Electrogeneration of a free-standing cytochrome c – silica matrix at a soft electrified interface

Interactions of a protein with a solid-liquid or a liquid-liquid interface may destabilise its conformation and hence result in a loss of biological activity. We propose here a method for the immobilisaiton of protein at an electrified liquid-liquid interface while maintaining it’s native conformation. Cytochrome c (Cyt c) is encapsulated in a silica matrix through an electrochemical process at an electrified liquid-liquid interface. Silica condensation is triggered by the interfacial transfer of cationic surfactant, cetyltrimethylammonium, at the lower end of the interfacial potential window. Cyt c is then adsorbed on the previously electrodeposited silica layer, when the interfacial potential is at the positive end of the potential window. By cycling the potential window back and forth, silica electrodeposition and Cyt c adsorption occurs sequentially as demonstrated by in situ UV-vis absorbance spectroscopy. After collection from the liquid-liquid interface, the Cyt c – silica matrix is characterised ex situ by UV-vis diffuse reflectance spectroscopy, confocal Raman microscopy and fluorescence microscopy, showing that the protein maintained its tertiary structure during the encapsulation process. The absence of denaturation is further confirmed in situ by the absence of electrocatalytic activity towards O₂, signalling Cyt c denaturation. This method of protein encapsulation may be used for other proteins in the development of biphasic bioelectrosynthesis or bioelectrocatalysis applications.