CN109307697B - Preparation method and application of electrochemiluminescence sensing electrode for detecting praziquantel - Google Patents

Preparation method and application of electrochemiluminescence sensing electrode for detecting praziquantel Download PDF

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CN109307697B
CN109307697B CN201811306541.1A CN201811306541A CN109307697B CN 109307697 B CN109307697 B CN 109307697B CN 201811306541 A CN201811306541 A CN 201811306541A CN 109307697 B CN109307697 B CN 109307697B
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praziquantel
electrode
template
detecting
electrochemiluminescence
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CN109307697A (en
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程荣琦
张勇
杜斌
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University of Jinan
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • 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 an electrochemiluminescence sensing electrode for detecting praziquantel. Belongs to the technical field of novel nanometer functional materials and chemical biosensors. According to the method, firstly, a cobalt nitride nanosheet array is prepared on a disposable throwable electrode, a polydopamine film and a molecular imprinting polymer which is coated with luminol in situ and takes praziquantel molecules as template molecules are sequentially and directly prepared on the cobalt 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 molecular imprinting polymer of the template molecules is eluted, and therefore the electrochemiluminescence sensing electrode for detecting the praziquantel is prepared.

Description

Preparation method and application of electrochemiluminescence sensing electrode for detecting praziquantel
Technical Field
The invention relates to a preparation method and application of an electrochemiluminescence sensor. Belongs to the technical field of novel nanometer functional materials and chemical biosensors.
Background
Praziquantel (Praziquantel) is an anthelmintic used in humans and animals, and is used exclusively for the treatment of tapeworms and trematodes. However, due to its high toxicity, the medication must be used strictly as required. Otherwise, dizziness, headache, nausea, abdominal pain, diarrhea, asthenia, soreness of limbs, etc. may be caused, and palpitation, chest distress, etc. may occur in severe cases, which may occasionally induce mental disorder or gastrointestinal hemorrhage. Animals need to be slaughtered and marketed in the drug withdrawal period of more than 5-7 days. When people eat animal food with residual anthelmintic, the drug toxicity can be transferred to human beings, thereby harming human health. At present, methods for detecting praziquantel molecules mainly comprise an enzyme-linked immunosorbent assay, a high performance liquid chromatography, a mass spectrometry method 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 and selectively detecting the praziquantel is very important to public health and has wide market application prospect.
The electroanalytical chemical sensors include electrochemical sensors, electrochemiluminescence sensors, photoelectrochemical sensors and the like, and the sensors have high specificity selectivity, excellent stability, excellent reproducibility, wide detection range and bottom detection limit. The sensor has the advantages of simple preparation, convenient detection, high sensitivity, low cost and the like, and can be widely applied to the fields of chromatographic separation, membrane separation, solid-phase extraction, drug controlled release, chemical sensing and the like. Molecularly Imprinted Polymers (MIPs), also known as "plastic antibodies", are capable of specifically recognizing and selectively adsorbing a specific target molecule (i.e., template molecule). The molecular imprinting technology has many advantages, such as corrosion resistance of organic reagents, good stability, high temperature resistance and simple preparation. Thus, MIP electroanalytical chemical sensors (MIP-ECS) based on the combination of MIPs with electroanalytical chemical sensors have attracted a great deal of interest in the field of electroanalytical chemistry, particularly the detection of small molecule contaminants, over the last few years. However, in the preparation process of the traditional MIP-ECS, the defects of difficult elution of template molecules, difficult control of the thickness of the imprinted membrane, poor reproducibility and the like exist, and the application of the molecularly imprinted membrane in an electroanalytical chemical sensor is limited. The problems, especially the technical problems that the sensitivity of the electrochemical sensor is reduced due to the fact that the thickness of the molecularly imprinted membrane is not easy to control, and the stability and the reproducibility are reduced due to the fact that the molecularly imprinted membrane is easy to fall off from the surface of an electrode in the elution process, limit the application of MIP _ ECS, so that the research of a new molecularly imprinted polymer synthesis method, a new molecularly imprinted membrane electrode modification method and a method for combining the molecularly imprinted membrane and a substrate material for solving the preparation and application problems of MIP-ECS has important research significance and market value.
