CN109254062B - Preparation method and application of macrolide antibiotic molecularly imprinted electrochemical sensor - Google Patents
Preparation method and application of macrolide antibiotic molecularly imprinted electrochemical sensor 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
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- 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 macrolide antibiotic molecularly imprinted electrochemical sensor. Belongs to the technical field of novel nanometer functional materials and chemical biosensors. According to the invention, firstly, an iron nitride nanosheet array is prepared on a disposable throwable electrode, and by utilizing the large specific surface area and the high adsorption activity to amino groups of the iron nitride nanosheet array, an in-situ growth method is adopted, a polydopamine film containing an electron mediator and a molecularly imprinted polymer taking macrolide antibiotic molecules as template molecules are sequentially and directly prepared on the iron nitride nanosheet array, 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 macrolide antibiotic molecularly imprinted electrochemical sensor is prepared.
Description
Technical Field
The invention relates to a preparation method and application of an electrochemical sensor. Belongs to the technical field of novel nanometer functional materials and chemical biosensors.
Background
Macrolide Antibiotics (MA) are a generic name of antibacterial drugs having a 12-16 carbon lactone ring in a molecular structure, inhibit bacterial protein synthesis by blocking the activity of peptidyl transferase in 50s ribosome, and belong to rapid bacteriostats. Mainly used for treating infection of aerobic gram positive coccus and negative coccus, certain anaerobe, legionella, mycoplasma, chlamydia and the like. When people eat animal food with residual antibiotics, drug resistance generated by animals can be transferred to human beings, thereby harming human health. At present, methods for detecting macrolide antibiotic molecules mainly comprise an enzyme-linked immunosorbent assay, a high performance liquid chromatography, a mass spectrometry and the like. The method is expensive and complex in operation, and the detection can be carried out only after professional training is required for a laboratory worker. Therefore, the method for quickly developing the macrolide antibiotics with high selectivity and sensitivity 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 macrolide antibiotic molecularly imprinted electrochemical sensor with strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, an iron nitride nanosheet array is prepared on a disposable throwable electrode, a polydopamine film containing an electron mediator and a molecularly imprinted polymer taking macrolide antibiotic molecules as template molecules are sequentially and directly prepared on the iron nitride nanosheet array by utilizing the large specific surface area and the high adsorption activity to amino groups and adopting an in-situ growth method, 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 macrolide antibiotic molecularly imprinted electrochemical sensor is prepared. When the electrochemical sensor is used for detecting macrolide antibiotic molecules, the macrolide antibiotic molecularly imprinted electrochemical sensor is inserted into a solution to be detected, and the macrolide antibiotic molecules in the solution to be detected are adsorbed into cavities of NIP. The larger the concentration of macrolide antibiotic molecules in the solution to be tested is, the more macrolide antibiotic molecules are adsorbed into the cavities of the NIP. When electrochemical detection is carried out, the intensity of detection current is reduced along with the increase of macrolide antibiotic molecules adsorbed in the cavities of the NIP, so that the concentration of the macrolide antibiotic molecules 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 macrolide antibiotic molecularly imprinted electrochemical sensor is provided, wherein the macrolide antibiotic molecularly imprinted electrochemical sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on an iron nitride nanosheet array electrode FeN-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 a macrolide antibiotic molecule;
2. the preparation method of the iron nitride nanosheet array electrode FeN-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 of ferric nitrate hexahydrate Fe (NO)3)3·6H2O and 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 ammonia water for 5-30 seconds, taking out, heating to 340-400 ℃ in an ammonia environment, keeping for 4-8 hours, then naturally cooling to room temperature in the ammonia environment, then inserting the ferric hydroxide nanosheet array precursor electrode 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 performing immersion washing for 2-4 times by using deionized water to prepare a ferric nitride nanosheet array electrode FeN-nanoarray;
