CN105116149B - Electrochemical immunosensor for detecting pesticide azacyclotin and preparation method thereof - Google Patents
Electrochemical immunosensor for detecting pesticide azacyclotin and preparation method thereof Download PDFInfo
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- CN105116149B CN105116149B CN201510301196.2A CN201510301196A CN105116149B CN 105116149 B CN105116149 B CN 105116149B CN 201510301196 A CN201510301196 A CN 201510301196A CN 105116149 B CN105116149 B CN 105116149B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
Abstract
The invention provides an electrochemical immunosensor for detecting the pesticide azacyclotin, and a preparation method and application thereof. The electrochemical immunosensor comprises a working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode is obtained by successively immobilizing a naphthol film, a nanometer cobaltosic oxide/polyaniline composite film, glutaraldehyde and azacyclotin artificial antigen on the surface of a substrate electrode and blocking non-specific active sites with bovine serum albumin. The invention also provides the preparation method for the electrochemical immunosensor. The Co3O4 electrochemical immunosensor for detecting large-steric hindrance azacyclotin has the advantages of easy availability of raw materials, stability, firm marks, no interference in antigen-antibody reaction, simple sample treatment, fast detection speed, convenience in carrying, etc., has long shelf-life, can be stored under easily achievable storage conditions, and is applicable to on-site detection and capable of detecting a plurality of drugs at the same time.
Description
Technical field
The present invention relates to a kind of electrochemical immunosensor and preparation method thereof of detection pesticide residues is and in particular to a kind of
Detect electrochemical immunosensor of big steric hindrance pesticide azacyclotin and preparation method thereof.
Background technology
Azacyclotin (act) also known as again and seize by force, the demodicid mite stannum that goes out, sub- ring stannum etc., chemical name be 1- (thricyclohexyl stannyl)-
1- hydrogen -1,2,4- triazoles, molecular formula is c20h35n3Sn, No. cas is 41083-11-8, is the stronger broad spectrum activity of action of contace poison
Acaricide, can kill deutonymph, become demodicid mite and summer egg, the longevity of residure is longer.China's national standard gb16333-1996 regulation azacyclotin exists
MRL in fruit is 2.0mg/kg, and Codex Alimentary Commission (cac) specifies the highest residual of azacyclotin
Limit the quantity as 0.1mg/kg, the MRL of France's regulation azacyclotin is 0.20mg/kg.
At present, pesticide residue detection method mainly has instrument detection method, immunodetection etc..Wherein, instrument detection method master
Gas chromatography to be included, liquid chromatography, mass spectrography, gas chromatography mass spectrometry method and Liquid Chromatography/Mass Spectrometry etc., but typically require to sample
Carry out the pretreatment of very complicated, time and effort consuming, high cost, and instrument is costly, disposable input is larger, needs
Professional and technical personnel safeguards, is unsuitable for Site Detection.Enzyme-linked immunosorbent assay (elisa) has become present analysis now
Cutting edge technology in field, can develop many quick detection products, such as quick detection kit, quick detection test paper
Bar, immunosensor etc.；Test kit or test strips product generally are directed to a medicine, i.e. the detection of single residual, but actual raw
In product, the compounding use due to pesticide is very universal, and in agricultural product, the pesticide of residual often has multiple, single pesticide composition immunity point
Analysis method is difficult to meet actually detected needs, but is difficult in a test kit or test strips for inhomogeneous Multiple Antibodies
Upper realization detects simultaneously, and one kind is simple and the measure that is effectively improved is exactly to use biosensor.
The pesticide sensor of at present commercialization is based primarily upon the inhibitory action of ache to detect pesticide, but the life of ache current mode
The disadvantage of thing sensor is that the oxidation reaction of enzyme catalysiss product thiocholine needs higher current potential, can substantially reduce sensing
The stability of device；Simultaneously using medium, the poor selectivity of the biosensor of enzyme inhibition, in order to improve biosensor
Selectivity, be developed the immunosensor using antibody as identification receptor, nano material introduced analytical chemistry simultaneously
In research, become a study hotspot, achieved with many innovative research achievements.In recent years, immunosensor is as a kind of new
Emerging biosensor identifies the high degree of specificity of material, sensitivity and stability with it, clinical diagnosises in national economy,
Before having a wide range of applications in the research such as Industry Control, food and pharmaceutical analysiss, environmental conservation and biotechnology, biochip
Scape.At present, the domestic product that there is no the quick detections such as azacyclotin quick detection kit and immunosensor.
Content of the invention
The technical problem to be solved is to provide a kind of electrochemical immunosensor of detection pesticide azacyclotin.
Technical problem to be solved is to provide a kind of electro-chemistry immunity biography of detection pesticide azacyclotin to the present invention further
The preparation method of sensor.
The present invention further technical problem to be solved be to provide a kind of using detection pesticide azacyclotin electrochemistry exempt from
The method that epidemic disease sensor detects azacyclotin.
