CN109254053B - Preparation method and application of environmental estrogen electrochemical analysis sensor - Google Patents
Preparation method and application of environmental estrogen electrochemical analysis sensor Download PDFInfo
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Abstract
The invention discloses a preparation method of an environmental estrogen electrochemical analysis sensor. Belongs to the technical field of novel nanometer functional materials and biosensing analysis. According to the invention, firstly, a cobalt hydroxide nanosheet array is prepared on a disposable throwable electrode, a polydopamine film containing an electronic mediator and a molecularly imprinted polymer taking environmental estrogen as template molecules are sequentially and directly prepared on the cobalt hydroxide nanosheet array by utilizing a large specific surface area, a high-activity hydroxyl functional group and an amino functional group of the polydopamine in an in-situ growth method, and after the template molecules are eluted, the original positions of the template molecules are changed into cavities, namely the molecularly imprinted polymer of the template molecules is eluted, so that the preparation of the environmental estrogen electrochemical analysis sensor is completed.
Description
Technical Field
The invention relates to a preparation method and application of an electrochemical analysis sensor. Belongs to the technical field of novel nanometer functional materials and biosensing analysis.
Background
Environmental estrogen is a chemical substance existing in the environment, has the activity similar to that of estrogen in organisms, has the function of simulating estrogen after entering human bodies, generates various diseases such as cancer, damage to reproductive development systems, immune systems, nervous systems and the like by disturbing the normal functions of endocrine, immune, nervous and other systems of the human bodies, and is environmental toxin seriously harming the human health. Common environmental estrogen sources include, for example, pesticides (e.g., organochlorine pesticides), second-hand smoke, waste gases (e.g., toxic gases generated by burning plastic waste), food additives, and the like. The detection method for the environmental estrogen mainly comprises a high performance liquid chromatography, a gas chromatography, a chromatography-mass spectrometry combined method, an enzyme-linked immunosorbent assay, a radioimmunoassay and the like. However, most of the detection methods are expensive and complex in operation, and inspection personnel can perform detection after being strictly trained. Some simple methods such as electrochemical analysis methods have poor specificity although they are simple to operate, sensitive and rapid to detect. Therefore, the method for rapidly and sensitively detecting the environmental estrogen is very important to public health and has wide market application prospect.
The molecular imprinting electrochemical sensor has high specificity selectivity, excellent stability, excellent reproducibility, wide detection range and bottom detection limit. The sensor has the advantages of simple preparation, convenient detection, high sensitivity, low cost and the like, and can be widely applied to the fields of chromatographic separation, membrane separation, solid-phase extraction, drug controlled release, chemical sensing and the like. Molecularly Imprinted Polymers (MIPs), also known as "plastic antibodies", are capable of specifically recognizing and selectively adsorbing a specific target molecule (i.e., template molecule). The molecular imprinting technology has many advantages, such as corrosion resistance of organic reagents, good stability, high temperature resistance and simple preparation. Thus, MIP electrochemical sensors based on MIP in combination with electrochemical sensors (MIP-ECS) have attracted a hot interest in the field of electroanalytical chemistry, especially the detection of small molecule contaminants, over the last few years. However, in the preparation process of the traditional MIP-ECS, the defects of difficult elution of template molecules, difficult control of the thickness of the imprinted membrane, poor reproducibility and the like exist, and the application of the molecularly imprinted membrane in an electrochemical sensor is limited. The problems, especially the technical problems that the sensitivity of the electrochemical sensor is reduced due to the fact that the thickness of the molecularly imprinted membrane is not easy to control, and the stability and the reproducibility are reduced due to the fact that the molecularly imprinted membrane is easy to fall off from the surface of an electrode in the elution process, limit the application of MIP _ ECS, so that the research of a new molecularly imprinted polymer synthesis method, a new molecularly imprinted membrane electrode modification method and a method for combining the molecularly imprinted membrane and a substrate material for solving the preparation and application problems of MIP-ECS has important research significance and market value.
