We use protein film electrochemistry (PFE) to characterise a wide variety of proteins which in vivo are found in different biochemical environments.
In order to study a protein using PFE it is necessary to immobilise it on an electrode. We use a variety of electrode materials, including TiO2-modified FTO, SAM-modified (self-assembled monolayer) gold, and, most commonly, pyrolytic graphite.
Simple Physical Surface Modification
In the simplest of experiments, a graphite electrode is simply polished to a mirror finish using an aqueous alumina slurry suspended on cotton wool. This produces a variety of oxidised functional groups on the surface (including carboxylic acids, ketones, lactones etc), which are believed to aid protein absorption. Alternatively, the graphite surface can be abraded using commercially-available sandpaper to produce a very rough, largely hydrophobic surface. In general, proteins bind preferentially to one of these surfaces over the other.
Chemical Surface Modification
A method used in the group to modify carbon surfaces is diazonium coupling. This has been used in the group for creating a bilirubin oxidase-coated cathode for use in the fuel cell. A layer of an aromatic acid (2-naphthoic acid) on the electrode surface increases the amount of enzyme attachment and stability of the enzyme film. It is also possible that these groups play a role in the transfer of electrons between the electrode and the enzyme. The current for a modified electrode can be as much as four times that of an unmodified electrode.
The scheme below shows the reaction for the coupling. An aromatic amine is converted to the diazonium salt, and then binds to the carbon surface with loss of nitrogen.
We use a variety of techniques to characterise the surfaces of the electrodes that we use in our studies, including scanning electron microscopy (SEM), N2 porosimetry, electrochemistry, fluroescence microscopy, and FT-IR/Raman spectroscopies.