Method for detecting ultralow content of aflatoxin

Technical Field

The invention belongs to the field of detecting aflatoxin, in particular to a kind of detection of aflatoxin method for ultra-low content.

Background Art

Aflatoxin (aflatoxin, AFT) is mainly composed of Aspergillus flavus (Aspergillus   flavus) and parasitic aspegillus (Aspergillus   parasiticus), and the like of the secondary metabolite produced by the fungus. Aflatoxin is divided into B1, B2, G1, G2 and M1, M2 several subgenotypes, wherein AFB1 by the International agency for research on cancer as a class I carcinogen , toxicity most large, the potassium cyanide is 10 times, arsenic of 68 times, low doses of long-term intake of a large dose can be triggered a plurality of animal liver generating carcinogesis or acute poisoning. Low doses of long-term intake of a large dose can be triggered a plurality of animal liver generating carcinogesis or acute poisoning.

AFB1 the physico-chemical nature is very stable, in the high-temperature 268-269 the decomposition only when [...] , high-voltage 0.103 MPa (the 120 [...]) 4h treatment toxicity is reduced by one half only, molecular mass is 312.06u. AFB1 of the human body can be enriched through the food chain, to endanger the health of the human body, thus all countries in the world will AFB1 as a mandatory monitoring indicators.

The aflatoxin detection method is mainly composed of:

I, biological assay method (use of aflatoxin which can affect the microbial, aquatic animal, poultry and the like the organism to identify the cellular metabolism the presence of aflatoxin. Poor specificity of this method, the sensitivity is low, generally only as a chemical analysis method the corroboration, characterized in that the sample to be detected does not need to be very pure, is mainly used for qualitative);

II, the chemical analysis methods (the most commonly used for the thin-layer chromatography (TLC), mainly semiquantify);

III, instrument analysis method (high performance liquid chromatography (HPLC) and capillary electrophoresis technology, with automation, high separation efficiency, high detection efficiency, shortcoming is that the need of expensive equipment, complex operation);

IV, immune analysis method (radioimmunassay, affinity chromatography, enzyme-linked immunoradioassay and immune colloidal gold technology, has the advantages of simple operation, select-high, high specificity, and the like, is a relatively mature rapid diagnostic method, but sometimes the detecting sensitivity is not very ideal.

Content of the invention

The purpose of the embodiment of the invention is to provide a detection method for ultra-low content of aflatoxin, aiming at solving the current problem of detecting aflatoxin method sensitivity is not high, the problem of relatively narrow detection range.

The embodiment of the invention is realized like this, a kind of detection of aflatoxin method for ultra-low content, the method comprises the following steps:

Preparing glassy carbon electrode, the glassy carbon electrode on the chemical modification of a layer of single-walled carbon nanotube, by covalently linking agent;

Bovine serum albumin-aflatoxin AFB1-BSA coupling, forming the antigen;

Antigen AFB1-BSA and single-wall carbon nanotube is connected with the reaction of the activated carboxyl group;

The sandwich method competition using antigen-antibody reaction with the added aflatoxin monomer to competitive adsorption of a resistance to, obtain two anti-;

Two anti-alkaline phosphatase-labeled;

Determining alkaline phosphatase-labeled secondary anti-electrochemical signal, draw the standard curve, the actual sample aflatoxin concentration.

Furthermore, by using the method for covalently linking agent: using 3-dimethyl amino propyl imine (EDC)/ N-hydroxy succinimide (NHS) legitimate covalent bond linking agent.

Furthermore, antigen AFB1-BSA on the single-walled carbon nanotube is connected with the activated carboxyl group for reaction:

Antigen AFB1-BSA amino terminal through a protein with the single-walled carbon nanotubes is connected with the reaction of the activated carboxyl group.

