| Deoxynivalenol(DON)also named vomitoxin,is one of the mycotoxins widely present in food crops,such as corn,wheat and rice.DON can seriously threaten animal or human health through oral intake or skin contact because of its immunotoxicity,cytotoxicity and reproductive toxicity.Global food quality and safety issues caused by DON contamination have attracted much attention.Therefore,there is an urgent need to establish detection methods that can be used for rapid screening of large-scale samples.In this study,anti-DON monoclonal antibodies were prepared based on synthetic immunization antigens,colloidal gold nanoparticles(GNPs)and chrysanthemum gold(Chrysanthemum-like gold@polydopamine,AuNC@PDA)immunochromatography assays(ICAs)were established for the qualitative and quantitative detection of DON in wheat and rice.The main research methods and conclusions are as follows:Firstly,five DON immunization antigens and one detection antigen were synthesized.The mice were immunized with five DON immunization antigens.After the fifth immunization,four mice with higher positive and better inhibition rates were screened by enzyme-linked immunosorbent assay.Spleen cells from four mice were fused with myeloma cells.The culture supernatant of hybridoma cells was evaluated,positive cell wells were unsuccessfully screened from No.1,No.3,and No.4 mice,two positive cell wells were successfully screened from No.2 mice,named 4H4 and5H4,the inhibition rates were 41.2%and 35.8%at a spiked DON concentration of 200ng/mL,respectively.The cells in these two wells were subcloned,and the culture supernatant of cells was evaluated.The positive cells were lost,and the anti-DON monoclonal antibody was unsuccessfully obtained.Secondly,a colloidal gold immunochromatography assay(CG-ICA)was established for qualitative and quantitative detection of DON in wheat and rice.The GNPs were synthesized by trisodium citrate reduction.The pH of antibody conjugation,the amount of mAb,the concentration of DON-BSA,the concentration of goat anti-mouse Ig G and the spray volume of GNPs-mAb were optimized.CG-ICA was established under optimal conditions.CG-ICA was used for the qualitative detection of DON in wheat and rice with a qualitative limit of detection(LOD)of 1000.0μg/kg.The stability and detection performance of the method were better.CG-ICA was used for the quantitative detection of DON in wheat and rice,the linear equations were Y=5.148-1.357 lgX and Y=6.142-1.611 lgX,correlation coefficients were 0.991and 0.989,the quantitative LOD were 75.688 and 65.661μg/kg,the linear ranges were150.0-3000.0 and 150.0-4500.0μg/kg,respectively.The recovery rates in wheat and rice samples were 69.4%-91.9%and 94.1%-101.6%,the intra CV were 10.9%-12.9%and 8.9%-11.2%the inter CV were 8.0%-15.3%and 7.1%-9.4%.The CV of CG-ICA for detecting DON after accelerated aging was less than 10.2%,respectively.The CG-ICA has lower LOD and higher stability,it can be used for rapid screening of large-scale samples.Thirdly,a chrysanthemum-like gold@polydopamine immunochromatography assay(AuNC@PDA-ICA)was established for qualitative and quantitative detection of DON in wheat and rice.The AuNC@PDA were synthesized by one-step method.The pH of antibody conjugation,the amount of mAb,the concentration of DON-BSA,the concentration of goat anti-mouse Ig G and the spray volume of AuNC@PDA-mAb were optimized.AuNC@PDA-ICA was established under optimal conditions.AuNC@PDA-ICA was used for the qualitative detection of DON in wheat and rice with a LOD of 1000.0μg/kg.The stability and detection performance of the method were better.AuNC@PDA-ICA was used for the quantitative detection of DON in wheat and rice,the linear equations were Y=4.987-1.305 lgX and Y=4.229-1.095 lgX,R~2were 0.999 and 0.996,the quantitative LOD were 110.524 and 105.985μg/kg,the linear ranges were 150.0-3000.0 and 300-4500μg/kg,respectively.The recovery rates in wheat and rice samples were 71.1%-89.7%and 93.5%-99.6%,the intra CV were 6.7%-9.1%and 2.8%-7.3%,the inter CV were 4.1%-9.2%and 3.1%-8.8%,respectively.The CV of AuNC@PDA-ICA for detecting DON after accelerated aging was less than 4.1%.The AuNC@PDA-ICA has lower LOD and higher stability,it can be used for rapid screening of large-scale samples.In summary,this study established CG-ICA and AuNC@PDA-ICA,sample pretreatment and detection process can be completed within 20 min.The qualitative and quantitative LOD of the methods can meet the maximum level requirements for DON in wheat and other foods prescribed by national food safety standard"Maximum levels of mycotoxins in foods"(GB 2761-2017).Both two ICAs have lower LOD and higher stability,they can be used for the rapid screening of large-scale samples on the spot,providing effective rapid monitoring and screening methods for Chinese grain supervision departments,market supervision and management departments and enterprises,and providing guarantee for the quality and safety of Chinese grain. |
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