Disclosure of Invention
The invention aims to provide a preparation method of an electrochemiluminescence sensing electrode for detecting praziquantel, which has the advantages of strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, a cobalt nitride nanosheet array is prepared on a disposable throwable electrode, a polydopamine film and a molecular imprinting polymer which is coated with luminol in situ and takes a praziquantel molecule as a template molecule are sequentially and directly prepared on the cobalt 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 molecule is eluted, the original position of the template molecule is changed into a cavity, namely the molecular imprinting polymer of the template molecule is eluted, and therefore, the electrochemiluminescence sensing electrode for detecting the praziquantel is prepared. When the detection electrode is used for detecting praziquantel molecules, an electrochemiluminescence sensing electrode for detecting praziquantel is inserted into a solution to be detected, and the praziquantel molecules in the solution to be detected are adsorbed into a cavity of the NIP. The higher the concentration of the praziquantel molecules in the solution to be tested is, the more praziquantel molecules are adsorbed into the cavities of the NIP. When the electrochemiluminescence detection is carried out, the current intensity passing through the electrode is reduced along with the increase of the praziquantel molecules adsorbed in the holes of the NIP, and the corresponding electrochemiluminescence signal is reduced along with the current intensity, so that the concentration of the praziquantel molecules in the solution to be detected can be qualitatively and quantitatively determined according to the reduction degree of the electrochemiluminescence light signal intensity.
The technical scheme adopted by the invention is as follows:
1. the electrochemiluminescence sensing electrode for detecting the praziquantel is obtained by growing a template-free molecularly imprinted polymer NIP in situ on a cobalt nitride nanosheet array electrode CoN-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 praziquantel molecule;
2. the preparation method of the cobalt nitride nanosheet array electrode CoN-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 cobalt nitrate hexahydrate Co (NO)3)2·6H2O 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,then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting at the temperature of 100-130 ℃ for 9-12 hours to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into 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 cobalt 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 cobalt nitride nanosheet array electrode CoN-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 phosphate buffer solution PBS containing dopamine and ammonium persulfate: the concentration of dopamine is 2-5 mg/mL, the concentration of ammonium persulfate is 3-8 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2-8.5;
3. the preparation method of the MIP containing the template molecularly imprinted polymer grown in situ by CoN-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 CoN-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the CoN-nanoarray into the precursor mixed solution in the step (2), and adding N2Rotating and stirring at the speed of 5-200 r/s and the speed of 1-20 drops/s in the mixed solution at the temperature of 20-40 ℃ in the environment and water bathDropwise adding 1-3 mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN to initiate polymerization, and obtaining an in-situ grown MIP containing a template molecular imprinting polymer on the CoN-nanoarray;
4. the preparation steps of the template-free molecularly imprinted polymer NIP for in-situ CoN-nanoarray growth in the technical scheme 1 are as follows: immersing the MIP which is obtained in the technical scheme 3 and grows in situ on the CoN-nanoarray and contains the template molecularly imprinted polymer into an eluant, eluting the template molecule for 5-20 min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9 (1-5);
5. the preparation steps of the electrochemiluminescence sensing electrode for detecting praziquantel in the technical scheme 1 are as follows: washing the template-free molecularly imprinted polymer NIP which grows in situ on the CoN-nanoarray prepared in the technical scheme 2-4 with deionized water for 2-4 times, and airing at room temperature to prepare the electrochemiluminescence sensing electrode for detecting the praziquantel;
6. the electrochemiluminescence sensing electrode for detecting praziquantel prepared by the technical scheme 1-5 is applied to detection of praziquantel molecules, and comprises the following application steps:
(1) preparing a standard solution: preparing a group of praziquantel molecule standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: inserting an electrochemiluminescence sensing electrode for detecting praziquantel as a working electrode into the praziquantel molecule 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: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; working on the assembly by means of two-step pulsed voltammetryApplying a cyclic voltage to the electrodes to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard was recorded asA 0The intensity of the response light signal of the standard solution containing praziquantel at different concentrations is recordedA iThe difference in response to the decrease in optical signal intensity is ΔA = A 0-A i，ΔAAnd mass concentration of praziquantel standard solutionCWith a linear relationship therebetween, plotting ΔA－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 s;
(4) detection of praziquantel in a sample to be detected: replacing the praziquantel standard solution in the step (1) with a sample to be detected, detecting according to the methods in the steps (2) and (3), and detecting according to the difference delta of the reduction of the intensity of the response light signalAAnd working curve to obtain the content of the praziquantel in the sample to be detected.
