Structure-switching Signaling Aptamers Detection For Mycotoxins In Cereal Samples Based On Photonic Crystal Microsphere Suspension Array

Mycotoxins can easily contaminate cereal.They not only affect the quality of the cereal but also are hazardous to human health.There were many methods for myco-toxins detection.However,they were high cost and complicated pretreatment This ar-ticle was based on structure-switching signaling aptamers and pho-tonic crystal mi-crospheres suspension array technology to establish a new detection method for mul-tiplex mycotoxins in cereal samples.It is a low cost and high-throughput method.The main contents of this paper included self-assembly,characterization,and surface chemical modification for silica photonic crystal microspheres(SPCMs),de-tection condition optimization,the establishment of the standard curve and the recov-ery rate for mycotoxin;the multiplex assay condition optimization,standard curves and recovery rate for three mycotoxins(Aflatoxin B1,Ochratoxin A,Fumonisin B1)in cereal samples were performed.The comparation experiment for mycotoxins recovery rate with the traditional ELISA method had been carried out.The work details were as fol-lows:1.Three different kinds of SPCMs were prepared by the laboratory-made self-assembly platform and characterized.Then,the different modified methods for surface microspheres were compared and carboxyl group modification method showed the lowest background signal and the most surface binding sites.2.Mycotoxins in cereal sample were detected by SPCMs modified with ca-rboxyl as carrier.The detection conditions were optimized.The optimal conditio ns were showed as follows:AFB1-aptamer and its complementary strand conce-ntration were 800nmol/L and 2400nmol/L,respectively;hybridization conditions was 37? for lh;binding conditions of AFB1 was 37? for 1.5h.OTA-aptamer and its complementary strand concentration were 600nmol/L and 3000nmol/L,respectively;hybridization condition was 37? for 1h;binding condition of OTA was 45? for 1h.FB1-aptamer and its complementary strand concentration were 800nmol/L and 3200nmol/L,respectively;hybridization condition was 37? for 1h;binding condition of FB1 was 45? for 1h.According to the optimal conditions,we setup standard curves.The linear range of AFB1?OTA?FB1 were 0.001-1ng/mL?0.01-10ng/mL and 0.001-1ng/mL,respectively.The linear equations were y=846.83434x+3356.83023(R2=0.99294)?y=1962.2397x+9573.46534(R2=0.99149)and y=612.67559x+2591.61248(R2=0.9898),respectively.The AFB1-aptamer specifi-c test showed that there was no significant cross-reactivity with its structurally similar toxins.The recovery rate test of AFB1 in three kinds of cereal samples was performed and the recovery rate was between 76.84±4.14%and 112.77±3.80%.The result showed that this method is feasible.3.Then,multiplex detection of toxins in cereal samples were performed.The optimal binding condition of toxins was 45 ? for 1.5h.According optimal cond-itions,the linear range for multiplex detection of AFB1?OTA?FB1 were 0.0001-O.lng/mL?0.0001-0.1ng/mL and 0.1-10ng/mL,respectively.The linear equations were y=548.87374x+4195.94847(R2=0.99024)?y=582.946x+2399.90(R2=0.99789)and y=620.8157x+2654.2092(R2=0.99967),respectively.At last the recover-y test showed that the recovery rate were between 79.68±1.25%and 108.84±4.59%,71.20±9.01%and 113.19±1.20%,76.99±4.08%and 105.96±5.36%for AFB1?OTA?FB1 multiplex detection,respectively.The result showed that this method is feasible.4.Finally,the comparation experiment in recovery rate in three kinds of cereal samples was performed between the developed method and traditional ELISA method.The results showed that they were in agreement and this meth-od is feasible.