CN108445066B - Preparation method of glucose oxidase electrode - Google Patents
Preparation method of glucose oxidase electrode Download PDFInfo
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- CN108445066B CN108445066B CN201810213151.3A CN201810213151A CN108445066B CN 108445066 B CN108445066 B CN 108445066B CN 201810213151 A CN201810213151 A CN 201810213151A CN 108445066 B CN108445066 B CN 108445066B
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
Abstract
The invention provides a preparation method of a glucose oxidase electrode, and relates to the technical field of biomedical detection. The method comprises the following steps: filtering a ferrocene ethanol solution with the concentration of 18g/L, respectively taking filtrate and an acetic acid solution according to the volume ratio of 1:19, mixing, adding chitosan powder into the mixed solution according to the proportion of 5g/L, stirring to obtain ferrocene chitosan dispersion gel, adding glucose oxidase into the ferrocene chitosan solution according to the dosage of 0.5g/L, dripping the glucose oxidase onto the surface of an electrode, drying for at least 12h at the temperature of 4 ℃, dripping glutaraldehyde solution onto the surface of the electrode, washing by using PBS buffer solution after at least 15min, and drying to obtain a glucose oxidase electrode. The method increases the contact area of ferrocene and glucose oxidase, promotes the electron transfer in the glucose oxidase electrode, improves the sensitivity and widens the detection range.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a preparation method of a glucose oxidase electrode.
Background
The method for rapidly detecting the glucose concentration by using the glucose oxidase electrode is widely applied to the fields of medicines and foods. The method mainly adopts the principle that the prepared glucose oxidase electrode test paper generates current response to blood glucose concentration through a screen printing technology, detects current intensity by using electronic equipment, judges the blood glucose concentration according to the current intensity and further realizes the rapid detection of the blood glucose concentration.
In the prior art, three methods are used for detecting the concentration of glucose by using a glucose oxidase electrode, wherein the first method is to detect the concentration of glucose by detecting electrons transferred by a product of an enzymatic reaction of the glucose oxidase, namely hydrogen peroxide, during an oxidation-reduction reaction on the surface of the electrode; the second is to transmit the electrons transferred in the enzymatic reaction to the surface of an electrode through electron mediators such as potassium ferricyanide and ferrocene, and then to detect the concentration of the grape; and the third method is to realize direct electron transfer between the enzyme and the electrode by means of metal nano particles, graphene, carbon nano tubes and other materials, and detect the blood glucose concentration.
Among them, glucose oxidase electrodes using an electron mediator are most common. Among many electron mediators, ferrocene is used as a commonly used electron mediator in the manufacture of glucose oxidase electrodes because of its good redox reversibility and low tendency to lose by dissolution in water. However, because glucose oxidase is mainly dissolved in aqueous solution, but ferrocene is not dissolved in water, uniform dispersion of ferrocene in glucose oxidase solution is difficult to realize, according to the reports of the use mode of ferrocene in the preparation of glucose oxidase electrode described by a glucose sensor modified by ferrocene-Nafion reported in analytical chemistry 1994, a glucose sensor modified by nanogold and ferrocene reported in sensor technology 2005, and a glucose sensor of a platinum black/ferrocene modified MEMS electrode reported in MEMS device and technology 2017, in the glucose oxidase electrode using ferrocene as an electron mediator, the transfer of electrons in the glucose oxidase electrode is realized by a mode (namely a covering method) that the electrode surface is covered with ferrocene and then a layer of enzyme membrane is modified on the surface of a ferrocene covering layer. However, the contact between ferrocene modified by the covering method and glucose oxidase is limited to the interface boundary between the ferrocene covering layer and the enzyme membrane, which makes the electron transfer efficiency in the glucose oxidase electrode using ferrocene as the electronic enzyme mediator extremely limited, resulting in the problems of low sensitivity and narrow detection range of glucose concentration detection.
Disclosure of Invention
The invention provides a preparation method of a glucose oxidase electrode, and aims to solve the problems of limited contact area between an electron mediator and glucose oxidase, low sensitivity of glucose concentration detection and narrow detection range of the existing glucose oxidase electrode taking ferrocene as the electron mediator.
The invention provides a preparation method of a glucose oxidase electrode, which comprises the following steps:
filtering 15-20 g/L ferrocene ethanol saturated solution, and mixing the filtrate and an acetic acid solution according to the volume ratio of 1: 15-30, wherein the mass percentage of the acetic acid solution is 0.7-1.2%;
adding chitosan powder into the mixed solution according to the proportion of 4-6 g/L, and stirring to obtain ferrocene chitosan dispersion gel;
adding glucose oxidase into the ferrocene chitosan dispersion gel according to the dosage of 0.5-1.5 g/L, dripping the glucose oxidase onto the surface of an electrode, drying for at least 12h at the temperature of 2-5 ℃, dripping glutaraldehyde solution onto the surface of the electrode, washing by using PBS buffer solution after at least 15min, and drying to obtain the glucose oxidase electrode.
