A reusable flexible enzymatic glucose sensor

Abstract

The application discloses a reusable flexible enzyme glucose sensor, which comprises a flexible substrate and an electrode system arranged on the flexible substrate; the electrode system comprises a working electrode, a counter electrode and a reference electrode; a functional layer is modified on the working electrode, and the functional layer is formed by interlacing and supporting of a beta-cyclodextrin / carboxyl carbon nanotube composite material and gold nanoparticles; and chitosan immobilized enzyme is further modified on the functional layer. The sensor prepared by using the application can specifically detect glucose, can exclude the influence of interfering substances, such as urea and ascorbic acid, on the detection of glucose, and can be reused.

Description

Technical Field

[0001] This invention relates to a reusable flexible enzyme glucose sensor. Background Technology

[0002] Glucose is the most important carbohydrate for the human body. According to the World Health Organization, approximately 3.4 million people die from high blood sugar, and there are currently 143 million people worldwide with diabetes. Abnormally high levels of glucose in biofluids can lead to serious complications such as blindness, heart disease, high blood pressure, and kidney failure. Therefore, glucose sensors play a crucial role in diabetes diagnosis, the food industry, and biotechnology. Continuous monitoring of blood glucose levels is essential for their diagnosis and treatment.

[0003] Although electrochemical glucose sensors have emerged in large numbers in recent years, these novel materials exhibit certain drawbacks in glucose detection. For example, the oxidation selectivity of enzyme-free glucose sensors is not as good as that of enzyme electrode sensors, and corresponding response currents can still be observed when large amounts of ascorbic acid and urea are present in the sample. Furthermore, the stability of the sensor remains a challenge for enzyme sensors due to the easy inactivation of glucose oxidase. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a reusable flexible enzyme glucose sensor with high sensitivity and stability.

[0005] To solve the above problems, the present invention adopts the following technical solution:

[0006] A reusable flexible enzyme glucose sensor is characterized in that: the flexible enzyme glucose sensor includes a flexible substrate and an electrode system disposed on the flexible substrate; the electrode system includes a working electrode, a counter electrode and a reference electrode; a functional layer is modified on the working electrode, the functional layer being formed by interleaving and supporting β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles; and a chitosan immobilized enzyme is further modified on the functional layer.

[0007] Furthermore, the functional layer is obtained by first modifying the surface of the working electrode with a mixed solution of β-cyclodextrin and carboxylated carbon nanotubes by drop coating, and then modifying the carboxylated carbon nanotubes with gold nanoparticles by electrodeposition.

[0008] Furthermore, the chitosan immobilized enzyme is formed by drop-coating a mixed solution of glucose oxidase and chitosan onto the functional layer.

[0009] Furthermore, the mass ratio of carboxylated carbon nanotubes to β-cyclodextrin in the β-cyclodextrin / carboxylated carbon nanotube composite material is 1:0.3-3.

[0010] Furthermore, the flexible substrate is made of polyethylene terephthalate (PET).

[0011] Furthermore, the counter electrode and the working electrode are formed by printing conductive carbon paste on a flexible substrate, and the reference electrode is formed by printing conductive silver paste on a flexible substrate.

[0012] Furthermore, the method for modifying the working electrode is as follows:

[0013] Step 1: β-Cyclodextrin and carboxylated carbon nanotubes are added to an ethanol solution and ultrasonically mixed until homogeneous. The resulting mixture is then drop-coated onto the surface of the activated working electrode. After drying, a Nafion ethanol mixture is added drop-coated to form a working electrode modified with a β-cyclodextrin / carboxylated carbon nanotube composite material. Since the cyclodextrin and carboxylated carbon nanotube composite material is hydrophilic, and an auric acid solution is used for gold deposition, Nafion is added drop-coated to prevent the composite material from detaching during the deposition process. This forms a selectively permeable membrane without affecting the deposition process and also solves the problem of material detachment.

[0014] Step 2: The working electrode modified with the β-cyclodextrin / carboxylated carbon nanotube composite material obtained in Step 1 is electrodeposited in chloroauric acid solution to modify gold nanoparticles on the carboxylated carbon nanotubes, thereby obtaining an electrode material containing a multi-level structure of β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles; the concentration of chloroauric acid solution is 3-5 mM, the electrodeposition voltage range is between -0.3 and -0.05 V, and the deposition time range is between 10 and 60 min.

