CN105911123A - Electrochemical detection method for vomitoxin - Google Patents
Electrochemical detection method for vomitoxin Download PDFInfo
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- CN105911123A CN105911123A CN201610506059.7A CN201610506059A CN105911123A CN 105911123 A CN105911123 A CN 105911123A CN 201610506059 A CN201610506059 A CN 201610506059A CN 105911123 A CN105911123 A CN 105911123A
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- 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
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- 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 an electrochemical detection method for vomitoxin. The electrochemical detection method comprises the following steps: 1) modifying a single-wall carbon nanotube/chitosan compound on a glassy carbon electrode, and drying in air; 2) connecting antigen DON-BSA to the glassy carbon electrode, namely, washing the electrode by using a phosphate buffer liquid, soaking in a mixed solution of EDC and NHS, incubating, washing by using the phosphate buffer liquid, dropping and coating bovine serum albumin-vomitoxin coupling antigen to the surface of the electrode, incubating, washing, and sealing rest active sites by using a bovine serum albumin solution; 3) testing the content of the vomitoxin in a sample to be tested, namely, mixing an excessive vomitoxin antibody with a sample solution to be tested, dropping to the surface of the glassy carbon electrode, incubating, washing, adding a second antibody marked by alkaline phosphatase, incubating, washing, testing electrochemical signals, and calculating, thereby obtaining results. The electrochemical detection method is high in detection sensitivity, wide in detection range and capable of rapidly detecting vomitoxin of ultra-low content.
Description
Technical field
The invention belongs to technical field of electrochemical detection, be specifically related to the electrochemical detection method of a kind of vomitoxin.
Background technology
Vomitoxin (vomitoxin), also known as deoxynivalenol (DON), entitled 3 α of chemistry, 7 α, 15-trihydroxy grass Fusariumsp-9-alkene-8-ketone, molecular weight is 296.3, and molecular formula is C15H20O6, crystallize as colourless needles, fusing point is l5l～153 DEG C, α, and alpha, beta-unsaturated ketone base causes it to have absworption peak under short wavelength UV, is the one of trichothecene, for a kind of sesquiterpene derivant, owing to it can cause the vomiting of pig to gain the name.It is typically by being grown in the generation of frumentum article (such as Semen Tritici aestivi, Semen Maydis, Fructus Hordei Vulgaris and stover) mycete fusarubin.But this uv absorption overlaps with other many material uv absorption, the most distinctive.DON is heat-resisting, pressure, does not decomposes in weak acid, adds alkali and HIGH PRESSURE TREATMENT can destroy its part virulence.The resistance to Tibetan power of DON is the strongest.It is reported disease wheat through the storage of 4 years, DON therein remains to retain original toxicity.
DON is widely present in the whole world, mainly pollutes the cereal crops such as Semen Tritici aestivi, Fructus Hordei Vulgaris, Semen Maydis, also pollutes cereal product, and humans and animals can produce poisonous effect widely after eating by mistake by the Cereals class of this endotoxin contamination, and European Union's criteria for classification is three grades of carcinogens.It addition, it is often gone back and other mould halitoxin such as aflatoxin pollutes crops jointly, can influence each other after entering human body.Discovered in recent years DON may be relevant with human esophagus cancer, IgA nephropathy, constitutes a threat to the health of the mankind and animal.DON belongs to hypertoxic or medium poisonous substance, and research shows, DON may have certain accumulation in vivo, but without special target organ, has the strongest cytotoxicity.After people and animals have taken in the food/feedstuff polluted by DON, can cause anorexia, vomit, suffer from diarrhoea, have a fever, the acute poisoning symptom such as astasia, bradykinesia, time serious, infringement hemopoietic system causes death, but the sensitivity of DON is differed by different animals, and pig is most sensitive animal.Research shows, immune system may be had an impact by DON, has obvious fetal toxicity and certain teratogenesis, may have genetoxic, but without carcinogenic, mutagenic action.Owing to the harm of DON is serious, cause the most attention of various countries.In corn and feedstuff, the content of DON has strict limit standard.
