CN109254061B - Preparation method and application of sulfonamide molecule electrochemiluminescence sensor - Google Patents

Preparation method and application of sulfonamide molecule electrochemiluminescence sensor Download PDF

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CN109254061B
CN109254061B CN201811306729.6A CN201811306729A CN109254061B CN 109254061 B CN109254061 B CN 109254061B CN 201811306729 A CN201811306729 A CN 201811306729A CN 109254061 B CN109254061 B CN 109254061B
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sulfonamide
molecule
template
electrode
molecules
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CN109254061A (en
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程荣琦
张勇
魏琴
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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 a sulfonamide molecule electrochemiluminescence sensor. Belongs to the technical field of novel nanometer functional materials and chemical biosensors. Firstly, a nickel oxide nanosheet array is prepared on a disposable throwable electrode, an in-situ growth method is adopted by utilizing the large specific surface area of the nickel oxide nanosheet array, a polydopamine film and a molecular imprinting polymer which is coated with luminol in situ and takes sulfonamide molecules as template molecules are sequentially and directly prepared on the nickel oxide nanosheet array, 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 sulfonamide molecule electrochemiluminescence sensor is prepared.

Description

Preparation method and application of sulfonamide molecule electrochemiluminescence sensor
Technical Field
The invention relates to a preparation method and application of an electrochemiluminescence sensor. Belongs to the technical field of novel nanometer functional materials and chemical biosensors.
Background
Sulfanilamide drugs are a general name of artificially synthesized antibacterial drugs, and are derivatives with a para-aminobenzenesulfonamide (sulfanilamide for short) as a basic structure, and are commonly called sulfanilamide drugs because the sulfanilamide drugs all contain a sulfanilamide structure. Because of the hydrogen on the sulfonamide group, the sulfonamide can be substituted by different heterocycles to form different sulfonamides. Compared with parent sulfanilamide, the compound has the advantages of high potency, low toxicity, wide antibacterial spectrum, easy absorption by oral administration, and the like. For example, sulfapyridine is the most common, most effective and representative sulfapyridine, has a strong inhibitory effect on gram-positive bacteria and a part of gram-negative bacteria, and has been widely used for treating human intestinal and respiratory diseases.
The sulfonamide has wide antimicrobial spectrum, simple use and low use cost, and is widely applied to the fields of animal husbandry, aquaculture and the like. However, due to abnormal use by humans, approximately 20,000 tons of sulfonamide molecules enter the global environment each year. Abuse or indiscriminate use of sulfonamides can cause severe joint irritation, muscle pain, Stevens Johnson syndrome, asthma, and other allergic reactions, which seriously harm human health. And the sulfonamide has stable property, wide in vivo distribution and easy to generate drug resistance, the metabolite-acetylated sulfanilamide in the liver has low solubility, is easy to separate out crystal in urine to cause toxicity of the kidney, and the maximum content of the sulfonamide in edible animal tissues in various countries in the world is 100 mu g/kg. At present, methods for detecting sulfonamide 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 rapidly and sensitively detecting the sulfonamides with high selectivity 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 a sulfonamide molecule electrochemiluminescence sensor with strong specificity, simple preparation, convenient detection, high sensitivity and low cost. Based on the purpose, firstly, a nickel oxide nanosheet array is prepared on a disposable throwable electrode, an in-situ growth method is adopted by utilizing the large specific surface area of the nickel oxide nanosheet array, a polydopamine film and a molecular imprinting polymer which is coated with luminol in situ and takes sulfonamide molecules as template molecules are sequentially and directly prepared on the nickel oxide nanosheet array, 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 sulfonamide molecule electrochemiluminescence sensor is prepared. When the sensor is used for detecting sulfonamide molecules, the sulfonamide molecule electrochemiluminescence sensor is inserted into a solution to be detected, and the sulfonamide molecules in the solution to be detected are adsorbed into the NIP holes. The larger the concentration of the sulfanilamide drug molecules in the solution to be detected is, the more sulfanilamide drug molecules are adsorbed in the cavity of the NIP. When the electrochemiluminescence detection is carried out, the intensity of the current passing through the electrode is reduced along with the increase of the sulfonamide molecules adsorbed in the holes of the NIP, and the corresponding electrochemiluminescence signal is reduced along with the decrease of the intensity of the electrochemiluminescence light signal, so that the concentration of the sulfonamide molecules in the solution to be detected can be qualitatively and quantitatively determined according to the degree of the reduction of the intensity of the electrochemiluminescence light signal.
