CN109307697A - A kind of preparation method and application for the electrogenerated chemiluminescence sensing electrode detecting praziquantel - Google Patents
A kind of preparation method and application for the electrogenerated chemiluminescence sensing electrode detecting praziquantel Download PDFInfo
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- CN109307697A CN109307697A CN201811306541.1A CN201811306541A CN109307697A CN 109307697 A CN109307697 A CN 109307697A CN 201811306541 A CN201811306541 A CN 201811306541A CN 109307697 A CN109307697 A CN 109307697A
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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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses a kind of preparation methods of electrogenerated chemiluminescence sensing electrode for detecting praziquantel.Belong to Nano-function thin films and chemical biosensor technical field.The present invention is prepared for cobalt nitride nano-chip arrays can disposably throw first on electrode, using its big specific surface area and to the high adsorption activity of amino, using the method for growth in situ, directly be prepared in succession on cobalt nitride nano-chip arrays in succession poly-dopamine film and in-stiu coating luminol using praziquantel molecule as the molecularly imprinted polymer of template molecule, after by template molecule elution, the position of template molecule originally has become hole, that is the molecularly imprinted polymer of eluted template molecule, thus, it is a kind of detect praziquantel electrogenerated chemiluminescence sensing electrode just prepare completion.
Description
Technical field
The present invention relates to a kind of preparation method and applications of Electrochemiluminescsensor sensor.Belong to Nano-function thin films
With chemical biosensor technical field.
Background technique
Praziquantel (Praziquantel) is a kind of for the mankind and the anthelmintic of animal, specifically treats tapeworm and fluke.
However since it is more toxic, medication must in strict accordance with require using.Otherwise dizzy, headache, nausea, abdomen be may result in
Bitterly, diarrhea, it is out of strength, have aches in the limbs, serious person can occasionally induce psychiatric disorder or go out it is possible that the symptoms such as palpitaition, uncomfortable in chest
Existing hemorrhage of digestive tract.Animal need to can butcher listing at withdrawal time 5 ~ 7 days or more.Remain such anthelmintic when people are edible
After animal food, drug toxicity can also be transferred to the mankind, to endanger human health.Currently, detection praziquantel molecule
Method mainly has enzyme-linked immunization, high performance liquid chromatography, mass spectrography etc..Such method instrument is valuable, complicated for operation, assayer
Member just can be carried out detection after needing professional training.Therefore, it develops as early as possible a kind of quick, highly selective and Sensitive Detection praziquantel
Method is extremely important to publilc health, and has wide market application prospect.
Electroanalytical Chemistry sensor includes electrochemical sensor, Electrochemiluminescsensor sensor, optical electro-chemistry sensor etc.,
Such sensor has high specific selectivity, excellent stability, excellent reproducibility, wide detection range and floor detection
Limit.Due to the sensor prepare simple, easy to detect, high sensitivity, it is at low cost the advantages that be widely used in chromatographic isolation, film
Point, the fields such as Solid Phase Extraction, medicine controlled releasing, chemical sensitisation.Molecularly imprinted polymer (MIP), also referred to as " plastics antibody ", can
Specific recognition and the specific target molecule of selective absorption (i.e. template molecule).Since molecular imprinting technology has many advantages,
Such as organic reagent corrosion resistance, good stability, heat-resisting quantity and preparation are simple.Therefore, in the past few years, it is based on
The MIP Electroanalytical Chemistry sensor (MIP-ECS) that MIP is combined with Electroanalytical Chemistry sensor causes Electroanalytical Chemistry field
Focus, the especially detection of small molecule contaminants.However, having template point in the preparation process of traditional MIP-ECS
The disadvantages of sub- hardly possible elutes, the thickness of blotting membrane is difficult to control, reproducibility is poor, limits molecular engram film in Electroanalytical Chemistry sensor
In application.These problems, especially molecular engram film thickness are not easy to control cause electrochemical sensor sensitivity decrease and
Molecular engram film easily falls off from electrode surface during elution leads to the technical problem of stability and reproducibility reduction, limits
Therefore the application of MIP_ECS finds the modification side of new molecularly imprinted polymer synthetic method, new molecular engram film electrode
The combination method of method and molecular engram film and base material has important grind to solve preparation and the application problem of MIP-ECS
Study carefully meaning and market value.
