CN103941008A - Method for manufacturing immunosensor for detecting chlorpyrifos - Google Patents
Method for manufacturing immunosensor for detecting chlorpyrifos Download PDFInfo
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- CN103941008A CN103941008A CN201410142965.4A CN201410142965A CN103941008A CN 103941008 A CN103941008 A CN 103941008A CN 201410142965 A CN201410142965 A CN 201410142965A CN 103941008 A CN103941008 A CN 103941008A
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- aluminum hydrotalcite
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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
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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
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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
- G01N33/54393—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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- 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
- G01N2430/10—Insecticides
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- 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
Abstract
The invention discloses a method for manufacturing an immunosensor for detecting chlorpyrifos, which belongs to the technical field of biologic sensor manufacturing. The method comprises the following steps: dropwise coating the surface of a naked glassy carbon electrode, which is subjected to cleaning, activation and performance testing, with a nickel aluminum hydrotalcite-graphene compound liquid, subsequently modifying by hollow nanogold, further fixing a chlorpyrifos monoclonal antibody on the modified electrode, and finally, sealing by bovine serum albumin. The immunosensor manufactured by the method has the characteristics of high sensitivity, low cost, high detection speed, good stability and high sample recycling rate. The detection limit of the immunosensor manufactured by the method can be 0.052ng/L, the linear range of the immunosensor is 5ng/mL-2 micrograms/mL, the recycling rate of the immunosensor can be 82.0-118.0%, and therefore the immunosensor is applicable to rapid detection on the residual amount of chlorpyrifos in fruits and vegetables.
Description
Technical field
The present invention relates to a kind of preparation method of the immunosensor that detects chlopyrifos, belong to agricultural product security detection technique field.
Technical background
China is large agricultural country, and agricultural development has very important effect to national economy and people's lives.Agricultural chemicals is current agricultural production for anti-ly curing the disease, worm, weeds be to the indispensable material of murrain, to promoting that agricultural produce has important effect.But the unreasonable use of agricultural chemicals, has not only caused the pollution of water environment, soil, atmosphere, has also caused the Residual Pesticides in Farm Produce to exceed 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 China's living standards of the people improve 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 of use is organophosphorus and carbamate chemicals for agriculture, and its Chlorpyrifos is one of insecticide variety of current produced worldwide and sales volume maximum.Chlopyrifos is the efficient poisoning broad spectrum pesticide herbicide of holding concurrently, there is certain interior absorption, can be transferred to human body by the inrichment of food chain, human body is had to potential carcinogenesis, therefore, chlopyrifos is the big event that environment and Pesticide Residues detect always.Visible, reinforcement to preserving the ecological environment, especially ensures that human health has very profound significance to the detection of Residual Pesticides in Farm Produce.
Traditional Detecting Pesticide 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), gas-matter coupling (GC-MS), liquid-matter coupling (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 the technical merit requirement to analyst is very high, is unsuitable for field quick detection.Conventional Fast Determination of Pesticide Residue method has enzyme to suppress method (enzyme suppresses test paper method and enzyme inhibition-spectrophotometry), can realize the field quick detection of organophosphorus pesticide, has good practical value.But quick measuring card is by the change color of visual inspection card, therefore generally can only be used for the observational measurement of the vegetable sample of severe overweight.The principle of enzyme inhibition-spectrophotometry is that the variation based on absorbance detects, but in vegetable and fruit, a large amount of pigments 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, be difficult to meet low-residual and the shortcoming such as the requirement that quantitatively detects.Immunosensor is the sensor that the immunoreactive principle that causes based on antigen and antibody specific binding is developed, and compared with traditional analytical approach, it has the advantages such as high specificity, analysis speed be fast, simple in structure, with low cost.The key of immunosensor is the structure at responsive interface, and it directly affects the main performance such as immobilization, stability, sensitivity and the selectivity of immunosensor 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 of taking is:
A kind of preparation method of the immunosensor that detects chlopyrifos, it is characterized in that, to drip and be coated with nickel aluminum hydrotalcite-Graphene complex liquid on the naked glass-carbon electrode surface through cleaning, activation and performance test, and then modify by ghost nm of gold, on electrode after modification, fix Dursban monoclonal antibody again, finally, with bovine serum albumin sealing, obtain detecting the immunosensor of 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) prepare nickel aluminum hydrotalcite-Graphene complex liquid;
3) by step 2) the compound drop of nickel aluminum hydrotalcite-Graphene prepared is coated onto on the pre-service glass-carbon electrode of step 1), after being uniformly dispersed, utilizes ghost nm of gold to modify, and obtains modified electrode;
3) on the modified electrode of step 3) gained, fix the monoclonal antibody of chlopyrifos, obtain fixed electorde;
4) with the fixed electorde of bovine serum albumin sealing step 4) gained, obtain chlopyrifos immunosensor.
