Enzyme Immobilization Strategies and Electropolymerization Conditions to Control Sensitivity and Selectivity Parameters of a Polymer-Enzyme Composite Glucose Biosensor

Abstract: In an ongoing programme to develop characterization strategies relevant to biosensors for in-vivo monitoring, glucose biosensors were fabricated by immobilizing the enzyme glucose oxidase (GOx) on 125 µm diameter Pt cylinder wire electrodes (Pt_C), using three different methods: before, after or during the amperometric electrosynthesis of poly(ortho-phenylenediamine), PoPD, which also served as a permselective membrane. These electrodes were calibrated with H₂O₂ (the biosensor enzyme signal molecule), glucose, and the archetypal interference compound ascorbic acid (AA) to determine the relevant polymer permeabilities and the apparent Michaelis-Menten parameters for glucose. A number of selectivity parameters were used to identify the most successful design in terms of the balance between substrate sensitivity and interference blocking. For biosensors electrosynthesized in neutral buffer under the present conditions, entrapment of the GOx within the PoPD layer produced the design (Pt_C/PoPD-GOx) with the highest linear sensitivity to glucose (5.0 ± 0.4 μA cm⁻² mM⁻¹), good linear range (K_M = 16 ± 2 mM) and response time (< 2 s), and the greatest AA blocking (99.8% for 1 mM AA). Further optimization showed that fabrication of Pt_C/PoPD-GOx in the absence of added background electrolyte (i.e., electropolymerization in unbuffered enzyme-monomer solution) enhanced glucose selectivity 3-fold for this one-pot fabrication protocol which provided AA-rejection levels at least equal to recent multi-step polymer bilayer biosensor designs. Interestingly, the presence of enzyme protein in the polymer layer had opposite effects on permselectivity for low and high concentrations of AA, emphasizing the value of studying the concentration dependence of interference effects which is rarely reported in the literature.