CN109254051B - Preparation method and application of environmental estrogen electrochemiluminescence sensor - Google Patents

Preparation method and application of environmental estrogen electrochemiluminescence sensor Download PDF

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CN109254051B
CN109254051B CN201811306535.6A CN201811306535A CN109254051B CN 109254051 B CN109254051 B CN 109254051B CN 201811306535 A CN201811306535 A CN 201811306535A CN 109254051 B CN109254051 B CN 109254051B
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environmental estrogen
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environmental
estrogen
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CN109254051A (en
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程荣琦
张勇
魏琴
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University of Jinan
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    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

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Abstract

The invention discloses a preparation method of an environmental estrogen electrochemiluminescence sensor. Belongs to the technical field of novel nanometer functional materials and biosensing analysis. Firstly, a cobalt oxide nanosheet array is prepared on a disposable throwable electrode, an in-situ growth method is adopted by utilizing the large specific surface area of the cobalt oxide nanosheet array, a polydopamine film and an in-situ luminol-coated molecularly imprinted polymer which takes environmental estrogen as template molecules are sequentially and directly prepared on the iron oxide nanosheet array, 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, and therefore, the preparation of the environmental estrogen electrochemiluminescence sensor is completed.

Description

Preparation method and application of environmental estrogen electrochemiluminescence sensor
Technical Field
The invention relates to a preparation method and application of an electrochemiluminescence 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 electroanalytical chemical sensors include electrochemical sensors, electrochemiluminescence sensors, photoelectrochemical sensors and the like, and the sensors have 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 electroanalytical chemical sensors (MIP-ECS) based on the combination of MIPs with electroanalytical chemical sensors have attracted a great deal of interest in the field of electroanalytical chemistry, particularly 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 electroanalytical chemical 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 environmental estrogen electrochemiluminescence sensor which has the advantages of strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, a cobalt oxide nanosheet array is prepared on a disposable throwable electrode, an in-situ growth method is adopted by utilizing the large specific surface area of the cobalt oxide nanosheet array, a polydopamine film and an in-situ luminol-coated molecularly imprinted polymer which takes environmental estrogen as template molecules are sequentially and directly prepared on the iron oxide nanosheet array, 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, and therefore, the preparation of the environmental estrogen electrochemical luminescence sensor is completed. When the electrochemical luminescence sensor is used for detecting environmental estrogen, the electrochemical luminescence sensor for the 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 the 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 the electrochemiluminescence detection is carried out, the current intensity passing through the electrode is reduced along with the increase of the environmental estrogen absorbed in the cavity of the NIP, and the corresponding electrochemiluminescence signal is reduced, so that the concentration of the clenbuterol in the solution to be detected can be qualitatively and quantitatively determined according to the reduction degree of the intensity of the electrochemiluminescence light signal.
The technical scheme adopted by the invention is as follows:
1. the environmental estrogen electrochemical luminescence sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a cobalt oxide nanosheet array electrode CoO-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 oxide nanosheet array electrode CoO-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)2Put it into a 50mL beaker, add 30mL deionized water, stir until clear, then transfer to 50mL in a 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), reacting at the temperature of 100-130 ℃ for 9-12 hours, taking out, airing, and annealing at the temperature of 300-400 ℃ for 1-3 hours to prepare a cobalt oxide nanosheet array precursor electrode;
(4) inserting the cobalt oxide 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 oxide nanosheet array electrode CoO-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 preparation method of the MIP containing the template molecularly imprinted polymer grown in situ by CoO-nanoarray in the technical scheme 1 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 CoO-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the CoO-nanoarray into the precursor mixed solution in the step (2), and adding N2Rotating and stirring at the speed of 5-200 r/s at the temperature of 20-40 ℃ in an environment and water bath, simultaneously dropwise adding 1-3 mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 drops/s for initiating polymerization, and obtaining the product on the CoO-nanoarrayMIP containing template molecular engram polymer growing in situ;
4. the preparation steps of the template-free molecularly imprinted polymer NIP for CoO-nanoarray in-situ growth 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 CoO-nanoarray and contains the template molecularly imprinted polymer into 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);
5. the preparation steps of the environmental estrogen electrochemiluminescence sensor in the technical scheme 1 are as follows: washing the template-free molecularly imprinted polymer NIP which grows in situ on the CoO-nanoarray prepared in the technical scheme 2-4 with deionized water for 2-4 times, and airing at room temperature to obtain the environmental estrogen electrochemiluminescence sensor;
