Although enzyme-based electro-chemical bio-sensors show significant potential towards the construction of a sensitive and selective sensor, a direct quantitative detection of the enzyme’s catalytic activity still remains challenging. Distinct properties of the enzyme are required as well as an electrode in order to enhance the direct electron transfer (DET) between the enzyme’s catalytic active site and the electrode.
Enzymes that contribute to these requirements are the cytochrome P450 enzymes (CYPs) that comprise for about 80% of the phase-I drug-metabolizing enzymes in the human liver. The isoenzyme cytochrome P450 2D6 (CYP2D6) is responsible for the metabolisation of 25% of all clinically prescribed drugs; therefore the interaction potential of xenobiotics with this isoenzyme is of utmost importance. Due to the fact that the CYP catalysed metabolisation reactions are electron dependent oxidative processes, CYPs are suitable candidates for the construction of a small-scale bio-sensor.
In our study, a screen printed working electrode with an additional carbon nanotube (CNT) layer was used for the immobilisation of the CYP2D6 enzyme. Two fundamentally different immobilisation techniques were investigated: (i) non-covalent immobilisation and (ii) covalent immobilisation, where either physical interactions or direct chemical bonds were exploited for the stable and electronically efficient connection between the enzymes and CNT electrode. The immobilisation was characterised by testing the activity of the enzymes through enzymatic demethylation of a model substrate, i.e. dextromethorphan, before and after an intensive wash of the working electrode. The concentration of the substrate and its product, i.e. dextrorphan, were determined using a high-performance liquid chromatography (HPLC). The stability, selectivity and sensitivity of the biosensor were further evaluated using a cyclic-voltammetrical processing of the induced signal.