CN109254049B - Preparation method and application of ampicillin sensor - Google Patents

Preparation method and application of ampicillin sensor Download PDF

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CN109254049B
CN109254049B CN201811306531.8A CN201811306531A CN109254049B CN 109254049 B CN109254049 B CN 109254049B CN 201811306531 A CN201811306531 A CN 201811306531A CN 109254049 B CN109254049 B CN 109254049B
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ampicillin
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张勇
刘召壹
魏琴
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University of Jinan
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    • 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

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Abstract

The invention discloses a preparation method of an ampicillin sensor. Belongs to the technical field of novel nanometer functional materials and biosensing analysis. Firstly, a ferronickel bimetal layered hydroxide nanosheet array is prepared on a disposable throwable electrode, a polydopamine film containing an electron mediator and a molecularly imprinted polymer taking ampicillin as a template molecule are directly and sequentially prepared on the ferronickel bimetal layered hydroxide nanosheet array by utilizing the large specific surface area, the high-activity hydroxyl functional group and the amino functional group of the polydopamine in an in-situ growth method, 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, and therefore the ampicillin sensor is prepared.

Description

Preparation method and application of ampicillin sensor
Technical Field
The invention relates to a preparation method and application of an electrochemical analysis sensor. Belongs to the technical field of novel nanometer functional materials and biosensing analysis.
Background
Ampicillin is also known as ampicillin, a beta-lactam antibiotic, a semi-synthetic broad-spectrum penicillin, which is used to treat a variety of bacterial infections. Indications include respiratory infections, urinary tract infections, meningitis, salmonella infections, and endocarditis. Because of convenient use and low cost, the medicine is multi-purpose for treating infectious diseases caused by chicken sensitive bacteria, such as escherichia coli, salmonella, pasteurella, staphylococcus, streptococcus and the like. In 2017, 10 and 27, the list of carcinogens published by the international agency for research on cancer of the world health organization, ampicillin is on the list of 3 types of carcinogens. Therefore, the development of a method for quickly, highly selectively and sensitively detecting ampicillin is very important to public health and has wide market application prospect.
The molecular imprinting electrochemical sensor has high specificity selectivity, excellent stability, excellent reproducibility, wide detection range and bottom detection limit. The sensor has the advantages of simple preparation, convenient detection, high sensitivity, low cost and the like, and can be widely applied to the fields of chromatographic separation, membrane separation, solid-phase extraction, drug controlled release, chemical sensing and the like. Molecularly Imprinted Polymers (MIPs), also known as "plastic antibodies", are capable of specifically recognizing and selectively adsorbing a specific target molecule (i.e., template molecule). The molecular imprinting technology has many advantages, such as corrosion resistance of organic reagents, good stability, high temperature resistance and simple preparation. Thus, MIP electrochemical sensors based on MIP in combination with electrochemical sensors (MIP-ECS) have attracted a hot interest in the field of electroanalytical chemistry, especially the detection of small molecule contaminants, over the last few years. However, in the preparation process of the traditional MIP-ECS, the defects of difficult elution of template molecules, difficult control of the thickness of the imprinted membrane, poor reproducibility and the like exist, and the application of the molecularly imprinted membrane in an electrochemical sensor is limited. The problems, especially the technical problems that the sensitivity of the electrochemical sensor is reduced due to the fact that the thickness of the molecularly imprinted membrane is not easy to control, and the stability and the reproducibility are reduced due to the fact that the molecularly imprinted membrane is easy to fall off from the surface of an electrode in the elution process, limit the application of MIP _ ECS, so that the research of a new molecularly imprinted polymer synthesis method, a new molecularly imprinted membrane electrode modification method and a method for combining the molecularly imprinted membrane and a substrate material for solving the preparation and application problems of MIP-ECS has important research significance and market value.
Disclosure of Invention
The invention aims to provide a preparation method of an ampicillin sensor, which has the advantages of strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, a nickel-iron bimetal layered hydroxide nanosheet array is prepared on a disposable throwable electrode, a poly dopamine film containing an electronic mediator and a molecularly imprinted polymer taking ampicillin as a template molecule are sequentially and directly prepared on the nickel-iron bimetal layered 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 poly dopamine, 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 preparation of the ampicillin sensor is completed. When the ampicillin sensor is used for detecting ampicillin, the ampicillin sensor is inserted into a solution to be detected, and ampicillin in the solution to be detected is adsorbed into a cavity of the NIP. The greater the ampicillin concentration in the solution to be tested, the more ampicillin is adsorbed in the cavity of the NIP. When electrochemical detection is carried out, the intensity of detection current is reduced along with the increase of ampicillin adsorbed in the hole of the NIP, so that the concentration of ampicillin in the solution to be detected can be qualitatively and quantitatively determined according to the reduction degree of the current intensity.