The detection method of vomitoxin mainly has thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), T infrared spectrum analysis (IR), elisa (ELISA) at present.Although TLC is simple to operate, but the method processes sample workload greatly, needs to use in a large number organic solvent, all can adversely affect operator and environment；HPLC, GC and IR have automatization's height, high detection usefulness, vomitoxin can carry out qualitative and quantitative analysis, but equipment price is expensive, complex pretreatment, is not easy to popularization and application；ELISA have quick, sensitive, can the feature such as quantitative, easy and simple to handle, less demanding to sample purity, high specificity, but reaction reagent needs cryopreservation, result otherwise can be caused not accurate enough, and the repeatability of result is easily affected by operating process, the method detection time is longer in addition.
Summary of the invention
It is an object of the invention to for problem above provide a kind of detection sensitivity height, detection range width, quick, be able to detect that the electrochemical detection method of super low loading vomitoxin.
The electrochemical detection method of the technical scheme is that a kind of vomitoxin, comprises the steps:
1) modifying SWCN/chitosan complexes on glass-carbon electrode: by glass-carbon electrode polishing grinding, ultra-pure water cleans, and then cleans with acetone and nitric acid mixed liquor, again with ultrapure water, dry, by SWCN/chitosan dispersion drop coating in electrode surface, dry；
2) antigen DON-BSA is connected on glass-carbon electrode: by step 1) the electrode phosphate buffered solution dried rinses, it is soaked in the mixed solution of EDC and NHS, hatch 50～70 minutes for 33～40 DEG C, then glass-carbon electrode is rinsed by phosphate buffered solution, immediately by bovine serum albumin-vomitoxin coupled antigen drop coating in electrode surface, hatch 50～70 minutes for 33～40 DEG C, rinse by phosphate buffered solution, hatch the remaining avtive spot of closing with bovine serum albumin solution；
3) the vomitoxin content in testing sample is measured: by vomitoxin antibody and the testing sample solution mixing of excess, dropping in step 2) process after glassy carbon electrode surface, hatch 75～100 minutes for 33～40 DEG C, rinse by phosphate buffered solution, add the two of alkali phosphatase enzyme mark to resist, hatch 75～100 minutes for 33～40 DEG C, phosphate buffered solution is rinsed, carry out differential pulse voltammetry scanning, measure two electrochemically resistant signals of alkali phosphatase enzyme mark, i.e. can get the vomitoxin content in testing sample according to standard curve.
In technique scheme, described step 3) in standard curve preparation method be: by the vomitoxin antibody of excess and be mixed to get different mixed liquor respectively containing the mark liquid of variable concentrations vomitoxin, respectively by mixed liquor drip in abovementioned steps 2) process after glassy carbon electrode surface, hatch 75～100 minutes for 33～40 DEG C, phosphate buffered solution is rinsed, add the two of alkali phosphatase enzyme mark to resist, hatch 75～100 minutes for 33～40 DEG C, phosphate buffered solution carries out differential pulse voltammetry scanning after rinsing in the diethanolamine solution containing 0.75mg/mL alpha-phosphate naphthalene ester, measure two electrochemically resistant signals of alkali phosphatase enzyme mark, linear regression method is used to obtain vomitoxin content measuring standard curve.
As preferably, described step 1) in a diameter of 3mm, the polishing grinding of the described glass-carbon electrode Al of a diameter of 0.05 μm of glass-carbon electrode2O3Polishing powder is polished.
As preferably, described step 1) in SWCN/chitosan dispersion the concentration of SWCN/chitosan complexes be 1mg/mL, usage amount 2 μ L.
As preferably, the concentration of described phosphate buffered solution be 0.01mol/L, pH be 7.4.
As preferably, described step 2) in the mixed solution usage amount of EDC and NHS be 50 μ L, wherein containing 5mmol/L EDC, 2mmol/L NHS；Bovine serum albumin-vomitoxin coupled antigen concentration is 10 μ g/mL, and usage amount is 10 μ L.
