Characterization of oxidoreductase–redox polymer electrostatic film assembly on gold by surface plasmon resonance spectroscopy and Fourier transform infrared–external reflection spectroscopy

Abstract

The electrostatic assembly of nanocomposite thin films consisting of alternating layers of an organometallic redox polymer (RP) and oxidoreductase enzymes, glucose oxidase (GOX), lactate oxidase (LOX) and pyruvate oxidase (PYX), was investigated. Multilayer nanostructures were fabricated on gold surfaces by the deposition of an anionic self-assembled monolayer of 11-mercaptoundecanoic acid, followed by the electrostatic attachment of a cationic RP, poly(vinylpyridine Os(bis-bipyridine) 2Cl-co-allylamine) (PVP-Os-AA), and anionic oxidoreductase enzymes. Surface plasmon resonance (SPR) spectroscopy, Fourier transform infrared external reflection spectroscopy (FT-IR–ERS) and electrochemistry were employed to characterize the assembly of these nanocomposite films. The surface concentration of GOX was found to be 2.4 ng/mm 2 for the first enzyme layer and 1.96 ng/mm 2 for the second enzyme layer, while values of 10.7 and 1.3 ng/mm 2 were obtained for PYX and LOX, respectively. The apparent affinity constant for GOX adsorption was found to be 8×10 7 M −1. FT-IR–ERS was used to verify the incorporation of GOX and its conformational stability inside of these nanocomposite thin films. An SPR instrument with a flow-through cell was modified by additions of Ag/AgCl reference and Pt counter electrodes, with the gold-coated SPR surface film serving as the working electrode. This enabled real-time observation of the assembly of sensing components and immediate, in situ electrochemical verification of substrate-dependent current upon the addition of enzyme to the multilayer structure. A glucose-dependant amperometric response with sensitivity of 0.197 μA/cm 2/mM for a linear range of 1–10 mM of glucose was obtained. The SPR and FT-IR–ERS studies also showed no desorption of polymer or enzyme from the nanocomposite RP–GOX structure when stored in aqueous environment occurred over the period of 3 weeks, suggesting that decreasing substrate sensitivity with time was due to loss of enzymatic activity rather than loss of film compounds from the nanostructure.