CN108051489A - A kind of electrochemical sensor for detecting Acetamiprid and its preparation method and application - Google Patents
A kind of electrochemical sensor for detecting Acetamiprid and its preparation method and application Download PDFInfo
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- CN108051489A CN108051489A CN201710834305.6A CN201710834305A CN108051489A CN 108051489 A CN108051489 A CN 108051489A CN 201710834305 A CN201710834305 A CN 201710834305A CN 108051489 A CN108051489 A CN 108051489A
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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Abstract
The present invention provides a kind of electrochemical sensor for detecting Acetamiprid and preparation method thereof, the electrochemical sensor includes detecting electrode and matching reagent；The detecting electrode includes gold electrode and the DHLA active layers, Acetamiprid antibody layer and the MCH confining beds that are wrapped in successively from the inside to the outside outside the gold electrode；The matching reagent includes Acetamiprid haptens probe solution and methylene blue probe solution.Electrochemical sensor of the present invention realizes the immune detection of small molecule, and detection speed is fast；Detection is limited to 3.2ng L‑1, detect more sensitive.Application method is simple, quick, the on-line checking of Acetamiprid suitable for food security.
Description
Technical field
The present invention relates to electrochemical sensor technology field more particularly to it is a kind of detect Acetamiprid electrochemical sensor and
Its preparation method.
Background technology
Novel pesticide Acetamiprid belongs to Nitromethylene heterocycle compound, may act on insect nerve system cynapse portion
The nicotinic acetylcholine receptor of position, the stimulation conduction of interference insect nervous system, causes nervous system path blockade, causes nerve
Transmitter acetylcholine synapses accumulation, it is final dead so as to cause insect paralysis.Acetamiprid has substituted common insecticides
Such as organophosphor, carbamate extensive use in the world.Acetamiprid is similar with other numerous pesticides, in the mistake of application
Residual is inevitably present in journey, and the food-safety problem caused by pesticide residue has become whole world public attention
Focus.
Detection for Acetamiprid, traditional method include high performance liquid chromatography (HPLC), gas-chromatography (GC), mass spectrum
(MS), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrum (GC-MS).Though these methods have high sensitivity, stablize
Property it is good, it is reproducible the advantages of, but generally require to carry out complicated sample pre-treatments, expensive detection during atual detection
Instrument and reagent, and also have higher requirement to the specialist of testing staff.Therefore, there is an urgent need to develop it is simple, quick,
Highly sensitive effective analysis method detects Acetamiprid.
Immuno analytical method is that the specific recognition effect based on antigen-antibody is measured, and high sensitivity, specificity is good,
But the research based on immunological technique focuses mostly in the detection of the large biological molecules such as DNA, protein, microorganism.These objects to be detected
Matter generally comprises multiple recognition sites, can be detected by conventional sandwich immunoassays, but pesticide such as Acetamiprid due to
It is small to molecular weight, it is not enough in combination with two kinds of antibody, it is impossible to be detected with sandwich immunoassays.Simply, sensitive, reliable point
Analysis method detection acetamiprid residue is the technical issues of Detecting Pesticide field is urgently to be resolved hurrily.
The content of the invention
In view of this, it is an object of the invention to provide a kind of electrifications easy to operate, fast and accurately detecting Acetamiprid
Learn sensor and preparation method thereof.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of for detecting the detecting electrode of Acetamiprid electrochemical sensor, the detecting electrode includes
The gold electrode and dihydrolipoic acid active layer being wrapped in successively from the inside to the outside outside the gold electrode, Acetamiprid antibody layer and sulfydryl oneself
Alcohol confining bed.
Preferably, the dihydrolipoic acid active layer is to pass through Au-S keys by the S in dihydrolipoic acid and gold electrode surfaces
Assembling is formed.
The present invention also provides the preparation methods of the detecting electrode, comprise the following steps：
1) gold electrode after cleaning is soaked in the ethanol solution of dihydro zinc sulfate, Au-S key self assemblies are carried out, described
Gold electrode surfaces form dihydro zinc sulfate Iy self-assembled layer；
2) the dihydro zinc sulfate Iy self-assembled layer surface obtained in the step 2) coats the MES bufferings containing NHS and EDC
Liquid carries out modification activation to the dihydro zinc sulfate Iy self-assembled layer, and dihydrolipoic acid active layer is formed in the gold electrode surfaces；
3) it is incubated in dihydro zinc sulfate activation layer surface cladding Acetamiprid capture antibody-solutions, in the DHLA
Active layer outer surface forms Acetamiprid antibody layer；
4) in the Acetamiprid antibody layer outer surface, cladding MCH solution is closed, in the Acetamiprid antibody layer appearance
Face forms MCH confining beds, obtains detecting electrode.
Preferably, the concentration of the ethanol solution of dihydrolipoic acid described in step 1) is 0.8~1.2mmol/L；The leaching
The time of bubble is 1.5~2.5h.
Preferably, the pH value of the MES buffer solutions containing NHS and EDC described in step 3) is 6.4~6.6；In the buffer solution
The concentration of MES is 0.09~0.11mol/L；The concentration of NHS is 4.5~5.5mmol/L；The concentration of EDC for 2.0~
3.0mmol/L。
Preferably, the concentration that antibody-solutions are captured described in step 4) is 8~12 μ g mL-1；The time of the incubation is
1.5~2.5h.
Preferably, further included after the incubation：The obtained decorative layer that is incubated using PBS is washed, obtains Acetamiprid
Antibody layer.
Preferably, the concentration of MCH solution described in step 5) is 1.5~2.5mmol/L；The time of the closing for 25~
35min。
The present invention also provides a kind of electrochemical sensor for including the detecting electrode, the electrochemical sensor also wraps
Matching reagent is included, the matching reagent includes Acetamiprid haptens probe solution and methylene blue probe solution.
Preferably, the Acetamiprid haptens probe solution concentration is 5~10 μm of ol/L.