As preferably, described step 3) in glass-carbon electrode in the diethanolamine solution containing 0.75mg/mL alpha-phosphate naphthalene ester, carry out differential pulse voltammetry scanning.
As preferably, described acetone and nitric acid mixed liquor are acetone and nitric acid 1:1 by volume is mixed to get.
The invention has the beneficial effects as follows: utilize SWCN/chitosan-modified electrochemical immunosensor that vomitoxin is carried out Electrochemical Detection first, the detection to super low loading vomitoxin can be realized, detection is limited to 5pg/mL, and the range of linearity is 10pg/mL～1000ng/mL.This method detection sensitivity is high, the detection range of linearity is wide, simple to operate, high specificity, detection is quick, result is accurate, and equipment needed thereby is simple, testing cost is low, has great actual application value.
Accompanying drawing explanation
Fig. 1 is the inventive method principle schematic.
Fig. 2 is the DPV curve chart of the electrochemical immunosensor of SWCN/chitosan-modified；Vomitoxin concentration is respectively as follows: (a) 0, (b) 10pg/mL, (c) 100pg/mL, (d) 500pg/mL, (e) 1ng/mL, (f) 10ng/mL, (g) 100ng/mL, (h) 1000ng/mL.
Fig. 3 is vomitoxin and the current signal linear graph of a relation of the inventive method.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
Main agents and Producer:
SWCN: Nanometer Port Co., Ltd., Shenzhen (Chinese)
Vomitoxin standard solution, chitosan, bovine serum albumin, 3-dimethylaminopropyl imines, N-hydroxy-succinamide, two anti-, alpha-phosphate naphthalene esters of alkali phosphatase enzyme mark: Sigma-Aldrich company (U.S.)
Bovine serum albumin-vomitoxin coupled antigen, vomitoxin antibody: Beijing Huaan Magnech Bio-Tech Co., Ltd. (Chinese)
Embodiment 1 electrochemical method detection vomitoxin
This method schematic diagram is as shown in Figure 1.Detection operation in accordance with the following steps:
1) on glass-carbon electrode, SWCN/chitosan complexes is modified: by the glass-carbon electrode of a diameter of 3mm Al of a diameter of 0.05 μm2O3Polishing powder is polished, then with ultra-pure water ultrasonic cleaning 5 minutes.Then with the acetone of volume ratio 1:1 and nitric acid mixed liquor ultrasonic cleaning 5 minutes cleaner with ultrapure water.After drying under room temperature, by 2 μ L 1mg/mL SWCNs/chitosan dispersion drop coating in the electrode surface cleaned up, naturally dry under room temperature, make dispersion liquid be fully contacted with glass-carbon electrode.SWCN/chitosan dispersion compound method: 5mg single pipe powder is added in the chitosan solution of 5mL 1mg/mL, to obtain homogeneous mixture, be stored in 4 DEG C before using in ultrasonic 2 hours.
2) antigen DON-BSA is connected on glass-carbon electrode: by step 1) the electrode 0.01mol/L of middle SWCN/chitosan complexes modified, after pH is the phosphate buffered solution flushing of 7.4, it is soaked in the mixed solution of 50 μ L 3-dimethylaminopropyl imines Han 5mmol/L (EDC) and 2mmol/L N-hydroxy-succinamide (NHS), 37 DEG C of baking ovens is hatched 1 hour with the carboxylic group on activation SWCN.Then rinse glass-carbon electrode by the phosphate buffered solution that 0.01mol/L, pH are 7.4, immediately 10 μ L 10 μ g/mL bovine serum albumin-vomitoxin coupled antigen (antigen DON-BSA) drop coating, in electrode surface, is hatched 1 hour in 37 DEG C of baking ovens.Then, after rinsing by the phosphate buffered solution that 0.01mol/L, pH are 7.4, it is further incubated for 30 minutes with 10 μ L 5mg/mL bovine serum albumin (BSA) solution, to close remaining avtive spot under room temperature.Antigen DON-BSA is by protein amino terminal and the activated carboxyl reaction forming on chitosan-modified SWCN.
