CN104931692A - Electrochemical immunosensor for chlorpyrifos detection, and preparation method and application thereof - Google Patents

Electrochemical immunosensor for chlorpyrifos detection, and preparation method and application thereof Download PDF

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Publication number
CN104931692A
CN104931692A CN201510307269.9A CN201510307269A CN104931692A CN 104931692 A CN104931692 A CN 104931692A CN 201510307269 A CN201510307269 A CN 201510307269A CN 104931692 A CN104931692 A CN 104931692A
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electrode
chlopyrifos
electrochemical immunosensor
chlorpyrifos
detection
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王伟华
韩占江
向延菊
田木星
宋周林
马政彪
李春燕
赵海蓉
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Tarim University
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Tarim University
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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction

Abstract

The invention provides an electrochemical immunosensor for chlorpyrifos detection, 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 prepared by sequentially fixing a naphthol film, a nano cobalt oxide/polyaniline composite film and a chlorpyrifos artificial antigen on the surface of a substrate electrode and using bovine serum albumin to block non-specific active sites. The invention further discloses the preparation method and application of the electrochemical immunosensor. The electrochemical immunosensor for chlorpyrifos detection has the advantages of being readily available in raw materials, stable, firm in marking, free of interference on antigen-antibody reaction, simple in sample treatment, high in detection speed, convenient to carry and the like; in addition, the Co3O4 biofilm of the electrochemical immunosensor is unlikely to be oxidized in air, so that the electrochemical immunosensor is long in shelf life, the storage condition can be met easily, and the electrochemical immunosensor is suitable for field detection as well as simultaneous detection of various pesticides.

Description

A kind of electrochemical immunosensor detecting chlopyrifos and preparation method thereof and application
Technical field
The present invention relates to a kind of electrochemical immunosensor detecting residues of pesticides and preparation method thereof and application, be specifically related to a kind ofly detect electrochemical immunosensor of agricultural chemicals chlopyrifos and preparation method thereof and application.
Background technology
Chlopyrifos [chlorpyrifos, O, O-diethyl-O-(3,5,6-trichloro-2-pyridyl) thiophosphate] be the efficient pesticides of widespread use in a kind of world wide, can be used on the control of insect of grain, veterinary antibiotics and industrial crops, because chlopyrifos is widely used in agricultural production, the residue problem caused in grain, environmental sample etc. is existing to be reported, constitutes potential threat to the health of people.At present, gas chromatography-mass spectrum and Liquid Chromatography-Tandem Mass Spectrometry and enzyme linked immune assay to the detection main method of medicament residue, these detection methods must have high-grade detecting instrument and sample pre-treatments is complicated, although enzyme linked immunosorbent detection technology can accelerate the method detected, but testing cost is higher, detect still need will be higher requirement, cannot realize a large amount of sample rapid field detect.And electrochemical immunosensor has sensitive, quick, high specificity, to the advantage of sample without the need to special processing, therefore, the electrochemical immunosensor in recent years for agricultural chemicals chlopyrifos residue has had more research application, Fe 3o 4the biological membrane of electrochemical immunosensor is put easily oxidized in atmosphere, and the shelf-life is short, and storage requirement is not easy to reach, such as prior art CN201410142965.4 discloses a kind of preparation method detecting the immunosensor of chlopyrifos, passing through cleaning, the naked glassy carbon electrode surface of activation and performance test is dripped and is coated with nickel aluminum hydrotalcite-Graphene complex liquid, then modify by ghost nm of gold, electrode again after modification fixes Dursban monoclonal antibody, finally close with bovine serum albumin, obtain the immunosensor detecting chlopyrifos, immunosensor prepared by this invention has highly sensitive, cost is low, detection speed is fast, good stability, and the feature that sample recovery rate is high, be applicable to the quick detection of fruits and vegetables Chlorpyrifos Residue amount,
Summary of the invention
Technical matters to be solved by this invention is, provides a kind of electrochemical immunosensor detecting chlopyrifos.
The further technical matters to be solved of the present invention provides a kind of preparation method detecting the electrochemical immunosensor of chlopyrifos.
The further technical matters to be solved of the present invention is, provides a kind of method utilizing the electrochemical immunosensor detecting chlopyrifos to detect chlopyrifos.
The technical scheme that the present invention solves the employing of its technical matters is, a kind of electrochemical immunosensor detecting chlopyrifos, comprise working electrode, contrast electrode, auxiliary electrode, described working electrode fixes naphthols film successively by basal electrode surface, nano-cobaltic-cobaltous oxide/polyaniline composite film (nano-Co 3o 4/ PAN), chlorpyrifos artificial antigen, and close nonspecific activity site with bovine serum albumin (BSA).