[0015] Step 3: Disperse glucose oxidase in chitosan solution and ultrasonically disperse until uniform. Then, drop-coat the resulting mixed solution onto the electrode surface prepared in Step 2 to obtain the modified working electrode. The pH value of the chitosan solution is between 4.2 and 6.3, and the mass concentration is between 0.1% and 1.0%. The concentration of glucose oxidase in the resulting mixed solution is 15–30 mg / mL.

[0016] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0017] 1. This invention introduces β-cyclodextrin, carboxylated carbon nanotubes and gold nanoparticles into the working electrode. The β-cyclodextrin-dispersed carboxylated carbon nanotubes provide more active sites, while the gold nanoparticles have an affinity for carboxyl groups, which allows more gold nanoparticles to be deposited on the electrode surface, thereby improving the electron transfer rate and conductivity of the sensing process.

[0018] 2. The β-cyclodextrin-carboxylated carbon nanotube-gold nanoparticle-chitosan composite material can serve to carry enzymes, enhance or maintain enzyme activity, and increase electron transfer performance, thereby improving the sensitivity and stability of the sensor and enabling its reusability. The electrochemical glucose sensor prepared using this invention can specifically detect glucose, eliminate the influence of interfering substances such as urea and ascorbic acid on glucose detection, and is reusable; even after multiple uses within two months, its current response remains excellent. Attached Figure Description

[0019] Figures 1-3 The images shown are scanning electron microscope (SEM) images of the modified electrode surfaces prepared in steps 1 to 3 of Example 1 of this invention.

[0020] Figure 4 The image shows a comparison of the current response of the working electrode obtained in step 2 under different mass ratios of β-cyclodextrin and carboxylated carbon nanotubes.

[0021] Figure 5 The image shows a comparison of the current response of the working electrode obtained in step 3 when the electrodeposition voltages are -0.3V, -0.2V, and -0.1V, respectively.

[0022] Figure 6 This is a comparison of the current response of the working electrode obtained in step 3 at different electrodeposition times.

[0023] Figure 7 This is a stability diagram of the glucose sensor prepared in Example 1 of the present invention.

[0024] Figure 8 This is a reproducibility diagram of the glucose sensor prepared in Example 1 of the present invention.

[0025] Figure 9 This is a graph showing the anti-interference performance of the glucose sensor prepared in Example 1 of the present invention.

[0026] Figure 10 This is a cyclic voltammogram of the glucose sensor prepared in Example 1 of the present invention.

[0027] Figure 11 The linear relationship between the reduction peak value of the glucose sensor prepared in Example 1 of this invention and the glucose concentration in the range of 0.2mM-3mM is shown. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below through specific embodiments. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0029] Example 1

[0030] The flexible enzyme glucose sensor provided in this embodiment includes a flexible PET substrate and an electrode system disposed on the flexible substrate; the electrode system includes a working electrode, a counter electrode, and a reference electrode; the counter electrode and the working electrode are formed by printing conductive carbon paste on the flexible substrate, and the reference electrode is formed by printing conductive silver paste on the flexible substrate.

[0031] A functional layer is modified onto the working electrode. This functional layer is composed of an interwoven and supported β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles. Chitosan-immobilized enzymes are also modified onto the functional layer. The specific modification method for the working electrode is as follows:

[0032] Step 1, Electrode activation: Soak the electrode in 0.05 mol / mL sulfuric acid solution for 2 hours, then use sulfuric acid solution as electrolyte to scan 20 times by cyclic voltammetry. After the peak value of the CV graph is basically stable, take it out, rinse it with pure water, air dry it at room temperature, and store it at 4℃.

[0033] Step 2: Add 2 mg of β-cyclodextrin and 2 mg of carboxylated carbon nanotubes (mass ratio of 1:1) to 1 mL of ethanol solution, mix thoroughly by ultrasonication, and drop the resulting mixed solution onto the surface of the activated working electrode. After standing at room temperature until dry, add 10 μL of Nafion ethanol mixed solution (Nafion volume percentage of 0.5%) to form a working electrode modified with β-cyclodextrin / carboxylated carbon nanotube composite material.

[0034] Step 3: The working electrode modified with the β-cyclodextrin / carboxylated carbon nanotube composite material obtained in Step 2 is electrodeposited in 4mM chloroauric acid solution. The electrodeposition voltage is -0.3V and the deposition time is 40min, so that gold nanoparticles are modified on the carboxylated carbon nanotubes, and an electrode material containing a multi-level structure of β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles is obtained.