The technical scheme adopted by the invention is as follows:
1. the ampicillin sensor is obtained by growing a template-free molecularly imprinted polymer NIP in situ on a nickel-iron bimetal layered hydroxide nanosheet array electrode NiFe LDH-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 ampicillin;
2. the preparation method of the NiFe LDH-nanoarray electrode with the nickel-iron bimetal layered hydroxide nanosheets, which is described 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 Ni (NO)3)2And Fe (NO)3)3And 3 to 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 treated 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 ferronickel bimetal layered hydroxide nanosheet array;
(4) inserting the precursor electrode of the nickel-iron double-metal layered hydroxide nanosheet array 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 NiFe LDH-nanoarray of the nickel-iron double-metal layered 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 cobalt sheets, pure silicon sheets and conductive carbon cloth; said Ni (NO)3)2And Fe (NO)3)3In a mixture of 1: 1;
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 with NiFe LDH-nanoarray in-situ growth described 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 NiFe LDH-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the NiFe LDH-nanoarray into the precursor mixed solution in the step (2), and adding the NiFe LDH-nanoarray into N2Under the temperature of an environment and a water bath of 20-40 ℃, rotationally stirring at the speed of 5-200 r/s, simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 drops/s to initiate polymerization, and obtaining the in-situ grown MIP on NiFe LDH-nanoarray;
4. the preparation steps of the NiFe LDH-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 NiFe LDH-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 ampicillin sensor in the technical scheme 1 is prepared by the following steps: washing the template-free molecularly imprinted polymer NIP which grows on the NiFe LDH-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 ampicillin sensor;
6. the ampicillin sensor prepared by the technical scheme 1-5 is applied to ampicillin detection, and comprises the following application steps:
(1) preparing a standard solution: preparing a group of ampicillin standard solutions with different concentrations including a blank standard sample;
(2) modification of a working electrode: taking an ampicillin sensor as a working electrode, inserting the ampicillin sensor into ampicillin 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 0The response current intensities of ampicillin standard solutions containing different concentrations were recordedI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIAnd ampicillin 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, the sampling time is 0.016s, and the pulse period is 0.5 s;
(4) detecting ampicillin in a sample to be detected: replacing the ampicillin standard solution in the step (1) with a sample to be tested, performing detection according to the methods in the steps (2) and (3), and determining the difference delta of the decrease of the response current intensityIAnd obtaining the content of ampicillin in the sample to be detected according to the working curve.
Advantageous results of the invention
(1) The ampicillin 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 NiFe LDH-nanoarray electrode of the nickel-iron bimetal layered hydroxide nanosheet, on one hand, more and more uniform molecularly imprinted polymer can be grown by utilizing the large specific surface area of the NiFe LDH-nanoarray electrode, and the NiFe LDH-nanoarray electrode has excellent electron transfer capacity, so that the detection sensitivity is greatly improved; on the other hand, when dopamine is polymerized on the nickel-iron bimetal layered 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, by utilizing the combination of the high-activity hydroxyl functional group rich in the nickel-iron bimetal layered hydroxide nanosheet array and the large specific surface area with dopamine, dopamine is uniformly covered on the nickel-iron bimetal layered hydroxide nanosheet array while a sufficiently thin polydopamine film is formed when the dopamine is polymerized in situ on the surface of the nickel-iron bimetal layered hydroxide nanosheet array, so that a better bedding is made for a better polymerized molecularly imprinted polymer in the next step; then, strong connection action of polydopamine on hydroxyl functional groups and amino groups rich in the molecularly imprinted polymer is utilized, NiFe LDH-nanoarray is skillfully used as a stirrer, immersion stirring is carried out in the molecularly imprinted precursor mixed solution, and the molecularly imprinted polymer with the membrane thickness can be controlled by directly growing in situ on the surface of the NiFe LDH-nanoarray by controlling the stirring speed, the dropping speed of a polymerization reaction initiator and the polymerization reaction temperature, so that the NiFe LDH-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 NiFe LDH-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 Ni (NO)3)2And Fe (NO)3)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 ferronickel bimetal layered hydroxide nanosheet array;
(4) inserting the precursor electrode of the nickel-iron bimetal layered hydroxide nanosheet array 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 washing with deionized water for 2 times to prepare a NiFe LDH-nanoarray electrode of the nickel-iron bimetal layered hydroxide nanosheet array;
wherein the disposable throwable electrode is foamed nickel; said Ni (NO)3)2And Fe (NO)3)3In a mixture of 1: 1; 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 NiFe LDH-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 of Ni (NO)3)2And 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 ferronickel bimetal layered hydroxide nanosheet array;