3) standard curve is drawn: 5 μ L vomitoxin antibody (being diluted by finished product 1:150 by volume) and the 5 μ L mark liquid (preparing by phosphate buffered solution) containing 8 kinds of variable concentrations vomitoxins are mixed to get 8 portions of mixed liquors respectively, 8 kinds of concentration of vomitoxin mark liquid are: 0,10pg/mL, 100pg/mL, 500pg/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL.Respectively 8 portions of mixed liquors are dripped in step 2) process after glassy carbon electrode surface, 37 DEG C of baking ovens are hatched 90 minutes.Then rinse by the phosphate buffered solution that 0.01mol/L, pH are 7.4, add two anti-(being diluted by finished product 1:200 by volume) of 10 μ L alkali phosphatase enzyme marks, 37 DEG C of baking ovens are hatched 90 minutes.With 0.01mol/L, pH be 7.4 phosphate buffered solution rinse after carry out in diethanolamine (DEA) solution of the alpha-phosphate naphthalene ester containing 0.75mg/mL (α-NP) differential pulse voltammetry (DPV) scanning, measure alkali phosphatase enzyme mark two electrochemically resistant signals (as shown in Figure 2).With ip, vomitoxin concentration (C) mapping can be obtained ip-C working curve.Linear regression method is used to obtain ip-C equation of linear regression y=7.28107-1.58141x, wherein y is current signal strength, x is vomitoxin concentration, the concentration of vomitoxin is inversely proportional to ip in the range of 10pg/mL～1000ng/mL, linearly dependent coefficient is 0.9979, obtains vomitoxin content measuring standard curve as shown in Figure 3.With the concentration corresponding more than the current signal of noise signal 3 times as minimum detectability, being repeated 5 times above experiment and draw, the minimum detectability obtaining said method is 5pg/mL.
4) the vomitoxin content in testing sample is measured: by 5 μ L vomitoxin antibody (being diluted by finished product 1:150 by volume) and 5 μ L testing sample solutions (testing sample solution uses existing common technology to obtain testing sample extraction) mixing, dropping in step 2) process after glassy carbon electrode surface, 37 DEG C of baking ovens are hatched 90 minutes.Then rinse by the phosphate buffered solution that 0.01mol/L, pH are 7.4, add two anti-(being diluted by finished product 1:200 by volume) of 10 μ L alkali phosphatase enzyme marks, 37 DEG C of baking ovens are hatched 90 minutes.With 0.01mol/L, pH be 7.4 phosphate buffered solution rinse after carry out in diethanolamine (DEA) solution of the alpha-phosphate naphthalene ester containing 0.75mg/mL (α-NP) differential pulse voltammetry (DPV) scanning, measure two electrochemically resistant signals of alkali phosphatase enzyme mark, i.e. can get the vomitoxin content in testing sample according to standard curve.
Utilize said method, measure the vomitoxin in wheat samples: wheat flour to be measured first adds methanol-water and dissolves, add PBS again, then cross immune affinity column purification extraction and obtain testing sample solution, detect in aforementioned manners after testing sample solution is mixed with vomitoxin antibody.In actual sample, the determination of recovery rates of vomitoxin the results are shown in Table 1, and seen from the response rate from table, this detection method is accurately and reliably.
Table 1 Semen Tritici aestivi recovery of standard addition measures
* data are that 3 actual measurements are average.
