CN107422013A - A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application - Google Patents
A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application Download PDFInfo
- Publication number
- CN107422013A CN107422013A CN201710513697.6A CN201710513697A CN107422013A CN 107422013 A CN107422013 A CN 107422013A CN 201710513697 A CN201710513697 A CN 201710513697A CN 107422013 A CN107422013 A CN 107422013A
- Authority
- CN
- China
- Prior art keywords
- solution
- aflatoxin
- preparation
- ptc
- room temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- 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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- 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
Abstract
The invention discloses a kind of immunity biosensor for determining aflatoxin B1 and preparation method and application, basal electrode surface is successively through GO PB PTC NH2Solution, AuNPs solution and the modification of aflatoxin b1 antibody solution, the basal electrode are that glass-carbon electrode, the nanogold and aflatoxin b1 antibody are covalently attached by Au S keys.Due to big with specific surface area, electrical conductivity is high for graphene oxide and nanogold, the features such as good biocompatibility, can reach the sensitivity for improving immunoelectrode.Compared to conventional antibodies, nano antibody has small volume, and dissolubility is good, and interface stability is good, and affinity is good, optimization customization, the advantages of simple hommization.The immunity biosensor is simple to operate, and cost is low, can be applied to the detection of aflatoxin B1 in food, and be with a wide range of applications in fields such as Food Safety Analysis.
Description
Technical field
The invention belongs to pathomycete rapid detection technical field, and in particular to a kind of to determine the immune of aflatoxin B1
Biology sensor and preparation method and application.
Background technology
Aflatoxin (AFT) is aspergillus flavus, aspergillus parasiticus and Aspergillus mainly caused secondary metabolites, is currently most
One of strong carcinogen.AFT is not single compound, but the compound that one group of chemical constitution is similar.It is separated to identify
12 kinds, toxin and the poisonous alcohol such as including B1, B2, G1, G2, M1, M2.Because the formation condition of these compounds is different, therefore it
There is different distributions and toxicity source, wherein M1 and M2 are mainly had found in milk.Aflatoxin B1 (AFB1) is mesh
Preceding known most strong carcinogen, AFB1 toxicity is 10 times of potassium cyanide, is 68 times of arsenic.Meanwhile AFB1 has mutagenesis, cause
Cancer and teratogenesis, in some instances it may even be possible to cause human and animal's acute poisoning dead.It is reported that AFB1 exposure may to human body and
Animal causes the toxic action of multiple systems, including digestive system toxicity, hepatotoxicity, hematotoxicity, immunotoxicity, reproduction and
Development toxicity etc..The mankind are fever, vomiting, stomachache and poor appetite exposed to AFB1 clinical symptoms, but observe that liver and spleen swells
Greatly, hepatodynia, xanthochromia, ascites, edema of lower extremity, dysfunction of liver, heart enlargement and pulmonary edema.Even fainted from fear when serious
Symptom, stupor and death.AFT poisonings can cause anaemia, jaundice, and enterogastric diseases and fecundity decline, particularly hepatic injury.
At present, the main method for detecting AFB1 is that thin-layer chromatography (TLC), enzyme linked immunosorbent assay (ELISA) (ELISA), fluorescence are exempted from
Epidemic disease determines and high performance liquid chromatography-tandem mass (HPLC-MS).Because these equipment prices are expensive, technical requirements are high, before sample
Complicated deficiency is handled, these methods can not realize AFB1 quick detection.Electrochemical analysis method can be simple, in real time, soon
The Screening and Identification of speed, have recently become analysis detection AFB1 study hotspot.
The advantages that graphene oxide (GO) is big with specific surface area, good conductivity, and biocompatibility is strong, therefore in sensor
After the use of field, the novel nano electrode with excellent properties can be obtained.PTC-NH2Chemistry and electrochemical properties with uniqueness
So as to reduce background current signal, strengthen electric conductivity for modified electrode.Prussian blue (PB) is used as a kind of molecule membrane material
Material, because of good, the advantage of the electron transport rate of enhancing and high surface-active with stability, therefore it is widely used in photoelectricity
The fields such as conversion, molecular recognition, ion-selective electrode, biology sensor, anticorrosion.
Nano antibody is a kind of missing light chain only CH2 and CH3 area comprising a weight chain variable district (VHH) and two routines
Natural antibody, be primarily present in the peripheral bloods such as camel and alpaca, the antibody is unlike artificial reconstructed single chain antibody fragments
(scFv) easy adhesion is even reunited like that.The VHH structures more importantly individually cloned and expressed have and original weight chain
The suitable structural stability of antibody and the binding activity with antigen, be currently known can combining target antigen minimum it is single
Position.It has small volume, and dissolubility is good, and interface stability is good, and affinity is good, easily passes through blood-brain barrier and targeting effect, optimization
Customization, simple hommization, nano antibody is in tumour, infectious disease, inflammatory bowel disease, Alzheimer disease, thrombosis and artery congee
There are extensive Utilization prospects in the diagnosis and treatment of a variety of diseases such as sample hardening illness.
