CN109254060B - Clenbuterol electrochemical sensing electrode and preparation method thereof - Google Patents
Clenbuterol electrochemical sensing electrode and preparation method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses a preparation method of a clenbuterol electrochemical sensing electrode. Belongs to the technical field of novel nanometer functional materials and food safety analysis. The method comprises the steps of firstly preparing an iron hydroxide nanosheet array on a disposable throwable electrode, utilizing a large specific surface area and a high-activity hydroxyl functional group of the iron hydroxide nanosheet array and an amino functional group of polydopamine, adopting an in-situ growth method, and directly and successively preparing a polydopamine film containing an electronic mediator and a molecularly imprinted polymer taking clenbuterol as a template molecule on the iron hydroxide nanosheet array, wherein after the template molecule is eluted, the original position of the template molecule is changed into a cavity, namely the molecularly imprinted polymer of the template molecule is eluted, so that the clenbuterol electrochemical sensing electrode is prepared.
Description
Technical Field
The invention relates to a preparation method and application of an electrochemical sensing electrode. Belongs to the technical field of novel nanometer functional materials and food safety analysis.
Background
Clenbuterol is a class of drugs called beta-agonists (beta-agonst), rather than a specific drug. The catalog of the 'clenbuterol' varieties specified in the 'clenbuterol' special treatment scheme (Shi 'an' 2011) of the office of the food safety committee of the state department is 16 varieties, which comprises the following steps: ractopamine, clenbuterol, salbutamol sulfate, dopamine hydrochloride, cimaterol, terbutaline sulfate, phenylethanolamine A, bambuterol, zilpaterol hydrochloride, chloropropanol hydrochloride, mabuterol, sibutrol, brombutamol, arformoterol tartrate, formoterol fumarate. Because they can promote the protein deposition and fat decomposition of animal body, inhibit fat deposition, can obviously raise lean meat percentage of carcass, increase weight and raise feed conversion rate, and can be used as growth-promoting agent and feed additive for livestock and poultry of cattle, sheep and pig, etc. Clenbuterol, however, has dangerous side effects, which can cause damage to the cardiovascular system and may present serious neurological symptoms when people eat meat products containing clenbuterol. Therefore, the method for quickly and sensitively detecting the clenbuterol 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 a clenbuterol electrochemical sensing electrode, which has the advantages of strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, an iron hydroxide nanosheet array is prepared on a disposable throwable electrode, a polydopamine film containing an electron mediator and a molecularly imprinted polymer taking clenbuterol as a template molecule are sequentially and directly prepared on the iron 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 iron hydroxide nanosheet array, and after the template molecule is eluted, the original position of the template molecule is changed into a cavity, namely the molecularly imprinted polymer of the template molecule is eluted, so that the clenbuterol electrochemical sensing electrode is prepared. When the electrochemical sensing electrode is used for detecting clenbuterol, the clenbuterol electrochemical sensing electrode is inserted into a solution to be detected, and clenbuterol in the solution to be detected is adsorbed into a cavity of the NIP. The higher the concentration of clenbuterol in the solution to be tested is, the more clenbuterol is adsorbed into the cavity of the NIP. When electrochemical detection is carried out, the intensity of the detection current is reduced along with the increase of the clenbuterol adsorbed in the NIP hole, 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 current intensity.
The technical scheme adopted by the invention is as follows:
1. a preparation method of a clenbuterol electrochemical sensing electrode is provided, wherein the clenbuterol electrochemical sensing electrode is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a ferric hydroxide nanosheet array electrode Fe-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 clenbuterol;
2. the preparation method of the ferric hydroxide nanosheet array electrode Fe-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 Fe (NO)3)3And 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 precursor electrode of the ferric hydroxide nanosheet array;
(4) inserting the ferric 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 ferric hydroxide nanosheet array electrode Fe-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 iron 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 Fe-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 Fe-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the Fe-nanoarray into the precursor mixed solution in the step (2), and adding the Fe-nanoarray into the precursor mixed solution in N2Rotating and stirring at the temperature of 20-40 ℃ in an environment and a water bath at the speed of 5-200 r/s, and simultaneously dropwise adding 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 drops/s to initiate polymerization to obtain the in-situ grown MIP on the Fe-nanoarray;
4. the preparation steps of the Fe-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 Fe-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 clenbuterol electrochemical sensing electrode in the technical scheme 1 is prepared by the following steps: washing the template-free molecularly imprinted polymer NIP which grows on the Fe-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 clenbuterol electrochemical sensing electrode;
6. the clenbuterol electrochemical sensing electrode prepared by the technical scheme 1-5 is applied to clenbuterol detection, and comprises the following application steps:
(1) preparing a standard solution: preparing a group of clenbuterol standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: inserting the clenbuterol electrochemical sensing electrode serving as a working electrode into the clenbuterol standard solutions with different concentrations prepared in the step (1), incubating 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 the standard solution containing clenbuterol at different concentrations is recorded asI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIAnd the mass concentration of the clenbuterol standard solutionCWith a linear relationship therebetween, plotting ΔI－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, pH, and the 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, and the sampling time is 0.016s and a pulse period of 0.5 s;
(4) and (3) detecting clenbuterol in the sample to be detected: replacing the standard clenbuterol solution in the step (1) with a sample to be detected, detecting according to the methods 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 clenbuterol in the sample to be detected;
7. the clenbuterol hydrochloride of technical scheme 1-6 is one of the following clenbuterol hydrochlorides: ractopamine, clenbuterol, salbutamol sulfate, dopamine hydrochloride, cimaterol, terbutaline sulfate, phenylethanolamine A, bambuterol, zilpaterol hydrochloride, chloropropanol hydrochloride, mabuterol, sibutrol, brombutamol, arformoterol tartrate, formoterol fumarate.