Summary of the invention
The purpose of the present invention is to provide a kind of high specificity, prepare simple, easy to detect, high sensitivity, at low cost
Detect the preparation method of the electrogenerated chemiluminescence sensing electrode of praziquantel, prepared Electrochemiluminescsensor sensor, preparation letter
List, favorable reproducibility, stability are strong, can be used for quick, the Sensitive Detection of praziquantel molecule.Based on this purpose, the present invention exists first
It can disposably throw and be prepared for cobalt nitride nano-chip arrays on electrode, live using its big specific surface area and to the high absorption of amino
Property, using the method for growth in situ, directly it is prepared for poly-dopamine film and original in succession on cobalt nitride nano-chip arrays in succession
Position cladding luminol using praziquantel molecule as the molecularly imprinted polymer of template molecule, it is former after by template molecule elution
The position of the template molecule come has become hole, the i.e. molecularly imprinted polymer of eluted template molecule, as a result, a kind of detection pyrrole quinoline
The electrogenerated chemiluminescence sensing electrode of ketone just prepares completion.When for will test praziquantel when detecting to praziquantel molecule
Electrogenerated chemiluminescence sensing electrode be inserted into solution to be measured, the praziquantel molecule in solution to be measured can be adsorbed onto the hole of NIP
In.Praziquantel molecular concentration in solution to be measured is bigger, and it is more to be adsorbed onto praziquantel molecule in the hole of NIP.It is electroluminescent when carrying out
When chemiluminescence detection, it can be become with increasing for praziquantel molecule in the hole of NIP is adsorbed by the current strength of electrode
Small, corresponding electrochemiluminescence signal can also become smaller therewith, thus reduced according to the light signal strength of electrogenerated chemiluminescence
Degree is capable of the concentration of praziquantel molecule in qualitative, quantitative solution to be measured.
The technical solution adopted by the invention is as follows:
1. a kind of preparation method for the electrogenerated chemiluminescence sensing electrode for detecting praziquantel, described detects the electroluminescent of praziquantel
Chemiluminescence sensing electrode is printed by growth in situ on cobalt nitride nano-chip arrays electrode CoN-nanoarray without template molecule molecule
Mark polymer NIP is obtained；The molecular engram that template molecule is free from without template molecule molecularly imprinted polymer NIP
Polymer；The molecularly imprinted polymer without containing template molecule is passed through by the MIP of molecularly imprinted polymer containing template molecule
Cross what eluted template molecule obtained；The MIP of molecularly imprinted polymer containing template molecule is the molecule print containing template molecule
Mark polymer；The template molecule is praziquantel molecule；
2. the preparation method of cobalt nitride nano-chip arrays electrode CoN-nanoarray described in technical solution 1 includes following system
Standby step:
(1) it will can disposably throw electrode and carry out ultrasonic cleaning processing using dilute hydrochloric acid, dehydrated alcohol and deionization respectively, to go
Except the oxide layer and surface impurity that can disposably throw electrode；
(2) 1 ~ 3 mmol cabaltous nitrate hexahydrate Co (NO is weighed3)2·6H2O and 3 ~ 9 mmol urea CO (NH2)2, it is put into
In 50 mL beakers, 30 mL deionized waters are added and stir to clarify, are then transferred into 50 mL ptfe autoclaves；
(3) in the disposable solution thrown in the reaction kettle that electrode is put into step (2) for handling step (1) well, 100 ~
It is reacted 9 ~ 12 hours at a temperature of 130 DEG C, cobalt hydroxide nano-chip arrays presoma electrode is prepared；
(4) the cobalt hydroxide nano-chip arrays presoma electrode that step (3) obtains is inserted into ammonium hydroxide and is taken out after 5 ~ 30 seconds, in ammonia
Under compression ring border, after being heated to 340 ~ 400 DEG C and being kept for 4 ~ 8 hours, continuation naturally rings to room temperature under ammonia environment, then by it
Be inserted into the phosphate buffer solution PBS containing dopamine and Ammonium Persulfate 98.5 in, 20 ~ 40 DEG C at a temperature of react 4 ~ 6 hours
Afterwards, it takes out and is embathed 2 ~ 4 times with deionized water, cobalt nitride nano-chip arrays electrode CoN-nanoarray is prepared；
The described disposable electrode of throwing is selected from one of cycle unit: foamed iron, foam copper, pure iron piece, pure copper sheet, pure iron piece,
Pure silicon piece, conductive carbon cloth；
