CN106770571A - A kind of preparation method of the aptamer sensor for Pesticides Testing - Google Patents
A kind of preparation method of the aptamer sensor for Pesticides Testing Download PDFInfo
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- CN106770571A CN106770571A CN201611197950.3A CN201611197950A CN106770571A CN 106770571 A CN106770571 A CN 106770571A CN 201611197950 A CN201611197950 A CN 201611197950A CN 106770571 A CN106770571 A CN 106770571A
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 72
- 238000012360 testing method Methods 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000000575 pesticide Substances 0.000 title claims description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 72
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 46
- 239000003905 agrochemical Substances 0.000 claims abstract description 28
- 238000001548 drop coating Methods 0.000 claims abstract description 25
- 230000004048 modification Effects 0.000 claims abstract description 17
- 238000012986 modification Methods 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 238000004140 cleaning Methods 0.000 claims abstract description 13
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 9
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 9
- 239000006229 carbon black Substances 0.000 claims abstract description 7
- 230000004913 activation Effects 0.000 claims abstract description 5
- 238000001994 activation Methods 0.000 claims abstract description 5
- -1 ferroferric oxide compound Chemical class 0.000 claims abstract 3
- 229910052799 carbon Inorganic materials 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 30
- 229920001661 Chitosan Polymers 0.000 claims description 28
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 24
- 239000002131 composite material Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 229940056319 ferrosoferric oxide Drugs 0.000 claims description 15
- 238000002484 cyclic voltammetry Methods 0.000 claims description 13
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- 239000012456 homogeneous solution Substances 0.000 claims description 2
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 claims description 2
- 238000007781 pre-processing Methods 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 239000012888 bovine serum Substances 0.000 claims 2
- 235000019241 carbon black Nutrition 0.000 claims 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
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- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
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- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 239000003987 organophosphate pesticide Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
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- QYMMJNLHFKGANY-UHFFFAOYSA-N profenofos Chemical compound CCCSP(=O)(OCC)OC1=CC=C(Br)C=C1Cl QYMMJNLHFKGANY-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The invention discloses a kind of preparation method of the aptamer sensor for detecting agricultural chemicals, belong to agricultural product security detection technique field.The present invention is in the bare glassy carbon electrode surface drop coating carbon black shitosan complex liquid through over cleaning, activation and performance test, then modified with graphene oxide ferroferric oxide compound, agricultural chemicals aptamers are fixed on the electrode after modification again, is finally closed with bovine serum albumin.Aptamer sensor prepared by the present invention has sensitivity high, low cost, and detection speed is fast, good stability, and sample recovery rate it is high the characteristics of.Up to 0.033 ng/mL, the range of linearity is 0.1 ~ 10 to the test limit of aptamer sensor prepared by the present invention5Ng/mL, the rate of recovery is 96.0% ~ 106.0%, it is adaptable to the quick detection of fruits and vegetables Pesticide Residues.
Description
Technical field
The present invention relates to a kind of preparation method for detecting the aptamer sensor of agricultural chemicals, belong to agricultural product security detection
Technical field.
Technical background
As our people's living standard is improved constantly, the quality security problem of agricultural product increasingly attracts attention, especially
Fruit, Pesticide Residues in Vegetables problem have become the focus of public attention, common pesticides such as chlopyrifos, parathion, Profenofos
Deng with certain absorbability, human body can be transferred to by the enrichment of food chain, there is potential carcinogenesis to human body,
Therefore, agricultural chemicals is always the big event of environment and food safety detection, is strengthened to the detection of Residual Pesticides in Farm Produce to protecting
Shield ecological environment, especially ensures that human health has very profound significance.
Conventional Fast Determination of Pesticide Residue method has inhibiting AChE and immunosensor method, it is possible to achieve organophosphorus pesticide
Field quick detection, with preferable practical value, however, inhibiting AChE exist the low rate of recovery, false retrieval, missing inspection ratio compared with
Height, poor repeatability, the shortcomings of be difficult to meet the requirement of low-residual and quantitative determination, immunosensor is special based on antigen and antibody
The opposite sex combines the sensor that the principle of triggered immune response is developed, but bio-sensing material antibody used is obtained and compared
Costliness, processing environment is harsher, and compared with traditional immunoassay method, aptamer sensor has high specificity, analysis
Speed is fast, simple structure, it is with low cost the advantages of, the sensitive interface of aptamer sensor is table of the fixed adaptation body to electrode
The main performances such as face, stability, sensitivity and selectivity that it directly affects aptamer sensor.
