solution, and then constant potential deposition is performed, the constant potential voltage is 0.1V, after 10 minutes of electrodeposition, the carbon cloth substrate is taken out and rinsed with deionized water, and dried in an oven at a constant temperature of 40°C for 1 hour to obtain a carbon cloth substrate modified with a three-dimensional gold nanoarray; wherein the cleaned carbon cloth substrate refers to a carbon cloth substrate obtained by placing the carbon cloth substrate in acetone, 10% sodium hydroxide, ethanol and distilled water in sequence for ultrasonic cleaning;

[0085] S2) Preparation of Prussian blue / 3D gold nanoflower array modified conductive substrate:

[0086] 2-1) 20 mL of a NiCl2 solution having a density of 0.0023 g / mL and 0.078 g of sodium citrate were mixed to obtain a mixed solution, wherein the molar concentration of NiCl2 in the mixed solution was 10 mM, and the molar concentration of sodium citrate was 15 mM, and then the mixed solution was mixed with 20 mL of a transition metal potassium cyanide reaction solution to obtain a Prussian blue reaction solution; wherein the transition metal potassium cyanide reaction solution was prepared in the following manner;

[0087] 0.146 g of potassium ferrocyanide was dissolved in 19.8 ml of deionized water, and then 0.2 ml of 1 mol / L sulfuric acid solution was added and mixed evenly to obtain a transition metal cyanide potassium solution. The molar concentration of potassium ferrocyanide in the transition metal cyanide potassium solution was 20 mM, and the molar concentration of H2SO4 was 20 mM.

[0088] During the preparation of the Prussian blue reaction solution, the side of the carbon cloth substrate modified with the three-dimensional gold nanoarray was always kept facing upward.

[0089] 2-2) The three-dimensional gold nanoflower array-modified carbon cloth substrate obtained in step 1-2) fixed on the three-electrode system is immersed in a Prussian blue reaction solution, and then cyclic voltammetry electrodeposition is performed, the electrodeposition potential range is 0-0.6V, the deposition number is 30 times, and then the carbon cloth substrate is taken out and rinsed with deionized water for more than 10s, and then dried in an oven at a constant temperature of 30°C for 1h to obtain a carbon cloth substrate modified with Prussian blue / three-dimensional gold nanoflower array;

[0090] S3 Preparation of conductive substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modification:

[0091] The carbon cloth substrate modified with Prussian blue / three-dimensional gold nanoflower array prepared in step 2-2) was immersed in a 0.2M L-cysteine ​​solution, and taken out after immersion for 1 hour, and dried naturally at room temperature to obtain a modified electrode based on Prussian blue / gold nanoflower / L-cysteine; the obtained modified electrode based on Prussian blue / gold nanoflower / L-cysteine ​​was stored in a refrigerator at 4°C;

[0092] Preparation of S4 ethanol biosensor electrode:

[0093] 0.9585 g of EDC·HCl and 1.15 g of NHS were dissolved in 500 ml of deionized water and mixed evenly to obtain a crosslinker, wherein the molar concentration of EDC·HCl in the crosslinker was 10 mM and the molar concentration of NHS was 20 mM; then the modified electrode based on Prussian blue / gold nanoflowers / L-cysteine ​​was immersed in the crosslinker containing EDC·HCl and NHS, taken out after 1 hour, and stored in a refrigerator at 4°C;

[0094] Then, 50 U of ethanol oxidase (purchased from Sigma Biotechnology Co., Ltd.) was added to 250 μL of PBS buffer to obtain an ethanol oxidase solution with a concentration of 0.2 U / μL;

[0095] Then, 10 μL of ethanol oxidase solution was dripped onto the surface of the treated modified electrode and dried in a refrigerator at 4°C.

[0096] Then prepare 5% (V / V) Nafion membrane solution, add 5 μL of Nafion membrane solution after the ethanol oxidase solution dries on the electrode surface, and dry and store in a 4°C refrigerator to obtain an ethanol biosensor electrode, which can be stored at 4°C for use.