The content of the invention
Goal of the invention：To solve the defects of prior art is present, the invention provides a kind of aflatoxin B1 residual to examine
Immunity biosensor of survey and preparation method thereof.Present invention also offers a kind of immune life of aflatoxin B1 residue detection
Thing sensor detecting method, described method high sensitivity, specificity is good, has the very wide range of linearity and relatively low test limit
And cost is cheap, the detection of aflatoxin B1 in food can be applied to.
The abbreviation of technical term is as follows in the present invention：
Aflatoxin B1:AFB1；Graphene oxide:GO；It is Prussian blue:PB；Glass-carbon electrode:GCE；Nanogold:AuNPs
Or GNP；Tetracarboxylic dianhydride:PTCDA；Aflatoxin B1 nano antibody：Nbs
Technical scheme：A kind of preparation method of immunity biosensor for determining aflatoxin B1 of the present invention,
Basal electrode surface is successively through GO-PB-PTC-NH2Solution, AuNPs solution and the modification of aflatoxin b1 antibody solution.
The AuNPs and aflatoxin b1 antibody are covalently attached by Au-S keys.
Preferably, the basal electrode is glass-carbon electrode.
The GO-PB-PTC-NH2Solution is prepared as follows：
(1) PTCDA is dissolved in acetone, adds ethylenediamine, 20~60min is stirred at room temperature, centrifuged, by precipitation second
Alcohol, water cleaning, drying at room temperature, obtain red powder PTC-NH2；
(2) GO is dissolved in ultra-pure water and forms suspension, FeCl is added in suspension3、K3[Fe(CN)6], KCl and
HCl, 12h is stirred at room temperature；Add the PTC-NH that step (1) obtains2, 12h is stirred at room temperature, obtains GO-PB-PTC-NH2Solution；
The ratio of PTCDA and ethylenediamine is 1g in the step (1):15mL~1g:The ratio of 5mL, PTCDA and acetone is
1g:3mL~1g:7mL.
GO concentration is 0.5~1mg/mL, GO, FeCl in GO suspension in the step (2)3、K3[Fe(CN)6] and KCl
Mass ratio is 2.1：1：1：46~8.4：1：4：46.
Hydrochloric acid is added in the step (2) to play a part of maintaining sour environment.
GO and PTC-NH in the step (2)2Mass ratio be 3:1~1:3.
Preferably, GO and PTC-NH in the step (2)2Mass ratio be 1:1.
The AuNPs solution is prepared as follows：0.2~2wt.% HAuCl is added in ultra-pure water4·3H2O is molten
Liquid, 90~100 DEG C are heated with stirring to, add 0.2~2wt.% sodium citrate solutions thereto, stir and boil 5~25min,
Cooling is taken out after changing colour, obtains AuNPs solution.
Preferably, the AuNPs solution is prepared as follows：1wt.% chlorauric acid solution is added in ultra-pure water, is stirred
Mix and be heated to 95 DEG C, 1wt.% sodium citrate solution is added into chlorauric acid solution, stirs and boils 15min, after changing colour
Cooling is taken out, obtains AuNPs solution.
The ultra-pure water, HAuCl4·3H2The volume ratio of O solution and sodium citrate solution is 100：1：1~100：1：3.
The HAuCl4·3H2The solvent of O solution is ultra-pure water, and the solvent of sodium citrate solution is ultra-pure water.
Preferably, the aflatoxin b1 antibody is aflatoxin B1 nano antibody.
A kind of preparation method for the immunity biosensor for determining aflatoxin B1, comprises the following steps：
(1) electrode pre-processes：Basal electrode is polished, polished and is cleaned by ultrasonic；
(2) by GO-PB-PTC-NH2Solution is added drop-wise to the electrode surface that step (1) obtains, and room temperature places 0.5~2h, directly
To GO-PB-PTC-NH2Electrode surface is entirely incorporated in, is cleaned with PBS solution, is dried；
(3) AuNPs fixation：The electrode that step (2) obtains is immersed in AuNPs solution, 1~10 DEG C of 1~5h of placement,
Cleaned, dried with PBS solution；
(4) it is covalently attached aflatoxin b1 antibody：The electrode surface obtained to step (3) is added dropwise aflatoxin B1 and resisted
Liquid solution, 1~10 DEG C of 6~18h of placement, makes aflatoxin b1 antibody and AuNPs fully connect, is cleaned with PBS solution, dried in the air
It is dry；
(5) non-specific sites are closed：The electrode surface dropwise addition BSA solution obtained to step (4), room temperature placement 10~
60min, the site that closing is not combined by nano antibody, is cleaned with PBS solution, dried.
Basal electrode is respectively with 0.05 and 0.03 μm of Al in the step (1)2O3Powder polishes, then respectively with anhydrous second
Alcohol, ultra-pure water are cleaned by ultrasonic 5min.
GO-PB-PTC-NH in the step (2)2The concentration of solution is 1~2mg/mL.GO-PB-PTC-NH2Solution was
Amount, uncombined GO-PB-PTC-NH is cleaned with PBS solution2。
The concentration of AuNPs solution is 0.2~1mg/mL in the step (3).AuNPs solution is excessive, uses PBS solution
The uncombined AuNPs of cleaning.