Preferably, the Acetamiprid haptens probe is to utilize mixed anhydride method by Acetamiprid haptens and amination probe
What coupling obtained, the sequence of the amination probe is as shown in Seq ID No.1.
Preferably, the nucleotide sequence of the methylene blue probe is as shown in Seq ID No.2.
Preferably, the concentration of the methylene blue probe solution is 5~15 μM.
The present invention provides application of the electrochemical sensor in Acetamiprid is detected.
Preferably, the detection Acetamiprid comprises the following steps：
A the mixed liquor of measuring samples and haptens probe) is modified into detecting electrode surface, obtains electrode to be checked；
B methylene blue probe solution, the methylene blue probe solution and haptens) is added dropwise to the electrode surface to be checked
Probes complementary is modified, and obtains band signal electrode；
C) using the band signal electrode as working electrode, Ag/AgCl is reference electrode, and Pt electrodes are to electrode, and it is poor to carry out
Sectors rushes Voltammetric detection, according to obtained electric signal and predetermined standard curve, obtains containing for Acetamiprid in the measuring samples
Amount.
Beneficial effects of the present invention：Electrochemical sensor provided by the invention uses the detection model of competitive type, Acetamiprid
Haptens probe, to capturing on antibody, realizes the immune detection of small molecule, detection speed is fast with Acetamiprid competitive binding；Detection
It is limited to 3.2ng L-1, detection is more sensitive, and testing result is accurately and reliably；Using gold electrode, electrode is easy, miniaturization, it is portable,
It is used multiple times；And the Acetamiprid electrochemical sensor application method is simple, quick, the pyridine worm suitable for food security
The on-line checking of amidine.
The preparation method of detecting electrode provided by the invention is combined by self assembly chemical bond and biological immune to golden electricity
Pole is modified, and forms dihydrolipoic acid active layer, Acetamiprid antibody layer and sulfydryl hexanol confining bed in gold electrode surfaces successively.
Electrode preparation method is simple, and performance is stablized, reproducible；It is low to make the process costs of electrode, it is inexpensive suitable for industrialization
It is required that；Detecting electrode is prepared using surface modification technology, and prepare detecting electrode material dosage and concentration it is appropriate so that inspection
Survey the requirement unobvious of electrode pair environment temperature, at room temperature use.
Description of the drawings
Fig. 1 is detection Acetamiprid electrochemical sensor schematic diagram of the present invention, and wherein A is Acetamiprid haptens probe
Preparation flow figure, B are to detect Acetamiprid sample and the flow diagram of blank control respectively；
Fig. 2 is the testing result figure of electrochemical impedance spectroscopy (EIS) in embodiment 1；
Fig. 3 is gold electrode CV testing result figures in embodiment 1；
Fig. 4 is the testing result figure that experiment is controlled in embodiment 2；
Fig. 5 is the testing result figure that Acetamiprid haptens concentration and probe concentration optimizes in embodiment 3；
Fig. 6 is the testing result figure that antibody concentration optimizes in embodiment 4；
Fig. 7 is the change in electric figure read in embodiment 5 using differential pulse voltammetry technology；
Fig. 8 is 5 standard curve testing result figure of embodiment；
Fig. 9 is the testing result figure of specificity analysis in embodiment 6.
Specific embodiment
The present invention provides a kind of for detecting the detecting electrode of Acetamiprid electrochemical sensor, the detecting electrode includes
The gold electrode and dihydrolipoic acid active layer being wrapped in successively from the inside to the outside outside the gold electrode, Acetamiprid antibody layer and sulfydryl oneself
Alcohol confining bed.
In the present invention, the detecting electrode modifies the basic electrode surface using gold electrode as basic electrode
It obtains, the gold electrode outer surface is enclosed with DHLA active layers, and the DHLA active layers are that DHLA passes through with gold electrode surfaces
Au-S keys assemble to be formed；In the present invention, the carboxyl on the DHLA active layers is the state in activation；The carboxyl exists
It is combined under the state of activation with Acetamiprid antibody.
In the present invention, the detecting electrode is wrapped in the Acetamiprid antibody layer of the DHLA active layers outer surface, institute
It is to capture antibody by Acetamiprid to be formed with the DHLA modification connections activated to state Acetamiprid antibody layer, and Acetamiprid capture resists
Body is can be with the antibody of the anti-raw specific immunity identification of Acetamiprid；The Fc sections and DHLA active layers of the Acetamiprid capture antibody
On carboxyl combine；The present invention is not particularly limited the source of the capture antibody.
In the present invention, the detecting electrode further includes the MCH confining beds for being wrapped in the Acetamiprid antibody layer outer surface,
The MCH confining beds are to be closed the DHLA for the activation not combined with Acetamiprid antibody with MCH, to reduce non-specific inhale
It is attached.Detecting electrode of the present invention can occur specific immunity with Acetamiprid or Acetamiprid haptens probe and be combined.
The present invention also provides the preparation methods of the detecting electrode, comprise the following steps：1) by the gold electrode after cleaning
The ethanol solution of dihydro zinc sulfate is soaked in, carries out Au-S key self assemblies, dihydro zinc sulfate is formed certainly in the gold electrode surfaces
Assembled layers；2) the dihydro zinc sulfate Iy self-assembled layer surface obtained in the step 1) coats the MES buffer solutions containing NHS and EDC,
Modification activation is carried out to the dihydro zinc sulfate Iy self-assembled layer, dihydrolipoic acid active layer is formed in the gold electrode surfaces；3)
It is incubated in dihydro zinc sulfate activation layer surface cladding Acetamiprid capture antibody-solutions, in the DHLA active layers appearance
Face forms Acetamiprid antibody layer；4) in the Acetamiprid antibody layer outer surface, cladding MCH solution is closed, in the Acetamiprid
Antibody layer outer surface forms MCH confining beds, obtains detecting electrode.