(4) inserting the precursor electrode of the nickel-iron bimetal layered hydroxide nanosheet array 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 with deionized water for 3 times to prepare a NiFe LDH-nanoarray electrode of the nickel-iron bimetal layered hydroxide nanosheet array;
wherein the disposable throwable electrode is a pure copper sheet; said Ni (NO)3)2And Fe (NO)3)3In a mixture of 1: 1; 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 NiFe LDH-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 3mmol Ni (NO)3)2And Fe (NO)3)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 ferronickel bimetal layered hydroxide nanosheet array;
(4) inserting the precursor electrode of the nickel-iron bimetal layered hydroxide nanosheet array 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 a NiFe LDH-nanoarray electrode of the nickel-iron bimetal layered hydroxide nanosheet array;
wherein the disposable throwable electrode is a conductive carbon cloth; said Ni (NO)3)2And Fe (NO)3)3In a mixture of 1: 1; 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 of ampicillin 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) NiFe LDH-nanoarray prepared in example 1 was clamped to spinInserting the mixture into the precursor mixed solution in the step (2) on a stirrer, and adding 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 NiFe LDH-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the NiFe LDH-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecules for 5 min at room temperature, and then taking out to obtain the NIP which is the template-free molecularly imprinted polymer; continuously washing with deionized water for 2 times, and air drying at room temperature to obtain ampicillin 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 of ampicillin 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 NiFe LDH-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the NiFe LDH-nanoarray into the precursor mixed solution in the step (2), and adding the NiFe LDH-nanoarray into N2Under the temperature of environment and water bath 30 ℃, stirring in a rotating way at the speed of 60 r/s, and simultaneously dripping 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 10 d/s to initiate polymerization to obtain the in-situ grown MIP containing the template molecularly imprinted polymer on the NiFe LDH-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the NiFe LDH-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecules 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 ampicillin 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 ampicillin 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 NiFe LDH-nanoarray prepared in the technical scheme 2 onto a rotary stirrer, inserting the NiFe LDH-nanoarray into the precursor mixed solution in the step (2), and adding the NiFe LDH-nanoarray into 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 d/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecular imprinting polymer on NiFe LDH-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the NiFe LDH-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecules for 20min at room temperature, and then taking out to obtain the NIP which is the template-free molecularly imprinted polymer; continuously washing with deionized water for 4 times, and air drying at room temperature to obtain ampicillin 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 ampicillin sensors prepared in examples 1 to 6 were used for ampicillin detection, and the following procedure was followed:
(1) preparing a standard solution: preparing a group of ampicillin standard solutions with different concentrations including a blank standard sample;
(2) modification of a working electrode: taking an ampicillin sensor as a working electrode, inserting the ampicillin sensor into ampicillin 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 intensities of ampicillin standard solutions containing different concentrations were recordedI iThe difference in response to the decrease in current intensity is ΔI = I 0-I i，ΔIAnd ampicillin standard solutionCWith a linear relationship therebetween, plotting ΔI－CA working curve; the PBS is 10mmol/L phosphate buffer solution, and the pH value of the phosphate buffer solution is 7.4; the parameters during DPV detection are set as follows: the range and the direction are 0-1V, the step is 0.05V, the pulse time is 0.05s, the sampling time is 0.016s, and the pulse period is 0.5 s;
(4) detecting ampicillin in a sample to be detected: replacing the ampicillin standard solution in the step (1) with a sample to be tested, performing detection according to the methods in the steps (2) and (3), and determining the difference delta of the decrease of the response current intensityIAnd obtaining the content of ampicillin in the sample to be detected according to the working curve.
EXAMPLE 8 ampicillin sensors prepared in examples 1 to 6 were used for ampicillin detection according to the detection procedure of example 7, with a linear range of 0.0003 to 100 mmol/L and a detection limit of 100 nmol/L.
Example 9 detection of ampicillin in a Water sample
Accurately transferring a certain water sample, adding an ampicillin standard solution with a certain mass concentration, taking the water sample without ampicillin as a blank, performing a labeling recovery experiment, detecting the ampicillin sensor prepared in examples 1-6 according to the steps of example 7, and determining the recovery rate of ampicillin in the water sample, wherein the detection results are shown in table 1:
TABLE 1 detection results of ampicillin in water samples
Figure 706443DEST_PATH_IMAGE001
The detection results in table 1 show that the Relative Standard Deviation (RSD) of the results is less than 3.0%, and the average recovery rate is 98.2-100.4%, which indicates that the method can be used for detecting ampicillin in a water sample, and the method has the advantages of high sensitivity, strong specificity and accurate and reliable results.

