Graphite counter electrode

Protein film voltammetry was performed at 25 °C inside an anaerobic glove box (Coy) using a double-walled vessel thermostated using an external water bath. A potentiostat (CH Instruments 1201A) was connected to a pyrolitic graphite electrode (PGE) working electrode, a graphite counter electrode, and an Ag/AgCl reference electrode (CH Instruments). The working electrode was treated for 5 min in 1 M nitric acid and thoroughly washed with double distilled water. It was then polished with Alumina (1 μm and 0.3 μm) washed thoroughly with double distilled water, and left to dry inside the glove box. Ten microliters of the 650 μM protein solution was deposited in the electrode and left to dry for at least 1 h. The electrode was placed in the buffer solution (50 mM phosphate equilibrated at various pH values from 6.5 to 8.2) and three complete scans were collected at a scan rate of 50 and 100 mV/s. The pH of the solutions was checked after each experiment. Raw voltammograms were processed with SOAS (84 (link)) to subtract the capacitive current and the position of the anodic and cathodic peaks was averaged and is reported versus SHE by adding 210 mV to the value obtained versus Ag/AgCl.

Nitric acid (HNO₃, 68%) and hydrogen peroxide (H₂O₂, 30%) were purchased from Sigma-Aldrich Sigma, (Darmstadt, Germany). Potassium phosphate monobasic (KH₂PO₄) and sodium hydroxide (NaOH, 99.9%) were purchased from Fisher Scientific (Waltham, MA, USA). Phosphate-buffered saline (PBS, 20 mmol∙L⁻¹ KH₂PO₄ + 20 mmol∙L⁻¹ K₂HPO₄ + 0.1 mol∙L⁻¹ KCl, pH 6.8) was used as a supporting electrolyte. Potassium ferricyanide (K₃[Fe(CN)₆]), iron trichloride (FeCl₃), and potassium chloride (KCl) were from BDH Chemicals (Philadelphia, PA, USA), hydrochloric acid (HCl, 37%) was from Fisher Scientific (Waltham, MA, USA), carbon nanotubes were from Nanocyl (Sambreville Belgium), glassy carbon (GC), silver/silver chloride reference electrode, and graphite counter-electrode were from CH-Instruments (Austin, TX, USA), sulfuric acid (H₂SO₄, 98%) was from Fisher Scientific (Waltham, MA, USA), titanium isopropoxide, isopropanol (99%), and 1 µm, 0.3 µm, and 0.05 µm alumina powder were from CH-Instruments (Austin, TX, USA), dimethylformamide (DMF) was from BDH Chemicals (Philadelphia, PA, USA), and sodium dodecyl sulfate (SDS) and poly(diallyldimethylammonium chloride) (PDDA, 4% w/w in water) were from Sigma. Solutions (Darmstadt, Germany) were prepared using distilled/deionized water (18 MΩ resistivity).