Advantageous results of the invention
(1) The clenbuterol electrochemical sensing electrode 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 molecularly imprinted polymer is grown in situ on the Fe-nanoarray electrode of the ferric hydroxide nanosheet array electrode 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 Fe-nanoarray electrode, and the Fe-nanoarray electrode has excellent electron transfer capacity, so that the detection sensitivity is greatly improved; on the other hand, when dopamine is polymerized onto the ferric 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 ferric 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 ferric hydroxide nanosheet array, a thin enough polydopamine film is formed and simultaneously the polydopamine film is uniformly covered on the ferric hydroxide nanosheet array, thereby laying a better polymerized molecularly imprinted polymer for the next step; then utilizing strong connection effect of polydopamine on hydroxyl functional groups and amino groups rich in the molecularly imprinted polymer, skillfully using Fe-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 Fe-nanoarray in situ by controlling the stirring speed, the dropping speed of a polymerization reaction initiator and the polymerization reaction temperature, so that the Fe-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 Fe-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) 1mmol of Fe (NO) was weighed3)3And 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 precursor electrode of the ferric hydroxide nanosheet array;
(4) inserting the ferric 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 ferric hydroxide nanosheet array electrode Fe-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 Fe-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 Fe (NO)3)3And 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 precursor electrode of the ferric hydroxide nanosheet array;
(4) inserting the ferric 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 washing for 3 times with deionized water to prepare the ferric hydroxide nanosheet array electrode Fe-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 Fe-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 Fe (NO) are weighed3)3And 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 precursor electrode of the ferric hydroxide nanosheet array;
(4) inserting the ferric hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 6 hours at the temperature of 40 ℃, taking out and washing with deionized water for 4 times to prepare the ferric hydroxide nanosheet array electrode Fe-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 0.5mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.5.
Example 4 preparation method of clenbuterol electrochemical sensing electrode
(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 Fe-nanoarray prepared in example 1 was clamped to a rotary stirrer, inserted into the precursor mixed solution in step (2), and subjected to 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 Fe-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the Fe-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 which is the template-free molecularly imprinted polymer; continuously washing with deionized water for 2 times, and air drying at room temperature to obtain clenbuterol electrochemical sensing electrode;
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 preparation method of clenbuterol electrochemical sensing electrode
(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 Fe-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the Fe-nanoarray into the precursor mixed solution in the step (2), and adding the Fe-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 Fe-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the Fe-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 which is the template-free molecularly imprinted polymer; continuously washing with deionized water for 3 times, and air drying at room temperature to obtain clenbuterol electrochemical sensing electrode;
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 preparation method of clenbuterol electrochemical sensing electrode
(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 Fe-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the Fe-nanoarray into the precursor mixed solution in the step (2), and adding the Fe-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 Fe-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the Fe-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 which is the template-free molecularly imprinted polymer; continuously washing with deionized water for 4 times, and air drying at room temperature to obtain clenbuterol electrochemical sensing electrode;
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 clenbuterol sensor prepared in embodiments 1-6 is applied to clenbuterol detection, and includes the following steps:
(1) preparing a standard solution: preparing a group of clenbuterol standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: inserting the clenbuterol electrochemical sensing electrode serving as a working electrode into the clenbuterol standard solutions with different concentrations prepared in the step (1), incubating 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; 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 the standard solution containing clenbuterol at different concentrations is recorded asI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIAnd the mass concentration of the clenbuterol 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) and (3) detecting clenbuterol in the sample to be detected: replacing the standard clenbuterol solution in the step (1) with a sample to be detected, detecting according to the methods in the steps (2) and (3), and responding to the difference delta of the reduction of the current intensityIAnd obtaining the content of the clenbuterol in the sample to be detected according to the working curve.