Further, described basal electrode is ito glass electrode, and described contrast electrode is Ag/AgCl electrode, and described auxiliary electrode is platinum electrode; Described chlorpyrifos artificial antigen is chlorpyrifos artificial immunizing antigen or envelope antigen.
The technical scheme that the present invention solves the employing of its technical matters is further that a kind of preparation method detecting the electrochemical immunosensor of chlopyrifos, comprises the following steps: first drip naphthol solution on pretreated basal electrode surface, dry; Then drip nano-cobaltic-cobaltous oxide/polyaniline composite material, dry; Drip glutaraldehyde solution activation again, cleaning, nitrogen dries up; Then drip chlorpyrifos artificial antigen, hatch respectively, spend the night, cleaning, dry, finally drip bovine serum albumin buffer blind.
Further, the preparation method of the electrochemical immunosensor of described detection chlopyrifos comprises the following steps:
(1) pre-service of basal electrode: basal electrode is cut into 4 × 1cm, EtOH Sonicate cleans, and dries, and seals probe area to 1cm with insulating gel 2, then use each ultrasonic cleaning 5 ~ 10min in liquid detergent, acetone, ethanol, deionized water successively, nitrogen dries up, for subsequent use;
(2) preparation of nano-cobaltic-cobaltous oxide/polyaniline composite material: 0.3 ~ 0.4g cetyl trimethyl ammonium bromide is dissolved in 45 ~ 55mL distilled water, add the cobaltosic oxide nano particle of 0.2 ~ 0.4g, ultrasonic disperse, 150 ~ 200 μ L aniline are added after stirring 4.5 ~ 5.5h, Keep agitation 0.5 ~ 1.5h, add the citric acid being equivalent to aniline amount of substance 1.5 times, stir 9.5 ~ 10.5h, add the ammonium sulfate with aniline amount of substance equivalent, reaction is stopped after continuing stirring 11.5 ~ 12.5h, reacted product is carried out suction filtration, retain filter cake, repeatedly wash to filtrate colourless with deionized water and absolute ethyl alcohol successively, filter cake is placed in vacuum drying oven 60 DEG C of dryings and namely obtains nano-Co in 4 hours 3o 4/ PAN compound substance, for subsequent use,
(3) finishing of basal electrode: first drip dropping 50 ~ 60 μ L 1.25% naphthol solution in the basal electrode surface that step (1) is for subsequent use, dry; Then drip nanometer cobalt-cobaltosic oxide/polyaniline composite material for subsequent use in the step (2) of 100 ~ 120 μ L 1:800 dilutions, dry; Then 100 ~ 120 μ L 0.25% glutaraldehyde solutions are dripped again, after activating 2 ~ 3h under normal temperature, rinse well with water and dry up with nitrogen, then the chlorpyrifos artificial antigen that 100 ~ 120 μ L 1:1600 dilute is dripped, at 37 DEG C, hatch 3 ~ 4h, spend the night, with 0.01mol/L, the PBS wash buffer of pH7.2 ~ 7.6 is clean, dries;
(5) nonspecific activity site is closed: the basal electrode after finishing is dripped 100 ~ 120 μ L 5mg/mL bovine serum albumin damping fluids and be put in the closed 5 ~ 6h of 37 DEG C of constant temperature, taking-up 0.01mol/L, the PBS wash buffer of pH7.2 ~ 7.6 is clean, obtains electrochemical immunosensor.
The technical scheme that the present invention solves the employing of its technical matters is further, a kind of method utilizing the electrochemical immunosensor detecting chlopyrifos to detect chlopyrifos, comprises the following steps:
(1) modify working electrode: the mixed liquor first working electrode of electrochemical immunosensor being dripped Dursban monoclonal antibody solution and chlopyrifos standard items composition, hatch 50 ~ 70min at being put in 37 DEG C, take out with 0.008 ~ 0.012mol.L -1the PBS damping fluid of pH 7.2 ~ 7.6 is rinsed well repeatedly, drips the IgG-HRP antibody of nanometer copper rod mark subsequently, hatches 30 ~ 45min at 37 DEG C, takes out with 0.008 ~ 0.012mol.L -1the PBS damping fluid of pH 7.2 ~ 7.6 is rinsed well repeatedly, for subsequent use;
(2) working curve is set up: by the working electrode after modification in step (1) and contrast electrode, it is 1: 1: 1 that auxiliary electrode inserts volume ratio respectively, the ratio of amount of substance meets the potassium chloride of 0.1: 5: 5, potassium ferrocyanide and the potassium ferricyanide are mixed into the three-electrode system be assembled in pH 7.4 damping fluid, the hydrogen peroxide of the 0.4mol/L ~ 0.5mol/L of 0.1 times of the mixeding liquid volume being equivalent to Dursban monoclonal antibody solution and chlopyrifos standard items composition is added under constantly stirring, measure the current value of variable concentrations chlopyrifos standard items, funtcional relationship between chlopyrifos standard concentration and corresponding current value, be electrochemical immunosensor working curve,
(3) chlopyrifos is measured: the working electrode after utilizing step (1) to modify carries out cyclic voltammetry scan in the scope of-0.8 ~ 0.8V, measure the current value in chlopyrifos solution to be measured, then according to the working curve that step (2) obtains, the concentration of solution Chlorpyrifos to be measured can be calculated.