The technical scheme adopted by the invention is as follows:
1. a preparation method of a sulfonamide molecule electrochemiluminescence sensor is provided, wherein the sulfonamide molecule electrochemiluminescence sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a nickel oxide nanosheet array electrode NiO-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 sulfonamide molecule;
2. the preparation method of the NiO-nanoarray electrode of the nickel oxide nanosheet array 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 nickel nitrate hexahydrate Ni (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), reacting at the temperature of 100-130 ℃ for 9-12 hours, taking out, airing, and annealing at the temperature of 300-400 ℃ for 1-3 hours to prepare a nickel oxide nanosheet array precursor electrode;
(4) inserting the nickel oxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 4-6 hours at the temperature of 20-40 ℃, taking out, and performing immersion cleaning for 2-4 times by using deionized water to prepare a NiO-nanoarray of the nickel oxide nanosheet array electrode;
the disposable and disposable electrode is selected from one of the following electrodes: foam nickel, foam copper, pure nickel sheets, pure copper sheets, pure iron sheets, pure silicon sheets and conductive carbon cloth;
in 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 template-containing molecularly imprinted polymer MIP with NiO-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 NiO-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the NiO-nanoarray into the precursor mixed solution in the step (2), and adding N2Under the temperature of 20-40 ℃ in an environment and a water bath, rotationally stirring at the speed of 5-200 r/s, 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 NiO-nanoarray;
4. the preparation steps of the NiO-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 NiO-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 which is the template-free molecularly imprinted polymer; the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9 (1-5);
5. the preparation steps of the sulfonamide molecule electrochemiluminescence sensor in the technical scheme 1 are as follows: washing the template-free molecularly imprinted polymer NIP which grows on the NiO-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 sulfonamide molecule electrochemiluminescence sensor;
6. the sulfonamide molecule electrochemiluminescence sensor prepared by the technical scheme 1-5 is applied to detection of sulfonamide molecules, and comprises the following application steps:
(1) preparing a standard solution: preparing a group of sulfonamide molecule standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking a sulfonamide molecule electrochemiluminescence sensor as a working electrode, inserting the sulfonamide molecule standard solution with different concentrations prepared in the step (1), hatching for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard was recorded asA 0The intensity of the response optical signal of the standard solution containing sulfonamide molecules with different concentrations is recorded asA iThe difference in response to the decrease in optical signal intensity is ΔA = A 0-A i，ΔAMass concentration of standard solution of sulfanilamide medicine moleculesCWith a linear relationship therebetween, plotting ΔA－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 s;
(4) detecting sulfonamide molecules in a sample to be detected: replacing the standard solution of the sulfanilamide drug molecules 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 optical signalAAnd working curve, obtaining the content of sulfonamide molecules in the sample to be detected;
7. the sulfanilamide medicine molecule in the technical scheme 1-6 is one of the following sulfanilamide medicine molecules: sulfamethazine SM2, sulfamethoxazole SIZ, sulfamethoxypyrimidine SMD, sulfamethoxazole SDM, phthalylsulfathiazole PST, sulfadiazine SD, sulfamethoxazole SMZ, sulfacetamide SA, sulfadiazine silver salt SD-Ag, and sulfamethazine SML.
Advantageous results of the invention
(1) The sulfonamide molecule electrochemiluminescence sensor is simple to prepare, convenient to operate, low in cost, applicable to portable detection and has market development prospect, and rapid, sensitive and high-selectivity detection of a sample is realized;
(2) according to the invention, the molecular imprinting polymer is grown in situ on the NiO-nanoarray electrode for the first time, on one hand, more and more uniform molecular imprinting polymers can be grown by utilizing the large specific surface area of the NiO-nanoarray, and the NiO-nanoarray has excellent electron transfer capacity, so that the detection sensitivity is greatly improved; on the other hand, NiO-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 large specific surface area of the nickel oxide nanosheet array is combined with dopamine, so that when dopamine is polymerized in situ on the surface of the nickel oxide nanosheet array, a sufficiently thin polydopamine film is formed and simultaneously uniformly covers the nickel oxide nanosheet array, thereby laying a better polymerized molecularly imprinted polymer for the next step; then utilizing the strong connection effect of polydopamine on amino groups rich in the molecularly imprinted polymer, skillfully using NiO-nanoarray as a stirrer, immersing and stirring in the molecularly imprinted precursor mixed solution, creatively coating luminol in situ, and directly growing the molecularly imprinted polymer with membrane thickness in situ on the surface of the NiO-nanoarray by controlling the stirring speed, the dropping speed of a polymerization reaction initiator and the polymerization reaction temperature, so that the NiO-nanoarray can firmly load the molecularly imprinted polymer and the luminol, thereby obviously improving the stability and the reproducibility of the prepared electrochemical sensor; 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 NiO-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 of nickel nitrate hexahydrate Ni (NO)3)2·6H2O and 3mmol Urea CO (NH)2)2Placing the mixture into a 50mL beaker, adding 30mL deionized water, stirring until the mixture is clear, and then transferring the mixture into a 50mL polytetrafluoroethylene reaction kettle;
(3) putting the disposable throwable electrode processed in the step (1) into the solution in the reaction kettle in the step (2), reacting at the temperature of 100 ℃ for 12 hours, taking out and airing, and annealing at the temperature of 300 ℃ for 3 hours to prepare a nickel oxide nanosheet array precursor electrode;
(4) inserting the nickel oxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 4 hours at the temperature of 20 ℃, taking out and washing for 2 times with deionized water to prepare a NiO-nanoarray of the nickel oxide nanosheet array electrode;
wherein the disposable throwable electrode is foamed nickel; the concentration of dopamine is 2 mg/mL, the concentration of ammonium persulfate is 3 mg/mL, the concentration of phosphate buffer solution PBS is 0.1mol/L, and the pH value is 7.2.
