CN103941008B - A kind of preparation method detecting the immunosensor of chlopyrifos - Google Patents

A kind of preparation method detecting the immunosensor of chlopyrifos Download PDF

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CN103941008B
CN103941008B CN201410142965.4A CN201410142965A CN103941008B CN 103941008 B CN103941008 B CN 103941008B CN 201410142965 A CN201410142965 A CN 201410142965A CN 103941008 B CN103941008 B CN 103941008B
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electrode
immunosensor
carbon electrode
aluminum hydrotalcite
glass
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CN103941008A (en
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郭业民
乔璐
孙霞
王相友
刘君峰
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Shandong University of Technology
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • 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
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/20—Herbicides, e.g. DDT

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Abstract

The invention discloses a kind of preparation method detecting the immunosensor of chlopyrifos, belong to biology sensor preparing technical field.The present invention is coated with nickel aluminum hydrotalcite-Graphene complex liquid dripping through cleaning, activation and the naked glassy carbon electrode surface of performance test, then modify by ghost nm of gold, electrode again after modification fixes Dursban monoclonal antibody, finally closes with bovine serum albumin.Immunosensor prepared by the present invention has highly sensitive, and cost is low, and detection speed is fast, good stability, and the feature that sample recovery rate is high.The detectability of immunosensor prepared by the present invention can reach 0.052ng/L, and the range of linearity is 5ng/mL-2 μ g/mL, and the recovery can reach 82.0%-118.0%, is applicable to the quick detection of fruits and vegetables Chlorpyrifos Residue amount.

Description

A kind of preparation method detecting the immunosensor of chlopyrifos
Technical field
The present invention relates to a kind of preparation method detecting the immunosensor of chlopyrifos, belong to agricultural product security detection technique field.
Background technology
China is large agricultural country, and agricultural development has very important effect to national economy and people's lives.Agricultural chemicals is that current agricultural is produced for anti-to be cured the disease, worm, weeds to the indispensable material of murrain, have important effect to promotion agricultural produce.But the unreasonable use of agricultural chemicals, not only causes the pollution of water environment, soil, air, result also in Residual Pesticides in Farm Produce and exceeds standard, cause the poisoning and trade barrier of human body.In addition, China is every year because the exceed standard economic loss that causes of residues of pesticides is huge.Along with our people's living standard improves constantly, the quality security problem of agricultural product more and more receives publicity, and especially fruit, Pesticide Residues in Vegetables problem have become the focus of public attention.Current, China produces and the pesticide overwhelming majority that uses is Organophosphorus and carbamate pesticides class agricultural chemicals, and its Chlorpyrifos is one of maximum insecticide variety of current produced worldwide and sales volume.Chlopyrifos is that efficiently poisoning broad spectrum pesticide is held concurrently herbicide, there is certain interior absorption, human body can be transferred to by the inrichment of food chain, to human body, there is potential carcinogenesis, therefore, chlopyrifos is the big event that environment and Pesticide Residues detect always.Visible, strengthening to the detection of Residual Pesticides in Farm Produce to preserving the ecological environment, especially ensureing that human health has very profound significance.
Traditional pesticide residue detection method mainly contains: spectroscopic methodology (infra-red sepectrometry, fluorescent spectrometry, ultraviolet-visible light, chemoluminescence method etc.), chromatography (gas chromatography (GC), liquid chromatography (LC), high performance liquid chromatography (HPLC), compounds GC-MS (GC-MS), liquid-mass chromatography (LC-MS), enzyme linked immunological (ELISA) etc.Although these method selectivity are good, highly sensitive, accuracy is high, detection limit is low, can detect multiple element or compound, but it needs expensive instrument and equipment, sample pretreatment process is loaded down with trivial details, time-consuming simultaneously, and require very high to the technical merit of analyst, be unsuitable for field quick detection.Conventional Fast Determination of Pesticide Residue method has inhibiting AChE (enzyme level test paper method and enzyme level spectrophotometric method), can realize the field quick detection of organophosphorus pesticide, have good practical value.But quick measuring card is changed by the color of visual inspection card, therefore generally can only be used for the observational measurement of the vegetable sample of severe overweight.Enzyme level spectrophotometric ratio juris carries out detecting based on the change of absorbance, but pigments a large amount of in vegetable and fruit can affect the accuracy of testing result.In addition, said method exists that the recovery is low, false retrieval, undetected ratio are higher, poor repeatability, is difficult to meet the shortcomings such as low-residual and the requirement that quantitatively detects.Immunosensor is the sensor that the immunoreactive principle caused based on antigen and antibody specific binding is developed, compared with traditional analytical approach, and the advantages such as it has high specificity, analysis speed is fast, structure is simple, with low cost.The key of immunosensor is the structure of sensitive interface, and it directly affects immobilization, the stability of immunosensor, the main performance such as sensitivity and selectivity of biomolecule.
