| Ochratoxin A(OTA) is one of the most widely distributed, most toxic and the greatest threat to humankind during the ochratoxins. OTA may occur in a large variety of foods, farm produces, and traditional Chinese medicines(TCMs), posing great potential risks to human health. Most countries and organizations have set guidelines and recommendations for the maximum residue levelsfor OTA, but a shortage of the maximum residue levels in TCMs. TCMs is susceptible to contaminants mycotoxins during the processes of planting, harvesting, processing and transportation. Most detection methods require expensive equipments, trained personnel as well as complicated operation, which are unsuitable for on-site detection. Therefore,developing a reliablemethod for easily, sensitive and accuratedetection of OTA in TCMs are in urgent demand.Gold nanoparticle immunochromatographic assay are suitable for the rapid detection because of the easy operation and on-site detection. However, this assay has disadvantages such as synthesis difficulties, poor stability and high rate of false negative owing to the antibody depended. Aptamer, a “chemical antibody”, has a stable structure with high affinity and specificity, and can be synthesized in vitro,improved the deficiency of the immunochromatographic assay. Hence, we develop an aptamer based lateral flow strip for on-site screening of OTA in real TCM samples(Astragalus membranaceus, Poria cocos and Gastrodia elata), and the results were confirmed by liquid chromatography tandem mass spectrometry(LC-MS/MS) to ensure the accuracy and reliability. The main contents and conclusions are as follows:1. Preparation of Au NPs and Au NPs-aptamer conjugateBy investigated on the optimal conditions for preparing Au NPs by sodium citrate method, the results showed that a 2.5 m L 1% sodium citrate, 8 min heating time, 100 rpm stirring rate and chloroauric acid added beforesodium citrate, monodispersion colloidal gold could be obtained at size of 10 nm. During the conjuation between Au NPs and aptamer, we inspired on the optimum amount of label and the p H value,and the results showed that the optimum amount of label was 1m L Au NPs with 6 μL10μM aptamer, and the optimum p H value was obtained with10 μL 0.1 mol/L K2CO3 solution. The obtained Au NPs-aptamer conjugate was valued by UV-vis, TEM and Mey’s Stabilization method, all these methods were defineded the success of conjugation.2. Self-assembly of the aptamer based lateral flow stripResearch on the optimum factors of the lateral flow strip: the most suitable formulations of blocking buffer and dilution of Au NPs-aptamer conjugate, dissolving solutions and concentration range of DNA probe 1 and 2, the type of NC membrane,the optimum formula of running buffer and the tolerance of methanol in the analysis.According to the optimized conditions, the aptamer-based lateral flow strip was built by ourselves and be used to detect OTA standard solutions. The results showed that the sensitivity of the aptamerbased strip was considered to be 1 ng/m L to OTA, with good selectivity and stability.3. Screening of OTA pollution situation in TCMs by the aptamer based lateral flow strip and confirmation by LC/MS-MSThe samples were extracted with methanol-water(80:20, v/v), and diluted 4times to be applied to the strip. With OTA solution in the extract of Astragalus membranaceus, Poria cocos and Gastrodia elata, the matrixs were detected by the the aptamer based lateral flow strip, we got the matrix detection limit of 1 ng/m L both of Poria cocos and Gastrodia elates, the matrix effect were basically eliminated.However, a matrix effect still exists for Astragalus membranaceus with a matrix detection limit of 2.5 ng/m L. Detection of OTA pollution situation on the 9 batches of Astragalus membranaceus, 6 batches of Poria cocos and 8 batches of Gastrodia elata by the aptamer based lateral flow strip, only the A. membranaceus sample sample collected from Gansu was positively with OTA, and the result were confirmed by LC/MS-MS method with 3.98 μg/kg OTA in the sample. It proved that the aptamer based lateral flow strip could be applied for rapid screening of OTA in TCMs and applied to other mycotoxins in more TCMs in the future. |
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