In the phosphate buffer solution PBS containing dopamine and Ammonium Persulfate 98.5: dopamine concentration is 2 ~ 5 mg/mL, mistake
The concentration of amine sulfate is 3 ~ 8 mg/mL, and the concentration of phosphate buffer solution PBS is 0.1 mol/L, and pH value is 7.2 ~ 8.5；
3. the MIP's of molecularly imprinted polymer containing template molecule of CoN-nanoarray growth in situ described in technical solution 1
Preparation method includes following preparation step:
(1) 0.25 ~ 0.45mmol template molecule and 3 ~ 5 mmol 2- methacrylic acid MAA are weighed respectively in peace times bottle, are added
Enter 8 ~ 15 mL acetonitriles, 30 min of ultrasound to whole dissolutions；
(2) 15 ~ 25 mmol ethylene glycol dimethacrylate EDMA are added in the solution of step (1), 30 min of ultrasound
To being uniformly mixed, precursor mixed solution is obtained；
(3) CoN-nanoarray prepared in technical solution 2 is clipped on Stirring device, the forerunner being inserted into step (2)
In body mixed solution, in N2At a temperature of environment and 20 ~ 40 DEG C of water-bath, with 5 ~ 200 revolutions per seconds of speed Stirring, while with 1
1 ~ 3 mL of luminol solution of 1 mmol/L of dropwise addition and 1 mmol azo two are different simultaneously into mixed solution for ~ 20 drops/sec of speed
Butyronitrile AIBN carries out initiation polymerization, and the molecularly imprinted polymer containing template molecule of growth in situ is obtained on CoN-nanoarray
MIP；
4. CoN-nanoarray growth in situ described in technical solution 1 without template molecule molecularly imprinted polymer NIP's
Preparation step are as follows: by the molecular engram containing template molecule of growth in situ gathers on CoN-nanoarray obtained in technical solution 3
It closes object MIP to be immersed in eluant, eluent, template molecule is subjected to 5 ~ 20 min of elution at room temperature, is then taken out, no mould is obtained
Plate molecular imprinted polymer NIP；The eluant, eluent is the mixed liquor of formic acid and methanol, wherein the volume of formic acid and methanol
Than for 9:(1 ~ 5)；
5. detecting the preparation step of the electrogenerated chemiluminescence sensing electrode of praziquantel described in technical solution 1 are as follows: by technical side
In case 2 ~ 4 the obtained growth in situ on CoN-nanoarray without template molecule molecularly imprinted polymer NIP, use deionization
Water logging is washed 2 ~ 4 times, is dried at room temperature, and the electrogenerated chemiluminescence sensing electrode of detection praziquantel is obtained；
6. using the electrogenerated chemiluminescence sensing electrode for detecting praziquantel prepared by technical solution described in technical solution 1 ~ 5,
Applied to the detection of praziquantel molecule, including following applying step:
(1) standard solution is prepared: preparing the praziquantel molecular criteria solution of one group of various concentration including blank standard specimen；
(2) working electrode is modified: the electrogenerated chemiluminescence sensing electrode that will test praziquantel is working electrode, inserting step (1)
The praziquantel molecular criteria solution of the various concentration of middle preparation takes out after hatching 10 min, is embathed 3 times with deionized water；
(3) working curve is drawn: using saturated calomel electrode electrode as reference electrode, platinum electrode is used as to electrode, with step
(2) the working electrode composition three-electrode system modified, is connected in electrochemiluminescdetection detection equipment；In a cell first
Be added afterwards 15 mL phosphate buffer solution PBS and 1mL 2 mmol/L hydrogen peroxide (H2O2) solution；With double rank Pulse Voltammetries
Method applies cyclical voltage to the working electrode of assembling, detects the light signal strength of electrogenerated chemiluminescence；The response light of blank standard specimen
Signal strength is denoted asA 0, the response light signal strength of the praziquantel standard solution containing various concentration is denoted asA i, it is strong to respond optical signal
Spending reduced difference is ΔA = A 0-A i, ΔAWith the mass concentration of praziquantel standard solutionCBetween it is linear, draw ΔA?CWorking curve；The phosphate buffer solution PBS concentration is 10 mmol/L, pH value 7.4；Double rank arteries and veins
Rush parameter setting when voltammetry detection are as follows: initial potential is 0 V, and pulse potential is 0.9 V, and the burst length is 0.1 s, pulse
Period is 30 s；
(4) in sample to be tested praziquantel detection: the praziquantel standard solution in step (1) is replaced with sample to be tested, according to step
Suddenly the method in (2) and (3) is detected, the difference DELTA that light signal strength reduces according to responseAAnd working curve, it obtains to be measured
The content of praziquantel in sample.
