Enzyme glucose electrochemical biosensor based on triple stable structure and preparation method thereof
By employing a triple-stabilizing structure combining MXene nanomaterials and enzyme nanoflowers in the enzyme glucose electrochemical sensor, the problems of sensor inactivation and insufficient sensitivity were solved, achieving long-term stability and high sensitivity of the sensor.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JILIN UNIVERSITY
- Filing Date
- 2026-02-05
- Publication Date
- 2026-04-21
AI Technical Summary
Existing enzyme-based electrochemical glucose sensors are prone to deactivation during use and have unstable structures, resulting in insufficient sensitivity to meet the requirements for precise glucose measurement.
Using MXene nanomaterials as a substrate, enzyme nanoflowers are grown in situ, combined with tannic acid modification and Nafion encapsulation, to form a triple-stable enzyme glucose electrochemical biosensor, enhancing the stability and sensitivity of the enzyme.
The enzyme-based glucose electrochemical biosensor achieves long-term stability and high sensitivity, making it suitable for glucose monitoring in various applications.
Smart Images
Figure CN121899223A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of glucose monitoring technology, specifically relating to an enzyme glucose electrochemical biosensor based on a triple stable structure and its preparation method. Background Technology
[0002] Diabetes is a prevalent chronic disease. Monitoring blood glucose levels is crucial for diabetic patients. Compared to methods like gas chromatography, liquid chromatography, and mass spectrometry, electrochemical biosensors offer advantages such as high specificity, portability, low cost, and rapid response, making them widely used in continuous and self-monitoring of blood glucose. Glucose electrochemical sensors are categorized into enzyme-based and non-enzyme-based sensors. The core advantage of enzyme-based glucose electrochemical sensors compared to non-enzyme-based sensors stems from the biological specificity and catalytic efficiency of enzymes, overcoming the shortcomings of non-enzyme sensors such as poor anti-interference, demanding reaction conditions, and insufficient biocompatibility. They are suitable for accurate and continuous monitoring of glucose in bodily fluids and are currently the mainstream technology choice for clinical blood glucose monitoring and wearable blood glucose devices. Despite the achievements of enzyme-based glucose electrochemical sensors in these areas, two issues remain: first, the inactivation of glucose oxidase and the loose structure of the sensor during use lead to sensor instability; second, the sensor sensitivity is insufficient for precise glucose measurement, and its sensitivity needs improvement.
[0003] Based on the above background, it is meaningful to develop a long-term stable enzyme glucose electrochemical sensor. Therefore, this invention provides a glucose electrochemical biosensor based on a triple stable structure and its preparation method. Summary of the Invention
[0004] To address the issues of long-term stability and high sensitivity in enzyme-glucose electrochemical sensors, this invention provides an enzyme-glucose electrochemical biosensor based on a triple-stable structure and its preparation method. This sensor uses MXene as a substrate and grows enzyme nanoflowers in situ, endowing the sensor with long-term stability and excellent sensitivity. Moreover, the preparation method is simple and can meet the needs of glucose monitoring in various applications.
[0005] The present invention discloses an enzyme glucose electrochemical biosensor based on a triple stable structure, comprising an electrode material, an enzyme active region, and an outer membrane restriction layer. MXene nanomaterials are selected as the electrode material. First, tannic acid (TA) is modified onto the surface of the MXene nanomaterials. This modification serves two purposes: firstly, it provides antioxidant protection; secondly, the abundant hydroxyl groups in tannic acid allow it to form a cross-linked network with the amino groups of enzyme molecules through hydrogen bonds and coordination bonds, thus solving the leakage problem of traditional enzymes. Next, glucose oxidase-copper nanoflowers (MXene@TA / Cu-GOxNF) are grown in situ using a one-pot method. After lyophilization, an MXene@TA / Cu-GOxNF solution is prepared using PBS buffer. This material serves as the enzyme active region, with the copper nanoflowers providing sites for GOx loading and enhancing the stability of glucose oxidase. Finally, the prepared MXene@TA / Cu-GOxNF solution is dropped onto a glassy carbon electrode, dried, and then a layer of Nafion (a perfluorosulfonic acid ionomer) is encapsulated on the glassy carbon electrode. Nafion effectively protects the enzyme layer. In summary, through the design of the material structure, a nanoflower-modified glucose biosensor working electrode is obtained. Copper nanoflowers enhance the structural stability and loading capacity of glucose oxidase. MXene's large specific surface area provides numerous loading sites for the copper nanoflowers, effectively immobilizing GOx. Through the design of this sensor structure, it exhibits excellent selectivity, stability, and sensitivity in glucose detection.
