Stock solutions of 10 mM HP and 100 mM AA were prepared in distilled water and 100 mM HCl, respectively.2.2. Site URL List 1|]# Instrumentation and SoftwareCalibrations for HP, AA and glucose were performed in a standard three-electrode cell containing 20 mL PBS at room temperature, a saturated calomel reference electrode (SCE), a stainless steel auxiliary electrode and either bare or modified platinum-iridium (90:10) working electrodes. Constant potential amperometry was performed at an applied potential of +0.7 V versus SCE, using Chart (v 5.2) software (AD Instruments Ltd., Oxford, UK) and a low-noise potentiostat (Biostat IV, ACM Instruments, Cumbria, UK). The working electrodes were allowed to settle in quiescent PBS to give a steady background current before the addition of small known aliquots of the analyte of interest.
2.3. Working Electrode PreparationCylinder electrode preparation has been described in detail recently [44]. Briefly, 125 ��m diameter Teflon-coated Pt-Ir wire (90:10, Advent Research Materials Ltd., Eynsham, England) was stripped of 1 mm Teflon to expose the bare metal, which displays many of the electrochemical properties of pure Pt [44]. Electropolymerization was carried out in oPD solutions (of varied monomer concentration, background electrolyte and enzyme concentration) at +0.7 V versus SCE for 15 minutes for these PtC electrodes [39,41]. Three main enzyme immobilization protocols were used in this work.
In the first, the enzyme was immobilized by adsorption and dip-evaporation before PoPD deposition [23].
Each electrode was dipped in a 200 U?mL?1 solution of GOx for 5 minutes, allowed to dry for 5 min, and then dipped quickly into the GOx solution four more times Batimastat with 5 minutes drying between each dip, followed by electropolymerization. This protocol was previously found to optimize enzyme loading for biosensors of the type PtC/EOx/PoPD [24]. The second design immobilized the enzyme by adsorption and dip-evaporation after PoPD deposition followed by exposure to glutaraldehyde (GA) vapour for GSK-3 15 min to crosslink the enzyme [23], and are termed PtC/PoPD/GOx-GA. The third method used co-immobilization, whereby either 1 mg?mL?1 (~650 U?mL?1; ~5 ��M) or 5 mg?mL?1 GOx was dissolved in oPD, and electropolymerized at +0.
7 V vs. SCE for 15 min [30,31] to give PtC/PoPD-GOx; see Figure 1.Figure 1.Sample steady-state calibration data and nonlinear regression analysis for the biosensor design, PtC/PoPD-GOx [Equation (4), R2 = 0.998, n = 8; left], illustrating the graphical significance of the Michaelis-Menten constants, Jmax and KM. The linear region …2.4. Enzyme Kinetic ParametersFirst generation biosensors of the general design PtC/PoPD~EOx (i.e.