Summary of the invention
The object of the present invention is to provide a kind of immunosensor preparation method that can overcome above-mentioned defect and detection chlorpyrifos pesticide residue simple to operate, highly sensitive, selectivity is good.The technical scheme taked is: a kind of preparation method detecting the immunosensor of chlopyrifos, it is characterized in that, be coated with nickel aluminum hydrotalcite-Graphene complex liquid dripping through cleaning, activation and the naked glassy carbon electrode surface of performance test, and 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.
The step of described method is as follows:
1) clean, activate and test naked glass-carbon electrode, obtain pre-service glass-carbon electrode;
2) nickel aluminum hydrotalcite-Graphene complex liquid is prepared;
3) by step 2) nickel aluminum hydrotalcite-Graphene compound drop of preparing is coated onto on the pre-service glass-carbon electrode of step 1), and after being uniformly dispersed, utilizing ghost nm of gold to modify, obtain modified electrode;
4) on the modified electrode of step 3) gained, fix the monoclonal antibody of chlopyrifos, obtain fixed electorde;
5) close the fixed electorde of step 4) gained with bovine serum albumin, obtain chlopyrifos immunosensor.
Described method step 1) described cleaning, activation to test naked glass-carbon electrode be, after utilizing hot piranha solution to soak naked glass-carbon electrode, use Al 2o 3slurry polishing, then use deionized water, HNO successively 3, absolute ethyl alcohol and deionized water ultrasonic cleaning, with cyclic voltammetry activation after drying, and by detecting glass-carbon electrode cyclic voltammetry curve spike potential difference test glass-carbon electrode.
Described method step 2) describedly prepare nickel aluminum hydrotalcite-Graphene complex liquid, adopt Hummers method to prepare graphite oxide, ultrasonic process after with mixed alkali liquor dissolved oxygen graphite, prepare suspension joining in the salt solusion containing nickel nitrate and aluminium nitrate, after regulating pH to 10.5, crystallization 6 h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, reflux after 4 h and cool, to spend after deionized water three times dry 24 h at 60 DEG C, finally dissolve with shitosan-acetum, and ultrasonic disperse evenly after, obtain nickel aluminum hydrotalcite-Graphene complex liquid.
Described method step 3) described dripping on pre-service electrode surface be coated with nickel aluminum hydrotalcite-Graphene complex liquid, get 6 μ L finely dispersed nickel aluminum hydrotalcite-graphene nano compound drops to be coated in electrode surface, 3 h under normal temperature, then use the phosphate buffer (PBS) of pH 7.5 to rinse surface, nitrogen dries up.
Described method step 3) describedly utilize ghost decorated by nano-gold, refer to that getting 6 μ L ghost nano-Au solutions drips and be coated on electrode, leave standstill 2 h at 26 DEG C, then rinse electrode surface with the phosphate buffer of pH 7.5, nitrogen dries up.
Described method step 4) monoclonal antibody of described fixing chlopyrifos, that modified electrode is immersed in the glutaraldehyde solution of 5%, take out after 30 min, flushing dries up, be immersed in Dursban monoclonal antibody solution, soak 8 h under 4 DEG C of conditions, with the PBS wash buffer surface of pH 7.5 after taking out, dry stand-by.
Described method step 5) described bovine serum albumin closes fixed electorde, refers to fixed electorde to immerse in bovine serum albumin (BSA) solution of 0.5% to leave standstill 2 h at 26 DEG C.