the disposable and disposable electrode is selected from one of the following electrodes: foam iron, foam copper, pure iron sheets, pure copper sheets, pure iron sheets, pure silicon wafers 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 template-containing molecularly imprinted polymer MIP grown in situ by FeN-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 FeN-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the FeN-nanoarray into the precursor mixed solution in the step (2), and adding the FeN-nanoarray into the precursor mixed solution in the step (2) to obtain the FeN-nanoarray2Rotating and stirring at the speed of 5-200 r/s in the environment and the temperature of 20-40 ℃ in a water bath, simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 d/s to initiate polymerization, and obtaining the in-situ grown on the FeN-nanoarrayMIP containing template molecular engram polymer;
4. the preparation steps of the FeN-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 FeN-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 macrolide antibiotic molecularly imprinted electrochemical sensor in the technical scheme 1 are as follows: washing the template-free molecularly imprinted polymer NIP which grows in situ on the FeN-nanoarray prepared in the technical scheme 2-4 with deionized water for 2-4 times, and airing at room temperature to obtain the macrolide antibiotic molecularly imprinted electrochemical sensor;
6. the macrolide antibiotic molecularly imprinted electrochemical sensor prepared by the technical scheme 1-5 is applied to detection of macrolide antibiotic molecules, and comprises the following application steps:
(1) preparing a standard solution: preparing a group of macrolide antibiotic molecular standard solutions with different concentrations including blank standards;
(2) modification of a working electrode: taking a macrolide antibiotic molecularly imprinted electrochemical sensor as a working electrode, inserting into macrolide antibiotic molecular standard solutions with different concentrations prepared in the step (1), incubating for 10min, taking out, and washing with deionized water for 3 times;
(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 0Containing macrolide antibiotic molecules in varying concentrationsThe response current intensity of the standard solution was recorded asI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIMass concentration of the standard solution of macrolide antibiotic moleculesCWith 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, the sampling time is 0.016s, and the pulse period is 0.5 s;
(4) detection of macrolide antibiotic molecules in the sample to be tested: replacing the macrolide antibiotic molecular standard solution in the step (1) with a sample to be tested, and detecting according to the difference delta of the reduction of the response current intensity by the method in the steps (2) and (3)IAnd working curve, obtaining the content of macrolide antibiotic molecules in the sample to be detected;
7. the macrolide antibiotic molecules described in technical schemes 1-6 are one of the following macrolide antibiotic molecules: erythromycin, midecamycin, azithromycin, clarithromycin, roxithromycin, tylosin, and ivermectin.
Advantageous results of the invention
(1) The macrolide antibiotic molecularly imprinted electrochemical 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 molecularly imprinted polymer is grown in situ on the FeN-nanoarray electrode for the first time, on one hand, more and more uniform molecularly imprinted polymers can be grown by utilizing the large specific surface area of the FeN-nanoarray electrode, and the FeN-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 iron nitride 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 adsorption activity of the nitride on amino and the large specific surface area of the nano array electrode are combined with dopamine, so that when dopamine is polymerized in situ on the surface of the iron nitride nanosheet array, a sufficiently thin poly dopamine film is formed and simultaneously the poly dopamine film is uniformly covered on the iron nitride nanosheet array, thereby laying a better foundation for more and better polymerized molecularly imprinted polymers in the next step; then, strong adsorption and connection effects of polydopamine on amino groups rich in the molecularly imprinted polymer are utilized, then FeN-nanoarray is skillfully used as a stirrer to be immersed and stirred in the molecularly imprinted precursor mixed solution, and the molecularly imprinted polymer with the film thickness can be directly grown in situ on the surface of the FeN-nanoarray by controlling the stirring speed, the dropping speed of a polymerization reaction initiator and the polymerization reaction temperature, so that the FeN-nanoarray can firmly load the molecularly imprinted polymer on one hand, and the stability and the reproducibility of the prepared electrochemical sensor are remarkably improved; on the other hand, the film forming thickness of the molecularly imprinted polymer on the surface of the electrode and the in-situ coating of the electronic mediator 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 fully improving the sensitivity and detection limit of the electrochemical sensor based on the molecular imprinting.