The content of the invention
Can overcome drawbacks described above it is an object of the invention to provide one kind and simple to operate, sensitivity is high, selectivity is good
Detection residues of pesticides aptamer sensor preparation method, the technical scheme taken is：
A kind of preparation of aptamer sensor for detecting agricultural chemicals, it is characterised in that the sensing interface group of aptamer sensor
Fitted into including conductive black-shitosan, graphene oxide composite ferroferric oxide nano-complex, and then the nucleic acid of fixed agricultural chemicals
Part, is finally that BSA is closed with bovine serum albumin, obtains detecting the aptamer sensor of agricultural chemicals.
The step of methods described, is as follows：
1) clean, activate and test bare glassy carbon electrode, obtain pretreatment glass-carbon electrode；
2) prepare carbon black-shitosan complex liquid, drop coating to step 1) pretreatment glass-carbon electrode on；
3) graphene oxide-ferroso-ferric oxide composite nano materials are prepared, and is disperseed with chitosan solution, this is disperseed
Drop is coated onto in step 2) obtained by modified electrode on；
4) in step 3) obtained by modified electrode on fix agricultural chemicals aptamers；
5) with bovine serum albumin close step 4) obtained by electrode, obtain aptamers biology sensor.
According to step 1) cleaning, to activate and test bare glassy carbon electrode be to soak naked glass using hot piranha solution
After carbon electrode, Al is used2O3Slurry polishing, then deionized water, HNO are used successively3, absolute ethyl alcohol and deionized water be cleaned by ultrasonic, after drying
Activated with cyclic voltammetry, and by detecting bare electrode cyclic voltammetry curve spike potential difference test electrode.
According to step 2) it is described in pretreatment electrode surface layering drop coating conductive black-chitosan solution, it is first by 0.25
G shitosans are dissolved in 50 mL acetic acid solutions to continue stirring until to be completely dispersed and obtain chitosan solution, then 1.0 mg are led
The min of ultrasonic Separation 50 obtains conductive black-chitosan solution during electric carbon black is added to the above-mentioned chitosan solutions of 1.0 mL, takes 7
The carbon black of μ L-chitosan solution drop coating is dried in atmosphere on pretreatment glass-carbon electrode, uses ultrapure water surface, will not
The liquid wash for being fixed on electrode surface is fallen, and obtains the glass-carbon electrode of carbon black-chitosan-modified.
According to step 3) it is described in electrode surface drop coating graphene oxide-ferroso-ferric oxide composite nano materials, it is first to adopt
Graphene oxide is prepared with Hummers methods, recycles hot solvent method to prepare graphene oxide-ferroferric oxide compound,
By the graphene oxide of 7 μ L-ferroso-ferric oxide composite nano materials drop coating on electrode, dry in atmosphere, obtain aoxidizing stone
The electrode of black alkene-ferroferric oxide compound modification.
According to step 4) the drop coating agricultural chemicals aptamers on the working electrode (s, be by step 3) 20 μ L of the electrode obtained immersion
2.5 μM of adaptation liquid solutions in, and 12 h are stood at 4 DEG C, washed off with the PBS of pH 7.5 afterwards
Aptamers on unadsorbed, obtain the electrode of aptamers modification.
According to step 5) bovine serum albumin closes fixed electrode, refers to that the BSA of fixed electrode immersion 0.5% is molten
2 h are stood in liquid at 26 DEG C.