EXAMPLE 2 preparation of NiO-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 nickel nitrate hexahydrate Ni (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), reacting for 11 hours at the temperature of 110 ℃, taking out and airing, and annealing for 2 hours at the temperature of 350 ℃ to prepare a nickel hydroxide nanosheet array precursor electrode;
(4) inserting the nickel hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 5 hours at the temperature of 30 ℃, taking out and washing for 3 times with deionized water to prepare a nickel oxide nanosheet array electrode NiO-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 NiO-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 nickel nitrate hexahydrate Ni (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), reacting for 9 hours at the temperature of 130 ℃, taking out and airing, and annealing for 1 hour at the temperature of 400 ℃ to prepare a nickel hydroxide nanosheet array precursor electrode;
(4) inserting the nickel hydroxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine, ammonium persulfate and cobalt nitrate, reacting for 6 hours at the temperature of 40 ℃, taking out and washing with deionized water for 4 times to prepare a NiO-nanoarray of the nickel oxide nanosheet array electrode;
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 sulfonamide molecule electrochemiluminescence sensor
(1) Respectively weighing 0.25 mmol of template molecules and 3mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 8 mL of acetonitrile, and performing ultrasonic treatment for 30min until all the template molecules and the 3mmol of 2-methacrylic acid MAA are dissolved;
(2) adding 15 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) the NiO-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 at 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 on the NiO-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the NiO-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 sulfonamide molecule electrochemiluminescence 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 sulfonamide molecule electrochemiluminescence sensor
(1) Respectively weighing 0.35mmol of template molecules and 4 mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 12 mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 2-methacrylic acid MAA are dissolved;
(2) adding 18 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the NiO-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the NiO-nanoarray into the precursor mixed solution in the step (2), and adding N2Under the temperature of environment and water bath 30 ℃, stirring in a rotating way at the speed of 60 r/s, simultaneously dripping 2 mL of 1mmol/L luminol solution and 1mmol of azobisisobutyronitrile AIBN into the mixed solution at the speed of 10 drops/s to initiate polymerization, and obtaining the in-situ grown MIP on the NiO-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the NiO-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 sulfonamide molecule electrochemiluminescence 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 method of sulfonamide molecule electrochemiluminescence sensor
(1) Respectively weighing 0.45mmol of template molecules and 5mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 15mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 5mmol of 2-methacrylic acid MAA are dissolved;
(2) adding 25mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the NiO-nanoarray prepared in the technical scheme 2 on a rotary stirrer, inserting the NiO-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 dropwise adding 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 on NiO-nanoarray;
(4) immersing the MIP which is obtained in the step (3) and grows in situ on the NiO-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 sulfonamide molecule electrochemiluminescence sensor;
the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9: 5.