6. the electrochemical luminescence 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 environmental estrogen electrochemical luminescence sensor as a working electrode, inserting the environmental estrogen electrochemical luminescence sensor 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: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard was recorded asA 0Containing different concentrationsThe intensity of the response light signal of the environmental estrogen standard solution is recordedA iThe difference in response to the decrease in optical signal intensity is ΔA = A 0-A i，ΔAAnd the mass concentration of the environmental estrogen standard solutionCWith a linear relationship therebetween, plotting ΔA－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 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 detecting according to the difference delta responding to the reduction of the optical signal intensityAAnd 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 environmental estrogen electrochemiluminescence sensor is simple to prepare, convenient to operate, low in cost, applicable to portable detection and has market development prospect, and rapid, sensitive and high-selectivity detection of a sample is realized;
(2) according to the invention, the molecular imprinting polymer is grown in situ on the cobalt oxide nanosheet array electrode CoO-nanoarray for the first time, on one hand, more and more uniform molecular imprinting polymers can be grown by utilizing the large specific surface area of the CoO-nanoarray, and the CoO-nanoarray has excellent electron transfer capacity, so that the detection sensitivity is greatly improved; on the other hand, CoO-nanoarray has electrocatalytic activity on hydrogen peroxide, and can realize stable and efficient reaction of a luminol-hydrogen peroxide electrochemiluminescence system without adding horseradish peroxidase, so that the prepared sensor does not need to consider the problem of inactivation of biological enzyme, the use and storage of the sensor can be more stable and the conditions are loose, the signal background is further reduced, the detection sensitivity is improved, and meanwhile, the detection cost is greatly reduced and the environmental pollution is reduced;
(3) the large specific surface area of the iron oxide nanosheet array is combined with dopamine, so that when dopamine is polymerized in situ on the surface of the iron oxide nanosheet array, a sufficiently thin polydopamine film is formed and simultaneously uniformly covers the iron oxide nanosheet array, and further, a better polymerized molecularly imprinted polymer is laid; secondly, strong connection effect of polydopamine on amino groups rich in the molecularly imprinted polymer is utilized, CoO-nanoarray is skillfully used as a stirrer, the mixture is immersed and stirred in the molecularly imprinted precursor mixed solution, and the molecularly imprinted polymer with membrane thickness can be creatively coated with luminol in situ and directly grown in situ on the surface of CoO-nanoarray by controlling stirring speed, dropping speed of a polymerization reaction initiator and polymerization reaction temperature, so that the CoO-nanoarray can firmly load the molecularly imprinted polymer and the luminol on one hand, and the stability and the preparation reproducibility of the electrochemical sensor are remarkably 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 film forming thickness and quantitatively coating luminol in situ, and fully improving the sensitivity and detection limit of the molecular imprinting-based electrochemiluminescence sensor.
Detailed Description
EXAMPLE 1 preparation of CoO-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)2Put it into a 50mL beaker, add 30mL deionized water, stir until clear, then transfer to 50mL polytetramethyleneIn a vinyl fluoride reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), reacting at the temperature of 100 ℃ for 12 hours, taking out and airing, and annealing at the temperature of 300 ℃ for 3 hours to prepare a cobalt oxide nanosheet array precursor electrode;
(4) inserting the cobalt oxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 4 hours at the temperature of 20 ℃, taking out and washing for 2 times by using deionized water to prepare a cobalt oxide nanosheet array electrode CoO-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 phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2.
EXAMPLE 2 preparation of CoO-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 2mmol 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), reacting for 11 hours at the temperature of 110 ℃, taking out and airing, and annealing for 2 hours at the temperature of 350 ℃ to prepare a cobalt oxide nanosheet array precursor electrode;
(4) inserting the cobalt oxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 5 hours at the temperature of 30 ℃, taking out and washing with deionized water for 3 times to prepare a cobalt oxide nanosheet array electrode CoO-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 phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.0.
EXAMPLE 3 preparation of CoO-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), reacting for 9 hours at the temperature of 130 ℃, taking out and airing, and annealing for 1 hour at the temperature of 400 ℃ to prepare a cobalt oxide nanosheet array precursor electrode;
(4) inserting the cobalt oxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 6 hours at the temperature of 40 ℃, taking out and washing with deionized water for 4 times to prepare a cobalt oxide nanosheet array electrode CoO-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 phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.5.
