Study On Novel Electrochemical Immunosensors Based On Nanomaterials And Their Application In Food Safty Detection

Electrochemical immunosensors, combining immunoassay and electrochemical sensor technology, serve as an interdisciplinary frontier related to chemistry, biology, physics and electronics. Owing to their high sensitivity and selectivity, fast responses and advantages in miniaturization and online detection potentials, electrochemical immunosensors have been extensively studied and applied in clinical medicine, environment protection and food inspection. Recently, nanomaterials, due to their unique properties, are widely used for developing electrochemical immunosensors. The application of nanomaterials has brought a great momentum to electrochemical immunosensors and open new horizons for highly sensitive detection, which provides an avenue for high-throughput analysis of both small molecules and biological macromolecules.With the growth of Chinese economy, the increasing of national income and the changes in consumption concept and concept of health, food safety has become the focus of public concern. However, series of serious food safety related accidents occurred recently have been seriously endanger people’s health, and have become a major public health problem and social problems. To this end, public health concerns highlight the need for developing effective, rapid, sensitive, and selective food safety detection methods.In this dissertation, we studied on the development of the novel electrochemical immunosensors based on nanomaterials, the detection mechanism of the immunosensors and their applications in the analysis of sudan I, clenbuterol and chloramphenicol in food stuffs. The dissertation is divided into six parts:Chapter one:PrefaceThis chapter reviews the main aspects of application of electrochemical immunosensor in food safety detection, including basic knowledge of electrochemical immunosensors, synthesis of the nanomaterials and its applications in the construction of electrochemical immunosensors. Then present situation and sources of food safety problems in China, the harm of the representative toxic and hazardous compounds (such as sudan I, clenbuterol and chloramphenicol) residues in food stuffs and the present analytical techniques for their detection are briefly introduced. Finally, the work and significance of this dissertation are brought forward.Chapter two:A monoclonal antibody-based immunosensor for detection of Sudan I using electrochemical impedance spectroscopySudan I monoclonal antibodies (Mabs) were prepared by hybridoma technique and firstly used to develop a Sudan I immunosensor by immobilizing the Mabs on a gold electrode. O-mercaptobenzoic acid (MBA) was covalently conjugated on the gold electrode to form a self-assembled monolayer (SAM). The immobilization of Sudan I Mabs to the SAM was carried out through a stable acyl amino ester intermediate generated by1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the SAM. The changes of the electrode behavior after each assembly step were investigated by cyclic voltammetric (CV) technique. The Sudan I concentration was measured through the increase of impedance values in the corresponding specific binding of Sudan I and Sudan I antibody. A linear relationship between the increased electron-transfer resistance (Ret) and the logarithmic value of Sudan I concentrations was found in the range of0.05to50ng mL-1with the detection limit of0.03ng mL-1. Using hot chili as a model sample, acceptable recovery of96.5-107.3%was obtained. The results were validated by conventional HPLC method with good correlation. The proposed method was proven to be a feasible quantitative method for Sudan I analysis with the properties of stability, highly sensitivity and selectivity.Chapter three:Construction of Dual Signal Amplification Competitive Immunosensor for Detection of ClenbuterolA dual signal amplification immunosensor based on glucose oxidase (GOD)-functionalized nanocomposites was developed for the detection of clenbuterol. The characteristics of the nanocomposites were confirmed by scanning electron microscopy (SEM) and photometry. The results indicate GOD in the nanocomposites keeps good enzyme dynamic response and electro-catalysis performance. The detection mechanism of the immunosensor was investigated by electrochemical methods of differential pulse voltammetry (DPV). A set of experimental conditions were also optimized for the detection of clenbuterol. The developed immunosensor exhibited high sensitivity, selectivity and reproducibility, which had a wide linear range of0.01～100ng mL-1and a low detection limit of4.50pg mL-1for clenbuterol. Accurate detection of clenbuterol in pig liver samples was demonstrated by standard addition method.Chapter four:Development of an electrochemical immunosensor for rapid detection of clenbuterol using anodic stripping voltammetry based on Cu@Au nanoparticles as antibody labelsAn electrochemical immunosensor for sensitive and rapid detection of clenbuterol (CB) using anodic stripping voltammetry (ASV) has been developed based on core-shell Cu@Au nanoparticles (NPs) as anti-clenbuterol antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM). A competitive immunoreaction system, which is defined as clenbuterol-carboxylic multi-walled carbon nanotubes (CMWCNTs) conjugates immobilized on electrode compete with the free clenbuterol for Cu@Au bimetallic NPs labeled antibody, was applied to determine the free CB in PBS by detection of the Cu@Au NPs labels. Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu2+were determined at GC/Nafion/Hg modified electrode by ASV. Under the optimal conditions, the proposed immunoassay could detect CB with a detection limit of0.03ng mL-1and the overall analysis could be completed in2h. Accurate detection of clenbuterol in spiked pig liver samples was demonstrated by comparison with conventional ELISA method.Chapter five:Label-free immunoassay for Chloramphenicol based on hollow gold nanospheres/chitosan compositeA novel label-free electrochemical immunosensor for rapid determination of chloramphenicol (CAP) was fabricated by entrapping monoclonal antibody to chloramphenicol (anti-CAP) in hollow gold nanospheres (HGNs)/chitosan composite modified on a glassy carbon electrode. The hollow gold nanospheres (HGNs) were prepared by using Co nanoparticles as sacrificial templates and characterized by transmission electron microscopy (TEM). The changes of the electrode behavior after each fabrication step were investigated by electrochemical impedance spectroscopy (EIS) technique. Under optimal conditions, the proposed immunosensor has a sensitive response to CAP in a linear range of0.1～1000ng mL-1with the detection limit of0.06ng mL-1. Accurate detection of CAP in real meat samples was demonstrated by comparison with conventional HPLC method. The proposed method was proven to be a feasible quantitative method for CAP analysis with the properties of simple preparation, stability, high sensitivity and selectivity.Chapter six:Photoelectrochemical immunosensor array based on thioglycolic acid capped CdS quantum dots for multiplexed detection of veterinary drug residuesA photoelectrochemical immunosensor based on multi-electrode array was developed for simultaneous and sensitive determination of veterinary drug residues. In this system, poly (dimethyldiallylammonium chloride)(PDDA), Au nanoparticles (Au NPs) and thioglycolic acid (TGA)-capped CdS quantum dots (QDs) were layer-by-layer assembled onto the home-made Au electrode array. The assembling process of the (CdS/PDDA/Au NPs/PDDA)n multilayer was characterized by electrochemical impedance spectroscopy. And then the antibodies for clenbuterol (CB), ractopamine (RAC) and chloramphenicol (CAP) were covalently immobilized onto the Au electrode array by1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) coupling reaction, respectively. The concentrations of CB, RAC and CAP were measured based on the photoelectrochemical effects of CdS QDs, Under the optimal conditions, the limits of detection (LOD) for CB, RAC and CAP were25,50and2.2pg mL-1(3σ), respectively, with acceptable recovery over the range of95.40-105.5%in pig liver samples. All results indicate that the immunosensor array system has potential application for practical, effective and high throughput analysis of veterinary drugs residues.