Further, in step (1), the IgG-HRP antibody of described nanometer copper rod mark is by pH to 8 ~ 10 regulating CuNRs solution with alkali lye, then IgG-HRP and the 1mg/mLHRP mixed liquor of volume ratio 1:1 is added dropwise in CuNRs solution, after stirring 10 ~ 20min, centrifugal 15 ~ 25min under 8000 ~ 12000rpm, then carries out 2 ~ 5 washings with the PBS of 0.01mol/L pH 7.4, is finally dispersed in 1ml PBS damping fluid by gained CuNRs-IgG-HRP and is prepared from.
Further, in step (3), described cyclic voltammetry scan speed is 50mV/s.
The sensitivity of immunochemical selectivity and zymochemistry combines together by the electrochemical immunosensor of the detection agricultural chemicals chlopyrifos of the present invention, using cobaltosic oxide as nano material, this raw material easily obtains, have stable, mark firmly, does not disturb antigen-antibody reaction, sample preparation is simple, detection speed is fast, carry the advantages such as convenient, and Co 3o 4the biological membrane of electrochemical immunosensor put be not easy in atmosphere oxidized, long shelf-life, storage requirement easily reaches, with large-scale instrument detection technique ratio, be more suitable for Site Detection, detect the medicine of multiple low-residual simultaneously, be expected to be combined with the high flux detection technique of biochip, thus create quick, convenient, high-level efficiency, many residual new instrument simultaneously detected, detection technique is an epoch-making breakthrough.
Accompanying drawing explanation
Fig. 1 is chlopyrifos hapten synthesis mass spectrogram.
Fig. 2 is chlorpyrifos artificial antigen ultraviolet full wavelength scanner figure.
Fig. 3 is the preparation flow figure (based on ito glass electrode, modify layer by layer by above-mentioned steps, prepare and have bioactive immunoelectrode) of immunoelectrode.
Fig. 4 is chlopyrifos Cleaning Principle figure of the present invention.
Fig. 5 is cyclic voltammetric schematic diagram.
Fig. 6 is Optimal Curve figure of the present invention.
Fig. 7 is chlopyrifos electrochemical immunosensor working curve diagram.
Fig. 8 is triangular scan of potential method experimental line of the present invention.Wherein, (a) is the type of attachment of three kinds of electrodes, and (b) is triangular scan of potential experimental line, is realized the triangular scanning voltage required for circuit applying cyclic voltammetry by G external control.Working electrode (WE), contrast electrode (RE) and auxiliary electrode (CE).Contrast electrode is used for fixed point position zero point, and electric current flows through working electrode and auxiliary electrode, and working electrode and contrast electrode form the system of an obstructed or basic few energising, utilize the stability of contrast electrode current potential to carry out the electrode potential of surveying work electrode.Working electrode and auxiliary electrode form the system of an energising, are used for the electric current that surveying work electrode passes through.Utilize three electrode measurement systems, come the some position of the electrode of research work simultaneously and the relation of electric current.
Fig. 9 is cyclic voltammetry proof diagram.Wherein, (a) bare electrode; B electrode that () naphthols is modified; (c) nano-Co 3o 4the electrode that/PAN modifies; The electrode of (d) Modified antigen; The electrode of (e) chlopyrifos antibody modification; Each modified electrode volume ratio be the 0.1mol/L potassium chloride of 1:1:1,5mmol/L potassium ferrocyanide and the 5mmol/L potassium ferricyanide be mixed and made into 1/15mol.L -1, cyclic voltammetry scan (CV) figure in pH 7.4 damping fluid, experimental result meets the principle of design, reaches anticipation.
Figure 10 is sensor assembly drawing.Wherein, (a) is overall layout chart; B () is operational platform design figure; C () is detection cell; D () is testing process.C () figure explains: working method: by tab, selects its working method [comprising: both positive and negative polarity is changed, load is selected, break-make road, the options such as oneself setting].
Viewing area: mainly show electric current (range 0 ~ 100 μ A), voltage (range 0 ~ 10v)
Correction zone: carry out correcting current by blank value, the data display of voltage table.(playing school zero, deduction blank value).
