Measurement method for corn aflatoxin
Aflatoxin, abbreviated as AFT in English, is a compound composed of a difuranyl ring and coumarin (oxanaphthoquinone). It is produced by strains such as Aspergillus flavus and Aspergillus parasiticus, with a molecular weight of 312-346 and a melting point of 200-300 ℃. It has stable and high temperature resistant properties and can produce fluorescence under long wave ultraviolet light. It is divided into B1, B2, G1, G2, M1, M2, P1, R1, GM, and toxic alcohols according to different fluorescence colors, RF values, and structures.
AFT is insoluble in water, ethane, ether, and petroleum ether, but easily soluble in organic solvents such as methanol, ethanol, chloroform, acetonitrile, and dimethylformamide. AFT can decompose slightly in acidic solvents with a pH of 1-3, rapidly decompose and break down in solutions with a pH of 9-10, rapidly decompose when exposed to alkali, and decompose into almost non-toxic salts in alkaline solutions with a pH of 9-10. It can be reduced under acidic conditions.
Objective of the experiment
Aflatoxin refers to some highly toxic carcinogenic substances produced by fungi. Short term intake of high doses (1 mg/kg body weight) can cause acute poisoning, common liver dysfunction symptoms such as fever, vomiting, jaundice, or liver necrosis. Long term exposure to low doses (20 micrograms/kg body weight) can significantly increase the risk of liver cancer, gastric cancer, and other diseases. As it is widely present in crops and food and poses great harm to humans and animals, establishing rapid and accurate detection methods is of great significance for ensuring food safety and public health.
The purpose of this experiment is to use the principle of solid-phase enzyme-linked immunosorbent assay (ELISA), namely enzyme-linked immunosorbent assay (ELISA), to limit and quantify the content of aflatoxin B1 in samples, providing scientific basis for food contamination risk assessment.
Experimental apparatus
① ST-2000A mycotoxin analyzer
② Corn, homogenizer, nitrogen drying device, oscillator, centrifuge, graduated pipette, balance (sensitivity 0.01g), micropipette, methanol, n-hexane, chloroform or dichloromethane reagent
Experimental Procedure
① Check the equipment used in the experiment to ensure that it is clean, dry, and free from contamination
② Mix methanol and deionized water in a 7:3 ratio to prepare the sample extraction solution
③ Take 2g of crushed corn sample and place it in a 50ml centrifuge tube. Add 5ml of sample extraction solution and shake for 5 minutes at room temperature of 4000 rpm for 10 minutes. After that, take 0.5ml of supernatant and add 0.5ml of deionized water. Mix well
④ Remove the required reagents from the 4 ℃ refrigerated environment, let them equilibrate at room temperature for 30 minutes, and shake well. Take out the required number of microplates and frames, and dilute 20 times the concentrated washing solution with deionized water to make the working washing solution.
⑤ Number the corresponding micropores of the sample and standard in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Add 50 µ l/well of standard or sample to their respective micropores, then add 50 µ l/well of enzyme marker, and then add 50 µ l/well of antibody working solution. Seal the plate with a cover membrane, gently shake for 5 seconds, and mix well. React at 25 ℃ for 30 minutes.
⑥ Remove the cover film, shake off the liquid inside the hole, and thoroughly wash it 5 times with 250 µ l of working detergent per hole, with a 30 second interval between each wash. Use absorbent paper to pat dry
⑦ Add substrate solution A (50 µ l) to each well, followed by substrate solution B (50 µ l). Shake for 5 seconds and mix well. Color at 25 ℃ in the dark for 15 minutes.
⑧ Add 50 µ l of termination solution to each well, gently shake and mix well to terminate the reaction.
⑨ Measure the absorbance value of each well at 450nm using an aflatoxin analyzer (dual wavelength 450/630nm is recommended). The measurement should be completed within 10 minutes after the termination of the reaction
⑩ The instrument completes the measurement and automatically provides the result
Experimental results
After testing, the aflatoxin content of the sample is approximately 0.19ug/kg, which meets the GB 2761-2017 "National Food Safety Standard for Fungal Toxins Limits in Food".