Electrode material for ascorbic acid sensor, biosensor, preparation method and application of biosensor
阅读说明：本技术 一种用于抗坏血酸传感器的电极材料、生物传感器及其制备方法和应用 (Electrode material for ascorbic acid sensor, biosensor, preparation method and application of biosensor ) 是由 李勇 张玉新 周鱼跃 薛伟 周应继 张瑞珠 于 2020-04-07 设计创作，主要内容包括：本发明涉及生物传感器技术领域,具体来说是一种用于抗坏血酸传感器的电极材料、生物传感器及其制备方法和应用,由以下方法制成：将表面去油处理后的泡沫镍通过水热氧化法进行表面氧化,制备得到Ni@NiO复合材料,再将氧化后的碳纳米管通过电喷法对Ni@NiO复合材料表面修饰,得到Ni@NiO复合材料/碳纳米管,再将抗坏血酸氧化酶负载在Ni@NiO复合材料/碳纳米管表面,制备得到了用于检测抗坏血酸的电极材料。本发明还将该电极材料和参比电极及对电极构成了生物传感器,能够采用电化学方法对抗坏血酸进行检测,且对抗坏血酸的检测具有高的灵敏度和低的检测限,响应时间迅速且选择性高。(The invention relates to the technical field of biosensors, in particular to an electrode material for an ascorbic acid sensor, the biosensor, a preparation method and an application thereof, wherein the electrode material is prepared by the following steps: the method comprises the steps of carrying out surface oxidation on foamed nickel subjected to surface deoiling treatment through a hydrothermal oxidation method to prepare a Ni @ NiO composite material, modifying the surface of the Ni @ NiO composite material through an electric spraying method by using an oxidized carbon nano tube to obtain a Ni @ NiO composite material/carbon nano tube, and loading ascorbic acid oxidase on the surface of the Ni @ NiO composite material/carbon nano tube to prepare the electrode material for detecting ascorbic acid. The electrode material, the reference electrode and the counter electrode form a biosensor, ascorbic acid can be detected by adopting an electrochemical method, and the ascorbic acid detection has high sensitivity, low detection limit, quick response time and high selectivity.)
1. An electrode material for detecting ascorbic acid, which is prepared by the following method: firstly, carrying out surface oxidation on foamed nickel subjected to surface deoiling treatment through a hydrothermal oxidation method to prepare a Ni @ NiO composite material, then modifying the surface of the Ni @ NiO composite material through an electro-spraying method by using an oxidized carbon nano tube to obtain a Ni @ NiO composite material/carbon nano tube, and finally loading ascorbic acid oxidase on the surface of the Ni @ NiO composite material/carbon nano tube to prepare the electrode material for detecting ascorbic acid.
2. The preparation method of the electrode material for detecting the ascorbic acid as claimed in claim 1, characterized by comprising the following steps:
(1) preparation of Ni @ NiO composite material:
after the surface of the foamed nickel is subjected to deoiling treatment, performing surface oxidation by a hydrothermal oxidation method to prepare a Ni @ NiO composite material;
(2) preparation of electrode material for detecting ascorbic acid: transferring the oxidized carbon nanotube prepared by the chemical oxidation method to the surface of the Ni @ NiO composite material by adopting an electronic spraying method, carrying out heat treatment to obtain the Ni @ NiO composite material/carbon nanotube, and fixing ascorbic acid oxidase on the surface of the Ni @ NiO composite material/carbon nanotube to prepare the electrode material for detecting ascorbic acid.
3. The preparation method of the electrode material for detecting ascorbic acid as claimed in claim 2, wherein the surface degreasing treatment method in step (1) is that the foamed nickel is soaked in acetone and subjected to ultrasonic treatment, then is subjected to ultrasonic treatment in deionized water, is soaked in HCl solution with concentration of 0.1 mol/L and subjected to ultrasonic treatment, and then is subjected to ultrasonic treatment in deionized water, and is dried at normal temperature until the quality of the electrode material does not change.
4. The method for preparing an electrode material for detecting ascorbic acid according to claim 2, wherein the hydrothermal oxidation method of step (1) is: putting the foamed nickel and the deionized water into a reaction kettle, reacting for 12h at the temperature of 100-140 ℃ in the reaction kettle, then reacting for 12h at the temperature of 130-140 ℃, cooling to room temperature, taking out and drying, finally sintering for 5-7h at the temperature of 300-400 ℃, and drying in vacuum.
