CN103175874B - Aflatoxin B1 detecting method - Google Patents

Aflatoxin B1 detecting method Download PDF

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Publication number
CN103175874B
CN103175874B CN201310053018.3A CN201310053018A CN103175874B CN 103175874 B CN103175874 B CN 103175874B CN 201310053018 A CN201310053018 A CN 201310053018A CN 103175874 B CN103175874 B CN 103175874B
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solution
afb
impedance
organic solvent
testing sample
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CN103175874A (en
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贾能勤
于利利
张洋
吴慧
杨亚云
黄楚森
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Shanghai Normal University
University of Shanghai for Science and Technology
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Abstract

The invention belongs to the field of electrochemical detection and particularly relates to an aflatoxin B1 detecting method. The method comprises the steps of: drawing a standard curve: co-incubating an aflatoxin B1 sample and organic solvent to prepare aflatoxin B1 standard substances with different concentrations, respectively adding an impedance solution in the standard substances and measuring the corresponding impedance values of the impedance solution with different AFB1 concentrations by a multi-walled carbon nanotube/ionic liquid/antibody modified electrode, thus drawing the standard curve; and purifying a to-be-detected sample, co-incubating the to-be-detected sample and the organic solvent to prepare a to-be-detected sample solution, then adding the impedance solution, measuring the impedance value of the impedance solution by a pre-treated modified electrode, and then calculating the AFB1 concentration in the to-be-detected sample according to the standard curve. According to the method, time for detecting the aflatoxin B1 is short, and the detecting steps, detecting instruments and the pre-treatment of the to-be-detected sample are simple. Besides, the method is good in selectivity, high in response speed, high in sensitivity, and wide in detecting range.

