Study On Novel Electrochemical Immunosensor Of E.coli In Dairy Products Based On Nanocomposites

Electrochemical immunoassay, taking the advantages of inherent simplicity and fast response time, has become one of the predominant analytical techniques in biological sample analysis. Recently, the application of nanocomposites in the immunosensor fabrication is one of the most popular techniques for their numerous signal amplifications and excellent electrocatalytic effects.The development of rapid and sensitive method for E.coli detection is very important in the fields of dairy industry and health security of human. In this work, three kind of electrochemical immunosensor were developed for E.coli analysis in dairy products. The results demonstrated that the proposed methods were highly sensitive and selective, and thus could become a promising technique for E.coli detection in dairy products. The details are listed below: 1. Highly Sensitive Electrochemical Immunoassay of E. coli in Dairy Product by Using Gold NanorodsAn electrochemical immunoassay strategy was proposed for E. coli detection in dairy product based on gold nanorod(AuNR)-labels({dAb-AuNR-FCA}) for signal amplification. The {dAb-AuNR-FCA} was fabricated by exploiting silica-functionalized AuNRs(AuNRs@SiO2) as the carriers for immobilization of detection antibody(d Ab) and ferrocenecarboxylic acid(FCA), in which d Ab was used for recognition of E. coli and FCA served as signal-generating molecule. In the case of the electrochemical assay, L-cysteine was first self-assembled on a gold electrode surface through Au-S bonding, and the carboxyl group of L-cysteine was activated by using EDC/NHS, which introduced a high density of capture antibody(cAb). After further treated with BSA to block the nonspecific binding, the modified-electrode could effectively capture E. coli by the specific interaction between surface-confined cAb and E. coli, followed by the attachment of the {dAb-AuNR-FCA} to form a sandwich-type system. The FCA in the bound labels was recorded by differential pulse voltammetry(DPV). Since FCA was only present when the {dAb-AuNR-FCA} immunoreacted with E. coli, the peak current reflected E. coli concentration in incubation solution. Here, the AuNRs@SiO2 as carriers had larger surface area to load enormous FCA, and thus played important roles in signal amplification to improve detection sensitivity. Under optimal experimental conditions, it showed wide linear relationship and low detection limit. The proposed strategy was also used to determine E. coli in dairy product, and the recoveries of standard additions were in the range of 95.6% to 106%. The results confirmed that the electrochemical immunoassay gave a useful protocol for E. coli analysis with high sensitivity, specificity and acceptable accuracy, and thus could potentially become a promising technique for the assay of E. coli in dairy product. 2. Optimized Dendrimer-encapsulated Gold Nanoparticles and Enhanced Carbon Nanotube Nanoprobes for Amplified Electrochemical Immunoassay of E. coli in Dairy Product Based on Enzymatically Induced Deposition of PolyanilineA highly sensitive immunosensor was reported for E. coli assay in dairy product based on electrochemical measurement of polyaniline(PAn) that was catalytically deposited by horseradish peroxidase(HRP) labels. Herein, the immunosensor was developed by using poly(amidoamine) dendrimer-encapsulated gold nanoparticles(PAMAM(Au)) as sensing platform. Importantly, the optimal HAu Cl4/PAMAM ratio was investigated to design the efficient PAMAM(Au) nanocomposites. The nanocomposites were proven to not only increase the amount of immobilized capture antibody(cAb), but also accelerate the electron transfer process. Moreover, the {d Ab-CNT-HRP} nanoprobes were prepared by exploiting the amplification effect of multiwalled carbon nanotubes(CNTs) for loading detection antibody(dAb) and enormous HRP labels. After a sandwich immunoreaction, the quantitatively captured nanoprobes could catalyze oxidation aniline to produce electroactive PAn for electrochemical measurement. On the basis of signal amplification of the PAMAM(Au)-based immunosensor and the {dAb-CNT-HRP} nanoprobes, the electrochemical detection of E. coli could be achieved in 3 h. The electrochemical immunosensor was also used to determine E. coli in dairy product(pure fresh milk, infant milk powder, yogurt in shelf-life and expired yogurt), and the recoveries of standard additions were in the range of 96.8% ~ 108.7%. Overall, this method gave a useful protocol for E. coli assay with high sensitivity, acceptable accuracy and satisfying stability, and thus provided a powerful tool to estimate the quality of dairy product. 3. Development of an Electrochemical Immunoassay of E.coli in Dairy Products Based on Dendrimers and Functional Gold NanoparticlesA novel electrochemical immunosensor for sensitive detection of E.coli in dairy products was developed. Electrode was first modified with thionine-chitosan film to provide an immunosensing platform for PAMAM(Au) nanocomposites to bind capture antibody(cAb). Gold nanoparticls(AuNPs) were prepared for the fabrication of the enhanced labels({dAb-AuNPs-HRP}), which provided a large capacity for loading enormous HRP and detection antibody(dAb). Using a sandwich immunoassay format, the {dAb-AuNPs-HRP} labels were introduced onto the electrode surface, and then produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide(H2O2) in the presence of thionine. The results confirmed that the electrochemical immunoassay gave a useful protocol for E.coli analysis with rapid response, high sensitivity and specificity, and thus provided a powerful tool for estimating the quality of dairy products.