CN109060918B - Hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase and preparation and application thereof - Google Patents

Hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase and preparation and application thereof Download PDF

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CN109060918B
CN109060918B CN201811006266.1A CN201811006266A CN109060918B CN 109060918 B CN109060918 B CN 109060918B CN 201811006266 A CN201811006266 A CN 201811006266A CN 109060918 B CN109060918 B CN 109060918B
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林芷琪
朴金花
黄彩弯
李晓霞
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South China University of Technology SCUT
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Abstract

The invention belongs to the technical field of electrochemical biosensors, and discloses a hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase, and preparation and application thereof. The biosensor is composed of a reference electrode, a counter electrode and a modified working electrode, wherein the modified working electrode is composed of a substrate electrode and a substance recognition membrane solidified on the surface of the substrate electrode, and the substance recognition membrane is mainly prepared by mixing nitrogen-doped graphene dispersion liquid, horseradish peroxidase solution and chitosan solution to form a membrane. The invention also discloses a preparation method of the sensor. The preparation method is simple and low in cost; the obtained sensor has good selectivity, sensitivity and stability, and good electrocatalysis performance; the hydroquinone is accurately detected, and the anti-interference capability is strong; meanwhile, the method has a wider detection range and a lower detection limit.

Description

Hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase and preparation and application thereof
Technical Field
The invention belongs to the technical field of electrochemical biosensors, and particularly relates to a biosensor for detecting hydroquinone based on nitrogen-doped graphene composite horseradish peroxidase, and a preparation method and application thereof.
Background
The phenolic compounds are chemical raw materials with wide application, wherein hydroquinone is used as a basic raw material of fine chemicals and is widely applied to the industries of cosmetics, dyes, pesticides, medicines, leather making and the like. However, hydroquinone is considered to be an environmental pollutant because of its high toxicity and difficult degradability. The effect of hydroquinone on human body is not negligible, and low concentration of hydroquinone can cause symptoms such as headache and tachycardia. Therefore, the development of a rapid and sensitive method for detecting hydroquinone is of great significance. There are many methods for detecting hydroquinone, such as high performance liquid chromatography, gas chromatography, pH flow injection analysis, simultaneous fluorescence, spectrophotometry, etc. However, most of the above mentioned methods have the disadvantages of long time consumption, expensive instrument, low sensitivity, complicated sample pretreatment, etc., and in contrast, the electrochemical sensor has the advantages of high identification capability for target objects, small sample usage amount, fast response, low cost, small volume and convenient popularization, and the electrochemical enzyme biosensor is widely applied by the characteristics of high catalytic capability, mild reaction conditions, good specificity, etc., so the hydroquinone electrochemical enzyme biosensor which is simple, fast, accurate and high in sensitivity has great application prospect.
In the preparation process of the electrochemical enzyme biosensor, a very important problem influencing the performance of the electrochemical enzyme biosensor is that a layer of non-conductive protein shell is coated outside the redox active center of an enzyme catalyst, and the protein shell prevents the enzyme active center from carrying out electron transfer with the surface of an electrode, so that the performance of the electrode is influenced; another important factor influencing the performance of enzyme biosensors is the stability and service life of enzyme modified electrodes, which is closely related to the immobilization mode of enzyme catalysts, and the preparation of stable enzyme modified electrodes by adopting a proper enzyme immobilization method is always a hot problem for researches of biosensors researchers.
Disclosure of Invention
In order to solve the defects and shortcomings of the prior art, the invention mainly aims to provide a hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase, which has good selectivity, sensitivity and stability.
The invention also aims to provide a preparation method of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase.
The invention further aims to provide an application of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase in hydroquinone detection.
The purpose of the invention is realized by the following technical scheme:
a hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase is composed of a reference electrode, a counter electrode and a modified working electrode, wherein the modified working electrode is composed of a substrate electrode and a substance recognition membrane solidified on the surface of the substrate electrode, and the substance recognition membrane is mainly prepared by mixing nitrogen-doped graphene dispersion liquid (N-GN), horseradish peroxidase solution (HRP) and Chitosan Solution (CS) to form a membrane;
the nitrogen-doped graphene dispersion liquid (N-GN) is obtained by dispersing nitrogen-doped graphene in water; the horse radish peroxidase solution (HRP) is obtained by dissolving horse radish peroxidase in PBS solution; the chitosan solution is obtained by preparing chitosan into a solution.