5. The method for preparing an electrode material for detecting ascorbic acid according to claim 2, wherein the step (2) of chemical oxidation of the carbon nanotubes comprises: adding 45 volume percent concentrated nitric acid into the carbon nano tube, refluxing for 10-12h at the temperature of 100-120 ℃, then cooling to room temperature, washing by deionized water until the deionized water is colorless and transparent, and drying until the mass does not change any more.
6. The method for preparing an electrode material for detecting ascorbic acid as in claim 2, wherein the step (2) of immobilizing ascorbate oxidase on the surface of the Ni @ NiO composite/carbon nanotube comprises: dripping ascorbic acid oxidase solution on the Ni @ NiO composite material/carbon nano tube repeatedly for 3-5 times, drying at normal temperature and then refrigerating at low temperature;
the preparation method of the ascorbic acid oxidase solution comprises the step of dissolving 500 units of ascorbic acid oxidase in a chitosan acetic acid solution with the mass percentage concentration of 100 mu L of 1%.
7. The method for preparing an electrode material for detecting ascorbic acid as claimed in claim 2, wherein the electrospray method in step (2) is specifically performed by: ultrasonically dispersing carbon oxide nanotubes in N, N' -dimethylformamide or N-methylpyrrolidone to form dispersion liquid, injecting the dispersion liquid into an injection container with a spray head, and injecting the dispersion liquid in the injection container to the surface of the Ni @ NiO composite material through the spray head after electrifying;
the electric spraying method adopts the spraying conditions that the temperature is 80-120 ℃, the distance between the surface of the Ni @ NiO composite material and a spray head is 10-14cm, the spraying flow is 0.05-0.1m L/h, the voltage is 8-12kV, and the inner diameter of the spray head is 0.4-0.6 mm.
8. The method for preparing an electrode material for detecting ascorbic acid as claimed in claim 2, wherein the heat treatment operation in the step (2) is: treating at 300-450 deg.C for 10-20min in inert atmosphere.
9. The biosensor of the electrode material for detecting ascorbic acid as claimed in claim 2, wherein the biosensor comprises the electrode material for detecting ascorbic acid prepared in step (2), a reference electrode, an auxiliary electrode and an electrolyte, wherein the electrode material for detecting ascorbic acid is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum sheet electrode is used as an auxiliary electrode to form a three-electrode system, and the electrode material for detecting ascorbic acid, the reference electrode and the auxiliary electrode are placed in the electrolyte to perform cyclic voltammetry scanning on an electrochemical workstation;
wherein the electrolyte comprises a buffer solution with pH 7 and ascorbic acid, and the buffer solution comprises CuH2PO4And CuHPO4Ascorbic acid was dissolved in a buffer solution and prepared to a concentration of 5 × 10-8-5×10-3mol/L of electrolyte.
10. Use of the biosensor of claim 9 in ascorbic acid concentration determination.
Technical Field
The invention relates to the technical field of biosensors, in particular to an electrode material for an ascorbic acid sensor, the biosensor, a preparation method and an application thereof.
Background
The sensor (english name: transducer/sensor) is a detection device, which can sense the measured information and convert the sensed information into electric signals or other information in required form according to a certain rule to output, so as to meet the requirements of information transmission, processing, storage, display, recording, control and the like.
Ascorbic acid (vitamin C) is absorbed by the small intestine and widely distributed in internal organs and tissues by ingestion of food, and is involved in the synthesis of collagen, the synthesis of l-carnitine, the synthesis of adrenocortical hormone, the synthesis of catecholamine, the decomposition of peroxidized lipids, the decomposition of active oxygen, and the like in biochemistry, and plays an important role in the living body. In recent years, attention has been paid to anticancer effects, immunity enhancement, and skin-beautifying and whitening effects due to the strong reducibility of vitamin C, and the vitamin C is used in high-concentration vitamin C spot therapy and the like.
Examples of the measurement of ascorbic acid include measurement for diagnosing ascorbic acid deficiency, measurement of ascorbic acid in food, and the like. In addition, in the high-concentration vitamin C spot therapy, it is necessary to monitor the vitamin C concentration in blood in real time, and in the self-blood glucose measurement of diabetes, the glucose value is affected by ascorbic acid in blood, and therefore, it is necessary to correct the glucose value based on the measurement value of the ascorbic acid concentration.