The concrete steps of described method are as follows:
1), after utilizing hot piranha solution to soak naked glass-carbon electrode, Al is used 2o 3slurry polishing, then use deionized water, HNO successively 3, absolute ethyl alcohol and deionized water ultrasonic cleaning, with cyclic voltammetry activation after drying, and by detecting glass-carbon electrode cyclic voltammetry curve spike potential difference test glass-carbon electrode, obtain pre-service electrode;
2) Hummers method is adopted to prepare graphite oxide, ultrasonic process after with mixed alkali liquor dissolved oxygen graphite, prepare suspension joining in the salt solusion containing nickel nitrate and aluminium nitrate, after regulating pH to 10.5, crystallization 6 h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, reflux after 4 h and cool, to spend after deionized water three times dry 24 h at 60 DEG C, finally dissolve with shitosan-acetum, and ultrasonic disperse evenly after, obtain nickel aluminum hydrotalcite-Graphene complex liquid;
3) get 6 μ L finely dispersed nickel aluminum hydrotalcite-graphene nano compound drops and be coated in step 1) gained pre-service electrode surface, 3 h under normal temperature, then the PBS wash buffer surface of pH 7.5 is used, nitrogen dries up, get 6 μ L ghost nano-Au solutions again to drip and be coated on electrode, leave standstill 2 h at 26 DEG C, then rinse electrode surface with the PBS of pH 7.5, after nitrogen dries up, obtain modified electrode;
4) immersed in the glutaraldehyde solution of 5% by step 3) gained modified electrode, take out after 30 min, flushing dries up, and is immersed in Dursban monoclonal antibody solution, soaks 8 h under 4 DEG C of conditions, with PBS wash buffer surface after taking out, obtains fixed electorde;
5) step 4) gained fixed electorde is immersed left at room temperature 2 h in the BSA solution of 0.5%.
Described a kind of preparation detecting the immunosensor of chlopyrifos, is characterized in that: the sensitive interface composition of immunosensor comprises nickel aluminum hydrotalcite-graphene nano composite membrane and ghost nm of gold, and then the monoclonal antibody of fixing anti-chlopyrifos.
Described a kind of preparation detecting the immunosensor of chlopyrifos, it is characterized in that: the cleaning of glass-carbon electrode (d=3mm), the structure of sensitive interface of immunosensor and process characterize (to be prepared nickel aluminum hydrotalcite-graphene nanocomposite material and ghost nm of gold material and disperses as spreading agent using shitosan, utilize the common modified electrode of synergy of nickel aluminum hydrotalcite-Graphene and ghost nm of gold), the foundation of immunosensor working curve, the detection of immunosensor performance, immunosensor is to the detection of actual sample.
Described a kind of preparation detecting the immunosensor of chlopyrifos, is characterized in that: the optimization of experiment condition, mainly comprises antibody concentration, test end liquid pH and incubation time; The working curve of prepared immunosensor is: % Δ I=-7+14.932 LgC (ng/mL) (R 2=0.9913,5-150 ng/mL), % Δ I=-57.544+38.724 LgC (ng/mL) (R 2=0.9926,150-2000 ng/mL); Immunosensor Performance Detection comprises specificity, reappearance, stability, reproducibility and the immunosensor mensuration to the vegetable sample recovery.
Its preparation principle is: immunosensor is using immune biomolecule as recognition component, by immobilization technology, immune protein is attached to susceptor surface, after there is Immune discrimination reaction, the immune complex of generation is associated with the physics of generation or chemical signal, by transducer be translated into relevant with test substance concentration (or activity) can quantitative or accessible physiochemical signal, amplify through secondary instrument again and output signal, thus realizing the detection to test substance.The present invention adopts nickel aluminum hydrotalcite-Graphene of preparing using shitosan as spreading agent and ghost nm of gold to modify glass-carbon electrode.Nickel aluminum hydrotalcite-graphene composite material, grows nickel aluminum hydrotalcite by traditional coprecipitation method and prepares nickel aluminum hydrotalcite-graphene composite material through reduction again in graphite oxide template.Hydrotalcite is the multi-functional nanometer material that a kind of anionic is similar to oxyhydroxide, kind and the quantity of interlayer anion have Modulatory character, Graphene improves electronic conduction speed as two-dimentional substrate, increases electrode conduction surface and maintain structural stability, and nickel aluminum hydrotalcite-graphene composite material shows excellent chemical property.Shitosan (CS) belongs to polysaccharide, it has excellent film forming, adsorbability, gas penetration potential and perviousness, have good adsorbability, stability and good biocompatibility after film forming, its abundant amino, cellular structure make it be widely used in the preparation of the fixing of biomolecule and modified electrode.The aminoterminal of shitosan connects ghost nm of gold by golden ammonia key, it has good bio-compatibility, can be used as the immobilization carrier of antibody, for antibody fixedly provides a good bioelectric interface at electrode surface, the biologically active maintaining antibody is to be used for and agricultural chemicals carries out effective specific binding.In addition, ghost nm of gold has higher specific surface area, and good stability, density are little, cost is low, as small conductive center, fully expose avtive spot, between inside and outside shell, there is electric potential difference, the transmission of electronics in electrochemical reaction can be promoted, and then increase the response current of immunosensor.Before adopting the immunosensor made of the present invention can gather at vegetables and fruits, going on the market, carry out the Fast Measurement of residues of pesticides, directly whether persticide residue is exceeded standard and detect, avoid the vegetables and fruits because eating containing remains of pesticide and cause poisoning, for crop production safety and consumption provide the technical support of residue detection.