The solvent of aflatoxin b1 antibody is 0.1M PBS in the step (4), and concentration is 1~3mg/mL.Aspergillus flavus
Toxin B1 antibody-solutions are excessive, and uncombined aflatoxin b1 antibody is cleaned with PBS solution.
The solvent of BSA solution is 0.1M PBS in the step (5), and concentration is 2~20mg/mL.BSA solution is excessive
, uncombined BSA is cleaned with PBS solution.
The immunity biosensor that any of the above-described preparation method is prepared.
The immunity biosensor that any of the above-described preparation method is prepared answering in quantitative detection aflatoxin B1
With.
Beneficial effect：Compared with prior art, the invention has the advantages that：
(1) the features such as graphene oxide and nanogold be due to big with specific surface area, and electrical conductivity is high, can improve immunoelectrode
Sensitivity.
(2) this experiment uses green non-poisonous harmful chemical reagent, to detection sample nondestructive evil.
(3) conventional antibodies are compared, nano antibody has small volume, and dissolubility is good, and interface stability is good, and affinity is good, excellent
Change customization, the advantages of simple hommization.
(4) immunity biosensor of the invention is simple to operate, and cost is low, can be applied to aflatoxin B1 in food
Detection, and be with a wide range of applications in fields such as Food Safety Analysis.
Brief description of the drawings
Fig. 1 prepares schematic diagram for the present invention's；
Fig. 2 is the cyclic voltammogram of the present invention；
Fig. 3 is the linear relationship chart between the peak current intensity of the present invention and antigen concentration；
Fig. 4 is the selectivity of the present invention and special Journal of Sex Research.
Embodiment
According to following embodiments, the present invention may be better understood.As it will be easily appreciated by one skilled in the art that embodiment institute
The content of description is merely to illustrate the present invention, without should be also without limitation on the present invention described in detail in claims.
The GO-PB-PTC-NH of embodiment 12Preparation
(1)PTC-NH2Preparation
1g PTCDA are dissolved in 5mL acetone, 10mL ethylenediamines are added dropwise, reaction is stirred at room temperature in fume hood
40min.Centrifuge, gained precipitated product drying at room temperature after ethanol, deionized water are washed respectively, produce red powder
PTC-NH2。
The building-up process is as shown in formula I：
(2)GO-PB-PTC-NH2Preparation
Weigh 10.15mg GO and be dissolved in 15mL ultra-pure waters and form suspension, 4.05mg is added in above-mentioned suspension
FeCl3·6H2O, 4.94mg K3[Fe(CN)6], 111.825mg KCl and 0.075mL 2M HCl (offer sour environment),
PB is prepared in above-mentioned suspension and is combined with GO.By PB and GO magnetic agitation 12h at room temperature, expected mixed liquor is in darkcyan within second day
And suspension is evenly distributed.Add 10.15mg PTC-NH2, 12h, the 3rd day mixed liquor for collecting preparation are stirred overnight at room temperature
(color is in kermesinus), produces required GO-PB-PTC-NH2Solution (1.64mg/mL), room temperature storage is stand-by.
The calculating process of concentration:K3[Fe(CN)6] quality 4.94mg obtain [Fe (CN)6]3-Molal weight be
0.015mM.It is Prussian blue (molecular formula according to the PB that mass conservation law generates：Fe4[Fe(CN)6]3, 859.25g/mol)
Its molal weight is that 0.005mM is 4.30mg, along with GO (10.15mg) and PTC-NH2 (10.15mg) its compound gross mass
For 24.60mg, cumulative volume 15ml, GO-PB-PTC-NH is calculated2Concentration is 1.64mg/ml.
The building-up process is as shown in Equation 2：
The GO-PB-PTC-NH of embodiment 22Preparation
(1)PTC-NH2Preparation
1g PTCDA are dissolved in 3mL acetone, 5mL ethylenediamines are added dropwise, reaction is stirred at room temperature in fume hood
20min.Centrifuge, gained precipitated product drying at room temperature after ethanol, deionized water are washed respectively, produce red powder
PTC-NH2。
(2)GO-PB-PTC-NH2Preparation
Weigh 5.25mg GO and be dissolved in 10.5mL ultra-pure waters and form suspension, 4.05mg is added in above-mentioned suspension
FeCl3·6H2O, 2.47mg K3[Fe(CN)6], 111.825mg KCl and 0.075mL 2M HCl (offer sour environment),
PB is prepared in above-mentioned suspension and is combined with GO.By PB and GO magnetic agitation 12h at room temperature, expected mixed liquor is in darkcyan within second day
And suspension is evenly distributed.Add 15.75mg PTC-NH2, 12h, the 3rd day mixed liquor for collecting preparation are stirred overnight at room temperature
(color is in kermesinus), produces required GO-PB-PTC-NH2Solution, room temperature storage are stand-by.
