| Mycotoxins are highly toxic and often found in agricultural products,which are harmful to human health.There are many methods for the detection of mycotoxins,but they have some disadvantages such as high cost,low sensitivity,long detection time,complex operation,large volume of reagent,complicated sample pretreatment and so on.Therefore,it is very important to develop a simple,fast,low cost,high sensitivity,enrichment,purification and detection technology.In this paper,a set of methods for the mycotoxin detection based on three-dimensional(3D)large porous inverse opal microsphere immunomagnetic beads enrichment,purification and high performance liquid chromatography(HPLC)fluorescence detection have been developed.Using the microfluidic self-assembly device home-made in the laboratory,the 3D large porous magnetic silica inverse opal microspheres were prepared by self-assembly method and high temperature calcination method of polystyrene(PS)droplet template.The experimental conditions for the preparation of microspheres were preliminarily studied.The experimental results were as follows:10-50nm 2.5%Fe3O4 magnetic particles dispersion;PS and SiO2(5nm ultrafine SiO2 particles)concentrations were 10%and 18%;the ratio of SiO2:PS(v/v)=1:5 and Fe3O4:SiO2(v/v)1:4.The results of metallographic and scanning electron microscopy showed that the microspheres prepared under these conditions have the best effect.Finally,a large number of magnetic silica inverse-opal microspheres with different pore sizes were prepared by using PS nanoparticles with different reflection peaks.From the characterization results,it can be concluded that the microspheres have good magnetic properties,bright color,uniform size,smooth edges,no large internal defects,neat arrangement,complete structure,and a wide range of ordered pore structure.A method for the enrichment,purification and HPLC-FLD detection for OTA based on 3D photonic crystal immunomagnetic microspheres with large pore size inverse opal structure has been established.The microspheres were chemically modified to bind the functional groups and conjugate antibodies of mycotoxins.The antigen-antibody specific reaction was used to enrich and purify the toxin in samples.After elution,the mycotoxins were detected by HPLC-FLD.Several key conditions are optimized and the experimental results are obtained:The best functional group is the aldehyde group;It takes only 20 minutes for the toxin to bind to the antibody;The eluant is a mixed solution(Methanol:Acetonitrile = 1:1);When the concentration of OTA-Ab is 60 ?g/mL,the maximum concentration of toxin is 200 ng/mL,and the recovery rate is over 80%.The recoveries of three kinds of cereals were tested and the results were as follows:76.75%±3.53?79.16%%±1.30 for rice,70.01%±5.05%?72.31%±3.39 for wheat,74.64%±3.97?82.14%±3.53%for corn.The enrichment and purification of the AFB1 and ZEN toxin in samples by the above method and the elution time have been performed.The results show that the time of each elution is only 20 minutes,and the elution takes 60 minutes for 3 times.Besides that,the best elution effect of AFB1 and ZEN was obtained by using mixed solution(Methanol:Acetonitrile = 1:1).The immobilized concentration of AFB1 antibody on the surfaces of microspheres was at 60 ?g/mL.The maximum column capacity concentration of toxin was at 200 ng/mL for AFB1.The ZEN immobilized antibody concentration was at 120?g/mL,and the maximum column capacity concentration of toxin was at 400 ng/mL.The recoveries of AFB1 in three kinds of cereals were as follows:76.12%±1.67%?82.04%±4.46%for rice,70.23%±1.78%?73.76%±1.45%for wheat,70.37%±5.58%?74.17%±2.23%for corn.The recoveries of ZEN in three kinds of cereals were as follows:72.57%±4.24%?76.02%±5.37%for rice,80.60%±1.32%?83.14%±7.09%for wheat,82.89%±1.79%?100.12%±5.58%for corn.It has been confirmed that this method is fast,simple and efficient,high accuracy,good precision,stable and feasible.It is suitable for the enrichment and purification of different kinds of Mycotoxins in cereal samples. |
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