The present invention solves its technical problem and employed technical scheme comprise that, a kind of electro-chemistry immunity of detection pesticide azacyclotin passes
Sensor, including working electrode, reference electrode, auxiliary electrode, described working electrode fixes naphthols film successively by basal electrode surface,
Nano-cobaltic-cobaltous oxide/polyaniline composite film (nano-co3o4/ pan), glutaraldehyde, azacyclotin artificial antigen, and with Ox blood serum egg
(bsa) closing nonspecific activity site in vain.
Further, described basal electrode is ito glass electrode, and described reference electrode is ag/agcl electrode, described auxiliary electricity
Extremely platinum electrode；Described azacyclotin artificial antigen is with plictran and 5- sulfydryl -3- amino-1,2,4-triazol as initial feed
First synthesize azacyclotin hapten, then be coupled, with bovine serum albumin, the azacyclotin artificial immunity antigen being prepared from.
The present invention solves its technical problem further and employed technical scheme comprise that, a kind of electrochemistry of detection pesticide azacyclotin
The preparation method of immunosensor, first in the basal electrode surface Deca naphthol solution of pretreatment, dries；Then Deca nanometer
Cobalto-cobaltic oxide/polyaniline composite material, dries；Distinguish the activation of Deca glutaraldehyde solution, cleaning again, nitrogen dries up；Then drip
Plus azacyclotin artificial antigen, incubation, overnight, and cleaning, dry, last Deca bovine serum albumin buffer blind.
Further, the electrochemical immunosensor preparation method of described pesticide azacyclotin comprises the following steps:
(1) preparation of nano-cobaltic-cobaltous oxide/polyaniline composite material: by 0.3～0.4g cetyl trimethyl bromination
Ammonium is dissolved in 45～55ml distilled water, add 0.2～0.4g cobaltosic oxide nano granule, ultrasonic disperse, stirring 4.5～
Add 150～200 μ l aniline, continuously stirred 0.5～1.5h after 5.5h, add the Fructus Citri Limoniae of 1.5 times of amount being equivalent to aniline material
Acid, stirs 9.5～10.5h, adds the ammonium sulfate with the amount equivalent of aniline material, stops anti-after continuing stirring 11.5～12.5h
Should, reacted product is carried out sucking filtration, retains filter cake, deionized water and dehydrated alcohol repeatedly wash to filtrate no successively
Color, filter cake is placed in 60 DEG C of dryings in vacuum drying oven and obtains final product nano-co within 4 hours3o4/ pan composite, standby；
(2) synthesis of azacyclotin artificial antigen: the plictran weighing 193g～195g is dissolved with 24～26ml methanol,
Then slowly it is added drop-wise to the 5- sulfydryl -3- amino -1,2,4- three of 80 DEG C of 58g～59g 48～52ml～85 DEG C of hot water dissolvings
(5- sulfydryl -3- amino -1,2,4- triazoles are all insoluble in cold water and methanol because of it, and dissolubility is very in the hot water for nitrogen azoles
Height is so pyroreaction is selected in reaction), there are a large amount of white precipitates to generate after stirring 1～2h, after white precipitate is filtered, use boiled water
Washing 3～4 times, carries out recrystallization with 95% ethanol after being dried, obtains final product azacyclotin hapten act-h1；By 8mg～10mg triazole
Stannum hapten 2.5ml～3.5ml dmf dissolves, and the concussion that is vortexed is lower slowly to add 9ml～11ml distilled water, is subsequently adding
38mg～42mg bsa, until completely dissolved, Deca 30～50 μ l glutaraldehyde solution, it is stirred overnight at 4 DEG C, solution is by milky
It is changed into slightly yellow；Dialysed 45h～48h with normal saline solution at 4 DEG C, every 3～4h changes a water；Obtain azacyclotin immunity anti-
Former act-h1-bsa, with normal saline dilution to 1mg/ml～1.5mg/ml, -20 DEG C of preservations；
(3) pretreatment of basal electrode: basal electrode is cut into 4 × 1cm, EtOH Sonicate cleans, dries, will with insulating cement
Probe area is sealed to 1cm2, then use each ultrasonic cleaning 5～10min in liquid detergent, acetone, ethanol, deionized water successively, nitrogen blows
Dry, standby；
(4) surface modification of basal electrode: first in basal electrode surface Deca 50～60 μ l that step (3) is standby
0.75% naphthol solution, dries；Then standby nanometer cobalt-four oxygen in the step (1) of Deca 100～120 μ l 1:400 dilution
Change three cobalts/polyaniline composite material, dry；Followed by Deca 100～120 μ l 0.25% glutaraldehyde solution, under room temperature, activate 2
After～3h, rinsed well with water and dried up with nitrogen, then the azacyclotin of Deca 100～120 μ l 1:1600 dilution manually resists
Former, it is incubated 3～4h at 37 DEG C, overnight, with 0.01mol/l, the pbs wash buffer of ph7.2～7.6 is clean, dries；
(5) closing in nonspecific activity site: by basal electrode Deca 100～120 μ l 5mg/ml after surface modification
Bovine serum albumin buffer is put in 37 DEG C of constant temperature and closes 5～6h, takes out and uses 0.01mol/l, the pbs buffer punching of ph7.2～7.6
Wash clean, obtains final product electrochemical immunosensor.