Methods described is comprised the following steps that：
1) after using hot Piranha solution immersion bare glassy carbon electrode, Al is used2O3Slurry polishing, then deionized water, HNO are used successively3, nothing
Water-ethanol and deionized water are cleaned by ultrasonic, and are activated with cyclic voltammetry after drying, and by detecting bare electrode cyclic voltammetry curve
Spike potential difference test electrode, obtains pre-processing electrode；
2) 0.25 g shitosans are added in the acetic acid solutions of 50 mL 1.0%, 0.5% chitosan solution is configured to, by 1.0 mg
Conductive black is added in 0.5% chitosan solution that 1.0 mL are newly prepared, and by ultrasonically treated until dispersed, will be prepared
Good carbon black-chitosan complexes are stored at 4 DEG C；
3) graphene oxide is prepared using Hummers methods, recycles hot solvent method to prepare graphene oxide-ferroso-ferric oxide multiple
Compound, 40 mg graphene oxides are added in 30 mL ethylene glycol, then are separately added into 3 g iron chloride, 9 g sodium acetates and
30 mL ethylenediamines (EDA), ultrasonic disperse obtains homogeneous solution, is then refluxed for heating 8 h, by washing number after centrifugation
Secondary, vacuum drying obtains graphene oxide-ferroferric oxide compound；
4) by 7 μ L the step of 2) prepare conductive black-chitosan complexes drop coating in step 1) obtained by pretreatment glass carbon electricity
Extremely go up, dry in atmosphere, use ultrapure water surface, the compound that will be not fixed to electrode surface is rinsed out, and obtains conduction
The electrode of carbon black-chitosan-modified；
5) the step of 7 μ L of drop coating 3) obtained in graphene oxide-ferroso-ferric oxide composite nano materials drop coating to step 4) gained
On the surface of modified electrode, dry in atmosphere, use ultrapure water surface, the compound that will be not fixed to electrode surface is rinsed
Fall, obtain the composite modified electrode of graphene oxide-ferroso-ferric oxide；
6) by step 5) electrode that obtains immerses in 2.5 μM of agricultural chemicals adaptation liquid solution of 20 μ L and 12 h stood at 4 DEG C,
The aptamers on unadsorbed are washed off with the PBS of pH 7.5 afterwards, the electrode of aptamers modification is obtained；
7) by step 6) 2 h are stood at 26 DEG C in the BSA solution of gained fixed electrode immersion 0.5%.
A kind of preparation of the aptamer sensor for examining agricultural chemicals, it is characterised in that：The sensitive interface of aptamer sensor
Composition includes conductive black-chitosan complex film and graphene oxide-ferroferric oxide compound, then fixed adaptation body.
A kind of preparation of the aptamer sensor for detecting agricultural chemicals, it is characterised in that：Glass-carbon electrode (d=3 mm) it is clear
Wash, the structure and process at sensor senses interface characterize and (prepare carbon black-shitosan, graphene oxide composite ferroferric oxide to receive
Nano composite material is simultaneously disperseed using shitosan as dispersant, using the common modified electrode of the synergy of nano material), fit
The foundation of part working sensor curve, the detection of aptamer sensor performance, detection of the aptamer sensor to actual sample.
A kind of preparation of the aptamer sensor for detecting agricultural chemicals, it is characterised in that：The optimization of experiment condition, main bag
Include test bottom liquid pH and incubation time；The working curve of prepared aptamer sensor is：ΔI=14.82×logC+13.26
(ng/mL) (R2=0.993,0.1-105ng/mL)；Aptamer sensor performance detection include specificity, reappearance, stability,
The measure of reproducibility and aptamer sensor to the vegetable sample rate of recovery.