Therefore, the determination of ascorbic acid is always an important index for detecting the health of a human body in medicine, and the determination of ascorbic acid generally adopts a titration analysis method, a spectrophotometry method, a chromatographic analysis method and a fluorescence analysis method, but the methods in the prior art require higher experimental conditions and operation techniques and have lower detection sensitivity.
Disclosure of Invention
In view of the technical defects, the invention aims to provide an electrode material for an ascorbic acid sensor, a biosensor, a preparation method and an application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
an electrode material for detecting ascorbic acid, which is prepared by the following method: firstly, carrying out surface oxidation on foamed nickel subjected to surface deoiling treatment through an oxidation method to prepare a Ni @ NiO composite material, then modifying the surface of the Ni @ NiO composite material through an electro-spray method by using an oxidized carbon nano tube to obtain a Ni @ NiO composite material/carbon nano tube, and finally loading ascorbic acid oxidase on the surface of the Ni @ NiO composite material/carbon nano tube to prepare the electrode material for detecting ascorbic acid.
The invention also provides a preparation method of the electrode material for detecting the ascorbic acid, which comprises the following steps:
(1) preparation of Ni @ NiO composite material:
after the surface of the foamed nickel is subjected to deoiling treatment, performing surface oxidation by an oxidation method to prepare a Ni @ NiO composite material;
(2) preparation of electrode material for detecting ascorbic acid: transferring the oxidized carbon nanotube prepared by the chemical oxidation method to the surface of the Ni @ NiO composite material by adopting an electronic spraying method, carrying out heat treatment to obtain the Ni @ NiO composite material/carbon nanotube, and fixing ascorbic acid oxidase on the surface of the Ni @ NiO composite material/carbon nanotube to prepare the electrode material for detecting ascorbic acid.
Preferably, the surface deoiling treatment method in the step (1) comprises the steps of soaking foamed nickel in acetone and performing ultrasonic treatment, then performing ultrasonic treatment in deionized water, then soaking the foamed nickel in an HCl solution with the concentration of 0.1 mol/L for ultrasonic treatment, then performing ultrasonic treatment in the deionized water, and drying at normal temperature until the quality does not change any more.
Preferably, the hydrothermal oxidation method in step (1) is: putting the foamed nickel and the deionized water into a reaction kettle, reacting for 12h at the temperature of 100-140 ℃ in the reaction kettle, then reacting for 12h at the temperature of 130-140 ℃, cooling to room temperature, taking out and drying, finally sintering for 5-7h at the temperature of 300-400 ℃, and drying in vacuum.
Preferably, the preparation method of the chemical oxidation method of the carbon nanotube in the step (2) comprises: adding 45 volume percent concentrated nitric acid into the carbon nano tube, refluxing for 10-12h at the temperature of 100-120 ℃, then cooling to room temperature, washing by deionized water until the deionized water is colorless and transparent, and drying until the mass does not change any more.
Preferably, the fixing operation of ascorbate oxidase on the surface of the Ni @ NiO composite/carbon nanotube in the step (2) is as follows: dripping ascorbic acid oxidase solution on the Ni @ NiO composite material/carbon nano tube repeatedly for 3-5 times, drying at normal temperature and then refrigerating at low temperature;
the preparation method of the ascorbic acid oxidase solution comprises the step of dissolving 500 units of ascorbic acid oxidase in a chitosan acetic acid solution with the mass percentage concentration of 100 mu L of 1%.
Preferably, the electrospray method in step (2) specifically operates as follows: ultrasonically dispersing carbon oxide nanotubes in N, N' -dimethylformamide or N-methylpyrrolidone to form dispersion liquid, injecting the dispersion liquid into an injection container with a spray head, and injecting the dispersion liquid in the injection container to the surface of the Ni @ NiO composite material through the spray head after electrifying;
the electric spraying method adopts the spraying conditions that the temperature is 80-120 ℃, the distance between the surface of the Ni @ NiO composite material and a spray head is 10-14cm, the spraying flow is 0.05-0.1m L/h, the voltage is 8-12kV, and the inner diameter of the spray head is 0.4-0.6 mm.
Preferably, the heat treatment operation in the step (2) is: treating at 300-450 deg.C for 10-20min in inert atmosphere.