Example 8 the clenbuterol sensors prepared in examples 1-6 were used for the detection of different clenbuterols according to the detection procedure of example 7, with the linear range and detection limits shown in table 1:
TABLE 1 detection technical index of clenbuterol hydrochloride
Example 9 detection of clenbuterol in pig urine samples
Accurately transferring a swine urine sample, adding a clenbuterol standard solution with a certain mass concentration, taking the swine urine sample without clenbuterol as a blank, performing a standard addition recovery experiment, detecting by using the clenbuterol sensors prepared in embodiments 1-6 according to the steps of embodiment 7, determining the recovery rate of clenbuterol in the swine urine sample, wherein the detection result is shown in table 2:
TABLE 2 results of clenbuterol assay in swine urine samples
The detection results in table 2 show that the Relative Standard Deviation (RSD) of the results is less than 3.5%, the average recovery rate is 98.4-101%, and the method can be used for detecting various clenbuterol in pig urine, and is high in sensitivity, strong in specificity, and accurate and reliable in result.
Example 10 detection of clenbuterol in sheep urine samples
Accurately transferring a certain amount of sheep urine samples, adding a clenbuterol standard solution with a certain mass concentration, taking the sheep urine samples without clenbuterol as blanks, performing a labeling recovery experiment, detecting by using the clenbuterol sensors prepared in examples 1-6 according to the steps of example 7, and determining the recovery rate of clenbuterol in the sheep urine samples, wherein the detection results are shown in table 3:
TABLE 3 results of clenbuterol assay in sheep urine samples
The detection results in the table 3 show that the Relative Standard Deviation (RSD) of the results is less than 3.5%, the average recovery rate is 98.4-1012%, and the method can be used for detecting various clenbuterol in sheep urine, and is high in sensitivity, strong in specificity, and accurate and reliable in result.
Claims (3)
1. The preparation method of the clenbuterol electrochemical sensing electrode is characterized in that the clenbuterol electrochemical sensing electrode is obtained by growing a template-free molecularly imprinted polymer NIP in situ on an iron hydroxide nanosheet array electrode; 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 clenbuterol; the MIP containing the template molecular imprinting polymer directly grows on the ferric hydroxide nanosheet array electrode 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 ferric hydroxide nanosheet array electrode on a rotary stirrer, inserting the ferric hydroxide nanosheet array electrode into the precursor mixed solution obtained in the step (2), and adding N2Under the temperature of an environment and a water bath of 20-40 ℃, rotationally stirring at the speed of 5-200 r/s, simultaneously dropwise adding 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 drops/s for initiating polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the iron hydroxide nanosheet array electrode;
the preparation method of the template-free molecularly imprinted polymer NIP comprises the following steps: immersing the obtained MIP which grows in situ on the ferric hydroxide nanosheet array electrode 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 which is a template-free 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);
the preparation method of the clenbuterol electrochemical sensing electrode comprises the following steps: and (3) washing the prepared template-free molecularly imprinted polymer NIP growing in situ on the ferric hydroxide nanosheet array electrode with deionized water for 2-4 times, and airing at room temperature to obtain the clenbuterol electrochemical sensing electrode.
2. The method for preparing a clenbuterol electrochemical sensing electrode according to claim 1, wherein the method for preparing the ferric hydroxide nanosheet array electrode 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 Fe (NO)3)3And 3 &9mmol 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 precursor electrode of the ferric hydroxide nanosheet array;
(4) inserting the ferric 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 the ferric hydroxide nanosheet array electrode;
the disposable and disposable electrode is selected from one of the following electrodes: foam nickel, foam copper, pure nickel sheets, pure copper sheets, pure iron 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 clenbuterol electrochemical sensing electrode prepared by the preparation method according to any one of claims 1-2, wherein the clenbuterol electrochemical sensing electrode is applied to clenbuterol detection, and the detection steps are as follows:
(1) preparing a standard solution: preparing a group of clenbuterol standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking a clenbuterol electrochemical sensing electrode as a working electrode, inserting the clenbuterol standard solution with different concentrations prepared in the step (1), incubating for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the three-electrode system is connected with the working electrode modified in the step (2) to form a three-electrode systemAs a station, adding 15mL of phosphate buffer solution PBS into an electrolytic cell in sequence; 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 the standard solution containing clenbuterol at different concentrations is denoted as IiThe difference of response current intensity is Δ I ═ I0-IiThe delta I and the mass concentration C of the clenbuterol standard solution form a linear relation, 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) and (3) detecting clenbuterol in the sample to be detected: and (3) replacing the clenbuterol standard solution in the step (1) with a sample to be detected, detecting according to the methods in the steps (2) and (3), and obtaining the clenbuterol content 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.
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