The Protein Chip Technology Based On TiO₂-porous Silicon Detects Multiple Mycotoxins In Grains

Mycotoxin contamination of food is an ongoing global concern.Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi,which cause disease in man and animals at low concentrations.They are cytotoxic,nephrotoxic,hepatotoxic,teratogenic,mutagenic,carcinogenic,and immunosuppressive.In addition,there may be multiplex mycotoxins in feeds and foods,which are more harmful.Generally,mycotoxins are detected by chromatography,immunoassays,or sensors.While chromatographic methods offer high sensitivity and accuracy,they are expensive,time-consuming,requirement of highly skilled personnel and tedious sample pretreament.Porous silicon has a large specific surface area and more binding sites.When they are applied in the detection of mycotoxins,precess of sample pretreatment is simple.In this paper,a protein microarray technology was established to detect the multiplex mycotoxins in cereals by using porous silicon as the carrier.In this paper,porous silicon was prepared by electrochemical etching with P-type monocrystalline silicon.First,the etched waveform was selected and determined as a square wave etch.Then,in order to increase the stability of the porous silicon,a layer of TiO2 was coated on the surface.and the effection of immobilization of protein before and after coating TiO2 was compared.The porous silicon was modified with aldehyde groups,carboxyl groups,and epoxy groups,respectively.The results showed that the epoxy-modified porous silicon is better than other groups modified porous silicon.Subsequently,the complete antigens,monoclonal antibodies and Cy3-IgG of OTA,AFBi and FB1 were optimized respectively.Results showed that the optimal concentrations of OTA-BSA,AFBi-BSA,FB1-BSA were at 200,100 and 200 μg/mL,respectively.The concentrations of OTA-Ab,AFBi-Ab and FBi-Ab were at 200,100 and 200μg/mL,respectively.The concentration of Cy3-IgG was at 60 μg/mL.And then,according to the optimized conditions,the standard curves of three mycotoxins were obtained.The result showed that:the detection linear range of OTA was 0.01-10 ng/mL,the linear equation was y=-1576.3675 log(x)+ 8761.3079,R2 = 0.999,and the detection limit was 32.6 pg/mL;the detection linear range of AFB1 was 0.001-1 ng/mL,and the linear equation was y=-1584.1929 log(x)+6743.3798,R2 = 0.985,and the detection limit was 0.314 ng/mL;the detection linear range of FB1 was 0.01-10 ng/mL,and the linear equation was y=-1187.7643 log(x)+ 9179.6908,R2 = 0.992,and the detection limit was 0.197 ng/mL.The precision of this method is below 13%.In addition,the specificity was analyzed using OTA as an example.The results showed that this method has good specificity for OTA and there was no obvious cross reaction with its structural analog OTB.The spiked recovery of OTA in rice,wheat,and corn was determined using this method and ELISA method,respectively.Among the 9 samples tested,the recoveries of 7 samples were in the range of 80-120%by this method,which was agreement with that of classic ELISA method.This shows that this method is feasible for the detection of mycotoxins in cereals.Based on the detection method of single mycotoxin,a new simultaneous detection method for multiplex mycotoxins was established.The influences of two different mixed reaction systems on the results were compared.The results showed that the linear range of the standard curve measured by the mixture mycotoxin with their corresponsing antibodies was reached to 2-3 orders of magnitude.The linear detection ranges of OTA,AFB1 and FB1 for simultaneous detection were 0.01-1,0.001-1 and 0.01-1 ng/mL,respectively.The multiplex specificity was analyzed and the results showed that the specificity of antigen and antibody was good.Then,the spiked recovery of multiplex mycotoxins in rice,wheat,and corn was determined using this method and ELISA method,respectively.The results showed that the recoveries determined by the two methods were basically the same,indicating that the method for the simultaneous detection of multiplex mycotoxins established in this chapter was applicable to the detection of multiplex mycotoxins in cereals.