Thionine covalently tethered to multilayer horseradish peroxidase in a self-assembled monolayer as an electron-transfer mediator

A new approach to construct a reagentless enzyme biosensor is described. Based on multilayer horseradish peroxidase in a self-assembled monolayer configuration, the biosensor was constructed using multilayer thionine covalently tethered to the enzyme as an electron-transfer mediator. The multilayer enzyme and the multilayer mediator were stepwisely synthesized onto an L-cysteine-assembled gold electrode using glutaraldehyde as a bifunctional reagent. The multilayer mediator tethered to the multilayer enzyme could effectively and stably shuttle electrons between the electrode and the multilayer enzyme linked onto the monolayer. The sensitivity of the resulting enzyme biosensor with eight layers of enzyme and three layers of mediator was more than 250 mu A cm(-2) for 1.0 x 10(-4) mol/L hydrogen peroxide under optimal conditions, whereas such a modified electrode with one layer of enzyme and one layer of mediator did not yield a detectable response to 1.0 x 10(-4) mol/L hydrogen peroxide.