[0035] Step 4: Disperse glucose oxidase in a 0.2% (pH=5) chitosan solution and ultrasonically disperse until homogeneous. Then, drop-coat the resulting mixed solution onto the electrode surface prepared in Step 3 and allow it to stand for 12 hours to allow glucose oxidase to deposit on the electrode surface, thus obtaining the modified working electrode. The concentration of glucose oxidase in the mixed solution is 30 mg / mL. The prepared electrode is stored at 4°C.

[0036] I. Morphological Characterization

[0037] Figures 1-3 The images shown are scanning electron microscope (SEM) images of the modified electrode surfaces prepared in steps 1 to 3 of Example 1 of this invention. Figure 1 This confirms that the β-cyclodextrin / multi-walled carbon nanotube composite material modified on the electrode surface in step 2 was indeed present. Figure 2This confirmed that the morphology of the electrode surface changed as gold was electrodeposited in step 3, and gold nanoparticles were successfully deposited on the surface of the interlaced carbon nanotubes. Figure 3 This confirmed that a chitosan-immobilized glucose oxidase membrane was successfully modified on the electrode surface after step 4.

[0038] II. Optimization of the mass ratio of β-cyclodextrin and carboxylated carbon nanotubes

[0039] The mass ratio of β-cyclodextrin to carboxylated carbon nanotubes in step 2 of Example 1 was set to 1:1 (2 mg β-cyclodextrin and 2 mg carboxylated carbon nanotubes), 1:3 (2 mg β-cyclodextrin and 6 mg carboxylated carbon nanotubes), and 3:1 (6 mg β-cyclodextrin and 2 mg carboxylated carbon nanotubes), respectively, to prepare a working electrode modified with β-cyclodextrin / carboxylated carbon nanotube composite material.

[0040] The modified electrode was tested by cyclic voltammetry on a CHI660E electrochemical workstation. The electrolyte was a PBS solution with pH=7.0 containing 1mM glucose. Figure 4 The image shows a comparison of the current response of the working electrode modified with the β-cyclodextrin / carboxycarbon nanotube composite material obtained in step 2 under different mass ratios of β-cyclodextrin and carboxycarbon nanotubes. It can be seen that the sensor with a mass ratio of β-cyclodextrin to carboxycarbon nanotubes of 1:1 has a higher current response for glucose detection.

[0041] III. Optimization of Electrodeposition Voltage

[0042] The electrodeposition voltage in step 3 of Example 1 was set to -0.3, -0.2, and -0.1V respectively to prepare electrode materials containing a multi-level structure of β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles.

[0043] The modified electrode was tested by cyclic voltammetry on a CHI660E electrochemical workstation. The electrolyte was a PBS solution with pH=7.0 containing 1mM glucose. Figure 5 The image shows a comparison of the current response of the working electrode obtained in step 3 when the electrodeposition voltages are -0.3, -0.2, and -0.1V, respectively. The sensor with gold nanoparticles deposited at a potential of -0.3V has a higher current response for glucose detection.

[0044] IV. Optimization of Electrodeposition Time

[0045] The electrodeposition time in step 3 of Example 1 was set to 10 min, 20 min, 40 min and 60 min respectively to obtain electrode materials containing β-cyclodextrin / carboxylated carbon nanotube composite materials and gold nanoparticle hierarchical structures.

[0046] The modified electrode was tested by cyclic voltammetry on a CHI660E electrochemical workstation. The electrolyte was a PBS solution with pH=7.0 containing 1mM glucose. Figure 6 The graph shows a comparison of the current response of the working electrode obtained in step 3 at different electrodeposition times. The sensor has a higher current response to glucose detection when the electrodeposition time is 40 min.

[0047] V. Response of the sensor obtained in Example 1 to glucose

[0048] The sensor described in Example 1 was tested by cyclic voltammetry on a CHI660E electrochemical workstation. The electrolyte was a PBS solution with pH=7.0 containing 1 mM glucose. Figure 7 The graph shows the stability of the glucose sensor prepared in Example 1. It can be seen from the graph that the current intensity of the electrode remained at 97% of the original value after one week and the RSD was 3.1%. After two months, the current intensity was 80% of the original value, which confirms that the modified electrode has high stability and can be reused.