The present invention solves its technical problem further and employed technical scheme comprise that, a kind of electricity using detection pesticide azacyclotin
The method that chemo-immunity sensor detects azacyclotin, comprises the following steps:
(1) modify working electrode: first will be molten for the working electrode Deca azacyclotin monoclonal antibody of electrochemical immunosensor
Liquid and the mixed liquor of azacyclotin standard substance composition；It is put in incubation 50～70min at 37 DEG C, take out with 0.008～0.012mol.l- 1The pbs buffer of ph 7.2～7.6 is rinsed well repeatedly, the igg-hrp antibody of subsequent Deca nanometer silver labelling, incubates at 37 DEG C
Educate 30～45min, take out with 0.008～0.012mol.l-1The pbs buffer of ph 7.2～7.6 is rinsed well repeatedly, standby；
(2) set up working curve: by the working electrode after modifying in step (1) and reference electrode, auxiliary electrode insertion body
Long-pending ratio is 1: 1: 1, and the potassium chloride of ratio satisfaction 0.1: 5: 5 of the amount of material, potassium ferrocyanide and the potassium ferricyanide are mixed into ph 7.4
The three-electrode system being assembled in buffer, is equivalent to azacyclotin monoclonal antibody solution and azacyclotin being stirred continuously lower addition
The hydrogen peroxide of 0.1 times of the 0.4mol/l～0.5mol/l of mixed liquor of standard substance composition, measures variable concentrations azacyclotin standard
The current value of product, the functional relationship between azacyclotin standard concentration and corresponding current value, as electrochemical immunosensor
Working curve；
(3) measure azacyclotin: the working electrode after being modified using step (1) is circulated in the range of -0.8～0.8v
Voltammetric scan, measures the current value in azacyclotin solution to be measured, the working curve then obtaining according to step (2), you can calculate
Go out the concentration of azacyclotin in solution to be measured.
Further, in step (1), described azacyclotin monoclonal antibody is conventionally prepared by azacyclotin artificial antigen
Form；The igg-hrp antibody of described nanometer silver labelling is by the ph to 8～10 adjusting agnrs solution with alkali liquor, then by volume
Igg-hrp and 1mg/mlhrp mixed liquor than 1:1 is added dropwise in agnrs solution, after stirring 10～20min, 8000～
It is centrifuged 15～25min under 12000rpm, then carry out 2～5 washings with the pbs of 0.01mol/l ph 7.4, finally by gained
Agnrs-igg-hrp is dispersed in 1ml pbs buffer and is prepared from.
Further, in step (3), described cyclic voltammetry scan speed is 50mv/s.
The electrochemical immunosensor of the detection pesticide azacyclotin of the present invention is by immunochemical specificity and enzymology
Susceptiveness combines together, and using Cobalto-cobaltic oxide as nano material, this raw material easily obtains, and has stable, labelling firmly, do not disturb
Antigen antibody reaction, sample treatment is simple, detection speed is fast, the advantages of carry convenient, and co3o4Electrochemistry immuno-sensing
It is oxidized that the biomembrane of device is placed in the air to be not easy, long shelf-life, and storage requirement easily reaches, and sensor technology is with large-scale instrument
Device detection technique ratio, more suitable for Site Detection, detects multi-medicament simultaneously, is expected to the high throughput testing technology with biochip
In conjunction with thus create the new instrument that quick, convenient, high efficiency, many residuals detect simultaneously, detection technique being epoch-making
Breakthrough.
Brief description
Fig. 1 is azacyclotin hapten synthesis mass spectrum.
Fig. 2 is azacyclotin artificial antigen's ultraviolet full wavelength scanner figure.
Fig. 3 is the canonical plotting of azacyclotin monoclonal antibody.
Fig. 4 is azacyclotin detection principle diagram of the present invention.
Fig. 5 is azacyclotin electrochemical immunosensor reaction condition optimization figure.
Fig. 6 is azacyclotin electrochemical immunosensor working curve diagram.
Fig. 7 is the preparation flow figure of immunoelectrode.
Fig. 8 is cyclic voltammetric schematic diagram.
Fig. 9 is cyclic voltammetry proof diagram [wherein, (a) bare electrode；B electrode that () naphthols is modified；(c)nano-co3o4/
The electrode that pan modifies；The electrode of (d) Modified antigen；The electrode of (e) azacyclotin antibody modification；Each modified electrode in volume ratio is
The 0.1mol/l potassium chloride of 1:1:1,5mmol/l potassium ferrocyanide and the 5mmol/l potassium ferricyanide are mixed and made into 1/15mol/l, ph
Cyclic voltammetry scan (cv) figure in 7.4 buffer].
