Here, we describe the formation of a hybrid biointerface consisting of gold nanoparticle (AuNP) and cytochrome c (cyt c) on indium tin oxide (ITO) electrodes using a two-step immobilization procedure. The Au nanoparticles were attached to the ITO electrodes by 3-mercaptopropyl trimethoxysilane (3-MPTMS). The electrode was then incubated with 11-mercapundecanoic acid (11-MUA) and the nanoparticles were activated to allow for coupling to cyt c. This process resulted in the formation of the AuNP/cyt c hybrid on the ITO electrode. The ITO/AuNP/cyt c substrate surfaces were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction analysis (XRD), and cyclic voltammetry (CV) techniques. Further analysis regarding the surface roughness properties of ITO, ITO/AuNP and ITO/AuNP/cyt c were also performed. The ITO/AuNP/cyt c immobilized ITO electrode displayed a pair of well-defined redox peaks (E(pa) at 0.09 V and E(pc) at 0.02 V) at pH 7.0 in HEPES buffer solution. Differential pulse voltammetry (DPV) and amperometric i-t measurements on the modified electrode showed a linear response after the addition of hydrogen peroxide (H(2)O(2)). The developed electrode sensor had an electron transfer rate constant (k(s)) of 0.69 s(-1) with a detection limit of 0.5 μM. The results of this study suggest that the hybrid layers were well fabricated on the ITO surface and the developed ITO/AuNP/cyt c electrode displayed an excellent electrocatalytic response for the detection of H(2)O(2).
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