Preparation method and application of an enzyme-free electrochemical sensor with a modified electrode

Abstract

The present invention is applicable to the field of electrochemical sensors and provides a method for preparing an enzyme-free electrochemical sensor with a modified electrode and its application, comprising the following steps: drawing a capillary quartz tube into a needle-shaped nanoquartz tube, uniformly distributing carbon nanoparticles on the tip of the nanoneedle tip to form a carbon nanotube (CNE); dissolving HAP, sodium hexametaphosphate, and potassium chloride in deionized water at a concentration ratio of 0.01 to 0.1:1:1, and ultrasonically forming a dispersion; placing the CNE as a working electrode, Ag / AgCl as a reference electrode, and a platinum wire as an auxiliary electrode in an electrolytic cell containing a HAP suspension solution, applying an external positive voltage to the working electrode CNE, and completing constant potential deposition to prepare an nHAP-CNE modified sensor. The sensor prepared based on the nHAP modified electrode has the advantages of high sensitivity, low sample requirement, high biological activity, and suitability for in vivo cell detection in detecting ascorbic acid.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical sensors, and in particular relates to a preparation method and application of an enzyme-free electrochemical sensor with modified electrodes. Background Art

[0002] Ascorbic acid, a product of biological metabolism, is a small biological molecule with strong biological activity and is susceptible to denaturation. Rapid, accurate, and highly resistant to interference are needed for its detection and analysis. Common methods for ascorbic acid detection include: oxidant dye titration, such as 2,6-dichlorophenol indophenol. This method is simple, rapid, and provides relatively accurate results, but is susceptible to interference from other reducing substances and has weak resistance to interference. Colorimetric methods, such as the 2,4-dinitrophenylhydrazine method, are currently standard methods for determining AA and offer high accuracy, but require a long operation time. Ultraviolet spectrophotometry is a rapid and low-cost method, but its detection process involves multiple steps and is not straightforward. Fluorescence quenching is a newer method for detecting ascorbic acid, but it requires high reagent requirements, is complex, and is expensive. Furthermore, ascorbic acid is easily oxidized in the presence of oxygen, metal ions, and high temperatures, resulting in shortcomings in all of these methods.

[0003] Since the 20th century, electrochemical methods have become one of the most popular detection methods due to their low cost, high efficiency and precision. Electrochemical methods are a fast and accurate detection method with many unique advantages, such as simple sample processing, low experimental temperature, easy micro-control and excellent anti-interference performance. Therefore, they have become one of the most popular detection methods currently being studied.

[0004] As a biomaterial, HAP has a unique crystal structure, numerous adsorption sites, and strong adsorption capacity. At the same time, HAP has specific recognition and selective response capabilities for biological molecules. It has been used to manufacture biosensors for detecting organic molecules, biological molecules, DNA, and viruses. Because organisms are complex organisms, ascorbic acid is highly active and low in content. As a biomaterial, nHAP has good biocompatibility and specific recognition. As an electrochemical catalytic material, it can ensure the biological activity of the substance to be tested with high precision. At the same time, the performance of nHAP's multiple adsorption sites makes it an excellent modified electrode material. In the past, electrochemical sensors for ascorbic acid detection generally used glassy carbon electrodes, which are at the macroscopic electrode dimension and difficult to detect trace substances. To date, there have been no public reports on nHAP-modified CNE as an electrochemical sensor. Summary of the Invention

[0005] The purpose of the embodiments of the present invention is to provide a method for preparing and applying an enzyme-free electrochemical sensor with a modified electrode, aiming to solve the problem that electrochemical sensors for ascorbic acid detection generally use glassy carbon electrodes, which are in the macroscopic electrode dimension and difficult to detect trace substances.

[0006] The present invention is achieved by a method for preparing an enzyme-free electrochemical sensor with a modified electrode and its application, comprising the following steps:

[0007] Step 1: Preparation of CNE electrode: A cleaned capillary quartz tube is drawn into a needle-shaped nano-quartz tube using a laser drawing instrument. Carbon nanoparticles are evenly distributed on the tip of the nano-needle tip using a vapor deposition method to form a CNE.