The present invention pre-processes gold electrode basic electrode, and the pretreatment preferably includes：1.1) by gold electrode in oxygen
Change and surface is polished in aluminium paste in minute surface, obtain polishing gold electrode；1.2) by the polishing gold electrode in sulfuric acid and hydrogen peroxide
Mixed solution in CV verifications are carried out after washing by soaking.
In the present invention the gold electrode polishing process for successively using the oxidation aluminium paste that grain size is 0.3 μm and 0.05 μm into
Row polishes twice；The time polished every time is preferably 2~5min, more preferably 3~4min.The present invention is in the gold electricity
Pole is polished to surface in being preferably rinsed after minute surface using secondary water to it, rinses out the aluminium oxide for floating over gold electrode surfaces
Particle.
The present invention is after gold electrode is polished, by obtained polishing gold electrode in the mixed solution of sulfuric acid and hydrogen peroxide
CV verifications are carried out after washing by soaking.The sulfuric acid preferred mass fraction described in the present invention be 98% sulfuric acid, the peroxidating
Hydrogen preferred mass fraction is 30% hydrogen peroxide；The volume ratio of heretofore described sulfuric acid and hydrogen peroxide is preferably 3:1；Institute
It is preferably 15~25min to state soaking time, more preferably 20min.
Heretofore described washing is preferably secondary water supersound washing, and the number of the washing is preferably 3~5 times；This
Invention is after washing preferably using nitrogen drying polishing gold electrode.
Heretofore described CV verifications are preferably using containing KCl and K3[Fe(CN)6] PBS for bottom liquid, to polish gold
Electrode is working electrode, and using Ag/AgCl as reference electrode, using Pt electrodes as to electrode, K is read using CV technologies3[Fe(CN)6]
The variation of signal.Concentration of the KCl in the liquid of bottom is preferably 0.2~0.3mM, more preferably 0.25mM；The K3[Fe
(CN)6] concentration be preferably 4.5~5.5mM, more preferably 5.0mM；The concentration of the PBS is preferably 8~12mM, more excellent
Choosing is 10mM.It is preferably -0.6~0.2V that current potential of the present invention in CV verification process, which is set, and sweep speed is preferably 50mV
S-1.K in CV verifications of the present invention3[Fe(CN)6] redox peaks be less than 85mV for qualification.If CV verifies K3[Fe
(CN)6] redox peaks be more than 85mV, then need to handle gold electrode again.
Pretreated gold electrode is soaked in the ethanol solution of DHLA by the present invention after completing to pre-process gold electrode,
The DHLA is self-assembled to gold electrode surfaces by Au-S keys, forms DHLA Iy self-assembled layers.The second of the DHLA in the present invention
The concentration of alcoholic solution is preferably 0.8~1.2mmol/L, more preferably 1.0mmol/L；The time of the immersion is preferably 1.5~
2.5h, more preferably 2h.
After gold electrode surfaces form DHLA Iy self-assembled layers, the present invention the DHLA Iy self-assembled layers outer cladding containing NHS and
The MES buffer solutions of EDC, the carboxyl on modification activation DHLA surfaces, DHLA active layers are formed in the gold electrode surfaces.In the present invention
In, the concentration of MES is preferably 0.09~0.11mol/L in the MES buffer solutions containing NHS and EDC, more preferably 0.1mol/
L；The pH value of the MES buffer solutions is preferably 6.4~6.6, and more preferably 6.5.In the present invention, in the MES buffer solutions
The concentration of NHS is preferably 4.5~5.5mM, more preferably 5mM；The concentration of EDC is preferably 2.0~3.0mM, more preferably
2.5mM.For MES buffer solutions are added dropwise to modified electrode surface, the MES is buffered the specific mode of cladding in the present invention
The amount that drop adds is preferably 5~10 μ l, more preferably 8 μ l.
The present invention after DHLA active layers are obtained, the DHLA activation layer surface cladding Acetamiprid capture antibody-solutions into
Row is incubated, and Acetamiprid antibody layer is formed in the DHLA active layers outer surface.The Fc sections and DHLA of the Acetamiprid capture antibody
Carboxyl on active layer combines.In the present invention, the concentration of the capture antibody-solutions is preferably 8~12 μ g mL-1, more preferably
For 10 μ g mL-1；The time of the incubation is preferably 1.5~2.5h, more preferably 2h.
The present invention preferably washs obtained modified gold electrode with PBS buffer solution after the incubation, forms pyridine worm
Amidine antibody layer.In the present invention, the concentration of the PBS buffer solution is preferably 10mmol/L, and pH value is preferably 7.4.
The present invention is closed after Acetamiprid antibody layer is obtained in Acetamiprid antibody layer surface cladding MCH solution,
MCH confining beds are formed in the Acetamiprid antibody layer surface, obtain detecting electrode.The concentration of heretofore described MCH is preferably
1.5~2.5mmol/L, more preferably 2mmol/L.In the present invention, the time of the closing is preferably 25~35min, more preferably
For 30min；The temperature of heretofore described closing is preferably 20~30 DEG C, more preferably 25 DEG C.
The present invention preferably cleans obtained modified gold electrode with PBS buffer solution, is examined after the closing
Survey electrode.In the present invention, the number of the cleaning is preferably 2~4 times, more preferably 3 times.The PBS buffer solution it is dense
Degree is preferably 10mmol/L, and pH value is preferably 7.4.
The present invention is during detecting electrode is prepared, and after often completing one layer of package, is intended to carry out CV verifications, institute to electrode
State K in CV verifications3[Fe(CN)6] redox peaks be less than 85mV for qualification.If CV verifies K3[Fe(CN)6] redox
Peak is more than 85mV, then needs to handle again.
The present invention also provides a kind of electrochemical sensor for including the detecting electrode, the electrochemical sensor also wraps
Matching reagent is included, the matching reagent includes Acetamiprid haptens probe solution and methylene blue probe solution.