Function indicator: whether lighting of display lamp, whether the duty showing various piece is normal.
Switch: for unlatching or the closedown of instrument.
Electrode plug wire district: associated connections post is inserted in manual operation, selects whether to use triple electrode circuit or two telegraph circuits.
Compensate: by instrument itself by regulating the resistance of control survey circuit (mainly in order to reach the requirement of measurement of correlation precision.
Embodiment
Below in conjunction with embodiment, the present invention is illustrated further.
One, reagent
Naphthols (DuPont); Co 3o 4(Tianjin great Mao chemical reagent factory); Aniline (Jiangsu Qiangsheng Chemical Co., Ltd.); Nano-Co3O4/PAN (laboratory oneself synthesis); Chlorpyrifos artificial antigen (laboratory oneself synthesis); Bovine serum albumin (BSA) (Shanghai Sheng Gong bioengineering company limited); Chlopyrifos monoclonal antibody (by Shenzhen prepared by remarkable bio tech ltd); Variable concentrations chlopyrifos standard items (Inst. of Environment Protection & Scientific Research Monitor, Ministry of Agric (Tianjin)); The sheep anti-mouse igg antibody (SouthernBiotech company) of HRP mark
Two, instrumentation and testing system
Potentiostat one
Alternating current-direct current digital voltmeter one
Electrochemical workstation (CHI660D) one
Glass three-electrode system is a set of
Electrode: ito glass electrode S=8 (2x4) cm 2
Auxiliary electrode: platinum electrode S=1 (1x1) cm 2
Contrast electrode: Ag/AgCl electrode, electrode potential is 0-0.8v
Electrolytic solution: 0.1mol/L KCl+5mmol/L K 4[Fe (CN) 6]+5mmol/L K 3[Fe (CN) 6
Embodiment 1: the preparation method detecting the electrochemical immunosensor of chlopyrifos
1, the synthesis of chlorpyrifos artificial antigen
(1) the haptenic synthesis of chlopyrifos: 3.75g 3-mercaptopropionic acid and 1.82g NaOH are added absolute ethyl alcohol 100mL (3-mercaptopropionic acid: absolute ethyl alcohol=35 ~ 40g/L; , heating stirring reaction, until all dissolve NaOH: absolute ethyl alcohol=15 ~ 20g/L); Then add the former medicine of 8.15g chlopyrifos (chlopyrifos: absolute ethyl alcohol=0.1 ~ 0.2g/mL) being dissolved in 50mL absolute ethyl alcohol, after back flow reaction 1.5 ~ 2.5h, filter reaction mixture, reduced pressure concentration; Then with being transferred in separating funnel after acetic acid ethyl dissolution, after adding saturated saline solution, solution ph being adjusted to 4 ~ 5, being extracted with ethyl acetate 2 ~ 3 times, organic phase evaporated under reduced pressure after drying agent anhydrous sodium sulfate drying, cross post purifying, for subsequent use; Reaction equation is as follows:
(2) synthesis of chlorpyrifos artificial antigen: take haptens 0.15mmoL ~ 0.25mmoL and be dissolved in 0.4mL ~ 0.6mL DMF, stirring adds 0.08mmoL ~ 0.12mmoL DCC and 0.08mmoL ~ 0.12mmoL NHS, 4 DEG C of lower magnetic force stirring reactions spend the night, and getting supernatant after centrifugal is A liquid; Take the PBS (10mmoL/L that appropriate BSA or OVA (mol ratio of haptens and BSA or OVA is approximately 10:1) is dissolved in 1mL ~ 1.5mL, pH 7.4) in, stirring and dissolving prepares B liquid, under magnetic agitation, A liquid is instilled gradually in B liquid, react 11 ~ 13h at 4 DEG C, after centrifugal, get supernatant, with PBS dialysis 2.5 ~ 3.5d at 4 DEG C, change 2 ~ 3 dislysates every day, the comlete antigen CPF-H1-BSA obtained, frozen stand-by in-20 DEG C of refrigerators.
Mass Spectrometric Identification is carried out to the chlopyrifos haptens of synthesis, determines its structure, as shown in Figure 1; Ultraviolet full wavelength scanner is carried out to the chlopyrifos antigen of synthesis, as shown in Figure 2.As shown in Figure 1, ESI-MS (-) m/z:209 [M-H]-, prove the success of chlopyrifos hapten synthesis; When strengthening its uv absorption after protein molecule on the hapten conjugation containing structure, simultaneously also may because the molecular structure destroyed around conjugation sites and weakened part uv absorption, as shown in Figure 2, the UV scanning figure of CPF-H1-BSA and CPF-H1 and BSA all there occurs skew, illustrates that chlopyrifos antigen synthesizes successfully.