Claims (8)
1. the electrochemical detection method of a vomitoxin, it is characterised in that comprise the steps:
1) on glass-carbon electrode, SWCN/chitosan complexes is modified: by glass-carbon electrode polishing grinding,
Ultra-pure water cleans, and then cleans with acetone and nitric acid mixed liquor, then with ultrapure water, dries, by single wall
Carbon Nanotubes/Chitosan dispersant liquid drop is applied to electrode surface, dries；
2) antigen DON-BSA is connected on glass-carbon electrode: by step 1) the electrode phosphate that dries delays
Dissolved liquid rinses, and is soaked in the mixed solution of EDC and NHS, hatches 50～70 minutes for 33～40 DEG C, so
Rinse glass-carbon electrode by phosphate buffered solution afterwards, immediately bovine serum albumin-vomitoxin coupled antigen is dripped
It is applied to electrode surface, hatches 50～70 minutes for 33～40 DEG C, rinse by phosphate buffered solution, pure with Sanguis Bovis seu Bubali
Protein solution hatches the remaining avtive spot of closing；
3) the vomitoxin content in testing sample is measured: by molten to vomitoxin antibody and the testing sample of excess
Liquid mix, dropping in step 2) process after glassy carbon electrode surface, hatch 75～100 minutes for 33～40 DEG C,
Rinse by phosphate buffered solution, add the two of alkali phosphatase enzyme mark and resist, hatch 75～100 points for 33～40 DEG C
Clock, phosphate buffered solution is rinsed, and carries out differential pulse voltammetry scanning, measures the two of alkali phosphatase enzyme mark
Electrochemically resistant signal, i.e. can get the vomitoxin content in testing sample according to standard curve.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
Step 3) in standard curve preparation method be: by excess vomitoxin antibody and containing variable concentrations vomiting poison
The mark liquid of element is mixed to get different mixed liquor respectively, respectively mixed liquor is dripped the most described step
Rapid 2) glassy carbon electrode surface after processing, hatches 75～100 minutes for 33～40 DEG C, and phosphate buffered solution is rushed
Wash, add the two of alkali phosphatase enzyme mark and resist, hatch 75～100 minutes for 33～40 DEG C, phosphate buffered solution
In the diethanolamine solution containing 0.75mg/mL alpha-phosphate naphthalene ester, differential pulse voltammetry scanning is carried out after flushing,
Measure two electrochemically resistant signals of alkali phosphatase enzyme mark, use linear regression method to obtain vomitoxin containing measuring
Calibration directrix curve.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
Step 1) in a diameter of 3mm of glass-carbon electrode, the polishing grinding of described glass-carbon electrode is by a diameter of 0.05 μm
Al2O3Polishing powder is polished.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
Step 1) in SWCN/chitosan dispersion the concentration of SWCN/chitosan complexes be
1mg/mL, usage amount 2 μ L.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
The concentration of phosphate buffered solution be 0.01mol/L, pH be 7.4.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
Step 2) in the mixed solution usage amount of EDC and NHS be 50 μ L, wherein containing 5mmol/L EDC, 2
mmol/LNHS；Bovine serum albumin-vomitoxin coupled antigen concentration is 10 μ g/mL, and usage amount is 10 μ L.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
Step 3) in glass-carbon electrode in the diethanolamine solution containing 0.75mg/mL alpha-phosphate naphthalene ester, carry out difference arteries and veins
Rush voltammetric scan.
The electrochemical detection method of vomitoxin the most according to claim 1, it is characterised in that described
Acetone and nitric acid mixed liquor are acetone and nitric acid 1:1 by volume is mixed to get.
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| CN106645344A (en) * | 2016-11-08 | 2017-05-10 | 湖南科技大学 | Preparation method and application of deoxynivalenol (DON) electrochemical sensor |
| CN106896147A (en) * | 2017-04-26 | 2017-06-27 | 重庆医科大学 | A kind of electrochemical sensor of Rapid Determination of Plasma indoles |
| CN112362710A (en) * | 2020-10-19 | 2021-02-12 | 湖北工业大学 | Preparation method and application of electrochemical immunosensor based on enzyme induction |
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Cited By (5)
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| CN106645344A (en) * | 2016-11-08 | 2017-05-10 | 湖南科技大学 | Preparation method and application of deoxynivalenol (DON) electrochemical sensor |
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| CN106896147A (en) * | 2017-04-26 | 2017-06-27 | 重庆医科大学 | A kind of electrochemical sensor of Rapid Determination of Plasma indoles |
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| CN112362710A (en) * | 2020-10-19 | 2021-02-12 | 湖北工业大学 | Preparation method and application of electrochemical immunosensor based on enzyme induction |
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Application publication date: 20160831 |