The GO-PB-PTC-NH of embodiment 32Preparation
(1)PTC-NH2Preparation
1g PTCDA are dissolved in 7mL acetone, 15mL ethylenediamines are added dropwise, reaction is stirred at room temperature in fume hood
60min.Centrifuge, gained precipitated product drying at room temperature after ethanol, deionized water are washed respectively, produce red powder
PTC-NH2。
(2)GO-PB-PTC-NH2Preparation
Weigh 20.3mg GO and be dissolved in 20.3mL ultra-pure waters and form suspension, 4.05mg is added in above-mentioned suspension
FeCl3·6H2O, 9.88mg K3[Fe(CN)6], 111.825mg KCl and 0.075mL 2M HCl (offer sour environment),
PB is prepared in above-mentioned suspension and is combined with GO.By PB and GO magnetic agitation 12h at room temperature, expected mixed liquor is in darkcyan within second day
And suspension is evenly distributed.Add 6.77mg PTC-NH2, 12h, the 3rd day mixed liquor for collecting preparation are stirred overnight at room temperature
(color is in kermesinus), produces required GO-PB-PTC-NH2Solution, room temperature storage are stand-by.
The AuNPs of embodiment 4 preparation
0.5mL HAuCl are added in 50mL ultra-pure waters4·3H2O (1wt.%) solution, stirs, is heated to 95 DEG C,
1.25mL sodium citrate solutions (1wt.%) are added, stirs and continues 15min, liquid color to be mixed becomes aubergine, stops
Only boil, mixed liquor stirred into 15min to cooling down, obtains the nanogold particle of 15nm or so size, be stored in 4 DEG C it is standby.
The building-up process is as shown in Equation 3：
The preparation method of the nano antibody of embodiment 5
After being immunized using AFB1-BSA six camels of progress, the immune phage library for AFB1 is constructed.Afterwards, with
The biological screening of AFB1 specific nanos antibody selection is carried out based on display technique of bacteriophage.Filamenlous phage surface display libraries exist
Grown in 2 × TY culture mediums (tryptone and yeast extract containing 100 μ g/mL ampicillins and 2% glucose), and
30min VCSM13 helper phage infections are incubated at room temperature.Culture is centrifuged and removes supernatant.Precipitation is resuspended in
In 2 × TY culture mediums, 37 DEG C of overnight incubation 12h, phage library is enriched with.Then, to microtiter plate (every hole of antigen coat
20 μ g AFB1-BSA) carry out library selection.Negative control is coated with 20 μ g BSA.It will be washed from antigen coat with negative hole
De- phage particle serial dilution and for infecting TG1 Bacillus coli cells.By counting in square Petri dish
The number of the bacterium colony of upper growth come determine it is each biology translation wheel relative enrichment.Random pick out self enrichment wheel 95 are solely
Vertical bacterium colony, and enzyme linked immunosorbent assay (ELISA) (PE-ELISA) is extracted by pericentral siphon and analyzed.Eventually through positive bacterium colony
Sequencing confirms AFB1 specific nano antibody, and nano antibody is purified, and obtains the antibody of high-purity and high yield.
The nano antibody of embodiment 6 and the AFB1 monoclonal antibody performance comparisions of commercialization
(1) heat endurance of the AFB1 monoclonal antibodies with AFB1 nano antibodies of commercialization is studied.Respectively at 0 DEG C, 50 DEG C, 65 DEG C,
80 DEG C, 90~100 DEG C of water-bath 10min, the results showed that nano antibody bioactivity is higher than AFB1 monoclonal antibodies, the thermostabilization of nano antibody
Property is better than AFB1 monoclonal antibodies.In addition, AFB1 nano antibodies are placed in into 90 DEG C of processing for carrying out different durations respectively from AFB1 monoclonal antibodies, adopt
Antibody activity is analyzed with ELISA method, its absorbance is surveyed in 405nm, the activity of antibody is evaluated with absorbance size.Knot
Fruit also shows the high thermal stability of nano antibody.The result of the AFB1 monoclonal antibodies thermostabilization of AFB1 nano antibodies and commercialization experiment is shown in
Tables 1 and 2.
(2) tolerance of the AFB1 monoclonal antibodies of commercialization with AFB1 nano antibodies to organic solvent is studied.Using different organic
Solvent such as methanol, ethanol, acetone and dmso treatment, in the case of high concentration, the activity of AFB1 nano antibodies is above
AFB1 monoclonal antibodies, are analyzed antibody activity using ELISA method, and its absorbance is surveyed in 405nm, anti-to evaluate with absorbance size
The activity of body.As a result show that nano antibody has the AFB1 monoclonal antibodies of more preferable organic solvent tolerance, nano antibody and commercialization
3 are shown in Table to the tolerability results of organic solvent.