2, nano-Co 3o 4the preparation of/PAN compound substance: be dissolved in 45 ~ 55mL distilled water by 0.3 ~ 0.4g cetyl trimethyl ammonium bromide, adds the Co of 0.2 ~ 0.4g 3o 4nano particle, 150 ~ 200 μ L aniline are added after ultrasonic disperse, stirring 4.5 ~ 5.5h, Keep agitation 0.5 ~ 1.5h, add the citric acid being equivalent to aniline amount of substance 1.5 times, stir 9.5 ~ 10.5h, add the ammonium sulfate with aniline amount of substance equivalent, reaction is stopped after continuing stirring 11.5 ~ 12.5h, reacted product is carried out suction filtration, retain filter cake, repeatedly wash to filtrate colourless with deionized water and absolute ethyl alcohol successively, filter cake is placed in vacuum drying oven 60 DEG C of dryings and namely obtains nano-Co in 4 hours 3o 4/ PAN compound substance, for subsequent use;
3, the preparation of electrochemical immunosensor:
(1) pre-service of basal electrode: basal electrode is cut into 4 × 1cm, EtOH Sonicate cleans, and dries, and seals probe area to 1cm with insulating gel 2, then use each ultrasonic cleaning 5 ~ 10min in liquid detergent, acetone, ethanol, deionized water successively, nitrogen dries up, for subsequent use;
(2) finishing of basal electrode: first drip 50 ~ 60 μ L 0.75% naphthol solution in the basal electrode surface that step (1) is for subsequent use, dry; Then drip nanometer cobalt-cobaltosic oxide/polyaniline composite material for subsequent use in the step (1) of 100 ~ 120 μ L 1:500 dilutions, dry; Then drip 100 ~ 120 μ L 0.25% glutaraldehyde solutions again, after activating 2 ~ 3h under normal temperature, rinse well with water and dry up with nitrogen; Then drip the chlorpyrifos artificial antigen that 100 ~ 120 μ L 1:500 dilute, at 37 DEG C, hatch 3 ~ 4h, spend the night, clean with the PBS wash buffer of 0.01mol/L, pH7.2 ~ 7.6, dry;
(3) nonspecific activity site is closed: the basal electrode after finishing is dripped 100 ~ 120 μ L 5mg/mL bovine serum albumin damping fluids and be put in the closed 5 ~ 6h of 37 DEG C of constant temperature, taking-up 0.01mol/L, the PBS wash buffer of pH7.2 ~ 7.6 is clean, obtain electrochemical immunosensor, finally will prepare sensor probe is kept in 4 DEG C of environment stand-by, and immunoelectrode preparation process as shown in Figure 3.
Embodiment 2: utilize electrochemical immunosensor to detect the method for chlopyrifos
1, the IgG-HRP antibody of Nanometer Copper mark is prepared: pH to 8 ~ 10 first regulating CuNRs solution with alkali lye, then IgG-HRP and the 1mg/mLHRP mixed liquor of volume ratio 1:1 is added dropwise in CuNRs solution, after stirring 10 ~ 20min, centrifugal 15 ~ 25min under 8000 ~ 12000rpm, then 2 ~ 5 washings are carried out with the PBS of 0.01mol/L pH 7.4, finally gained CuNRs-IgG-HRP is dispersed in 1ml PBS damping fluid, preserves at 4 DEG C, for subsequent use.
2, chlopyrifos is detected: this experiment adopts indirect competition immunoassay, comprises the following steps:
(1) first the working electrode of described electrochemical immunosensor is dripped the mixed liquor of Dursban monoclonal antibody solution and chlopyrifos standard items composition, hatch 60min at being put in 37 DEG C, taking-up 0.01mol.L -1the PBS damping fluid of pH 7.4 is rinsed well repeatedly, drips the IgG-HRP antibody of nanometer copper rod mark subsequently, hatches 45min, taking-up 0.01mol.L at 37 DEG C -1the PBS damping fluid of pH 7.4 is rinsed well repeatedly, for subsequent use;
(2) working curve is set up: by the working electrode after modification in step (1) and contrast electrode, it is 1: 1: 1 that auxiliary electrode inserts volume ratio respectively, the ratio of amount of substance meets the potassium chloride of 0.1: 5: 5, potassium ferrocyanide and the potassium ferricyanide are mixed in pH 7.4 damping fluid and are assembled into three-electrode system, the hydrogen peroxide of the 0.4mol/L ~ 0.5mol/L of 0.1 times of the mixeding liquid volume being equivalent to Dursban monoclonal antibody solution and chlopyrifos standard items composition is added under constantly stirring, measure the current value of variable concentrations chlopyrifos standard items, funtcional relationship between chlopyrifos standard concentration and corresponding current value, be electrochemical immunosensor working curve, as shown in Figure 4, measuring principle schematic diagram as shown in Figure 5,
(3) chlopyrifos is measured: the working electrode after utilizing step (1) to modify carries out cyclic voltammetry scan in the scope of-0.8 ~ 0.8V, sweep speed is 50mV/s, measure the current value in chlopyrifos solution to be measured, then according to the working curve that step (2) obtains, the concentration of solution Chlorpyrifos to be measured can be calculated, as shown in Figure 9, Figure 10.