Description

A kind of AFB 1detection method
Technical field
The invention belongs to Electrochemical Detection field, particularly a kind of AFB 1detection method.
Background technology
Aflatoxin is mainly by multiple mycetogenetic toxic metabolites such as Aspergillus flavus and aspergillus parasiticus bacterium, contains a bifuran and a coumarin in its structure.Aflatoxin is the strongest class biotoxin of toxicity of finding so far, has very strong toxicity, carcinogenicity, mutagenicity and teratogenesis.Separated aflatoxin and derivant thereof have kind more than 20, wherein AFB at present 1(AFB 1) toxicity, carcinogenicity, pollution rate maximum, and be the most stable a kind of of its chemical property in various mycotoxins, general food processing means are minimum to the destructiveness of its generation, are extensively present in foodstuff grain and processed food, very big to human health risk.A large amount of research datas show: grain, edible oil (peanut oil, rapeseed oil, soybean oil, corn oil), extraordinary oil, and as all found AFB in the seed of olive oil, camellia wet goods oil crops and processed goods, fruit, dry fruit, vegetables, flavouring, tobacco and herbal medicine, dairy products and fermentation series products 1existence.
Each state has all stipulated the limit standard of various mycotoxins and has strengthened supervision.European Union is defined in total aflatoxin content in the food such as corn, peanut, nut and cereal can not surpass 4ppb, wherein AFB 1can not surpass 2ppb; AFB in China's food 1in allowance vegetable oil (except corn, peanut oil), 10 μ g/kg must not be surpassed, in other grains, beans, fermented food and dispensed food for baby, 5 μ g/kg must not be surpassed.Although countries in the world are not quite similar to aflatoxin limit standard in food, be all tending towards lower control line.The common technology that detects at present aflatoxin is HPLC, ELISA, and TLC etc., but these detection techniques are all existing expensive, consuming time long, the deficiency such as operating process is complicated of sample pre-treatments complexity, instrument and equipment in varying degrees.And AC impedance electrochemical technology is carried out Electrode system as a kind of electrochemical detection method of rapid sensitive because of its impedance spectrum that can realize in wide frequency ranges very, and the Electrochemical Measurement Technology as a kind of not damaged, original position electrode process, is widely used in the research of electrode interface characteristic.Carbon nano-tube, because there being the features such as good electric conductivity, high specific surface area and thermodynamic stability, is widely used; Ionic liquid is the complete liquid by negative ion and cation composition, the material consisting of ion being in a liquid state at room temperature or near room temperature temperature.
Summary of the invention
The object of this invention is to provide a kind of AFB 1(AFB 1) detection method, the method selectivity is good, fast response time, highly sensitive, sensing range is wide.
The object of invention can realize by following scheme:
A kind of AFB 1detection method, its step comprises:
(1) drawing standard curve: aflatoxin B1 sample and organic solvent are hatched altogether, be configured to the AFB of variable concentrations 1standard items add respectively impedance solution in standard items; With multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, record different AFBs in impedance solution 1the corresponding resistance value of concentration, then take impedance difference as ordinate, AFB 1standard sample concentration is horizontal ordinate, drawing standard curve;
(2) testing sample purification is rear and organic solvent is hatched altogether, be made into testing sample solution, and then add impedance solution, with pretreated multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, record the resistance value in solution, according to the typical curve of gained in step (1), calculate AFB in testing sample again 1concentration.
On calibration curve, by linear equation, be Ret=1651.9C+600.56
In formula: Ret--impedance difference (ohm)
C--aflatoxin concentration (ng/ml)
Impedance solution in described step (1) and step (2) is phosphate buffered solution, and the pH value of this solution is 6.8-8.0, and concentration is 0.5-1.5mmol/L; In described phosphate buffered solution, also contain the K that concentration is 0.5-1.5mmol/L 4fe (CN) 6or K 3fe (CN) 6kCl with 0.05-0.15mol/L.Preferably, the pH value of described phosphate buffered solution is 7.0-7.8, and concentration is 0.8-1.2mmol/L, also contains the K that concentration is 0.8-1.2mmol/L in described phosphate buffered solution 4fe (CN) 6or K 3fe (CN) 6kCl with 0.08-1.12mol/L.Preferred, the pH value of described phosphate buffered solution is 7.4, and concentration is 1mmol/L, also contains the K that concentration is 1mmol/L in described phosphate buffered solution 4fe (CN) 6or K 3fe (CN) 6kCl with 0.1mol/L.
In described step (1), by the known AFB of variable concentrations 1sample solution is hatched altogether 20-30 minute with organic solvent respectively under 35 ℃ of-40 ℃ of conditions, wherein, and AFB 1with the proportioning that adds of organic solvent be: 1-10ng:1mL; In described step (2), testing sample solution and organic solvent after purifying are hatched altogether to 20-30 minute under 35 ℃ of-40 ℃ of conditions, wherein, the proportioning that adds of testing sample and organic solvent is 2-6ng:1mL; Described organic solvent is methyl alcohol, methenyl choloride or acetone.
In described step (1), the volume ratio of standard items and impedance solution is 1:5-20; In described step (2), the volume ratio of testing sample solution and impedance solution is 1:5-20.
In described step (2), the method for purification of testing sample is, testing sample is cleaned with sherwood oil, adds methanol aqueous solution stratification, takes off a layer mixed liquor and adds metabisulfite solution dilution, then add methenyl choloride standing, by lower floor's mixed liquor processed.
In described step (1), the volume ratio of standard items and impedance solution is 1:5-20; In described step (2), the volume ratio of testing sample solution and impedance solution is 1:5-20.
The proportioning that adds of described testing sample, methyl alcohol, sodium sulphate, methenyl choloride is 10g:40-45mL:1-2g:8-12mL.
The preparation method of the multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode in described step (1) and step (2) is
(a) multi-walled carbon nano-tubes that is 100-300nm by length and 1-butyl-3-methyl-imidazoles hexafluorophosphate mix, magnetic agitation, then add AFB 1the reaction of monoclonal antibody shaking table, makes the multi-walled carbon nano-tubes mixed liquor that ionic liquid wraps up;
(b) mixed liquor making in step (a) is added drop-wise to through pretreated glass-carbon electrode surface, standby after rinsing by phosphate buffered solution.Preferably, the preprocess method of described glass-carbon electrode for by metallographic paper polishing glass-carbon electrode for, polish after at the Al that uses respectively 0.3 μ m and 0.05 μ m 2o 3burnishing powder polishing; Then by electrode successively at the HNO of 7.25mol/L 3ultrasonic processing in solution, absolute ethyl alcohol and redistilled water; Finally electrode is dried up with nitrogen.