The relative activity of the AFB1 nano antibodies of table 1 and the AFB1 monoclonal antibodies of commercialization at different temperatures
The relative activity of the AFB1 nano antibodies of table 2 and the AFB1 monoclonal antibodies of commercialization under 90 DEG C of different times
The relative activity of the AFB1 nano antibodies of table 3 and the AFB1 monoclonal antibodies of commercialization to the tolerance test of organic solvent
Embodiment 7 determines the preparation of the immunity biosensor of aflatoxin B1
Schematic diagram is prepared as shown in figure 1, comprising the following steps：
(1) electrode pre-processes：By glass-carbon electrode respectively with 0.05 and 0.03 μm of Al2O3Powder-processed, nothing is then used respectively
Water-ethanol, ultra-pure water are cleaned by ultrasonic 5min；
(2)GO-PB-PTC-NH2Modified electrode：10 μ L 1.64mg/mL GO-PB-PTC-NH are taken with liquid-transfering gun2Solution drips
Pretreated glassy carbon electrode surface is added in, room temperature places 2h, and uncombined composite is cleaned with PBS solution, is dried, and utilizes
The larger specific surface area advantage of carbon nano-composite material makes AuNPS more be enriched on electrode；
(3) AuNPS fixation：The electrode that step (2) obtains is immersed in AuNPS solution, placed in 4 DEG C of refrigerators
3h, uncombined AuNPS is cleaned with PBS solution, is dried；
(4) it is covalently attached nano antibody：It is added dropwise what 10 μ L2mg/mL aflatoxin B1s nano antibodies obtained to step (3)
On electrode, aflatoxin B1 nano antibody is fixed on electrode surface using Au-S keys, is stood overnight in 4 DEG C of refrigerators, makes Huang
Aspertoxin B1 nano antibodies are fully connected with AuNPS, and uncombined nano antibody is cleaned with PBS solution, is dried；
(5) non-specific sites are closed：It is added dropwise on the electrode that 10 μ L 10mg/mLBSA solution obtain to step (4), room temperature
Lower placement 30min, some sites not combined by aflatoxin B1 nano antibody are closed, prevent Cucumber in testing sample
Non-specific binding occurs therewith, improves background signal, the BSA being not associated with PBS, dries；
(6) antigen is detected：It is added dropwise on the electrode that the μ L of determined antigen solution 10 obtain to step (5), reacts 12h at 4 DEG C, make
Fully with aflatoxin B1 nano antibody specific reaction occurs for antigen, and the peak point current obtained by immunoelectrode is strong and weak
Change to detect the concentration of antigen.
Embodiment 8 determines the preparation of the immunity biosensor of aflatoxin B1
Schematic diagram is prepared as shown in figure 1, comprising the following steps：
(1) electrode pre-processes：By glass-carbon electrode respectively with 0.05 and 0.03 μm of Al2O3Powder-processed, nothing is then used respectively
Water-ethanol, ultra-pure water are cleaned by ultrasonic 5min；
(2)GO-PB-PTC-NH2Modified electrode：20 μ L 1mg/mL GO-PB-PTC-NH are taken with liquid-transfering gun2Solution is added dropwise
Glassy carbon electrode surface after the pre-treatment, room temperature place 0.5h, and uncombined composite is cleaned with PBS solution, is dried, and utilize
The larger specific surface area advantage of carbon nano-composite material makes AuNPS more be enriched on electrode；
(3) AuNPS fixation：The electrode that step (2) obtains is immersed in 0.2mg/mL AuNPS solution, put at 10 DEG C
5h is put, uncombined AuNPS is cleaned with PBS solution, is dried；
(4) it is covalently attached nano antibody：20 μ L 1mg/mL aflatoxin B1s nano antibodies are added dropwise to obtain to step (3)
Electrode on, aflatoxin B1 nano antibody is fixed on electrode surface using Au-S keys, 10 DEG C of placement 6h, makes aspergillus flavus malicious
Plain B1 nano antibodies are fully connected with AuNPS, and uncombined nano antibody is cleaned with PBS solution, is dried；
(5) non-specific sites are closed：It is added dropwise on the electrode that 10 μ L 20mg/mL BSA solution obtain to step (4), room
Temperature is lower to place 10min, closes some sites not combined by aflatoxin B1 nano antibody, prevents some things in testing sample
Non-specific binding occurs therewith for matter, improves background signal, the BSA being not associated with PBS, dries；
(6) antigen is detected：It is added dropwise on the electrode that the μ L of determined antigen solution 10 obtain to step (5), reacts 12h at 4 DEG C, make
Fully with aflatoxin B1 nano antibody specific reaction occurs for antigen, and the peak point current obtained by immunoelectrode is strong and weak
Change to detect the concentration of antigen.
