| Objective:To develop the colloidal gold test strip based on the lateral flow immune chromatography technique for rapid detection of aflatoxin B1 in lotus seed. angelica sinensis, ginger powder, pericarpium citri reticulatae, panax quinquefolium and radix paeoniae alba. The established method for detecting aflatoxin B1 was simple, fast, economic and efficient.Methods:①The antibody of AFB1 was evaluated titer by using enzyme-linked immunosorbent assay (ELLISA);②Nano gold particles (NGP) were prepared for using the method of citric acid three sodium reduction, colloidal gold solution was used to identify the morphology by ocular estimate, observe distribution of apparent by the transmission electron microscope and determinate the maximum absorption wavelength by UV Spectrophotometry; ③The indirect competitive colloidal gold immune chromatography technology (GICA) was apply to optimize of the coupling concentration of colloidal gold labeled with monoclonal antibody, the solution pH and the concentration of AFB1-BSA (Text line) and Goat anti-Mouse IgG (Control line); ④The preparation of the colloidal gold strip was preliminary tested the content of AFB1 and determine the minimum detection limit. The performance of the strip was inspected (included specificity, stability and reproducibility). ⑤The strip method was used to detected AFB1 in different batch samples of 6 kinds of traditional Chinese medicine (TCM) and UFLC-MS/MS method was prepared to determinated in the content of AFB1 in TCM.Results: ①The results of preparation strip:The monoclonal antibodies (McAb) of AFB1 were determined with coating antigen and the results indicated the content of McAb was 3.8 mg/mL. The titer of the McAb was 1:1.0×104 and the 50% inhibition concentration was 0.01 mg/mL. Gold particles diameter was deteminated about 40 nm by TEM. The colloidal gold solution get maximum absorption wavelength at 518 nm. The coupling concentration of colloidal gold labeled with McAb was 6 μg/mL, under the condition of pH 8.2 and colloidal gold solution was coated with McAb were obtained the maximum absorbance. The concentration of AFB1-BSA (Text line) and Goat anti-Mouse IgG (Control line) were 0.3 mg/mL and 0.5 mg/mL, respectively. The limit of detection of the constructed test strip was 2.5 ng/mL, the time of detection was within 10 min. Inspection for the specificity of the strip results shows that the strip good specificity.②The results of the preparation strip was used to preliminary screening AFB1 in 6 kinds of traditional Chinese medicine (TCM):The preparation of the strip was used to primary screening of commercially available in 6 kinds of TCM, The number of positive samples was 6 copies lotus seed,9 copies in angelica sinensis,23 copies in ginger powders,1 copies in radix paeoniae alba and 9 copies in dried tangerine or orange peel, respectively. The content of AFB1 was excced 2.5 ng/mL,10 samples of American ginseng samples were not detected AFB1.③The strip results compared with the LC-MS/MS results:These positive samples were confirmed by UFLC-MS/MS. the content of AFB1 was ranged from 4.37 μg/kg to 7.82 μg/kg in lotus seed,3.85-10.82 μg/kg in angelica sinensis, 2.53-6.82 μg/kg in ginger powders,2.90-10.21 μg/kg in dried tangerine or orange peel. There was only 1 positive sample in radix paeoniae alba, and the content of AFB1 was 2.75 μg/kg. American ginseng was not detected positive sample. There were no false results and the constructed colloidal gold test strip is simple, quick and accurate for detecting AFB1 in lotus seed.The strip test results compared with the LC-MS/MS results, the strip of sensitivity is good, which could satisfy the purpose of preliminary screening for aflatoxin B1 in Chinese traditional medicines.Conclusions:The major advantages of the constructed test strip were economic and reasonable, easy operation, intuitive results, high sensitivity. Compared with conventional AFB1 analysis methods, strip testing was omitted the cumbersome, expensive instruments, equipment operation and suitable for rapid detection of Chinese medicinal materials at real-time, real-field, and it will be provide the basis for rapid screening of AFB1 in traditional Chinese medicine.But, due to the complexity of traditional Chinese medicine (TCM) chemical composition, matrix interference was serious. The preparation strips was only applicable to detect components and structure difference with AFB1 in TCM. Therefore, it is significance emphasis on optimizing the tracer immune protein for the future research. |
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