Embodiment 3: the optimization of chlopyrifos electrochemical immunosensor reaction conditions
In immunosensor, the performance be optimized for playing sensor the condition in immunologic process is extremely important, for the dilution ratio of fixing Antigen buffer, the dilution ratio of the antibodies buffer be combined with antigen, with the amount of haptens binding antibody, with the immunoreactive time, temperature etc. all have a great impact the accuracy of sensor and sensitivity.This experiment is focused on naphthol solution concentration, nano-Co 3o 4/ Pan solution dilution ratio, antigen-antibody dilution ratio, the pH value of potassium ferricyanide damping fluid dilution is optimized, and three parallel laboratory tests of the same Setup Experiments in optimizing process, data are got three and tested to obtain mean value.As shown in Figure 6, that is: this experiment determines 1.25% as the optium concentration of Nafion in immunosensor to experimental result; 1:800 is best dilution ratio; 1:1600 is as the fixed concentration dilution ratio of artificial coupled antigen; 1:1000 is as the best dilution ratio of chlopyrifos antibody; The PBS (1/15mol/L) of pH=4 is as Matrix Solution.
Embodiment 4: the typical curve (working curve) of chlopyrifos electrochemical immunosensor and detection limit
Under the optimum experimental condition obtained after optimization, adopt the chlopyrifos standard items of 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), draw out typical curve, see Fig. 7.Shown by Fig. 7, the linear detection range of this sensor is 0.05ng/mL ~ 100ng/mL, and equation of linear regression is y=-0.5309x+57.4, R 2=0.9931, lowest detection is limited to 0.05ng/mL.
Embodiment 5: the performance evaluation of chlopyrifos electrochemical immunosensor
1, the reappearance of chlopyrifos electrochemical immunosensor
Get fine horse jujube and each 6 parts of grey jujube sample (commercially available), 110 DEG C ~ 120 DEG C oven dry, pulverize, be dissolved in absolute ethyl alcohol, detect with chlopyrifos electrochemical immunosensor obtained sample solution, every increment product repeat 6 times, and result is as shown in table 1 below.
Table 1-chlopyrifos electrochemical immunosensor reappearance detected value
As shown in Table 1, the reappearance of every increment product is all fine.
2, the stability of chlopyrifos electrochemical immunosensor
Get fine horse jujube and each 6 parts of grey jujube sample (commercially available), 110 DEG C ~ 120 DEG C oven dry, pulverize, be dissolved in absolute ethyl alcohol, obtained sample solution detected with chlopyrifos electrochemical immunosensor, detects 1 every day, detect 6 days, result is as shown in table 2 below.
Table 2-chlopyrifos electrochemical immunosensor Detection of Stability value
As shown in Table 2, within 6 days, the change of current-responsive value is little, and this sensor has higher stability.
3, the recovery experiment of chlopyrifos electrochemical immunosensor
Get fine horse jujube and grey jujube two kinds of samples (commercially available), 110 DEG C ~ 120 DEG C oven dry, pulverize, be dissolved in absolute ethyl alcohol, chlopyrifos standard items ethanol dissolves, and adds chlopyrifos preparation of standard sample mark-on sample respectively by 10 μ g/g, 20 μ g/g and 50 μ g/g concentration in sample, often kind of sample parallel measures three times, the results are shown in following table 3.Meanwhile, high effective liquid chromatography for measuring mark-on sample is adopted to test in contrast.
The recovery control value of table 3-chlopyrifos electrochemical immunosensor
As shown in Table 3, spiked levels is at 10 ~ 50 μ g/g, and the fine horse jujube Chlorpyrifos recovery is 88.7% ~ 94.9%, and standard deviation is 3.2% ~ 4.7%, and the method accuracy is high, can be used for the analysis of actual sample Chlorpyrifos.