[0008] Step 2: Preparation of HAP suspension: HAP, sodium hexametaphosphate, and potassium chloride were dissolved in deionized water at a concentration ratio of (0.01-0.1):1, and ultrasonicated to form a dispersion;

[0009] Step 3: The CNE prepared in step 1 was used as the working electrode, Ag / AgCl was used as the reference electrode, and platinum wire was used as the auxiliary electrode. The three electrodes together formed a three-electrode system. The three-electrode system was placed in an electrolytic cell containing the HAP suspension solution prepared in step 2. An external positive voltage was applied to the working electrode CNE using an electrochemical workstation to complete constant potential deposition to prepare an nHAP-CNE modified sensor.

[0010] According to a further technical solution, the capillary quartz tube in step 1 is cleaned by soaking the capillary quartz tube in piranha solution for 20-40 minutes and then rinsing it with deionized water.

[0011] According to a further technical solution, the nanopore size of the needle-shaped nano-quartz tube in step 1 is 30 nm to 80 nm.

[0012] According to a further technical solution, the vapor precipitation method in step 1 selects high-temperature pyrolysis of butane to obtain pyrolytic carbon.

[0013] According to a further technical solution, the protective gas for the vapor deposition method in step 1 is nitrogen or hydrogen.

[0014] According to a further technical solution, the ultrasonic dispersion time in step 2 is 20-60 minutes.

[0015] According to a further technical solution, the constant potential deposition method in step three is chronoamperometry, and the applied voltage of the constant potential deposition is 1-10 mV.

[0016] According to a further technical solution, the time for constant potential deposition in step three is 1000-2000s.

[0017] A method for preparing an enzyme-free electrochemical sensor of a modified electrode is provided, and the nHAP-CNE modified sensor is used in the electrocatalytic oxidation of ascorbic acid.

[0018] The invention discloses an application of an nHAP-CNE modified sensor prepared by a method for preparing an enzyme-free electrochemical sensor of a modified electrode in a PBS buffer solution with a pH of 2 to 10.

[0019] The present invention provides a method for preparing an enzyme-free electrochemical sensor with a modified electrode and its application. The present invention uses nHAP as a modifying material for the sensing working electrode, thereby overcoming the disadvantage of ascorbic acid being easily unstable on traditional electrodes; using nHAP as a modifying material for the sensing working electrode overcomes the disadvantage of requiring an enzyme as a reaction catalyst; using nHAP as a modifying material for the sensing working electrode increases the specific surface area of the modified electrode, enhances the charge transfer rate, and improves the disadvantage of weak adsorption capacity of traditional electrodes; the sensor prepared based on the nHAP modified electrode of the present invention has the advantages of high sensitivity, small sample requirements, high biological activity, and suitability for cell live detection in detecting ascorbic acid; the nHAP-CNE modified sensor prepared by the present invention has low cost, high efficiency, no need for enzyme mediator participation, high biological activity, and is suitable for cell live detection; the nHAP-CNE modified sensor can be used in 1×10 -5 ~2×10 -3 mol / L, and the detection limit of ascorbic acid (AA) can reach 1.3×10 -7 mol / L, with excellent AA sensing detection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of a scanning electron microscope (SEM) image of the unmodified sensor prepared in Example 2 of the present invention;

[0021] Figure 2 This is a SEM schematic diagram of the nHAP-CNE modified sensor prepared in Example 2 of the present invention;

[0022] Figure 3 Characteristic curves of the nHAP-CNE modified sensor and the unmodified sensor prepared in Example 2 of the present invention;

[0023] Figure 4 CV curves of ascorbic acid detected by the nHAP-CNE modified sensor and the unmodified sensor prepared in Example 4 of the present invention;

