Establishment Of A New Method For Electrochemical Immunosensor And LC-MS/MS Detection Of Mycotoxins In Malt

ObjectiveAffected by the matrix itself and external factors,Chinese medicinal materials are highly susceptible to mildew and contamination of mycotoxins during growth,harvesting,processing,storage and transportation,affecting the quality and safety of medicinal materials and threatening the physical and mental health of users.Among them,aflatoxin B₁?AFB₁?and Ochratoxin A?OTA?are two mycotoxins with high detection rate and pollution level in Chinese herbal medicine,and have strong liver and kidney toxicity.Carcinogenic,teratogenic,mutagenic and other effects.As a precursor of AFB₁,streptomycin?ST?has a good predictive effect on the production and residue of AFB₁.Therefore,to explain the relationship between ST and AFB₁ in Chinese herbal medicines,and to establish a sensitive and reliable analytical method to quickly detect the contamination level of mycotoxins in Chinese herbal medicines,which is of great significance for ensuring the quality and safety of medicinal materials.Therefore,this topic takes the"medication and food dual-use"malt as the research object,and investigates the relationship between ST and AFB₁ during storage,and establish a sensitive electrochemical method to quickly detect the pollution level of OTA in malt,and toxin monitoring provides technical support and theoretical basis.MethodsSince ST is a precursor substance of AFB₁,the relationship between the growth of AFB₁ and ST under the optimal conditions of bacterial growth and bacterial growth was studied by using reverse culture technique.The raw malt was cultured in two batches for 20 days,and the powder was sampled every two days.The sample extraction solvent,pretreatment method and liquid pHase flow comparison were optimized.LC-MS detection method was established under the optimal conditions to detect the growth relationship between ST and AFB₁.Self-assembled films were prepared by using thioglycolic acid?TGA?based on self-assembly?SAM?principle,and self-assembled films were characterized by cyclic voltammetry?CV?and electrochemical impedance spectroscopy?EIS?.The coupling time,optimum concentration and coupling temperature of the self-assembled membrane were optimized,and finally the optimal conditions for stable self-assembled membrane were obtained.Using the prepared Au/TGA self-assembled membrane,the bovine serum albumin-conjugated OTA antigen?BSA-Ag?was coupled,and the optimal time,optimal concentration and optimal temperature were investigated.The optimal coupling time and concentration of Au/TGA-BSA/Ag coupled antibody?Ab?.The Au/TGA-BSA/Ag-Ab electrochemical immunosensor was established under the optimal conditions,and the OTA was detected.The standard curve was established and the malt sample was added and recovered.ResultsThis experiment determined that the best mobile phase of AFB₁ and ST in liquid quality was aqueous solution containing 0.1%formic acid and 5 mM ammonium acetate?phase A?,methanol solution containing 0.1%formic acid?phase B?,and established AFB₁ and ST.Standard curve:AFB₁ was in the range of 0.1-50 ng/mL,linear equation was y=55600x-1600?r=0.9999?,test limit is 0.03 ng/mL;ST was in the range of 0.25-100 ng/mL,linear equation was y=64600x+4210?r=0.9993?,the detection limit was 0.04 ng/mL.Experiments have shown that the ST content was proportional to the AFB₁content during malt production.The optimum self-assembled film for Au/TGA was determined as follows:at 4?,the TGA concentration of 4 mmol/L,and a coupling time of 90 min with a gold electrode,TGA forms a stable single molecule on the gold electrode.membrane.Then,using the established Au/TGA self-assembled film,the optimal experimental conditions for the Au/TGA-BSA/Ag-Ab sensor were determined:at 37?,the EDC/NHS concentration ratio was 2:1,and the activation time was 20 min.At the same time,the Au/TGA activation effect was the best,then the BSA-Ag was coupled,and the optimal coupling conditions were 200?g/mL,45 min and 25?,finally,the optimal coupling condition was200?g/mL,coupled for 4 h.The standard curve of the OTA standard solution was established using the Au/TGA-BSA/Ag-Ab sensor obtained under the optimal conditions.The OTA concentration range was 0.1-1 ng/mL,and the linear equation was y=0.2219x+0.1888?r>0.99?,the detection limit is?LOD?0.08 ng/mL.The malt samples were spiked and recovered.The recovery of OTA was 71.8-97.2%,and the RSD value was less than 3%,which correspond to the experimental requirements.ConclusionsThis experiment proved that when the ST content was above 1?g/kg,the AFB₁ content was seriously exceeded.Monitoring the ST content was essential for effective control of AFB₁ content.The Au/TGA-BSA/Ag-Ab immunosensor was established by Au/TGA self-assembled membrane to detect the reproducibility,stability and specificity of malt matrix,and it has a great application prospect.