Non-labeled Fluorescence Immunodetection Of Multiple Mycotoxins In Cereals Based On Photonic Crystal Microspheres

Mycotoxins are secondary metabolites produced by fungi growing in food or feed.Due to the strong carcinogenicity,teratogenicity and mutagenicity of mycotoxins,they can not only affect agricultural products and animals,but also do harm to human life and health.Nowadays,many methods have been used for detecting mycotoxins,however,most of them have the disadvantages of long detection time,high cost,large amounts of reagents,and tedious sample processing.Due to these reasons,it is critical to develop a new method to detect mycotoxins with rapid,high sensitivity and strong specificity.Therefore,a method based on photonic crystal microsphere-based label-free fluorescent immunoassay for the detection of multiple mycotoxins in grains was established in this paper.In this paper,photonic crystal microspheres were prepared by using our la boratory platform and characterized by metallographic microscope,scanning ele ctron microscope,and transmission electron microscopy.The results showed tha t the photonic crystal microspheres were uniform in size and stable in structure.Then we respectively modified these groups on the surface of photonic crystal microspheres with epoxy group,aldehyde group and carboxyl group.Comparin g the fluorescence signals,the results show that the modified epoxy group is b etter.At the same time,gold nanoparticles were prepared by the traditional tris odium citrate reduction method,and the group was modified and activated.Fin ally,the scanning electron microscope characterization of the photonic crystal microspheres after the immune competition reaction was performed to determin e the combination of the photonic crystal and the gold nanoparticles.A method for the detection of aflatoxin B₁(AFB₁)in cereals based on la bel-free fluorescent immunoassay of photonic crystal microspheres was establish ed in this paper.First,the concentrations of AFB₁ antigen and antibody were optimized.The results show:the optimal concentrations of AFB₁-BSA and AF B₁-Ab were respectively determined to be 80?g/m L and 60?g/m L.Accor ding to the optimized conditions,a standard curve for AFB₁ was prepared.The results show that the detection range of AFB₁ is 0.1-10ng/m L The detection linear equation is y=-1189.43949logx+5292.31172,the linear correlation co efficient(R²)is 0.995,the detection limit is 0.151ng/m L.Afterwards,the AF B₁ spiked recovery in rice,wheat,and corn was measured by this method and traditional ELISA method.The AFB₁ spike recovery measured by this method were as follows:84.07%±5.71%-98.59%±7.47%for rice,93.88%±10.02%-100.43%±5.83%for wheat,92.78%±14.74%-101.02%±5.13%for co rn.The overall recovery rate measured by this method was 84.07%±5.71%-101.02%±5.13%.The AFB₁ spiked recoveries measured by ELISA were as f ollows:86.54%±6.37%-97.19%±1.84%for rice,89.55%±1.11%-117.26%±5.77%for wheat,84.99%±3.53%-100.04%±3.24%for corn.The overal l recovery rate measured by ELISA method was 84.99%±3.53%-117.26%±5.77%.The results of the two methods were consistent,indicating that the met hod established we developed is feasible.Then the method we developed was applied to the detection of ochratoxin A(OTA)and fumonisin B₁(FB₁).The results showed that the linear equati on of OTA detection was y=-560.78811logx+1062.86894,the linear correlat ion coefficient(R²)was 0.999,and the detection limit of OTA was 0.0227ng/m L,the linear equation of FB₁ detection was y=-92.98732logx+603.51418,the linear correlation coefficient(R²)was 0.994,and the detection limit of FB₁ was 0.159ng/m L.The OTA spiked recoveries measured by this method wer e as follows:80.90%±5.82%-115.92%±7.28%for rice,67.79%±4.04%-97.79%±9.40%for wheat,85.15%±4.38%-93.37%±4.61%for corn.The overall recovery rate range was in:67.79%±4.04%-115.92%±7.28%.The FB₁ spiked recovery rates measured by this method were as follows:88.96%±12.26%-97.24%±12.65%for rice,86.64%±2.54%-97.78%±11.22%for wheat,85.15%±4.38%-93.37%±4.61%for corn.The overall recovery rate range was in 85.15%±4.38%-97.78%±11.22%.By comparing the establish ed method with the recovery rate measured by ELISA method,it was found th at the recovery rates of the two methods remained consistent.It can be seen t hat the established method can be applied to the detection of different types of mycotoxins,and has laid a good foundation for the detection of different type s of mycotoxins in future foods.