According to the preparation method of the glucose oxidase electrode, the ferrocene is dispersed in the chitosan gel by virtue of ethanol, then the glucose oxidase is dissolved in the chitosan gel, and the glucose oxidase electrode with the ferrocene dispersed in the enzyme membrane is prepared by utilizing the characteristic that the ethanol has both polar hydroxyl groups and non-polar hydrocarbyl groups and can volatilize in the gel film forming process, so that the contact area of the ferrocene and the glucose oxidase is successfully increased, the electron transfer in the glucose oxidase electrode is promoted, and the glucose oxidase electrode with higher sensitivity and wider detection range is obtained.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention.
FIG. 1 is a cyclic voltammogram of a glucose oxidase electrode prepared by a method (dispersion method) used in example 1 of the present invention and a method (coverage method) in which a saturated solution of ferrocene alcohol is covered on the surface of an electrode and then a layer of enzyme membrane is modified on the surface of the electrode, in a 6mmol/L glucose solution, under the premise that the concentration of glucose oxidase, the concentration of chitosan, the concentration of ferrocene alcohol solution and the sample loading amount are the same;
fig. 2 and fig. 3 are time-current graphs obtained by detecting glucose oxidase electrodes prepared by a dispersion method and a coverage method under a voltage of 0.45V for glucose solutions with different concentrations by a time-current method respectively on the premise that the concentration of glucose oxidase, the concentration of chitosan, the concentration of ferrocene ethanol solution and the sample addition amount are the same.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of a glucose oxidase electrode, which comprises the following steps:
filtering a 15-20 g/L ferrocene ethanol saturated solution, and mixing a filtrate and an acetic acid solution according to a volume ratio of 1: 15-30, wherein the mass percent of the acetic acid solution is 0.7-1.2%;
step two, adding chitosan powder into the mixed solution according to the proportion of 4-6 g/L, and stirring to obtain ferrocene chitosan dispersion gel;
and step three, adding glucose oxidase into the ferrocene chitosan dispersion gel according to the dosage of 0.5-1.5 g/L, dripping the glucose oxidase onto the surface of the electrode, drying for at least 12 hours at the temperature of 2-5 ℃, dripping glutaraldehyde solution onto the surface of the electrode, washing by using PBS buffer solution after at least 15 minutes, and drying to obtain the glucose oxidase electrode.
Specifically, in the step one, a nylon filter element with the diameter of 0.22 μm can be adopted for filtering the ferrocene ethanol solution.
Preferably, the concentration of the ferrocene ethanol solution is 18g/L, the volume ratio of the filtrate to the acetic acid solution is 1:19, and the mass percent of the acetic acid solution is 1%. Specifically, in the second step, chitosan is purchased from Shanghai-derived leafy organisms, and the deacetylation degree is 90%. Preferably, chitosan powder is added into the mixed solution according to the proportion of 5g/L to obtain the ferrocene chitosan dispersion gel.
Further, before step three, the method further comprises: the electrode is pretreated, and the pretreatment method specifically comprises the following steps:
polishing the electrode with 1mm, 0.3mm and 0.05mm alumina powder in sequence, and sequentially polishing the electrode with HNO3And (3) performing ultrasonic treatment on the solution and the mixture of ethanol and water for at least 3min, and cleaning.
In particular, HNO3The solution is HNO3Mixing the solution with water according to the volume ratio of 1:1, wherein the volume ratio of ethanol to water in the mixture of ethanol and water is 1: 1.
Specifically, in step three, glucose oxidase was purchased from a leaf organism of Shanghai origin, obtained from Aspergillus niger, at 200U/mg. The pH of the PBS buffer was 7.4 and the concentration was 0.05 mol/L.
According to the preparation method of the glucose oxidase electrode, the ferrocene is dispersed in the chitosan gel by virtue of the ethanol, and then the glucose oxidase is dissolved in the chitosan gel, so that the contact area of the ferrocene and the glucose oxidase is successfully increased, the transfer of electrons in the glucose oxidase electrode is promoted, and the glucose oxidase electrode with higher sensitivity and wider detection range is obtained.
