Determination of the biomolecular composition of an enzyme-polymer biosensor

Previously there has been considerable research in producing biosensors by the entrapment of proteins in electrochemically formed conducting polymer films. In this paper we describe a method enabling the amount of the enzyme glucose oxidase, entrapped at the surface of a poly(pyrrole) film to be quantified using X-ray photoelectron spectroscopy (XPS). Components of the biosensor XPS spectrum were deconvoluted as a linear combination of characteristic polymer and enzyme spectra, using primarily the high-energy C(1s) signals (288.2 and 286.4 eV) due to functional groups in the enzyme and the N(1s) signal present in both enzyme and polymer samples at 400.2 eV, together with the lesser shoulder at 398.0 eV, related to iminic nitrogen species in the polymer alone. The O(1s) peak at 531.7 eV, associated with the immobilized enzyme, provided additional compositional information, although here the effects of surface contamination were more pronounced. Further, and in parallel with this XPS study, corroborative results were obtained using reflectance Fourier transform infrared spectroscopy in order to monitor the total enzyme concentration within the bulk of the film. Finally, electrochemical measurements were made in order to assess the catalytic activity of the immobilized enzyme. When used together, the three methods proved successful in probing the structure/composition of the biosensor film, illustrating a potential application in determining absolute amounts of protein within a complex copolymeric matrix.