In the present invention, the concentration of the Acetamiprid haptens probe solution is preferably 5~10 μM, and more preferably 6
~9 μM, most preferably 7.5 μM.In the present invention, the Acetamiprid haptens probe solution is to utilize mixed anhydride method by pyridine
Worm amidine haptens is obtained with amination probe conjugate, and the sequence of the amination probe is NH2-TTTTTAGCATCGGACA.
In the present invention, the Acetamiprid haptens probe preferably with mixed anhydride method by Acetamiprid haptens
It is obtained with amination probe conjugate, specifically includes following steps：Synthesize Acetamiprid haptens；By Acetamiprid haptens and amination
Probe conjugate obtains Acetamiprid haptens probe HPP.
In the present invention, the building-up process of specific Acetamiprid haptens includes：A is replaced with sulphur atom in Acetamiprid heterocycle
Chlorine atom, b from sulphur atom extension linking group introduce carboxyl.The structure of obtained haptens described in the present invention is such as formula
Shown in I：
Specific hapten synthesis route is as shown in Figure 1A in the present invention：By Acetamiprid, 3- mercaptopropionic acids and hydroxide
After the mixing reflux of potassium in ethanol, filtering obtains head product.The substance of the Acetamiprid, 3- mercaptopropionic acids and potassium hydroxide
The ratio of amount is preferably 1:1:2；The amount of the Acetamiprid substance is preferably 2~6mmol, more preferably 4mmol；The 3-
The amount of mercaptopropionic acid substance is preferably 2~6mmol, more preferably 4mmol；The amount of the substance of the potassium hydroxide is preferred
For 6~10mmol, more preferably 8mmol.In the present invention, the time of the mixing reflux is preferably 4~8h, more preferably
It is 6h.
It is currently preferred the head product is washed with ethyl alcohol after be dissolved in water, obtain head product aqueous solution；The water
Dosage is preferably 40~60ml, more preferably 50ml；The head product aqueous solution is acidified with HCl, obtains head product acid
Change solution.The concentration of the hydrochloric acid is preferably 5~7mol/L, more preferably 6mol/L；The head product souring soln
PH value is preferably 2.5~3.5, and more preferably 3.
The head product souring soln is extracted, washed, dried and recrystallized through ethyl acetate by the present invention, obtains Acetamiprid
Haptens.The number of the extraction is preferably 2~4 times, more preferably 3 times；The dosage of ethyl acetate used in extraction every time
Preferably 25~35ml；More preferably 30ml.Solution organic phase is collected, after tune pH is 3, extracting solution is done through anhydrous sodium sulfate
After dry, concentration, yellow viscous liquid is obtained, is target compound.The drying preferably uses desiccant dryness；In the present invention
Embodiment in, the drier can be specifically chosen anhydrous sodium sulfate；The recrystallization uses the recrystallization hand of this field routine
Section, without other particular/special requirements.
The present invention is total to after Acetamiprid haptens is obtained using the carboxyl on haptens and amination probe 5' terminal amino groups
Conjugated Acetamiprid haptens the probe HPP, the HPP of being formed of valency can extremely be captured by specific recognition and Acetamiprid competitive binding
Antibody surface.
Acetamiprid haptens and tri-n-butylamine are preferably dissolved in dimethyl by the present invention after Acetamiprid haptens is prepared
In formamide, mixed solution is obtained；Under agitation, isobutyl chloroformate is added drop-wise in the mixed solution, then
It is reacted, obtains Acetamiprid haptens connection solution.In the present invention, the speed of the stirring is preferably 200rpm.At this
In invention, the temperature of the reaction is preferably 20~30 DEG C, more preferably 25 DEG C；The time of the reaction is preferably 1.5
~2.5h, more preferably 2h.
After obtaining Acetamiprid haptens connection solution, the present invention is preferably stirring Acetamiprid haptens connection solution
Under be added drop-wise in the PBS solution (pH7.4) of the probe containing amination, after dialysis obtain Acetamiprid haptens probe solution.The PBS
The concentration of amination probe is preferably 80~120nmol/L in solution, more preferably 100nmol/L.The body of the PBS solution
Product is preferably 2ml.
In the present invention, the temperature of the dialysis is preferably 4 DEG C, and the time of the dialysis is preferably 60~84h,
More preferably 72h.
Heretofore described Acetamiprid haptens probe solution is preferably maintained in -20 DEG C.
In the present invention, the matching reagent further includes methylene blue probe solution, the methylene blue probe solution
Concentration be preferably 5~15 μM, more preferably 8~12 μM, most preferably 10 μM.In the present invention, the methylene blue
The sequence of probe is TGTCCGATGCTAAAAA-MB.
The preparation method of heretofore described methylene blue probe solution is molten using the methylene blue probe of this field routine
The preparation method of liquid, without other particular/special requirements.
In use, the methylene blue probe and Acetamiprid in the methylene blue probe solution described in the present invention
Haptens probe hybridizes, and by the power of the electrochemical signals of acquisition testing electrode surface methylene blue probe, reaches to pyridine worm
The quantitative purpose of amidine.The methylene blue probe is by artificial synthesized acquisition, the solvent of the methylene blue probe solution
Preferably ultra-pure water.
The electrochemical sensor of detection Acetamiprid of the present invention is real by detecting electrode and matching reagent collective effect
Now to the detection of Acetamiprid.The preparation flow of the electrochemical sensor of specific detection Acetamiprid of the present invention and work are former
Reason is as shown in Figure 1B.First, gold electrode is incubated in dihydrolipoic acid (DHLA) and forms DHLA Iy self-assembled layers by Au-S keys,
Then it is living using 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC)/n-hydroxysuccinimide (NHS)
The carboxyl of HDLA forms DHLA active layers in polarizing electrode.By Acetamiprid capture antibody modification on the electrode with DHLA active layer knots
It closes, then closes 1- mercapto hexanes (MCH) in electrode surface to reduce non-specific adsorption.Sample to be tested and Acetamiprid half are anti-
Former probe (HPP) induces the Immune discrimination of competitive type.Methylene blue probe (MBP) can hybridize with HPP, by gathering methylene
Blue electrochemical signals, achieve the purpose that quantitative to Acetamiprid.If there is substantial amounts of Acetamiprid in sample, HPP will be minimal amount of
Competitive binding is to electrode surface, and corresponding less with reference to methylene blue on the electrode, this causes electrochemical signals relatively low；If sample
Without Acetamiprid, HPP will largely competitive binding be to electrode surface, thus methylene blue is largely bound to electrode, electrochemistry
Signal is higher；The electrochemical sensor can realize determining for the highly sensitive high specific of Acetamiprid according to the strong and weak of electrochemical signals
Amount.