Beneficial achievement of the invention
(1) the electrogenerated chemiluminescence sensing electrode preparation of detection praziquantel of the present invention is simple, easy to operate, realizes pair
Quick, sensitive, the highly selective detection of sample, and it is at low cost, it can be applied to portable inspectiont, there is market development prospect；
(2) the growth in situ molecularly imprinted polymer on cobalt nitride nano-chip arrays electrode CoN-nanoarray for the first time of the invention,
On the one hand more, molecularly imprinted polymer more evenly can be grown using the big specific surface area of CoN-nanoarray, and
CoN-nanoarray has excellent electron transmission ability, to greatly improve detection sensitivity；On the other hand, CoN-
Nanoarray has electro catalytic activity to hydrogen peroxide, and may not need addition horseradish peroxidase can be realized luminol-mistake
The stabilization of hydrogen oxide electrogenerated chemiluminescence system, highly effective reaction, so that prepared sensor is without considering that biological enzyme inactivation is asked
Topic, so that the use and storage of sensor can be more stable loose with condition, thus further decreasing signal back
While scape, raising detection sensitivity, greatly reduces testing cost and reduce environmental pollution；
(3) present invention specific surface area and dopamine big using high adsorption activity and nano-array electrode of the nitride to amino
It combines, so that dopamine in cobalt nitride nano-chip arrays in situ Polymerization, is forming sufficiently thin poly-dopamine film
While, on uniform fold to cobalt nitride nano-chip arrays, thus for more preferably polymerizable molecular imprinted polymers in next step
Carry out place mat；Later using poly-dopamine to the strong absorption connection function for the amino being rich on molecularly imprinted polymer, then ingeniously
It uses CoN-nanoarray as blender wonderfully, immersion stirring is carried out in molecular engram precursor mixed solution, pass through control
The rate of addition and polymeric reaction temperature of mixing speed processed, initiators for polymerization, in the surface CoN-nanoarray direct in-situ
Growth can control the molecularly imprinted polymer of film thickness, print the secured supporting molecular of CoN-nanoarray
Mark polymer and luminol, to significantly improve the stability and reproducibility of prepared Electrochemiluminescsensor sensor；It is another
Aspect can effectively control molecularly imprinted polymer in the film forming thickness of electrode surface, solve and be unable to control molecular engram film and exist
The technical problem that electrode surface film forming thickness is unable to control so as to cause poor reproducibility；In addition, more due to preparation side of the invention
Method coats effective control of film forming thickness and the in situ quantitation of luminol, can sufficiently improve electroluminescent based on molecular engram
The sensitivity of chemiluminescence sensor and detection limit have important scientific meaning and application value.
Specific embodiment
The preparation of 1 CoN-nanoarray of embodiment
(1) it will can disposably throw electrode and carry out ultrasonic cleaning processing using dilute hydrochloric acid, dehydrated alcohol and deionization respectively, to go
Except the oxide layer and surface impurity that can disposably throw electrode；
(2) 1 mmol cabaltous nitrate hexahydrate Co (NO is weighed3)2·6H2O and 3 mmol urea CO (NH2)2, it is put into 50 mL
In beaker, 30 mL deionized waters are added and stir to clarify, are then transferred into 50 mL ptfe autoclaves；
(3) in the disposable solution thrown in the reaction kettle that electrode is put into step (2) for handling step (1) well, 100
It is reacted 12 hours at a temperature of DEG C, cobalt hydroxide nano-chip arrays presoma electrode is prepared；
(4) the cobalt hydroxide nano-chip arrays presoma electrode that step (3) obtains is inserted into ammonium hydroxide and is taken out after 5 seconds, in ammonia
Under environment, be heated to 340 DEG C and keep 8 hours after, continuation naturally ring to room temperature under ammonia environment, be then inserted into containing
In the phosphate buffer solution PBS of dopamine and Ammonium Persulfate 98.5,20 DEG C at a temperature of reaction 4 hours after, take out and spend from
Sub- water logging is washed 2 times, and cobalt nitride nano-chip arrays electrode CoN-nanoarray is prepared；
The electrode therein that can disposably throw is foamed iron；Dopamine concentration is 2 mg/mL, and the concentration of Ammonium Persulfate 98.5 is 3 mg/mL,
The concentration of phosphate buffer solution PBS is 0.1 mol/L, pH value 7.2.
The preparation of 2 CoN-nanoarray of embodiment
(1) it will can disposably throw electrode and carry out ultrasonic cleaning processing using dilute hydrochloric acid, dehydrated alcohol and deionization respectively, to go
Except the oxide layer and surface impurity that can disposably throw electrode；
(2) 2 mmol cabaltous nitrate hexahydrate Co (NO are weighed3)2·6H2O and 6 mmol urea CO (NH2)2, it is put into 50 mL
In beaker, 30 mL deionized waters are added and stir to clarify, are then transferred into 50 mL ptfe autoclaves；
(3) in the disposable solution thrown in the reaction kettle that electrode is put into step (2) for handling step (1) well, 110
It is reacted 11 hours at a temperature of DEG C, cobalt hydroxide nano-chip arrays presoma electrode is prepared；
(4) the cobalt hydroxide nano-chip arrays presoma electrode that step (3) obtains is inserted into ammonium hydroxide and is taken out after 15 seconds, in ammonia
Under environment, be heated to 370 DEG C and keep 6 hours after, continuation naturally ring to room temperature under ammonia environment, be then inserted into containing
In the phosphate buffer solution PBS of dopamine and Ammonium Persulfate 98.5,30 DEG C at a temperature of reaction 5 hours after, take out and spend from
Sub- water logging is washed 3 times, and cobalt nitride nano-chip arrays electrode CoN-nanoarray is prepared；
The electrode therein that can disposably throw is pure copper sheet；Dopamine concentration is 3.5 mg/mL, and the concentration of Ammonium Persulfate 98.5 is 6.2
The concentration of mg/mL, phosphate buffer solution PBS are 0.1 mol/L, pH value 8.0.
