Studies On Electrochemistry Immunosensors Based On Gold Nanoparticles As A Carrier

Electrochemical immunosensor has the advantages of immunity technology and electrochemical technology. The detection of the immunoassay is based on the change in the amperometric or impedimetric response before and after antigen-antibody reaction. The shift of response exhibits a linear dependence on the concentration of analytes. With their potential utility as high sensitivity, nice selectivity, miniaturization, easy automation and potential ability for real-time and on-site analysis, electrochemical immunosensor can be applied to a wide range of analytical tasks, such as clinical diagnosis, bioassay, environmental monitoring and industial analysis. Antigen-antibody immobilization plays an important role in design and fabrication of electrochemical immunosensors, which usually decides the performance of immunosensor, such as sensitivity, selectivity, response time and lifetime. A good immobilization method should meet the following requirements: (1) produce immobilized antigen-antibody that retain their biocatalytic activities; (2) produce immunosensors that have a low mass-transfer resistance and a rapid response time; (3) produce immobilized antigen-antibody that are stable and do not leach from the substrate. In this paper, we developed three novel amperometric immunosensors and a mediator-free impedimetric immunosensor, based on efficient immobilization of biomolecule and gold nanoparticles (nano-Au), could retain efficiently activity of loading immunoreactants and increase the assembled proteins.(1) Amperometric immunosensor based on layer-by-layer assembly of thiourea, gold nanoparticles and methylene blueAn amperometric immunosensor with highly sensitivity has been proposed based on layer-by-layer assembly of gold nanoparticles and methylene blue on thiourea modified glassy carbon electrode. Thiourea (TU) could be immobilized onto surface of the pretreated GCE through cyclic voltammetry and thiourea (NH₂CSNH₂) has two amino groups (R-NH₂) that can be used for coupling of gold nanoparticles. Methylene blue (MB) is positively charged in pH 6.5 PBS buffer and can be easily absorbed by the charged negatively gold nanoparticles as a result of the adsorption of citrate in the fabrication process to obtain the desired number of {nano-Au/MB}_n mutilayer films. Nano-Au has a large surface area to increase the immobilization amount of anti-HCG. The characteristics of the modified electrode at different stages of modification was studied by using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The performances of the immunosensor were studied in detail. The cathodic peak current response of CVs were proportional to the concentration of HCG in the range from 1.0 to 100.0mIU/mL and correlation coefficient of 0.9960 with a detection limit of 0.3 mIU/mL at 3σ. The application of the immunosensor in human serum were excellent.(2) Amperometric immunosensor based on anthranilic acid, gold nanoparticles and methylene blue as mediatorA highly sensitive amperometric immunosensor has been developed for the detection of HBsAg based on anthranilic acid, gold nanoparticles and methylene blue as mediator on a glassy carbon electrode through electrostatic adsorption. Anthranilic acid was electropolymerized to glassy carbon electrode surface for the first time, which used to electrostatic interaction with the positively charged methylene blue (MB). MB can be easily absorbed by the charged negatively gold nanoparticles. Finally, HBsAg was firmly adsorbed on the nano-Au monolayer. The characteristics of the modified electrode at different stages of modification was studied by using cyclic voltammetry (CV). The effects of experimental conditions such as pH, temperature and scan rates have been investigated. Under optimal conditions, the cathodic peak current response of CVs were proportional to the concentration of HBsAg in the range from 3.0-250 ng/mL with a detection limit of 1.0 ng/mL. Moreover, the immunosensor system could provide high sensitivity, low detection limit, good selectivity and specificity for the assay of HBsAg.(3) Amperometric immunosensor based on layer-by-layer assembly of thiourea, prussian blue nanoparticles and gold nanoparticlesA novel human chorionic gonadotrophin (HCG) immunosensor has been developed by self-assembling thiourea (TU), prussian blue nanoparticles (nano-PB) and nano-structure gold (NG) film on gold electrode. Firstly, TU was formed on gold electrode (GE) by the self-assembled method and PB nanoparticles were immobilized on thiourea modified electrode surface by electrostatic adsorption. Then nano-structure gold film was formed on nano-PB/TU modified electrode by electrodeposition method, and finally the human chorionic gonadotrophin antibody (anti-HCG) were immobilized onto modified electrode. The characteristics of the modified electrode at different stages of modification was studied by using cyclic voltammetry (CV) and scanning electron microscopy (SEM), The performances of the immunosensor were studied in detail. Under optimal conditions, the reduction current of the amperometric response were proportional to the HCG concentration in two ranges from 0.2 to 2.0 mIU/mL and from 2.0 to 75.0 mIU/mL with a detection limit of 0.07 mIU/mL at a signal-to-noise ratio of 3.(4) Empedimetric immunosensor based on layer-by-layer (LBL) self-assembly of thiourea (TU) and gold nanoparticles (nano-Au) on gold electrodeAn impedimetric immunosensor based on layer-by-layer self-assembly of thiourea (TU) and gold nanoparticles (nano-Au) on gold electrode for detection ofα-1-fetoprotein (AFP) has been developed. Thiourea are formed on gold electrode (GE) by the self-assembled method and thiourea are functionalized with NH₂ and can be easily absorbed gold nanoparticle by charged -NH₂ groups on the gold electrode surface, while gold nanoparticle charged negatively as a result of the adsorption of citrate in the fabrication process can be easily absorbed by thiourea to form layered, ordered, and electroactive mutilayer films. Nano-Au has a large surface area to increase the immobilization amount of antibody. Then, we could monitor the variation of electrochemical impedance response to detect the antibody-antigen interaction. Compared with the nano-Au/TU monolayer films, the {nano-Au/TU}_n multilayer films has several attractive advantages, such as high electroactivity and high stability which contribute to the high sensitivity, good linearity and and long life time of the proposed immunosensor. Under optimal conditions, the logarithm of impedance was proportional to the AFP concentration in the range from 5.0 to 300.0 ng/mL with a detection limit of 1.7ng/mL.