[0049] The sensor described in Example 1 was tested by cyclic voltammetry on a CHI660E electrochemical workstation. The electrolyte was a PBS solution with pH=7.0 containing 1mM glucose. The starting voltage was set to 0.2V, the voltage range was between -0.6V and 1.2V, and the scan rate was set to 0.05V / s. Figure 8 The graph shows the reproducibility of the glucose sensor prepared in Example 1. As can be seen from the graph, after 20 scans, the curves basically overlap, showing good reproducibility.

[0050] Figure 9 The graph shows the anti-interference performance of the glucose sensor prepared in Example 1. First, the response to 1mM glucose was detected. Then, when 1mM ascorbic acid and 1mM urea were added to the 1mM glucose solution, the sensor did not show a large current response, indicating that the prepared sensor has strong anti-interference performance.

[0051] Figure 10 The image shows the cyclic voltammetry curves of the glucose sensor prepared in Example 1 within a glucose concentration range of 0.2 mM to 3 mM. From bottom to top, the glucose concentrations are 0.2, 0.4, 0.6, 0.8, 1.0, 2, and 3 mM. It can be seen that the reduction current increases with increasing glucose concentration. Simultaneously, it can be obtained... Figure 11 The linear relationship.

[0052] Figure 11 The linear relationship between the reduction peak value and glucose concentration of the glucose sensor prepared in Example 1 in the glucose concentration range of 0.2 mM-3 mM is shown. Based on y = 17.645x - 38.469, R... 2=0.993 indicates that the sensor exhibits good linearity in the 0.2mM-1mM range. The calculated detection limit is 6.5μM, and the sensitivity is 716.402μA / mM / cm. 2 .

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reusable flexible enzyme glucose sensor, characterized in that: The flexible enzyme glucose sensor includes a flexible substrate and an electrode system disposed on the flexible substrate; the electrode system includes a working electrode, a counter electrode, and a reference electrode; a functional layer is modified on the working electrode, the functional layer being formed by interleaving and supporting β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles, wherein the mass ratio of carboxylated carbon nanotubes to β-cyclodextrin in the β-cyclodextrin / carboxylated carbon nanotube composite material is 1:0.3-3; a chitosan-immobilized enzyme is also modified on the functional layer; The functional layer is obtained by first modifying the surface of the working electrode with a mixed solution of β-cyclodextrin and carboxylated carbon nanotubes by drop coating, and then modifying the carboxylated carbon nanotubes with gold nanoparticles by electrodeposition. The chitosan-immobilized enzyme is formed by drop-coating a mixed solution of glucose oxidase and chitosan onto a functional layer.

2. The flexible enzyme glucose sensor according to claim 1, characterized in that: The flexible substrate is made of polyethylene terephthalate (PET).

3. The flexible enzyme glucose sensor according to claim 1, characterized in that: The counter electrode and the working electrode are formed by printing conductive carbon paste on a flexible substrate, and the reference electrode is formed by printing conductive silver paste on a flexible substrate.

4. The flexible enzyme glucose sensor according to claim 1, characterized in that, The modification method for the working electrode is as follows: Step 1: Add β-cyclodextrin and carboxylated carbon nanotubes to an ethanol solution, mix them evenly by ultrasonication, and drop the resulting mixed solution onto the surface of the activated working electrode. After drying, add Nafion ethanol mixed solution to form a working electrode modified with β-cyclodextrin / carboxylated carbon nanotube composite material. Step 2: Electrodeposit the β-cyclodextrin / carboxylated carbon nanotube composite material modified working electrode obtained in Step 1 in chloroauric acid solution to modify gold nanoparticles on the carboxylated carbon nanotubes, thereby obtaining an electrode material containing a multi-level structure of β-cyclodextrin / carboxylated carbon nanotube composite material and gold nanoparticles. Step 3: Disperse glucose oxidase in chitosan solution and ultrasonically disperse it evenly. Then, drop the resulting mixed solution onto the electrode surface prepared in step 2 to obtain the modified working electrode.

5. The flexible enzyme glucose sensor according to claim 4, characterized in that: In step 2, the concentration of the chloroauric acid solution is 3~5mM, the electrodeposition voltage range is -0.3~-0.05V, and the deposition time range is 10~60min.

6. The flexible enzyme glucose sensor according to claim 4, characterized in that: In step 3, the pH value of the chitosan solution is between 4.2 and 6.3, the mass concentration is between 0.1 and 1.0%, and the concentration of glucose oxidase in the resulting mixed solution is 15 to 30 mg / mL.

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