Detailed Description
EXAMPLE 1 preparation of FeN-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 iron nitrate hexahydrate Fe (NO) was weighed3)3·6H2O and 3mmol Urea CO (NH)2)2Put it into a 50mL beakerAdding 30mL of 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 ammonia water for 5 seconds, then taking out, heating to 340 ℃ in an ammonia environment, keeping for 8 hours, then continuing to naturally cool to room temperature in the ammonia environment, then inserting the ferric hydroxide nanosheet array precursor electrode 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 nitride nanosheet array electrode FeN-nanoarray;
wherein the disposable throwable electrode is foam iron; 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 FeN-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) 2 mmol of iron nitrate hexahydrate Fe (NO) was weighed3)3·6H2O and 6 mmol 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 ammonia water for 15 seconds, then taking out, heating to 370 ℃ in an ammonia environment, keeping for 6 hours, then continuing to naturally cool to room temperature in the ammonia environment, then inserting the ferric hydroxide nanosheet array precursor electrode 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 ferric nitride nanosheet array electrode FeN-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 FeN-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 iron nitrate hexahydrate Fe (NO) was weighed3)3·6H2O and 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 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 ammonia water for 30 seconds, then taking out, heating to 400 ℃ in an ammonia environment, keeping for 4 hours, then continuing to naturally cool to room temperature in the ammonia environment, then inserting the ferric hydroxide nanosheet array precursor electrode into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 6 hours at the temperature of 40 ℃, taking out, and performing immersion washing for 4 times by using deionized water to prepare a ferric nitride nanosheet array electrode FeN-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 preparation method of macrolide antibiotic molecularly imprinted electrochemical 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 FeN-nanoarray prepared in example 1 was inserted into the precursor mixed solution of step (2) while being clamped on a rotary stirrer, and stirred in 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 the FeN-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the FeN-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 macrolide antibiotic molecularly imprinted electrochemical 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 preparation method of macrolide antibiotic molecularly imprinted electrochemical sensor
(1) Respectively weighing 0.35mmol of template molecules and 4mmol 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 FeN-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, and inserting the FeN-nanoarray into the precursor mixed solution in the step (2)In N at2Under 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 d/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the FeN-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the FeN-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 macrolide antibiotic molecularly imprinted electrochemical 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 preparation of macrolide antibiotic molecularly imprinted electrochemical 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 FeN-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the FeN-nanoarray into the precursor mixed solution in the step (2), and adding the FeN-nanoarray into the precursor mixed solution in the step (2) to obtain the FeN-nanoarray2Under 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 the FeN-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the FeN-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 macrolide antibiotic molecularly imprinted electrochemical 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.
Example 7 the macrolide antibiotic molecularly imprinted electrochemical sensor prepared in example 1-6 is applied to detection of macrolide antibiotic molecules, and comprises the following steps:
(1) preparing a standard solution: preparing a group of macrolide antibiotic molecular standard solutions with different concentrations including blank standards;
(2) modification of a working electrode: taking a macrolide antibiotic molecularly imprinted electrochemical sensor as a working electrode, inserting into macrolide antibiotic standard solutions with different concentrations prepared in the step (1), incubating for 10min, taking out, and washing with deionized water for 3 times;
(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 standard solutions containing macrolide antibiotic molecules at different concentrations is recordedI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIMass concentration with macrolide antibiotic 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) detection of macrolide antibiotics in the sample to be tested: replacing the macrolide antibiotic standard solution in step (1) with the sample to be tested, and performing the test according to the methods in steps (2) and (3)Measuring, according to the difference Delta of decrease of response current intensityIAnd working curve to obtain the content of macrolide antibiotics in the sample to be detected.
Example 8 macrolide antibiotic molecularly imprinted electrochemical sensors prepared in examples 1 to 6, applied to the detection of different macrolide antibiotics according to the detection procedure of example 7, have the linear range and detection limit shown in table 1:
TABLE 1 technical indices for the detection of macrolide antibiotics
Example 9 detection of macrolide antibiotics in pig urine samples
Accurately transferring a pig urine sample, adding a macrolide antibiotic standard solution with a certain mass concentration, taking the pig urine sample without macrolide antibiotic as a blank, carrying out a labeling recovery experiment, detecting by using macrolide antibiotic molecular imprinting electrochemical sensors prepared in examples 1-6 according to the steps of example 7, and determining the recovery rate of the macrolide antibiotic in the pig urine sample, wherein the detection results are shown in a table 2:
TABLE 2 detection of macrolide antibiotics in pig urine samples
The detection results in Table 2 show that the Relative Standard Deviation (RSD) of the results is less than 3.2%, the average recovery rate is 98.2-101%, and the method can be used for detecting multiple macrolide antibiotics in pig urine, and is high in sensitivity, strong in specificity, accurate and reliable in result.