The nitrogen-doped graphene: horse radish peroxidase: the mass ratio of the chitosan is (2-20): (15-35): 10.
the nitrogen-doped graphene material is prepared by the following method:
(a) uniformly dispersing graphene oxide and aniline; under the action of an oxidant and in an acidic medium, aniline is subjected to polymerization reaction, and is placed in a hydrothermal reaction kettle for hydrothermal reaction to obtain a polyaniline-graphene oxide compound;
(b) and calcining the polyaniline-graphene oxide compound to obtain the nitrogen-doped graphene composite material.
The mass-volume ratio of the graphene oxide to the aniline is (50-200) mg: 0.5 mL;
the oxidant is ammonium persulfate, the acidic medium is hydrochloric acid, and the molar ratio of the oxidant to the acidic medium is (2-2.5): 1, the volume ratio of aniline to an acidic medium is 1 (10-20); adding an acidic medium in the form of an aqueous solution, wherein the concentration of the acidic medium is 0.5 mol/L; the temperature of the polymerization reaction is 4-10 ℃, and the time of the polymerization reaction is 6-12 h; the temperature of the hydrothermal reaction is 160-200 ℃, and the time of the hydrothermal reaction is 12-20 h.
The calcining atmosphere in the step (b) is protective atmosphere, the protective atmosphere is nitrogen or inert atmosphere, the calcining refers to calcining at 400-500 ℃, and the calcining time is 1.5-2.5 hours; and then continuously calcining at 700-800 ℃, wherein the continuous calcining time is 0.5-1.5 hours.
Ultrasonically stripping graphene oxide in water, and adding aniline for ultrasonic dispersion to be uniform; and then adding a mixture of an oxidant and an acidic medium at a low temperature, carrying out polymerization reaction and hydrothermal reaction to obtain the polyaniline-graphene oxide compound. The mass-volume ratio of the graphene oxide to water is (50-200) mg: 50mL, ultrasonic stripping time of 1-4 h, and reaction time of 6-12 h.
And after the reaction is finished, filtering, washing, drying in vacuum, and grinding to obtain the polyaniline-graphene oxide compound.
The preparation method of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase comprises the following steps:
(1) carrying out surface pretreatment on the substrate electrode;
(2) dispersing the nitrogen-doped graphene composite material in water to obtain a dispersion liquid; dissolving horseradish peroxidase in a PBS solution to obtain a horseradish peroxidase solution; preparing chitosan into a solution to obtain a chitosan solution; uniformly mixing the dispersion liquid, a horseradish peroxidase solution and a chitosan solution to obtain a composite solution;
(3) dripping the composite solution on the surface of the substrate electrode subjected to surface pretreatment, and airing at room temperature to obtain an enzyme modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system by the enzyme modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting hydroquinone.
The surface pretreatment in the step (1) specifically comprises the following steps: the surface of the substrate electrode was coated with Al having diameters of 0.3 μm and 0.05 μm in this order2O3Polishing the powder into a mirror surface, and washing with water; then ultrasonic cleaning is carried out in absolute ethyl alcohol and water in sequence, and the mixture is taken out, cleaned by water and dried.
The concentration of the doped graphene material dispersion liquid in the step (2) is 2-20 mg/mL.
The concentration of the horseradish peroxidase solution in the step (2) is 15-35 mg/mL, and the enzyme solution is prepared by adopting a PBS solution (pH is 6.5, and 0.1M).
The chitosan solution in the step (2) is prepared from 0.1-2wt% acetic acid solution, and the concentration is 10 mg/mL.
The volume ratio of the doped graphene dispersion liquid, the horseradish peroxidase solution and the chitosan solution in the step (2) is 1:1: 1.
The dropping amount of the composite solution in the step (3) is 3-10 mu L.