The present invention also provides the application methods of above-mentioned detection Acetamiprid electrochemical sensor, comprise the following steps：A) will
The mixed liquor of measuring samples and Acetamiprid haptens probe modification detecting electrode surface obtains electrode to be checked；B) to electrode table to be checked
Methylene blue probe solution is added dropwise in face, and methylene blue probe solution with haptens probes complementary by modifying in electrode surface to be checked
Obtain band signal electrode；C) by band signal electrode connect electrochemical workstation, using Ag/AgCl as reference electrode, using Pt electrodes as
To electrode, the variation of electric signal is read using differential pulse voltammetry technology, detects the Acetamiprid content in sample.
In the present invention, the mixed liquor of measuring samples and Acetamiprid haptens probe is added drop-wise to detecting electrode surface, obtained
Obtain electrode to be checked.In the present invention, the volume for being added drop-wise to the mixed liquor of detecting electrode surface is preferably 5~10 μ L, more excellent
Choosing is 8 μ L.Heretofore described measuring samples are preferably food or the cleaning solution of crops, as food or crops are applied
Used Acetamiprid pesticide, can there are Acetamiprids in its cleaning solution.The mixed liquor is preferably with above-mentioned haptens probe solution
Based on, measuring samples are added in thereto.The volume ratio of heretofore described measuring samples and haptens probe is preferably 1:
1, the measuring samples volume is preferably 4 μ L.The solvent of the mixed liquor of the measuring samples and Acetamiprid haptens probe is excellent
Choosing is PBS.Final concentration of the Acetamiprid haptens probe in mixed solution is preferably 7.5 μM.
After the currently preferred electrode to be checked for obtaining mixed liquor modification is sufficiently stirred cleaning in PBS buffer solution, into
Row operates in next step.
Methylene blue probe solution is added dropwise after electrode to be checked is obtained, to electrode surface to be checked in the present invention, and methylene blue is visited
Pin solution by with haptens probes complementary, modify in electrode surface to be checked, obtain band signal electrode.In the present invention, the drop
The volume for being added to the methylene blue probe solution of electrode surface to be checked is preferably 5~10 μ L, more preferably 8 μ L；The methylene
The concentration of blue probe solution is preferably 8~12 μM, more preferably 10 μM.
It is currently preferred the band signal electrode of acquisition is placed in PBS buffer solution be sufficiently stirred cleaning after, carry out it is next
Step operation.
The present invention is after band signal electrode is obtained, and using the band signal electrode as working electrode, Ag/AgCl is reference
Electrode, Pt electrodes are to electrode, carry out differential pulse voltammetry scanning, according to electric signal and predetermined working curve is obtained, obtain
The content of Acetamiprid into detection sample.In the present invention, the current potential of differential pulse voltammetry scanning be preferably -0.1 to -
0.4V, pulse width are preferably 0.05V, and pulse width scanning is preferably 0.06S.The electrolyte of the scanning is preferably
PBS buffer solution, the concentration of the PBS buffer solution are preferably 8~12mmol/L.
In the present invention, the predetermined working curve preparation specifically comprises the following steps：A) by the pyridine of various concentration
The mixed liquor of worm amidine and HPP are modified in electrode surface；B) by the complementary modification of MBP and HPP in electrode surface, in PBS buffer solution
Cleaning electrode；C) electrochemical workstation is connected, scans the change for reading electric signal in PBS buffer solution using differential pulse voltammetry
Change, draw predetermined working curve I=Xlog CAcetamiprid+Y。
In the present invention, the Acetamiprid concentration of the various concentration is respectively preferably:0,5,10,50,102, 5 × 102,
103, 5 × 103, 104, 5 × 104, 105, 5 × 105ng L-1；The volume of the mixed liquor of the Acetamiprid and HPP is preferably 5~
10 μ l, more preferably 8 μ l；The concentration of HPP is preferably 7~8 μM in the mixed liquor of the Acetamiprid and HPP, more preferably
For 7.5 μM.The volume of the MBP is preferably 5~10 μ l, more preferably 8 μ l；The concentration of the MBP is preferably 8~12
μM, more preferably 10 μM.
The present invention is scanned in PBS buffer solution using differential pulse voltammetry and reads telecommunications under different Acetamiprid concentration conditions
Number variation, according to the variation of electric signal, draw predetermined working curve I=Xlog CAcetamiprid+ Y.The PBS buffer solution it is dense
Degree is preferably 8~12mmol/L；More preferably 10mmol/L.
The size for the electric current that electrochemical sensor of the present invention is generated according to electrode surface methylene blue is played to mesh
The effect of analyte detection is marked, the amount of Acetamiprid has direct relation in the amount of fixed MBP and measuring samples on electrode, and Acetamiprid is got over
More, the amount of fixed MBP is fewer, and signal is weaker.