For reaching above object, following technical scheme is taked to realize: a kind of preparation detecting the immunosensor of chlopyrifos, it is characterized in that: the cleaning of naked glass-carbon electrode before the preparation of (1) immunosensor, activation and performance test, if the spike potential difference in test loop volt-ampere curve is at 120 below mV, oxidation peak and reduction peak symmetry, then described glass-carbon electrode can use, otherwise will return in cleaning step, until meet the requirements.(2) cleaned naked glassy carbon electrode surface is dripped and is coated with finely dispersed nickel aluminum hydrotalcite-Graphene complex liquid, then ghost nm of gold is modified, then fix Dursban monoclonal antibody, finally use bovine serum albumin(BSA) (BSA) to close nonspecific binding site.After immunosensor preparation terminates, to put in refrigerator 4 DEG C and save backup.
For reaching above object, following technical scheme is taked to realize: a kind of preparation detecting the immunosensor of chlopyrifos, it is characterized in that: the above-mentioned immunosensor prepared is done the scanning of cyclic voltammetric method with different speed of sweeping by (1) in work end liquid, peak value can be obtained and sweep fast relation, obtain being control by diffusion.(2) configure a series of chlopyrifos titer, carry out cyclic voltammetry scan, be inhibited rate, draws the working curve of the immunosensor of above-mentioned preparation, sensing range and detectability further; (3) a series of the pesticide solution often used in combination is configured, to detect the selectivity of prepared immunosensor; In refrigerator, place a period of time again measure by cyclic voltammetry scan multistage the stability verifying above-mentioned immunosensor, by dissociating and its regenerability of immune detection again; (5) recovery drawing this immunosensor is analyzed to actual fruits and vegetables sample.
The present invention adopts traditional coprecipitation method in graphite oxide template, grow nickel aluminum hydrotalcite to prepare nickel aluminum hydrotalcite-graphene nanocomposite material through reduction again, disperse using the shitosan with good biocompatibility and film forming as spreading agent again, the nano-complex film obtained can promote the transmission of electronics in electrochemical reaction, improve the response current on electrode, in order to prepare the strong and highly sensitive immunosensor of response signal; There is the golden ammono-system that a large amount of amino nano composite membranes and ghost nm of gold formed there is higher stability and high-sequential, increase the current-responsive of immunosensor, and improve the microenvironment of electrode surface, as carrier material by glutaraldehyde covalent cross-linking antibody, improve the effectively fixing of sensor surface antibody, thus increase accuracy of detection.
The preparation technology of described immunosensor is as follows: (1) is got the finely dispersed nickel aluminum hydrotalcite-graphene nano composite membrane of 6 μ L and dripped and be coated in the good glassy carbon electrode surface of pre-service, leave standstill 3 h under normal temperature, rinse electrode surface with the PBS of pH 7.5; (2) after electrode surface dries, get 6 μ L ghost nm of gold and drip and be coated in electrode surface, leave standstill 2 h under normal temperature, then use the PBS wash buffer surface of pH 7.5, nitrogen dries up; (3) immerse in the glutaraldehyde solution of 5% by above-mentioned through nanometer-material-modified good electrode, take out after 30 min, rinse with the PBS of pH 7.5 and dry up, be immersed in Dursban monoclonal antibody solution, 8 h are soaked, with the PBS wash buffer surface of pH 7.5 after taking out under 4 DEG C of conditions; (4) be finally dipped in the BSA solution of 0.5% by the above-mentioned electrode prepared, left at room temperature 2 h, and then closed nonspecific binding site, immunosensor completes, for subsequent use under being kept at 4 DEG C of conditions.