Figure 10 is that [wherein, (a) is the type of attachment of three kinds of electrodes to triangular scan of potential method experimental line of the present invention；(b)
It is triangular scan of potential experimental line, realize by g external control circuit is applied with the triangular scanning required for cyclic voltammetry
Voltage；Working electrode (we), reference electrode (re) and auxiliary electrode (ce)；Reference electrode is used for pinpointing a zero point, and electric current flows through
Working electrode and auxiliary electrode, working electrode and reference electrode constitute an obstructed or substantially few system being energized, using reference
The stability of electrode potential is measuring the electrode potential of working electrode；Working electrode and auxiliary electrode constitute a body being energized
System, for measuring the electric current that working electrode passes through.Using three electrode measurement systems, come the point position of research work electrode and electricity simultaneously
The relation of stream].
Figure 11 is sensor assembling figure.Wherein, (a) is overall layout chart；B () is operational platform design figure；C () is detection
Pond；D () is detection process.B () diagram is released: working method: by tab, selects its working method [to include: both positive and negative polarity turns
Change, load selects, break-make road, the option such as oneself setting].
Viewing area: mainly show electric current (range 0～100 μ a), voltage (range 0～10v)
Correction zone: by blank value come correcting current, the data display of voltmeter.(play the effect in school zero, deduction is blank
Value).
Function indicates area: whether lighting of display lamp, to show whether the working condition of various pieces is normal.
Switch: being turned on and off for instrument.
Electrode plug wire area: manual operation insertion associated connections post, to choose whether using triple electrode circuit or two electrodes
Circuit.
Compensate: by instrument itself by adjusting the resistance to control measuring circuit (primarily to reaching measurement of correlation
The requirement of precision.
Specific embodiment
With reference to embodiment, the present invention is illustrated further.
First, reagent
Naphthols (dupont company of the U.S.)；co3o4(Tianjin great Mao chemical reagent factory)；(Jiangsu prosperity chemical industry is limited for aniline
Company)；nano-co3o4/ pan (laboratory oneself synthesis)；Azacyclotin artificial antigen (laboratory oneself synthesis)；Bovine serum albumin
(bsa) (Shanghai Sheng Gong biological engineering company limited)；Azacyclotin monoclonal antibody (laboratory oneself synthesis)；Variable concentrations triazole
Stannum standard substance (Inst. of Environment Protection ＆ Scientific Research Monitor, Ministry of Agric (Tianjin))；The sheep anti mouse igg antibody of hrp labelling
(southernbiotech company)
2nd, instrumentation and testing system
Reference electrode: ag/agcl electrode, electrode potential is 0-0.8v
Electrolyte: 0.1mol/l kcl+5mmol/l k4[fe(cn)6]+5mmol/l k3[fe(cn)6Embodiment 1: detection
The preparation method of the electrochemical immunosensor of pesticide azacyclotin
1st, the synthesis of azacyclotin artificial antigen
(1) the haptenic synthesis of azacyclotin: the plictran weighing 193g～195g is dissolved with 24～26ml methanol, so
Slowly it is added drop-wise to 5- sulfydryl -3- amino -1,2,4- three nitrogen of 80 DEG C of 58g～59g 48～52ml～85 DEG C of hot water dissolvings afterwards
(5- sulfydryl -3- amino -1,2,4- triazoles are all insoluble in cold water and methanol because of it, and dissolubility is very high in the hot water for azoles
So pyroreaction is selected in reaction), there are a large amount of white precipitates to generate after stirring 1～2h, washed with boiled water after white precipitate is filtered
Wash 3～4 times, carry out recrystallization with 95% ethanol after being dried, obtain final product azacyclotin hapten act-h1；Reaction equation is as follows:
(2) synthesis of azacyclotin artificial antigen: will be molten with 2.5ml～3.5ml dmf for 8mg～10mg azacyclotin hapten
Solution, the concussion that is vortexed is lower slowly to add 9ml～11ml distilled water, is subsequently adding 38mg～42mg bsa, until completely dissolved, drips
Plus 30～50 μ l glutaraldehyde solutions, be stirred overnight at 4 DEG C, solution is changed into slightly yellow from milky；Normal saline solution is used at 4 DEG C
Dialysis 45h～48h, every 3～4h change a water；Obtain azacyclotin immunizing antigen act-h1-bsa, with normal saline dilution extremely
1mg/ml～1.5mg/ml, -20 DEG C of preservations；
Mass Spectrometric Identification is carried out to the azacyclotin hapten of synthesis, determines its structure, as shown in Figure 1；Azacyclotin to synthesis
Antigen carries out ultraviolet full wavelength scanner, as shown in Figure 2.As shown in Figure 1, esi-ms (+) m/z:446.6 [m+h]+it was demonstrated that triazole
The success of stannum hapten synthesis；When its uv absorption will be strengthened after protein molecule on the hapten conjugation contain structure, simultaneously
Be likely to because destroy conjugation sites around molecular structure and weakened part uv absorption, as shown in Figure 2, act-h1-bsa with
The UV scanning figure of act-h1 and bsa all there occurs skew, illustrates that azacyclotin artificial immunity antigen synthesizes successfully.
