Establishment Of An Immunological Detection Method For Zearalenone Based On Metal Nanomaterials

Zearalenone(ZEN)is a mycotoxin with estrogenic properties synthesized by fusarium through polyketide pathway.It is widely distributed and exists in a variety of contaminated cereals,such as corn,barley,oats and rice.ZEN had potential risks to human and animal health.Detection method for ZEN can induce acute and chronic poisoning,resulting in damage to liver,kidney,reproductive system and immune system.Therefore,it is urgent to establish the simple,convenient and ultra-sensitive detection method for ZEN.At present,the instrumental detection methods of ZEN include high performance liquid chromatography(HPLC),liquid chromatography tandem mass spectrometry(LC-MS/MS),gas chromatography(GC)and thin layer chromatography(TCL).The disadvantages of the instrumental detection methods are expensive,complex sample preparation and long analysis time,so they are not suitable for on-site rapid detection.Immunoassay had the advantages of simplicity,rapidity and high sensitivity.It has gradually become an important technique for the detection of ZEN.In this study,in order to detect ZEN in corn samples,three immunoassay methods were established.1.Indirect competitive ELISA(ic-ELSIA).The complete antigen coated on the bottom of the 96-well plates competed with the free ZEN in samples to bind the monoclonal antibody.And the HRP labeled secondary antibody binds with the monoclonal antibody.HRP catalyzed the substrate TMB to produce oxidized TMB.The ZEN in samples was blue.After the reaction was terminated with acid,the OD₄₅₀ value was read by the microplate spectrometer for analysis.After optimizing the conditions,the standard curve equation was y=42.190 x-23.994(R₂=0.992).The linear detection range(IC₂₀?IC₈₀)was 11.04 pg/mL–291.68 pg/mL,the sensitivity(IC₅₀)was 57 pg/ml,and the minimum detection limit was 2.5?g/kg,the recovery of spiked standard in corn flour was 81.29%–105.80%.2.Digital colorimetric detection method based on alkaline phosphatase(ALP)-mediated growth of prussian blue nanoparticles(PBNPs)Based on the ic-ELSIA,biotin-conjugated secondary antibody was used to bind Mc Ab and ALP-conjugated avidin to biotin.ALP hydrolyzes ascorbic acid 2-phosphate(AAP)to produce ascorbic acid(AA).AA reduce potassium ferricyanide(K₃[Fe(CN)₆])to potassium ferrocyanide(K₄[Fe(CN)₆]),and(K₄[Fe(CN)₆])reacted with Fe Cl₃ to produce blue PB NPs.Meanwhile,AAP reacts with Fe Cl₃ to form a brown AAP-Fe³⁺complex.When blue and brown were mixed in different ratios,there were colorful color changes.Using the color recognition software on the mobile phone could read the Red,Green and Blue(RGB)value of the solution and quantitatively analyze ZEN.After optimizing the conditions,the standard curve equation was y=51.280-47.020 x(R~2=0.995),the linear detection range was 7.81 pg/mL?500 pg/mL and the minimum detection limit was 4.47 pg/mL,and the recovery of spiked standard in corn flour was 104.63%–118.64%.3.Ratio fluorescence detection method based on ALP-mediated copper nanoclusters(CuNCs)to catalyze o-phenylenediamine(OPD).Based on ic-ELSIA,using ALP labeled Ig G as enzyme labeled secondary antibody,Cu CNs catalyzes OPD to generate2,3-diaminophenazine(DAP)(Em=570 nm)with yellow fluorescence Meanwhile,Cu CNs had AA oxidase activity.After ALP hydrolyzed AAP to produce AA,Cu CNs catalyzed AA to produce dehydrogenated AA(DHAA),and DHAA reacted with OPD to produce3-(dihydroxyethyl)furan[3,4-b]quinoxolin-1-one(DFQ)with blue fluorescence(Em=430 nm).Finally,the concentration of ZEN was detected quantitatively by the ratio of fluorescence intensity(F₄₃₀/F₅₇₀).After condition optimization,the linear regression equation of ZEN was y=8.354-2.818 x(R₂=0.955),the linear detection range was 15.63pg/mL?250 pg/mL,and the minimum detection limits was 0.13 pg/mL,The average recoveries of ZEN in corn flour were 104.28–118.24%.The three ZEN immunoassay methods established in this experiment were characterized by high sensitivity,high accuracy and good accuracy.The ic-ELISA method had the advantages of simple operation and low cost.The digital colorimetric method was based on alkaline phosphatase-mediated growth of Prussian blue nanoparticles and RGB color values using smartphone for results reading.It was more convenient to use and suitable for field detection.The ratio fluorescence method was based on alkaline phosphatase-mediated copper nanoclusters catalyzing o-phenylenediamine.The interference of background signal could be effectively eliminated by reading the ratio of fluorescence intensity.The three methods are suitable for different detection scenarios and lay a foundation for subsequent real sample assays.