The Study Of High-throughput Detection Of Mycotoxins Based On Three-dimensional Silica Colloidal Crystal Beads

Mycotoxin is a very important influential factor in food safety, and there are various detection methods. In this article we used a novel nanomaterial-three dimensional silica colloidal crystal beads (SCCBs) as the carriers, developed a high-throughput immunoassay for the detection of mycotoxins in grains.We used Stober method to synthesis monodisperse silica sol and then to fabricate three dimensional silica colloidal crystal beads, then annealed under high temperature after thoroughly scrubbing to increase it’s mechanical strength. SCCBs were immersed in piranha solution(30%hydrogen peroxide and70%concentrated sulfuric acid) after ultrasonic cleaning in distilled water, the surface of SCCBs occurred lytic response and generated more hydroxyl groups, then silylanized with GPTMS by immersing it in toluene solution of1%GPTMS. Then coated SCCBs with PBS solution of AFB1-OV with different concentration (60ng/ml,120ng/ml,240ng/ml,480ng/ml,200ng/ml), and the optimal coating concentration was confirmed at240ng/ml. we also studied the influence of temperature on the modifying of SCCBs, the SCCBs were dried at110℃for45-60mins and the single molecular self-assembly membrane with terminal epoxy group combined fastness with the SCCBs. To establish the standard curve of AFB1the reaction conditions(incubation temperature and incubation time) were optimized, and the optimal incubation temperature and incubation time were defined at37℃and lh respectively. The linear range of the standard curve of AFB1was0.001～1ng/ml, the curvilinear equation was y=170.074-106.329x, RA2=0.98741. Recovery tests were carried out in various grains(peanut、corn、wheat) to validate the reliability of the method, the recoveries were75.68%～134.46%in peanut、76.13%～121.37%in corn and77.34%～109.86%in wheat.We utilize the difference of the maximal reflection peak to encode the SCCBs, then multiplexed immunoassay was carried out with the SCCBs coated with different coupled antigen(AFB1-OV、FB1-OV、CIT-OV). Through light spectrum decoding the SCCBs coated with different coupled antigen were distinguished and the standard curves of each mysotoxin were established. The standard curve of AFB1was y=175.572-103.562x, RA2=0.98735, the linear range of the standard curve of AFB1was0.001～1ng/ml; the standard curve of FB1was y=186.802-58.56x, RA2=0.99197, the linear range of the standard curve of FB1was0.001～lOng/ml; the standard curve of CIT was y=170.88333-89.78x, RA2=0.99988. To validate the reliability of the multiplexed immunoassay, recovery test were carried out in corn and the three mycotoxins were74.69%～127.87%for AFB1,86.59%～105.84%for FB1,88.30%～124.20%for CIT, respectively...