Disclosure of Invention
The invention aims to provide a preparation method of an electrochemical analysis sensor for environmental estrogen, which has the advantages of strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, a cobalt hydroxide nanosheet array is prepared on a disposable throwable electrode, a polydopamine film containing an electron mediator and a molecularly imprinted polymer taking environmental estrogen as template molecules are sequentially and directly prepared on the cobalt hydroxide nanosheet array in an in-situ growth method by utilizing the large specific surface area and the high-activity hydroxyl functional group of the cobalt hydroxide nanosheet array and the amino functional group of the polydopamine, and after the template molecules are eluted, the original positions of the template molecules are changed into cavities, namely the molecularly imprinted polymer of the template molecules is eluted, so that the preparation of the environmental estrogen electrochemical analysis sensor is completed. When the electrochemical analysis sensor for environmental estrogen is used for detecting environmental estrogen, the electrochemical analysis sensor for environmental estrogen is inserted into a solution to be detected, and the environmental estrogen in the solution to be detected can be adsorbed into a cavity of NIP. The greater the concentration of environmental estrogen in the solution to be tested, the more environmental estrogen is adsorbed into the cavities of the NIP. When electrochemical detection is carried out, the intensity of the detection current is reduced along with the increase of the environmental estrogen adsorbed in the hole of the NIP, so that the concentration of the environmental estrogen in the solution to be detected can be qualitatively and quantitatively determined according to the reduction degree of the current intensity.
The technical scheme adopted by the invention is as follows:
1. the environmental estrogen electrochemical analysis sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a cobalt hydroxide nanosheet array electrode Co-nanoarray; the template-free molecularly imprinted polymer NIP is a molecularly imprinted polymer without template molecules; the molecularly imprinted polymer without the template molecule is obtained by eluting the template molecule from a MIP containing the template molecularly imprinted polymer; the MIP containing the template molecule engram polymer is the MIP containing the template molecule; the template molecule is environmental estrogen;
2. the preparation method of the cobalt hydroxide nanosheet array electrode Co-nanoarray in the technical scheme 1 comprises the following preparation steps:
(1) carrying out ultrasonic cleaning treatment on the disposable throwable electrode by respectively using dilute hydrochloric acid, absolute ethyl alcohol and deionized water so as to remove an oxide layer and surface impurities of the disposable throwable electrode;
(2) weighing 1-3 mmol Co (NO)3)2And 3-9 mmol of urea CO (NH)2)2Placing the mixture into a 50mL beaker, adding 30mL deionized water, stirring until the mixture is clear, and then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting at the temperature of 100-130 ℃ for 9-12 hours to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 4-6 hours at the temperature of 20-40 ℃, taking out and washing with deionized water for 2-4 times to prepare a cobalt hydroxide nanosheet array electrode Co-nanoarray;
the disposable and disposable electrode is selected from one of the following electrodes: foam nickel, foam copper, pure nickel sheets, pure copper sheets, pure cobalt sheets, pure silicon sheets and conductive carbon cloth;
in the phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate: the concentration of dopamine is 2-5 mg/mL, the concentration of ammonium persulfate is 3-8 mg/mL, the concentration of cobalt nitrate is 0.1-0.5 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2-8.5;
3. the preparation method of the Co-nanoarray in-situ grown MIP containing the template molecularly imprinted polymer comprises the following preparation steps:
(1) respectively weighing 0.25-0.45 mmol of template molecules and 3-5 mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 8-15 mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 2-methacrylic acid MAA are dissolved;
(2) adding 15-25 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the Co-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the Co-nanoarray into the precursor mixed solution in the step (2), and adding the Co-nanoarray into the precursor mixed solution in N2Under the temperature of environment and water bath of 20-40 ℃, rotationally stirring at the speed of 5-200 r/s, simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 drops/s to initiate polymerization, and obtaining the in-situ grown MIP on Co-nanoarray;
4. the preparation steps of the Co-nanoarray in-situ grown template-free molecularly imprinted polymer NIP in the technical scheme 1 are as follows: immersing the MIP which is obtained in the technical scheme 3 and grows in situ on the Co-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecules for 5-20 min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9 (1-5);
5. the preparation steps of the electrochemical sensor for analyzing environmental estrogen in the technical scheme 1 are as follows: washing the template-free molecularly imprinted polymer NIP which grows on the Co-nanoarray in situ prepared in the technical scheme 2-4 with deionized water for 2-4 times, and airing at room temperature to prepare the environmental estrogen electrochemical analysis sensor;
6. the electrochemical analysis sensor for environmental estrogen prepared by the technical scheme 1-5 is applied to the detection of environmental estrogen, and comprises the following application steps:
(1) preparing a standard solution: preparing a group of environmental estrogen standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking an electrochemical analysis sensor of environmental estrogen as a working electrode, inserting the electrochemical analysis sensor of environmental estrogen into the environmental estrogen standard solutions with different concentrations prepared in the step (1), hatching for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, forming a three-electrode system with the modified working electrode in the step (2), connecting the three-electrode system with an electrochemical workstation, and sequentially adding 15mL phosphate buffer solution PBS into an electrolytic bath; detecting a current response of the assembled working electrode by Differential Pulse Voltammetry (DPV); the response current intensity of the blank sample is recordedI 0The response current intensity of standard solution containing different concentrations of environmental estrogen is recordedI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIAnd the mass concentration of the environmental estrogen standard solutionCWith a linear relationship therebetween, plotting ΔI－CA working curve; the phosphate buffer solution PBThe S concentration is 10mmol/L, pH value is 7.4; the parameters during DPV detection are set as follows: the range and the direction are 0-1V, the step is 0.05V, the pulse time is 0.05s, the sampling time is 0.016s, and the pulse period is 0.5 s;
(4) detecting environmental estrogen in a sample to be detected: replacing the environmental estrogen standard solution in the step (1) with a sample to be detected, detecting according to the method in the steps (2) and (3), and responding to the difference delta of the reduction of the current intensityIAnd working curve, obtaining the content of environmental estrogen in the sample to be tested;
7. the environmental estrogen in the technical scheme 1-6 is one of the following environmental estrogens: estradiol, estriol, diethylstilbestrol, bisphenol A and nonyl phenol.
Advantageous results of the invention
(1) The electrochemical analysis sensor for environmental estrogen is simple to prepare, convenient to operate, low in cost, applicable to portable detection and promising in market development prospect, and realizes quick, sensitive and high-selectivity detection on a sample;
(2) according to the invention, the molecularly imprinted polymer is grown in situ on the Co-nanoarray electrode of the cobalt hydroxide nanosheet array for the first time, on one hand, more and more uniform molecularly imprinted polymer can be grown by utilizing the large specific surface area of the Co-nanoarray, and the Co-nanoarray has excellent electron transfer capacity, so that the detection sensitivity is greatly improved; on the other hand, when dopamine is polymerized onto a cobalt hydroxide nanosheet array in situ, cobalt ions are creatively doped as an electronic mediator, and electrochemical response current is directly generated during detection, so that the sensor can directly detect in a buffer solution without adding other mediator substances, thereby further reducing the signal background, improving the detection sensitivity, greatly reducing the detection cost and reducing the environmental pollution;
(3) according to the invention, the high-activity hydroxyl functional groups rich in the cobalt hydroxide nanosheet array and the large specific surface area are combined with dopamine, so that when dopamine is polymerized in situ on the surface of the cobalt hydroxide nanosheet array, a thin enough polydopamine film is formed and simultaneously the polydopamine film is uniformly covered on the cobalt hydroxide nanosheet array, thereby laying a better polymerized molecularly imprinted polymer for the next step; then utilizing the strong connection effect of polydopamine on hydroxyl functional groups and amino groups rich in the molecularly imprinted polymer, skillfully using Co-nanoarray as a stirrer, immersing and stirring in the molecularly imprinted precursor mixed solution, and directly growing the molecularly imprinted polymer capable of controlling the film thickness on the surface of the Co-nanoarray in situ by controlling the stirring speed, the dropping speed of a polymerization reaction initiator and the polymerization reaction temperature, so that the Co-nanoarray can firmly load the molecularly imprinted polymer on one hand, and the stability and the reproducibility of the prepared electrochemical sensor are obviously improved; on the other hand, the film forming thickness of the molecularly imprinted polymer on the surface of the electrode can be effectively controlled, and the technical problem of poor reproducibility caused by the fact that the film forming thickness of the molecularly imprinted film on the surface of the electrode cannot be controlled is solved; in addition, the preparation method of the invention has important scientific significance and application value for effectively controlling the thickness of the formed film and coating the electronic mediator in situ, and fully improving the sensitivity and detection limit of the electrochemical sensor based on the molecular imprinting.
Detailed Description
EXAMPLE 1 preparation of Co-nanoarray
(1) Carrying out ultrasonic cleaning treatment on the disposable throwable electrode by respectively using dilute hydrochloric acid, absolute ethyl alcohol and deionized water so as to remove an oxide layer and surface impurities of the disposable throwable electrode;
(2) weighing 1mmol Co (NO)3)2And 3mmol of Urea CO (NH)2)2Placing the mixture into a 50mL beaker, adding 30mL deionized water, stirring until the mixture is clear, and then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting at the temperature of 100 ℃ for 12 hours to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 4 hours at the temperature of 20 ℃, taking out, and performing immersion washing for 2 times by using deionized water to prepare a cobalt hydroxide nanosheet array electrode Co-nanoarray;
wherein the disposable throwable electrode is foamed nickel; the concentration of dopamine is 2 mg/mL, the concentration of ammonium persulfate is 3 mg/mL, the concentration of cobalt nitrate is 0.1 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2.