2、nano-co3o4The preparation of/pan composite: 0.3～0.4g cetyl trimethylammonium bromide is dissolved in 45
In～55ml distilled water, add the co of 0.2～0.4g3o4Nano-particle, ultrasonic disperse, stirring 4.5～5.5h after add 150～
200 μ l aniline, continuously stirred 0.5～1.5h, add be equivalent to aniline material 1.5 times of amount citric acid, stirring 9.5～
10.5h, adds the ammonium sulfate with the amount equivalent of aniline material, continues stopped reaction after stirring 11.5～12.5h, will be reacted
Product carries out sucking filtration, retains filter cake, and deionized water and dehydrated alcohol repeatedly wash colourless to filtrate successively, and filter cake is placed in very
In empty baking oven, 60 DEG C of dryings obtain final product nano-co in 4 hours3o4/ pan composite, standby；
3rd, the preparation of electrochemical immunosensor:
(1) pretreatment of basal electrode: basal electrode is cut into 4 × 1cm, EtOH Sonicate cleans, dries, will with insulating cement
Probe area is sealed to 1cm2, then use each ultrasonic cleaning 5～10min in liquid detergent, acetone, ethanol, deionized water successively, nitrogen blows
Dry, standby；
(2) surface modification of basal electrode: first in basal electrode surface Deca 50～60 μ l that step (1) is standby
0.75% naphthol solution, is placed in drying baker and dries；Then standby nanometer cobalt-four oxygen of Deca 100～120 μ l 1:500 dilution
Change three cobalts/polyaniline composite material, be again placed in drying baker and dry；Molten followed by Deca 100～120 μ l, 0.25% glutaraldehyde
Liquid, activates to it, after activation 2～3h under room temperature, is rinsed with water and dries up twice and with nitrogen；It is incubated 3～4h at 37 DEG C,
Overnight, with 0.01mol/l, the pbs wash buffer of ph7.2～7.6 five times, dry；
(3) closing in nonspecific activity site: by basal electrode Deca 100～120 μ l 5mg/ml after surface modification
Bovine serum albumin buffer, to eliminate non-specific adsorption, is put in 37 DEG C of constant temperature and closes 5～6h, take out and use 0.01mol/l,
The pbs wash buffer of ph7.2～7.6 is clean, obtains final product electrochemical immunosensor, finally will prepare sensor probe and protect
Exist stand-by, as shown in Figure 7 in 4 DEG C of environment.
Embodiment 2: the method detecting azacyclotin using electrochemical immunosensor
1st, the preparation of azacyclotin monoclonal antibody: by azacyclotin antigen act-h1-bsa immunity 6-8 week old spf level female
Balb/c mice, animal immune process is as follows: takes the good azacyclotin antigen of 100 μ l subpackages and equal-volume freundShi Freund's complete adjuvant
Emulsion is emulsified into using the mutual pushing manipulation of syringe, emulsifying, to dripping, drops to and can keep in water completely not disperseing, becomes drop-wise to bubble through the water column,
I.e. emulsifying is complete；Using dorsal sc, abdominal cavity and foot pad multi-point injection immunity 0.2ml～0.3ml/ only (containing antigen 1 00 μ g～
150 μ g), after 3 weeks, same dose artificial's antigen and equal-volume freundShi Freund's incomplete adjuvant are mixed into Emulsion, same dose and side
Method injects booster immunization；Then, every 2 weeks booster immunizations 1 time, and dosage is identical, immunity 4 times altogether, and Indirect Elisa detects blood
Clear antibody titer, potency is antiserum a490nm/ comparison a490nmSero-fast maximum dilution multiple when >=2.1, selects potency high
Person is used for the preparation of monoclonal antibody；Take reinforced immune mouse boosting cell and sp2/0 myeloma cell fusion (cell before 3d
Number ratio is about 6 1), positive hybridoma cell is screened using indirect elisa and indirect competition elisa, and adopts micro- gram
Grand method carries out sub-clone to positive cell, can build strain when the positives rate of Tissue Culture Plate is 100%, -70 DEG C of refrigerators are frozen；
By the hybridoma centrifugation of culture, supernatant discarded, hybridoma is suspended with serum-free medium, and cell number is adjusted to (1
～2) × 106Individual/ml, every mouse peritoneal injection 0.5ml；Extract ascites after 2 weeks, Dan Ke is carried out using caprylic acid-ammonium
The precipitation separation of grand antibody, spectrophotometry antibody concentration, such as Fig. 3.
2nd, prepare the igg-hrp antibody of nanometer silver labelling: adjust the ph to 8～10 of agnrs solution first with alkali liquor, then
Igg-hrp the and 1mg/mlhrp mixed liquor of volume ratio 1:1 is added dropwise in agnrs solution, after stirring 10～20min, 8000
It is centrifuged 15～25min under～12000rpm, then carry out 2～5 washings with the pbs of 0.01mol/l ph 7.4, finally by institute
Obtain agnrs-igg-hrp to be dispersed in 1ml pbs buffer, preserve at 4 DEG C, standby.