Described method step 1) described cleaning, activation to test naked glass-carbon electrode be to utilize hot piranha solution to soak after naked glass-carbon electrode, use Al
2o
3slurry polishing, then use successively deionized water, HNO
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, dry after with cyclic voltammetry activation, and by detecting the poor test gold electrode of gold electrode cyclic voltammetry curve spike potential.
Described method step 2) described nickel aluminum hydrotalcite-Graphene complex liquid of preparing, to adopt Hummers method to prepare graphite oxide, using ultrasonic processing after mixed alkali liquor dissolved oxygen graphite, prepare suspension joining in the salt solusion that contains nickel nitrate and aluminium nitrate, regulate after pH to 10.5, crystallization 6h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, cooling after backflow 4h, dry 24h at 60 DEG C after washing three times with deionized water, finally dissolve with shitosan-acetum, and after ultrasonic being uniformly dispersed, obtain nickel aluminum hydrotalcite-Graphene complex liquid.
Described method step 3) described painting nickel aluminum hydrotalcite-Graphene complex liquid that drips on pre-service electrode surface, to get the compound drop of the finely dispersed nickel aluminum hydrotalcite-graphene nano of 6 μ L to be coated in electrode surface, 3h under normal temperature, then use the phosphate buffer (PBS) of pH7.5 to rinse surface, nitrogen dries up.
Described method step 3) the described ghost decorated by nano-gold that utilizes, 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.
Described method step 4) monoclonal antibody of described fixing chlopyrifos, by in the glutaraldehyde solution of modified electrode immersion 5%, after 30min, take out, flushing dries up, be immersed in Dursban monoclonal antibody solution, under 4 ° of C conditions, soak 8h, the PBS damping fluid with pH7.5 after taking out rinses surface, dries stand-by.
Described method step 5) describedly seal fixed electorde with bovine serum albumin, refer in bovine serum albumin (BSA) solution that will fixed electorde immerses 0.5% and leave standstill 2h at 26 DEG C.
The concrete steps of described method are as follows:
1) utilize hot piranha solution to soak after naked glass-carbon electrode, use Al
2o
3slurry polishing, then use successively deionized water, HNO
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, dry after with cyclic voltammetry activation, and by detecting the poor test gold electrode of gold electrode cyclic voltammetry curve spike potential, obtain pre-service electrode;
2) adopt Hummers method to prepare graphite oxide, with ultrasonic processing after mixed alkali liquor dissolved oxygen graphite, prepare suspension joining in the salt solusion that contains nickel nitrate and aluminium nitrate, regulate after pH to 10.5, crystallization 6h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, cooling after backflow 4h, dry 24h at 60 DEG C after washing three times with deionized water, finally dissolve with shitosan-acetum, and after ultrasonic being uniformly dispersed, obtain nickel aluminum hydrotalcite-Graphene complex liquid;
3) get the compound drop of the finely dispersed nickel aluminum hydrotalcite-graphene nano of 6 μ L and be coated in step 1) gained pre-service electrode surface, 3h under normal temperature, then rinse surface with the PBS damping fluid of pH7.5, nitrogen dries up, getting 6 μ L ghost nano-Au solutions drips and is coated on electrode again, at 26 DEG C, leave standstill 2h, then rinse electrode surface with the PBS of pH7.5, after nitrogen dries up, obtain modified electrode;
4) by the glutaraldehyde solution of step 3) gained modified electrode immersion 5%, after 30min, take out, flushing dries up, and is immersed in Dursban monoclonal antibody solution, under 4 ° of C conditions, soaks 8h, surperficial with the flushing of PBS damping fluid after taking out, and obtains fixed electorde;
5) will in the BSA solution of step 4) gained fixed electorde immersion 0.5%, under room temperature, leave standstill 2h.