Beneficial effect of the present invention:
First, invention introduces a kind of electrode material for super capacitor: nickel aluminum hydrotalcite-graphene nanometer composite, nickel aluminum hydrotalcite is grown using Graphene as base material, effectively improve electric conductivity and electric capacity, this nano composite material and the synergistically modified electrode of ghost nm of gold, enhance signal response significantly;
Secondly, immunosensor sensing range prepared by the present invention is wider, and the range of linearity is 5 ng/mL-150 ng/ mL and 150 ng/mL-2 μ g/mL, and detectability is lower, reaches 0.052 ng/mL.With chlopyrifos titer for contrast, detect the blended solution adding chaff interference, testing result does not have significant change, and can ignore, specificity is good.Detect after preserving three weeks to the immunosensor prepared, its current-responsive still can remain on more than 82% of initial current, has good stability, and this immunosensor can bring back to life 5 times effectively, and reproducibility is better;
3rd, the testing result of the immunosensor prepared by the present invention is consistent with the testing result of vapor-phase chromatography, can be used as the supplementary means of the front Fast Determination of Pesticide Residue of vegetables listing.
Accompanying drawing explanation
The FFIR figure of Fig. 1 graphite oxide, nickel aluminum hydrotalcite and nickel aluminum hydrotalcite-graphene complex.
The transmission electron microscope picture of Fig. 2 ghost nm of gold.
The scanning electron microscope (SEM) photograph of Fig. 3 nickel aluminum hydrotalcite-graphene complex and ghost nm of gold;
(a, the scanning electron microscope (SEM) photograph of nickel aluminum hydrotalcite-graphene complex under low range; B, the scanning electron microscope (SEM) photograph of nickel aluminum hydrotalcite-graphene complex under high magnification; C, ghost nm of gold scanning electron microscope (SEM) photograph).
The cyclic voltammogram of Fig. 4 glass-carbon electrode modification;
(a, glass-carbon electrode; B, the electrode after nickel aluminum hydrotalcite-graphene complex modification; C, the electrode after ghost decorated by nano-gold; D, the electrode after sessile antibody; E, bovine serum albumin(BSA) close after electrode; F, hatches the electrode after agricultural chemicals).
The working curve diagram of Fig. 5 immunosensor.
Embodiment
The preparation of embodiment 1 nickel aluminum hydrotalcite-graphene nano complex liquid
Hummers method is adopted to prepare graphite oxide.Concrete technological process: the reaction bulb assembling 250 mL in ice-water bath, add 1 g dag, 1 g sodium nitrate and 46 mL sulfuric acid, stir 30 min, then gradation adds 5 g potassium permanganate, control temperature of reaction and be no more than 20 DEG C, stirring reaction a period of time, be then warming up to about 35 DEG C, continue stirring 30 min, slowly add 80 mL deionized waters again, continue after mixing 20 min, add the oxygenant that the hydrogen peroxide reduction of 6 mL 30% is residual, make solution become glassy yellow.Filter while hot, and wash until sulfate radical-free is detected in filtrate with 5% HCl solution and deionized water.The vacuum drying chamber finally filter cake being placed in 60 DEG C is fully dry, obtains graphite oxide.30 mg graphite oxides add ultrasonic 30 min in mixed ammonium/alkali solutions that 50 mL contain NaOH (0.2 M) and natrium carbonicum calcinatum (0.05 M), add the salt solusion that 50 mL contain nickel nitrate (0.075 M) and aluminium nitrate (0.025 M) and obtain suspending liquid under room temperature vigorous stirring.The sodium hydroxide solution of 0.2 M joins and regulates the pH value of solution to 10.5 in above-mentioned suspending liquid.Then suspending liquid crystallization 6 hours at 60 DEG C, then solution is continued to be heated to 83 DEG C under vigorous stirring, and the sodium sulfide solution of 34 mg/mL joins in above-mentioned solution fast, and the solution after 4 hours that refluxes is cooled to room temperature, spend deionized water three times centrifugal, dry 24 h at 60 DEG C.Meanwhile, as a comparison, nickel aluminum hydrotalcite material is synthesized when not adding base oxidation