3rd, detect azacyclotin: this experiment adopts indirect competition immunoassay, comprises the following steps:
(1) first by the working electrode Deca azacyclotin monoclonal antibody solution of electrochemical immunosensor and azacyclotin standard
The mixed liquor of product composition；It is put in incubation 60min at 37 DEG C, take out and use 0.01mol.l-1The pbs buffer of ph 7.4 rinses repeatedly
Totally, the igg-hrp antibody of subsequent Deca nanometer silver labelling, is incubated 45min at 37 DEG C, takes out and uses 0.01mol.l-1Ph's 7.4
Pbs buffer is rinsed well repeatedly, standby；
(2) set up working curve: the working electrode after modifying in step (1) is inserted respectively with reference electrode, auxiliary electrode
Entering volume ratio is 1: 1: 1, and the potassium chloride of ratio satisfaction 0.1: 5: 5 of the amount of material, potassium ferrocyanide and the potassium ferricyanide are mixed into ph
The three-electrode system being assembled in 7.4 buffer, is equivalent to azacyclotin monoclonal antibody solution and three being stirred continuously lower addition
The hydrogen peroxide of 0.1 times of the 0.4mol/l～0.5mol/l of mixed liquor of azoles stannum standard substance composition, measures variable concentrations azacyclotin
The current value of standard substance, the functional relationship between azacyclotin standard concentration and corresponding current value, as electro-chemistry immunity pass
Sense device working curve, as shown in Figure 6；
(3) measure azacyclotin: the working electrode after being modified using step (1) is circulated in the range of -0.8～0.8v
Voltammetric scan, sweep speed is 50mv/s, measures the current value in azacyclotin solution to be measured, is then obtained according to step (2)
Working curve, you can calculate the concentration of azacyclotin in solution to be measured, such as Fig. 4, shown in Fig. 9, Figure 10.
Embodiment 3: the optimization of azacyclotin electrochemical immunosensor reaction condition
In immunosensor, the performance condition in immunologic process being optimized for playing sensor is extremely important,
For the thinner ratio of fixing Antigen buffer, the thinner ratio with the antibodies buffer of antigen binding, with hapten binding antibody
Amount, the time with immunoreation, temperature etc. all has a great impact to the accuracy of sensor and sensitivity.This experiment is focused on
To naphthol solution concentration, nano-co3o4/ pan solution thinner ratio, antigen-antibody thinner ratio, the ph of potassium ferricyanide buffer dilution
Value is optimized, three parallel laboratory tests of same Setup Experiments in optimization process, and data takes three to test to obtain meansigma methodss, experiment
Result is as shown in figure 5, this experiment determines azacyclotin: 0.75% as naphthols in immunosensor optium concentration；1:400 is
nano-co3o4The optimal thinner ratio of/pan solution；1:1600 is as the fixed concentration thinner ratio of artificial coupled antigen；1:400 conduct
The optimal thinner ratio of azacyclotin antibody；The pbs (1/15mol/l) of ph=7.4 is as Matrix Solution.
Embodiment 4: the standard curve (working curve) of azacyclotin electrochemical immunosensor and detection limit
Under the optimum experimental condition obtaining after optimization, using cyclic voltammetry variable concentrations (10ng/ml, 20ng/
Ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml and 100ng/ml) azacyclotin
Standard substance, draw out standard curve, and result is shown in Fig. 6.Result shows, the linear detection range of this sensor be 0.05ng/ml～
100ng/ml, equation of linear regression is y=-0.5267x+54.667, r2=0.9932, lowest detection is limited to 0.05ng/ml
(0.05ug/kg).
Embodiment 5: the performance evaluation of azacyclotin electrochemical immunosensor
1st, the repeatability of azacyclotin electrochemical immunosensor
Take fine horse Fructus Jujubae and each 6 parts of grey Fructus Jujubae sample (commercially available), 110 DEG C～120 DEG C drying, pulverize, be dissolved in 95% ethanol
In, prepared sample solution is detected with azacyclotin electrochemical immunosensor, every part of sample is repeated 6 times, result such as table 1
Shown, as shown in Table 1, the repeatability of every part of sample is all fine.
Table 1 is the repeatability result of azacyclotin electrochemical immunosensor
2nd, the stability of azacyclotin electrochemical immunosensor
Take fine horse Fructus Jujubae and each 6 parts of grey Fructus Jujubae sample (commercially available), 110 DEG C～120 DEG C drying, pulverize, be dissolved in 95% ethanol
In, prepared sample solution is detected with azacyclotin electrochemical immunosensor, daily detection 1 time, detect 6 days, result
As shown in table 2, as shown in Table 2, within 6 days, current-responsive value changes are little, and this sensor has higher stability.
Table 2 is the stability result of azacyclotin electrochemical immunosensor
3rd, the response rate experiment of azacyclotin electrochemical immunosensor
Take fine horse Fructus Jujubae and grey two kinds of samples (commercially available) of Fructus Jujubae, 110 DEG C～120 DEG C drying, pulverize, be dissolved in 95% ethanol
In, azacyclotin standard substance are dissolved with 95% ethanol, press 10 μ g/g respectively, and 20 μ g/g and 50 μ g/g concentration add triazole in sample
Stannum standard sample prepares mark-on sample, every kind of sample parallel assay three times, the results are shown in Table 3；Meanwhile, using high performance liquid chromatography
Measure mark-on sample as control experiment, result shows that the method accuracy is high, can be used for the analysis of azacyclotin in actual sample.