[0006] The preparation method of an enzyme glucose electrochemical biosensor based on a triple stable structure according to the present invention comprises the following steps:
[0007] (1) Preparation of MXene
[0008] First, add 5-10 mL of deionized water to the reaction vessel under ice-water bath conditions. Then, add 15-25 mL of 8-12 M HCl in 3-5 portions, stirring and cooling to room temperature. Next, add 1-2 g of LiF. After the LiF is completely dissolved, continue to add 1-2 g of Ti3AlC2 in small amounts several times under ice-water bath conditions. After sealing the reaction vessel, react for 20-30 hours at 30-40℃ and 300-500 rpm in a water bath. Then, centrifuge the resulting solution with deionized water at 3000-4000 rpm for 3-10 seconds. min; take the lower layer precipitate, and wash it with deionized water at 3000~4000r for 3~10min; repeat the operation of "take the lower layer precipitate and wash it with deionized water" several times until the pH of the supernatant is 5.8~6.2, then add 50~100mL of deionized water to the obtained precipitate, sonicate for 1~1.5h, and then centrifuge at 3000~4000r for 40~60min. The obtained supernatant is the MXene monolayer nanosheet solution. After dilution or concentration, the concentration of MXene monolayer nanosheets is 8~15mg / mL.
[0009] (2) Preparation of MXene@TA solution
[0010] Dissolve 40-60 mg of tannic acid (TA) in 10-20 mL of deionized water to obtain an aqueous solution of tannic acid; then add 10-20 mL of MXene monolayer nanosheet solution with a concentration of 8-15 mg / mL to the aqueous solution of tannic acid, and stir the reaction at room temperature in the dark for 10-20 h to obtain an MXene@TA solution;
[0011] (3) Preparation of MXene@TA / Cu-GOxNF
[0012] Take 5-10 mL of the MXene@TA solution prepared in step (2), add 5-10 mL of PBS buffer, then add 200-400 μL of 0.1-0.2 mol / L CuSO4 aqueous solution, mix and sonicate at 30-40℃ for 1-2 h; then add 15-30 mg of glucose oxidase (GOx), let stand at room temperature for 2-4 days, wash and freeze dry to obtain MXene@TA / Cu-GOxNF;
[0013] (4) Preparation of MXene@TA / Cu-GOxNF electrode
[0014] Add PBS buffer to the MXene@TA / Cu-GOxNF obtained in step (3) to make the concentration of MXene@TA / Cu-GOxNF 8~15mg / mL; take 5~10μL of the solution and drop it onto a glassy carbon electrode with a diameter of 3~5mm, dry it under N2 conditions, then drop 5~10μL of Nafion solution onto the electrode surface, dry it under N2 conditions, and obtain the MXene@TA / Cu-GOxNF electrode;
[0015] (5) Preparation of enzyme-glucose electrochemical biosensor
[0016] Using the MXene@TA / Cu-GOxNF electrode obtained in step (4) as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode, an enzyme glucose electrochemical biosensor based on a triple stable structure was assembled.
[0017] The enzyme glucose electrochemical biosensor based on a triple stable structure described in this invention is prepared by the above method.
[0018] The present invention adopts the above technical solution and has the following advantages.
[0019] 1) MXene possesses a large specific surface area and excellent conductivity, providing numerous attachment sites for enzymes. Copper nanoflowers not only provide these sites but also enhance electron transfer efficiency, resulting in a significant increase in sensitivity.
[0020] 2) The sensor has a triple stable structure, namely, the stability of the MXene nanomaterial film itself, the preparation of glucose oxidase-copper nanoflowers to improve the stability of glucose oxidase to the surrounding environment, and the bond between the nanoflowers and MXene nanomaterials to further improve the stability of the sensor.
[0021] 3) The reaction conditions are mild, the preparation is simple, and the operation is easy. Attached Figure Description
[0022] Figure 1 SEM image of the MXene@TA / Cu-GOxNF material obtained in Example 1;
[0023] Figure 2 The CV curves of the electrochemical sensor obtained in Example 1 under different electrolytes were measured by an electrochemical workstation. The horizontal axis represents the voltage value and the vertical axis represents the current value.
