Development And Application Of An Electrochemical Immunosensor-based Method For The Detection Of Ochratoxin A

Ochratoxin A(OTA)is listed as one of the most important mycotoxins with potential risks to human health and food safety.Otas are hepatotoxic,nephrotoxic,immunosuppressive,teratogenic and fetal toxic and are widely found in coffee,beans,meat,milk,eggs,grapes,grains and grain products.To ensure food safety,it is important to establish an efficient and accurate ochratoxin A test method.The specific experimental content of this study is as follows:(1)An electrochemical immunosensor was prepared using OTA antibody and diazo salt grafted on the electrode surface for OTA rapid detection.In this work,the Screen-printed carbon electrodes(SPCE)are modified with diazo salt by electrodeposition.The grafted diazo film serves as the bottom layer,exposing the carboxyl functional groups on the electrode surface.Then carbodiimide is used to activate the carboxyl group,and the antibody and carboxyl group are crosslinked to conduct amide bonding so as to realize the immobilization of the antibody.Cyclic voltammetry(CV)and Electrochemical impedance spectroscopy(EIS)were used to characterize immune sensors.The immunosensor presented excellent electrochemical performance for OTA detection in the spiked samples.Under optimal working conditions,the linear detection range of the developed immune sensor is20-200 ng/m L(R~2=0.997),and the detection limit is 0.5 ng/m L,which is suitable for the detection of OTA in food.In addition,the sensor is highly selective to OTA in the presence of interfering compounds and has stability for up to half a month.The electrochemical immunosensor developed in this study is easy to construct,sensitive,fast and stable,with low detection limit for OTA in actual samples,low analysis time and cost,and will be widely used in the rapid detection of OTA.(2)An electrochemical immunosensor was prepared using OTA antibodies,gold nanoparticles and multi-walled carbon nanotubes modified on the electrode surface for OTA rapid detection.In this work,gold nanoparticles were synthesized by citric acid reduction method,and then gold nanoparticles and multi-walled carbon nanotubes were deposited on the surface of composite film screen printing electrode to study the electrochemical performance of the immunosensor.The results showed that gold nanoparticles and multi-walled carbon nanotubes significantly promoted the electron transfer between the immune protein and the underlying electrode,and the resulting bioelectrode retained its biocatalytic activity and realized rapid and sensitive OTA quantitative detection.Under the optimized experimental conditions,the detection concentration of OTA was 0.2～500 ng/m L(R~2=09991),S/N=3,and the detection limit was 0.05 ng/m L.The sensor has the advantages of short response time,high sensitivity and good reproducibility,which will provide A new method for the detection of ochratoxin A.(3)Based on the principle of colloidal gold immunochromatography,A colloidal gold test strip for the rapid detection of ochratoxin A was established,including the preparation of colloidal gold,the preparation of gold-labeled probe,the test and optimization of the condition parameters of the test strip,etc.The visual detection limit of the strip for OTA detection was 0.25 ng/m L and the detection time was 8min.The strip could specifically identify OTA without cross-reaction with other mycotoxins,and the strip had good repeatability,which could be used for semi-quantitative detection of OTA in food.However,compared with electrochemical method,the latter has a lower detection limit and is a more sensitive detection means in detection.