Construction Of Electrochemical Immunosensor Based On Enzyme-induced Cu²⁺/Cu⁺ Conversion

Ethyl carbamate(EC)is mainly a toxic compound produced by urea and ethanol in the process of fermenting alcoholic beverages.Due to the low content of EC and many interfering components in alcoholic beverages,there is an urgent need for a fast and sensitive detection method to detect EC in alcoholic beverages.In this paper,an electrochemical immunosensor was developed based on enzyme-induced Cu²⁺/Cu⁺conversion for sensitive and selective detection of EC.In this strategy,alkaline phosphatase(Alkaline-phosphatase,ALP)firstly catalyzed the hydrolysis of2-phospho-L-ascorbic acid(2-phospho-L-ascorbic acid,AAP)to L-ascorbic acid(Ascorbic acid,AA).Then AA interacted with Cu²⁺to mediate Cu²⁺/Cu⁺conversion.When EC was present,it can trigger the above-mentioned reaction through a"sandwich"immunoassay model and connect with the ALP enzyme,so more Cu⁺will be produced and less Cu²⁺will remain.The conversion rate of Cu²⁺/Cu⁺can be detected by the square wave voltammetry(Square wave voltammetry,SWV)technique to indicate the concentration level of EC.This electrochemical immunoassay method integrates the advantages of enzyme labeling,signal amplification,signal conversion and output,which can selectively and sensitively detect EC in food samples.The specific research content is as follows:(1)The electrochemical behaviors of Au nanoparticles(Au nanoparticles,Au NPs),graphene(Graphene,GN),graphene-Au nanoparticles(GN-Au NPs)modified electrodes were explored,mainly involving five aspects:the characterization of the synthesis of Au NPs and GN-Au NPs,optimization of the concentration of Au NPs,GN,GN-Au NPs modified electrodes,characterization of the scan rate of Au NPs,GN,GN-Au NPs modified electrodes,and electrochemical characterization of AA reduction Cu²⁺by GN-Au NPs modified electrodes.It can be concluded that the electrochemical behavior of Au NPs,GN,GN-Au NPs modified on the electrode is a typical diffusion control process.Au NPs,GN,and GN-Au NPs can all increase the conductivity of the electrode.GN-Au NPs behave higher-conductivity than the Au NPs and GN counter electrodes.And the optimal concentrations of Au NPs,GN,and GN-Au NPs modified on the electrode were 30 mg/L,0.8 mg/m L,and 0.8 mg/m L,respectively.Finally,according to the electrode modified with GN-Au NPs optimal concentration was used to determine the reduction of Cu²⁺by AA.It was found that GN-Au NPs had a greater influence on the electrochemical process of AA reduction of Cu²⁺.(2)Based on enzyme-induced Cu²⁺/Cu⁺ conversion proposed an electrochemical immunosensor was used to detect EC sensitively and selectively.The electrochemical immunoassay method showed good analytical performance for EC,with a linear range of 2.5 nmol/L?2.5×10⁴ nmol/L,R²=0.9928 in the linear range,and a limit of detection(A limit of detection,LOD)2.28 nmol/L(S/N=3).The recovery rate experiment showed that the recovery rates of vodka and red wine were respectively 88.1%?98.5%,72.9%?107.8%and Coefficient of Variation 3.6%?12.0%and 3.0%?10.6%,which were comparable to other reported methods.The Cu⁺cascade reaction caused by Cu²⁺reduction greatly improves the detection sensitivity.By combining enzyme-mediated immunoassay with electrochemical technology,it can provide a flexible and solid platform for detecting harmful substances in food.