To electrochemical sensor of detection Acetamiprid provided by the invention and preparation method thereof and make with reference to embodiment
It is described in detail with method, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
A kind of preparation and verification of interlayer type electrochemical sensor of the present invention, comprise the following steps：
A, gold electrode is processed by shot blasting in 0.3 and 0.05 μm of oxidation aluminium paste first, until being in minute surface, uses secondary water
It rinses；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
After using secondary water supersound washing several times after 20min, with 0.25mM KCl and 5.0mM K3[Fe(CN)6] 10mM PBS for bottom liquid,
K is read using EIS and CV technologies3[Fe(CN)6] signal variation；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L10 μ g mL-1Capture antibody be added dropwise in electrode surface, and at room temperature in DHLA/Au electrode surfaces
Be incubated 2 it is small when, then washed with 10mM PBS (pH=7.4), with 0.25mM KCl and 5.0mM K3[Fe(CN)6] 10mM
PBS is bottom liquid, and K is read using EIS and CV technologies3[Fe(CN)6] signal variation；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary, with 0.25mM KCl and 5.0mM K3[Fe(CN)6] 10mM PBS for bottom liquid, K is read using EIS and CV technologies3[Fe
(CN)6] signal variation；
F, mixed liquor modifications of the 8 μ L containing Acetamiprid and 7.5 μM of HPP is triggered into Immune competition reaction, electricity in electrode surface
Pole is sufficiently stirred cleaning in PBS buffer solution, with 0.25mM KCl and 5.0mM K3[Fe(CN)6] 10mM PBS for bottom liquid, adopt
K is read with EIS and CV technologies3[Fe(CN)6] signal variation；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing three times, with 0.25mM KCl and 5.0mM K3[Fe(CN)6] 10mM PBS for bottom liquid, using EIS and CV technologies
Read K3[Fe(CN)6] signal variation.
H, the results are shown in Figure 2 by its EIS, it can be seen that with electrode surface antibody, MCH, Acetamiprid and HPP, MBP
Modification, impedance is increasing, illustrates that these modifications hinder the electron transmission of electrode surface, at the same also demonstrate these molecules into
Work(is modified.The results are shown in Figure 3 by CV, K3[Fe(CN)6] redox current repairing with antibody, MCH, Acetamiprid and HPP, MBP
It adorns and gradually reduces, it is similar to the trend that EIS is observed, further demonstrate the successful preparation of electrochemical sensor.
Embodiment 2
A kind of control experimental verification of interlayer type electrochemical sensor of the present invention, comprises the following steps：
A, 6 gold electrodes (being named as a, b, c, d, e, f) are polished place in 0.3 and 0.05 μm of oxidation aluminium paste first
Reason, until being in minute surface, is rinsed with secondary water；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
With after secondary water supersound washing several times after 20min, nitrogen drying is spare；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L10 μ g mL-1Capture antibody be added dropwise in electrode surface, and at room temperature in DHLA/Au electrode surfaces
Be incubated 2 it is small when, then washed with 10mM PBS (pH=7.4)；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary；
F, mixed liquor modifications of the 8 μ L containing Acetamiprid and 7.5 μM of HPP is triggered into Immune competition reaction, electricity in electrode surface
Pole is sufficiently stirred cleaning in PBS buffer solution；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing is three times.
H, the difference of a-f Electrode treatments is：(a) without Acetamiprid, (b) 50 μ g L-1 Acetamiprids, (c) unmodified antibody,
(d) unmodified HPP, (e) unmodified MBP, (f) 16mg L-1 chlopyrifos replace Acetamiprid.Electrochemical workstation is connected, with Ag/
AgCl is reference electrode, and using Pt electrodes as to electrode, current potential is arranged to -0.1 and arrives -0.4V, pulse width 0.05V, pulse width
It scans as 0.06S, using the variation that electric signal is read in differential pulse voltammetry technology 10mM PBS.
F, its results are shown in Figure 4, for blank sample at the significant strong DPV peaks of about -0.25V, show on the electrode
Combine substantial amounts of HPP and MBP (curve a).DPV peaks very little in the case of there are Acetamiprid shows almost do not have in electrode surface
Capture MBP (curve b).In no antibody, (curve c) HPP (in the case of curve d) and MBP (curve e)), are measured
DPV peak of curve electric current very littles.This means the competitive reaction is the specific effect initiation of antibody and Acetamiprid, HPP, and
Non- other non-specific interactions induction.In addition, after non-targeted chlopyrifos is replaced Acetamiprid, DPV curves obtain and blank
The similar peak point current of sample, it was demonstrated that the specificity of this method (curve f).These are integrated as a result, the inventive method can be special
The detection Acetamiprid of the opposite sex.
Embodiment 3
In order to realize the optimized analysis performance of biosensor, HPP concentration is optimized.Comprise the following steps：
A, 8 gold electrodes (being named as a-h) are processed by shot blasting in 0.3 and 0.05 μm of oxidation aluminium paste first, until
In minute surface, rinsed with secondary water；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
With after secondary water supersound washing several times after 20min, nitrogen drying is spare；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L10 μ g mL-1Capture antibody be added dropwise in electrode surface, and at room temperature in DHLA/Au electrode surfaces
Be incubated 2 it is small when, then washed with 10mM PBS (pH=7.4)；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary；
F, 8 μ L to be modified containing the mixed liquor of Acetamiprid and HPP in electrode surface, wherein b, d, f, h Acetamiprid concentration is 0,
A, the HPP concentration of b modifications is 2.5 μM, and the HPP concentration of c, d modification is 5 μM,
E, the HPP concentration of f modifications is 7.5 μM, and the HPP concentration of g, h modification is 10 μM；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing is three times.
H, electrochemical workstation is connected, using Ag/AgCl as reference electrode, using Pt electrodes as to electrode, current potential is arranged to-
0.1 arrives -0.4V, pulse width 0.05V, and pulse width scanning is 0.06S, using being read in differential pulse voltammetry technology 10mM PBS
Take the variation of electric signal.The results are shown in Figure 5 for it, and it is no target to calculate every group of HPP concentration corresponding (I0-I)/I0, wherein I0
Response and I are containing excessive target response, as Fig. 5 can be seen that (I0-I)/I0 obtains maximum under 7.5 μM of concentration.Therefore, select
The optimal HPP concentration of 7.5 μM of conducts.