EXAMPLE 2 preparation of Co-nanoarray
(1) Carrying out ultrasonic cleaning treatment on the disposable throwable electrode by respectively using dilute hydrochloric acid, absolute ethyl alcohol and deionized water so as to remove an oxide layer and surface impurities of the disposable throwable electrode;
(2) weighing 2 mmol Co (NO)3)2And 6 mmol of Urea CO (NH)2)2Placing the mixture into a 50mL beaker, adding 30mL deionized water, stirring until the mixture is clear, and then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting for 11 hours at the temperature of 110 ℃ to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 5 hours at the temperature of 30 ℃, taking out, and performing immersion washing for 3 times by using deionized water to prepare a cobalt hydroxide nanosheet array electrode Co-nanoarray;
wherein the disposable throwable electrode is a pure copper sheet; the concentration of dopamine is 3.5 mg/mL, the concentration of ammonium persulfate is 6.2 mg/mL, the concentration of cobalt nitrate is 0.3 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.0.
EXAMPLE 3 preparation of Co-nanoarray
(1) Carrying out ultrasonic cleaning treatment on the disposable throwable electrode by respectively using dilute hydrochloric acid, absolute ethyl alcohol and deionized water so as to remove an oxide layer and surface impurities of the disposable throwable electrode;
(2) 3mmol of Co (NO) are weighed3)2And 9mmol of Urea CO (NH)2)2Placing the mixture into a 50mL beaker, adding 30mL deionized water, stirring until the mixture is clear, and then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting at the temperature of 130 ℃ for 9 hours to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting at the temperature of 40 ℃ for 6 hours, taking out, and performing immersion washing with deionized water for 4 times to prepare a cobalt hydroxide nanosheet array electrode Co-nanoarray;
wherein the disposable throwable electrode is a conductive carbon cloth; the concentration of dopamine is 5mg/mL, the concentration of ammonium persulfate is 8mg/mL, the concentration of cobalt nitrate is 0.5 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.5.
Example 4 method for preparing environmental estrogen electrochemical analysis sensor
(1) Respectively weighing 0.25 mmol of template molecules and 3mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 8 mL of acetonitrile, and performing ultrasonic treatment for 30min until all the template molecules and the 3mmol of 2-methacrylic acid MAA are dissolved;
(2) adding 15 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) the Co-nanoarray prepared in example 1 was clamped to a rotary stirrer and inserted into the precursor mixed solution in step (2) under N2Under the temperature of environment and water bath 20 ℃, stirring in a rotating way at the speed of 200 r/s, simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1 d/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on Co-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the Co-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecules for 5 min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; continuously washing with deionized water for 2 times, and air drying at room temperature to obtain environmental estrogen electrochemical analysis sensor;
the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9: 1.
Example 5 method for preparing environmental estrogen electrochemical analysis sensor
(1) Respectively weighing 0.35mmol of template molecules and 4 mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 12 mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 2-methacrylic acid MAA are dissolved;
(2) adding 18 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the Co-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the Co-nanoarray into the precursor mixed solution in the step (2), and adding the Co-nanoarray into the precursor mixed solution in N2Under the temperature of environment and water bath 30 ℃, stirring in a rotating way at the speed of 60 r/s, simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 10 drops/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on Co-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the Co-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecule for 10min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; continuously washing with deionized water for 3 times, and air drying at room temperature to obtain environmental estrogen electrochemical analysis sensor;
the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9: 3.
Example 6 method for preparing environmental estrogen electrochemical analysis sensor
(1) Respectively weighing 0.45mmol of template molecules and 5mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 15mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 5mmol of 2-methacrylic acid MAA are dissolved;
(2) adding 25mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the Co-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the Co-nanoarray into the precursor mixed solution in the step (2), and adding the Co-nanoarray into the precursor mixed solution in N2Under the temperature of environment and water bath 40 ℃, rotationally stirring at the speed of 5 r/s, simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 20 drops/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on Co-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the Co-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecules for 20min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; continuously washing with deionized water for 4 times, and airing at room temperature to obtain the environmental estrogen electrochemical analysis sensor;
the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9: 5.
