Electrochemical dynamics of cytochrome p450-3a4 isoenzyme biosensor for protease inhibitor antiretroviral drug

• Main Author: Hendricks, Nicolette Rebecca
• Format: Others
• Language: English
• Published: 2010
• Subjects: Antiretroviral therapy
• Online Access: http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3467_1361368134

<p>The highly active antiretroviral therapy (HAART) drug agent, indinavir, and the endocrine disruptor compound, 2,4-dichlorophenol (2,4-DCP), are directly related to two of South Africa&rsquo === s, and in fact, two of the globe&rsquo === s most fundamentally important and comprehensively researched subjects areas, which includes, HIV/AIDS and water pollution. In fact&nbsp === these two compounds share multiple significant commonality factors. Firstly, they have a profound effect on the health aspects of humans, albeit from opposite sides of the&nbsp === &lsquo === equation&rsquo === . Secondly, in the context of metabolism, they both share the same rout of biotransformation, and as such, both have a profound effect on the main first pass&nbsp === metabolising hepatic enzyme, CYP450 3A4, as well as xenobiotics sharing the same metabolic athway. Thirdly and perhaps more importantly, in direct relation to the human mortality, their levels preferentially require constant or regular monitoring, a process, at this stage, is still only officially possible with complex specialized analytically-based techniques. Moreover, these techniques are only based on centralized detection and quantification, which essentially means expensive procedures, and long waiting periods for results. 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+&ndash === 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.&nbsp === In addition to this, the so-formed biosensor was re-inforced by a thin nafion layer as outer-film. The complete biosensor may be denoted as GCE||naf|CMECo( Sep)3+|flCYP3A4|naf. Biosensor platform 2 on the&nbsp === other hand, was constructed by entrapment of the N-terminally modified human recombinant CYP3A4 (consisting only of the heme domain and the surrounding apoprotein), prepared locally through genetic engineering, as a histidine-tagged, catalytically active soluble construct, denoted nCYP3A4, in a biocompatible ionically crosslinked hydrogel-composite membrane. Enzyme immbilization in this case was also realized on a pre-formed nafion-Co(Sep)3+ carrier matrix film, however, in this case the electroactive carrier&nbsp === matrix was prepared by integrating the electroactive Co(Sep)3+ species deep within the nafion microstructure through potentiostatic electrodeposition method at a costant&nbsp === potential of +450 mV for 1200 sec. The so prepared biosensor, is denoted GCE||naf|El- Co(Sep)3+|nCYP3A4|Agrs-PEI-PVA. In this regard, biosensor for platform 2, different variables affecting the performance and stability of the biosensor were evaluated. Selected ex-situ characaterization methods, including scanning electrochemical microscopy (SEM), Fourier Transform Infrared (FTIR) and UVVis spectroscopy was used as complimentary characterization methods , morphological and structural charaterization, revealed&nbsp === the formation of a highly stable electroactive composite film for the carrier matrix in biosensor platform 2 , exhibiting a compact nature and a smooth consistancy in which the&nbsp === electroactive Co(Sep)3+ mediating species was embedded deep within the microstructure of the pre-formed nafion film. Moreover, the method of preparation was highly reproducible, while voltammetric studies also corroborated the stability of the carrier matrix film. Overall, the design path used for this method was shown to be an improvement&nbsp === as compared to the design path used for biosensor platform 1, particularly with regard to the carrier matrix. Nevertheless, the proposed substrates were successfully detected&nbsp === and quantified by the individual biosensor plaforms. In this regard, the dynamic linear range of the GCE||naf|CMECo( Sep)3+|flCYP3A4|naf biosensor, for 2,4-DCP exhibited an&nbsp === upper limit of 45&nbsp === A, with the sensitivity determined as 0.038 A M-1. In addition to this, the LOD was calculated as 0.043 g L-1, which was lesser than the USA Environmental Protection Agency&rsquo === s (EPA) drinking water equivalent level (DWEL) for 2.4-DCP. In the case of the GCE||naf|El-Co(Sep)3+|nCYP3A4|Agrs-PEI-PVA biosensor, the linear&nbsp === concentration range for indinavir was shown to be between 2.183 M 3.552 M, while the sensitivity was determined as 0.035 A M-1. Morover, the LOD value, determined as 59.72 mg L-1 was suggested to be of signifiance with regard to the maximum plasma concentration, CMax, with respect to the ritonavir-boosted regimen, which is the proposed method of administering indinavir. This can also be of value for HIV/AIDS patients who are poor metabolizers, as they will have significantly elevated concentration of the drug,&nbsp === when administered with ritonavir as booster. Above and beoynd these results, the overpotential for the reduction of dioxygen, which is a crucial step in the catalytic cycle of the&nbsp === CYP3A4 enzyme, was significantly reduced by the GCE||naf|El-Co(Sep)3+|nCYP3A4|Agrs-PEI-PVA biosesnor, as compared to the other biosensor.</p>