The preparation of described a kind of immunosensor that detects chlopyrifos, is characterized in that: the responsive 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.
The preparation of described a kind of immunosensor that detects chlopyrifos, it is characterized in that: the cleaning of glass-carbon electrode (d=3mm), the structure at the responsive 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, the detection of immunosensor to actual sample.
The preparation of described a kind of immunosensor that detects 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, occur after immune recognition reaction, the immune complex generating is associated with physics or the chemical signal of generation, by transducer be translated into relevant with test substance concentration (or activity) can quantitative or accessible physical chemistry signal, amplify and output signal through secondary instrument again, thereby realize the detection to test substance.The present invention adopts nickel aluminum hydrotalcite-Graphene and the ghost nm of gold prepared as spreading agent using shitosan to modify glass-carbon electrode.Nickel aluminum hydrotalcite-graphene composite material, the nickel aluminum hydrotalcite of growing in graphite oxide template by traditional coprecipitation method is prepared nickel aluminum hydrotalcite-graphene composite material through reduction again.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, increases electrode conduction surface and maintain structural stability as two-dimentional substrate, and nickel aluminum hydrotalcite-graphene composite material shows good chemical property.Shitosan (CS) belongs to polysaccharide, it has excellent film forming, adsorbability, gas penetration potential and perviousness, after film forming, have good adsorbability, stability and good biocompatibility, its abundant amino, cellular structure make it be widely used in the preparation of the fixing and modified electrode of biomolecule.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 providing of electrode surface a good bioelectric interface, the biologically active that has kept 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 is little, cost is low, as small conductive center, fully expose avtive spot, between inside and outside shell, exist electric potential difference, can promote the transmission of electronics in electrochemical reaction, and then increase the response current of immunosensor.Before the immunosensor that adopts the present invention to make can be gathered at vegetables and fruits, be gone on the market, carry out the Fast Measurement of residues of pesticides, directly whether persticide residue is exceeded standard and detected, avoid because of the edible vegetables and fruits that contain remains of pesticide cause poisoning, for crop production safety and consumption provide the technical support of residue detection.
For reaching above object, take following technical scheme to realize: a kind of preparation of the immunosensor that detects chlopyrifos, it is characterized in that: cleaning, activation and the performance test of naked glass-carbon electrode before the preparation of (1) immunosensor, if the spike potential in test loop volt-ampere curve is poor below 120mV, oxidation peak and reduction peak symmetry, described glass-carbon electrode can use, otherwise will return in cleaning step, until meet the requirements.(2) cleaned naked glass-carbon electrode surface is dripped and is coated with finely dispersed nickel aluminum hydrotalcite-Graphene complex liquid, then modify ghost nm of gold, then fix Dursban monoclonal antibody, finally use bovine serum albumin(BSA) (BSA) sealing nonspecific binding site.After immunosensor preparation finishes, put in refrigerator 4 DEG C and save backup.
For reaching above object, take following technical scheme to realize: a kind of preparation of the immunosensor that detects chlopyrifos, it is characterized in that: (1) does cyclic voltammetric method scanning with the different speed of sweeping by the above-mentioned immunosensor preparing in work end liquid, can obtain peak value and sweep fast relation, obtaining being subject to diffusion control.(2) configure a series of chlopyrifos titer, carry out cyclic voltammetry scan, the rate that is inhibited, further draws working curve, sensing range and the detectability of the immunosensor of above-mentioned preparation; (3) configure the pesticide solution that a series of frequent mixing are used, to detect the selectivity of prepared immunosensor;
by cyclic voltammetry scan multistage and place a period of time and again measure the stability of the above-mentioned immunosensor of checking in refrigerator, by dissociating and its regenerability of immune detection again; (5) actual fruits and vegetables sample is analyzed to the recovery that draws this immunosensor.