The preparation of 3 CoN-nanoarray of embodiment
(1) it will can disposably throw electrode and carry out ultrasonic cleaning processing using dilute hydrochloric acid, dehydrated alcohol and deionization respectively, to go
Except the oxide layer and surface impurity that can disposably throw electrode；
(2) 3 mmol cabaltous nitrate hexahydrate Co (NO are weighed3)2·6H2O and 9 mmol urea CO (NH2)2, it is put into 50 mL
In beaker, 30 mL deionized waters are added and stir to clarify, are then transferred into 50 mL ptfe autoclaves；
(3) in the disposable solution thrown in the reaction kettle that electrode is put into step (2) for handling step (1) well, 130
It is reacted 9 hours at a temperature of DEG C, cobalt hydroxide nano-chip arrays presoma electrode is prepared；
(4) the cobalt hydroxide nano-chip arrays presoma electrode that step (3) obtains is inserted into ammonium hydroxide and is taken out after 30 seconds, in ammonia
Under environment, be heated to 400 DEG C and keep 4 hours after, continuation naturally ring to room temperature under ammonia environment, be then inserted into containing
In the phosphate buffer solution PBS of dopamine and Ammonium Persulfate 98.5,40 DEG C at a temperature of reaction 6 hours after, take out and spend from
Sub- water logging is washed 4 times, and cobalt nitride nano-chip arrays electrode CoN-nanoarray is prepared；
The electrode therein that can disposably throw is conductive carbon cloth；Dopamine concentration is 5 mg/mL, and the concentration of Ammonium Persulfate 98.5 is 8 mg/
The concentration of mL, phosphate buffer solution PBS are 0.1 mol/L, pH value 8.5.
Embodiment 4 detects the preparation method of the electrogenerated chemiluminescence sensing electrode of praziquantel
(1) 0.25 mmol template molecule and 3 mmol 2- methacrylic acid MAA are weighed respectively in peace times bottle, and 8 mL second are added
Nitrile, 30 min of ultrasound to whole dissolutions；
(2) 15 mmol ethylene glycol dimethacrylate EDMA are added in the solution of step (1), 30 min of ultrasound are to mixed
It closes uniformly, obtains precursor mixed solution；
(3) CoN-nanoarray prepared in embodiment 1 is clipped on Stirring device, the presoma being inserted into step (2)
In mixed solution, in N2At a temperature of environment and 20 DEG C of water-bath, with 200 revolutions per seconds of speed Stirring, while with 1 drop/sec
Speed into mixed solution simultaneously be added dropwise 1 mmol/L 1 mL of luminol solution and 1 mmol azodiisobutyronitrile AIBN into
Row causes polymerization, and the MIP of molecularly imprinted polymer containing template molecule of growth in situ is obtained on CoN-nanoarray；
(4) MIP of molecularly imprinted polymer containing template molecule of growth in situ on CoN-nanoarray for obtaining step (3)
It is immersed in eluant, eluent, template molecule is subjected to 5 min of elution at room temperature, then takes out, obtains no template molecule molecule
Imprinted polymer NIP；Continue to be embathed 2 times with deionized water, dry at room temperature, obtains the electrogenerated chemiluminescence of detection praziquantel
Sensing electrode；
Eluant, eluent therein is the mixed liquor of formic acid and methanol, and wherein the volume ratio of formic acid and methanol is 9:1.
