For the preparation of platinum microelectrodes (see Figure 1), a tungsten substrate with a diameter of 125 μm and AC resistance of 0.5 MΩ was insulated with parylene-C via a vacuum deposition (A-M Systems; Sequim, WA). The exposed tips (typically 50 – 60 μm length) were cleaned for 10 s in hydrofluoric acid (48 wt % in water), electrolyzed for 30 s at 50 °C in an electrocleaning solution at an applied potential of −5 V (vs a platinum electrode), and rinsed with water.58 (link) The conical electrode was then transferred into an acidic platinum electroplating solution, plated for 5 s at 50 °C at an applied potential of −0.5 V (vs a platinum electrode), and rinsed with water and ethanol.57 The ensuing platinum-deposited working electrode was platinized in 3% chloroplatinic acid (v/v in water) by cycling the potential from +0.6 to −0.35 V (vs Ag/AgCl) at a scan rate of 20 mV/sec using a CH Instruments 730B bipotentiostat.40 (link),54 (link) Finally, the multilayered microelectrode (i.e., platinum black/platinum/tungsten, Pt-B/Pt/W) was modified with the optimized fluorinated xerogel-derived permselective membrane by dip-coating the sensor tip into a sol solution consisting of 60 μL of MTMOS, 15 μL of 17FTMS, 300 μL of ethanol, 80 μL of water, and 5 μL of 0.5 M HCl. After allowing the xerogel “film” to cure for 10 min, the process was repeated to yield a ~2.5 μm thick final membrane. The xerogel-modified electrode was then allowed to dry for 24 h under ambient conditions.
Response and calibration curves were obtained by injecting aliquots of the standard NO solution (1.9 mM or 41 nM) into 100 mL of PBS (0.01 M, pH 7.4; not deoxygenated) at room temperature under constant stirring. All microelectrodes were pre-polarized for 30 min to 5 h. Currents were recorded at an applied potential of +0.7 and +0.8 V (vs Ag/AgCl) for the platinized and non-platinized working electrodes, respectively.