Philosophiae Doctor - PhD / This thesis firstly reports on the development and characterization of reagent-less and cobalt(III) sepulchrate[Co(Sep)³⁺] mediated biosensor platforms (biosensor platform 1 and biosensor platform 2), with human recombinant heme thiolate, cytochrome P450 3A4 isoenzyme (CYP3A4), as biorecognition component. Secondly, each biosensor platform was evaluated by using an entirely different category of compound as model substrate, with the overall objective being the development of alternative analytical method for the detection and quantification of each of these substrates,by amperometric transduction method. In this regard biosensor platform 1 was evaluated for the detection of 2,4-dichlorophenol, whereas biosensor platform 2 was evaluated for the detection of protease inhibitor (PI) HAART drug, indinavir. Fourthly, this dissertation also reports on the use of genetic engineering as complimentary method during biosensor investigations, as source for continuous supply of catalytically active biological recognition component. With respect to the preparation of the biosensors in particular, biosensor platform 1 was constructed by entrapping the commercially sourced full-length, wild type CYP3A4 on a pre-formed electroactive carrier matrix, consisting of Co(Sep)³⁺–modified nafion membrane on a glassy carbon electrode. In this regard, the nafion-Co(Sep)³⁺ composite was prepared by integrating the Co(Sep)³⁺ species into a pre-formed nafion film through manual drop-coating and mixing methods.