CN104374913B - A kind of preparation method of competitive type Aspergillus flavus toxin immuno sensor and application - Google Patents
A kind of preparation method of competitive type Aspergillus flavus toxin immuno sensor and application Download PDFInfo
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- CN104374913B CN104374913B CN201410622503.2A CN201410622503A CN104374913B CN 104374913 B CN104374913 B CN 104374913B CN 201410622503 A CN201410622503 A CN 201410622503A CN 104374913 B CN104374913 B CN 104374913B
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Abstract
The invention belongs to food inspection and biosensor technique field, it discloses a kind of competitive type immunosensor of quick detection aflatoxin.Its production program and detection method are: take screen printing electrode as working electrode, one deck Nano silver grain is modified by electrodeposition process, then the antigen mixed solution of aflatoxin antibody, bovine serum albumin(BSA), aflatoxin and supported copper ion hydroxyapatite mark is added successively, with dilute sulfuric acid solution load copper ion hydroxyapatite, the copper ion of its inside is discharged, the detection of aflatoxin is made to be converted into the detection of copper ion, achieve higher sensitivity, detectability can be low to moderate 0.1ng/kg.
Description
Technical field
The invention belongs to food inspection and biosensor technique field, be specifically related to a kind of screen printing electrode immunosensor of quick detection aflatoxin.
Background technology
Aflatoxin (Aflatoxins) is the metabolic product of aspergillus flavus and aspergillus parasiticus, mainly contains B1, B2, G1, G2 and other two kinds of metabolic products M1, M2.Wherein M1 and M2 separates from milk.B1, B2, G1, G2, M1 and M2 are very close on molecular structure.
Aflatoxin is the strongest carcinogen found at present, larger than the ability of induced by dimethylnitrosamine liver cancer 75 times of its carcinogenicity, and the median lethal dose of AFB l is 0.36mg/kg body weight, belongs to the poison range of special severe toxicity.It causes the poisoning of people mainly to damage liver, hepatitis cirrhosis occurs, hepatonecrosis etc.Clinical manifestation is had a stomach upset, anorexia, n and V, abdominal distension and hepatic region tenderness etc., and severe patient occurs that oedema is gone into a coma, dead down to twitching.
Aflatoxin almost cannot be avoided in agricultural product, and in agricultural production, the corn that aflatoxin exceeds standard is much.National health department forbids that enterprise uses the grain be heavily polluted to carry out food processing and production, and formulates relevant standard supervision enterprise execution.Even if but aflatoxin content is very low still can cause infringement to health, be difficult to control.
Nineteen ninety-five, the food aflatoxin maximum permissible concentration that the World Health Organization (WHO) formulates is 15 μ g/kg; Federal Government relevant laws specify that aspergillus flavus poison content (referring to the total amount of B1+B2+G1+G2) in human consumption's food and milk cow forage can not more than 15 μ g/kg; Content in the milk of human consumption can not more than 0.5 μ g/kg, and the content in other animal feeds can not more than 300 μ g/kg.And European Union member countries' regulation is more strict, in peanut and nut and converted products, all cereal preparations and converted products, aflatoxin B1 limitation is 2.0 μ g/kg; In former milk, thermal treatment milk and processing dairy products, M1 limitation is 0.050 μ g/kg; In baby food (comprising infant's milk), M1 limitation is 0.025 μ g/kg.
The detection that most domestic testing agency is all using thin film chromatography and liquid phase chromatography to carry out aflatoxin.Because its sense cycle is long, the shortcomings such as reagent needed for program complexity is various can not meet modern measure requirement far away.The present invention adopts screen printing electrode, cheap, combined with electrochemical technology and competitive immunoassay method, improves the selectivity of sensor and substantially increases the sensitivity of detection, saving detection time, reduce testing cost.
Summary of the invention
The object of the invention is to avoid that the instrument and equipment of traditional detection method is complicated, operating process is loaded down with trivial details, testing staff requires the shortcomings such as high, testing cost is high, provide the preparation method of screen printing electrode immunosensor of a kind of highly sensitive, high specificity, favorable reproducibility, detection aflatoxin easy and simple to handle and cheap and easy to get.