Example 1
1) Weighing 0.018g of ferrocene, dissolving in 1mL of absolute ethyl alcohol to prepare a ferrocene ethyl alcohol saturated solution, filtering the ferrocene ethyl alcohol solution by using a 0.22 mu m nylon filter element, and mixing 50 mu L of filtrate with 950 mu L of acetic acid solution (1% wt);
2) adding 0.005g of chitosan powder with deacetylation degree of 90% into 1mL of the prepared mixed solution, and uniformly stirring to completely dissolve the chitosan powder to prepare ferrocene chitosan dispersion gel;
3) adding 1mg of glucose oxidase (200U/mg) into 100 mu L of the mixed solution prepared in the step 2, and uniformly stirring to completely dissolve the glucose oxidase;
4) taking a gold disc electrode with the diameter of 2mm, polishing the electrode by using alumina powder with the grain diameters of 1mm, 0.3mm and 0.05mm in sequence, and respectively polishing the electrode on HNO3Solution (HNO)31:1) and mixed solution of ethanol and water (the volume ratio of ethanol to water is 1:1) for 3min, and rinsing the surface of the electrode by using a large amount of deionized water to finish the pretreatment of the electrode;
5) and (3) vertically dropwise adding the mixed solution prepared in the step (4) to the surface of a pretreated gold disc electrode with the diameter of 2mm, putting the gold disc electrode into a refrigerator at 4 ℃ for airing for 12 hours, standing the gold disc electrode at room temperature for 15 minutes, then sucking 3 mu L of glutaraldehyde solution (prepared by mixing 50% glutaraldehyde and 0.05mol/L PBS buffer solution (pH7.4) according to the volume ratio of 2.5: 97.5) to be dropwise added to the surface of the electrode, washing the glutaraldehyde solution by using the 0.05mol/L PBS buffer solution (pH7.4) after 15 minutes, and airing the gold disc electrode at room temperature to obtain the glucose oxidase electrode.
The glucose oxidase electrode prepared in example 1 was used as a working electrode, a saturated calomel electrode as a reference electrode, and a platinum wire electrode as a counter electrode, a three-electrode system was constructed and connected to an electrochemical workstation (CHI660E, usa), and glucose standard solution was detected by selecting a time current method and a voltage of 0.45V.
Example 2
1) Weighing 0.015g of ferrocene, dissolving the ferrocene into 1mL of absolute ethyl alcohol to prepare a ferrocene ethyl alcohol saturated solution, filtering the ferrocene ethyl alcohol solution by using a 0.22 mu m nylon filter element, and mixing 50 mu L of filtrate with 750 mu L of acetic acid solution (1% wt);
2) adding 0.004g of chitosan powder with deacetylation degree of 90% into 1mL of the prepared mixed solution, and uniformly stirring to completely dissolve the chitosan powder to prepare ferrocene chitosan dispersion gel;
3) adding 0.5mg of glucose oxidase (200U/mg) into 100 mu L of the mixed solution prepared in the step 2, and uniformly stirring to completely dissolve the glucose oxidase;
4) taking a gold disc electrode with the diameter of 2mm, polishing the electrode by using alumina powder with the grain diameters of 1mm, 0.3mm and 0.05mm in sequence, and respectively polishing the electrode on HNO3Solution (HNO)3Volume ratio of ethanol to water 1:1) and mixed solution of ethanol and water (volume ratio of ethanol to water)1:1) carrying out ultrasonic treatment for 3min, and finishing electrode pretreatment after washing the surface of the electrode by using a large amount of deionized water;
5) and (3) vertically dropwise adding the mixed solution prepared in the step (4) to the surface of a pretreated gold disc electrode with the diameter of 2mm, putting the gold disc electrode into a refrigerator at the temperature of 2 ℃ for airing for 16 hours, standing the gold disc electrode at the room temperature for 15 minutes, then sucking 3 mu L of glutaraldehyde solution (prepared by mixing 50% glutaraldehyde and 0.05mol/L PBS buffer solution (pH7.4) according to the volume ratio of 2.5: 97.5) to be dropwise added to the surface of the electrode, washing the glutaraldehyde solution by using the 0.05mol/L PBS buffer solution (pH7.4) after 20 minutes, and airing the gold disc electrode at the room temperature to obtain the glucose oxidase electrode.
Example 3
1) Weighing 0.02g of ferrocene, dissolving in 1mL of absolute ethyl alcohol to prepare a ferrocene ethyl alcohol saturated solution, filtering the ferrocene ethyl alcohol solution by using a 0.22 mu m nylon filter element, and mixing 50 mu L of filtrate with 1000 mu L of acetic acid solution (1% wt);
2) adding 0.006g of chitosan powder with deacetylation degree of 90% into 1mL of the prepared mixed solution, and uniformly stirring to completely dissolve the chitosan powder to obtain ferrocene chitosan dispersion gel;
3) adding 1.5mg of glucose oxidase (200U/mg) into 100 mu L of the mixed solution prepared in the step 2, and uniformly stirring to completely dissolve the glucose oxidase;
4) taking a gold disc electrode with the diameter of 2mm, polishing the electrode by using alumina powder with the grain diameters of 1mm, 0.3mm and 0.05mm in sequence, and respectively polishing the electrode on HNO3Solution (HNO)31:1) and mixed solution of ethanol and water (the volume ratio of ethanol to water is 1:1) for 3min, and rinsing the surface of the electrode by using a large amount of deionized water to finish the pretreatment of the electrode;
5) and (3) vertically dropwise adding the mixed solution prepared in the step (4) to the surface of a pretreated gold disc electrode with the diameter of 2mm, putting the gold disc electrode into a refrigerator with the temperature of 5 ℃ for airing for 15 hours, standing the gold disc electrode at room temperature for 15 minutes, then sucking 3 mu L of glutaraldehyde solution (prepared by mixing 50% glutaraldehyde and 0.05mol/L PBS buffer solution (pH7.4) according to the volume ratio of 2.5: 97.5) to be dropwise added to the surface of the electrode, washing the glutaraldehyde solution by using the 0.05mol/L PBS buffer solution (pH7.4) after 20 minutes, and airing the gold disc electrode at room temperature to obtain the glucose oxidase electrode.