Table 3 is the response rate experimental result of azacyclotin electrochemical immunosensor
4th, electrochemical properties experimental result during electrode modification
With electrochemical properties in self assembling process for the cyclic voltammetry Electrode, see Fig. 7, Fig. 8 and Fig. 9.Can by Fig. 9
Know, when naphthols film modified to electrode on after, the current value of cyclic voltammetry curve reduces, and illustrates that naphthols film hinders the biography of electronics
Pass；Work as nano-co3o4After/pan and naphthols film there occurs ion exchange, oxidoreduction in potassium ferricyanide solution for the modified electrode
Peak current increases, and the nano-co in naphthols film is described3o4/ pan can transmit electronics effectively；When electrode modifies azacyclotin again
After antigen-bsa, redox peaks decline compared with polyaniline reduction peak, and this is because protein molecular is adsorbed onto on electrode thus hindering
Electric transmission.Particularly after azacyclotin antibody and azacyclotin antigen-bsa specificity combine, the Ag-Ab of generation is combined
Thing blocks the more aperture passage in modified electrode surface, increased resistance when by film so that response current further under
Fall.Therefore, can detect that the poisonous and harmful substances such as the azacyclotin in food whether there is with this electrochemical immunosensor.
5th, the detection method of azacyclotin electrochemical immunosensor
Coordination electrode potential, with different speed, is repeatedly scanned with triangular waveform one or many, potential range in time
It is to make different reduction and oxidation reaction can alternately occur on electrode, and record current-potential curve (current-responsive curve),
One linear ramp is applied on electrode, with constant pace of change scanning, when reaching the termination current potential of certain setting, more instead
To the take-off potential being returned to a certain setting.According to cyclic voltammetry curve in figure peak current ip, peak potential and peak electric potential difference δ and
Relation between sweep speed is it can be determined that the reversibility of electrode reaction.When electrode reaction completely reversibility, at 25 DEG C, this
The quantitative expression of a little parameters has:
(1) ipc=2.69 × 105n3/2d01/2υ1/2(a·cm-2), that is, ipc is just become with the bulk concentration of reactant o
Ratio is directly proportional to υ 1/2.Wherein: do is the diffusion coefficient (cm of o2/ s), c is the bulk concentration of o, and υ is sweep speed (v/s).
That is: react with antibody with testing sample in the present invention, lead to amount of antibody to change thus immune electric current generation is above-mentioned
Change.
(2) │ ipc │=│ ipa │, i.e. │ ipc/ipa │=1, and unrelated with electrical potential scan rate υ.
(3)And With scanning speed υ and unrelated, it is certain value.
Wherein (2) and (3) are the key characters of the reversible system cyclic voltammetry curve that diffusion mass transfer step controls, and are detections
The most useful criterion of reversible electrode reaction.
May determine that the degree of reversibility of electrode reaction according to curve shape, intermediate, phase boundary absorption or New phase formation can
Energy property, and the property of coupled chemical reactions etc..It is commonly used to measuring electrode response parameter, judge its rate-determining steps and reaction machine
Reason, and observe in the range of whole potential scan, which reaction, and its property is how.
Claims (4)
1. a kind of detection pesticide azacyclotin electrochemical immunosensor it is characterised in that include working electrode, reference electrode,
Auxiliary electrode, described working electrode fixes naphthols film successively by basal electrode surface, and nano-cobaltic-cobaltous oxide/polyaniline is combined
Film, glutaraldehyde, azacyclotin artificial antigen, and nonspecific activity site is closed with bovine serum albumin, described basal electrode is ito
Glass electrode, described reference electrode is ag/agcl electrode, and described auxiliary electrode is platinum electrode；Described azacyclotin artificial antigen is
With plictran and 5- sulfydryl -3- amino -1,2,4- triazoles first synthesize azacyclotin hapten for initial feed, then with Ox blood serum egg
It is coupled the azacyclotin artificial antigen being prepared from vain.
2. a kind of preparation method of the electrochemical immunosensor of detection pesticide azacyclotin as claimed in claim 1, exists first
The basal electrode surface Deca naphthol solution of pretreatment, dries；Then Deca nano-cobaltic-cobaltous oxide/polyaniline composite material,
Dry；Deca glutaraldehyde solution activation again, cleaning, nitrogen dries up；Then Deca azacyclotin artificial antigen, incubation, overnight, clearly
Wash, dry, Deca bovine serum albumin buffer blind.
