Electrophoretic protein deposition: a new enzyme immobilization method for the development of amperometric biosensors

Electrodeposition is an enzyme immobilization method based on the well-known electrophoretic phenomena of proteins under the influence of an electrical field. In the original method, the enzyme is mixed to a collagen dispersion at a pH value different from their isoelectric points to form macromolecular complexes which migrate to, and deposit on, an electrode surface held at an appropriate electric potential. In spite of the interest of such a method, quite similar to the enzyme entrapment in electrosynthesized polymers, the so-called "electrochemical immobilization", up to now few papers have been devoted on this subject. These studies do not deal with the understanding of both chemical and electrochemical processes involved in protein electrodeposition, which are particularly significant for the proper development of biosensors. For example, a study of a suitable electrochemical technique, able to control the protein deposition while minimizing the undesirable but collateral faradaic processes (i.e. O2 evolution), cannot be found in the relevant literature. More important, the realization of an useful biosensor, free of interference and fouling problems (which arise in real matrices analysis) has not yet been achieved with this approach. The electrodeposition method, herewith called "electrophoretic protein deposition" (EPD), has been investigated in our laboratory with the aim to develop a novel approach in amperometric biosensor realization. The influence of some chemical and electrochemical parameters on the protein deposition has been studied with several electrochemical methodologies. Galvanodynamic and potentiodynamic techniques have been compared in terms of membrane quality, thickness and spatial control of protein deposition. An electrochemical quartz crystal microbalance study permitted further insights about the growth of proteic deposit on the electrode surface. The enzyme electrodes so obtained have been further characterized to realize the feasibility of EPD procedure for the development of an useful biosensor. In this respect, the realization of amperometric biosensors using the hybrid approach has been drawn out to this novel enzyme immobilization procedure. In particular, EPD of co-crosslinked bovine serum albumin/glucose oxidase membranes coupled with electrosynthesized non-conducting films of poly-2-naphthol or poly-o-aminophenol permitted the realization of glucose biosensors with anti-interference and anti-fouling performances so interesting to assure a future employment for real sample analysis