4, electrochemical properties experimental result in electrode modification process
With the electrochemical properties of cyclic voltammetry Electrode in self assembling process, see Fig. 8, Fig. 9.As shown in Figure 9, when naphthols film modified to electrode after, the current value of cyclic voltammetry curve reduces, and illustrates that naphthols film hinders the transmission of electronics; Work as nano-Co 3o 4after/PAN and naphthols film there occurs ion-exchange, the redox peak current of modified electrode in potassium ferricyanide solution increases, and the nano-Co in naphthols film is described 3o 4/ Pan can transmit electronics effectively; After electrode modifies chlopyrifos antigen-BSA again, redox peak comparatively polyaniline reduction peak declines, and this to be adsorbed onto on electrode due to protein molecular thus to hinder electric transmission.Particularly after chlopyrifos antibody and chlopyrifos antigen-BSA selectivity combine, the antigen-antibody complex of generation blocks modified electrode surface more multiple aperture passage, adds making response current decline further by resistance during film.Therefore, use this electrochemical immunosensor can detect the poisonous and harmful substances such as chlopyrifos in food whether to exist.
5, the detection method of the electrochemical immunosensor of chlopyrifos
Control electrode electromotive force is with different speed, repeatedly scan with triangular waveform one or many in time, as shown in Figure 8, potential range makes electrode different reduction and oxidation reaction can alternately occur, and record current-potential curve (current-responsive curve), as shown in Figure 5, electrode applies a linear ramp, with the scanning of constant pace of change, when reaching the termination current potential of certain setting, be more oppositely returned to the take-off potential of a certain setting.According to peak current Ip, peak electromotive force and the relation between peak electric potential difference Δ and sweep speed in cyclic voltammetry curve figure, the reversibility of electrode reaction can be judged.When electrode reaction completely reversibility, at 25 DEG C, the quantitative expression of these parameters has:
(1) Ipc=2.69 × 105n 3/ 2D01/2 υ 1/2 (Acm -2), namely Ipc is directly proportional to the bulk concentration of reactant O, is directly proportional to υ 1/2.Wherein: DO is the coefficient of diffusion (cm of O 2/ s), C is the bulk concentration of O, and υ is sweep speed (V/s).That is: in the present invention along with testing sample and antibody react, cause antibody amount to change thus above-mentioned change occurs immune electric current.
(2) | Ipc|=|Ipa|, namely | Ipc/Ipa|=1, and have nothing to do with electrical potential scan rate υ.
(3) and with sweep velocity υ with irrelevant, 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 the most useful criterions detecting reversible electrode reaction.
The degree of reversibility of electrode reaction can be judged, the possibility of the absorption of intermediate, phase boundary or New phase formation, and the character etc. of coupled chemical reactions according to curve shape.Be commonly used to potential electrode response parameter, judge its rate-determining steps and reaction mechanism, and observe within the scope of whole potential scan which reaction can occur, and character how.

Claims (7)

1. one kind is detected the electrochemical immunosensor of chlopyrifos, it is characterized in that, comprise working electrode, contrast electrode, auxiliary electrode, described working electrode fixes naphthols film successively by basal electrode surface, nano-cobaltic-cobaltous oxide/polyaniline composite film, chlorpyrifos artificial antigen, and close nonspecific activity site with bovine serum albumin.
2. the electrochemical immunosensor of detection chlopyrifos according to claim 1, it is characterized in that, described basal electrode is ito glass electrode, and described contrast electrode is Ag/AgCl electrode, and described auxiliary electrode is platinum electrode; Described chlorpyrifos artificial antigen is chlorpyrifos artificial immunizing antigen or envelope antigen.
3. the preparation method detecting the electrochemical immunosensor of chlopyrifos as claimed in claim 1 or 2, is characterized in that, comprise the following steps: first drip naphthol solution on pretreated basal electrode surface, dry; Then drip nano-cobaltic-cobaltous oxide/polyaniline composite material, dry; Drip glutaraldehyde solution activation again, cleaning, nitrogen dries up; Then drip chlorpyrifos artificial antigen, hatch respectively, spend the night, cleaning, dry, finally drip bovine serum albumin buffer blind.