Embodiment 5 detects the preparation method of the electrogenerated chemiluminescence sensing electrode of praziquantel
(1) 0.35mmol template molecule and 4 mmol 2- methacrylic acid MAA are weighed respectively in peace times bottle, and 12 mL second are added
Nitrile, 30 min of ultrasound to whole dissolutions；
(2) 18 mmol ethylene glycol dimethacrylate EDMA are added in the solution of step (1), 30 min of ultrasound are to mixed
It closes uniformly, obtains precursor mixed solution；
(3) CoN-nanoarray prepared in technical solution 2 is clipped on Stirring device, the forerunner being inserted into step (2)
In body mixed solution, in N2At a temperature of environment and 30 DEG C of water-bath, with 60 revolutions per seconds of speed Stirring, while with 10 drops/sec
Speed into mixed solution simultaneously be added dropwise 1 mmol/L 2 mL of luminol solution and 1 mmol azodiisobutyronitrile AIBN into
Row causes polymerization, and the MIP of molecularly imprinted polymer containing template molecule of growth in situ is obtained on CoN-nanoarray；
(4) MIP of molecularly imprinted polymer containing template molecule of growth in situ on CoN-nanoarray for obtaining step (3)
It is immersed in eluant, eluent, template molecule is subjected to 10 min of elution at room temperature, then takes out, obtains no template molecule molecule
Imprinted polymer NIP；Continue to be embathed 3 times with deionized water, dry at room temperature, obtains the electrogenerated chemiluminescence of detection praziquantel
Sensing electrode；
Eluant, eluent therein is the mixed liquor of formic acid and methanol, and wherein the volume ratio of formic acid and methanol is 9:3.
Embodiment 6 detects the preparation method of the electrogenerated chemiluminescence sensing electrode of praziquantel
(1) 0.45mmol template molecule and 5 mmol 2- methacrylic acid MAA are weighed respectively in peace times bottle, and 15 mL second are added
Nitrile, 30 min of ultrasound to whole dissolutions；
(2) 25 mmol ethylene glycol dimethacrylate EDMA are added in the solution of step (1), 30 min of ultrasound are to mixed
It closes uniformly, obtains precursor mixed solution；
(3) CoN-nanoarray prepared in technical solution 2 is clipped on Stirring device, the forerunner being inserted into step (2)
In body mixed solution, in N2At a temperature of environment and 40 DEG C of water-bath, with 5 revolutions per seconds of speed Stirring, while with 20 drops/sec
Speed into mixed solution simultaneously be added dropwise 1 mmol/L 3 mL of luminol solution and 1 mmol azodiisobutyronitrile AIBN into
Row causes polymerization, and the MIP of molecularly imprinted polymer containing template molecule of growth in situ is obtained on CoN-nanoarray；
(4) MIP of molecularly imprinted polymer containing template molecule of growth in situ on CoN-nanoarray for obtaining step (3)
It is immersed in eluant, eluent, template molecule is subjected to 20 min of elution at room temperature, then takes out, obtains no template molecule molecule
Imprinted polymer NIP；Continue to be embathed 4 times with deionized water, dry at room temperature, obtains the electrogenerated chemiluminescence of detection praziquantel
Sensing electrode；
Eluant, eluent therein is the mixed liquor of formic acid and methanol, and wherein the volume ratio of formic acid and methanol is 9:5.
The electrogenerated chemiluminescence sensing electrode of the detection praziquantel of 7 embodiment 1 ~ 6 of embodiment preparation, is applied to praziquantel
The detection of molecule, steps are as follows:
(1) standard solution is prepared: preparing the praziquantel molecular criteria solution of one group of various concentration including blank standard specimen；
(2) working electrode is modified: the electrogenerated chemiluminescence sensing electrode that will test praziquantel is working electrode, inserting step (1)
The praziquantel standard solution of the various concentration of middle preparation takes out after hatching 10 min, is embathed 3 times with deionized water；
(3) working curve is drawn: using saturated calomel electrode electrode as reference electrode, platinum electrode is used as to electrode, with step
(2) the working electrode composition three-electrode system modified, is connected in electrochemiluminescdetection detection equipment；In a cell first
Be added afterwards 15 mL phosphate buffer solution PBS and 1mL 2 mmol/L hydrogen peroxide (H2O2) solution；With double rank Pulse Voltammetries
Method applies cyclical voltage to the working electrode of assembling, detects the light signal strength of electrogenerated chemiluminescence；The response light of blank standard specimen
Signal strength is denoted asA 0, the response light signal strength of the praziquantel standard solution containing various concentration is denoted asA i, it is strong to respond optical signal
Spending reduced difference is ΔA = A 0-A i, ΔAWith the mass concentration of praziquantel standard solutionCBetween it is linear, draw ΔA?CWorking curve；The phosphate buffer solution PBS concentration is 10 mmol/L, pH value 7.4；Double rank arteries and veins
Rush parameter setting when voltammetry detection are as follows: initial potential is 0 V, and pulse potential is 0.9 V, and the burst length is 0.1 s, pulse
Period is 30 s；
(4) in sample to be tested praziquantel detection: the praziquantel standard solution in step (1) is replaced with sample to be tested, according to step
Suddenly the method in (2) and (3) is detected, the difference DELTA that light signal strength reduces according to responseAAnd working curve, it obtains to be measured
The content of praziquantel in sample.