Its preparation principle is：Aptamer sensor using aptamers as recognition component, by immobilization technology by aptamers
Susceptor surface is attached to, after there is specific recognition reaction, the compound of generation is related to the physically or chemically signal for producing
Connection, relevant with test substance concentration (or activity) quantifying or accessible physical chemistry letter is translated into by transducer
Number, then amplify and output signal by secondary meter, so as to realize the detection to test substance, the present invention is used with shitosan
The conductive black and graphene oxide composite ferroferric oxide nano-complex prepared as dispersant are repaiied to glass-carbon electrode
Decorations, graphene oxide composite ferroferric oxide nano material, by traditional solvent thermal process by heating, backflow, purification,
It is prepared by cleaning, drying and other steps.Conductive black is a kind of typical special carbon black, because of its good electric conductivity and property higher
Valency ratio, is widely used in conductive and antistatic product, and graphite oxide dilute (graphene oxide, GO) is dilute as graphite
Derivative, is the spall of graphite oxide, can process original graphite with strong protonic acid, then carry out oxidation generation graphite oxide, oxygen
Graphite is dilute cheap, is easily obtained, while it has preferable hydrophily and relatively low bio-toxicity, therefore is cured in biology
The fields such as, bioanalysis and bio-sensing have potential application value, and shitosan (CS) belongs to polysaccharide, and it has
Excellent film forming, adsorptivity, gas permeability and permeability, have good adsorptivity, stability and good biology after film forming
Compatibility, its abundant amino, cellular structure make it be widely used in the fixation of biomolecule and the preparation of modified electrode, shell
The good biocompatibility of glycan, can provide as the immobilization carrier of aptamers, the fixation for aptamers in electrode surface
One good bioelectric interface, maintains the bioactivity of aptamers for effectively being specifically bound with agricultural chemicals, adopts
The aptamer sensor being made of the present invention can carry out the quick measure of residues of pesticides before vegetables and fruits harvesting, listing, directly right
Whether persticide residue is exceeded to be detected, it is to avoid cause poisoning containing the vegetables and fruits of remains of pesticide because edible, is agricultural product peace
Full production provides the technical support of residue detection with consumption.
To achieve the above objectives, following technical scheme is taken to realize：A kind of preparation of the aptamer sensor for detecting agricultural chemicals,
It is characterized in that：(1) cleaning of bare glassy carbon electrode, activation and performance test before prepared by aptamer sensor, if test loop
, in 100 below mV, oxidation peak and reduction peak are symmetrical, then the glass-carbon electrode can be used, otherwise for spike potential difference in volt-ampere curve
In returning to cleaning step, until meeting the requirements；(2) the cleaned finely dispersed charcoal of bare glassy carbon electrode surface drop coating
Black-shitosan complex liquid, then modifies graphene oxide-ferroferric oxide compound, and then fixed adaptation body, finally uses ox blood
Pure albumen (BSA) closes nonspecific binding site, and aptamer sensor is prepared after terminating, and is put into 4 DEG C of preservations in refrigerator standby
With.
To achieve the above objectives, following technical scheme is taken to realize：A kind of preparation of the aptamer sensor for detecting agricultural chemicals,
It is characterized in that：(1) the above-mentioned aptamer sensor for preparing is done into cyclic voltammetric side in the bottom liquid that works with different speed of sweeping
Method is scanned, and can be obtained peak value and be swept the relation of speed, obtain being controlled by diffusion；(2) a series of pesticide standard is configured
Liquid, is circulated voltammetric scan, and be inhibited rate, from which further follows that working curve, the inspection of the aptamer sensor of above-mentioned preparation
Survey scope and test limit；(3) a series of the pesticide solutions being often used in mixed way are configured, to detect that prepared aptamers are sensed
The specificity of device；(4) a period of time is placed by cyclic voltammetry scan multistage and in refrigerator, and measurement checking is above-mentioned suitable again
Stability with body sensor, by dissociation and its regenerability of immune detection again；(5) actual fruits and vegetables sample is divided
Analysis draws the rate of recovery of the aptamer sensor.
The graphene oxide that the present invention is used is to process original graphite with strong protonic acid, then carries out oxidation generation oxidation stone
Ink, then prepares graphite oxide using traditional solvent thermal process by heating, backflow, purification, cleaning, drying and other steps preparation
Alkene-ferriferrous oxide nano composite, then entered as dispersant using the shitosan with good biocompatibility and film forming
Row dispersion, the nano-complex film for obtaining can promote the transmission of electronics in electrochemical reaction, improve the response current on electrode,
It is used to prepare that response signal is strong and sensitivity aptamer sensor high.
The preparation technology of the aptamer sensor is as follows：(1) the finely dispersed conductive black-chitosan nanos of 7 μ L are taken
Rice composite membrane drop coating stands 3 h under the glassy carbon electrode surface for having pre-processed, normal temperature, and electrode surface is rinsed with the PBS of pH7.5；
(2) after electrode surface dries, the chitosan dispersion drop coating of 7 μ L graphene oxides-ferriferrous oxide nano composite is taken
2 h are stood under electrode surface, normal temperature, then surface, nitrogen drying is rinsed with the PBS of pH7.5；(3) by above-mentioned warp
Nanometer-material-modified good electrode is immersed in agricultural chemicals adaptation liquid solution, and 12 h are soaked under the conditions of 4 DEG C, with pH7.5's after taking-up
PBS rinses surface；(4) finally the above-mentioned electrode for preparing is dipped in 0.5% BSA solution, 2h is stood at room temperature,
And then nonspecific binding site is closed, aptamer sensor completes, standby under the conditions of being stored in 4 DEG C.