The present invention adopts traditional coprecipitation method nickel aluminum hydrotalcite of growing in graphite oxide template to prepare nickel aluminum hydrotalcite-graphene nanocomposite material through reduction again, disperse as spreading agent using the shitosan with good biocompatibility and film forming again, the nano-complex film obtaining 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 the nano composite membrane of a large amount of amino and ghost nm of gold form and there is higher stability and high-sequential, increase the current-responsive of immunosensor, and improve the microenvironment of electrode surface, pass through glutaraldehyde covalent cross-linking antibody as carrier material, improve the effectively fixing of sensor surface antibody, thereby increased accuracy of detection.
The preparation technology of described immunosensor is as follows: (1) is got the finely dispersed nickel aluminum hydrotalcite-graphene nano of 6 μ L composite membrane and dripped and be coated in the glass-carbon electrode surface that pre-service is good, leaves standstill 3h under normal temperature, rinses electrode surface with the PBS of pH7.5; (2) after electrode surface dries, get 6 μ L ghost nm of gold and drip and be coated in electrode surface, leave standstill 2h under normal temperature, then rinse surface with the PBS damping fluid of pH7.5, nitrogen dries up; (3) by above-mentioned in nanometer-material-modified good electrode immerses 5% glutaraldehyde solution, after 30min, take out, rinse and dry up with the PBS of pH7.5, be immersed in Dursban monoclonal antibody solution, under 4 ° of C conditions, soak 8h, the PBS damping fluid with pH7.5 after taking out rinses surface; (4) finally the above-mentioned electrode preparing is dipped in 0.5% BSA solution, under room temperature, leaves standstill 2h, and then sealing nonspecific binding site, immunosensor completes, and is kept under 4 ° of C conditions for subsequent use.
Beneficial effect of the present invention:
First, the present invention has introduced a kind of electrode material for super capacitor: nickel aluminum hydrotalcite-graphene nanometer composite, the growth nickel aluminum hydrotalcite using Graphene as base material, electric conductivity and electric capacity are effectively improved, this nano composite material and the synergistically modified electrode of ghost nm of gold, strengthened signal response significantly;
Secondly, immunosensor sensing range prepared by the present invention is wider, and the range of linearity is 5ng/mL-150ng/ mL and 150ng/mL-2 μ g/mL, and detectability is lower, reaches 0.052ng/mL.Taking chlopyrifos titer as contrast, detect the blended solution that adds chaff interference, testing result does not have significant change, can ignore, and specificity is good.The immunosensor for preparing is preserved to detection after three weeks, and its current-responsive still can remain on the more than 82% of initial current, have good stability, and this immunosensor can bring back to life 5 times effectively, and reproducibility is better;
The 3rd, the testing result of immunosensor and the testing result of vapor-phase chromatography that the present invention is prepared are consistent, can be used as the supplementary means of the front Fast Determination of Pesticide Residue of vegetables listing.