The electrogenerated chemiluminescence sensing electrode of the detection praziquantel of 8 embodiment 1 ~ 6 of embodiment preparation, according to embodiment 7
Detecting step be applied to praziquantel detection, the range of linearity be 5 × 10-5~ 800 mmol/L, detection are limited to 2 nmol/L.
The detection of praziquantel in 9 pig urine samples of embodiment
Pig urine samples are accurately pipetted, the praziquantel standard solution of certain mass concentration are added, the pig urine sample of praziquantel not to be added
Product are blank, carry out recovery testu, the electrogenerated chemiluminescence sensing electrode for detecting praziquantel prepared with embodiment 1 ~ 6,
It is detected according to the step of embodiment 7, measures the rate of recovery of praziquantel in pig urine samples, testing result is shown in Table 2:
The testing result of praziquantel in 1 pig urine samples of table
For 2 testing result of table it is found that the relative standard deviation (RSD) of result is less than 3.2 %, average recovery rate is 99.0 ~ 101%,
Show the present invention can be used for pig urine in praziquantel detection, the high sensitivity of method, high specificity, as a result accurately and reliably.
The detection of praziquantel in 10 sheep urine samples of embodiment
Certain sheep urine samples are accurately pipetted, the praziquantel standard solution of certain mass concentration are added, the sheep of praziquantel not to be added
Urine samples are blank, carry out recovery testu, and the electrogenerated chemiluminescence of the detection praziquantel prepared with embodiment 1 ~ 6 senses electricity
Pole is detected according to the step of embodiment 7, measures the rate of recovery of praziquantel in sheep urine samples, and testing result is shown in Table 3:
The testing result of praziquantel in 2 sheep urine samples of table
3 testing result of table it is found that the relative standard deviation (RSD) of result less than 3.3 %, average recovery rate is 98.0 ~
101.4%, show that the present invention can be used for the detection of praziquantel in sheep urine, as a result the high sensitivity of method, high specificity accurately may be used
It leans on.
Claims (6)
1. a kind of preparation method for the electrogenerated chemiluminescence sensing electrode for detecting praziquantel, which is characterized in that the detection pyrrole
The electrogenerated chemiluminescence sensing electrode of quinoline ketone is by growth in situ on cobalt nitride nano-chip arrays electrode CoN-nanoarray without template
Molecular imprinted polymer NIP is obtained；Described has been free from template molecule without template molecule molecularly imprinted polymer NIP
Molecularly imprinted polymer；The molecularly imprinted polymer without containing template molecule is gathered by molecular engram containing template molecule
Close what object MIP was obtained by eluted template molecule；The MIP of molecularly imprinted polymer containing template molecule is containing template molecule
Molecularly imprinted polymer；The template molecule is praziquantel molecule.
2. cobalt nitride nano-chip arrays electrode CoN-nanoarray as described in claim 1 is it is characterized in that, the CoN-
The preparation method of nanoarray includes following preparation step:
(1) it will can disposably throw electrode and carry out ultrasonic cleaning processing using dilute hydrochloric acid, dehydrated alcohol and deionization respectively, to go
Except the oxide layer and surface impurity that can disposably throw electrode；
(2) 1 ~ 3 mmol cabaltous nitrate hexahydrate Co (NO is weighed3)2·6H2O and 3 ~ 9 mmol urea CO (NH2)2, it is put into 50
In mL beaker, 30 mL deionized waters are added and stir to clarify, are then transferred into 50 mL ptfe autoclaves；
(3) in the disposable solution thrown in the reaction kettle that electrode is put into step (2) for handling step (1) well, 100 ~
It is reacted 9 ~ 12 hours at a temperature of 130 DEG C, iron hydroxide nano-chip arrays presoma electrode is prepared；
(4) the cobalt hydroxide nano-chip arrays presoma electrode that step (3) obtains is inserted into ammonium hydroxide and is taken out after 5 ~ 30 seconds, in ammonia
Under compression ring border, after being heated to 340 ~ 400 DEG C and being kept for 4 ~ 8 hours, continuation naturally rings to room temperature under ammonia environment, then by it
Be inserted into the phosphate buffer solution PBS containing dopamine and Ammonium Persulfate 98.5 in, 20 ~ 40 DEG C at a temperature of react 4 ~ 6 hours
Afterwards, it takes out and is embathed 2 ~ 4 times with deionized water, cobalt nitride nano-chip arrays electrode CoN-nanoarray is prepared；
The described disposable electrode of throwing is selected from one of cycle unit: nickel foam, foam copper, pure nickel piece, pure copper sheet, pure iron piece,
Pure silicon piece, conductive carbon cloth；
In the phosphate buffer solution PBS containing dopamine and Ammonium Persulfate 98.5: dopamine concentration is 2 ~ 5 mg/mL, mistake
The concentration of amine sulfate is 3 ~ 8 mg/mL, and the concentration of phosphate buffer solution PBS is 0.1 mol/L, and pH value is 7.2 ~ 8.5.