Embodiment 4
In order to realize the optimized analysis performance of biosensor, HPP concentration is optimized.Comprise the following steps：
A, 5 gold electrodes (being named as a-e) are processed by shot blasting in 0.3 and 0.05 μm of oxidation aluminium paste first, until
In minute surface, rinsed with secondary water；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
With after secondary water supersound washing several times after 20min, nitrogen drying is spare；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L0.01 μ g mL-1Capture antibody be added dropwise in a electrode surfaces, 8 μ L, 0.1 μ g mL-1Capture antibody drop
It is added in b electrode surfaces, 8 μ L, 1 μ g mL-1Capture antibody be added dropwise in c electrode surfaces, 8 μ L, 10 μ g mL-1Capture antibody drop
It is added in d electrode surfaces, 8 μ L, 100 μ g mL-1Capture antibody be added dropwise in e electrode surfaces, and at room temperature in DHLA/Au electrodes
When surface incubation 2 is small, then washed with 10mM PBS (pH=7.4)；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary；
F, mixed liquors of the 8 μ L containing Acetamiprid and 7.5 μM of HPP is modified in electrode surface；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing is three times.
H, electrochemical workstation is connected, using Ag/AgCl as reference electrode, using Pt electrodes as to electrode, current potential is arranged to-
0.1 arrives -0.4V, pulse width 0.05V, and pulse width scanning is 0.06S, using being read in differential pulse voltammetry technology 10mM PBS
Take the variation of electric signal.The results are shown in Figure 6 for it, and DPV signals increase with the increase of antibody concentration, then reaches 10 μ g
The maximum of mL-1.Therefore, using 10 μ g mL-1 as optimum antibody concentration.
Embodiment 5
To study the analysis ability of the biosensor, a series of Acetamiprids are detected.Comprise the following steps：
A, 12 gold electrodes (being named as a-l) are processed by shot blasting in 0.3 and 0.05 μm of oxidation aluminium paste first, until
In minute surface, rinsed with secondary water；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
With after secondary water supersound washing several times after 20min, nitrogen drying is spare；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L, 10 μ g mL-1Capture antibody be added dropwise in electrode surface, and at room temperature in DHLA/Au electrode surfaces
Be incubated 2 it is small when, then washed with 10mM PBS (pH=7.4)；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary；
F, by mixed liquor modifications of the 8 μ L containing Acetamiprid and 7.5 μM of HPP in electrode surface, wherein a to l corresponds to the pyridine of modification
Worm amidine concentration is respectively:0,5,10,50,102,5×102,103,5×103,104,5×104,105, 5×105ng L-1；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing is three times.
H, electrochemical workstation is connected, using Ag/AgCl as reference electrode, using Pt electrodes as to electrode, current potential is arranged to-
0.1 arrives -0.4V, pulse width 0.05V, and pulse width scanning is 0.06S, using being read in differential pulse voltammetry technology 10mM PBS
Take the variation of electric signal.Its result is as shown in Fig. 7~8, in Fig. 7 it will be seen that in Acetamiprid concentration 5 to 105ng L-1
When, DPV peak value of response is reduced with the increase of Acetamiprid concentration.It may be seen that the logarithm of Acetamiprid concentration and electricity in Fig. 8
Flow size inversely, matched curve I=-0.213log CAcetamiprid+ 1.246, the range of linearity is 5ng L-1To 105ng L-1,
Detection is limited to 3.2ng L-1。
Embodiment 6
In order to study the specificity of biosensor, different pesticides is analyzed.Comprise the following steps：A, 7
Gold electrode (being named as a-g) is processed by shot blasting in 0.3 and 0.05 μm of oxidation aluminium paste first, and until being in minute surface, use is secondary
Water rinses；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
With after secondary water supersound washing several times after 20min, nitrogen drying is spare；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L, 10 μ g mL-1Capture antibody be added dropwise in electrode surface, and at room temperature in DHLA/Au electrode surfaces
Be incubated 2 it is small when, then washed with 10mM PBS (pH=7.4)；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary；
F, by mixed liquor modifications of the 8 μ L containing pesticide and 7.5 μM of HPP in electrode surface, wherein a to g corresponds to the pesticide of modification
It is Acetamiprid, chlopyrifos, acephatemet, omethoate, 2,4- dichlorphenoxyacetic acids (2,4-D), imidacloprid and carbofuran respectively；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing is three times.
H, electrochemical workstation is connected, using Ag/AgCl as reference electrode, using Pt electrodes as to electrode, current potential is arranged to-
0.1 arrives -0.4V, pulse width 0.05V, and pulse width scanning is 0.06S, using being read in differential pulse voltammetry technology 10mM PBS
Take the variation of electric signal.The results are shown in Figure 9 for it, and in Fig. 9 in addition to Acetamiprid has apparent current reduction, other pesticides are without bright
Display signals change, and show that the sensor has good specificity.
Embodiment 7
It can be applied to actual sample to study biosensor to detect, cabbage and strawberry sample carried out point
Analysis.Using standard samples recovery, comprise the following steps：
A, gold electrode is processed by shot blasting in 0.3 and 0.05 μm of oxidation aluminium paste first, until being in minute surface, uses secondary water
It rinses；
B, gold electrode is immersed in H2SO4(98%) and H2O2(30%) ratio is 3:In 1 mixed liquor (volume ratio),
With after secondary water supersound washing several times after 20min, nitrogen drying is spare；
C, the ethanol solution of 1mM DHLA is configured, pretreated gold electrode is soaked in the ethanol solution of DHLA two
Hour, DHLA is self-assembled to gold electrode surfaces by Au-S keys；
D, the 0.1M MES buffer solutions (pH=6.5) of NHS containing 5mM and 2.5mM EDC are configured, take 8uL modifications in electrode
The carboxyl on surface active DHLA surfaces；
D, by 8 μ L, 10 μ g mL-1Capture antibody be added dropwise in electrode surface, and at room temperature in DHLA/Au electrode surfaces
Be incubated 2 it is small when, then washed with 10mM PBS (pH=7.4)；
E, the modification of 8 μ L 2mM MCH solution at the electrode surface, and is incubated at room temperature 30 minutes, after with PBS wash three
It is secondary；
F, the mixed liquor of 8 μ L actual samples containing pesticide and 7.5 μM of HPP are modified in electrode surface；
G, 8uL 10 μM of MBP, MBP are added dropwise by being modified with HPP complementations in electrode surface, electrode is in PBS buffer solutions
Stirring and washing is three times.