Embodiment 7 the environmental estrogen sensor prepared in embodiments 1 to 6 is applied to the detection of environmental estrogens, and comprises the following steps:
(1) preparing a standard solution: preparing a group of environmental estrogen standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking an electrochemical analysis sensor of environmental estrogen as a working electrode, inserting the electrochemical analysis sensor of environmental estrogen into the environmental estrogen standard solutions with different concentrations prepared in the step (1), hatching for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, forming a three-electrode system with the modified working electrode in the step (2), connecting the three-electrode system with an electrochemical workstation, and sequentially adding 15mL PBS into an electrolytic bath; detection of current of assembled working electrode by Differential Pulse Voltammetry (DPV)Responding; the response current intensity of the blank sample is recordedI 0The response current intensity of standard solution containing different concentrations of environmental estrogen is recordedI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIAnd the mass concentration of the environmental estrogen standard solutionCWith a linear relationship therebetween, plotting ΔI－CA working curve; the PBS is 10mmol/L phosphate buffer solution, and the pH value of the phosphate buffer solution is 7.4; the parameters during DPV detection are set as follows: the range and the direction are 0-1V, the step is 0.05V, the pulse time is 0.05s, the sampling time is 0.016s, and the pulse period is 0.5 s;
(4) detecting environmental estrogen in a sample to be detected: replacing the environmental estrogen standard solution in the step (1) with a sample to be detected, detecting according to the method in the steps (2) and (3), and responding to the difference delta of the reduction of the current intensityIAnd obtaining the content of the environmental estrogen in the sample to be detected according to the working curve.
Example 8 the environmental estrogen sensors prepared in examples 1 to 6 were applied to the detection of different environmental estrogens according to the detection procedure of example 7, and the linear range and detection limit are shown in table 1:
TABLE 1 technical index for detection of environmental estrogens
Example 9 detection of environmental estrogens in milk
Accurately transferring a milk sample, adding a standard solution of environmental estrogen with a certain mass concentration, taking the milk without the environmental estrogen as a blank, performing a standard addition recovery experiment, detecting the environmental estrogen sensor prepared in the embodiments 1-6 according to the steps of the embodiment 7, and determining the recovery rate of the environmental estrogen in the milk sample, wherein the detection result is shown in a table 2:
TABLE 2 detection results of environmental estrogens in milk
The detection results in table 2 show that the Relative Standard Deviation (RSD) of the results is less than 3.0%, the average recovery rate is 98.0-101.6%, and the method can be used for detecting multiple environmental estrogens in milk, and is high in sensitivity, strong in specificity, and accurate and reliable in result.
Example 10 detection of environmental estrogens in Water samples
Accurately transferring a certain water sample, adding a standard solution of environmental estrogen with a certain mass concentration, taking the water sample without the environmental estrogen as a blank, performing a standard addition recovery experiment, detecting the environmental estrogen sensor prepared in the embodiments 1-6 according to the steps of the embodiment 7, determining the recovery rate of the environmental estrogen in the water sample, and obtaining a detection result shown in table 3:
TABLE 3 detection results of environmental estrogens in water samples
The detection results in table 3 show that the Relative Standard Deviation (RSD) of the results is less than 3.0%, the average recovery rate is 98.4-102%, and the method can be used for detecting multiple environmental estrogens in a water sample, and is high in sensitivity, strong in specificity, and accurate and reliable in result.
Claims (7)
1. The preparation method of the environmental estrogen electrochemical analysis sensor is characterized in that the environmental estrogen electrochemical analysis sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a cobalt hydroxide nanosheet array electrode Co-nanoarray; the template-free molecularly imprinted polymer NIP is a molecularly imprinted polymer without template molecules; the molecularly imprinted polymer without the template molecule is obtained by eluting the template molecule from a MIP containing the template molecularly imprinted polymer; the MIP containing the template molecule engram polymer is the MIP containing the template molecule; the template molecule is environmental estrogen; the MIP containing the template molecular engram polymer is directly grown on Co-nanoarray in situ, and the preparation method comprises the following preparation steps:
(1) respectively weighing 0.25-0.45 mmol of template molecules and 3-5 mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 8-15 mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 2-methacrylic acid MAA are dissolved;
(2) adding 15-25 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the Co-nanoarray on a rotary stirrer, inserting the Co-nanoarray into the precursor mixed solution in the step (2), and adding N2And (2) rotationally stirring at the speed of 5-200 r/s in the environment and at the temperature of 20-40 ℃ in a water bath, and simultaneously dropwise adding 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 d/s to initiate polymerization to obtain the in-situ grown MIP on the Co-nanoarray.