Embodiment 7 the sulfonamide molecule electrochemiluminescence sensor prepared in embodiments 1 to 6 is applied to detection of sulfonamide molecules, and includes the following steps:
(1) preparing a standard solution: preparing a group of sulfonamide molecule standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking a sulfonamide molecule electrochemiluminescence sensor as a working electrode, inserting the sulfonamide molecule standard solution with different concentrations prepared in the step (1), hatching for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using double-order pulse voltammetry to detect the voltage(ii) the intensity of the chemiluminescent light signal; the intensity of the response light signal of the blank standard was recorded asA 0The intensity of the response optical signal of the standard solution containing sulfonamide molecules with different concentrations is recorded asA iThe difference in response to the decrease in optical signal intensity is ΔA = A 0-A i，ΔAMass concentration of standard solution of sulfanilamide medicine moleculesCWith a linear relationship therebetween, plotting ΔA－CA working curve; the concentration of the phosphate buffer solution PBS is 10mmol/L, and the pH value is 7.4; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 s;
(4) detecting sulfonamide molecules in a sample to be detected: replacing the standard solution of the sulfanilamide drug molecules 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 optical signalAAnd working curve to obtain the content of sulfonamide molecules in the sample to be detected.
Embodiment 8 the sulfonamide molecule electrochemiluminescence sensor prepared in embodiments 1 to 6 is applied to detection of different sulfonamide molecules according to the detection steps of embodiment 7, and the linear range and the detection limit are shown in table 1:
TABLE 1 detection technical index of sulfonamide molecules
Figure 771374DEST_PATH_IMAGE002
Example 9 detection of sulfonamide molecules in pig urine samples
Accurately transferring a pig urine sample, adding a standard solution of sulfanilamide drug molecules with a certain mass concentration, taking the pig urine sample without the sulfanilamide drug molecules as a blank, carrying out a labeling recovery experiment, detecting by using the sulfanilamide drug molecule electrochemiluminescence sensor prepared in the embodiment 1-6 according to the steps of the embodiment 7, and determining the recovery rate of the sulfanilamide drug molecules in the pig urine sample, wherein the detection result is shown in a table 2:
TABLE 2 detection results of sulfonamide molecules in pig urine samples
Figure 688514DEST_PATH_IMAGE004
The detection results in table 2 show that the Relative Standard Deviation (RSD) of the results is less than 3.1%, the average recovery rate is 98.2-101.2%, and the method can be used for detecting various sulfonamide molecules in pig urine, and is high in sensitivity, strong in specificity, and accurate and reliable in result.
Example 10 detection of sulfonamide molecules in Water samples
Accurately transferring a certain water sample, adding a standard solution of the sulfanilamide drug molecules with a certain mass concentration, taking the water sample without the sulfanilamide drug molecules as a blank, performing a labeling recovery experiment, detecting by using the sulfanilamide drug molecule electrochemiluminescence sensor prepared in the embodiment 1-6 according to the steps of the embodiment 7, determining the recovery rate of the sulfanilamide drug molecules in the water sample, and obtaining a detection result shown in a table 3:
TABLE 3 detection results of sulfonamide molecules in water sample
Figure 468252DEST_PATH_IMAGE006
The detection results in table 3 show that the Relative Standard Deviation (RSD) of the results is less than 3.1%, the average recovery rate is 98.0-101.4%, and the method can be used for detecting various sulfonamide molecules in a water sample, and is high in sensitivity, strong in specificity, and accurate and reliable in result.

Claims (7)

1. The preparation method of the sulfonamide molecule electrochemiluminescence sensor is characterized in that the sulfonamide molecule electrochemiluminescence sensor is obtained by in-situ growth of a template-free molecularly imprinted polymer NIP on a nickel oxide nanosheet array electrode NiO-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 sulfonamide molecule; the MIP containing the template molecular engram polymer is directly grown on NiO-nanoarray in situ, and the preparation method comprises the following preparation steps:
(1) respectively weighing 0.25-0.45 mmol of template molecules and 3-5 mmol of 2-methacrylic acid MAA in an ampoule bottle, adding 8-15 mL of acetonitrile, and carrying out ultrasonic treatment for 30min until all the template molecules and the 2-methacrylic acid MAA are dissolved;
(2) adding 15-25 mmol of ethylene glycol dimethacrylate EDMA into the solution obtained in the step (1), and carrying out ultrasonic treatment for 30min until the mixture is uniformly mixed to obtain a precursor mixed solution;
(3) clamping the NiO-nanoarray on a rotary stirrer, inserting the NiO-nanoarray into the precursor mixed solution in the step (2), and adding N2And (2) rotationally stirring at the speed of 5-200 r/s at the temperature of 20-40 ℃ in an environment and 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 NiO-nanoarray.