[0024] Figure 3 The electrochemical sensor obtained in Example 1 was used to measure the time-current response curves of solutions with different concentrations of glucose added in an electrochemical workstation. The horizontal axis represents time and the vertical axis represents current value.
[0025] Figure 4 : for the basis Figure 3 The obtained linear fitting curves of different glucose concentrations and response currents;
[0026] Figure 5 The graph shows the anti-interference test results of the electrochemical sensor obtained in Example 1 measured on an electrochemical workstation. The horizontal axis represents time, and the vertical axis represents the current value of the sensor when different substances are added to the electrolyte.
[0027] Figure 6 The electrochemical sensor obtained in Example 1 was used to measure the time-current response of the sensor in PBS buffer over 15 days using an electrochemical workstation. The horizontal axis represents time, and the vertical axis represents current value. Detailed Implementation
[0028] The following embodiments are further illustrations of the present invention, but the present invention is not limited thereto.
[0029] Example 1. Fabrication of a glucose electrochemical biosensor based on a triple-stable structure
[0030] (1) Preparation of MXene
[0031] First, add 5 mL of deionized water to the reaction vessel under ice-water bath conditions. Then, add 15 mL of 9 M HCl in three portions, stirring and cooling to room temperature. Next, add 1 g of LiF. After the LiF is completely dissolved, continue to add 1 g of Ti3AlC2 in small amounts several times under ice-water bath conditions. After sealing the reaction vessel, react for 24 h at 35 °C and 400 rpm in a water bath. Then, centrifuge the resulting solution with deionized water at 3500 rpm for 5 min. Take the lower precipitate and wash it with deionized water at 3500 rpm for 5 min. Repeat the operation of "taking the lower precipitate and washing with deionized water" several times until the pH of the supernatant is 6. Then, add 50 mL of deionized water to the obtained precipitate, sonicate for 1.5 h, and centrifuge at 3500 rpm for 50 min. The resulting supernatant is the MXene monolayer nanosheet solution. Dilute it with deionized water to make the concentration of MXene monolayer nanosheets 10 mg / mL.
[0032] (2) Preparation of MXene@TA solution
[0033] Dissolve 50 mg of tannic acid (TA) in 10 mL of deionized water to obtain an aqueous solution of tannic acid; then add 10 mL of MXene monolayer nanosheet solution with a concentration of 10 mg / mL to the aqueous solution of tannic acid, and stir the mixture at room temperature in the dark for 16 h to obtain an MXene@TA solution.
[0034] (3) Preparation of MXene@TA / Cu-GOxNF
[0035] Take 5 mL of the MXene@TA solution prepared in step (2), add 5 mL of PBS buffer, then add 300 μL of 0.12 mol / L CuSO4 solution, mix and sonicate at 35 °C for 1 h; then add 20 mg of glucose oxidase (GOx), let stand at room temperature for 3 days, wash and freeze dry to obtain 80 mg of MXene@TA / Cu-GOxNF;
[0036] (4) Preparation of MXene@TA / Cu-GOxNF electrode
[0037] Add PBS buffer to the MXene@TA / Cu-GOxNF obtained in step (3) to make the concentration of MXene@TA / Cu-GOxNF 10 mg / mL; take 5 μL of the solution and drop it onto a glassy carbon electrode with a diameter of 3 mm, dry it under N2 conditions, then drop 5 μL of Nafion onto the electrode surface and dry it under N2 conditions to obtain the MXene@TA / Cu-GOxNF electrode.
[0038] (5) Preparation of enzyme-glucose electrochemical biosensor
[0039] Using the MXene@TA / Cu-GOxNF electrode obtained in step (4) as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode, an enzyme glucose electrochemical biosensor was assembled.
[0040] Figure 1 The image shows the SEM image of the MXene@TA / Cu-GOxNF material obtained in step (3) of Example 1. It can be seen that the nanoflowers are grown in situ on the surface of the MXene nanosheets, and the diameter of a single nanoflower is about 4 micrometers.
[0041] Figure 2 The CV curves of the sensor obtained in step (5) of Example 1 are shown in 5mM Glu solution and PBS buffer, respectively. It can be seen that the current value of the sensor in Glu solution (dark curve) is much greater than the current value in PBS buffer (light curve), showing that the sensor has a good signal response to glucose.