Beneficial effects of the present invention：
First, invention introduces a kind of electrode material for super capacitor：Conductive black-chitosan complexes, using conductive charcoal
Black excellent electric conductivity and the excellent biocompatibility of shitosan, film forming, are effectively improved electric conductivity and electric capacity, utilize
The shitosan mixed solution of graphene oxide-ferroferric oxide compound improves the specific surface area of electrode, collaboration carbon black modification electricity
Pole, significantly enhances signal response；
Secondly, the aptamer sensor detection range that prepared by the present invention is wider, and the range of linearity is 0.1-105Ng/mL, test limit
It is relatively low, 0.033 ng/mL is reached, the aptamer sensor to preparing detects that its current-responsive can still keep after preserving three weeks
More than the 80% of initial current, have good stability, and the aptamer sensor can effectively bring back to life 5 times, reproducibility is preferable；
3rd, the testing result of the aptamer sensor prepared by the present invention is consistent with the testing result of gas chromatography, can make
It is the supplementary means of Fast Determination of Pesticide Residue before vegetables listing.
Brief description of the drawings
Fig. 1 conductive black transmission electron microscope pictures；(A, conductive black transmission electron microscope picture under low power；B, conductive black is saturating under high power
Penetrate electron microscope)；
Fig. 2 graphene oxides, graphene oxide-ferroferric oxide compound scanning electron microscope (SEM) photograph；(A, graphene oxide under low power
Scanning electron microscope (SEM) photograph；B, graphene oxide-ferroferric oxide compound scanning electron microscope (SEM) photograph under high power)；
Fig. 3 electrode assembling process impedance spectrums；(a, bare glassy carbon electrode；B, the electricity after conductive black-chitosan complexes modification
Pole；C, the electrode after graphene oxide-ferroso-ferric oxide modification；D, the electrode after fixed adaptation body；E, bovine serum albumin(BSA) envelope
Electrode after closing)；
Fig. 4 aptamer sensors condition of work optimizes：The optimization of pH value and incubation time；
The working curve of Fig. 5 aptamer sensors；(A, from a to h, the concentration of pesticide standard solution is respectively 105, 104,
1000, 100, 10, 1, 0.1 ng/mL；B, the Working calibration curve of aptamer sensor).
Specific embodiment
The preparation of 1 conductive black of embodiment-chitosan nano complex liquid
Shitosan (CS) is dissolved in 1.0% acetic acid solution, 0.5% CS solution is prepared, magnetic agitation is complete to shitosan at room temperature
CL, 1.0 mg conductive black powder are dissolved in the above-mentioned CS solution of 1.0 mL, and ultrasonic disperse is until be uniformly dissolved
Composite solution, Fig. 1 for conductive black-chitosan complexes transmission electron microscope picture, A figures are scanning figure under low power, and B figures are high power
Mirror projection figure；
The preparation of 2 graphene oxides of embodiment-ferroferric oxide compound
Graphene oxide is obtained by improved Hummers methods, and specific preparation process is as follows：Powdered graphite is added
H2SO4(12mL）、K2S2O8 (2.5 g)、P2O5In the mixed liquor of (2.5 g), 80 DEG C of 4.5 h of reaction are subsequently adding 0.5
L water dilutes, and filtration washing is to remove unnecessary acid, the pre-product of gained dried overnight at room temperature, then will pre-oxidize
Graphite adds H2SO4In (120 mL), KMnO is gradually added under ice-water bath protection4(15 g), this mixture is at 35 DEG C
The min of stirring reaction 30, react 90 min at 90 DEG C after, add the dilution of 250 mL water, make its boiling reaction 25 min, then stir
After mixing 2 hours, 0.7 L water and 20 mL 30%H are added2O2Solution terminating reaction, with 1 after mixture filtering:10 it is dilute