In described step (a), the proportioning that adds of multi-walled carbon nano-tubes, 1-butyl-3-methyl-imidazoles hexafluorophosphate and aflatoxin B1 monoclonal antibody is: 1.2-2.4mg:1mL:0.6-1 μ g.
Compared with prior art, the invention has the beneficial effects as follows: 1, the detection time of aflatoxin B1 provided by the invention short, detecting step is simplified, whole testing process is 10 minutes, detecting instrument is simple, cheap, and testing sample pre-treatment is also fairly simple.2, good, the fast response time of this detection method selectivity, highly sensitive, sensing range are 0.1ng/mL-10ng/mL.
Accompanying drawing explanation
Fig. 1 is AFB 1the canonical plotting of standard sample.
Fig. 2 is the AFB of multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode and variable concentrations 1ac impedance spectroscopy.
Fig. 3 is impedance difference and AFB 1the linear relationship chart of concentration.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
The present embodiment equipment used: electrochemical workstation CHI660B type.
Embodiment 1
1, the preparation of multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode:
With 2 #, 5 #abrasive paper for metallograph polishing grinding glass-carbon electrode, follows the Al with 0.3 μ m, 0.05 μ m 2o 3burnishing powder polishing glass-carbon electrode, washes away surface contaminants, the HNO that is then 7.25mol/L in concentration successively 3, in absolute ethyl alcohol, redistilled water ultrasonic 2 minutes respectively, finally by electrode with nitrogen dry up, standby.
By length, be 1-2 μ m, the multi-walled carbon nano-tubes that caliber is 10-20nm is placed in (dense H among mixed acid solution 2sO 4with the mixed acid solution of dense HNO3, dense H wherein 2sO 4with dense HNO 3volume ratio be 3:1) continuous ultrasound 16 hours.Then, washing multi-walled carbon nano-tubes mixed liquor centrifugal with redistilled water be to neutral, obtains the multi-walled carbon nano-tubes of length distribution between 100-300nm after dry, standby; 1-butyl-3-methyl-imidazoles hexafluorophosphate of multi-walled carbon nano-tubes after 0.5mg is interrupted and 0.25mL mixes and magnetic agitation 1 hour, adding concentration is the aflatoxin B1 monoclonal antibody 0.25mL of 1.64 μ g/mL again, shaking table reaction 0.5 hour, makes the multi-walled carbon nano-tubes mixed liquor that ionic liquid wraps up; The multi-walled carbon nano-tubes mixed liquor 4 μ L of the ionic liquid parcel making are dripped to the glass-carbon electrode surface of handling well, at (the Na that wherein contains 87mmol/L in phosphate buffered solution of the phosphate buffered solution with 10mmol/L 2hPO 4, 14mmol/L KH 2pO 4, the NaCl of 137mmol/L and the KCl of 2.7mmol/L) rinse out unconjugated antibody, finally glass-carbon electrode is placed in refrigerator to two hours.
2, Specification Curve of Increasing method:
By the AFB of Different Weight 1sample, adds methenyl choloride, under 37 ℃ of conditions, hatches altogether 25 minutes, makes respectively the AFB that concentration is 1ng/mL, 3ng/mL, 5ng/mL, 7ng/mL, 10ng/mL 1standard sample, at AFB 1in standard sample, add impedance solution 15mL, then the above-mentioned multi-walled carbon nano-tubes/ionic liquid making/antibody modification electrode is placed on in impedance solution, (this impedance solution is the phosphate buffered solution of pH value=7.4, concentration is 1mmol/L, also contains the K that concentration is 1mmol/L in described phosphate buffered solution 4fe (CN) 6or K 3fe (CN) 6kCl with 0.1mol/L), by electrochemical AC impedance method, measure resistance value, the AFB1 standard sample concentration of take is horizontal ordinate, take impedance difference as ordinate, drawing standard curve.Specific standards curve as shown in Figure 1.
3, the AFB in testing sample 1the assay method of concentration:
(1) in testing sample, extract AFB 1
Testing sample of the present invention is olive oil, takes and mixes olive oil sample 10g in the beaker of 20mL, uses 10ml sherwood oil, pours in separating funnel repeated washing 5 times after sample is cleaned into; Add 50mL methanol aqueous solution (wherein the volume ratio of methyl alcohol and water is 4:1) and shake 2-3 minute, stratification, taking off a layer methanol aqueous solution proceeds in another separating funnel, add 50mL aqueous sodium persulfate solution (concentration is 20g/L) dilution, add methenyl choloride 10mL, jog 2-3 minute, stratification, take off layer mixed liquor and (with filter paper, clog funnel eck by the little funnel dehydration of 5g anhydrous slufuric acid sodium solution is housed, and wetting with a small amount of methenyl choloride), with a small amount of methenyl choloride, wash funnel and filter in volumetric flask, in mixed liquor, the concentration of institute's test sample product is 1g/mL.
(2) measure AFB 1concentration
Solution to be measured in step (1) 4 μ L and 1mL methenyl choloride are hatched altogether and within 25 minutes, made testing sample solution under 37 ℃ of conditions, add impedance solution 15mL, then the above-mentioned multi-walled carbon nano-tubes/ionic liquid making/antibody modification electrode is placed on in impedance solution, (this impedance solution is the phosphate buffered solution of pH value=7.4, concentration is 1mmol/L, also contains the K that concentration is 1mmol/L in described phosphate buffered solution 4fe (CN) 6or K 3fe (CN) 6kCl with 0.1mol/L), by electrochemical AC impedance method, measure resistance value, according to typical curve, by the impedance difference recording, calculate the AFB in institute's test sample product 1concentration.
The precision test of method of testing provided by the invention:
(1) AFB 1quantitative detection
According to detection method of the present invention, the sensing range of AFB1 is 0.1-10ng/mL, and specifically as shown in Figures 2 and 3, wherein Fig. 2 is multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode and AFB 1concentration is a:0.1ng/mL, b:1ng/mL, c:3ng/mL, d:5ng/mL, e:7ng/mL, f:10ng/mL, 7 ac impedance spectroscopies of g:13ng/mL, Fig. 3 is converted into Fig. 2 the linear relationship chart of impedance difference and AFB1 concentration, as can be seen from Figure, in the concentration range of electronics transfer resistance from 0.1ng/mL to 10ng/mL, be good linear relationship, linearly dependent coefficient is 0.994, detection is limited to 0.1ng/mL, when AFB1 concentration arrives after 13ng/mL, keeping stable no longer increases, illustrate that upper limit of detection arrives 10ng/mL, reach the interior requirement that detects actual sample of scope of GB regulation.Do same batch and test with the precision of different batches, repeat 5 times, the relative standard deviation in batch and between criticizing is all less than 5.0%.
(2) AFB 1the recovery detect
In olive oil extraction sample, adding respectively concentration is the AFB of 2ng/mL, 4ng/mL, 7ng/mL 1after standard items, hatch 25 minutes with 37 ℃ of methenyl cholorides, measure AC impedance value, on calibration curve, by linear equation Ret=1651.9C+600.56, calculate corresponding aflatoxin concentration, with measuring concentration and adding the ratio of concentration to draw actual sample recovery of standard addition, specifically see the following form.
As can be seen from the table, the mean value of the recovery of standard addition of three samples is 105%, illustrate this detection method can be used for detecting in sample AFB 1content, and this method is highly sensitive, sensing range is wide.