To achieve these goals, the present invention is realized by following measures.Technical scheme of the present invention, comprises the following steps.
1. a preparation method for competitive type Aspergillus flavus toxin immuno sensor, comprises the following steps:
(1) preparation of the hydroxyapatite labelled antigen of supported copper ion
Be the ratio mixing of 1:1 ~ 5 with mass ratio by the nanometer hydroxyapatite of particle diameter 20 ~ 60nm and copper sulphate, be dissolved in 5 ~ 15mL water, shake 12 ~ 24 hours, centrifuging;
Get the nanometer hydroxyapatite of the supported copper ion that 5 ~ 10 mg obtain, be dissolved in 5 ~ 15mL phosphate buffered solution, add the aflatoxin antigenic solution that 0.1 ~ 0.2ml concentration is 1 μ g/mL, hatching concussion 4 ~ 8 hours, centrifuging;
The hydroxyapatite labelled antigen of the supported copper ion of acquisition is rejoined 5 ~ 15mL phosphate buffered solution, and adds the bovine serum albumin(BSA) of 1mg, hatching concussion 4 ~ 8 hours, centrifuging;
Precipitation is re-dispersed in 5 ~ 10mL phosphate buffered solution, namely obtains the storing solution of the hydroxyapatite labelled antigen of supported copper ion;
(2) preparation of competitive type Aspergillus flavus toxin immuno sensor
Screen printing electrode adds the aqueous solution that 50 μ L contain 0.05mmol/L silver nitrate, 0.2mmol/L sodium citrate and 0.05mol/L potassium nitrate, connect electrochemical workstation, potentiostatic electrodeposition is carried out with-0.2V, sedimentation time is 30 ~ 180s, dry, the screen printing electrode that obtained Nano silver grain is modified;
Screen printing electrode drips the aflatoxin antibody-solutions that 5 ~ 10 μ L concentration are 1 μ g/mL, hatches 1 ~ 4 hour, and clean up by phosphate buffered solution;
The concentration that screen printing electrode drips 5 ~ 10 μ L is 1% bovine serum albumin solution, hatches 1 ~ 4 hour, and cleans up by phosphate buffered solution;
The volume ratio that screen printing electrode drips 5 ~ 10 μ L is the aflatoxin of 1 ~ 5:1, the hydroxyapatite labelled antigen mixed solution of supported copper ion, hatch 1 ~ 4 hour, and clean up by phosphate buffered solution, i.e. obtained competitive type Aspergillus flavus toxin immuno sensor.
2. a preparation method for competitive type Aspergillus flavus toxin immuno sensor, the detection for aflatoxin comprises the following steps:
(1) drafting of working curve
The aflatoxin of variable concentrations is adopted to prepare a series of screen printing electrode immunosensor according to the method described in 1;
By contrast electrode, be connected on electrochemical workstation to electrode and working electrode, add the acetate buffer solution of 50 μ L in a cell, then add 5 ~ 10 μ L 1% dilution heat of sulfuric acid, by the hydroxyapatite of supported copper ion dissolve, discharge copper ion;
Set potential range as-0.6 ~ 0.4V, sedimentation potential is-0.8V, and sedimentation time is 100 ~ 300s, is detected the current-responsive of the screen printing electrode modified by Anodic Stripping linear voltammetry;
According to the relation of gained current-responsive and aflatoxin concentration, drawing curve;
(2) detection of aflatoxin
Adopt the aflatoxin in 5 ~ 10 μ L testing samples step of replacing (1), other is identical with step (1);
According to measured numerical value, the working curve of contrast aflatoxin, calculates the content of aflatoxin in sample.
the present invention possesses following advantage:
(1) the supported copper ion hydroxyapatite that the present invention adopts has good biocompatibility, it dissolves in acid solution simultaneously, original position discharges copper ion, do not need transfer and dilution, thus improve detection sensitivity, make the detectability of aflatoxin can be low to moderate 0.1 ng/kg, far below testing requirement
(2) the present invention does not need pre-treatment, fast, accurately, can be used for the detection of the aflatoxin in complex sample.