Example 4 method for preparing environmental estrogen electrochemiluminescence 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) CoO-nanoarray prepared in example 1 was clamped to a rotating stirrer and inserted into the procedureIn the precursor mixed solution in the step (2), in N2Under the temperature of environment and water bath 20 ℃, stirring in a rotating way at the speed of 200 r/s, simultaneously dripping 1mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1 drop/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the CoO-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the CoO-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecule 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 the environmental estrogen electrochemical luminescence 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 electrochemiluminescence 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 CoO-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the CoO-nanoarray into the precursor mixed solution in the step (2), and adding N2Under the temperature of environment and water bath 30 ℃, rotationally stirring at the speed of 60 revolutions per second, simultaneously dripping 2 mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 10 drops per second to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the CoO-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the CoO-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 the environmental estrogen electrochemical luminescence 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 electrochemiluminescence 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 CoO-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the CoO-nanoarray into the precursor mixed solution in the step (2), and adding N2Under the temperature of environment and water bath 40 ℃, rotationally stirring at the speed of 5 r/s, simultaneously dripping 3mL of 1mmol/L luminol solution and 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 the CoO-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the CoO-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecule 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 air drying at room temperature to obtain the environmental estrogen electrochemical luminescence 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 environmental estrogen electrochemical luminescence sensor as a working electrode, inserting the environmental estrogen electrochemical luminescence sensor 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: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard was recorded asA 0The intensity of the response light signal of the standard solution containing different concentrations of environmental estrogen is recordedA iThe difference in response to the decrease in optical signal intensity is ΔA = A 0-A i，ΔAAnd the mass concentration of the environmental estrogen standard solutionCWith a linear relationship therebetween, plotting ΔA－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 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 detecting according to the difference delta responding to the reduction of the optical signal intensityAAnd 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
Figure 87261DEST_PATH_IMAGE002
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
Figure 535560DEST_PATH_IMAGE004
The detection results in table 2 show that the Relative Standard Deviation (RSD) of the results is less than 3.0%, and the average recovery rate is 98.4-101%, which indicates that the method can be used for detecting multiple environmental estrogens in milk, and has the advantages of high sensitivity, strong specificity, and accurate and reliable results.
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
Figure 315297DEST_PATH_IMAGE006
The detection results in table 3 show that the Relative Standard Deviation (RSD) of the results is less than 3.5%, the average recovery rate is 98.8-101.4%, and the method can be used for detecting multiple environmental estrogens in water samples, 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 luminescence sensor is characterized in that the environmental estrogen electrochemical luminescence sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a cobalt oxide nanosheet array electrode CoO-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 the CoO-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 CoO-nanoarray on a rotary stirrer, inserting the CoO-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 an environment and at the temperature of 20-40 ℃ in a water bath, simultaneously dropwise adding 1-3 mL of 1mmol/L luminol solution and 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 containing the template molecularly imprinted polymer on the CoO-nanoarray.
2. The method for preparing an environmental estrogen electrochemiluminescence sensor as claimed in claim 1, wherein the method for preparing CoO-nanoarray comprises 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), reacting at the temperature of 100-130 ℃ for 9-12 hours, taking out, airing, and annealing at the temperature of 300-400 ℃ for 1-3 hours to prepare a cobalt oxide nanosheet array precursor electrode;
(4) inserting the cobalt oxide 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 oxide nanosheet array electrode CoO-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 electrochemiluminescence sensor as claimed in claim 1, wherein the preparation of the template-free molecularly imprinted polymer NIP comprises the steps of: immersing the obtained MIP which grows in situ on the CoO-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 electrochemiluminescence sensor as claimed in claim 1, wherein the environmental estrogen electrochemiluminescence sensor is prepared by the steps of: and (3) washing the obtained template-free molecularly imprinted polymer NIP growing in situ on the CoO-nanoarray with deionized water for 2-4 times, and airing at room temperature to obtain the environmental estrogen electrochemical luminescence sensor.
5. The method according to claim 1, 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 luminescence sensor for 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 environmental estrogen electrochemical luminescence sensor as a working electrode, inserting the environmental estrogen electrochemical luminescence sensor 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: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard is recorded as A0The intensity of the response light signal of the standard solution containing different concentrations of environmental estrogen is recorded as AiThe difference in response to the decrease in the optical signal intensity is Δ a ═ a0-AiThe linear relation between delta A and the mass concentration C of the environmental estrogen standard solutionDrawing a delta A-C working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 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 A of the reduction of the intensity of the response optical signal 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.
CN201811306535.6A 2018-11-05 2018-11-05 Preparation method and application of environmental estrogen electrochemiluminescence sensor Expired - Fee Related CN109254051B (en)

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