Embodiment 9 determines the preparation of the immunity biosensor of aflatoxin B1
Schematic diagram is prepared as shown in figure 1, comprising the following steps：
(1) electrode pre-processes：By glass-carbon electrode respectively with 0.05 and 0.03 μm of Al2O3Powder-processed, nothing is then used respectively
Water-ethanol, ultra-pure water are cleaned by ultrasonic 5min；
(2)GO-PB-PTC-NH2Modified electrode：10 μ L 2mg/mL GO-PB-PTC-NH are taken with liquid-transfering gun2Solution is added dropwise
Glassy carbon electrode surface after the pre-treatment, room temperature place 2h, and uncombined composite is cleaned with PBS solution, dries, utilizes carbon
The larger specific surface area advantage of nano composite material makes AuNPS more be enriched on electrode；
(3) AuNPS fixation：The electrode that step (2) obtains is immersed in 1mg/mL AuNPS solution, in 10 DEG C of placements
1h, uncombined AuNPS is cleaned with PBS solution, is dried；
(4) it is covalently attached nano antibody：10 μ L 3mg/mL aflatoxin B1s nano antibodies are added dropwise to obtain to step (3)
Electrode on, aflatoxin B1 nano antibody is fixed on electrode surface using Au-S keys, 4 DEG C of placement 18h, makes aspergillus flavus malicious
Plain B1 nano antibodies are fully connected with AuNPS, and uncombined nano antibody is cleaned with PBS solution, is dried；
(5) non-specific sites are closed：It is added dropwise on the electrode that 100 μ L 2mg/mL BSA solution obtain to step (4), room
Temperature is lower to place 60min, closes some sites not combined by aflatoxin B1 nano antibody, prevents some things in testing sample
Non-specific binding occurs therewith for matter, improves background signal, the BSA being not associated with PBS, dries；
(6) antigen is detected：It is added dropwise on the electrode that the μ L of determined antigen solution 10 obtain to step (5), reacts 12h at 4 DEG C, make
Fully with aflatoxin B1 nano antibody specific reaction occurs for antigen, and the peak point current obtained by immunoelectrode is strong and weak
Change to detect the concentration of antigen.
Embodiment 10 detects the immunity biosensor cyclic voltammogram of aflatoxin B1
The glass-carbon electrode that each step obtains in embodiment 7 is placed in containing 2mM K3[Fe(CN)6] 0.01M PBS it is molten
Cyclic voltammetry scan is carried out with 0.1V/s speed in liquid, as a result as shown in Figure 2, with GO-PB-PTC-NH2- AuNPs is compound
Thing modifies the peak current signal in glassy carbon electrode surface, obtaining an increase.Because GO-PB-PTC-NH2- AuNPs composite woods
Material can reduce electrode surface impedance value, improve electron transmission, and then improve the sensitivity of sensor.However, as nanometer resists
Body, BSA closings and the dropwise addition of antigen, peak point current is less and less, is because above-mentioned is protein, is not conductive materials, increases
The big impedance value of electrode surface so that current signal gradually reduces.It these results suggest that the trim success in each modification stage
It is fixed on electrode surface.
Embodiment 11 detects the line between the immunity biosensor peak current intensity of aflatoxin B1 and antigen concentration
Sexual intercourse is studied
The preparation method of immunity biosensor is with embodiment 7, the difference is that antigen concentration in step (6).Prepare different
The aflatoxin B1 standard liquid of concentration, respectively 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL and 100ng/mL,
Each concentration parallel control three times, detects peak current intensity.By data analysis, obtain between peak current intensity and antigen concentration
Linear relationship, as a result see Fig. 3.It is in good linear relationship between the logarithm and peak current intensity of aflatoxin B1 concentration,
With the continuous increase of aflatoxin B1 concentration, the peak current intensity of immunosensor constantly reduces therewith, has relatively low
Monitoring lower-cut (10pg/mL).《Measure-colloidal gold method of aflatoxin B1 in NY/T 2550-2014 feeds》Middle aspergillus flavus poison
Plain B1 detection is limited to 1.0ng/mL.The test limit of sensor of the present invention is higher by three orders of magnitude than agriculture standard, says
The bright sensor has higher sensitivity.
Embodiment 12 detects the interference free performance of the immunity biosensor of aflatoxin B1
The shadow that investigation AFB1 analogue AFB2 and AFG1 and solvent PBS solution detects to aflatoxin B1
Ring.The preparation method of immunity biosensor is with embodiment 7, the difference is that antigen, is as a result shown in Fig. 4.Under same concentrations i.e.
100ng/mL AFB1 measure signals are AFB2 respectively, 5.2 times, 9.4 times and 23.5 of AFG1 signals and blank PBS control solution
Times, illustrate that immunosensor of the present invention has higher specific performance, and determining with very high to AFB1
Selectivity.
Claims (10)
- A kind of 1. preparation method for the immunity biosensor for determining aflatoxin B1, it is characterised in that basal electrode surface Successively through GO-PB-PTC-NH2Solution, AuNPs solution and the modification of aflatoxin b1 antibody solution.
- 2. preparation method according to claim 1, it is characterised in that the basal electrode is glass-carbon electrode.
- 3. preparation method according to claim 1, it is characterised in that the GO-PB-PTC-NH2Solution is made as follows It is standby：(1) PTCDA is dissolved in acetone, adds ethylenediamine, 20~60min of reaction is stirred at room temperature, centrifuged, by precipitation second Alcohol, water cleaning, drying at room temperature, obtain powdered PTC-NH2；(2) GO is dissolved in ultra-pure water and forms suspension, FeCl is added in suspension3、K3[Fe(CN)6], KCl and HCl, Reaction 12h is stirred at room temperature；The PTC-NH that step (1) obtains is added thereto2, reaction 12h is stirred at room temperature, obtains GO-PB-PTC- NH2Solution.
- 4. preparation method according to claim 3, it is characterised in that the ratio of PTCDA and ethylenediamine in the step (1) For 1g:15mL~1g:5mL.