The invention also protects a biosensor of an electrode material for detecting ascorbic acid, which comprises the electrode material for detecting ascorbic acid, a reference electrode, an auxiliary electrode and electrolyte, wherein the electrode material for detecting ascorbic acid, the reference electrode, the auxiliary electrode and the electrolyte are prepared in the step (2), the electrode material for detecting ascorbic acid is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum sheet electrode is used as an auxiliary electrode to form a three-electrode system, the electrode material for detecting ascorbic acid, the reference electrode and the auxiliary electrode are placed in the electrolyte, and cyclic voltammetry scanning is carried out on an electrochemical workstation;
wherein the electrolyte comprises a buffer solution with pH 7 and ascorbic acid, and the buffer solution comprises CuH2PO4And CuHPO4Ascorbic acid was dissolved in a buffer solution and prepared to a concentration of 5 × 10-8-5×10-3mol/L of electrolyte.
The invention also protects the application of the biosensor in the determination of the concentration of the ascorbic acid.
Compared with the prior art, the invention has the beneficial effects that:
1. the method adopts an electrochemical method to detect the ascorbic acid, and has the advantages of high sensitivity, low detection limit, quick response time and good selectivity.
2. The invention firstly carries out surface oxidation on the foam nickel after surface deoiling treatment by a hydrothermal oxidation method to prepare a Ni @ NiO composite material, the Ni @ NiO composite material has the advantages of high chemical stability, good electrocatalytic performance, strong electron transfer capability and the like, has potential application prospect in a biosensor, transfers the oxidized carbon nano tube to the surface of the Ni @ NiO composite material by an electric spraying method to ensure that the oxidized carbon nano tube can be stably positioned on the Ni @ NiO composite material to obtain the Ni @ NiO composite material/carbon nano tube, finally loads ascorbic acid oxidase on the surface of the Ni @ NiO composite material/carbon nano tube to prepare the electrode material of the ascorbic acid sensor, the modified ascorbic acid oxidase can efficiently catalyze the oxidation of the ascorbic acid, so that the electrode has extremely high detection sensitivity and reaches extremely high detection limit, is suitable for trace detection of ascorbic acid.
3. The carbon nano tube has the characteristics of small size, high mechanical strength, large specific surface area, high conductivity and strong interface effect, and has excellent electrical properties, so that the sensitivity and stability of biological detection can be improved, the overpotential of oxidation reduction is reduced, and direct electron transfer is carried out.
4. The invention takes the foam nickel as a matrix, nickel (Ni) is a magnetic metal, has unique properties and can be applied to magnetic storage media, fuel cell electrodes, catalysts and the like, a NiO nano structure has the advantages of high chemical stability, good electrocatalysis performance, strong electron transfer capacity and the like, and has potential application prospect in biosensors, and the Ni @ NiO composite material has excellent electrochemical biological activity, magnetic performance and good dispersibility in water, so that the Ni @ NiO composite material has wide application prospect in the fields of bioseparation, biosensors and the like, in addition, the foam nickel is spongy porous metal nickel, the porous foam nickel is convenient for the adhesion of carbon oxide nanotubes, and not only the solvent is decomposed and dissipated after heat treatment, but also the adhesion strength of the carbon oxide nanotubes on the Ni @ NiO composite material is deepened, and the porous Ni @ NiO composite material and the carbon nanotubes provide supported pore passages and sites for ascorbic acid oxidase, the magnetic Ni @ NiO composite material can adsorb the ascorbic acid oxidase, so that the ascorbic acid oxidase can be stably positioned on the Ni @ NiO composite material/carbon nano tube during electrochemical detection, and stable detection is performed.
Drawings
FIG. 1 is a cyclic voltammetry curve of the sensor electrode in ascorbic acid solutions with different concentrations, wherein a, b, c, d, e and f respectively represent the concentrations of the ascorbic acid solutions to be 1 × 10-3mol/L、1×10-4mol/L、1×10- 5mol/L、1×10-6mol/L、1×10-7mol/L、1×10-8；
Fig. 2 is a performance test chart of charge and discharge of the electrode material for detecting ascorbic acid prepared in inventive example 2.
Detailed Description
The following description of the preferred embodiments 1-3 and the accompanying drawings 1-2 will be made in detail with reference to the accompanying drawings.