2. The method for preparing sulfonamide molecule electrochemiluminescence sensor as claimed in claim 1, wherein the NiO-nanoarray is prepared by 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 nickel nitrate hexahydrate Ni (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), reacting at the temperature of 100-130 ℃ for 9-12 hours, taking out, airing, and annealing at the temperature of 300-400 ℃ for 1-3 hours to prepare a nickel oxide nanosheet array precursor electrode;
(4) inserting the nickel oxide nanosheet array precursor electrode obtained in the step (3) into phosphate buffer solution PBS containing dopamine and ammonium persulfate, reacting for 4-6 hours at the temperature of 20-40 ℃, taking out, and performing immersion cleaning for 2-4 times by using deionized water to prepare a NiO-nanoarray of the nickel oxide nanosheet array electrode;
the disposable and disposable electrode is selected from one of the following electrodes: foam nickel, foam copper, pure nickel sheets, pure copper sheets, pure iron sheets, pure silicon sheets and conductive carbon cloth;
in 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 sulfonamide molecule electrochemiluminescence sensor as claimed in claim 1, wherein the preparation of template-free molecularly imprinted polymer NIP comprises: immersing the obtained MIP which grows in situ on the NiO-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 which is a template-free molecularly imprinted polymer; the eluent is a mixed solution of formic acid and methanol, wherein the volume ratio of the formic acid to the methanol is 9 (1-5).
4. The method for preparing a sulfonamide molecule electrochemiluminescence sensor according to claim 1, wherein the sulfonamide molecule electrochemiluminescence sensor is prepared by the following steps: and (3) washing the obtained template-free molecularly imprinted polymer NIP growing in situ on the NiO-nanoarray with deionized water for 2-4 times, and airing at room temperature to obtain the sulfonamide molecule electrochemiluminescence sensor.
5. A method for preparing a sulfonamide molecule electrochemiluminescence sensor as claimed in any of claims 1 to 4, wherein the sulfonamide molecule is one of the following sulfonamide molecules: sulfamethazine SM2, sulfamethoxazole SIZ, sulfamethoxypyrimidine SMD, sulfamethoxazole SDM, phthalylsulfathiazole PST, sulfadiazine SD, sulfamethoxazole SMZ, sulfacetamide SA, sulfadiazine silver salt SD-Ag, and sulfamethazine SML.
6. The application of the sulfonamide molecule electrochemiluminescence sensor prepared by the preparation method according to any one of claims 1 to 5 in detection of sulfonamide molecules, wherein the detection steps are as follows:
(1) preparing a standard solution: preparing a group of sulfonamide molecule standard solutions with different concentrations including blank standard samples;
(2) modification of a working electrode: taking a sulfonamide molecule electrochemiluminescence sensor as a working electrode, inserting the sulfonamide molecule standard solution with different concentrations prepared in the step (1), hatching for 10min, taking out, and washing for 3 times by using deionized water;
(3) drawing a working curve: forming a three-electrode system by using a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the modified working electrode in the step (2), and connecting the three-electrode system to electrochemiluminescence detection equipment; 15mL of phosphate buffer PBS followed by 1mL of 2mmol/L hydrogen peroxide (H) was added to the cell2O2) A solution; applying cyclic voltage to the assembled working electrode by using a double-order pulse voltammetry to detect the intensity of an optical signal of electrochemiluminescence; the intensity of the response light signal of the blank standard is recorded as A0The intensity of the response optical signal of the standard solution containing sulfonamide molecules with different concentrations is recorded as AiThe difference in response to the decrease in the optical signal intensity is Δ a ═ a0-AiThe mass concentration C of the standard solution of sulfanilamide drugs and the mass concentration A of the sulfanilamide drugs are in a linear relation, and a working curve of Delta A-C is drawn; the phosphate buffer solution PBS has the concentration of 10mmol/L and the pH value of7.4 of the total weight of the mixture; the parameters of the double-order pulse voltammetry during detection are set as follows: the initial potential is 0V, the pulse potential is 0.9V, the pulse time is 0.1s, and the pulse period is 30 s;
(4) detecting sulfonamide molecules in a sample to be detected: and (3) replacing the standard solution of the sulfonamide molecules 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 sulfonamide molecules in the sample to be detected according to the difference value delta A of the reduction of the intensity of the response optical signal and the working curve.
7. Use according to claim 6, wherein the sulfonamide molecule is one of the following sulfonamide molecules: sulfamethazine SM2, sulfamethoxazole SIZ, sulfamethoxypyrimidine SMD, sulfamethoxazole SDM, phthalylsulfathiazole PST, sulfadiazine SD, sulfamethoxazole SMZ, sulfacetamide SA, sulfadiazine silver salt SD-Ag, and sulfamethazine SML.
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