Polyazetidine-based immobilization of redox proteins for electron-transfer-based biosensors

A highly stable functional composite film was prepared using polyazetidine prepolymer (PAP) with peroxidase from horseradish (HRP) and/or glucose oxidase (GOx). The good permeability of the PAP layer to classical electrochemical mediators, as evaluated by the determination of the diffusion coefficient of different redox molecules, is of great importance in view of the use of PAP as an immobilizing agent in second-generation biosensor development. Cyclic voltammetry of the HRP-PAP layer on a glassy carbon electrode (GCE) showed a pair of stable and quasi-reversible peaks for the HRP-Fe((III))/Fe((II)) redox couple at about -370 mV vs. Ag/AgCl electrode in pH 6.5 phosphate buffer. The electrochemical reaction of HRP entrapped in the PAP film exhibited a surface-controlled electrode process. This film and the successive modifications (HRP-PAP self-assembled monolayer (SAM) modified Au electrode) were used as a biological catalyst (hydrogen peroxide transducers) for glucose biosensors, after coupling to GOx. Both HRP/GOx-PAP and HRP/GOx-PAP SAM third generation biosensors were prepared and characterized. The use of PAP as immobilizing agent offers a biocompatible micro-environment for confining the enzyme and foreshadows the great potentiality of this immobilizing agent not only in theoretical studies on protein direct electron transfer but also from an applications point of view in the development of second- and third-generation biosensors.