The biosensor for detecting hydroquinone enzyme based on nitrogen-doped graphene composite horseradish peroxidase is applied to hydroquinone quantitative detection.
The principle of the invention is as follows:
firstly, preparing a doped graphene material, overcoming the defects of curling of graphene, interlayer stacking and poor dispersibility in a solvent by virtue of the synergistic effect of different components, and then powerfully increasing the fixed quantity and stability of an enzyme catalyst on the surface of an electrode by utilizing the film forming property of chitosan and the carrier characteristic of doped graphene so as to be beneficial to catalyzing a substrate; finally, taking a proper amount of mixed liquid to be dripped on the surface-pretreated working electrode to obtain a modified working electrode; and then the modified working electrode is utilized to form a three-electrode system by matching with a reference electrode and a counter electrode, so as to prepare the enzyme biosensor for detecting hydroquinone.
According to the invention, nitrogen-doped graphene and horseradish peroxidase are applied to the enzyme biosensor, and the sensor for detecting hydroquinone prepared by the method has good detection performance, wherein the detection range is 9 × 10-5～5.075×10-3mol/L, linear equation is I (mu A) ═ 11.37-5.89C (mmol/L), correlation coefficient is R20.998. detection limit of 1 × 10-5mol/L(S/N＝3)。
The preparation method and the obtained product have the following advantages and beneficial effects:
(1) the biosensor for detecting hydroquinone has good electron transfer performance, can transfer electrons generated by reaction well, can realize selective detection of biomolecules, and improves the reaction speed of the biosensor.
(2) The biosensor for detecting hydroquinone has good selectivity, reproducibility and stability, can accurately detect hydroquinone, and has strong anti-interference capability.
(3) The biosensor for detecting hydroquinone can be used for detecting hydroquinone in water or hydroquinone in soil, is simple to prepare, has a wide detection range and a low detection limit, can be used for reaction in a room-temperature neutral environment, is stable in performance, and has a good application prospect.
Drawings
FIG. 1 is a cyclic voltammogram of a nitrogen-doped graphene-based composite horseradish peroxidase modified working electrode in example 3 in different solutions; curve a is cyclic voltammetry curve of the enzyme modified working electrode in PBS, curve b is cyclic voltammetry curve of the enzyme modified working electrode in 0.5mM Hydroquinone (HQ), and curve c is cyclic voltammetry curve of the enzyme modified working electrode in 0.5mM hydroquinone and 0.5mM H2O2Cyclic voltammograms in the mixed solution;
fig. 2 is a cyclic voltammogram of the biosensor for detecting hydroquinone based on nitrogen-doped graphene composite horseradish peroxidase, prepared in example 3, in hydroquinone solutions of different concentrations, where the corresponding hydroquinone concentrations are in the square boxes in the chart;
fig. 3 is a standard curve diagram of response current of the biosensor for detecting hydroquinone based on nitrogen-doped graphene composite horseradish peroxidase, which is prepared in example 3, to different concentrations of hydroquinone.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
A preparation method of an electrochemical enzyme biosensor for detecting hydroquinone based on nitrogen-doped graphene composite horseradish peroxidase comprises the following steps:
(1) a glassy carbon electrode having a diameter of 3mm was successively coated with Al having a diameter of 0.3 μm and 0.05. mu.m2O3Polishing the powder into a mirror surface, washing with distilled water, then sequentially ultrasonically cleaning in absolute ethyl alcohol and distilled water for 1min, taking out, washing with distilled water, and airing at room temperature to obtain a pretreated glassy carbon electrode;
(2) mixing 100mg of graphene oxide with 50mL of deionized water, ultrasonically stripping for 4h, adding 0.5mL of aniline, continuously ultrasonically stirring for 30min after uniformly stirring, adding 5mL of 0.5MHCl containing 1.25g of Ammonium Persulfate (APS) under low-temperature stirring (below 10 ℃), continuously stirring for 6 h at low temperature, transferring into a reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 16 h, filtering the solution obtained after reaction by using a sand core filter, washing the solution with distilled water for a plurality of times, carrying out vacuum drying at 80 ℃ for overnight, grinding to obtain powder, placing the powder in a quartz boat, calcining in a tubular furnace in a nitrogen calcining atmosphere at 400 ℃ for 2h, subsequently heating to 700 ℃ for 1 h, taking out and grinding to obtain a nitrogen-doped graphene material;
(3) dispersing a nitrogen-doped graphene material into a dispersion liquid with the concentration of 10mg/mL in water, preparing a horseradish peroxidase solution with the concentration of 15mg/mL by adopting a PBS solution (0.1mol/L, pH6.5), preparing a 10mg/mL chitosan solution by adopting a 1 wt% acetic acid solution, mixing the three solutions in a volume ratio of 1:1:1 to obtain a mixed solution, dripping 6 mu L of the mixed solution on the surface of the electrode in the step (1), and airing at room temperature to obtain a modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system (a platinum electrode is used as a counter electrode, and saturated calomel is used as a reference electrode) by the modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting hydroquinone.
