A method of preparing an electrode for an electrochemical sensor and applications thereof

Abstract

The application relates to a preparation method and application of an electrode of an electrochemical sensor, and belongs to the technical field of electrochemical biological detection. The application aims to solve the problem that the stability of a thin film electrode and the electric signal conversion efficiency are simultaneously improved, and the influence of oxygen on ferrocene is reduced. The application uses chitosan grafted with ferrocene and chemically bonded glucose oxidase as a modification layer on the surface of the electrode. The modification electrode has electrochemical activity for glucose, and the stability of the sensing electrode is improved. The application aldehydezes glucose oxidase under mild experimental conditions, and covalently connects the chitosan grafted with ferrocene, so that the enzyme is stably fixed in the chitosan, a CHIT-Fc solution with enzyme fixation is obtained, the uniform dispersion of the glucose oxidase in the ferrocene and derivatives thereof is realized, the stability of the enzyme can be improved without affecting the enzyme activity, the improved electrochemical sensor can operate in a lower detection limit, and the high stability can be maintained to realize multiple uses.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemical biological detection, and particularly relates to a preparation method and application of an electrode of an electrochemical sensor. BACKGROUND

[0002] Electrochemical biosensors have shown great potential for application in environmental monitoring, disease diagnosis, food safety, and other fields due to their high sensitivity, low cost, fast response, and portability. In particular, in cases where continuous monitoring and on-site rapid diagnosis are required, such sensors provide a very effective solution.

[0003] Chitosan, a natural high-molecular-weight polymer extracted from crustaceans, has attracted much attention due to its good biocompatibility, biodegradability, and rich potential for chemical modification. Chitosan contains a large number of hydroxyl and amino groups, which allows it to be easily connected to various functional molecules through simple chemical methods. However, despite these advantages, the electrochemical activity of chitosan in its pure state is relatively low, which limits its application in certain high-demand electrochemical sensor fields. To overcome this limitation, researchers have explored methods to introduce electroactive molecules, such as ferrocene, into the chitosan molecular chain. Ferrocene has been widely used as an electron transfer mediator in electrochemical sensors due to its good electrochemical stability and reversible redox properties. By combining ferrocene and chitosan, the electrochemical performance of chitosan can be improved, and a sensing material with high sensitivity and good stability can be prepared. This chitosan-ferrocene derivative not only retains the biocompatibility and multifunctionality of chitosan, but also endows the material with excellent electrochemical activity, making it possible to develop new types of electrochemical biosensors. For example, in the field of glucose monitoring, this material can directly convert biological recognition events (such as enzyme catalytic reactions) into electrical signals through electrochemical methods, simplifying the complex signal conversion and amplification process required by traditional biosensors. However, due to the polarity difference between glucose oxidase and ferrocene and its derivatives, the solubility of the two is different, making it difficult to achieve uniform dispersion of glucose oxidase in ferrocene and its derivatives. This makes the electron transfer efficiency of glucose oxidase electrodes using ferrocene as an electron enzyme mediator extremely limited, resulting in low sensitivity and narrow detection range in glucose concentration detection.

[0004] Weiwei Yang et al. published "Synthesis of Ferrocene-Branched Chitosan Derivatives: Redox Polysaccharides and their Application to Reagentless Enzyme-Based Biosensors" in 2007, which discloses:

[0005] Ferrocene branched chitosan derivative was synthesized by n-alkylation reaction of chitosan with ferrocene carboxaldehyde. The structure of the product was determined by hydrogen nuclear magnetic resonance and infrared spectroscopy. CHIT-Fc was used as a functionalized matrix for GOD immobilization on a glassy carbon electrode.

[0006] Ferrocene CHIT-Fc exhibited excellent redox activity, establishing efficient electrical communication between GOD and the glucose oxidation electrode. In summary, we have demonstrated that ferrocene branched chitosan derivative can be effectively used for the fabrication of reagentless glucose biosensor by forming CHIT-Fc / GOD film on gce. This redox polysaccharide not only provides a very suitable environment for enzyme entrapment, but also establishes efficient electrical communication between GOD and the electrode. Amperometric experiments showed that CHITFc / GOD film electrode was exceptionally sensitive in the electroanalytical process of glucose. Due to the hydrophobicity of ferrocene group, the concentration of redox sites in this polymer is limited by the solubility of modified CHIT. To overcome this problem, more hydrophilic redox chitosan derivatives will be synthesized and investigated in the future. We believe that chitosan redox biopolymers have a broad application prospect in the field of biosensors and bioelectronic devices.

