CN103175874A - A kind of detection method of aflatoxin B1 - Google Patents
A kind of detection method of aflatoxin B1 Download PDFInfo
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- CN103175874A CN103175874A CN2013100530183A CN201310053018A CN103175874A CN 103175874 A CN103175874 A CN 103175874A CN 2013100530183 A CN2013100530183 A CN 2013100530183A CN 201310053018 A CN201310053018 A CN 201310053018A CN 103175874 A CN103175874 A CN 103175874A
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Abstract
本发明属于电化学检测领域，特别涉及一种黄曲霉毒素B1的检测方法,绘制标准曲线：将黄曲霉毒素B1样品与有机溶剂共孵育，配置成不同浓度的黄曲霉毒素B1标准品，在标准品中分别加入阻抗溶液；用多壁碳纳米管/离子液体/抗体修饰电极测得阻抗溶液中不同AFB1浓度的相应的阻抗值，绘制标准曲线；将待测样品提纯后与有机溶剂共孵育，配成待测样品溶液，然后再加入阻抗溶液，用预处理过的修饰电极测得阻抗溶液的阻抗值，再根据标准曲线，计算待测样品中AFB1的浓度。本发明检测黄曲霉毒素B1的时间短，检测步骤精简、检测仪器简单、待测样品前处理也比较简单。而且本检测方法的选择性好、响应速度快、灵敏度高、检测范围宽。
The invention belongs to the field of electrochemical detection, in particular to a detection method for aflatoxin B1 , drawing a standard curve: co-incubating aflatoxin B1 samples with organic solvents, and configuring aflatoxin B1 standard products with different concentrations , add impedance solution to the standard respectively; use multi-walled carbon nanotubes/ionic liquid/antibody modified electrode to measure the corresponding impedance values of different AFB 1 concentrations in the impedance solution, and draw a standard curve; Co-incubated with solvents to prepare the sample solution to be tested, then added the impedance solution, measured the impedance value of the impedance solution with the pretreated modified electrode, and calculated the concentration of AFB 1 in the sample to be tested according to the standard curve. The detection time of the aflatoxin B1 of the present invention is short, the detection steps are simplified, the detection instrument is simple, and the pretreatment of the samples to be tested is also relatively simple. Moreover, the detection method has good selectivity, fast response speed, high sensitivity and wide detection range.
Description
Technical field
The invention belongs to the 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.The aflatoxin separated at present and derivant thereof existing kind more than 20, wherein AFB
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, extensively are 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 in the seeds 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, all found AFB
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
110 μ g/kg must not be surpassed in allowance vegetable oil (except corn, peanut oil), 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, all be 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 realizing the impedance spectrum in wide frequency ranges very as a kind of electrochemical detection method of rapid sensitive because of it, AC impedance electrochemical technology carrys out the Electrode system, and the Electrochemical Measurement Technology as a kind of not damaged, original position electrode process, be widely used in the research of electrode interface characteristic.Carbon nano-tube, because the characteristics such as good electric conductivity, high specific surface area and thermodynamic stability are arranged, is widely used; Ionic liquid is the complete liquid by negative ion and cation composition, the material consisted of ion be in a liquid state at room temperature or near room temperature temperature.
Summary of the invention
The purpose 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 purpose 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; Record different AFBs in impedance solution with multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode
1the corresponding resistance value of concentration, then take the impedance difference as ordinate, AFB
1standard sample concentration is horizontal ordinate, the drawing standard curve;
(2) the testing sample purification is rear and organic solvent is hatched altogether, be made into testing sample solution, and then add impedance solution, record the resistance value in solution with pretreated multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, 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; Also contain the K that concentration is 0.5-1.5mmol/L in described phosphate buffered solution
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 the methanol aqueous solution stratification, takes off a layer mixed liquor and adds the 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 in described step (1) and step (2)/antibody modification electrode is
(a) multi-walled carbon nano-tubes that is 100-300nm by length and 1-butyl-3-methyl-imidazoles hexafluorophosphate mixes, magnetic agitation, then adds AFB
1the reaction of monoclonal antibody shaking table, make the multi-walled carbon nano-tubes mixed liquor that ionic liquid wraps up;
(b) mixed liquor made 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
3the burnishing 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 and gets final product 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 the 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.
