A novel third generation uric acid biosensor using uricase electro-activated with ferrocene on a Nafion coated glassy carbon electrode

A new uric acid biosensor was constructed using ferrocene (Fc) induced electro-activated uricase (UOx) deposited within Nation (Naf) on glassy carbon electrode (GCE). Electro-activation of UOx was successfully achieved by cyclic voltammetry through the electrostatic interaction of Fc with Trp residues within the hydrophobic pockets in UOx. The Naf/UOx/Fc composite was characterised by AFM, FTIR and EDX to ensure proper immobilisation. The interaction of Fc with the enzyme was analysed by Trp fluorescence spectroscopy and the alpha-helicity of the protein was measured by CD spectropolarimetry. The charge transfer resistance (R-ct), calculated from electrochemical impedance spectroscopy, for the modified sensor was lowered (1.39 k Omega) and the enzyme efficiency was enhanced by more than two fold as a result of Fc incorporation. Cyclic voltammetty, differential pulse voltammetty and amperometry all demonstrated the excellent response of the Naf/UOx/Fc/GCE biosensor to uric acid. The sensor system generated a linear response over a range of 500 nM to 600 mu M UA, with a sensitivity and limit of detection of 1.78 mu A mu M-1 and 230 nM, respectively. The heterogeneous rate constant (k(s)) for UA oxidation was much higher for Naf/UOx/Fc/GCE (1.0 x 10(-4) cm s(-1)) than for Naf/UOx/GCE (8.2 x 10(-5) cm s(-1)). Real samples, i.e. human blood, were tested for serum UA and the sensor yielded accurate results at a 95% confidence limit. (C) 2014 Elsevier B.V. All rights reserved.