[0097] The linear range of the ethanol biosensor electrode prepared in Example 2 was tested using the differential pulse technology of the Shanghai Chenhua CHI660E electrochemical workstation. The voltage variation range was -0.1-0.6 V with an increment of 0.005 V. The ethanol biosensor electrode (working electrode), platinum wire (counter electrode) and Ag / AgCl (reference electrode) prepared in Example 3 were connected to the corresponding electrode wires on the electrochemical workstation.

[0098] The linear range test results of the ethanol biosensor electrode prepared in Example 3 are as follows: Figure 5 As shown. Figure 5 It can be seen that the sensitivity of the ethanol biosensor electrode prepared in Example 3 of the present application to ethanol is 7.02 μA·mM -1 cm -2 When the ethanol concentration is greater than or equal to 0.05mM, the ethanol biosensor electrode prepared in Example 1 of the present application will have a more obvious current response, the linear range is 0.05mM-10mM, and the detection time is only 15s. In addition, after completing the standard curve test, the ethanol biosensor electrode prepared in Example 3 of the present application was stored in a PBS buffer solution at 4°C for 30 days, and its response signal was 90% of the initial signal, indicating that the ethanol biosensor electrode has excellent stability.

[0099] The present invention combines the beneficial properties of Prussian blue and gold nanoflowers, utilizes the dispersion effect of the gold nanoflower array to reduce the agglomeration of Prussian blue, and at the same time, cross-links L-cysteine. L-cysteine ​​has abundant carboxyl groups and sulfur groups, which can form peptide bonds with amino groups in enzymes, and can better fix the enzymes. The sulfur groups also have strong binding force with the three-dimensional gold nanoflower structure on the conductive substrate. Therefore, the ethanol biosensor electrode prepared by the present invention has very excellent anti-interference ability and good stability.

[0100] In addition, in order to verify the anti-interference performance of the ethanol biosensor electrode prepared in the present application, the present application specifically tests the anti-interference ability of the ethanol biosensor electrode prepared in Example 2. Specifically, first, ethanol, sucrose, glucose, fructose, glutamic acid, pyruvic acid and acetaldehyde solutions with a molar concentration of 1 M are respectively prepared, and then the ethanol biosensor electrode prepared in Example 2 is fixed on a three-electrode system for testing in PBS buffer, and then ethanol, sucrose, glucose, fructose, glutamic acid, pyruvic acid and acetaldehyde solutions with a molar concentration of 1 M are injected in turn, and then the ethanol biosensor electrode prepared in Example 2 is judged whether it responds to the substance according to the current response when different substances are injected. The test results of the anti-interference ability are shown in Figure 4 (B). As can be seen from FIG. 4(B), the ethanol biosensor electrode prepared in Example 2 has a current response only to ethanol, and has no current response to common interfering substances in the ethanol fermentation process, such as sucrose, glucose, fructose, glutamate, pyruvate, acetaldehyde, etc. Therefore, the ethanol biosensor electrode prepared in the present application has good anti-interference properties to common interfering substances in the ethanol fermentation process, and can meet the requirements of in-situ detection and detection accuracy in ethanol fermentation.