Example 10 detection of macrolide antibiotics in sheep urine samples
Accurately transferring a certain amount of sheep urine samples, adding a macrolide antibiotic standard solution with a certain mass concentration, taking the sheep urine samples without macrolide antibiotics as blanks, carrying out a labeling recovery experiment, detecting by using macrolide antibiotic molecularly imprinted electrochemical sensors prepared in examples 1-6 according to the steps of example 7, and determining the recovery rate of the macrolide antibiotic in the sheep urine samples, wherein the detection results are shown in Table 3:
TABLE 3 detection results of macrolide antibiotics in sheep urine samples
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 98.0-101.4%, and the method can be used for detecting multiple macrolide antibiotics in sheep urine, and is high in sensitivity, strong in specificity, and accurate and reliable in result.
Claims (7)
1. A preparation method of a macrolide antibiotic molecularly imprinted electrochemical sensor is characterized in that the macrolide antibiotic molecularly imprinted electrochemical sensor is obtained by growing a template-free molecularly imprinted polymer NIP in situ on a ferric nitride nanosheet array electrode FeN-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 a macrolide antibiotic molecule; the MIP containing the template molecular engram polymer is directly grown on the FeN-nanoarray in situ, and the preparation method comprises the following preparation steps:
(1) respectively weighing 0.35-0.45 mmol of template molecules and 3-4 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 FeN-nanoarray on a rotary stirrer, inserting the FeN-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, 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 FeN-nanoarray.
2. The method for preparing a macrolide antibiotic molecularly imprinted electrochemical sensor according to claim 1, wherein the method for preparing FeN-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 of ferric nitrate hexahydrate Fe (NO)3)3·6H2O and 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 ammonia water for 5-30 seconds, taking out, heating to 340-400 ℃ in an ammonia environment, keeping for 4-8 hours, then naturally cooling to room temperature in the ammonia environment, then inserting the ferric hydroxide nanosheet array precursor electrode 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 washing for 2-4 times by using deionized water to prepare a ferric nitride nanosheet array electrode FeN-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 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 a macrolide antibiotic molecularly imprinted electrochemical sensor according to claim 1, wherein the template-free molecularly imprinted polymer NIP is prepared by the steps of: immersing the obtained MIP which grows in situ on the FeN-nanoarray and contains the template molecularly imprinted polymer in 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).
4. The method for preparing a macrolide antibiotic molecularly imprinted electrochemical sensor according to claim 1, wherein the macrolide antibiotic molecularly imprinted electrochemical sensor is prepared by the steps of: and (3) washing the obtained template-free molecularly imprinted polymer NIP growing in situ on the FeN-nanoarray with deionized water for 2-4 times, and airing at room temperature to obtain the macrolide antibiotic molecularly imprinted electrochemical sensor.
5. A method for preparing a macrolide antibiotic molecularly imprinted electrochemical sensor as claimed in any of claims 1 to 4, wherein the macrolide antibiotic is one of the following macrolide antibiotics: erythromycin, midecamycin, azithromycin, clarithromycin, roxithromycin, tylosin, and ivermectin.
6. The application of the macrolide antibiotic molecularly imprinted electrochemical sensor prepared by the preparation method according to any one of claims 1 to 5 in macrolide antibiotic detection, wherein the detection steps are as follows:
(1) preparing a standard solution: preparing a group of macrolide antibiotic standard solutions with different concentrations including blank standards;
(2) modification of a working electrode: taking a macrolide antibiotic molecularly imprinted electrochemical sensor as a working electrode, inserting into macrolide antibiotic standard solutions with different concentrations prepared in the step (1), incubating for 10min, taking out, and washing with deionized water for 3 times;
(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 solutions containing macrolide antibiotics at different concentrations is denoted IiThe difference of response current intensity is Δ I ═ I0-IiThe Delta I and the mass concentration C of the macrolide antibiotic standard solution are in 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) detection of macrolide antibiotics in the sample to be tested: and (3) replacing the macrolide antibiotic 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 content of the macrolide antibiotic 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. Use according to claim 6, wherein the macrolide antibiotic is one of the following macrolide antibiotics: erythromycin, midecamycin, azithromycin, clarithromycin, roxithromycin, tylosin, and ivermectin.
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