[0024] Figure 5 CV curves of the nHAP-CNE modified sensor prepared in Example 4 of the present invention detecting ascorbic acid at different concentrations;

[0025] Figure 6 This is the CA curve of the nHAP-CNE modified sensor prepared in Example 5 of the present invention for detecting trace amounts of ascorbic acid;

[0026] Figure 7 This is the CA curve of the nHAP-CNE modified sensor prepared in Example 6 of the present invention when detecting ascorbic acid and resisting pH interference. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0029] Example 1, a method for preparing a non-enzymatic electrochemical sensor with a modified electrode provided in one embodiment of the present invention, comprises the following steps:

[0030] Step 1: Preparation of a CNE electrode: Soak a capillary quartz tube in piranha solution for 20 minutes and then rinse with deionized water. Use a laser puller to pull the cleaned capillary quartz tube into a needle-shaped nano-quartz tube with a nanopore size of 30 nm. Use vapor deposition to evenly distribute carbon nanoparticles on the tip of the nano-needle tip to form a CNE. Use a vapor deposition method to obtain pyrolytic carbon by pyrolyzing butane at high temperature. The protective gas for the vapor deposition method is nitrogen or hydrogen.

[0031] Step 2: Preparation of HAP suspension: HAP, sodium hexametaphosphate, and potassium chloride were dissolved in deionized water at a concentration ratio of 0.01:1:1, and ultrasonicated for 20-60 minutes to form a dispersion;

[0032] Step 3: The CNE prepared in step 1 was used as the working electrode, Ag / AgCl was used as the reference electrode, and platinum wire was used as the auxiliary electrode. The three electrodes together constituted a three-electrode system. The three-electrode system was placed in an electrolytic cell containing the HAP suspension solution prepared in step 2. An external positive voltage of 1 mV was applied to the working electrode CNE using an electrochemical workstation. The constant potential deposition time was 1000 s. The constant potential deposition was completed to prepare an nHAP-CNE modified sensor.

[0033] Example 2, a method for preparing a non-enzymatic electrochemical sensor with a modified electrode provided in one embodiment of the present invention, comprises the following steps:

[0034] Step 1: Preparation of a CNE electrode: Soak a capillary quartz tube in piranha solution for 30 minutes and then rinse with deionized water. Use a laser puller to pull the cleaned capillary quartz tube into a needle-shaped nano-quartz tube with a nanopore size of 55 nm. Use vapor deposition to evenly distribute carbon nanoparticles on the tip of the nano-needle tip to form a CNE. Use a vapor deposition method to obtain pyrolytic carbon by pyrolyzing butane at high temperature. The protective gas for the vapor deposition method is nitrogen or hydrogen.

[0035] Step 2: Preparation of HAP suspension: HAP, sodium hexametaphosphate, and potassium chloride were dissolved in deionized water at a concentration ratio of 0.05:1:1, and ultrasonicated for 40 minutes to form a dispersion;

[0036] Step 3: The CNE prepared in step 1 was used as the working electrode, Ag / AgCl was used as the reference electrode, and platinum wire was used as the auxiliary electrode. The three electrodes together constituted a three-electrode system. The three-electrode system was placed in an electrolytic cell containing the HAP suspension solution prepared in step 2. An external positive voltage of 5.5 mV was applied to the working electrode CNE using an electrochemical workstation. The constant potential deposition time was 1500 s. The constant potential deposition was completed to prepare the nHAP-CNE modified sensor.

[0037] Example 3, a method for preparing a non-enzymatic electrochemical sensor with a modified electrode provided in one embodiment of the present invention, comprises the following steps:

[0038] Step 1: Preparation of a CNE electrode: Soak a capillary quartz tube in piranha solution for 40 minutes and then rinse with deionized water. Use a laser puller to pull the cleaned capillary quartz tube into a needle-shaped nano-quartz tube with a nanopore size of 80 nm. Use vapor deposition to evenly distribute carbon nanoparticles on the tip of the nano-needle tip to form a CNE. Use a vapor deposition method to obtain pyrolytic carbon by pyrolyzing butane at high temperature. The protective gas for the vapor deposition method is nitrogen or hydrogen.