[0007] Weiwei Yang et al. published in 2016 in the article "Effect of film-forming solution pH on the properties of chitosan-ferrocene film electrodes" discloses:

[0008] Ferrocene branched chitosan (CHIT-Fc) film electrodes were prepared using CHIT-Fc solutions with different pH values as raw materials to study the effect of film-forming solution pH on ferrocene charge transport. The redox behavior of CHIT-Fc polymer film electrodes was studied using cyclic voltammetry. C*D ct l / 2 and membrane permeability increased with the decrease of film-forming solution pH. Glucose oxidase (GOD) was added to these films, proving the effective electrical communication between enzyme molecules and the glucose oxidation electrode. When the film-forming solution pH was 2.8, the film showed the best electrocatalytic response to glucose, with a sensitivity of 77.5 μA·mM -1 ·cm -2 .

[0009] The above technology gives the content of CHIT-Fc and glucose oxidase for preparing a film electrode for detecting glucose, but it does not give how to improve the stability and electrical signal conversion efficiency of the film electrode, and the influence of oxygen on ferrocene, how to solve the problem of improving the stability and electrical signal conversion efficiency of the film electrode, and reducing the influence of oxygen on ferrocene at the same time. SUMMARY

[0010] The present application aims at solving the problems of the current thin film electrode, improving the stability of the thin film electrode and the electrical signal conversion efficiency, and reducing the influence of oxygen on ferrocene, and provides a preparation method and application of an electrode of an electrochemical sensor.

[0011] The technical scheme of the preparation method of the electrode of the electrochemical sensor of the present application is as follows:

[0012] (1) using chitosan to graft ferrocene to obtain a ferrocene branched chitosan derivative CHIT-Fc;

[0013] (2) incubating the aldehyde-modified glucose oxidase solution with CHIT-Fc to realize chemical bonding and fixation of the two through Schiff base reaction to obtain an enzyme-fixed CHIT-Fc solution;

[0014] (3) coating the enzyme-fixed CHIT-Fc solution obtained in (2) on a substrate and drying to obtain an electrochemical sensor electrode CHIT-Fc-GOD for glucose detection;

[0015] wherein the grafting degree of the chitosan grafted ferrocene is 0.1-0.8; and the pH of the CHIT-Fc solution is 2-3.

[0016] Further, the grafting degree of the chitosan grafted ferrocene is 0.15-0.5.

[0017] Further, the grafting degree of the chitosan grafted ferrocene is 0.15-0.3.

[0018] Further, the pH of the CHIT-Fc solution is 2.7.

[0019] Further, the ferrocene branched chitosan derivative CHIT-Fc in step (1) is obtained by the following method:

[0020] Mixing chitosan / acetic acid aqueous solution and aldehyde ferrocene / methanol solution, stirring at room temperature for 0.5-24h, adding sodium borohydride cyanide, continuing to stir for 8-48h, after the reaction is completed, adding sodium hydroxide solution dropwise to the reaction liquid, recovering the precipitate to obtain CHIT-Fc; wherein the mass ratio of chitosan to aldehyde ferrocene is 1:0.01-1.1.

[0021] Further, the preparation method of the aldehyde-modified glucose oxidase solution in step (2) is as follows:

[0022] The glucose oxidase is dissolved in a phosphate buffer solution under light-proof condition, sodium periodate is added under ice water bath condition, after stirring, the temperature is increased to room temperature, glycol is added, the reaction is stirred, after the reaction is completed, the obtained reaction solution is transferred into a permeable membrane, and is permeated with a buffer solution to obtain aldehyde-modified glucose oxidase; wherein, the mass-volume ratio of the glucose oxidase and the phosphate buffer solution is 7-8 mg: 1 mL, the mass ratio of the glucose oxidase and the sodium periodate is 1.5-2: 1, and the mass-volume ratio of the glucose oxidase and the glycol is 7-8 mg: 3-4 μL.

[0023] Further, the incubation time in step (2) is 24 h.

[0024] Further, the substrate in step (3) is an activated glassy carbon electrode.

[0025] The application of the electrode of the electrochemical sensor prepared by the preparation method of the electrode of the electrochemical sensor.

[0026] Further, the electrode of the electrochemical sensor is a working electrode, Ag / AgCl is a reference electrode, and Pt is a counter electrode to form an electrochemical sensor.

[0027] Further limitation, the detection potential of the electrochemical sensor is less than 0.32 V.

