In this study we have tested the use of modified gold nanoparticles (GNPs) for introduction into cytochrome c (CytC) multilayer assemblies, constructed by the layer-by-layer deposition technique. For this purpose gold nanoparticles of different size are synthesized and the GNPs modified with mercaptopropionic acid (MPA). The particles are characterized by UV-vis-spectroscopy and transmission electron microscopy (TEM). The modified particles have been studied with respect to their binding capability on a CytC layer by SPR. The negatively charged particles exhibit rather fast adsorption kinetics on CytC. We can also show that CytC binds to MPA-modified gold nanoparticles. For the construction of electro-active CytC multilayers a negatively charged polyelectrolyte can be used. We demonstrated that the polyelectrolyte can be replaced by small-sized GNPs (5nm) in the multilayer assemblies, but assemblies consisting only of GNPs and CytC can be formed too. When these arrangements are prepared on gold electrodes, full electro-activity of the redox protein in the multiple layers can be obtained. CytC exhibits a formal potential of -24+/-12mV vs. Ag/AgCl. The electrochemical response is linearly increasing with the number of layers deposited, reaching a CytC surface concentration of 100pmol/cm(2) with 6 layers. The electron transfer mechanism through the layer system and the particle size influence are discussed.
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