[0039] Step 2: Preparation of HAP suspension: HAP, sodium hexametaphosphate, and potassium chloride were dissolved in deionized water at a concentration ratio of 0.1:1:1, and ultrasonicated for 60 minutes to form a dispersion;

[0040] Step 3: The CNE prepared in step 1 was used as the working electrode, Ag / AgCl was used as the reference electrode, and platinum wire was used as the auxiliary electrode. The three electrodes together constituted a three-electrode system. The three-electrode system was placed in an electrolytic cell containing the HAP suspension solution prepared in step 2. An external positive voltage of 10 mV was applied to the working electrode CNE using an electrochemical workstation. The constant potential deposition time was 2000 s. The constant potential deposition was completed to prepare the nHAP-CNE modified sensor.

[0041] Figure 1 and Figure 2 The SEM images of the unmodified sensor CNE and the nHAP-CNE modified sensor prepared in Example 2 are shown in FIG. Figure 1 and Figure 2 It can be seen that nHAP has been loaded onto CNE.

[0042] The nHAP-CNE modified sensor obtained in Example 2, the AgCl electrode and the Pt counter electrode constituted a three-electrode system, and a cyclic voltammetry test was performed in FcMeOH to obtain the characteristic curve of the electrode. The CV curve is as follows: Figure 3 .

[0043] Depend on Figure 3 It can be seen that the CV curves of the unmodified sensor CNE and the nHAP-CNE modified sensor are both "S"-shaped, indicating that both are nano-sized electrodes. According to the formula for calculating the effective area of nanoelectrodes:

[0044]

[0045] The ratio of the effective area of the nHAP-CNE-modified sensor to the bare carbon electrode was 100:1. This indicates that both the CNE and nHAP-CNE-modified sensors retained distinct nanoelectrode characteristics before and after modification. Furthermore, modification significantly increased the effective area and signal-to-noise ratio, indicating enhanced electrochemical activity of the nHAP-CNE-modified sensor.

[0046] Example 4, application of nHAP-CNE modified sensor in electrocatalytic oxidation of ascorbic acid, direct electrochemistry of ascorbic acid on nHAP-CNE modified electrode;

[0047] In Example 2, a three-electrode system consisting of an nHAP-CNE modified sensor or an unmodified sensor, an Ag / AgCl electrode, and a Pt counter electrode was obtained. Cyclic voltammetry was performed in a solution containing 0.1 mmol / L-10 mmol / L ascorbic acid. The CV curves of the nHAP-CNE modified sensor with a content of 0.1 mmol / L-10 mmol / L ascorbic acid were obtained as shown in FIG. Figure 4 and Figure 5 .

[0048] like Figure 4 As shown, no ascorbic acid oxidation peak current was observed on the unmodified sensor CNE. However, on the nHAP-CNE-modified sensor, an oxidation peak current of Ipa = 4.14 nA and an oxidation peak potential of Epa = 90 mV were observed, demonstrating the electrocatalytic properties of nHAP towards ascorbic acid. Therefore, the nHAP-CNE-modified sensor has a positive effect on the ascorbic acid oxidation reaction, improving detection sensitivity and signal-to-noise ratio.

[0049] like Figure 5 As shown in the figure, AA exhibits a good CV response on the nHAP-CNE modified sensor within the concentration range of 0.1 mmol / L to 10 mmol / L, and the oxidation peak current of AA increases with increasing concentration. Therefore, the HAP-CNE modified electrode has both detection capability and significant catalytic performance for the redox reaction of AA.