Claims (8)

1. an AFB 1detection method, its step comprises:
(1) drawing standard curve: by AFB 1sample and organic solvent are hatched altogether, are configured to the AFB of variable concentrations 1standard items add respectively impedance solution in standard items; With multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, record the corresponding resistance value of aflatoxin B1 concentration different in impedance solution, then take impedance difference as ordinate, AFB 1standard sample concentration is horizontal ordinate, drawing standard curve;
(2) testing sample purification is rear and organic solvent is hatched altogether, be made into testing sample solution, and then add impedance solution, with pretreated multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, record the resistance value of impedance solution, according to the typical curve of gained in step (1), calculate AFB in testing sample again 1concentration;
The preparation method of described multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode is,
(a) multi-walled carbon nano-tubes that is 100-300nm by length and 1-butyl-3-methyl-imidazoles hexafluorophosphate mix, then add AFB 1the reaction of monoclonal antibody shaking table, makes the multi-walled carbon nano-tubes mixed liquor that ionic liquid wraps up; Described multi-walled carbon nano-tubes, 1-butyl-3-methyl-imidazoles hexafluorophosphate and AFB 1the proportioning that adds of monoclonal antibody is: 1.2-2.4mg:1mL:0.6-1 μ g;
(b) mixed liquor making in step (a) is added drop-wise to through pretreated glass-carbon electrode surface, standby after rinsing by phosphate buffered solution.
2. AFB according to claim 1 1detection method, it is characterized in that: the impedance solution in described step (1) and step (2) is phosphate buffered solution, and the pH value of this solution is 6.8-8.0, and concentration is 0.5-1.5mmol/L; In described phosphate buffered solution, also contain the K that concentration is 0.5-1.5mmol/L 4fe (CN) 6or K 3fe (CN) 6kCl with 0.05-0.15mol/L.
3. AFB according to claim 2 1detection method, it is characterized in that: in described step (1), by the known AFB of variable concentrations 1sample solution is hatched altogether 20-30 minute with organic solvent respectively under 35 ℃ of-40 ℃ of conditions, and wherein, the proportioning that adds of aflatoxin B1 and organic solvent is: 1-10ng:1mL.
4. AFB according to claim 2 1detection method, it is characterized in that: in described step (2), testing sample solution and organic solvent after purifying are hatched altogether to 20-30 minute under 35 ℃ of-40 ℃ of conditions, wherein, the proportioning that adds of testing sample and organic solvent is 2-6 μ g:1mL.
5. according to the AFB described in claim 1,3 or 4 1detection method, it is characterized in that: described organic solvent is methyl alcohol, methenyl choloride or acetone.
6. AFB according to claim 2 1detection method, it is characterized in that: in described step (1), the volume ratio of standard items and impedance solution is 1:5-20; In described step (2), the volume ratio of testing sample solution and impedance solution is 1:5-20.
7. AFB according to claim 2 1detection method, it is characterized in that: in described step (2), the method of purification of testing sample is, testing sample is cleaned with sherwood oil, add methanol aqueous solution stratification, take off a layer mixed liquor and add metabisulfite solution dilution, then add methenyl choloride standing, by lower floor's mixed liquor processed.
8. AFB according to claim 7 1detection method, it is characterized in that: the proportioning that adds of described testing sample, methyl alcohol, sodium sulphate, methenyl choloride is 10g:40-45mL:1-2g:8-12mL.
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