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 destabilize its conformation and hence result in a loss of biological activity. We propose here a method for the immobilization of proteins at an electrified liquid-liquid interface. 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, Δ_o^wϕ, is at the positive end of the potential window. By cycling of the potential window back and forth, silica electrodeposition and Cyt c adsorption occur sequentially as demonstrated by in situ UV-vis absorbance spectroscopy. After collection from the liquid-liquid interface, the Cyt c-silica matrix is characterized 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 toward O₂ (observed in the case of Cyt c denaturation). This method of protein encapsulation may be used for other proteins (e.g., Fe-S cluster oxidoreductases, copper-containing reductases, pyrroloquinoline quinone-containing enzymes, or flavoproteins) in the development of biphasic bioelectrosynthesis or bioelectrocatalysis applications.