3. the preparation method of the electrochemical immunosensor of detection pesticide azacyclotin according to claim 2, its feature exists
In comprising the following steps:
(1) preparation of nano-cobaltic-cobaltous oxide/polyaniline composite material: will be molten for 0.3～0.4g cetyl trimethylammonium bromide
Solution, in 45～55ml distilled water, adds the cobaltosic oxide nano granule of 0.2～0.4g, ultrasonic disperse, stirring 4.5～5.5h
Add 150～200 μ l aniline, continuously stirred 0.5～1.5h afterwards, add the citric acid of 1.5 times of amount being equivalent to aniline material, stir
Mix 9.5～10.5h, add the ammonium sulfate with the amount equivalent of aniline material, continue stopped reaction after stirring 11.5～12.5h, will
Reacted product carries out sucking filtration, retains filter cake, and deionized water and dehydrated alcohol repeatedly wash colourless to filtrate successively, will filter
Cake is placed in 60 DEG C of dryings in vacuum drying oven and obtains final product within 4 hours nano-cobaltic-cobaltous oxide/polyaniline composite material, standby；
(2) synthesis of azacyclotin artificial antigen: the plictran weighing 193g～195g is dissolved with 24～26ml methanol, then
Slowly it is added drop-wise to 5- sulfydryl -3- amino -1 of 80 DEG C of 58g～59g 48～52ml～85 DEG C of hot water dissolvings, 2,4- triazoles
In, there are a large amount of white precipitates to generate after stirring 1～2h, washed with boiled water 3～4 times after white precipitate is filtered, use after being dried
95% ethanol carries out recrystallization, obtains final product azacyclotin hapten act-h1；By 8mg～10 mg azacyclotin hapten 2.5ml～
3.5ml dmf dissolves, and the concussion that is vortexed is lower slowly to add 9ml～11ml distilled water, is subsequently adding 38mg～42mg bsa, has treated
After CL, Deca 30～50 μ l glutaraldehyde solution, it is stirred overnight at 4 DEG C, solution is changed into slightly yellow from milky；Use at 4 DEG C
Normal saline solution dialysis 45h～48h, every 3～4h change a not good liquor；Obtain azacyclotin artificial antigen act-h1-bsa, with life
Reason saline is diluted to 1mg/ml～1.5mg/ml, -20 DEG C of preservations；
(3) pretreatment of basal electrode: basal electrode is cut into 4 × 1cm, EtOH Sonicate cleans, dries, will be popped one's head in insulating cement
Area is sealed to 1cm2, respectively it is cleaned by ultrasonic 5～10min with liquid detergent, acetone, ethanol, deionized water successively, nitrogen dries up, standby
With；
(4) surface modification of basal electrode: first in basal electrode surface Deca 50～60 μ l 0.75% that step (3) is standby
Naphthol solution, dries；Then standby nano-cobaltic-cobaltous oxide in the step (1) of Deca 100～120 μ l 1: 400 dilution/poly-
Aniline composite, dries；Followed by Deca 100～120 μ l 0.25% glutaraldehyde solution, after activation 2～3h under room temperature, use
Water is rinsed well and is dried up with nitrogen, then the azacyclotin artificial antigen of Deca 100～120 μ l1: 1600 dilution, at 37 DEG C
Incubation 3～4h, overnight, with 0.01mol/l, the pbs wash buffer of ph7.2～7.6 is clean, dries；
(5) closing in nonspecific activity site: by basal electrode Deca 100～120 μ l 5mg/ml Sanguis Bovis seu Bubali after surface modification
Albumin buffer is put in 37 DEG C of constant temperature and closes 5～6h, takes out and uses 0.01mol/l, and the pbs wash buffer of ph7.2～7.6 is done
Only, obtain final product electrochemical immunosensor.
4. the electrochemical immunosensor described in a kind of utilization claim 1 detects the method for azacyclotin it is characterised in that including
Following steps:
(1) modify working electrode: first by the working electrode Deca azacyclotin monoclonal antibody solution of electrochemical immunosensor and
Azacyclotin standard substance or the mixed liquor of solution composition to be measured；It is put at 37 DEG C incubation 50～70min, take out with 0.008～
0.012mol·l-1The pbs buffer of ph 7.2～7.6 is rinsed well repeatedly, and the igg-hrp of subsequent Deca nanometer silver labelling resists
Body, is incubated 30～45min at 37 DEG C, takes out with 0.008～0.012mol l-1The pbs buffer of ph 7.2～7.6 rushes repeatedly
Wash clean, standby；
(2) set up working curve: the working electrode after modifying in step (1) is inserted volume ratio with reference electrode, auxiliary electrode
For 1: 1: 1, the ratio of the amount of material meets the ph's 7.4 that 0.1: 5: 5 potassium chloride, potassium ferrocyanide and the potassium ferricyanide are mixed into
It is assembled into three-electrode system in buffer, be equivalent to azacyclotin monoclonal antibody solution and azacyclotin mark being stirred continuously lower addition
The hydrogen peroxide of 0.1 times of the 0.4mol/l～0.5mol/l of mixed liquor of quasi- product composition, measures variable concentrations azacyclotin standard substance
Current value, the functional relationship between azacyclotin standard concentration and corresponding current value, as electrochemical immunosensor work
Make curve；
(3) measure azacyclotin: the working electrode after modifying using step (1) is circulated volt-ampere in the range of -0.8～0.8v
Scanning, measures the current value in azacyclotin solution to be measured, the working curve then obtaining, you can calculate and treat according to step (2)
Survey the concentration of azacyclotin in solution.
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