Development Of Three Kinds Of Immunochromatographic Strips For Rapid Detection Of Aflatoxin B₁ In Fermented Food

Mold is the general term of filamentous fungi.Common molds include Aspergillus,Fusarium,Penicillium and Alternaria.In appropriate living conditions,mold will infect various foods,resulting in spoilage and quality deterioration.Mycotoxins are metabolites of fungi.When ingested or absorbed through the skin,mycotoxins can cause illness or death in humans or livestock,so they are considered one of the most dangerous food contaminants.Aflatoxins（AFs）,metabolites produced by Aspergillus flavus and Aspergillus parasiticus,are considered to be the most carcinogenic compounds.Aflatoxin B₁（AFB₁）is the most toxic,carcinogenic and widely distributed in nature.Fermented food is mainly fermented by molds,yeasts,bacteria and other microorganisms.Common fermented foods include fermented bean products（bean paste,soy sauce,etc.）and fermented grain products（vinegar,baijiu,yellow rice wine,etc.）.Aspergillus flavus and Aspergillus parasiticus capable of producing AFB₁ has been found in a variety of beans,grains and fermented food,and fermented food may be contaminated by AFB₁ in the process of production,storage and transportation.Therefore,it is necessary to establish a fast,sensitive and accurate method for the detection of AFB₁ in fermented food.Based on immunological principles,three lateral flow immunoassays（LFIA）were established for the rapid detection of AFB₁.The double T-lines LFIA using traditional gold nanoparticles（Au NPs）as signal labeled material was developed for the qualitative detection of AFB₁.A LFIA method using novel quantum dot nanobeads（QBs）as signal labeling materials was developed for the quantitative detection of AFB₁ in common fermented foods.Using the aggregation induced emission fluorescent microsphere（AIEFM）,which was used as signal labled material recently,we attempted to develop a LFIA method for the quantitative detection of AFB₁ in common fermented foods.The main research methods and conclusions are as follows:（1）A gold nanoparticles lateral flow immunoassay（Au NPs-LFIA）was established based on immunological principles.The method adopts the reading mode of two T-lines and can be used for the qualitative detection of AFB₁ in the naked eye.Au NPs were synthesized by sodium citrate reduction method.The p H,the amount of m Ab labeling,AFB₁-BSA concentration on test line,the volume of Au NPs-m Ab probe,and immune response time were optimized.Double T-lines Au NPs-LFIA was established and the sensitivity of this method was evaluated.In the optimal conditions,the detection limits of Au NPs-LFIA for AFB₁ were 0.125 ng/m L and 0.5 ng/m L,respectively.Double T-lines Au NPs-LFIA can be used as a preliminary rapid screening method for AFB₁detection,which can clearly distinguish weak positive（national minimum quantity standard）samples from strong positive samples in actual fermented food,so as to reduce the false positive rate of detection.（2）In order to detect AFB₁ in fermented food rapidly,a fluorescence quantitative immunochromatography method based on QBs was established.The p H,the amount of EDC and m Ab labeling,the concentration of AFB₁-BSA on the test line,the volume of QB_S-m Ab probe,and the immune reaction time were optimized.In optimal conditions,the linear equation of standard curve in buffer solution was y=-1.9303 lg（x）-0.7847（R²=0.994）.The IC₅₀ was 0.044 ng/m L while the linear range was 0.01-0.2ng/m L.The linear equation of the standard curve in the matrix mixture was y=-2.0374lg（x）-0.7519（R²=0.994）.The IC₅₀ was 0.0487μg/kg,the linear range was 0.01-0.2μg/kg.Ten kinds of fermented food were selected for standard recovery experiment and the concentration of AFB₁ was all 5μg/kg.The results showed that the recovery rates were 74.53%-117.73%,and the coefficient of variation（CV）were 1.55%-9.54%.The average recovery of 10 food samples was 102.59%,so the QBs-LFIA method established in this study is suitable for the quantitative determination of AFB₁ in fermented foods.Moreover,the specific experimental results of this method showed that the cross-reaction rate of the method was low for other five kinds of mycotoxins.The experimental results of accelerated preservation showed that this method was relatively stable.Therefore,the QBs-LFIA method,which is sensitive,specific,accurate and stable,can meet the requirements of practical application and has a good application prospect.（3）The LFIA method using QBs as signal labeling material has been studied widely.This study attempts to apply the AIEFM material with excellent optical properties for the rapid detection of AFB₁.It is useful for the practical application of AIEFM.This method established a fluorescence quantitative immunochromatography method based on AIEFM and applied it to the detection of AFB₁ in three fermented foods.Conditions of p H,the amount of EDC and m Ab labeling,AFB₁-BSA concentration on the test line,the volume of AIEFM-m Ab probe and immune response time were optimized.Under the optimal conditions,the quantitative standard curves was established.The linear equation of the standard curve in the buffer solution was y=-1.5255 lg（x）-0.4503（R²=0.986）,the IC₅₀ was 0.0397 ng/m L,and the linear range was0.01-0.1 ng/m L.The linear equation of the standard curve in the matrix mixture was y=-2.0673 lg（x）-0.1418,（R²=0.992）,the linear relationship was good,the IC₅₀ was0.0652μg/kg,and the linear range was 0.01-0.2μg/kg.Three kinds of common fermented food were selected to carry out the standard recovery experiment,and the results showed that the recovery rates were between 86.26%-110.32%,and CV value were between 1.31%-8.74%,indicating that the accuracy of the method was good.The accelerated preservation experiment of AIEFM-LFIA method was carried out,and the results showed that the method had good stability.The cross-reaction rate with 5mycotoxins was low,indicating good specificity.Therefore,AIEFM-LFIA established in this study can be used for actual detection.In summary,a double T-lines Au NPs-LFIA method was established in this study,which could realize the qualitative detection in the field.A QBs-LFIA method was developed for the quantitative detection of AFB₁ in common fermented foods.The AIEFM-LFIA method was established based on the AIEFM,and its practical application effect was evaluated,which could provide ideas for more extensive application of AIEFM.