Advantageous results of the invention
(1) The electrochemiluminescence sensing electrode for detecting the praziquantel is simple to prepare, convenient to operate, low in cost, applicable to portable detection and has market development prospect, and the sample can be quickly, sensitively and selectively detected;
(2) according to the invention, the molecularly imprinted polymer is grown in situ on the cobalt nitride nanosheet array electrode CoN-nanoarray 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 CoN-nanoarray, and the CoN-nanoarray has excellent electron transfer capacity, so that the detection sensitivity is greatly improved; on the other hand, CoN-nanoarray has electrocatalytic activity on hydrogen peroxide, and can realize stable and efficient reaction of a luminol-hydrogen peroxide electrochemiluminescence system without adding horseradish peroxidase, so that the prepared sensor does not need to consider the problem of inactivation of biological enzyme, the use and storage of the sensor can be more stable and the conditions are loose, the signal background is further reduced, the detection sensitivity is improved, and meanwhile, the detection cost is greatly reduced and the environmental pollution is reduced;
(3) 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 cobalt nitride nanosheet array, a sufficiently thin polydopamine film is formed and simultaneously the polydopamine film is uniformly covered on the cobalt nitride nanosheet array, thereby laying a better foundation for the next step of polymerizing the molecularly imprinted polymer; then utilizing strong adsorption and connection effects of polydopamine on amino groups rich in the molecularly imprinted polymer, skillfully using CoN-nanoarray as a stirrer, immersing and stirring the polydopamine in a molecularly imprinted precursor mixed solution, and directly growing the molecularly imprinted polymer with the film thickness in situ on the surface of the CoN-nanoarray by controlling the stirring speed, the dropping speed of a polymerization reaction initiator and the polymerization reaction temperature, so that the CoN-nanoarray can firmly load the molecularly imprinted polymer and luminol on one hand, and the stability and the reproducibility of the prepared electrochemiluminescence sensor are remarkably improved; on the other hand, the film forming thickness of the molecularly imprinted polymer on the surface of the electrode can be effectively controlled, and the technical problem of poor reproducibility caused by the fact that the film forming thickness of the molecularly imprinted film on the surface of the electrode cannot be controlled is solved; in addition, the preparation method of the invention has important scientific significance and application value for effectively controlling the film forming thickness and quantitatively coating luminol in situ, and fully improving the sensitivity and detection limit of the molecular imprinting-based electrochemiluminescence sensor.
Detailed Description
EXAMPLE 1 preparation of CoN-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 cobalt nitrate hexahydrate Co (NO) was weighed3)2·6H2O and 3mmol Urea CO (NH)2)2Put it into a 50mL beaker, add 30mL of deionized water, stir until clear, then transfer to 50mL of TeflonIn an alkene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), and reacting at the temperature of 100 ℃ for 12 hours to prepare a cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt hydroxide nanosheet array precursor electrode obtained in the step (3) into ammonia water for 5 seconds, then taking out, heating to 340 ℃ in an ammonia environment, keeping for 8 hours, then naturally cooling to room temperature in the ammonia environment, then inserting the cobalt hydroxide nanosheet array precursor electrode into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 4 hours at the temperature of 20 ℃, taking out, and performing immersion washing for 2 times by using deionized water to prepare a cobalt nitride nanosheet array electrode CoN-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 phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2.
EXAMPLE 2 preparation of CoN-nanoarray
(1) Carrying out ultrasonic cleaning treatment on the disposable throwable electrode by respectively using dilute hydrochloric acid, absolute ethyl alcohol and deionized water so as to remove an oxide layer and surface impurities of the disposable throwable electrode;
(2) weighing 2mmol cobalt nitrate hexahydrate Co (NO)3)2·6H2O 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 cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt 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 cobalt hydroxide nanosheet array precursor electrode into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 5 hours at the temperature of 30 ℃, taking out, and performing immersion washing for 3 times by using deionized water to prepare a cobalt nitride nanosheet array electrode CoN-nanoarray;
wherein the disposable throwable electrode is a pure copper sheet; the concentration of dopamine is 3.5 mg/mL, the concentration of ammonium persulfate is 6.2 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.0.