Electrochemical tests were performed at room temperature, all at 10mL containing 0.5mM H2O2In phosphate buffer (0.1mol/L, pH6.5) and tested by passing N through2And cyclic voltammetry is adopted in the test process. Wherein, the blank control is not added with hydroquinone solution, and 50 mu L of hydroquinone solution is added after the test is stable.
In this example, the reduction peak catalytic current was 7.921 μ A at a hydroquinone concentration of 0.5 mmol/L.
Example 2
A preparation method of a hydroquinone enzyme biosensor based on nitrogen-doped graphene composite horseradish peroxidase comprises the following steps:
(1) a glassy carbon electrode having a diameter of 3mm was successively coated with Al having a diameter of 0.3 μm and 0.05. mu.m2O3Polishing the powder to obtain mirror surface, washing with distilled water, sequentially ultrasonic cleaning with anhydrous ethanol and distilled water for 1min, taking out, and steamingWashing with distilled water, and airing at room temperature to obtain a pretreated glassy carbon electrode;
(2) mixing 100mg of graphene oxide with 50mL of deionized water, ultrasonically stripping for 4h, adding 0.5mL of aniline, continuously ultrasonically stirring for 30min after uniformly stirring, adding 5mL of 0.5MHCl containing 1.25g of Ammonium Persulfate (APS) under low-temperature stirring (below 10 ℃), continuously stirring for 6 h at low temperature, transferring into a reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 16 h, filtering the solution obtained after reaction by using a sand core filter, washing the solution with distilled water for a plurality of times, carrying out vacuum drying at 80 ℃ for overnight, grinding to obtain powder, placing the powder into a quartz boat, calcining in a tubular furnace under nitrogen atmosphere, calcining at 400 ℃ for 2h, subsequently heating to 700 ℃ for 1 h, taking out and grinding to obtain a doped graphene material;
(3) dispersing the doped graphene material in water to obtain a dispersion liquid with the concentration of 10mg/mL, preparing a horseradish peroxidase solution with the concentration of 20mg/mL by adopting a PBS solution (0.1mol/L, pH 6.5.5), preparing a chitosan solution with the concentration of 10mg/mL by adopting a 1 wt% acetic acid solution, mixing the three solutions in a volume ratio of 1:1:1 to obtain a mixed solution, dripping 6 mu L of the mixed solution on the surface of the electrode in the step (1), and airing at room temperature to obtain a modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system (a platinum electrode is used as a counter electrode, and saturated calomel is used as a reference electrode) by the modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting the hydroquinone.
Electrochemical tests were performed at room temperature, all at 10mL containing 0.5mM H2O2In phosphate buffer (0.1mol/L, pH6.5) and tested by passing N through2And cyclic voltammetry is adopted in the test process. Wherein, the blank control is not added with hydroquinone solution, and 50 mu L of hydroquinone solution is added in turn after the test is stable.
In this example, the reduction peak catalytic current was 9.819 μ A at a hydroquinone concentration of 0.5 mmol/L.