Under the premise that the concentration of glucose oxidase, the concentration of chitosan, the concentration of ferrocene ethanol solution and the sample addition amount are the same, 2 glucose oxidase electrodes are prepared by covering ferrocene ethanol saturated solution on the surface of an electrode, then modifying a layer of enzyme membrane on the surface of the electrode (covering method), and dispersing ferrocene in chitosan gel dissolved with glucose oxidase by using the method described in example 1 (dispersing method), and the electrochemical performances of the glucose oxidase electrode prepared by the covering method and the dispersing method in 6mmol/L glucose solution are compared by using a cyclic voltammetry method in an electrochemical detection method, wherein the scanning range is-0.2-0.6V, and the scanning rate is 50 mV/s. The results are shown in fig. 1, compared with the glucose oxidase electrode prepared by the covering method, the glucose oxidase electrode prepared by the dispersing method has higher current response intensity to the glucose solution of 6mmol/L than the glucose oxidase electrode prepared by the covering method, which indicates that the dispersing method successfully improves the response current intensity and better promotes the electron transfer.
Fig. 2 and 3 are time-current curves obtained by detecting glucose oxidase electrodes prepared by a dispersion method adopted in example 1 and a covering method in which a ferrocene ethanol saturated solution is covered on the surface of an electrode and then a layer of enzyme membrane is modified on the surface of the electrode by a time-current method at a voltage of 0.45V for glucose solutions of different concentrations under the premise that the concentrations of glucose oxidase, chitosan, ferrocene ethanol solution and sample addition are the same. In the graph of FIG. 2, the current intensity increases with the increase of the concentration of the glucose solution, 16mmol/L of glucose solution can be detected effectively at maximum, through calculation, the linear range of the detection of the glucose concentration covers the range of the glucose concentration in human blood (fasting blood glucose value is 3.9-6.1 mmol/L), the linear correlation coefficient reaches 0.99, and the sensitivity is 75 nA/mmol.L-1And the kit has the potential of being applied to blood sugar detection. FIG. 3 shows that the maximum effective glucose concentration of glucose oxidase electrode prepared by covering method is 8mmol/L, and the calculated sensitivity is 12 nA/mmol.L-1In contrast, the glucose oxidase electrode prepared by the dispersion method used in example 1 had better sensitivity and detection range than the glucose oxidase electrode prepared by the overlay method, so the dispersion method was used with the aid of BThe alcohol disperses the ferrocene in the chitosan gel, and then the glucose oxidase is dissolved in the chitosan gel, so that the detection range and the sensitivity of the glucose oxidase electrode can be improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A method of making a glucose oxidase electrode, comprising:
filtering 15-20 g/L ferrocene ethanol saturated solution, and mixing the filtrate and an acetic acid solution according to the volume ratio of 1: 15-30, wherein the mass percentage of the acetic acid solution is 0.7-1.2%;
adding chitosan powder into the mixed solution according to the proportion of 4-6 g/L, and stirring to obtain ferrocene chitosan dispersion gel;
adding glucose oxidase into the ferrocene chitosan dispersion gel according to the dosage of 0.5-1.5 g/L, dripping the glucose oxidase onto the surface of an electrode, drying for at least 12h at the temperature of 2-5 ℃, dripping glutaraldehyde solution onto the surface of the electrode, washing by using PBS buffer solution after at least 15min, and drying to obtain a glucose oxidase electrode;
the glutaraldehyde solution is prepared from 50% by mass of glutaraldehyde and a PBS buffer solution according to the volume ratio of 2.5: 97.5;
the concentration of the glucose oxidase is 150-250U/mg.
2. The method of claim 1, wherein the degree of deacetylation of the chitosan is 90%.
3. The method of claim 1, wherein the PBS buffer has a pH of 7.4 and a concentration of 0.05 mol/L.
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