Furthermore, mark two anti-alkaline phosphatase (EC3.1.3 . 1) can be adopted: glutaraldehyde cross-linked labeling method

The present invention provides detection of aflatoxin method of ultra low content of simple operation, high selectivity, high specificity, can be simple and rapid detection of aflatoxin content, the necessary apparatus is simple and easy, low cost, is of great practical use value and good development of space.

Description of drawings

Figure 1 is method flowchart of the embodiment of the invention provides detection of aflatoxin ultra low content.

Figure 2 is a schematic diagram of the chemical reaction process of the embodiment of the invention provides detection of aflatoxin ultra low content;

Figure 3 is aflatoxin B1 content measuring standard curve a schematic diagram of the embodiment of the invention provides.

Mode of execution

For the purpose of the present invention, the technical scheme and more clearly understand, the embodiment of the following combination, the further detailed description of the invention. It should be understood, the specific embodiment described in order to explain the invention only, is not used to limit the invention.

Figure 1 shows the present invention provides ultra-low the detection of aflatoxin content of the flow of the method. In order to facilitate the description, only shown the part of the connection with the present invention.

As shown in Figure 1, an embodiment of the present invention provides detection of aflatoxin ultra low content of the method comprises the following steps:

Preparing glassy carbon electrode, the glassy carbon electrode on the chemical modification of a layer of single-walled carbon nanotube, by covalently linking agent;

Bovine serum albumin-aflatoxin AFB1-BSA coupling, forming the antigen;

Antigen AFB1-BSA and single-wall carbon nanotube is connected with the reaction of the activated carboxyl group;

The sandwich method competition using antigen-antibody reaction with the added aflatoxin monomer to competitive adsorption of a resistance to, obtain two anti-;

Two anti-alkaline phosphatase-labeled;

Determining alkaline phosphatase-labeled secondary anti-electrochemical signal, draw the standard curve, the actual sample aflatoxin concentration.

As the embodiment of the invention optimized scheme, the method of using linking agent is covalently bonded: using 3-dimethyl amino propyl imine (EDC)/ N-hydroxy succinimide (NHS) legitimate covalent bond linking agent.

As the embodiment of the invention optimized scheme, antigen AFB1-BSA on the single-walled carbon nanotube is connected with the activated carboxyl group for reaction:

Antigen AFB1-BSA amino terminal through a protein with the single-walled carbon nanotubes is connected with the reaction of the activated carboxyl group.

As the embodiment of the invention optimized scheme, mark two anti-alkaline phosphatase can adopt: glutaraldehyde cross-linked labeling method.

Table 1 peanut and determining the recovery rate

* Data is 3 times the average measured

In conjuction with the lower and the specific embodiment of the principle of the application of this invention for further description.

Use of glassy carbon electrode, and is modified by a chemical method on the electrode a layer of single-walled carbon nanotube, and utilizing 3-dimethyl amino propyl imine (EDC)/ N-hydroxy succinimide (NHS) covalently linking agent. The conductive properties of the use of single-walled carbon nanotubes, so that the modified glassy carbon electrode can measure ultra-low content aflatoxin. Bovine serum albumin-aflatoxin coupling, forming the antigen (AFB1-BSA) with the single-walled carbon nanotubes by protein amino activation of the carboxyl reaction connection, and at the same time through the antigen-antibody reaction of the aflatoxin monomer to competitive adsorption an anti-(sandwich method competition law), then adding and determining alkaline phosphatase-labeled secondary anti-electrochemical signal draw the standard curve and measured sample aflatoxin concentration. At the same time optimizing AFB1-BSA this method, an anti-, two anti-equal concentration detection limit of this invention can reach 5pg/ml, at the same time from the standard curve 10pg/ml to 100ng/ml, compared with the method in the past with the detection limit is low, wide range of concentration.

For this invention the stated above the better practical example , is not used to limit the invention, where in the present invention within the spirit and principle of the any modifications made by the, equivalent replacement and improved, and the like, shall be included in the scope of protection of this invention.