Probing traces of hydrogen peroxide by use of a biosensor based on mediator-free DNA and horseradish peroxidase immobilized on silver nanoparticles

A new electrochemical biosensor for determination of hydrogen peroxide (H2O2) has been developed by immobilizing horseradish peroxidase (HRP) on silver colloids (nanosilver) and use of a DNA-functionalized interface. In the presence of the DNA and the nanosilver the immobilized HRP gives a pair of well-defined redox peaks with an electron-transfer rate constant of 3.27 +/- 0.91 s(-1) in pH 7.0 PBS. The presence of DNA also provides a biocompatible microenvironment for enzyme molecules, greatly amplifies the amount of HRP molecules immobilized on the electrode surface, and improves the sensitivity of the biosensor. Under optimum conditions the biosensor has electrocatalytic activity in the reduction of hydrogen peroxide with linear dependence on H2O2 concentration in the range 1.5 x 10(-6) to 2.0 x 10(-3) mol L-1; the detection limit is 5.0 x 10(-7) mol L-1 at a signaltonoise ratio of 3. The K-m(app) m value of HRP in the composite membrane was found to be 1.62 mmol L-1. These results suggest that the properties of the complex film, with its bioelectrochemical catalytic activity, could make it useful for development of bioelectronic devices and for investigation of protein electrochemistry at functional interfaces.