[0028] The application comprises the following beneficial effects:

[0029] The application uses chitosan grafted with ferrocene and chemically bonded glucose oxidase as a modification layer on the surface of the electrode, the modified electrode has electrochemical activity for glucose, and the stability of the sensing electrode is improved, the efficiency of the electrical signal conversion is improved, and the influence of oxygen on ferrocene is reduced.

[0030] (1) The glucose oxidase is aldehyde-modified by mild experimental conditions, and is covalently connected with chitosan grafted with ferrocene, so that the enzyme is stably fixed in the chitosan, and the enzyme-fixed CHIT-Fc solution is obtained, the uniform dispersion of the glucose oxidase in the ferrocene and its derivatives is realized, the stability of the glucose oxidase can be improved without affecting the enzyme activity, the improved electrochemical sensor can operate within a lower detection limit, and the high stability can be maintained for multiple uses.

[0031] (2) The enzyme-fixed CHIT-Fc solution is uniformly coated on the surface of the electrode, and only by controlling the temperature and humidity conditions in the drying process, the formation of the film can be optimized, and the preparation process is simple, the cost is low, and the method is suitable for large-scale production. In addition, the sensor is easy to use, and is suitable for non-professional users to operate without complex equipment, so that it has a significant advantage in on-site rapid detection applications.

[0032] (3) The sensor provided by the application combines the biocompatibility of chitosan and the excellent electrochemical stability of ferrocene, exhibits high sensitivity and good selectivity, can accurately detect the glucose content in complex biological samples, and is suitable for applications that require rapid and accurate detection, such as environmental monitoring, food safety inspection, and preclinical diagnosis.

[0033] (4) The application promotes the development of electrochemical sensing technology, and also helps to promote the progress of biomedical research and public health monitoring, and is expected to have a profound social and economic impact worldwide.

[0034] Compared with Synthesis of Ferrocene-Branched Chitosan Derivatives: Redox Polysaccharides and their Application to Reagentless Enzyme-Based Biosensors and Effect of film-forming solution pH on the properties of chitosan-ferrocene film electrodes, the application improves the grafting degree of ferrocene, adjusts the preparation process, realizes that the electrode of the prepared electrochemical sensor has good stability, and also improves the electrical signal conversion efficiency, reduces the influence of oxygen on detection, and can improve the sensitivity of glucose sensing. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a synthesis diagram of ferrocene branched chitosan derivative CHIT-Fc;

[0036] Figure 2 It is a comparison diagram of the glucose detection performance of the modified electrodes prepared in Examples 1, 3, 4, 5 and Comparative Example 1;

[0037] Figure 3 It is a cycle performance diagram of the modified electrode prepared in Example 1;

[0038] Figure 4 It is a cycle performance diagram of the modified electrode prepared in Example 3;

[0039] Figure 5 It is a cycle performance diagram of the modified electrode prepared in Example 4;

[0040] Figure 6 It is a cycle performance diagram of the modified electrode prepared in Example 5;

[0041] Figure 7Cyclic performance plot of the modified electrode prepared for Comparative Example 1;

[0042] Figure 8 Measurement linear range of the modified electrode prepared for Example 1;

[0043] Figure 9 Cyclic performance plot of the modified electrode prepared for Example 1 with and without oxygen removal;

[0044] Figure 10 Cyclic performance plot of the modified electrode prepared for Example 2 with and without oxygen removal. DETAILED DESCRIPTION

[0045] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the description of the examples.

[0046] In the following description, a lot of specific details are set forth in order to provide a thorough understanding of the present application, however, the present application can be practiced in other manners different from those described herein, and those skilled in the art can make similar generalizations without departing from the concept of the present application, therefore, the present application is not limited to the specific examples disclosed below.

[0047] In the following examples, the experimental methods used are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, which can be obtained by commercial channels by those skilled in the art, and the purity of the solid and liquid reagents used is analytical pure.

[0048] Example 1

[0049] The method for preparing the electrode CHIT-Fc-GOD of the electrochemical sensor for glucose detection in this example is carried out according to the following steps:

[0050] Step one, 150 mg of chitosan is dissolved in 60 mL of 0.1 M aqueous acetic acid, 30.6 mg of aldehyde ferrocene is dissolved in 30 mL of methanol solvent and then added to the chitosan solution, stirred at room temperature for 12 h, then 30.05 mg of excess sodium boron cyanide is added and stirred for 24 h, the solution gradually changes from dark red to orange and then to yellow, at the end of the reaction, 40 mL of 5% sodium hydroxide solution is added dropwise to the solution to precipitate the product completely, and the product is recovered, which is the ferrocene branched chitosan derivative CHIT-Fc, the degree of ferrocene branching is 0.15, and the synthesis schematic diagram is as shown in Figure 1