graphite according to above-mentioned identical synthesis condition.Be dissolved in the acetum of 1.0% by shitosan (CS), prepare 0.3% CS solution, room temperature lower magnetic force is stirred to shitosan and dissolves completely.Take 2 mg nickel aluminum hydrotalcite-Graphenes to join in 1 mL 0.3%CS solution, ultrasonic disperse 8 h obtains finely dispersed nickel aluminum hydrotalcite-graphene nano complex liquid.Fig. 1 is the FFIR figure of graphite oxide, nickel aluminum hydrotalcite and nickel aluminum hydrotalcite-graphene complex.Observe infrared signature absorption peak, can see that the absorption peak of nickel aluminum hydrotalcite-graphene complex is similar to nickel aluminum hydrotalcite, slightly skew is the existence due to Graphene, confirms the formation of nickel aluminum hydrotalcite-graphene complex
The preparation of embodiment 2 ghost nm of gold is using Co nano particle as sacrifice templated synthesis ghost nm of gold.First logical high-purity N in 50 mL water 2deoxygenation, adds 8.4 mg citric acids and 15 mg sodium borohydrides under stirring.Then 50 μ L 0.4 M CoCI2 solution (reacting the Co nano particle carrying out all the time preventing from generating under the protection of nitrogen oxidized) are injected.Logical N is stopped after 20 min 2, get 30 mL cobalt nano-sized colloidal solutions and join (1 mM) in the chlorauric acid solution of 18 mL, continue to whisk 30 min.Finally that product is centrifugal, abandoning supernatant, gained precipitation is scattered in 5 mL pH 7.5 PBS for subsequent use.Getting 2 mL ghost nano Au colloid liquid solutions joins in the chitosan solution of 5 mL 0.5%, and stirred at ambient temperature 2 h, in order to make electrode modified material.Fig. 2 is the transmission electron microscope picture of the ghost nm of gold prepared, and ghost nm of gold presents spherical as can be seen from FIG., and center is of light color, and edge color is dark, confirms its hollow-core construction, is conducive to the structure of immunosensor.Fig. 3 is the scanning electron microscope (SEM) photograph of nickel aluminum hydrotalcite-graphene complex and ghost nm of gold, and can see the hexagon bulk crystals that tactical rule, size are even, crystallinity is high from figure a and b, crystal structure is complete.Can see from figure c ghost nm of gold present be evenly distributed, fine and close spherical-like morphology
Before the cleaning of embodiment 3 glass-carbon electrode, activation and test glass-carbon electrode are modified, first immerse " piranha " solution (H of heat 2sO 4: 30% H 2o 2=3:1) in soak 15min, next clean up with water, with 0.3 μm, the Al of 30 nm 2o 3slurry is polished to minute surface on chamois leather, removes surface contaminants, then use the HNO of 6 mol/L successively after polishing by washed with de-ionized water 3, absolute ethyl alcohol and deionized water ultrasonic cleaning 5 min separately, dry under nitrogen environment.
The activation of glass-carbon electrode: thoroughly after cleaning, electrode is at 0.5 mol/L H 2sO 4with cyclic voltammetry activation in solution, sweep limit 1.0 V ~-1.0 V, scanning is till reaching stable cyclic voltammogram repeatedly.
The test of the glass-carbon electrode that pre-service is good: containing 1 × 10 -3mol/L K 3the 0.20 mol/L KNO of Fe (CN) 6 3run cyclic voltammetry curve in solution, sweep velocity 50 mV/S, sweep limit is-0.1 V ~ 0.6 V, to test the performance of glass-carbon electrode; When the spike potential difference in cyclic voltammetry curve is at 80 below mV, and as far as possible close to 64 mV, described glass-carbon electrode can use, otherwise will re-start cleaning, processes described glass-carbon electrode, until meet the requirements.Fig. 4 is the cyclic voltammogram of glass-carbon electrode modification.After glassy carbon electrode surface modifies nickel aluminum hydrotalcite-graphene nanometer composite and ghost nm of gold, electric current increases successively, because the two all has good electric conductivity.After sessile antibody, closed bovine serum albumin bletilla hatch agricultural chemicals, electric current progressively reduces, and is because it is inert protein molecule, is modified at the transmission that electrode surface can hinder electronics.