[0042] Figure 3 This is the time-current response curve of the sensor in Example 1 when different concentrations of Glu solution are continuously added. Figure 4 It is based on Figure 3 The obtained linear fitting curves of different glucose concentrations and response currents yielded a linear regression equation of y = 4.8469x + 0.04016, with a correlation coefficient R0. 2 The value is 0.9975. The sensitivity is calculated by dividing the slope by the electrode area, resulting in a final sensitivity of 68.57 μA mM. -1 ;
[0043] Figure 5 The diagram shows the anti-interference experiment of the sensor in Example 1. 1 mM glucose (Glu), ascorbic acid (AA), uric acid (UA), hydrogen peroxide (H2O2), and glucose (Glu) were added sequentially to the same sensor PBS solution. When ascorbic acid (AA), uric acid (UA), and hydrogen peroxide (H2O2) were added, the sensor did not show a significant current response. Only when Glu was added at the beginning and end did the sensor show a significant current response, demonstrating the sensor's excellent selectivity.
[0044] Figure 6 The graph shows the time-current response of the sensor in Example 1 over 15 days in PBS buffer. After 15 days of immersion in PBS buffer, the sensor current was 91.2% of the initial value, demonstrating the long-term stability of the sensor.
[0045] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, corresponding changes and substitutions can be made based on the technical solutions and inventive concepts of the present invention, and any modifications or substitutions with the same performance or purpose should be considered within the scope of protection of the present invention.
Claims
1. A method for preparing an enzyme-based glucose electrochemical biosensor with a triple stable structure, comprising the following steps: (1) Preparation of MXene First, add 5-10 mL of deionized water to the reaction vessel under ice-water bath conditions. Then, add 15-25 mL of 8-12 M HCl in 3-5 portions. Stir and cool to room temperature; then add 1-2 g of LiF. After the LiF is completely dissolved, continue to add 1-2 g of Ti3AlC2 in small amounts several times under ice-water bath conditions; after sealing the reaction vessel, react for 20-30 h at 30-40℃ and 300-500 rpm in a water bath. Then, centrifuge the resulting solution with deionized water at 3000-4000 rpm for 3-10 min; take the lower precipitate and centrifuge with deionized water at 3000-4000 rpm. Centrifuge and wash for 3-10 min; repeat the operation of "take the lower layer precipitate and centrifuge and wash with deionized water" multiple times until the pH of the supernatant is 5.8-6.
2. Then add 50-100 mL of deionized water to the obtained precipitate, sonicate for 1-1.5 h, and centrifuge at 3000-4000 r for 40-60 min. The obtained supernatant is the MXene monolayer nanosheet solution. After dilution or concentration, the concentration of MXene monolayer nanosheets is 8-15 mg / mL. (2) Preparation of MXene@TA solution Dissolve 40-60 mg of tannic acid in 10-20 mL of deionized water to obtain an aqueous solution of tannic acid; then add 10-20 mL of MXene monolayer nanosheet solution with a concentration of 8-15 mg / mL to the aqueous solution of tannic acid, and stir the mixture at room temperature in the dark for 10-20 h to obtain an MXene@TA solution. (3) Preparation of MXene@TA / Cu-GOxNF Take 5-10 mL of the MXene@TA solution prepared in step (2), add 5-10 mL of PBS buffer, then add 200-400 μL of 0.1-0.2 mol / L CuSO4 aqueous solution, mix and sonicate at 30-40℃ for 1-2 h; then add 15-30 mg of glucose oxidase, let stand at room temperature for 2-4 days, wash and freeze dry to obtain MXene@TA / Cu-GOxNF; (4) Preparation of MXene@TA / Cu-GOxNF electrode Add PBS buffer to the MXene@TA / Cu-GOxNF obtained in step (3) to make the concentration of MXene@TA / Cu-GOxNF 8~15mg / mL; take 5~10μL of the solution and drop it onto a glassy carbon electrode with a diameter of 3~5mm, dry it under N2 conditions, then drop 5~10μL of Nafion solution onto the electrode surface, dry it under N2 conditions, and obtain the MXene@TA / Cu-GOxNF electrode; (5) Preparation of enzyme-glucose electrochemical biosensor Using the MXene@TA / Cu-GOxNF electrode obtained in step (4) as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode, an enzyme glucose electrochemical biosensor based on a triple stable structure was assembled.
2. An enzyme glucose electrochemical biosensor based on a triple stable structure, characterized in that: It is prepared by the preparation method described in claim 1.