Brief description of the drawings
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 is modified; C, the electrode after ghost decorated by nano-gold; D, the electrode after sessile antibody; E, the electrode after bovine serum albumin(BSA) sealing; 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
Adopt Hummers method to prepare graphite oxide.Concrete technological process: the reaction bulb that assembles 250mL in ice-water bath, add 1g dag, 1g sodium nitrate and 46mL sulfuric acid, stir 30min, then gradation adds 5g potassium permanganate, control temperature of reaction and be no more than 20 DEG C, stirring reaction a period of time, is then warming up to 35 DEG C of left and right, continues to stir 30min, slowly add again 80mL deionized water, continuous mixing after 20min, adds the hydrogen peroxide of 6mL30% to reduce residual oxygenant, makes solution become glassy yellow.Filter while hot, and with 5%HCl solution and deionized water wash until in filtrate sulfate radical-free be detected.The vacuum drying chamber that finally filter cake is placed in to 60 DEG C is fully dry, obtains graphite oxide.30mg graphite oxide adds ultrasonic 30min in the mixed ammonium/alkali solutions that 50mL contains NaOH (0.2M) and natrium carbonicum calcinatum (0.05M), and the salt solusion that adds 50mL to contain nickel nitrate (0.075M) and aluminium nitrate (0.025M) under room temperature vigorous stirring obtains suspending liquid.The sodium hydroxide solution of 0.2M joins the pH value to 10.5 of regulator solution in above-mentioned suspending liquid.Then suspending liquid crystallization 6 hours under 60 C, then solution is continued to be heated to 83 C under vigorous stirring, and the sodium sulfide solution of 34mg/mL joins in above-mentioned solution fast, and the solution after 4 hours that refluxes is cooled to room temperature, centrifugal with deionized water washing three times, dry 24h under 60 C.Meanwhile, as a comparison, in the situation that not adding substrate graphite oxide, synthesize nickel aluminum hydrotalcite material according to above-mentioned identical synthesis condition.Shitosan (CS) is dissolved in 1.0% acetum, preparation 0.3% CS solution, room temperature lower magnetic force is stirred to shitosan and dissolves completely.Take 2mg nickel aluminum hydrotalcite-Graphene and join in 1mL0.3%CS solution, ultrasonic dispersion 8h 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, has confirmed the formation of nickel aluminum hydrotalcite-graphene complex
The preparation of embodiment 2 ghost nm of gold
Using Co nano particle as sacrificing the synthetic ghost nm of gold of template.First in 50mL water, lead to high-purity N
2deoxygenation, adds 8.4mg citric acid and 15mg sodium borohydride under stirring.Then inject 50 μ L 0.4M CoCI
2solution (reaction carries out preventing that the Co nano particle generating is oxidized all the time under the protection of nitrogen).After 20min, stop logical N
2,get in the chlorauric acid solution that 30mL cobalt nano-sized colloidal solution joins 18mL (1mM), continue to whisk 30min.Finally that product is centrifugal, abandoning supernatant, gained precipitation is scattered in 5mL pH7.5 PBS for subsequent use.Get 2mL ghost nm of gold colloidal solution and join in the chitosan solution of 5mL 0.5%, under room temperature, stir 2h, in order to make electrode modified material.Fig. 2 is the transmission electron microscope picture of the ghost nm of gold for preparing, and ghost nm of gold presents spherically as can be seen from FIG., and center is of light color, and edge color is dark, has confirmed 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, can see tactical rule, the hexagon bulk crystals that size is even, crystallinity is high from figure a and b, and crystal structure is complete.From figure can see c ghost nm of gold present be evenly distributed, fine and close spherical-like morphology
Cleaning, activation and the test of embodiment 3 glass-carbon electrodes
Before glass-carbon electrode is modified, first immerse " piranha " solution (H of heat
2sO
4: 30% H
2o
2=3:1) the middle 15min that soaks, water cleans up, and next uses the Al of 0.3 μ m, 30nm
2o
3slurry is polished to minute surface on chamois leather, after polishing, removes surface contaminants by washed with de-ionized water, then uses successively the HNO of 6mol/L
3, absolute ethyl alcohol and deionized water ultrasonic cleaning 5min separately, dry under nitrogen environment.
The activation of glass-carbon electrode: after thoroughly cleaning, electrode is at 0.5mol/L H
2sO
4in solution, with cyclic voltammetry activation, sweep limit 1.0V～-1.0V, scans repeatedly until reach stable cyclic voltammogram.
The test of the glass-carbon electrode that pre-service is good: containing 1 × 10
-3mol/L K
3fe (CN)
60.20 mol/L KNO
3in solution, run cyclic voltammetry curve, sweep velocity 50mV/S, sweep limit is-0.1V～0.6V, to test the performance of glass-carbon electrode; Poor below 80mV when the spike potential in cyclic voltammetry curve, and approach as far as possible 64mV, 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 glass-carbon electrode finishing 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, sealing bovine serum albumin bletilla are hatched agricultural chemicals, electric current progressively reduces, and is because it is inert protein molecule, is modified at electrode surface and can hinders the transmission of electronics.