The preparation of embodiment 4 immunosensor
Get 6 μ L finely dispersed nickel aluminum hydrotalcite-graphene nano compound drops and be coated in electrode surface, 3 h under normal temperature, then use the PBS wash buffer surface of pH 7.5, nitrogen dries up.Get ghost nano-Au solution that 6 μ L prepare again to drip and be coated on electrode, 2 h under normal temperature, then rinse electrode surface with the phosphate buffer of pH 7.5, nitrogen dries up.Immerse above-mentioned in the glutaraldehyde solution of 5% through nanometer-material-modified electrode, take out after 30 min, flushing dries up, and is immersed in the Dursban monoclonal antibody solution of 1.5 mL 5 μ g/mL again.Soak 8 h under 4 DEG C of conditions, with PBS wash buffer surface after taking out, dry stand-by.Finally above-mentioned electrode is immersed left at room temperature 2 h in the BSA solution of 0.5%, with nonspecific binding site on enclosed-electrode, immunosensor completes, for subsequent use under being kept at 4 DEG C of conditions.
The optimization of embodiment 5 immunosensor service condition and mensuration
Be optimized from antibody concentration, test end liquid pH and incubation time tripartite in the face of the experiment condition of prepared immunosensor, the scope of antibody concentration is the scope of 50 ng/mL-50 μ g/mL, pH is 5.0-8.5, and the scope of incubation time is 5-45 min.Through analysis of experiments, determine that optimum antibody concentration is 5 μ g/mL, the best pH value of test end liquid is 7.0, and best incubation time is 30 min.
The chlopyrifos standard solution of configuration 5-2000 ng/mL, the above-mentioned immunosensor prepared is immersed respectively the chlopyrifos standard solution of variable concentrations, hatch 30 min at normal temperatures, before and after detecting immune response, curent change obtains its working curve (Fig. 5).
Immunosensor is deposited at chaff interferences such as Azodrin, sevin, carbofuran, 3-hydroxyl carbofurans and in case the chlopyrifos of 200 ng/mL is tested, to detect its selectivity; 5 immunosensors prepared under the same terms are selected to detect its reappearance; The continuous chlopyrifos solution detecting same concentrations for 15 days is successively to detect its stability; Sensor Glycine-HCl buffer (pH 2.8) after immunity to be dissociated immunity again after chlopyrifos pesticides 5 min, detect its power of regeneration.
Vegetables are thoroughly cleaned up and uses washed with de-ionized water 3 times, certain density agricultural chemicals in sprinkling, after placing 5 h, by 10 mL acetone/0.1 M pH 7.5 phosphate buffered solution (1/9, v/v) solvent supersonic process 20 min is, and then centrifugal 10 min (10000 rpm), the supernatant obtained is used for detecting the recovery of actual sample, and its recovery can reach 82.0%-118.0%.The relative standard deviation that recovery of standard addition detects is 3.68-5.82%, and the reappearance of sensor is good, and within-run and between-run analysis coefficient is respectively 3.9% and 6.2%.
The detection method operating procedure of this kind of immunosensor detection chlorpyrifos pesticide residue is simple, detection time is shorter, detect pesticide concentration scope wide, highly sensitive, good stability, power of regeneration is high and have the good recovery and reappearance to actual sample analysis, meets China's Fast Determination of Pesticide Residue technical development and internationalization requirement.

Claims (9)

1. one kind is detected the preparation method of the immunosensor of chlopyrifos, it is characterized in that, be coated with nickel aluminum hydrotalcite-Graphene complex liquid dripping through cleaning, activation and the naked glassy carbon electrode surface of performance test, 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.
2. according to method described in claim 1, it is characterized in that, step is as follows:
1) clean, activate and test naked glass-carbon electrode, obtain pre-service glass-carbon electrode;
2) nickel aluminum hydrotalcite-Graphene complex liquid is prepared;
3) by step 2) nickel aluminum hydrotalcite-Graphene compound drop of preparing is coated onto on the pre-service glass-carbon electrode of step 1), and after being uniformly dispersed, utilizing ghost nm of gold to modify, obtain modified electrode;
4) on the modified electrode of step 3) gained, fix the monoclonal antibody of chlopyrifos, obtain fixed electorde;
5) close the fixed electorde of step 4) gained with bovine serum albumin, obtain chlopyrifos immunosensor.
