| Mycotoxins are common contaminants in agricultural products and various mycotoxins with strong toxicity have been found in agricultural products, which have a huge threat on human health. At present, many methods have been developed in the detection of mycotoxins, but they always have obvious limitations, such as a heavy workload, high cost and reagent consuming. Based on aptamer recognition technology and fluorescence analysis technology, the present study successfully established a novel, low cost and high-throughput method to detect multiplex mycotoxins simultaneously which used photonic crystal microspheres as a carrier of the suspension array. The work details are as follows:(1) A lab-made simple micro-fluidic device was used to produce photonic crystal microspheres, and fiber optic spectrometer was used to measure the reflection peaks of microspheres. In order to ensure the accuracy of subsequent detection, functionalization of the surface of the microspheres was studied, which mainly included the optimization of surface modification methods to enhance its binding ability with aptamers and to minimize the background fluorescence of substrate.(2) Based on aptamer recognition technology and fluorescence analysis technology, a detection method for OTA using photonic crystal microspheres as carrier was developed. The optimal conditions are as follows:the concentration of OTA-aptamer is400nmol/L and the concentration of FITC-labeled complementary DNA chain is500nmol/L; the efficient hybridization condition was at37℃for2h; the OTA binding reaction was conducted at37℃for1h. Under the optimal conditions, the linear range for the OTA concentration detection was1-0.001ng/mL, and the linear equation was y=992.61+224.35x, R2=0.994. The specific test showed that there was no significant cross-reaction with AFB1and FB1. At last the recovery rate experiment of OTA in three agricultural products was also performed, and the recovery rate was:73.73±2.39%～106.90±5.37%in rice,90.67±5.27%～125.17±2.31%in corn and87.91±5.26%～119.55±7.82%in wheat, respectively.(3) Based on above described technology, a novel method was established for detecting OTA and FB1in agricultural products simultaneously. Specifically, microspheres were used as a carrier and covalently bound with toxin-aptamer, and then fluorescence-labeled aptamer-complementary sequence was hybridized with aptamer on the surface of microspheres. Finally, these processed microspheres could be used to detect multiplex mycotoxins simultaneously. By experimentation, we confirmed the linear range is1-O.Olng/mL for OTA and1-0.001ng/mL for FB1and linear equations is y=1054.35+322.89x (R2=0.986), y=1491.68+410.88x (R2=0.994), respectively. The recovery rate experiment of OTA and FB1in cereal were performed and the recovery rate were81.8±6.28%～116.38±7.60%for OTA and76.58±5.96%～122.32±2.89%for FB1. At last, we also demonstrate a comparative study between the present method and traditional ELISA method, by detecting mycotoxins in21kinds of cereal samples using both methods. The detection results showed that there was no significant difference observed between each pair of experiments (P>0.05). |
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