Simultaneous Determination Technology For Multiplex Mycotoxins In Grain And Oilcrops Based On QDNBs

Mycotoxins are secondary metabolites mainly produced by Aspergillus,Pinicielium and Fusarium,found all around the world as natural contaminants in numerous commodities of plant origin.More than 300 compounds are now recognized as mycotoxins,of which approximately a dozen groups regularly receive attention as threats to the health of human and animal and environmental safety.Reseachers have found that most fungi are able to produce several mycotoxins concurrently and they could infect agro-products simultaneously or in quick succession,mycotoxins co-contaminate heavily in agro-products.Toxicologically,several mycotoxins co-exposure at one time has been confirmed significant synergy and combination effect on health damage.Thus,the demonstration of simultaneous sensing technologies for mycotoxins co-contamination with merits of rapidness is of great theoretical significance and has greatly utility value to state regulation of mycotoxins co-contaminants in agro-products.However,the current determination methodology for multiplex mycotoxins is mainly for a single mycotoxin and the available simultaneous determination methods developed for mycotoxins analysis such as multi-compounds immunochromatographic assay based on colloidal gold meet the qualitative or semi-quantitative analysis but weaken for poor simultaneous quantitation for several mycotoxins in a single run.Based on immunochromatography,this study was carried out:immunochromatographic assay for three mycotoxins detection in a quantitative way taking QDNBs as labels for their features as high quantum yield fluorescence and desirable chemical/colloidal stability,to offset the technical barrier of poor stability about QD based strips.This proposed method notably improves the throughput and sensitivity in simultaneous quantitation for multi mycotoxins and provides a new way for multi mycotoxins simultaneous screening in agro-products.Main research contents and innovative points are as follows.1.High sensitive immunochromatographic assay based on quantum dots nanobeads was developed for total aflatoxins and has been applied in the quantitative analysis for real sample such as rice and peanut.The coupling technique of QDNBs and anti-AFT mAb was optimized by the parameters of p H and antibody amounts.By optimizing the amounts of fluorescence probes,detection time and methanol concentraton,QDNBs based immunochromatography for total aflatoxins was developed and QDNBs based strips for AFT analysis were fabricated.The LODs for AFT in rice and peanut matrix were 1.4and 2.8 pg/mL,respectively.Dynamic ranges varied from 2 to 250,from 2 to 125 pg/mL.Recovery rates of standard solution addition ranged from 86.3%to 118.0%,from 91.3%to 106.3%for rice and peanut extracts with relative standard deviation?RSD?lower than 11.8%.The developed method shows good accuracy and precision.The inter-and intra-batch difference rates were below 11.2%,which verified this work with a desirable reproductivity.The proposed QDNBs based strip exhibited characteristics of easy-to–operate and could finish the quantitative determination for AFT within 10 min.Ten real sample from the market were chosen for AFT quantitative validation using the new method,whose results were in good correlation to those obtained from HPLC.2.Highly selective and specific immunochromatographic assay based on quantum dots nanobeads were developed for AFB₁,ZEA and FB₁ detection.Three antibody fluorescence probes were prepared with QDNBs and test strips with three test lines were fabricated.By optimizing immunoreagent amounts and immunoreaction time,a highly sensitive and point-of-care immunochromatographic assay for 3-plex mycotoxin determination in a single run was introduced.LODs of2.4,2.5,4.5;20.4,28.1,36.3;2.5,3.2,4.0 ng/kg for AFB₁,ZEA and FB₁ respectively were obtained in methanol solution,,maize extracts and peanut extracts.Linear ranges were varied up to 2 orders of magnitude.The average recovery of three different spiked mycotoxins at three concentration level ranged from 86.3%to 103.7%,with the relative standard deviation lower than 8.8%,which indicated desirable accuracy and precision.Compared to the existing methods,this method has its advantages of high specificity and high sensitivity and could quantify three mycotoxins within 15 min,which provided a powerful tool for rapid and quantitative screening of co-contamination in agro-products.