[0051] ​Step two, 74.4 mg of glucose oxidase was dissolved in 10 mL of phosphate buffer solution (pH 6.9), the system temperature was controlled at about 4°C by ice water bath, 120 mg of sodium periodate was added carefully, stirred for 90 min (4°C), then the system temperature was raised to 25°C, 31.1 uL of ethylene glycol was added to inhibit polymerization, stirred for 45 min, the reaction was stopped, the product was a yellow solution, the solution product was moved into a permeable membrane, and the buffer was permeated to remove small molecular impurities. The whole reaction should be carried out in the dark, and finally the aldehyde-modified glucose oxidase was obtained.

[0052] Step three, 10 mg of CHIT-Fc and 7.4 mg of aldehyde-modified glucose oxidase were incubated together in the dark for 24 h to obtain an enzyme-immobilized CHIT-Fc solution.

[0053] Step four, the cleaned and activated glassy carbon electrode was used as the substrate, the enzyme-immobilized CHIT-Fc solution was uniformly coated on the surface of the electrode and dried under vacuum at 35°C to form a continuous film, and an electrode CHIT-Fc-GOD for the electrochemical sensor for glucose detection was obtained.

[0054] Example 2

[0055] The difference between this example and example 1 is that the amount of aldehyde ferrocene used is 20.4 mg, and the rest of the process steps and parameter settings are the same as example 1, and CHIT-Fc with a grafting degree of 0.1 of ferrocene and CHIT-Fc-GOD electrode are obtained.

[0056] Example 3

[0057] The difference between this example and example 1 is that the amount of aldehyde ferrocene used is 61.2 mg, and the rest of the process steps and parameter settings are the same as example 1, and CHIT-Fc with a grafting degree of 0.31 of ferrocene and CHIT-Fc-GOD electrode are obtained.

[0058] Example 4

[0059] The difference between this example and example 1 is that the amount of aldehyde ferrocene used is 102.0 g, and the rest of the process steps and parameter settings are the same as example 1, and CHIT-Fc with a grafting degree of 0.50 of ferrocene and CHIT-Fc-GOD electrode are obtained.

[0060] Example 5

[0061] The difference between this example and example 1 is that the amount of aldehyde ferrocene used is 163.2 mg, and the rest of the process steps and parameter settings are the same as example 1, and CHIT-Fc with a grafting degree of 0.8 of ferrocene and CHIT-Fc-GOD electrode are obtained.

[0062] Comparative Example 1

[0063] Step one, 133.4 mg of chitosan, 16.6 mg of ferrocene and 111 mg of glucose oxidase were incubated together for 24 h under light-proof condition to obtain a mixed solution.

[0064] Step two, the cleaned and activated glassy carbon electrode was used as a substrate, the mixed solution was uniformly coated on the electrode surface and dried under vacuum condition at 35℃ to form a continuous film, thus obtaining the electrode of the electrochemical sensor for glucose detection, and the grafting degree of ferrocene was 0.01.

[0065] Application Example

[0066] (1) The sensitivity and cycle of the modified electrode prepared in Test Example 1 and Example 5 and Comparative Example 1 in glucose detection was tested, and the electrochemical performance in 6 mmol / L glucose solution was detected by cyclic voltammetry, the scanning range was-0.2-0.6 V, and the scanning rate was 50 mV / s, and the results were shown in FIGS. 1 and 2. Figures 2 to 5 As shown in the figures, the modified electrode prepared in Example 1 and Example 5 did not change significantly in 10 tests, indicating that the stability of the modified electrode was improved. In contrast, the modified electrode prepared in Comparative Example 1 was prepared by dispersion, and the electrode film was constructed by adsorption. The glucose signal decreased rapidly after multiple tests, which proved that the preparation method of the modified electrode provided by the application improved the stability. Figure 6 The measurement linear range of the modified electrode prepared in Example 1 was known from FIG. 3. Figure 6 As can be seen from FIG. 3, as the glucose concentration increased, the sensor could maintain a stable detection signal, and the current remained linearly related to the concentration.

[0067] (2) The sensitivity and cycle of the modified electrode prepared in Example 3 and Example 4 in glucose detection was tested, and the electrochemical performance in 6 mmol / L glucose solution was detected by cyclic voltammetry, the scanning range was-0.2-0.6 V, and the scanning rate was 50 mV / s, and the results were shown in FIGS. 4 and 5. Figure 7 and 8 As shown in the figures, Figure 7 As can be seen from FIG. 3, as the glucose concentration increased, the sensor could maintain a stable detection signal, and the current remained linearly related to the concentration.