Example 3
A preparation method of a hydroquinone enzyme biosensor based on nitrogen-doped graphene composite horseradish peroxidase comprises the following steps:
(1) a glassy carbon electrode having a diameter of 3mm was successively coated with Al having a diameter of 0.3 μm and 0.05. mu.m2O3Polishing the powder into a mirror surface, washing with distilled water, then sequentially ultrasonically cleaning in absolute ethyl alcohol and distilled water for 1min, taking out, washing with distilled water, and airing at room temperature to obtain a pretreated glassy carbon electrode;
(2) mixing 100mg of graphene oxide with 50mL of deionized water, ultrasonically stripping for 4h, adding 0.5mL of aniline, continuously ultrasonically stirring for 30min after uniformly stirring, adding 5mL of 0.5MHCl containing 1.25g of Ammonium Persulfate (APS) under low-temperature stirring (below 10 ℃), continuously stirring for 6 h at low temperature, transferring into a reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 16 h, filtering the solution obtained after reaction by using a sand core filter, washing the solution with distilled water for a plurality of times, carrying out vacuum drying at 80 ℃ for overnight, grinding to obtain powder, placing the powder into a quartz boat, calcining in a tubular furnace under nitrogen atmosphere, calcining at 400 ℃ for 2h, subsequently heating to 700 ℃ for 1 h, taking out and grinding to obtain a doped graphene material;
(3) dispersing the doped graphene material in water to obtain a dispersion liquid with the concentration of 10mg/mL, preparing a horseradish peroxidase solution with the concentration of 25mg/mL by adopting a PBS solution (0.1mol/L, pH 6.5.5), preparing a chitosan solution with the concentration of 10mg/mL by adopting a 1% acetic acid solution, mixing the three solutions in a volume ratio of 1:1:1 to obtain a mixed solution, dripping 6 mu L of the mixed solution on the surface of the electrode in the step (1), and airing at room temperature to obtain a modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system (a platinum electrode is used as a counter electrode, and saturated calomel is used as a reference electrode) by the modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting the hydroquinone.
Electrochemical tests were performed at room temperature, all at 10mL containing 0.5mM H2O2In phosphate buffer (0.1mol/L, pH6.5) and tested by passing N through2And cyclic voltammetry is adopted in the test process. Wherein, the blank control is not added with hydroquinone solution, and 50 mu L of hydroquinone solution is added in turn after the test is stable.
The biosensor of this example measured a reduction peak catalytic current of 10.89 μ A at a hydroquinone concentration of 0.5 mmol/L.
The cyclic voltammograms of the enzyme-modified working electrodes in different solutions in this example are shown in FIG. 1. Curve a is the cyclic voltammetry curve of the enzyme modified working electrode in PBS, curve b is the cyclic voltammetry curve of the enzyme modified working electrode in 0.5mM hydroquinone, and curve c is the cyclic voltammetry curve of the enzyme modified working electrode in 0.5mM hydroquinone and 0.5mM H2O2Cyclic voltammograms in mixed solution. As can be seen from FIG. 1, when hydroquinone solution was added alone, a pair of distinct redox peaks appeared when hydroquinone was added with H2O2The simultaneous existence of the two shows that the reduction peak is obviously increased and the oxidation peak is obviously reduced, which indicates that the H is added2O2Is favorable for the catalytic action of the enzyme on the p-dihydroxybenzene.
The cyclic voltammogram of the biosensor for detecting hydroquinone based on nitrogen-doped graphene composite horseradish peroxidase in hydroquinone solutions with different concentrations prepared in this example is shown in fig. 2. And continuously adding hydroquinone into a phosphate buffer solution with the pH value of 6.5 and the concentration of 0.1mo1/L to obtain a cyclic voltammetry curve, wherein the hydroquinone concentration at the right end of the oxidation curve is 0.09mmo1/L, 0.5mmo1/L, 1.5mmo1/L, 2mmo1/L, 3mmo1/L, 4mmo1/L and 5.075mmo1/L in sequence from bottom to top. The characteristics of the graphene-doped carrier are utilized, so that the fixation amount and stability of the enzyme catalyst on the surface of the electrode are powerfully increased, and the catalysis of the substrate is facilitated.