EXAMPLE 3 preparation of CoN-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 cobalt nitrate hexahydrate Co (NO)3)2·6H2O 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 cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt 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 naturally cooling to room temperature in the ammonia environment, then inserting the cobalt hydroxide nanosheet array precursor electrode into phosphate buffer solution PBS containing dopamine and ammonium persulfate, 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 cobalt nitride nanosheet array electrode CoN-nanoarray;
wherein the disposable throwable electrode is a conductive carbon cloth; the concentration of dopamine is 5mg/mL, the concentration of ammonium persulfate is 8mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 8.5.
Example 4 preparation method of electrochemiluminescence sensing electrode for detecting Praziquantel
(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) CoN-nanoarray prepared in example 1 was clamped to a rotary stirrer and inserted into the precursor mixed solution in step (2) in N2Under the temperature of environment and water bath 20 ℃, stirring in a rotating way at the speed of 200 r/s, simultaneously dripping 1mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1 drop/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the CoN-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the CoN-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 electrochemiluminescence sensing electrode for detecting praziquantel;
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 electrochemiluminescence sensing electrode for detecting Praziquantel
(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 CoN-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the CoN-nanoarray into the precursor mixed solution in the step (2), and adding N2Stirring at 60 rpm at the temperature of 30 ℃ in an environment and water bath, and simultaneously dropwise adding 2 mL of 1mmol/L luminol solution and 1mmol coupling into the mixed solution at the speed of 10 drops/secondCarrying out initiated polymerization on azodiisobutyronitrile AIBN, and obtaining a template-containing molecularly imprinted polymer MIP growing in situ on CoN-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the CoN-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 electrochemiluminescence sensing electrode for detecting praziquantel;
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 electrochemiluminescence sensing electrode for detecting Praziquantel
(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 CoN-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the CoN-nanoarray into the precursor mixed solution in the step (2), and adding N2Under the temperature of environment and water bath 40 ℃, rotationally stirring at the speed of 5 r/s, simultaneously dripping 3mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 20 drops/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the CoN-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the CoN-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 electrochemiluminescence sensing electrode for detecting praziquantel;
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 electrochemiluminescence sensing electrode for detecting praziquantel prepared in embodiments 1 to 6 is applied to detection of praziquantel molecules, and comprises the following steps:
(1) preparing a standard solution: preparing a group of praziquantel molecule standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: inserting an electrochemiluminescence sensing electrode for detecting praziquantel as a working electrode into the praziquantel 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: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard was recorded asA 0The intensity of the response light signal of the standard solution containing praziquantel at different concentrations is recordedA iThe difference in response to the decrease in optical signal intensity is ΔA = A 0-A i，ΔAAnd mass concentration of praziquantel standard solutionCWith a linear relationship therebetween, plotting ΔA－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 s;
(4) detection of praziquantel in a sample to be detected: replacing the praziquantel standard solution in the step (1) with a sample to be detected, detecting according to the methods in the steps (2) and (3), and detecting according to the difference delta of the reduction of the intensity of the response light signalAAnd working curve to obtain the content of the praziquantel in the sample to be detected.
Example 8 the electrochemiluminescence sensing electrode for detecting praziquantel prepared in examples 1 to 6 is applied to the detection of praziquantel according to the detection procedure of example 7, and the linear range is 5 × 10-5800 mmol/L, and the detection limit is 2 nmol/L.
Example 9 detection of Praziquantel in pig urine samples
Accurately transferring a pig urine sample, adding a praziquantel standard solution with a certain mass concentration, taking the pig urine sample without praziquantel as a blank, carrying out a standard addition recovery experiment, detecting by using the electrochemiluminescence sensing electrode for detecting praziquantel prepared in examples 1-6 according to the steps of example 7, and determining the recovery rate of praziquantel in the pig urine sample, wherein the detection results are shown in table 2:
TABLE 1 detection results of Praziquantel in pig urine samples
Figure 722103DEST_PATH_IMAGE002
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 99.0-101%, and the method can be used for detecting praziquantel in pig urine, and is high in sensitivity, strong in specificity and accurate and reliable in result.
