Application Of Aflatoxin B₁ Colloidal Gold Immunochromatography In Traditional Chinese Medicine

Aflatoxin B₁（aflatoxin B₁）is one of aflatoxin mainly produced by Aspergillus flavusand.and Aspergillus parasiticus.Among these mycotoxins,aflatoxin B₁ is the most widely distributed,most harmful and most closely related to human health.In1993,the International Agency for Research on Cancer（IARC）designated AFB₁ as the Group I carcinogen,which poses a serious threat to human health.At present,many countries in the world attach great importance to the detection and control of AFB₁.Many countries around the world have established limits to ensure the safety of foods and drugs to protect human health.Most of the current testing methods for AFB₁ require specialized equipment and technicians,and the operation is too cumbersome.Therefore,it is important to establish a simple,fast and accurate AFB₁detection method.Based on the colloidal gold chromatography technique,this paper established and perfected a colloidal gold immunochromatographic method of AFB₁,and detected the residue in four kinds of traditional Chinese medicines,including Codonopsis pilosula,lotus seed,malt and Coix seed,and the results of detection were confirmed by liquid chromatography tandem mass spectrometry（LC-MS/MS）.The main research contents and conclusions are as follows:1.Preparation of colloidal gold and colloidal gold-antibody conjugateThe preparation of colloidal gold by trisodium citrate reduction method and its influencing factors in the preparation process were investigated.Optimum preparation conditions were as follows:2.5 mL of trisodium citrate,reaction boiling time keeping for 15 min,stirring speed at 200 rpm.We prepared a particle size around 13 nm of colloidal gold particles.In the process of conjugation of colloidal gold and antibody,the optimum amount of labeled colloidal gold labeled antibody and the optimal pH value were determined as 6 mL of colloidal gold,6μL of antibody and 10μL of0.1mol/L K₂CO₃ solution used to adjust the optimum pH for colloidal gold labelling.The colloidal gold-antibody conjugate was characterized by UV spectrophotometry,transmission electron microscopy and Mey’s stabilization assay to determine the colloidal gold and antibody coupling success.2.Preparation of colloidal gold immunochromatography for measuring AFB1Colloidal gold immunoassay strip was constructed based on a competition principle method.Gold standard pad contains a certain amount of gold-labeled antibody conjugate,test line AFB₁-BSA antigen,quality control line for goat anti-mouse secondary antibody.The test strip was optimized,and the best preparation conditions were selected.The type of nitrocellulose membrane was determined,the treatment solution of sample pad and conjugate pad was optimized,and the solution of test solution and methanol content also was investigated.Finally,the AFB1standard solution and other mycotoxin standard solutions were tested with the optimized colloidal gold immunoassay strips.The results showed that the strips had good stability and had no cross reaction with other toxins,and had good specificity.Sensitivity of 0.1 ng/mL can be applied to the rapid screening of AFB1 in Chinese herbal medicines.3.Application of the gold nanoparticle immunochromatographic strip and confirmed by LC-MS/MSThe colloidal gold immunochromatography was used to detect AFB₁ from four kinds of Chinese herbal medicines,including Codonopsis,lotus seeds,malt and Coix seed.The samples were extracted with 80%methanol-water solution and the extract was diluted with methanol content of 20%prior to application in AFB₁ screening by the test strip.There are 9 batches of Codonopsis pilosula（Franch.）Nannf.samples,6batches of Nelumbo nucifera Gaertn.samples,6 batches of Codonopsis pilosula（Franch.）Nannf.samples and 4 batches of Coix lacryma-jobi L.var.Ma-yuen（Roman.）Stapf samples tested in the laboratory.The results of the test strip test were verified by ultra-performance liquid chromatography coupled with mass spectrometry.The results showed that one batch of Nelumbo nucifera Gaertn.sample（Hunan3）and one batch of Hordeurn vulgare L.sample（Hebei）and one of batch of Codonopsis pilosula（Franch.）Nannf.Sample（Guizhou）were tested as positive samples,and the rest were negative.The test results were consistent with the confirmatory results of LC-MS/MS,indicating that the prepared gold immunochromatography possessed a good accuracy.4.Application of aflatoxin B₁ colloidal gold immunoassay strip in animal medicinesA colloidal gold immunochromatographic assay was used to detect AFB₁ in four species of animal drugs:Agkistrodon acutus（Guenther）、Zaocw dhumnades（Cantor）、Buthus martensii Karsch、Whitmania acranulata Whitman.The samples were dissolved in methanol and diluted with 15%methanol-PBS.Sample strips were tested on 5 batches of Agkistrodon acutus（Guenther）samples,6 batches of Zaocw dhumnades（Cantor）samples,4 batches of Buthus martensii Karsch samples and 4batches of Whitmania acranulata Whitman samples.The test results showed that all four kinds of animal medicines in different batches showed negative results.