[0101] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing an ethanol biosensor electrode, characterized in that: The following steps are involved: S1 Preparation of conductive substrate modified with three-dimensional gold nanoarrays: 1-1) preparing an acidic electrodeposition solution from chloroauric acid, a complexing agent and a solvent; 1-2) immersing the conductive substrate fixed on the three-electrode system in an electrodeposition solution to perform constant potential electrodeposition, and then taking out the conductive substrate and rinsing it, and drying it at a constant temperature to obtain a conductive substrate modified with a three-dimensional gold nanoarray; Preparation of S2 Prussian blue / 3D gold nanoflower array modified conductive substrate: 2-1) preparing a Prussian blue reaction solution from a transition metal salt solution, a surfactant and a transition metal potassium cyanide reaction solution; 2-2) immersing the conductive substrate modified with the three-dimensional gold nanoflower array fixed on the three-electrode system in a Prussian blue reaction solution, and then performing cyclic voltammetry electrodeposition, and then taking out the conductive substrate, rinsing it, and drying it at a constant temperature to obtain a conductive substrate modified with Prussian blue / three-dimensional gold nanoflower array; S3 Preparation of conductive substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modification: The conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array is immersed in L-cysteine ​​solution, and then taken out and dried after a period of time to obtain a conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine; Preparation of S4 ethanol biosensor electrode: The ethanol oxidase solution is fixed on a conductive substrate modified with Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​by a cross-linking method or an embedding method, and then dried at low temperature to obtain a conductive substrate with ethanol oxidase on the surface, i.e., an ethanol biosensor electrode; In step S4, the step of fixing the ethanol oxidase solution on the Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modified conductive substrate obtained in step S3 by cross-linking is as follows: using PBS buffer as a solvent to prepare the ethanol oxidase solution, and then adding glutaraldehyde aqueous solution to obtain a cross-linking solution; Then, the cross-linking solution is dripped onto the surface of the conductive glass modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine, and dried at low temperature to obtain a conductive substrate with ethanol oxidase on the surface, namely, an ethanol biosensor electrode; in step S4, the steps of fixing the ethanol oxidase solution on the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​obtained in step S3 by cross-linking are as follows: preparing a solution containing EDC·HCl and NHS, and then immersing the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​in the solution containing EDC·HCl and NHS, taking it out after a period of time, obtaining a pretreated modified electrode, and storing it at low temperature; then, using PBS buffer as a solvent to prepare an ethanol oxidase solution, and then taking the ethanol oxidase solution and dripping it on the surface of the pretreated modified electrode, and drying it at low temperature; Then, Nafion membrane solution is added dropwise and dried at low temperature to obtain a conductive substrate having ethanol oxidase on the surface, namely, an ethanol biosensor electrode.

2. The method for preparing an ethanol biosensor electrode according to claim 1, characterized in that: In step 1-2), the three-electrode system is a working electrode / reference Ag|AgCl electrode / counter electrode system; during the constant potential deposition process, the constant potential voltage is one of -0.3V, -0.1V, 0.1V and 0.3V, and the electrodeposition time is 2-15min.

3. The method for preparing an ethanol biosensor electrode according to claim 1, characterized in that: The three-electrode system in step 2-2) is the same as the three-electrode system in step 1-2); during the cyclic voltammetry electrodeposition process, the electrodeposition potential range is 0-0.6V, and the deposition times are 20-30 times.

4. The method for preparing an ethanol biosensor electrode according to claim 1, characterized in that: In step S4, in the solution containing EDC·HCl and NHS, the molar concentration of EDC·HCl is 5-20 mM, and the molar concentration of NHS is 10-30 mM; the storage temperature and the low-temperature drying temperature are both -5-10°C; the concentration of ethanol oxidase in the ethanol oxidase solution is 0.2- 4.0U / μL; the volume concentration of the Nafion membrane solution is 5%.

5. The method for preparing an ethanol biosensor electrode according to claim 1, characterized in that: In step S4, the step of fixing the ethanol oxidase solution on the conductive substrate modified by Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​by embedding method is as follows: dissolving chitosan and acetic acid in deionized water, mixing them evenly, and obtaining a chitosan fixing solution; Then, ethanol oxidase solution was prepared by using PBS buffer as solvent; Then, the chitosan fixation solution and the ethanol oxidase solution are mixed in a certain volume ratio to obtain an enzyme fixation solution; and then the enzyme fixation solution is drop-coated on the surface of a gold sheet substrate based on Prussian blue / three-dimensional gold nanoflower array / L-cysteine ​​modified.

6. An ethanol biosensor electrode prepared by using the preparation method of the ethanol biosensor electrode; The method for preparing the ethanol biosensor electrode is the method for preparing the ethanol biosensor electrode according to claim 1. 7 . An ethanol biosensor, comprising the ethanol biosensor electrode according to claim 6 .

8. Use of an ethanol biosensor for rapid detection of ethanol in a fermentation process, wherein the ethanol biosensor is the ethanol biosensor according to claim 7.

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