4. the preparation method of the electrochemical immunosensor of detection chlopyrifos according to claim 3, is characterized in that, comprise the following steps:
(1) pre-service of basal electrode: basal electrode is cut into 4 × 1cm, EtOH Sonicate cleans, and dries, and seals probe area to 1cm with insulating gel 2, then use each ultrasonic cleaning 5 ~ 10min in liquid detergent, acetone, ethanol, deionized water successively, nitrogen dries up, for subsequent use;
(2) preparation of nano-cobaltic-cobaltous oxide/polyaniline composite material: 0.3 ~ 0.4g cetyl trimethyl ammonium bromide is dissolved in 45 ~ 55mL distilled water, add the cobaltosic oxide nano particle of 0.2 ~ 0.4g, ultrasonic disperse, 150 ~ 200 μ L aniline are added after stirring 4.5 ~ 5.5h, Keep agitation 0.5 ~ 1.5h, add the citric acid being equivalent to aniline amount of substance 1.5 times, stir 9.5 ~ 10.5h, add the ammonium sulfate with aniline amount of substance equivalent, reaction is stopped after continuing stirring 11.5 ~ 12.5h, reacted product is carried out suction filtration, retain filter cake, repeatedly wash to filtrate colourless with deionized water and absolute ethyl alcohol successively, filter cake is placed in vacuum drying oven 60 DEG C of dryings and namely obtains nano-Co in 4 hours 3o 4/ PAN compound substance, for subsequent use,
(3) finishing of basal electrode: first drip dropping 50 ~ 60 μ L 1.25% naphthol solution in the basal electrode surface that step (1) is for subsequent use, dry; Then drip nanometer cobalt-cobaltosic oxide/polyaniline composite material for subsequent use in the step (2) of 100 ~ 120 μ L 1:800 dilutions, dry; Then 100 ~ 120 μ L 0.25% glutaraldehyde solutions are dripped again, after activating 2 ~ 3h under normal temperature, rinse well with water and dry up with nitrogen, then the chlorpyrifos artificial antigen that 100 ~ 120 μ L 1:1600 dilute is dripped, at 37 DEG C, hatch 3 ~ 4h, spend the night, with 0.01mol/L, the PBS wash buffer of pH7.2 ~ 7.6 is clean, dries;
(5) nonspecific activity site is closed: the basal electrode after finishing is dripped 100 ~ 120 μ L 5mg/mL bovine serum albumin damping fluids and be put in the closed 5 ~ 6h of 37 DEG C of constant temperature, taking-up 0.01mol/L, the PBS wash buffer of pH7.2 ~ 7.6 is clean, obtains electrochemical immunosensor.
5. utilize the electrochemical immunosensor described in claim 1 or 2 to detect a method for chlopyrifos, it is characterized in that, comprise the following steps:
(1) working electrode is modified: the mixed liquor first working electrode of electrochemical immunosensor described in claim 1 or 2 being dripped Dursban monoclonal antibody solution and chlopyrifos standard items composition, hatch 50 ~ 70min at being put in 37 DEG C, take out with 0.008 ~ 0.012mol.L -1the PBS damping fluid of pH 7.2 ~ 7.6 is rinsed well repeatedly, drips the IgG-HRP antibody of nanometer copper rod mark subsequently, hatches 30 ~ 45min at 37 DEG C, takes out with 0.008 ~ 0.012mol.L -1the PBS damping fluid of pH 7.2 ~ 7.6 is rinsed well repeatedly, for subsequent use;
(2) working curve is set up: by the working electrode after modification in step (1) and contrast electrode, it is 1: 1: 1 that auxiliary electrode inserts volume ratio respectively, the ratio of amount of substance meets the potassium chloride of 0.1: 5: 5, potassium ferrocyanide and the potassium ferricyanide are mixed into the three-electrode system be assembled in pH 7.4 damping fluid, the hydrogen peroxide of the 0.4mol/L ~ 0.5mol/L of 0.1 times of the mixeding liquid volume being equivalent to Dursban monoclonal antibody solution and chlopyrifos standard items composition is added under constantly stirring, measure the current value of variable concentrations chlopyrifos standard items, funtcional relationship between chlopyrifos standard concentration and corresponding current value, be electrochemical immunosensor working curve,
(3) chlopyrifos is measured: the working electrode after utilizing step (1) to modify carries out cyclic voltammetry scan in the scope of-0.8 ~ 0.8V, measure the current value in chlopyrifos solution to be measured, then according to the working curve that step (2) obtains, the concentration of solution Chlorpyrifos to be measured can be calculated.
6. the method for detection chlopyrifos according to claim 5, it is characterized in that, in step (1), the IgG-HRP antibody of described nanometer copper rod mark is by pH to 8 ~ 10 regulating CuNRs solution with alkali lye, then IgG-HRP and the 1mg/mLHRP mixed liquor of volume ratio 1:1 is added dropwise in CuNRs solution, after stirring 10 ~ 20min, centrifugal 15 ~ 25min under 8000 ~ 12000rpm, then carry out 2 ~ 5 washings with the PBS of 0.01mol/L pH 7.4, finally gained CuNRs-IgG-HRP is dispersed in 1ml PBS damping fluid and is prepared from.
7. the method for the detection chlopyrifos according to claim 5 or 6, is characterized in that, in step (3), described cyclic voltammetry scan speed is 50mV/s.
CN201510307269.9A 2015-06-04 2015-06-04 Electrochemical immunosensor for chlorpyrifos detection, and preparation method and application thereof Pending CN104931692A (en)

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