Claims (5)

1. The preparation method of the ampicillin sensor is characterized in that the ampicillin sensor is obtained by growing a template-free molecularly imprinted polymer NIP in situ on a nickel-iron bimetal layered hydroxide nanosheet array electrode NiFe LDH-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 ampicillin, the MIP containing the molecular engram polymer of the template is directly grown on NiFe LDH-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 NiFe LDH-nanoarray on a rotary stirrer, inserting the NiFe LDH-nanoarray into the precursor mixed solution in the step (2), and adding N2And (2) rotationally stirring at the speed of 5-200 r/s in the environment and at the temperature of 20-40 ℃ in a water bath, and simultaneously dropwise adding 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 d/s to initiate polymerization to obtain the in-situ grown MIP (molecularly imprinted polymer) containing the template on the NiFe LDH-nanoarray.
2. The method for preparing an ampicillin sensor as claimed in claim 1, wherein the method for preparing NiFe LDH-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 Ni (NO)3)2And Fe (NO)3)3And 3 to 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 treated 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 ferronickel bimetal layered hydroxide nanosheet array;
(4) inserting the precursor electrode of the nickel-iron bimetal layered hydroxide nanosheet array 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 washing for 2-4 times by using deionized water to prepare a NiFe LDH-nanoarray of the nickel-iron bimetal layered 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 cobalt sheets, pure silicon sheets and conductive carbon cloth; said Ni (NO)3)2And Fe (NO)3)3In a mixture of 1: 1;
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 ampicillin 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 NiFe LDH-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. A method for producing an ampicillin sensor as defined in any one of claims 1 to 3, wherein the ampicillin sensor is produced by: and (3) washing the prepared template-free molecularly imprinted polymer NIP growing in situ on the NiFe LDH-nanoarray for 2-4 times by using deionized water, and airing at room temperature to obtain the ampicillin sensor.
5. The use of the ampicillin sensor manufactured according to the manufacturing method of claims 1 to 4 for ampicillin detection, wherein the detection comprises the following steps:
(1) preparing a standard solution: preparing a group of ampicillin standard solutions with different concentrations including a blank standard sample;
(2) modification of a working electrode: taking an ampicillin sensor as a working electrode, inserting the ampicillin sensor into ampicillin 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 intensities of ampicillin standard solutions containing different concentrations are denoted as IiThe difference of response current intensity is Δ I ═ I0-IiDelta I and ampicillinThe mass concentration C of the forest standard solution is 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) detecting ampicillin in a sample to be detected: and (3) replacing the ampicillin 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 ampicillin 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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