The preparation of embodiment 4 immunosensors
Get the compound drop of the finely dispersed nickel aluminum hydrotalcite-graphene nano of 6 μ L and be coated in electrode surface, 3h under normal temperature, then rinses surface with the PBS damping fluid of pH7.5, and nitrogen dries up.Get ghost nano-Au solution that 6 μ L prepare again and drip and be coated on electrode, 2h under normal temperature, then rinses electrode surface with the phosphate buffer of pH7.5, and nitrogen dries up.After 30min, take out in the glutaraldehyde solution of nanometer-material-modified electrode immersion 5% above-mentioned, flushing dries up, and is immersed in the Dursban monoclonal antibody solution of 1.5mL 5 μ g/mL again.Under 4 ° of C conditions, soak 8h, after taking out, rinse surface with PBS damping fluid, dry stand-by.Finally will in the BSA solution of above-mentioned electrode immersion 0.5%, under room temperature, leave standstill 2h, with nonspecific binding site on enclosed-electrode, immunosensor completes, and is kept under 4 ° of C conditions for subsequent use.
Optimization and the mensuration of embodiment 5 immunosensor service conditions
From antibody concentration, test end liquid pH and incubation time three aspects:, the experiment condition of prepared immunosensor is optimized, the scope of antibody concentration is 50ng/mL-50 μ g/mL, and the scope of pH is 5.0-8.5, and the scope of incubation time is 5-45min.Through analysis of experiments, determine that optimum antibody concentration is 5 μ g/mL, the optimal pH of test end liquid is 7.0, best incubation time is 30min.
Configure the chlopyrifos standard solution of 5-2000 ng/mL, the above-mentioned immunosensor preparing is immersed respectively to the chlopyrifos standard solution of variable concentrations, hatch at normal temperatures 30min, detect immune response front and back curent change and obtain its working curve (Fig. 5).
Immunosensor in the situation that existing, is tested the chlopyrifos of 200ng/mL the chaff interferences such as Azodrin, sevin, carbofuran, 3-hydroxyl carbofuran, to detect its selectivity; Select 5 immunosensors that prepare under the same terms to detect its reappearance; The continuous chlopyrifos solution that detects successively same concentrations for 15 days is to detect its stability; By the immunity again after chlopyrifos pesticides 5min of dissociating of glycocoll-HCl damping fluid (pH2.8) for the sensor after immunity, detect its power of regeneration.
Vegetables are thoroughly cleaned up and are used washed with de-ionized water 3 times, certain density agricultural chemicals in sprinkling, place after 5h, by 10mL acetone/0.1M pH7.5 phosphate buffered solution (1/9, v/v) do solvent supersonic and process 20min, and then centrifugal 10min (10000rpm), the supernatant obtaining 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, in batch, is respectively 3.9% and 6.2% with interassay coefficient of variation.
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 analysis has the good recovery and reappearance to actual sample, 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, to drip and be coated with nickel aluminum hydrotalcite-Graphene complex liquid on the naked glass-carbon electrode surface through cleaning, activation and performance test, then modify by ghost nm of gold, on electrode after modification, fix Dursban monoclonal antibody again, finally, with bovine serum albumin sealing, obtain detecting the immunosensor of chlopyrifos.
2. method according to claim 1, is characterized in that, step is as follows:
Clean, activate and test naked glass-carbon electrode, obtain pre-service glass-carbon electrode;
Prepare nickel aluminum hydrotalcite-Graphene complex liquid;
By step 2) the compound drop of nickel aluminum hydrotalcite-Graphene prepared is coated onto on the pre-service glass-carbon electrode of step 1), after being uniformly dispersed, utilizes ghost nm of gold to modify, and obtains modified electrode;
On the modified electrode of step 3) gained, fix the monoclonal antibody of chlopyrifos, obtain fixed electorde;
With the fixed electorde of bovine serum albumin sealing step 4) gained, obtain chlopyrifos immunosensor.
3. method according to claim 2, is characterized in that, cleaning, activate and test naked glass-carbon electrode described in step 1) is to utilize hot piranha solution to soak after naked glass-carbon electrode, uses Al
2o
3slurry polishing, then use successively deionized water, HNO
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, dry after with cyclic voltammetry activation, and by detecting the poor test gold electrode of gold electrode cyclic voltammetry curve spike potential.