The standard curve graph of the response current of the biosensor for detecting hydroquinone based on nitrogen-doped graphene composite horseradish peroxidase, which is prepared in the embodiment, to hydroquinone with different concentrations is shown in fig. 3. the detection range of the modified electrode to a substrate is 9 × 10-5～5.075×10-3mol/L, linear equation is I (mu A) ═ 11.37-5.89C (mmol/L), correlation coefficient is R20.998. detection limit of 1 × 10-5mol/L(S/N＝3)。
Example 4
A preparation method of a hydroquinone enzyme biosensor based on nitrogen-doped graphene composite horseradish peroxidase comprises the following steps:
(1) a glassy carbon electrode with the diameter of 3mmUsing Al with diameter of 0.3 μm and 0.05 μm2O3Polishing the powder into a mirror surface, washing with distilled water, then sequentially ultrasonically cleaning in absolute ethyl alcohol and distilled water for 1min, taking out, washing with distilled water, and airing at room temperature to obtain a pretreated glassy carbon electrode;
(2) mixing 100mg of graphene oxide with 50mL of deionized water, ultrasonically stripping for 4h, adding 0.5mL of aniline, continuously ultrasonically stirring for 30min after uniformly stirring, adding 5mL of 0.5MHCl containing 1.25g of Ammonium Persulfate (APS) under low-temperature stirring (below 10 ℃), continuously stirring for 6 h at low temperature, transferring into a reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 16 h, filtering the solution obtained after reaction by using a sand core filter, washing the solution with distilled water for a plurality of times, carrying out vacuum drying at 80 ℃ for overnight, grinding to obtain powder, placing the powder into a quartz boat, calcining in a tubular furnace under nitrogen atmosphere, calcining at 400 ℃ for 2h, subsequently heating to 700 ℃ for 1 h, taking out and grinding to obtain a doped graphene material;
(3) dispersing the doped graphene material in water to obtain a dispersion liquid with the concentration of 10mg/mL, preparing a horseradish peroxidase solution with the concentration of 30mg/mL by adopting a PBS solution (0.1mol/L, pH 6.5.5), preparing a chitosan solution with the concentration of 10mg/mL by adopting a 1% acetic acid solution, mixing the three solutions in a volume ratio of 1:1:1 to obtain a mixed solution, dripping 6 mu L of the mixture on the surface of the electrode in the step (1), and airing at room temperature to obtain a modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system (a platinum electrode is used as a counter electrode, and saturated calomel is used as a reference electrode) by the modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting the hydroquinone.
Electrochemical tests were performed at room temperature, all at 10mL containing 0.5mM H2O2In phosphate buffer (0.1mol/L, pH6.5) and tested by passing N through2And cyclic voltammetry is adopted in the test process. Wherein, the blank control is not added with hydroquinone solution, and 50 mu L of hydroquinone solution is added in turn after the test is stable.
In this example, the reduction peak catalytic current was 9.466 μ A at a hydroquinone concentration of 0.5 mmol/L.
Example 5
A preparation method of a hydroquinone enzyme biosensor based on nitrogen-doped graphene composite horseradish peroxidase comprises the following steps:
(1) a glassy carbon electrode having a diameter of 3mm was successively coated with Al having a diameter of 0.3 μm and 0.05. mu.m2O3Polishing the powder into a mirror surface, washing with distilled water, then sequentially ultrasonically cleaning in absolute ethyl alcohol and distilled water for 1min, and then placing the glassy carbon electrode in 10mL potassium ferricyanide solution (5mmol/L K)3Fe(CN)6+0.1mol/L KCl) is scanned for 6 circles by adopting a cyclic voltammetry method at 0-0.8V for electrode activation, and the electrode is taken out and washed by distilled water and dried at room temperature to obtain a pretreated glassy carbon electrode;
(2) mixing 100mg of graphene oxide with 50mL of deionized water, ultrasonically stripping for 1-4 h, adding 0.5mL of aniline, continuously ultrasonically stirring for 30min after uniformly stirring, adding 5mL of 0.5MHCl containing 1.25g of Ammonium Persulfate (APS) under low-temperature stirring (below 10 ℃), continuously stirring for 6 h at low temperature, transferring into a reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 16 h, filtering the solution obtained after reaction by using a sand core filter, washing for a plurality of times by using distilled water, carrying out vacuum drying at 80 ℃ overnight, grinding to obtain powder, placing the powder into a quartz boat, calcining in a tubular furnace in a nitrogen atmosphere, calcining at 400 ℃ for 2h, subsequently heating to 700 ℃ for 1 h, taking out and grinding to obtain a doped graphene material;
(3) dispersing the doped graphene composite material in water to obtain a dispersion liquid with the concentration of 10mg/mL, preparing a horseradish peroxidase solution with the concentration of 35mg/mL by adopting a PBS solution (0.1mol/L, pH 6.5.5), preparing a chitosan solution with the concentration of 10mg/mL by adopting a 1% acetic acid solution, mixing the three solutions in a volume ratio of 1:1:1 to obtain a mixed solution, dripping 6 mu L of the mixture on the surface of the electrode in the step (1), and airing at room temperature to obtain a modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system (a platinum electrode is used as a counter electrode, and saturated calomel is used as a reference electrode) by using the modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting hydroquinone.