[0050] Example 5, trace detection of ascorbic acid using nHAP-CNE modified electrical sensor;

[0051] In Example 2, a three-electrode system consisting of a nHAP-CNE modified sensor, an Ag / AgCl electrode, and a Pt counter electrode was obtained. Ascorbic acid solution was added dropwise to an electrolyte solution of pH = 7, and a chronoamperometric test was performed. The CA curve was as follows: Figure 6 As can be seen from the figure, in 1×10 -5 mol / L-2×10 -3 In the mol / L range, the current increases with the increase of ascorbic acid concentration. It can be seen that the response of the nHAP-CNE modified electrical sensor (nanohydroxyapatite modified sensor) to ascorbic acid is a Nernst response. The current signal (y) is linearly related to the AA concentration (x), and the regression equation is y = 0.329x + 0.315 (nA). The response slope is 3.29×10 -7 A / mol / L, the linear correlation coefficient was 0.995, and the detection limit (S / N=3) was 1.3×10 -7 mol / L. Compared with other electrodes, the nHAP-CNE modified sensor has a lower detection limit. -5 mol / L-2×10 -3 Ascorbic acid can be effectively detected in the concentration range of 1 mol / L.

[0052] Example 6, anti-interference performance experiment of nHAP-CNE modified sensor in detecting ascorbic acid;

[0053] In Example 2, a three-electrode system consisting of an nHAP-CNE modified sensor, an Ag / AgCl electrode, and a Pt counter electrode was obtained. In the range of pH = 3 to 10, ascorbic acid solution was added dropwise to electrolyte solutions with different initial concentrations and pH values, and a chronoamperometric test was performed. The CA curve was as follows: Figure 7 .

[0054] Depend on Figure 7 It can be seen that the nHAP-CNE modified sensor can stably detect ascorbic acid in the experimental pH range of 3-10.

[0055] 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 in the scope of protection of the present invention.

Claims

1. Application of nHAP-CNE modified sensor prepared by a method for preparing a non-enzymatic electrochemical sensor of modified electrode in electrocatalytic oxidation of ascorbic acid, characterized in that: The method for preparing the enzyme-free electrochemical sensor of the modified electrode comprises the following steps: Step 1: Preparation of CNE electrode: A cleaned capillary quartz tube is drawn into a needle-shaped nano-quartz tube using a laser drawing instrument. Carbon nanoparticles are evenly distributed on the tip of the nano-needle tip using a vapor deposition method to form a CNE. Step 2: Preparation of HAP suspension: Dissolve HAP, sodium hexametaphosphate, and potassium chloride in deionized water at a concentration ratio of 0.01-0.1:1:1, and sonicate to form a dispersion. Step 3: The CNE prepared in step 1 was used as the working electrode, Ag / AgCl was used as the reference electrode, and platinum wire was used as the auxiliary electrode. The three electrodes together formed a three-electrode system. The three-electrode system was placed in an electrolytic cell containing the HAP suspension solution prepared in step 2. An external positive voltage was applied to the working electrode CNE using an electrochemical workstation to complete constant potential deposition to prepare an nHAP-CNE modified sensor.

2. The use according to claim 1, characterized in that In the step 1, the capillary quartz tube is cleaned by soaking the capillary quartz tube in piranha solution for 20-40 minutes and then rinsing it with deionized water.

3. The use according to claim 1, characterized in that The nanopore size of the needle-shaped nano-quartz tube in step 1 is 30 nm-80 nm.

4. The use according to claim 1, characterized in that The vapor deposition method in step 1 selects high-temperature pyrolysis of butane to obtain pyrolytic carbon.

5. The use according to claim 1, characterized in that The protective gas for the vapor deposition method in step 1 is nitrogen or hydrogen.

6. The use according to claim 1, characterized in that The ultrasonic dispersion time in step 2 is 20-60 min.

7. The use according to claim 1, characterized in that The constant potential deposition method in step three is chronoamperometry, and the constant potential deposition applied voltage is 1-10 mV.

8. The use according to claim 1, characterized in that The constant potential deposition time in step 3 is 1000-2000s.

9. The use according to claim 1, characterized in that The application is the application of the nHAP-CNE modified sensor in a PBS buffer solution with a pH of 2-10.

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