Faradic Peaks Enhanced by Carbon Nanotubes in Microsomal Cytochrome P450 Electrodes

Abstract

[a] 1IntroductionThei ncreasing needs of personalized therapies have recently stimulated impressive advances in research and development of portable point-of-care biosensors for realtime monitoring as well as biosensors for drug development [1,2].Enzymatic amperometric biosensors based on the enzyme family cytochrome P450 (CYP) have drawn increasing attention because CYP is am onooxygenase enzyme primarily involved in the metabolism of bioactive metabolites and hydrophobic xenobiotics such as drugs, environmental pollutants and steroids [3].T he 3D structure of the enzyme is illustrated in Scheme 1A.CYP catalyses the monooxygenase reaction, atwo-electron reaction where the electrocatalytic transformation of as ubstrate is coupled to the electrocatalytic reduction of oxygen, according to the general equation:As ubstrate (RH) is hydroxylated (ROH) through the insertion of one oxygen atom into the substrate,w hile the second atom of oxygen is reduced to water [4].T he two electrons necessary for the monooxygenation are provided by the redox reaction of the heme group in the active site of the CYP (Scheme 1A), which is in-vivo activated through electron transfer from its redox partners:t wo electrons derived from NAD(P)H are transferred to the CYP active site via the electron transport protein, cytochrome P450 reductase (CPR) [6].T he direct immobilization of CYP on an electrode overcomes the need for NAD(P)H and CPR, as the electrons needed for the redox reaction of the heme group are directly supplied by the electrode [4,5,7,8].Another approach is the electrode functionalization with microsomes containing both CYP and CPR (msCYPs). msCYPs are commonly used in the industry for drug development [2].A sr eported in [2,7,9],t he discovery that microsomes are as effective as recombinant CYPs in enabling direct electrochemistry on electrodes promoted an increasing interest in biosensors based on microsomes-CYP,a st he production of microsomes is cheaper than recombinant CYPs.I naprevious study [9] msCYPs were immobilized on ap olycation-coated electrode and the authors proved that the direct electron transfer occurred according to the natural electron transfer path (electrode!CPR!CYP). Moreover, am ore recent work [10] proved that, in presence of as ubstrate, the amount of metabolite formation in the microsomes containing the reductase was almostd ouble respect to what obtained with CYP alone.T he authors concluded that the electron transfer from the electrode to the CPR and then to the CYP is am ore efficient pathway than the direct electron supply to the CYP.I nt his work the authors also showed that ah ydrophobic electrode surface facilitates the immobilization of msCYP,b ecause of the presence of hydrophobic regions in the surface structure of CYP,C PR and the lipids that compose the microsome.With cyclic voltammetry (CV) or other electro-analytical techniques,i ti sp ossible to apply ap otential to the immobilized CYP and record the current that is produced. This current can be attributed to the reduction of Ab...

Enzymatic and Nonenzymatic Electrochemical Interaction of Abiraterone (Antiprostate Cancer Drug) with Multiwalled Carbon Nanotube Bioelectrodes

“…In order to identify these peaks, electrochemical responses of different biosensors working on 900 nM drug solutions were compared (Figure ). These peaks were identified by comparing the response of these different realized biosensors with each other and with independent observation published in the literature by other authors . Comparison of behavior in the presence and absence of abiraterone is presented in Figure a,b.…”

“…(D) The fourth peak (61 mV) was increasing over time and was stabilized with increasing the number of cycles, so it was identified as a background peak, as also confirmed by our group in other similar electrochemical systems. 6 The inhibition of CYP3A4 by increasing the concentration of abiraterone was observed in voltammograms acquired by carbon/MWCNT/micrCYP3A4 biosensor. The CYP3A4 faradaic peak (−270 mV) is related to the reduction of the active site of the enzyme (heme group).…”

“…(D) The fourth peak (61 mV) was increasing over time and was stabilized with increasing the number of cycles, so it was identified as a background peak, as also confirmed by our group in other similar electrochemical systems. 6 …”

“…These peaks were identified by comparing the response of these different realized biosensors with each other and with independent observation published in the literature by other authors. 6 Comparison of behavior in the presence and absence of abiraterone is presented in Figure 3a,b.…”

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone

“…Encapsulation of enzymes in polymers and gels to immobilize them on electrodes has also been used for microsomes [31,32]. Microsomes can be immobilized on electrodes modified with carbon nanotubes [33,34].…”