After hydrochloric acid is rinsed for several times, then rinsed to neutrality with water, then by the further dialysis purification of products therefrom remove remaining metal from
Son, dialysis one week after, the graphite oxide that will be obtained at ambient temperature ultrasound peel off 30 min, final GO storing solutions it is dense
Degree is about 0.5 mgmL-1, Fig. 2A is the low power scanning electron microscope (SEM) photograph of graphene oxide, stone aoxidized as we can see from the figure
The very thin lamellar structure of black alkene, graphene oxide composite ferroferric oxide is obtained by solvent-thermal method：40 mg graphite oxides
Alkene mixes 3.0 g FeCl3·6H2O, 9.0 g CH3COONa and 30 mL ethylenediamines (EDA) ultrasounds obtain homogeneous phase solution, will
This mixed solution is heated to reflux 8 h, and mixture is centrifuged, and then uses water and alcohol washes for several times respectively, and 60 DEG C true
Empty drying to obtain graphene oxide composite ferroferric oxide, Fig. 2 B are the scanning electricity of graphene oxide composite ferroferric oxide
Mirror figure, on spherical ferroso-ferric oxide ion load to lamella graphene oxide；
Cleaning, activation and the test of the glass-carbon electrode of embodiment 3
Before glass-carbon electrode modification, " piranha " solution (H of heat is first dipped into2SO4: H2O2= 3:1) 15 min of immersion in,
Cleaned up with water, next with 0.3 μm, the Al of 30 nm2O3Slurry is polished to minute surface on chamois leather, and deionized water is used after polishing
Cleaning removal surface contaminants, then successively with the HNO of 6 mol/L3, absolute ethyl alcohol and deionized water be each cleaned by ultrasonic 5 min,
Dried under nitrogen environment.
The activation of glass-carbon electrode：After thoroughly cleaning, electrode is in 0.5 mol/L H2SO4Activated with cyclic voltammetry in solution,
The V of V ~ -1.0 of sweep limits 1.0, is repeatedly scanned with untill the cyclic voltammogram of stabilization is reached.
The test of the glass-carbon electrode for having pre-processed：Containing 1 × 10-3 mol/L K3Fe(CN)60.20 mol/L KNO3
Cyclic voltammetry curve is run in solution, the mV/s of sweep speed 50, sweep limits is -0.1 V ~ 0.6 V, to test glass-carbon electrode
Performance；When the spike potential difference in cyclic voltammetry curve is in 100 below mV, the glass-carbon electrode can be used, otherwise re-start
Cleaning, processes the glass-carbon electrode, and until meeting the requirements, Fig. 3 is the impedance diagram of glass-carbon electrode modification, in glass-carbon electrode table
Face is modified after carbon black-chitosan complexes and graphene oxide-ferriferrous oxide nano compound respectively, and impedance value subtracts successively
Small, because the two is respectively provided with good electric conductivity, after fixed adaptation body, closing bovine serum albumin(BSA), impedance increases respectively, is
Because it is protein molecular, modification can form insulating barrier so as to hinder the transmission of electronics in electrode surface.
The preparation of the aptamer sensor of embodiment 4
Taking the compound drop-coated of the finely dispersed carbon black-shitosans of 7 μ L and being stood under electrode surface, normal temperature dries it, Ran Houyong
The PBS of pH7.5 rinses surface, nitrogen drying, then to take graphene oxide-ferriferrous oxide nano that 7 μ L prepare multiple
Polymer solution drop coating is dried on electrode under normal temperature, then rinses electrode surface with the phosphate buffer of pH7.5, and nitrogen blows
It is dry, then be immersed in above-mentioned in the agricultural chemicals of 2.5 μM of 20 μ L adaptation liquid solution through nanometer-material-modified electrode, under the conditions of 4 DEG C
Soak 12 h, surface rushed with PBS after taking-up, dry it is stand-by, finally by above-mentioned electrode immersion 0.5% BSA solution in room
Temperature is lower to stand 2 h, and with nonspecific binding site on enclosed-electrode, aptamer sensor completes, and is stored in 4 DEG C of conditions
Under it is standby.