4. method according to claim 2, it is characterized in that, step 2) described nickel aluminum hydrotalcite-Graphene complex liquid of preparing, to adopt Hummers method to prepare graphite oxide, use again ultrasonic processing after mixed alkali liquor dissolved oxygen graphite, join again in the salt solusion that contains nickel nitrate and aluminium nitrate and prepare suspension, regulate after pH to 10.5, crystallization 6h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, cooling after backflow 4h, dry 24h at 60 DEG C after washing three times with deionized water, finally dissolve with shitosan-acetum, and after ultrasonic being uniformly dispersed, obtain nickel aluminum hydrotalcite-Graphene complex liquid.
5. method according to claim 2, it is characterized in that, described in step 3), on pre-service electrode surface, drip and be coated with nickel aluminum hydrotalcite-Graphene complex liquid, to get the compound drop of the finely dispersed nickel aluminum hydrotalcite-graphene nano of 6 μ L to be coated in electrode surface, at 26 DEG C, leave standstill 3h, then rinse surface with the PBS damping fluid of pH7.5, nitrogen dries up.
6. method according to claim 2, is characterized in that, utilizes ghost decorated by nano-gold described in step 3), refer to that getting 6 μ L ghost nano-Au solutions drips and be coated on electrode, at 26 DEG C, leave standstill 2h, then rinse electrode surface with the phosphate buffer of pH7.5, nitrogen dries up.
7. method according to claim 2, it is characterized in that, described in step 4), fix the monoclonal antibody of chlopyrifos, be by the glutaraldehyde solution of modified electrode immersion 5%, after 30min, take out, flushing dries up, be immersed in Dursban monoclonal antibody solution, under 4 ° of C conditions, soak 8h, after taking out, rinse surface with PBS damping fluid, dry stand-by.
8. method according to claim 2, is characterized in that, seals fixed electorde described in step 5) with bovine serum albumin, refers in the BSA solution that will fixed electorde immerses 0.5% and leaves standstill 2h at 26 DEG C.
9. method according to claim 2, is characterized in that, concrete steps are as follows:
Utilize hot piranha solution to soak after naked glass-carbon electrode, use Al
2o
3slurry polishing, then use successively deionized water, HNO
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, dry after with cyclic voltammetry activation, and by detecting the poor test gold electrode of gold electrode cyclic voltammetry curve spike potential, obtain pre-service electrode;
Adopt Hummers method to prepare graphite oxide, with ultrasonic processing after mixed alkali liquor dissolved oxygen graphite, prepare suspension joining in the salt solusion that contains nickel nitrate and aluminium nitrate, regulate after pH to 10.5, crystallization 6h at 60 DEG C, after crystallization, be heated to 83 DEG C and add sodium sulfide solution, cooling after backflow 4h, dry 24h at 60 DEG C after washing three times with deionized water, finally dissolve with shitosan-acetum, and after ultrasonic being uniformly dispersed, obtain nickel aluminum hydrotalcite-Graphene complex liquid;
Get the compound drop of the finely dispersed nickel aluminum hydrotalcite-graphene nano of 6 μ L and be coated in step 1) gained pre-service electrode surface, 3h under normal temperature, then rinse surface with the PBS damping fluid of pH7.5, nitrogen dries up, getting 6 μ L ghost nano-Au solutions drips and is coated on electrode again, at 26 DEG C, leave standstill 2h, then rinse electrode surface with the phosphate buffer of pH7.5, after nitrogen dries up, obtain modified electrode;
By in the glutaraldehyde solution of step 3) gained modified electrode immersion 5%, after 30min, to take out, flushing dries up, and is immersed in Dursban monoclonal antibody solution, under 4 ° of C conditions, soaks 8h, surperficial with the flushing of PBS damping fluid after taking out, and obtains fixed electorde;
To in the BSA solution of step 4) gained fixed electorde immersion 0.5% 26 DEG C, leave standstill 2h.
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