The 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, follow the Al with 0.3 μ m, 0.05 μ m
2o
3burnishing powder polishing glass-carbon electrode, wash 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 the use redistilled water is centrifugal, washing multi-walled carbon nano-tubes mixed liquor is extremely neutral, obtains the multi-walled carbon nano-tubes of length distribution between 100-300nm after drying, standby; The 1-butyl of multi-walled carbon nano-tubes after 0.5mg is interrupted and 0.25mL-3-methyl-imidazoles hexafluorophosphate mixes and magnetic agitation 1 hour, add again the aflatoxin B1 monoclonal antibody 0.25mL that concentration is 1.64 μ g/mL, shaking table reaction 0.5 hour, make the multi-walled carbon nano-tubes mixed liquor that ionic liquid wraps up; The multi-walled carbon nano-tubes mixed liquor 4 μ L that the ionic liquid made is wrapped up drip to the glass-carbon electrode surface of handling well, at (the Na that wherein in phosphate buffered solution, contains 87mmol/L 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 on in refrigerator two hours and gets final product.
2, Specification Curve of Increasing method:
AFB by Different Weight
1sample, add 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
1add impedance solution 15mL in standard sample, then the above-mentioned multi-walled carbon nano-tubes/ionic liquid made/antibody modification electrode is placed on to (the phosphate buffered solution that this impedance solution is pH value=7.4 in impedance solution, 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 the electrochemical AC impedance method, measure resistance value, the AFB1 standard sample concentration of take is horizontal ordinate, take the impedance difference as ordinate, the drawing standard curve.The specific standards curve as shown in Figure 1.
3, the AFB in testing sample
1the assay method of concentration:
(1) extract AFB in testing sample
1
Testing sample of the present invention is olive oil, takes and mixes olive oil sample 10g in the beaker of 20mL, uses the 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 the funnel eck by the little funnel dehydration that 5g anhydrous slufuric acid sodium solution is housed, and wetting with a small amount of methenyl choloride), wash funnel and filter in volumetric flask with a small amount of methenyl choloride, in mixed liquor, the concentration of institute's test sample product is 1g/mL.
(2) measure AFB
1concentration
Solution to be measured 4 μ L in step (1) 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 made/antibody modification electrode is placed on to (the phosphate buffered solution that this impedance solution is pH value=7.4 in impedance solution, 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 the electrochemical AC impedance method, measure resistance value, according to typical curve, by the impedance difference recorded, 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, be good linear relationship in the concentration range of electronics transfer resistance from 0.1ng/mL to 10ng/mL, linearly dependent coefficient is 0.994, detection is limited to 0.1ng/mL, after AFB1 concentration arrives 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 with the experiment of the precision of different batches, repeat 5 times, in batch and batch between relative standard deviation all be less than 5.0%.
(2) AFB
1the recovery detect
Extract in sample and add respectively the AFB that concentration is 2ng/mL, 4ng/mL, 7ng/mL at olive oil
1after standard items, with 37 ℃ of methenyl cholorides, hatch 25 minutes, measure the AC impedance value, calculate corresponding aflatoxin concentration by linear equation Ret=1651.9C+600.56 on calibration curve, with measuring concentration and adding the ratio of concentration to draw the 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 (10)
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; Record the corresponding resistance value of aflatoxin B1 concentration different in impedance solution with multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, then take the impedance difference as ordinate, AFB
1standard sample concentration is horizontal ordinate, the drawing standard curve;
(2) the testing sample purification is rear and organic solvent is hatched altogether, be made into testing sample solution, and then add impedance solution, record the resistance value of impedance solution with pretreated multi-walled carbon nano-tubes/ionic liquid/antibody modification electrode, according to the typical curve of gained in step (1), calculate AFB in testing sample again
1concentration.
