On-site Visual Detection Of Aflatoxin B₁ In Traditional Chinese Medicines By The Colloidal Gold Test Strip

Colloidal gold immunochromatography is a rapidly developing technique which can be used to fix the specific antigen or antibody in the the nitrocellulose(NC) membrane. Colloidal gold is used as tracer and the results can be achieved by the naked eye observation. The technology showed on-site visual detection of target in non laboratory conditions, with simple operation, time saving, labor saving, and low cost advantages. It has been applied in the field of medicine, drugs, hormones, and food safety detection. In the detection of mycotoxins, it focus on food, feed and other substrates, however it has been little reported in detection of toxins in traditional Chinese medicine. As the most toxic among aflatoxins, aflatoxin B1(AFB1) is listed as a Ι carcinogen by World Health Organization and has been reported in a higher incidence and residual in TCM, thereby affecting the quality and safety of medicines. Traditional detection methods need to be carried out in the laboratory and specialized equipment, and time consuming, it is difficult to achieve on-site quick testing. And TCM is vulnerable to pollution risk in planting, harvesting, processing and storage process. Therefore, it is important to establish appropriate rapid screening analytical methods to evaluate the residual quantity of AFB1, so we can guarantee the quality and the safety and health of users. In this paper, an immune chromatography method was established and was used in the the rapid screening of AFB1 in the TCM for the first time. Continually, as novel alternative of antibody, aptamer show high specificity and affinity. Aptamer recognition element can overcome defects of antibody, on-site strip assay based on aptamer was setup, which can lay the foundation for later study and for on-site visual detection of aflatoxin B1 in TCM. The main research contents and conclusions are as follows:1. Preparation of colloidal gold particles and optimization AFB1 aptamer sequence lay the foundation for the construction of the visual test strip.The amount of trisodium citrate, the stirring speed, time after boiling heating, and other key parameters were optimized. The colloidal gold was characterized by UV visible spectrum scanning and transmission electron microscopy(TEM). Rapid visual detection was established by optimization of salt concentration and concentration of aptamer. DNA sequence with high affinity to AFB1 was successfully optimized, which laid the foundation for follow-up study.2. Colloidal gold strip was prepared for AFB1 of TCM by on-site visual inspection.Gold nanoparticles with different particle sizes of 16.70~40.10 nm were prepared. The colloidal gold particles with various particle sizes were then used to prepare test strips with colloidal gold-labeled anti-aflatoxin B1 antibodies. The preparation parameters of these test strips were optimized and their performance was compared. The test strip containing 17.4 nm colloidal gold particles exhibited a detection limit of 0.1 ng/mL. The detection limit was 0.1 ng / mL and cut-off value was 1 ng/mL. The detection time was 20 min.3. AFB1 contamination in 4 kinds of TCM was detected by self-assembled colloidal gold test strip.4 kinds of TCM were prepared by optimizing the extraction solvent and dilution ratio, and the effect of sample matrix on the detection of colloidal gold test paper was discussed. Sample preparation was optimized as follows: Gastrodia elata B1 and Poria cocos were extracted by 70% methanol-water and were diluted three-fold; 60% methanol was used as extraction solvent in Bletilla striata; Radix Angelicae Dahuricae was extracted by 70% methanol-PBS and were diluted four-fold. The method has been successfully applied to aflatoxin B1 detection. The sensitivity for detection of aflatoxin B1 was 5 μg/kg、5 μg/kg、3.125 μg/kg、1.25 μg/kg by eye in Gastrodia elata B1., Poria cocos., Bletilla striata., and Radix Angelicae Dahuricae. The test results of the test strip were confirmed by UPLC-MS/MS as well.4. On-site strip assay was developed based on aptamer, which has the advantages of the high specificity and strong affinity compapered new antibody substitution material.Colloidal gold labeled aptamer was used as probes. We developed quick and on-site strip assay for detection of AFB1 in the competion format. The best ratio between gold nanoparticles and DNA, best buffer, NC membrane was optimized. The best amount of aptamer is 3 μL, the best buffer is 10 mM PBS(1% surcrose,0.02% MgSO4,0.1% Tween20)and take the Vivid 90 NC membrane. The limit of detection with naked eyes is 0.5 μg/mL and detection time is 17 min. which can lay the foundation for in-depth study.