Embodiment
embodiment one
The preparation of the hydroxyapatite labelled antibody of supported copper ion of the present invention, step is as follows:
(1) by the nanometer hydroxyapatite of particle diameter 30nm and copper sulphate with mass ratio be the ratio mixing of 1:3, be dissolved in 5mL water, shake 12 hours, centrifuging;
(2) get the nanometer hydroxyapatite of the supported copper ion that 5mg obtains, be dissolved in 5mL phosphate buffered solution, add the aflatoxin antibody-solutions that 0.1ml concentration is 1 μ g/mL, hatching concussion 4 hours, centrifuging;
(3) the hydroxyapatite labelled antibody of the supported copper ion of acquisition is rejoined 5mL phosphate buffered solution, and add the bovine serum albumin(BSA) of 1mg, hatching concussion 4 hours, centrifuging;
(4) precipitation is re-dispersed in 5mL phosphate buffered solution, namely obtains the storing solution of the hydroxyapatite labelled antibody of supported copper ion.
embodiment two
The preparation of competitive type Aspergillus flavus toxin immuno sensor of the present invention, step is as follows
(1) on screen printing electrode, add the aqueous solution that 50 μ L contain 0.05mmol/L silver nitrate, 0.2mmol/L sodium citrate and 0.05mol/L potassium nitrate, connect electrochemical workstation, potentiostatic electrodeposition is carried out with-0.2V, sedimentation time is 120s, dry, the screen printing electrode that obtained Nano silver grain is modified;
(2) on screen printing electrode, drip the aflatoxin antibody-solutions that 10 μ L concentration are 1 μ g/mL, hatch 1 hour, and clean up by phosphate buffered solution;
(3) concentration dripping 10 μ L on screen printing electrode is 1% bovine serum albumin solution, hatches 1 hour, and cleans up by phosphate buffered solution;
(4) volume ratio dripping 10 μ L on screen printing electrode is the hydroxyapatite labelled antigen mixed solution of the aflatoxin of 2:1, supported copper ion, hatch 1 hour, and clean up by phosphate buffered solution, i.e. obtained competitive type Aspergillus flavus toxin immuno sensor.
embodiment three
Arbitrary described a kind of competitive type Aspergillus flavus toxin immuno sensor in embodiment 1 ~ 2, for the detection of aflatoxin, comprises the following steps:
(1) aflatoxin of variable concentrations is adopted to prepare a series of screen printing electrode immunosensor according to described method arbitrary in embodiment 1 ~ 2;
(2) by contrast electrode, be connected on electrochemical workstation to electrode and working electrode, add the acetate buffer solution of 50 μ L in a cell, add the dilution heat of sulfuric acid of 1% of 5 ~ 10 μ L again, the hydroxyapatite of supported copper ion is dissolved, discharges copper ion;
(3) set potential range as-0.6 ~ 0.4V, sedimentation potential is-0.8V, and sedimentation time is 100 ~ 300s, is detected the current-responsive of the screen printing electrode modified by Anodic Stripping linear voltammetry;
(4) according to the relation of gained current-responsive and aflatoxin concentration, drawing curve;
embodiment four
Adopt the aflatoxin in 10 μ L testing samples replacement embodiments three, other is identical with embodiment three;
According to measured numerical value, the working curve of contrast aflatoxin, calculates the content of aflatoxin in sample.