- 5. preparation method according to claim 3, it is characterised in that GO and PTC-NH in the step (2)2Mass ratio For 3:1~1:3.
- 6. preparation method according to claim 1, it is characterised in that the AuNPs solution is prepared as follows：Super 0.2~2wt.% HAuCl is added in pure water4·3H2O solution, be heated with stirring to 90~100 DEG C, thereto add 0.2~ 2wt.% sodium citrate solutions, stir and boil 5~25min, cooling is taken out after changing colour, obtains AuNPs solution.
- 7. preparation method according to claim 1, it is characterised in that the aflatoxin b1 antibody is aspergillus flavus poison Plain B1 nano antibodies.
- 8. preparation method according to claim 1, it is characterised in that comprise the following steps：(1) basal electrode polished, polished and be cleaned by ultrasonic；(2) by GO-PB-PTC-NH2Solution is added drop-wise to the electrode surface that step (1) obtains, and room temperature places 0.5~2h, molten with PBS Liquid cleans, and dries；(3) electrode that step (2) obtains is immersed in AuNPs solution, 1~10 DEG C of 1~5h of placement, is cleaned, dried in the air with PBS solution It is dry；(4) aflatoxin b1 antibody solution is added dropwise in the electrode surface obtained to step (3), 1~10 DEG C of 6~18h of placement, uses PBS solution is cleaned, and is dried；(5) BSA solution is added dropwise in the electrode surface obtained to step (4), and room temperature places 10~60min, cleaned, dried in the air with PBS solution It is dry.
- 9. the immunity biosensor that the preparation method described in claim 1-8 any one is prepared.
- 10. application of the immunity biosensor in quantitative detection aflatoxin B1 described in claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710513697.6A CN107422013A (en) | 2017-06-29 | 2017-06-29 | A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710513697.6A CN107422013A (en) | 2017-06-29 | 2017-06-29 | A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107422013A true CN107422013A (en) | 2017-12-01 |
Family
ID=60426665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710513697.6A Pending CN107422013A (en) | 2017-06-29 | 2017-06-29 | A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107422013A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318553A (en) * | 2018-02-06 | 2018-07-24 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
| CN109187963A (en) * | 2018-08-21 | 2019-01-11 | 成都师范学院 | A kind of method of preparation and use of the electrochemical immunosensor for aflatoxin B1 detection |
| CN110530853A (en) * | 2019-09-16 | 2019-12-03 | 南京工业大学 | Method based on visualization BPE-ECL technology detection aflatoxin B1 |
| CN111781263A (en) * | 2020-07-15 | 2020-10-16 | 桂林电子科技大学 | Preparation method of electrochemical immunosensor based on rGO/PB @ AuPtNPs nanocomposite |
| CN112179962A (en) * | 2020-09-29 | 2021-01-05 | 陕西科技大学 | Detection method of aflatoxin based on iron ion probe-nanogold/glassy carbon electrode modified electrode |
| CN113686935A (en) * | 2021-08-16 | 2021-11-23 | 江西农业大学 | Electrochemical sensing detection method and modified electrode for aflatoxin B1 in food |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105418760A (en) * | 2015-12-02 | 2016-03-23 | 南昌大学 | Nanobody aiming at aflatoxin |
| CN105466986A (en) * | 2015-11-17 | 2016-04-06 | 中国科学院电子学研究所 | Preparation method of electrochemical biosensor sensitive membrane |
| CN106324057A (en) * | 2016-08-01 | 2017-01-11 | 红河学院 | Method for detecting aflatoxin through immunosensor |
-
2017
- 2017-06-29 CN CN201710513697.6A patent/CN107422013A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105466986A (en) * | 2015-11-17 | 2016-04-06 | 中国科学院电子学研究所 | Preparation method of electrochemical biosensor sensitive membrane |
| CN105418760A (en) * | 2015-12-02 | 2016-03-23 | 南昌大学 | Nanobody aiming at aflatoxin |
| CN106324057A (en) * | 2016-08-01 | 2017-01-11 | 红河学院 | Method for detecting aflatoxin through immunosensor |
Non-Patent Citations (5)
| Title |
|---|
| DENG PAN,ET AL.: "Direct Immunoassay for Facile and Sensitive Detection of Small Molecule Aflatoxin B1 based on Nanobody", 《CHEM. EUR. J.》 * |
| JING ZHANG,ET AL.: "A novel immunosensor for detection of beta-galactoside alpha-2,6-sialyltransferase in serum based on gold nanoparticles loaded on Prussian blue-based hybrid nanocomposite film", 《ELECTROCHIMICA ACTA》 * |
| LIULIU GAO,ET AL.: "Ultrasensitive electrochemical biosensor based on graphite oxide, Prussian blue, and PTC-NH2 for the detection of α2,6-sialylated glycans in human serum", 《BIOSENSORS AND BIOELECTRONICS》 * |
| ZHONGYUAN LIU,ET AL.: "Highly sensitive, reagentless amperometric immunosensor based on a novel redox-active organic–inorganic composite film", 《SENSORS AND ACTUATORS B》 * |
| 高柳柳: "用于特殊糖型α2,6-唾液酸化聚糖检测的超灵敏电化学生物传感器的构建", 《重庆医科大学硕士学位论文》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318553A (en) * | 2018-02-06 | 2018-07-24 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