3. the MIP of molecularly imprinted polymer containing template molecule as described in claim 1, which is characterized in that described containing template point
Sub- molecularly imprinted polymer MIP is that direct in-situ is grown on CoN-nanoarray, and preparation method includes following preparation step
It is rapid:
(1) 0.25 ~ 0.45mmol template molecule and 3 ~ 5 mmol 2- methacrylic acid MAA are weighed respectively in peace times bottle, are added
Enter 8 ~ 15 mL acetonitriles, 30 min of ultrasound to whole dissolutions；
(2) 15 ~ 25 mmol ethylene glycol dimethacrylate EDMA are added in the solution of step (1), 30 min of ultrasound
To being uniformly mixed, precursor mixed solution is obtained；
(3) CoN-nanoarray is clipped on Stirring device, is inserted into the precursor mixed solution in step (2), in N2
At a temperature of environment and 20 ~ 40 DEG C of water-bath, with 5 ~ 200 revolutions per seconds of speed Stirring, while with 1 ~ 20 drop/sec of speed to
1 ~ 3 mL of luminol solution and 1 mmol azodiisobutyronitrile AIBN that 1 mmol/L is added dropwise simultaneously in mixed solution are caused
Polymerization, obtains the MIP of molecularly imprinted polymer containing template molecule of growth in situ on CoN-nanoarray.
4. as described in claim 1 without template molecule molecularly imprinted polymer NIP, it is characterised in that the no template point
The preparation step of sub- molecularly imprinted polymer NIP are as follows: by obtained in claim 3 on CoN-nanoarray growth in situ
The MIP of molecularly imprinted polymer containing template molecule be immersed in eluant, eluent, template molecule is subjected to elution 5 ~ 20 at room temperature
Min then takes out, and obtains no template molecule molecularly imprinted polymer NIP；The eluant, eluent is the mixing of formic acid and methanol
Liquid, wherein the volume ratio of formic acid and methanol is 9:(1 ~ 5).
5. the preparation step of the electrogenerated chemiluminescence sensing electrode as described in claim 1 based on detection praziquantel are as follows: will weigh
Benefit require in 2 ~ 4 the growth in situ obtained on CoN-nanoarray without template molecule molecularly imprinted polymer NIP, spend
Ionized water embathes 2 ~ 4 times, dries at room temperature, obtains the electrogenerated chemiluminescence sensing electrode of detection praziquantel.
6. sensing electricity using the electrogenerated chemiluminescence based on detection praziquantel prepared by preparation method described in claim 1 ~ 5
Pole, the detection applied to praziquantel, which is characterized in that the detecting step is as follows:
(1) standard solution is prepared: preparing the praziquantel standard solution of one group of various concentration including blank standard specimen；
(2) working electrode is modified: the electrogenerated chemiluminescence sensing electrode that will test praziquantel is working electrode, inserting step (1)
The praziquantel standard solution of the various concentration of middle preparation takes out after hatching 10 min, is embathed 3 times with deionized water；
(3) working curve is drawn: using saturated calomel electrode electrode as reference electrode, platinum electrode is used as to electrode, with step
(2) the working electrode composition three-electrode system modified, is connected in electrochemiluminescdetection detection equipment；In a cell first
Be added afterwards 15 mL phosphate buffer solution PBS and 1mL 2 mmol/L hydrogen peroxide (H2O2) solution；With double rank Pulse Voltammetries
Method applies cyclical voltage to the working electrode of assembling, detects the light signal strength of electrogenerated chemiluminescence；The response light of blank standard specimen
Signal strength is denoted asA 0, the response light signal strength of the praziquantel standard solution containing various concentration is denoted asA i, it is strong to respond optical signal
Spending reduced difference is ΔA = A 0-A i, ΔAWith the mass concentration of praziquantel standard solutionCBetween it is linear, draw ΔA?CWorking curve；The phosphate buffer solution PBS concentration is 10 mmol/L, pH value 7.4；Double rank arteries and veins
Rush parameter setting when voltammetry detection are as follows: initial potential is 0 V, and pulse potential is 0.9 V, and the burst length is 0.1 s, pulse
Period is 30 s；
(4) in sample to be tested praziquantel detection: the praziquantel standard solution in step (1) is replaced with sample to be tested, according to step
Suddenly the method in (2) and (3) is detected, the difference DELTA that light signal strength reduces according to responseAAnd working curve, it obtains to be measured
The content of praziquantel in sample.
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