## 3 Results and Discussion
### Amperometric Fatty Acid Biosensor Design and Characterisation  
As mentioned in the previous section, our cyclic voltammetric studies demonstrated that it should be feasible to fabricate a fatty acid amperometric biosensor, based on a CoPC-SPCE, by immobilising  
Figure 1: Cyclic voltammograms obtained for: (**a**) linoleic acid using a plain SPCE with LOX (blue), and blank buffer solution (dashed black); (**b**) linoleic acid using a CoPC-SPCE with LOX (red), and blank buffer solution (black); (**c**) \(\alpha\)-linolenic acid using a plain SPCE with LOX (blue) and a blank buffer solution (dashed black); (**d**) \(\alpha\)-linolenic acid using a CoPC-SPCE with LOX (red), and a blank buffer solution (black). SPCE = screen-printed carbon electrode; CoPC = cobalt phthalocyanine; LOX = lipoxygenase. LOX onto the electrode surface; a schematic diagram of the biosensor construction is shown in Figure 2. Figure 3 shows a schematic diagram of the sequence of reactions involved in the measurement of the fatty acids. The enzyme LOX is immobilised on to the surface of a CoPC-SPCE by cross-linking with glutaraldehyde. During the biosensor operation, the fatty acid (linoleic or \(\alpha\)-linolenic acid) from the bulk solution diffuses into the enzyme reaction layer which, in the presence of oxygen, produces the corresponding hydroperoxide (see previous section). This species chemically reduces Co\({}^{2+}\) to Co\({}^{+}\) in the cobalt phthalocyanine molecule, which then undergoes electrochemical oxidation to reform Co\({}^{2+}\). These reactions can be considered as the electrocatalytic oxidation of the hydroperoxide species to produce O\({}_{2}\), a proton and a free radical (R.). The transfer of one electron from Co\({}^{+}\) to the electrode surface (oxidation) constitutes the analytical response. This electrochemical response occurs at much lower oxidation potentials than required for the direct oxidation which occurs using a plain SPCE; therefore, better selectivity is achieved when exploiting the electrocatalytic reaction. It should be noted that hydrogen peroxide undergoes electrocatalytic oxidation using a cobalt phthalocyanine based sensor which involves the chemical reduction of Co\({}^{2+}\) to Co\({}^{+}\) followed by electrochemical oxidation of Co\({}^{+}\) back to Co\({}^{2+}\) (the latter constituting the analytical response) [18], whereas other biomolecules such as cysteine involve the Co\({}^{3+}\) to Co\({}^{2+}\) redox couple [19]. We believe it is likely the hydroperoxides of \(\alpha\)-linolenic and linoleic acid would undergo electrocatalytic oxidation involving the Co\({}^{2+}\)/Co\({}^{+}\) couple, due to the similarity of their functional groups to hydrogen peroxide and the similarity of their electrocatalytic oxidation potentials [20]. In order to deduce the surface morphology of the biosensor during the modification processes, we employed scanning electron microscopy. Figure 4 shows a series of three SEM images obtained during the construction of the amperometric biosensor: image (i) shows the initial CoPC-SPCE, as can be seen characteristic flakes of carbon are evident, and it is clear that the CoPC-SPCE has a porous, three dimensional structure; image (ii) was taken after the deposition of LOX, followed by a drying step; the enzyme layer is dispersed evenly in discrete structures; image (iii) was obtained after the deposition of a glutaraldehyde layer on top of the enzyme layer, followed by a drying/cross linking step. This latter step results in a cohesive coating that was considered to strongly immobilise the enzyme onto the SPCE-CoPC surface. At the stage, we considered that the biosensor should be suitable for our proposed method of operation, for fatty acid analysis, involving amperometry in stirred solution. This is discussed in the following section.