The Study On The Enrichment,Purification And Detection Of OTA,AFB₁ And DON Using The Inverse Opal Photonic Crystal Microspheres

Mycotoxins are secondary metabolites produced by the fungus in the growth process,which are harmful to human and animals.Mycotoxins showed their carcinogenicity,teratogenicity and mutagenicity.There are many methods for the detection of mycotoxins,but they have some disadvantages such as high cost,long detection time,large volume of reagent,complicated sample pretreatment.Therefore,it is very important to develop a fast,simple,low cost enrichment,purification technology.This paper proposes an enrichment,purification and detection of OTA,AFB1 and DON with inverse-opaline photonic crystal microspheres.Firstly,inverse-opaline photonic crystal microspheres were prepared by self-assembly with nanosilica and polystyrene(PS).The microspheres showed a regular structure and structural color.The experimental conditions for the preparation of microspheres were preliminarily studied.The experimental results were as follows:PS and SiO2 concentrations were at 10%and 18%(wt%);the ratio of PS:SiO2(v/v)=5:1.The microspheres were characterized by metallographic microscope and scanning electron microscope(SEM).They had smooth surfaces,uniform pore size and no collapse of the inner structure.Then the surface of the microspheres was modified with aldehyde,carboxyl and epoxy groups.The optimal modification group was aldehyde groups.The several conditions for immunoaffinity-HPLC to enrich and purify OTA,AFB1 and DON were optimized with inverse-opaline photonic crystal microspheres.The optimal eluent concentration and volume for OTA was methanol:acetonitrile(1:1)and 400?L,respectively.The immobilized antibody concentration of OTA on the surfaces of microspheres was at 60p,g/mL.20 ?L microspheres immobilized with OTA antibody could enrich 50ng OTA.The enrichment efficiency of inverse-opaline photonic crystal microsphere reached to 90%.The enrichment efficiency of solid nanosilica microsphere only had 20%.The recovery rates of OTA in rice,wheat and corn were 78.60 ± 0.71?81.67± 5.20%,75.00 ± 31.92?89.17 ± 1.44%,71.60 ± 3.70?84.58 ±2.60%,respectively.The above method was applied to AFB1.The optimal eluent concentration for AFB1 was 100%methanol.The immobilized antibody concentration of AFB1 on the surfaces of microspheres was at 60?g/mL.20 ?L microspheres immobilized with AFB1 antibody could enrich 40ng AFB1.The enrichment efficiency of inverse-opaline photonic crystal microsphere reached to 90%.The recovery rate of AFB1 in rice,wheat,and corn were 84.62±3.85?90.83±3.00%,70.51 ±2.22?87.70±0.89%,74.94±4.11?77.96± 2.79%,respectively.Finally,DON was enriched and detected by High Performance Liquid Chromatography-Diode array detector(HPLC-DAD).The optimal eluent concentration for DON was 30%methanol.The immobilized antibody concentration of DON on the surfaces of microspheres was at 100?g/mL.20?L microspheres immobilized with DON antibody could enrich 40ng DON.The enrichment efficiency of inverse-opaline photonic crystal microsphere reached to 90%.the recovery rate of DON in rice,wheat,and corn were 68.09±1.72?69.20±2.40%,60.63±0.27?63.17± 1.98%,56.19±1.26-63.49± 3.60%,respectively.In summary,it is feasible to enrich and purify and detect OTA,AFB1 and DON with inverse-opaline photonic crystal microsphere immunoaffinity-HPLC method.These results indicate the new developed method is suitable for mycotoxin assay.