EXAMPLE 10 detection of Praziquantel in sheep urine samples
Accurately transferring a certain amount of sheep urine samples, adding a praziquantel standard solution with a certain mass concentration, taking the sheep urine samples without praziquantel as blanks, carrying out a labeling recovery experiment, detecting the recovery rate of praziquantel in the sheep urine samples by using the electrochemiluminescence sensing electrodes for detecting praziquantel prepared in examples 1-6 according to the steps of example 7, and finding out the detection results shown in table 3:
TABLE 2 detection results of Praziquantel in sheep urine samples
Figure 639244DEST_PATH_IMAGE004
The detection results in the table 3 show that the Relative Standard Deviation (RSD) of the results is less than 3.3%, the average recovery rate is 98.0-101.4%, and the method can be used for detecting praziquantel in sheep urine, and is high in sensitivity, strong in specificity, and accurate and reliable in result.

Claims (5)

1. A preparation method of an electrochemiluminescence sensing electrode for detecting praziquantel is characterized in that the electrochemiluminescence sensing electrode for detecting praziquantel is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a cobalt nitride nanosheet array electrode CoN-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 praziquantel molecule; the MIP containing the template molecular engram polymer is directly grown on the CoN-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 CoN-nanoarray on a rotary stirrer, and inserting the CoN-nanoarray into the precursor mixed solution in the step (2), wherein N is2And (2) rotationally stirring at the speed of 5-200 r/s in an environment and at the temperature of 20-40 ℃ in a water bath, simultaneously dropwise adding 1-3 mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 1-20 drops/s to initiate polymerization, and obtaining the in-situ grown MIP containing the template molecularly imprinted polymer on the CoN-nanoarray.
2. The method for preparing an electrochemiluminescence sensing electrode for detecting praziquantel according to claim 1, wherein the method for preparing the CoN-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 cobalt nitrate hexahydrate Co (NO)3)2·6H2O 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 cobalt hydroxide nanosheet array precursor electrode;
(4) inserting the cobalt 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 cobalt 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 cobalt nitride nanosheet array electrode CoN-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 preparation method of the electrochemiluminescence sensing electrode for detecting praziquantel as claimed in claim 1, wherein the preparation steps of the template-free molecularly imprinted polymer NIP are as follows: immersing the obtained MIP which grows in situ on the CoN-nanoarray and contains the template molecularly imprinted polymer in an eluant, eluting the template molecule for 5-20 min at room temperature, and then taking out to obtain the NIP without the template molecularly imprinted polymer; the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9 (1-5).
4. The method for preparing an electrochemiluminescence sensing electrode for detecting praziquantel according to claim 1, wherein the preparation steps of the electrochemiluminescence sensing electrode for detecting praziquantel are as follows: and (3) washing the obtained template-free molecularly imprinted polymer NIP growing in situ on the CoN-nanoarray with deionized water for 2-4 times, and airing at room temperature to obtain the electrochemiluminescence sensing electrode for detecting the praziquantel.
5. The application of the electrochemiluminescence sensing electrode for detecting praziquantel prepared by the preparation method according to any one of claims 1 to 4, and the application of the prepared electrochemiluminescence sensing electrode to the detection of praziquantel, wherein the detection steps are as follows:
(1) preparing a standard solution: preparing a group of praziquantel standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: inserting an electrochemiluminescence sensing electrode for detecting praziquantel as a working electrode into the praziquantel 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: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard is recorded as A0The intensity of the response light signal of the standard solution containing praziquantel at different concentrations is recorded as AiThe difference in response to the decrease in optical signal intensity is Δ A＝A0-AiThe mass concentration C of the praziquantel standard solution is in a linear relation with the mass concentration A of the praziquantel standard solution, and a working curve of delta A-C is drawn; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 s;
(4) detection of praziquantel in a sample to be detected: and (3) replacing the praziquantel 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 praziquantel in the sample to be detected according to the difference delta A of the reduction of the intensity of the response optical signal and the working curve.
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