H, electrochemical workstation is connected, using Ag/AgCl as reference electrode, using Pt electrodes as to electrode, current potential is arranged to-
0.1 arrives -0.4V, pulse width 0.05V, and pulse width scanning is 0.06S, using being read in differential pulse voltammetry technology 10mM PBS
Take the variation of electric signal.The results are shown in Table 1 for it, and the rate of recovery of the method shows this in the range of 93.7-104.3%
Sensor can be applied to the detection of actual sample.
The actual sample analysis of 1 Acetamiprid of table
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (15)
1. a kind of detecting electrode for being used to detect Acetamiprid electrochemical sensor, which is characterized in that the detecting electrode includes gold
The electrode and dihydrolipoic acid active layer being wrapped in successively from the inside to the outside outside the gold electrode, Acetamiprid antibody layer and sulfydryl hexanol
Confining bed.
2. detecting electrode according to claim 1, which is characterized in that the dihydrolipoic acid active layer is pungent by dihydro sulphur
S in acid assembles to be formed by Au-S keys with gold electrode surfaces.
3. the preparation method of the detecting electrode of claim 1 or 2, comprises the following steps：
1) gold electrode after cleaning is soaked in the ethanol solution of dihydro zinc sulfate, carries out Au-S key self assemblies, in the gold electricity
Pole surface forms dihydro zinc sulfate Iy self-assembled layer；
2) the dihydro zinc sulfate Iy self-assembled layer surface obtained in the step 2) coats the MES buffer solutions containing NHS and EDC, right
The dihydro zinc sulfate Iy self-assembled layer carries out modification activation, and dihydrolipoic acid active layer is formed in the gold electrode surfaces；
3) it is incubated in dihydro zinc sulfate activation layer surface cladding Acetamiprid capture antibody-solutions, is activated in the DHLA
Layer outer surface forms Acetamiprid antibody layer；
4) in the Acetamiprid antibody layer outer surface, cladding MCH solution is closed, in the Acetamiprid antibody layer outer surface shape
Into MCH confining beds, detecting electrode is obtained.
4. preparation method according to claim 3, which is characterized in that the ethanol solution of dihydrolipoic acid described in step 1)
Concentration be 0.8~1.2mmol/L；The time of the immersion is 1.5~2.5h.
5. preparation method according to claim 3, which is characterized in that the MES buffer solutions containing NHS and EDC described in step 3)
PH value be 6.4~6.6；The concentration of MES is 0.09~0.11mol/L in the buffer solution；The concentration of NHS for 4.5~
5.5mmol/L；The concentration of EDC is 2.0~3.0mmol/L.
6. preparation method according to claim 3, which is characterized in that described in step 4) capture antibody-solutions concentration be
8~12 μ g mL-1；The time of the incubation is 1.5~2.5h.
7. preparation method according to claim 6, which is characterized in that further included after the incubation：The incubation is obtained
Decorative layer using PBS wash, obtain Acetamiprid antibody layer.
8. preparation method according to claim 3, which is characterized in that the concentration of MCH solution described in step 5) for 1.5~
2.5mmol/L；The time of the closing is 25~35min.
9. a kind of electrochemical sensor for including detecting electrode described in claim 1, which is characterized in that matching reagent is further included,
The matching reagent includes Acetamiprid haptens probe solution and methylene blue probe solution.
10. electrochemical sensor according to claim 9, which is characterized in that the Acetamiprid haptens probe solution is dense
It spends for 5~10 μm of ol/L.
11. electrochemical sensor according to claim 9, which is characterized in that the Acetamiprid haptens probe is to utilize
Mixed anhydride method obtains Acetamiprid haptens and amination probe conjugate, the sequence such as Seq ID of the amination probe
Shown in No.1.
12. electrochemical sensor according to claim 9, which is characterized in that the nucleotides sequence of the methylene blue probe
Row are as shown in Seq ID No.2.
13. electrochemical sensor according to claim 9, which is characterized in that the concentration of the methylene blue probe solution
For 5~15 μM.
14. application of the electrochemical sensor in Acetamiprid is detected described in claim 9~13 any one.
15. application according to claim 14, the detection comprises the following steps：
A the mixed liquor of measuring samples and haptens probe) is modified into detecting electrode surface, obtains electrode to be checked；
B methylene blue probe solution, the methylene blue probe solution and haptens probe) is added dropwise to the electrode surface to be checked
Complementation modification, obtains band signal electrode；
C) using the band signal electrode as working electrode, Ag/AgCl is reference electrode, and Pt electrodes are to electrode, carry out difference arteries and veins
Voltammetric detection is rushed, according to obtained electric signal and predetermined standard curve, obtains the content of Acetamiprid in the measuring samples.
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| CN110261451B (en) * | 2019-05-28 | 2021-11-16 | 广州钰芯传感科技有限公司 | Preparation method and application of ferrocene-labeled hapten-polyethyleneimine conjugate aiming at pesticide coumaphos molecule recognition |
| CN112378977A (en) * | 2020-11-12 | 2021-02-19 | 济南大学 | Electrochemical sensor for detecting acetamiprid |
| CN113943248A (en) * | 2021-12-21 | 2022-01-18 | 信达安检测技术(天津)有限公司 | Acetamiprid hapten, acetamiprid complete antigen, and synthesis and application thereof |
| CN113943248B (en) * | 2021-12-21 | 2022-03-08 | 信达安检测技术(天津)有限公司 | Acetamiprid hapten, acetamiprid complete antigen, and synthesis and application thereof |
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