[0068] (3) Test the influence of dissolved oxygen of Example 1 and Example 2, Figure 9 For the glucose detection ability diagram of Example 1 before and after N2 deoxygenation, it can be found that the influence of oxygen on glucose detection is small. Figure 10 For the glucose detection ability diagram of Example 2 before and after N2 deoxygenation, the influence of oxygen on the 0.1 grafting degree ferrocene-chitosan system is larger, because in the cyclic voltammetry detection process, due to the small amount of ferrocene, the ratio of oxygen content to ferrocene is increased, so that the oxygen limits the redox reaction of ferrocene in the detection process. The 0.15 grafting degree increases the ratio of ferrocene, so that the ratio of oxygen is reduced in the detection process, so that the redox reaction of ferrocene is the main reaction, thereby reducing the influence of oxygen.

[0069] Therefore, Example 1 is the optimal grafting degree ratio, which not only meets the stability of the sensor, can reduce the influence of oxygen on the detection, and can improve the sensitivity of glucose sensing.

[0070] Example 4 modified electrode, in the grafting degree investigation, it is found that when the grafting degree continues to increase to 0.5, Figure 8 It can be seen that the signal continues to decrease, which is caused by the decrease of glucose oxidase loading amount, and the signal decreases in 10 cycles, and is more obvious than 0.3, which shows that the increase of grafting degree further leads to the decrease of sensor stability, which is also caused by the decrease of the site and space left for glucose oxidase, and most of the glucose oxidase does not exist on the electrode surface in the form of bonding.

[0071] Although the present application has been disclosed as above with preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be limited by the claims.

Claims

1. A method of preparing an electrode of an electrochemical sensor, characterized by, The preparation method is as follows: (1) using chitosan grafting ferrocene, obtaining ferrocene branched chitosan derivative CHIT-Fc; (2) incubating the aldehyde-modified glucose oxidase solution with CHIT-Fc, realizing chemical bonding and fixing of the two through Schiff base reaction, obtaining enzyme-fixed CHIT-Fc solution; (3) coating the enzyme-fixed CHIT-Fc solution obtained in (2) on a substrate, drying, obtaining an electrochemical sensor electrode CHIT-Fc-GOD for glucose detection; Wherein, the grafting degree of chitosan grafting ferrocene is 0.15; the pH of CHIT-Fc solution is 2-3.

2. The method of claim 1, wherein the method further comprises: The pH of CHIT-Fc solution is 2.

7.

3. The method of claim 1, wherein the electrode is an electrode for an electrochemical sensor. The ferrocene branched chitosan derivative CHIT-Fc in step (1) is obtained by the following way: Mixing chitosan / acetic acid aqueous solution and aldehyde ferrocene / methanol solution, stirring at room temperature for 0.5-24h, adding sodium borohydride cyanide, continuing to stir for 8-48h, after the reaction is completed, adding sodium hydroxide solution dropwise to the reaction liquid, recovering the precipitate to obtain CHIT-Fc; wherein, the mass ratio of chitosan to aldehyde ferrocene is 1:0.01-1.

1.

4. The method of claim 1, wherein the electrode is an electrode for an electrochemical sensor. The preparation method of aldehyde-modified glucose oxidase solution in step (2) is as follows: Under light-proof condition, dissolving glucose oxidase in phosphate buffer solution, under ice water bath condition, adding sodium periodate, stirring, then warming to room temperature, adding ethylene glycol, stirring and reacting, after the reaction is completed, transferring the obtained reaction liquid into a permeable membrane, permeating with buffer solution to obtain aldehyde-modified glucose oxidase; wherein, the mass-volume ratio of glucose oxidase to phosphate buffer solution is 7-8mg:1mL, the mass ratio of glucose oxidase to sodium periodate is 1.5-2:1, and the mass-volume ratio of glucose oxidase to ethylene glycol is 7-8mg:3-4μL.

5. The method of claim 1, wherein the electrode is an electrode for an electrochemical sensor. The incubation time in step (2) is 24h.

6. The method of claim 1, wherein the electrode is an electrode for an electrochemical sensor. The substrate in step (3) is an activated glassy carbon electrode.

7. Use of an electrochemical sensor electrode prepared according to the method of claim 1. The electrochemical sensor electrode is used for detecting glucose.

8. Use according to claim 7, characterized in that, The electrochemical sensor electrode is a working electrode, Ag / AgCl is a reference electrode, and Pt is a counter electrode to form an electrochemical sensor.

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