Electrochemical tests were performed at room temperature, all at 10mL containing 0.5mM H2O2In phosphate buffer (0.1mol/L, pH6.5) and tested by passing N through2And cyclic voltammetry is adopted in the test process. Wherein, the blank control is not added with hydroquinone solution, and 50 mu L of hydroquinone solution is added in turn after the test is stable.
In this example, the reduction peak catalytic current was 7.235 μ A at a hydroquinone concentration of 0.5 mmol/L.
Preparing the graphene oxide:
(a) cleaning graphite powder;
(b) and (3) taking the cleaned graphite powder, and preparing the graphene oxide by adopting a modified Hummers method.
The graphite powder cleaning process in the step (a) is operated as follows: weighing 2.5-10 g of graphite powder in a big beaker, adding 100mL of distilled water, adding 100mL of concentrated hydrochloric acid in a ventilation kitchen, heating in a water bath at 60-80 ℃, stirring for 2h, carrying out vacuum filtration, sequentially washing with distilled water, acetone and ethanol, after washing, drying in a vacuum drying oven at 100 ℃, and grinding with a quartz mortar into powder for later use.
The graphene oxide preparation operation process in the step (b) is as follows: (1) low-temperature reaction (0-4 ℃): placing 1000mL big beaker in ice water bath, adding 110mL concentrated H2SO4Simultaneously stirring to reduce the temperature to a low-temperature reaction region, and then sequentially adding 2.5-10 g of cleaned graphite powder and 2.5g of NaNO3，15g KMnO4. Timing after adding, stirring and reacting for 90min, wherein the solution is purple green; (2) medium-temperature reaction (30-40 ℃): raising the temperature, keeping the temperature at about 35 ℃ under stirring, reacting for 90min while timing, and keeping the solution in purple green; (3) high-temperature reaction (70-100 ℃): after the medium-temperature reaction, slowly adding 220mL of deionized water into a beaker, heating to control the temperature to be 85 ℃, and then slowly adding a certain amount of hydrogen peroxide (5 percent about 13mL) until the solution gradually becomes golden yellow; (4) after the preparation reaction is finished, when the reaction solution is warm, the solution after the reaction is washed by deionized water and filtered, and is repeatedly centrifuged by a centrifugal machine with the rotating speed of 4000rpm and then is cleaned and filtered until SO can not be detected in the filtrate4 2-Till now (with BaCl)2Examination of). And ultrasonically cleaning the obtained black product by using deionized water for 40min, and drying at 40 ℃ for 24h to obtain black-brown graphene oxide.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (9)

1. A hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase is characterized in that: the material recognition membrane is mainly prepared by mixing nitrogen-doped graphene dispersion liquid, horseradish peroxidase solution and chitosan solution to form a membrane;
the nitrogen-doped graphene dispersion liquid is obtained by dispersing nitrogen-doped graphene in water; the horseradish peroxidase solution is obtained by dissolving horseradish peroxidase in a PBS solution; the chitosan solution is obtained by preparing chitosan into a solution; the nitrogen-doped graphene: horse radish peroxidase: the mass ratio of the chitosan is (2-20): (15-35): 10;
the nitrogen-doped graphene is prepared by the following method:
(a) uniformly dispersing graphene oxide and aniline; under the action of an oxidant and in an acidic medium, aniline is subjected to polymerization reaction, and is placed in a hydrothermal reaction kettle for hydrothermal reaction to obtain a polyaniline-graphene oxide compound;
(b) calcining the polyaniline-graphene oxide compound to obtain nitrogen-doped graphene; the calcining is to calcine at 400-500 ℃ and then continue calcining at 700-800 ℃;
in the step (a), the mass-to-volume ratio of the graphene oxide to the aniline is (50-200) mg: 0.5 mL;
the molar ratio of the oxidant to the acidic medium is (2-2.5): 1, the volume ratio of aniline to an acidic medium is 1 (10-20); the temperature of the polymerization reaction is 4-10 ℃;
the calcining atmosphere in the step (b) is protective atmosphere, and the calcining time in the step (b) is 1.5-2.5 hours; the continuous calcining time is 0.5-1.5 hours.
