A Method to Construct a Third-Generation Horseradish Peroxidase Biosensor:  Self-Assembling Gold Nanoparticles to Three-Dimensional Sol−Gel Network

A novel method for fabrication of horseradish peroxidase biosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol−gel network. A cleaned gold electrode was first immersed in a hydrolyzed (3-mercaptopropyl)-trimethoxysilane (MPS) sol−gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol−gel network. Finally, horseradish peroxidase (HRP) was adsorbed onto the surface of the gold nanoparticles. The distribution of gold nanoparticles and HRP was examined by atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The performance and factors influencing the performance of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response (2.5 s) to H2O2. The detection limit of the biosensor was 2.0 μmol L-1, and the linear range was from 5.0 μmol L-1 to 10.0 mmol L-1. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.