This paper describes the development and evaluation of a generic method for the immobilization of enzymes onto a gold electrode and its application to amperometric biosensors. The surface of the gold electrode was modified with gold nano-particles (AuNP) and mixed self-assembled monolayers (SAMs) to form an enzyme biosensor matrix. Horseradish peroxidase (HRP) was immobilized on the modified surface to form a biosensor matrix on a gold electrode. After the deposition of gold nano-particles on a bare gold surface, the AuNP-deposited gold electrode and_x000D_ a bare electrode were compared for the surface area and electric current using AFM and cyclic voltammetry (CV). The AuNP strongly adhered to the surface of the gold electrode, had uniform distribution and was very stable. A mixed SAM, composed of two different monolayer molecules, dithiobis-N-succinimidyl propionate (DTSP) and inert tetradecane-1-thiol (TDT), was formed using reductive desorption technique and cyclic voltammetry was used to verify the formation of mixed deposition. First, 3-mercaptopropionic acid (MPA) and TDT were deposited with a specified deposition ratio between the two components. Then, MPA was desorbed by applying electric potential to the surface. Finally, DTSP was deposited where MPA was. The ratios of 20 : 80 and 50 : 50 between MPA and TDT were examined, and differences in the CV responses were discussed. HRP was immobilized on the mixed SAM surface. The investigated method is regarded as an effective way for stable enzyme attachment, while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of samples and enzymes and the SAM matrix helps avoid enzyme leaking. It is interesting that the mixed SAM shows unique CV characteristics compared to the uni-molecular SAMs. The reaction kinetics of the SAM-immobilized enzyme is discussed with the CV results and is observed to_x000D_ obey the Michaelis-Menten equation.

Gold Nano-particles Mixed Self-Assembled Monolayer Reductive Desorption Amperometric Sensor Horseradish Peroxidase Enzyme Sensor

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