Development Of Surface Plasmon Resonance Biosensor For The Detection Of Three Mycotoxins In Maize

Aflatoxin B1(AFB1),deoxynivalenol(DON)and zearalenone(ZEN)are three kinds of mycotoxin,which are harmful to food-producing animals.These mycotoxin can enter animal feed through contaminating raw plant.As the third most important factor other than bacteria and viruses,the mycotoxins can cause acute or chronic toxicity to animals,So,it is necessary to screen and control mycotoxins in animal feed.At present,there are many methods for mycotoxins detection,but these methods are time-consuming,requiring complex sample preparation and operation.Explorationn of new methods for mycotoxins detection is becoming more and more important.The surface plasmom resonance(SPR)technology has the advantages of simple operation,high sensitivity,short detection time,low sample consumption,labeling free,real-time monitoring of the reaction process,no need for purification of various biological components,and simple sample preparation.Hence,SPR has been widely used in the detection of hazardous chemicals.However,there are rarely reports for multi-mycotoxin detection.In this study,the mycotoxin haptens were coupled with bovine serum albunin(BSA)and ovalbumin(OVA)to obtain the three immunogens(AFB1-BSA,DON-BSA and ZEN-BSA)with immunogenicity and coating antigens(AFB1-OVA,DON-OVA and ZEN-OVA),respectively.The mice were immuned with three immunogens(AFB1-BSA,DON-BSA and ZEN-BSA)separately.The mouse spleen cells were fused with myeloma cells based on hybridoma technology.The monoclonal antibodies(mAbs)for AFB1,DON and ZEN were successfully prepared.The mAbs for AFB1,DON and ZEN have no cross-reactivity with other toxins and the corresponding mAb affinity is 5.4×107 mol/L,7.5×106 mol/L and 3.1×107mol/L,respectively.The coating antigen AFB,-OVA was bound to the carboxy-modified SPR chip.According to the binding interval of antigen coating,the AFB1 chip reaction condition was optimized and determined.The results show that the proportion of coupling are highest under the conditions of acetate buffering.According to the flow rate binding curve,the most suitable concentration of AFB1-OVA is 0.02 mg/ml,the optimal concentration of anti-AFB1 mAb is 1:10000.The direct competitive SPR method for AFB1 was established with the detection limit of 2.94 ?g/kg.Similarily,the SPR method for DON and ZEN were established with the detection limit of 96.22 ?g/kg and 9.32?g/kg,respectively.The detection results between SPR method and high performance liquid chromatography(HPLC)method are basically the same when used for detection AFB1,ZON and ZEN in corn.Finally,the SPR method for simultaneous detection AFB1,DON and ZEN was established in this study with the detection limit of 3.77?g/kg,109.08 ?g/kgand 11.11 ?g/kg,respectively,which could meet the requirements when used in field sample.When applicated in 50 field corn samples,the SPR method developed in this study has no obvious difference with HPLC method(P>0.05).