The optimization of the aptamer sensor use condition of embodiment 5 and measure
The experiment condition of prepared aptamer sensor is optimized from test bottom liquid pH and incubation time, tests bottom liquid
The scope of pH is 6.0 ~ 8.5, and the scope of incubation time is 10 ~ 60 min, by analysis of experiments as shown in figure 4, testing bottom liquid
Optimal pH is 7.5, and optimal incubation time is 40 min.
It is 10 to be respectively configured concentration5, 104, the pesticide standard solution of 1000,100,10,1,0.1 ng/mL will be upper
The pesticide standard solution that the aptamer sensor for preparing immerses various concentrations respectively is stated, 40 min are incubated at normal temperatures, detected
Curent change obtains its working curve before and after reaction（Fig. 5）.
5 are selected to detect its reappearance in the aptamer sensor prepared under the same terms；Phase is detected successively within continuous 15 days
With the pesticide solution of concentration detecting its stability；Reacted sensor is dissociated with Glycine-HCl buffer (pH2.8)
Its power of regeneration is detected again after the min of agricultural chemicals 5, as a result shows that the sensor has good reappearance, stability and regeneration
Property.
Vegetables thoroughly cleaning totally and with deionized water is cleaned 3 times, certain density agricultural chemicals in sprinkling, after placing 5 h,
With 10 mL acetone/0.1 M pH7.5 PBSs (1/9, v/v) do solvent supersonic process 20 min, then again from
The min of the heart 10 (10000 rpm), for detecting the rate of recovery of actual sample, its rate of recovery can reach the supernatant for obtaining
96.0% ~ 106.0%, the relative standard deviation of recovery of standard addition detection is 1.21% ~ 3.70%, and the reappearance of sensor is good.
The detection method operating procedure of this aptamer sensor detection agricultural chemicals residues of pesticides is simple, and detection time is shorter, inspection
Survey pesticide concentration scope wide, sensitivity is high, power of regeneration is high and has the preferable rate of recovery and reappearance to actual sample analysis,
Meet the development of China Fast Determination of Pesticide Residue technology and internationalization required, although the present invention with preferred embodiment disclose as
On, but it is not limited to the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention, all
Can do various changes and modification, therefore being defined of should being defined by claims of protection scope of the present invention.
Claims (8)
1. it is a kind of detect agricultural chemicals aptamer sensor preparation method, it is characterised in that be through over cleaning, activation and performance
The bare glassy carbon electrode surface drop coating conductive black-shitosan complex liquid of test, is then combined with graphene oxide-ferroso-ferric oxide
Nano material is modified electrode, then the aptamers of agricultural chemicals are fixed on electrode after modification, is finally sealed with bovine serum albumin
Close, obtain detecting the aptamer sensor of agricultural chemicals.
2. method according to claim 1, it is characterised in that step is as follows：
1）Clean, activate and test bare glassy carbon electrode, obtain pretreatment glass-carbon electrode；
2）Prepare conductive black-shitosan complex liquid, drop coating to step 1）Pretreatment glass-carbon electrode on；
3）Graphene oxide-ferroso-ferric oxide composite nano materials are prepared, and is disperseed with chitosan solution, by this dispersion liquid
Drop coating is in step 2）On the modified electrode of gained；
4）In step 3）Agricultural chemicals aptamers are fixed on the modified electrode of gained；
5）Step 4 is closed with bovine serum albumin）The electrode of gained, obtains the aptamer sensor for Pesticides Testing.
3. method according to claim 2, it is characterised in that step 1）The cleaning, activate and test bare glassy carbon electrode and be
After using hot piranha solution immersion bare glassy carbon electrode, Al is used2O3Slurry polishing, then deionized water, HNO are used successively3, anhydrous second
Alcohol and deionized water are cleaned by ultrasonic, and are activated with cyclic voltammetry after drying, and by detecting bare electrode cyclic voltammetry curve peak electricity
Potential difference tests electrode.