2. The hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to claim 1, which is characterized in that: the oxidant in the step (a) is ammonium persulfate, the acidic medium is hydrochloric acid, the acidic medium is added in the form of aqueous solution, and the concentration of the acidic medium is 0.5 mol/L; the polymerization reaction time is 6-12 h; the temperature of the hydrothermal reaction is 160-200 ℃, and the time of the hydrothermal reaction is 12-20 h.
3. The hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to claim 1, which is characterized in that: ultrasonically stripping graphene oxide in water, and adding aniline for ultrasonic dispersion to be uniform; and then adding a mixture of an oxidant and an acidic medium at a low temperature, carrying out polymerization reaction and hydrothermal reaction to obtain the polyaniline-graphene oxide compound.
4. The hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to claim 3, wherein the biosensor comprises: the mass-volume ratio of the graphene oxide to water is (50-200) mg: 50mL, and the ultrasonic stripping time is 1-4 h.
5. The preparation method of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to any one of claims 1 to 4, which is characterized by comprising the following steps: the method comprises the following steps:
(1) carrying out surface pretreatment on the substrate electrode;
(2) dispersing the nitrogen-doped graphene composite material in water to obtain a dispersion liquid; dissolving horseradish peroxidase in a PBS solution to obtain a horseradish peroxidase solution; preparing chitosan into a solution to obtain a chitosan solution; uniformly mixing the dispersion liquid, a horseradish peroxidase solution and a chitosan solution to obtain a composite solution;
(3) dripping the composite solution on the surface of the substrate electrode subjected to surface pretreatment, and airing at room temperature to obtain an enzyme modified working electrode based on the nitrogen-doped graphene composite horseradish peroxidase;
(4) and (3) forming a three-electrode system by the enzyme modified working electrode, the reference electrode and the counter electrode to obtain the biosensor for detecting hydroquinone.
6. The preparation method of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to claim 5, which is characterized by comprising the following steps: the concentration of the doped graphene material dispersion liquid in the step (2) is 2-20 mg/mL;
the concentration of the horseradish peroxidase solution in the step (2) is 15-35 mg/mL, and the enzyme solution is prepared by adopting a PBS solution;
the chitosan solution in the step (2) is prepared from 0.1-2wt% acetic acid solution, and the concentration is 10 mg/mL.
7. The preparation method of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to claim 6, which is characterized by comprising the following steps: the PBS solution was pH6.5, 0.1M PBS solution.
8. The preparation method of the hydroquinone biosensor based on the nitrogen-doped graphene composite horseradish peroxidase according to claim 5, which is characterized by comprising the following steps: the volume ratio of the doped graphene dispersion liquid, the horseradish peroxidase solution and the chitosan solution in the step (2) is 1:1: 1.
9. The application of the nitrogen-doped graphene composite horseradish peroxidase-based hydroquinone biosensor according to any one of claims 1-4 in hydroquinone detection.
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