Claims (2)
1. a preparation method for competitive type Aspergillus flavus toxin immuno sensor, is characterized in that, comprises the following steps:
(1) preparation of the hydroxyapatite labelled antigen of supported copper ion
Be the ratio mixing of 1:1 ~ 5 with mass ratio by the nanometer hydroxyapatite of particle diameter 20 ~ 60nm and copper sulphate, be dissolved in 5 ~ 15mL water, shake 12 ~ 24 hours, centrifuging;
Get the nanometer hydroxyapatite of the supported copper ion that 5 ~ 10 mg obtain, be dissolved in 5 ~ 15mL phosphate buffered solution, add the aflatoxin antigenic solution that 0.1 ~ 0.2ml concentration is 1 μ g/mL, hatching concussion 4 ~ 8 hours, centrifuging;
The hydroxyapatite labelled antigen of the supported copper ion of acquisition is rejoined 5 ~ 15mL phosphate buffered solution, and adds the bovine serum albumin(BSA) of 1mg, hatching concussion 4 ~ 8 hours, centrifuging;
Precipitation is re-dispersed in 5 ~ 10mL phosphate buffered solution, namely obtains the storing solution of the hydroxyapatite labelled antigen of supported copper ion;
(2) preparation of competitive type Aspergillus flavus toxin immuno sensor
Screen printing electrode adds the aqueous solution that 50 μ L contain 0.05mmol/L silver nitrate, 0.2mmol/L sodium citrate and 0.05mol/L potassium nitrate, connect electrochemical workstation, potentiostatic electrodeposition is carried out with-0.2V, sedimentation time is 30 ~ 180s, dry, the screen printing electrode that obtained Nano silver grain is modified;
Screen printing electrode drips the aflatoxin antibody-solutions that 5 ~ 10 μ L concentration are 1 μ g/mL, hatches 1 ~ 4 hour, and clean up by phosphate buffered solution;
The concentration that screen printing electrode drips 5 ~ 10 μ L is 1% bovine serum albumin solution, hatches 1 ~ 4 hour, and cleans up by phosphate buffered solution;
The volume ratio that screen printing electrode drips 5 ~ 10 μ L is the aflatoxin of 1 ~ 5:1, the hydroxyapatite labelled antigen mixed solution of supported copper ion, hatch 1 ~ 4 hour, and clean up by phosphate buffered solution, i.e. obtained competitive type Aspergillus flavus toxin immuno sensor.
2. the sensor that prepared by preparation method as claimed in claim 1 is detecting the application in aflatoxin, comprises the following steps:
(1) drafting of working curve
The aflatoxin of variable concentrations is adopted to prepare a series of competitive type Aspergillus flavus toxin immuno sensor according to the method described in claim 1;
By contrast electrode, be connected on electrochemical workstation to electrode and working electrode, add the acetate buffer solution of 50 μ L in a cell, then add 5 ~ 10 μ L 1% dilution heat of sulfuric acid, by the hydroxyapatite of supported copper ion dissolve, discharge copper ion;
Set potential range as-0.6 ~ 0.4V, sedimentation potential is-0.8V, and sedimentation time is 100 ~ 300s, is detected the current-responsive of the screen printing electrode modified by Anodic Stripping linear voltammetry;
According to the relation of gained current-responsive and aflatoxin concentration, drawing curve;
(2) detection of aflatoxin
Adopt the aflatoxin in 5 ~ 10 μ L testing samples step of replacing (1), other is identical with step (1);
According to measured numerical value, the working curve of contrast aflatoxin, calculates the content of aflatoxin in sample.
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| CN108593742B (en) * | 2018-05-03 | 2020-08-18 | 南京工业大学 | Electrochemical aptamer sensor for quantitatively detecting aflatoxin B1 and application thereof |
| CN110865185B (en) * | 2019-08-19 | 2023-03-14 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method for detecting ochratoxin A based on copper ion fluorescent probe indirect competition method |
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| CN103196984A (en) * | 2013-03-13 | 2013-07-10 | 济南大学 | Preparation method and application of sensor for simultaneous detection of multiple aflatoxins |
| CN103196973A (en) * | 2013-03-13 | 2013-07-10 | 济南大学 | Method for preparing seven-channel aflatoxin immunosensor and application |
| CN203204000U (en) * | 2013-04-23 | 2013-09-18 | 济南大学 | Immunosensor for simultaneously detecting aflatoxin B1 and ochratoxin A |
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| CN103196984A (en) * | 2013-03-13 | 2013-07-10 | 济南大学 | Preparation method and application of sensor for simultaneous detection of multiple aflatoxins |
| CN103196973A (en) * | 2013-03-13 | 2013-07-10 | 济南大学 | Method for preparing seven-channel aflatoxin immunosensor and application |
| CN203204000U (en) * | 2013-04-23 | 2013-09-18 | 济南大学 | Immunosensor for simultaneously detecting aflatoxin B1 and ochratoxin A |
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