2. The method for preparing an environmental estrogen electrochemical analysis sensor according to claim 1, wherein the Co-nanoarray is prepared by the following steps:
(1) carrying out ultrasonic cleaning treatment on the disposable throwable electrode by respectively using dilute hydrochloric acid, absolute ethyl alcohol and deionized water so as to remove an oxide layer and surface impurities of the disposable throwable electrode;
(2) weighing 1-3 mmol Co (NO)3)2And 3-9 mmol of urea CO (NH)2)2Placing the mixture into a 50mL beaker, adding 30mL deionized water, stirring until the mixture is clear, and then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting at the temperature of 100-130 ℃ for 9-12 hours to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 4-6 hours at the temperature of 20-40 ℃, taking out, and performing immersion cleaning for 2-4 times by using deionized water to prepare a cobalt hydroxide nanosheet array electrode Co-nanoarray;
the disposable and disposable electrode is selected from one of the following electrodes: foam nickel, foam copper, pure nickel sheets, pure copper sheets, pure cobalt sheets, pure silicon sheets and conductive carbon cloth;
in phosphate buffer solution PBS containing dopamine and ammonium persulfate: the concentration of dopamine is 2-5 mg/mL, the concentration of ammonium persulfate is 3-8 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2-8.5.
3. The method for preparing an environmental estrogen electrochemical analysis sensor according to claim 1, wherein the preparation of the template-free molecularly imprinted polymer NIP comprises the following steps: immersing the obtained MIP which grows in situ on the Co-nanoarray and contains the template molecularly imprinted polymer in an eluant, eluting the template molecule for 5-20 min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9 (1-5).
4. The method for preparing an environmental estrogen electrochemical analysis sensor according to claim 1, wherein the environmental estrogen electrochemical analysis sensor is prepared by the steps of: and (3) washing the obtained template-free molecularly imprinted polymer NIP growing in situ on the Co-nanoarray with deionized water for 2-4 times, and airing at room temperature to obtain the environmental estrogen electrochemical analysis sensor.
5. The method for preparing an environmental estrogen electrochemical analysis sensor according to any one of claims 1 to 4, wherein the environmental estrogen is one of the following environmental estrogens: estradiol, estriol, diethylstilbestrol, bisphenol A and nonyl phenol.
6. The application of the electrochemical sensor for analyzing environmental estrogen prepared by the preparation method according to any one of claims 1 to 5, which is applied to the detection of environmental estrogen, is characterized in that the detection steps are as follows:
(1) preparing a standard solution: preparing a group of environmental estrogen standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking an electrochemical analysis sensor of environmental estrogen as a working electrode, inserting the electrochemical analysis sensor of environmental estrogen into the environmental estrogen standard solutions with different concentrations prepared in the step (1), hatching for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as a counter electrode, forming a three-electrode system with the modified working electrode in the step (2), connecting the three-electrode system with an electrochemical workstation, and sequentially adding 15mL phosphate buffer solution PBS into an electrolytic bath; detecting a current response of the assembled working electrode by Differential Pulse Voltammetry (DPV); the response current intensity of the blank sample is recorded as I0The response current intensity of standard solution containing different concentrations of environmental estrogen is denoted as IiThe difference of response current intensity is Δ I ═ I0-IiThe mass concentration C of the standard solution of environmental estrogen is in linear relation with the Delta I, and a Delta I-C working curve is drawn; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters during DPV detection are set as follows: the range and the direction are 0-1V, the step is 0.05V, the pulse time is 0.05s, the sampling time is 0.016s, and the pulse period is 0.5 s;
(4) detecting environmental estrogen in a sample to be detected: and (3) replacing the environmental estrogen standard solution in the step (1) with the sample to be detected, detecting according to the methods in the steps (2) and (3), and obtaining the content of the environmental estrogen in the sample to be detected according to the difference value delta I of the reduction of the response current intensity and the working curve.
7. The use according to claim 6, wherein the environmental estrogen is one of the following environmental estrogens: estradiol, estriol, diethylstilbestrol, bisphenol A and nonyl phenol.
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