| CN108318553B (en) * | 2018-02-06 | 2019-11-12 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
| CN109187963A (en) * | 2018-08-21 | 2019-01-11 | 成都师范学院 | A kind of method of preparation and use of the electrochemical immunosensor for aflatoxin B1 detection |
| CN109187963B (en) * | 2018-08-21 | 2021-11-05 | 成都师范学院 | Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection |
| CN110530853A (en) * | 2019-09-16 | 2019-12-03 | 南京工业大学 | Method based on visualization BPE-ECL technology detection aflatoxin B1 |
| CN110530853B (en) * | 2019-09-16 | 2021-09-21 | 南京工业大学 | Method for detecting aflatoxin B1 based on visual BPE-ECL technology |
| CN111781263A (en) * | 2020-07-15 | 2020-10-16 | 桂林电子科技大学 | Preparation method of electrochemical immunosensor based on rGO/PB @ AuPtNPs nanocomposite |
| CN111781263B (en) * | 2020-07-15 | 2023-03-28 | 桂林电子科技大学 | Preparation method of electrochemical immunosensor based on rGO/PB @ AuPtNPs nanocomposite |
| CN112179962A (en) * | 2020-09-29 | 2021-01-05 | 陕西科技大学 | Detection method of aflatoxin based on iron ion probe-nanogold/glassy carbon electrode modified electrode |
| CN113686935A (en) * | 2021-08-16 | 2021-11-23 | 江西农业大学 | Electrochemical sensing detection method and modified electrode for aflatoxin B1 in food |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107422013A (en) | A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application | |
| Miao et al. | Ratiometric electrochemical immunosensor for the detection of procalcitonin based on the ratios of SiO2-Fc–COOH–Au and UiO-66-TB complexes | |
| Huang et al. | Portable and quantitative point-of-care monitoring of Escherichia coli O157: H7 using a personal glucose meter based on immunochromatographic assay | |
| CN108802133B (en) | A kind of preparation method and application detecting stomach neoplasms tumor markers interlayer type immunosensor | |
| Xu et al. | An electrochemical biosensor for rapid detection of E. coli O157: H7 with highly efficient bi-functional glucose oxidase-polydopamine nanocomposites and Prussian blue modified screen-printed interdigitated electrodes | |
| Yang et al. | Detection of Escherichia coli with a label-free impedimetric biosensor based on lectin functionalized mixed self-assembled monolayer | |
| Pan et al. | Direct immunoassay for facile and sensitive detection of small molecule aflatoxin B1 based on nanobody | |
| Zhuo et al. | Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification | |
| Liang et al. | A versatile nanozyme integrated colorimetric and photothermal lateral flow immunoassay for highly sensitive and reliable Aspergillus flavus detection | |
| Jain et al. | Development of an antibody functionalized carbon nanotube biosensor for foodborne bacterial pathogens | |
| CN106442994B (en) | A kind of preparation method and application of the electrochemical immunosensor based on Ag@Au nano composite materials | |
| Wang et al. | Rapid and ultrasensitive detection of Salmonella typhimurium using a novel impedance biosensor based on SiO2@ MnO2 nanocomposites and interdigitated array microelectrodes | |
| Hu et al. | Enzyme immunosensor based on gold nanoparticles electroposition and Streptavidin-biotin system for detection of S. pullorum & S. gallinarum | |
| Sun et al. | A novel electrochemical immunosensor for the highly sensitive and selective detection of the depression marker human apolipoprotein A4 | |
| CN104459124A (en) | Preparation method and application of electrochemical immunosensor based on HS-beta-CD-Ag-GOD conjugate | |
| CN104181299B (en) | A kind of preparation method of platinum hybrid cupric oxide multi-wall carbon nano-tube tube sensor and application | |
| CN107132260B (en) | A kind of electrochemical sensor based on nano material detection Ractopamine | |
| Taebi et al. | A novel method for sensitive, low-cost and portable detection of hepatitis B surface antigen using a personal glucose meter | |
| Ren et al. | An ultrasensitive squamous cell carcinoma antigen biosensing platform utilizing double-antibody single-channel amplification strategy | |
| CN109115855A (en) | A kind of preparation method and application for the electrochemical immunosensor detecting Alzheimer's disease marker | |
| Dong et al. | Broad-spectrum electrochemical immunosensor based on one-step electrodeposition of AuNP–Abs and Prussian blue nanocomposite for organophosphorus pesticide detection | |
| Wang et al. | Graphene-Prussian blue/gold nanoparticles based electrochemical immunoassay of carcinoembryonic antigen | |
| CN105067694B (en) | Preparation method and its detection method for the nano immune sensor of rapid detection of enterobacter sakazakii | |
| Koç et al. | Electrochemical investigation of gold based screen printed electrodes: an application for a seafood toxin detection | |
| Panhwar et al. | High-performance E. coli antibody-conjugated gold nanorods for the selective electrochemical detection of pathogens in drinking water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171201 |