3. according to method described in claim 2, it is characterized in that, cleaning, activate and test naked glass-carbon electrode described in step 1) is, after utilizing hot piranha solution to soak naked glass-carbon electrode, use Al 2o 3slurry polishing, then use deionized water, HNO successively 3, absolute ethyl alcohol and deionized water ultrasonic cleaning, with cyclic voltammetry activation after drying, and by detecting glass-carbon electrode cyclic voltammetry curve spike potential difference test glass-carbon electrode.
4. according to method described in claim 2, it is characterized in that, step 2) describedly prepare nickel aluminum hydrotalcite-Graphene complex liquid, adopt Hummers method to prepare graphite oxide, use ultrasonic process after mixed alkali liquor dissolved oxygen graphite again, join again in the salt solusion containing nickel nitrate and aluminium nitrate and prepare suspension, after regulating pH to 10.5, crystallization 6h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, cool after backflow 4h, to spend after deionized water three times dry 24h at 60 DEG C, finally dissolve with shitosan-acetum, and ultrasonic disperse evenly after, obtain nickel aluminum hydrotalcite-Graphene complex liquid.
5. according to method described in claim 2, it is characterized in that, described in step 3), the nickel of preparation aluminum hydrotalcite-Graphene compound drop is coated onto on pre-service glass-carbon electrode, get 6 μ L finely dispersed nickel aluminum hydrotalcite-graphene nano compound drops to be coated in electrode surface, 3h is left standstill at 26 DEG C, then use the PBS wash buffer surface of pH7.5, nitrogen dries up.
6. according to method described in claim 2, it is characterized in that, described in step 3), utilize ghost decorated by nano-gold, refer to that getting 6 μ L ghost nano-Au solutions drips and be coated on electrode, leave standstill 2h at 26 DEG C, then rinse electrode surface with the phosphate buffer of pH7.5, nitrogen dries up.
7. according to method described in claim 2, it is characterized in that, the monoclonal antibody of chlopyrifos is fixed described in step 4), be immersed in the glutaraldehyde solution of 5% by modified electrode, take out after 30min, flushing dries up, be immersed in Dursban monoclonal antibody solution, soak 8h under 4 DEG C of conditions, with PBS wash buffer surface after taking out, dry stand-by.
8. according to method described in claim 2, it is characterized in that, described in step 5), close fixed electorde with bovine serum albumin, refer to and leave standstill 2h by the BSA solution of fixed electorde immersion 0.5% 26 DEG C.
9. according to method described in claim 2, it is characterized in that, concrete steps are as follows:
1), after utilizing hot piranha solution to soak naked glass-carbon electrode, Al is used 2o 3slurry polishing, then use deionized water, HNO successively 3, absolute ethyl alcohol and deionized water ultrasonic cleaning, with cyclic voltammetry activation after drying, and by detecting glass-carbon electrode cyclic voltammetry curve spike potential difference test glass-carbon electrode, obtain pre-service electrode;
2) Hummers method is adopted to prepare graphite oxide, ultrasonic process after with mixed alkali liquor dissolved oxygen graphite, prepare suspension joining in the salt solusion containing nickel nitrate and aluminium nitrate, after regulating pH to 10.5, crystallization 6h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, cool after backflow 4h, to spend after deionized water three times dry 24h at 60 DEG C, finally dissolve with shitosan-acetum, and ultrasonic disperse evenly after, obtain nickel aluminum hydrotalcite-Graphene complex liquid;
3) get 6 μ L finely dispersed nickel aluminum hydrotalcite-graphene nano compound drops and be coated in step 1) gained pre-service electrode surface, 3h under normal temperature, then the PBS wash buffer surface of pH7.5 is used, nitrogen dries up, get 6 μ L ghost nano-Au solutions again to drip and be coated on electrode, leave standstill 2h at 26 DEG C, then rinse electrode surface with the phosphate buffer of pH7.5, after nitrogen dries up, obtain modified electrode;
4) immersed in the glutaraldehyde solution of 5% by step 3) gained modified electrode, take out after 30 min, flushing dries up, and is immersed in Dursban monoclonal antibody solution, soaks 8h under 4 DEG C of conditions, with PBS wash buffer surface after taking out, obtains fixed electorde;
5) step 4) gained fixed electorde is immersed in the BSA solution of 0.5% and leave standstill 2h at 26 DEG C.
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