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; Also contain the K that concentration is 0.5-1.5mmol/L in described phosphate buffered solution
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 claim 1,3 or 4 described AFBs
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 for purification of testing sample is, testing sample is cleaned with sherwood oil, add the methanol aqueous solution stratification, take off a layer mixed liquor and add the metabisulfite solution dilution, add again 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.
9. AFB according to claim 1
1detection method, it is characterized in that: the preparation method of the multi-walled carbon nano-tubes/ionic liquid in described step (1) and step (2)/antibody modification electrode is,
(a) multi-walled carbon nano-tubes that is 100-300nm by length and 1-butyl-3-methyl-imidazoles hexafluorophosphate mixes, then adds AFB
1the reaction of monoclonal antibody shaking table, make the multi-walled carbon nano-tubes mixed liquor that ionic liquid wraps up;
(b) mixed liquor made in step (a) is added drop-wise to through pretreated glass-carbon electrode surface, standby after rinsing by phosphate buffered solution.
10. AFB according to claim 9
1detection method, it is characterized in that: in described step (a), 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.
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| CN104330452A (en) * | 2014-11-14 | 2015-02-04 | 福州大学 | Soft material modified screen-printed electrode as well as preparation method and application of soft material modified screen-printed electrode |
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| CN106093139A (en) * | 2016-06-29 | 2016-11-09 | 中国科学院长春应用化学研究所 | A kind of detection method of concentration of methanol solution |
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| CN113686935A (en) * | 2021-08-16 | 2021-11-23 | 江西农业大学 | Electrochemical sensing method and modified electrode for aflatoxin B1 in food |
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| RU2534732C1 (en) * | 2013-07-26 | 2014-12-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Национальный исследовательский Томский политехнический университет" | Method for quantitative determination of aflatoxin b1 by differential voltammetry |
| CN104181294A (en) * | 2013-12-02 | 2014-12-03 | 重庆医科大学 | Method for detecting ultralow content of aflatoxin |
| US20170212104A1 (en) * | 2014-07-15 | 2017-07-27 | C2Sense, Inc. | Formulations for enhanced chemiresistive sensing |
| CN104330452A (en) * | 2014-11-14 | 2015-02-04 | 福州大学 | Soft material modified screen-printed electrode as well as preparation method and application of soft material modified screen-printed electrode |
| CN104330452B (en) * | 2014-11-14 | 2017-09-22 | 福州大学 | A kind of screen printing electrode of soft material modification and preparation method and application |
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| CN106093173A (en) * | 2016-06-13 | 2016-11-09 | 合肥工业大学 | A kind of electrochemical sensor, preparation method and the application in quickly detection AFB1 thereof |
| CN106093139A (en) * | 2016-06-29 | 2016-11-09 | 中国科学院长春应用化学研究所 | A kind of detection method of concentration of methanol solution |
| CN112179962A (en) * | 2020-09-29 | 2021-01-05 | 陕西科技大学 | A kind of detection method of aflatoxin based on iron ion probe-nano gold/glassy carbon electrode modified electrode |
| CN112179962B (en) * | 2020-09-29 | 2023-06-16 | 陕西科技大学 | A detection method of aflatoxin based on iron ion probe-nano gold/glassy carbon electrode modified electrode |
| CN113686935A (en) * | 2021-08-16 | 2021-11-23 | 江西农业大学 | Electrochemical sensing method and modified electrode for aflatoxin B1 in food |
| CN113686935B (en) * | 2021-08-16 | 2023-01-31 | 江西农业大学 | Electrochemical sensing and detection method and modified electrode of aflatoxin B1 in food |
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