Electron-transfer mediator microbiosensor fabrication based on immobilizing HRP-labeled au colloids on gold electrode surface by 11-mercaptoundecanoic acid monolayer

In this paper, a new strategy for constructing a mediator-type amperometric hydrogen peroxide (H2O2) microbiosensor was described. An electropolymerized thionine film (PTH) vas deposited directly onto a gold electrode surface. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. Consequently, horseradish peroxidase (HRP) was successfully immobilized on nanometer-sized Au colloids, which were supported by thiol-tailed groups of 11-mercaptoundecanoic acid (11-MUA) monolayer covalently bound onto PTH film. With the aid of the PTH mediator, HRP-labeled Au colloids microbiosensor displayed excellent electrocatalytical response to the reduction of H2O2. This matrix showed a biocompatible microenvironment for retaining the native activity of the covalent HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H2O2. The proposed H2O2 microbiosensor exhibited linear range of 3.5 mu M-0.7 mM with a detection limit of 0.05 mu M (S/N = 3). The response showed a Michaelis-Menten behavior at larger H2O2 concentrations. The K-M(app) value for the biosensors based on 24 nm Au colloids was found to be 47 mu M, which demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H2O2 with no loss of enzymatic activity. This microbiosensor possessed good analytical performance and storage stability.