4. method according to claim 2, it is characterised in that step 2）It is described conductive in pretreatment electrode surface layering drop coating
Carbon black-chitosan solution, is first to be dissolved in be continued stirring until in 50 mL acetic acid solutions by 0.25 g shitosans to be completely dispersed
To chitosan solution, then during 1.0 mg carbon blacks are added into the above-mentioned chitosan solutions of 1.0 mL, the min of ultrasonic disperse 50 obtains charcoal
Black-chitosan solution, takes the carbon black-chitosan solution drop coating of 7 μ L on pretreatment glass-carbon electrode, dries in atmosphere, with super
Pure water rinsing surface, the liquid wash that will be not fixed to electrode surface is fallen, and obtains the glass-carbon electrode of carbon black-chitosan-modified.
5. method according to claim 2, it is characterised in that step 3）It is described in electrode surface drop coating graphene oxide-four
Fe 3 O composite nano materials, are first to prepare graphene oxide using Hummers methods, recycle hot solvent method to prepare four
Fe 3 O composite graphite alkene, by the graphene oxide of 7 μ L-ferroso-ferric oxide composite nano materials drop coating on electrode, in sky
Dried in gas, obtain the electrode of ferroso-ferric oxide composite graphite alkene modification.
6. method according to claim 2, it is characterised in that step 4）The agricultural chemicals aptamers of drop coating on the working electrode (s,
It is in step 3) the electrode obtained is immersed in 2.5 μM of agricultural chemicals adaptation liquid solution of 20 μ L, and 12 h, Zhi Houyong are stood at 4 DEG C
The PBS of pH 7.5 washes off the aptamers on unadsorbed, obtains the electrode of aptamers modification.
7. method according to claim 2, it is characterised in that step 5）The bovine serum albumin closes fixed electrode, is
Referring to will stand 2 h in the bovine serum albumen solution of fixed electrode immersion 0.5% 26 DEG C.
8. method according to claim 2, it is characterised in that comprise the following steps that：
1）After using hot Piranha solution immersion bare glassy carbon electrode, Al is used2O3Slurry polishing, then deionized water, HNO are used successively3, nothing
Water-ethanol and deionized water are cleaned by ultrasonic, and are activated with cyclic voltammetry after drying, and by detecting bare electrode cyclic voltammetry curve
Spike potential difference test electrode, obtains pre-processing electrode；
2）0.25 g shitosans are added in the acetic acid solutions of 50 mL 1.0%, 0.5% chitosan solution is configured to, by 1 mg charcoals
It is black to be added in 0.5% chitosan solution that 1 mL is newly prepared, by ultrasonically treated until conduction that is dispersed, will preparing
Carbon black-chitosan complexes are stored at 4 DEG C；
3）Graphene oxide is prepared using Hummers methods, recycles hot solvent method to prepare graphene oxide-ferroso-ferric oxide multiple
Nano material is closed, 40 mg graphene oxides are added in 30 mL ethylene glycol, then be separately added into 3 g iron chloride, 9 g vinegar
Sour sodium and 30 mL ethylenediamines (EDA), ultrasonic disperse obtains homogeneous solution, is then refluxed for heating 8 h, by water after centrifugation
Wash for several times, vacuum drying obtains graphene oxide-ferroferric oxide compound；
4）The step of by 7 μ L 2）The carbon black of preparation-chitosan complexes drop coating is in step 1）The pretreatment glass-carbon electrode of gained
On, dry in atmosphere, ultrapure water surface is used, the compound that will be not fixed to electrode surface is rinsed out, and obtains carbon black-shell
The electrode of glycan modification；
5）Graphene oxide-ferroso-ferric oxide composite nano materials the drop coating to step 4 of the μ L of drop coating 7）The table of gained modified electrode
On face, dry in atmosphere, use ultrapure water surface, the compound that will be not fixed to electrode surface is rinsed out, and is aoxidized
The electrode of Graphene-ferroso-ferric oxide composite nano materials modification；
6）By step 5）2.5 μM of agricultural chemicals that the electrode for obtaining immerses 20 μ L are adapted in liquid solution and 12 h are stood at 4 DEG C,
The aptamers on unadsorbed are washed off with the PBS of pH 7.5 afterwards, the electrode of aptamers modification is obtained；
7）By step 6）In the bovine serum albumen solution of gained fixed electrode immersion 0.5% 2 h are stood at 26 DEG C.
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