Circuits Design and Nano-Structured Electrodes for Drugs Monitoring in Personalized Therapy

Drug development and personalized therapy require accurate and frequent monitoring of the metabolic response by living tissues to treatments. In case of high risk side effects, e.g. therapies with interfering anti-cancer molecule cocktails, direct monitoring of the patient’s drugs metabolism is essential as the metabolic pathways efficacy is highly variable on a patient-by-patient basis. Currently, there are no fully mature point-of-care bio-sensing systems for drugs metabolism monitoring directly in blood. The aim of the paper is to investigate solutions to develop point-of-care systems for drugs monitoring in personalized therapy. P450 enzymes are the considered probe molecules as they are key proteins directly involved in drugs metabolism of humans. Sensitivity improvement is ensured by means of enzyme integration onto electrodes structured with carbon nanotubes. Component Off-The-Shelf (COTS) based circuits design is investigated toward bio-chip development. Results show that the proposed circuitry is suitable for the aim and confirm that nanotubes are detection enhancers

Quantum Dots and Wires to improve Enzymes-Based Electrochemical Bio- sensing

An investigation on nano-structured electrodes to detect different metabolites is proposed in this paper. Three different metabolites are considered: glucose, lactate, and cholesterol. The direct detection of hydrogen peroxide is also considered since it does not involve any enzyme. The metabolites and the peroxide were detected by using screen-printed electrodes modified by using multi-walled carbon nanotubes. In all cases, improvements of orders of magnitude were registered both on detection sensitivity and on detection limit. A close comparison with data recently published in literature has shown the existence of an inverse linear correlation between detection sensitivity and detection limit when sensor performances improve due to nano- structured materials. This inverse linear relationship seems to be a general law as it is here demonstrated for all the considered detections on glucose, lactate, cholesterol, and hydrogen peroxide

Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6

Human Cytochrome P450 2C9 and Its Polymorphic Modifications: Electroanalysis, Catalytic Properties, and Approaches to the Regulation of Enzymatic Activity

The electrochemical properties of cytochrome P450 2C9 (CYP2C9) and polymorphic modifications P450 2C9*2 (CYP2C9*2) and P450 2C9*3 (CYP2C9*3) were studied. To analyze the comparative electrochemical and electrocatalytic activity, the enzymes were immobilized on electrodes modified with a membrane-like synthetic surfactant (didodecyldimethylammonium bromide (DDAB)). An adequate choice of the type of modified electrode was confirmed by cyclic voltammetry of cytochromes P450 under anaerobic conditions, demonstrating well-defined peaks of reduction and oxidation of the heme iron. The midpoint potential, Emid, of cytochrome P450 2C9 is −0.318 ± 0.01 V, and Emid = −0.324 ± 0.01 V, and Emid = −0.318 ± 0.03 V for allelic variant 2C9*2 and allelic variant 2C9*3, respectively. In the presence of substrate diclofenac under aerobic conditions, cytochrome P450 2C9 and its polymorphic modifications P450 2C9*2 and P450 2C9*3 exhibit catalytic properties. Stimulation of the metabolism of diclofenac by cytochrome P450 2C9 in the presence of antioxidant medications mexidol and taurine was shown

Screen-printed electrodes based on carbon nanotubes and cytochrome P450scc for highly sensitive cholesterol biosensors

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) immobilized on nanostructured rhodium\u2013graphite electrodes. Multi-walled carbon nanotubes (MWCNT) were deposited onto the rhodium\u2013graphite electrodes by drop casting. Cytochrome P450scc was deposited onto MWCNT-modified rhodium\u2013graphite electrodes. Cytochrome P450scc was also deposited onto both gold nanoparticle-modified and bare rhodium\u2013graphite electrodes, in order to have a comparison with our previous works in this field. Cyclic voltammetry indicated largest enhanced activity of the enzyme at the MWCNT-modified surface. The role of the nanotubes in mediating electron transfer to the cytochrome P450scc was verified as further improved with respect to the case of rhodium\u2013graphite electrodes modified by the use of gold nanoparticles. The sensitivity of our system in cholesterol sensing is higher by orders of magnitude with respect to other similar systems very recently published that are based on cholesterol oxidase and esterase. The electron transfer improvement attained by the use of MWCNT in P450-based cholesterol biosensors was demonstrated to be larger than 2.4 times with respect to the use of gold nanoparticles and 17.8 times larger with respect to the case of simple bare electrodes. The sensitivity was equal to 1.12 μA/(mM mm2) and the linearity of the biosensor response was improved with respect to the use of gold nanoparticles

Pharmacological regulation of the activity of cytochrome P450 3A4 AND P450 2C9 isoenzymes by vitamins and natural compounds

The influence of vitamins with antioxidant properties (vitamins A, E, C), B vitamins (B1, B2, B6) and vitamin-like substances (coenzyme Q10, taurine and L-carnitine) on the enzymes of the first phase of xenobiotic metabolism - cytochromes P450 3A4 and P450 2C9 has been studied. The experiments with informed volunteers have shown that B vitamins can shorten the duration of nonsteroidal anti-inflammatory drug diclofenac therapy and reduce the daily need for it. The positive effect of B vitamins in reducing the pain syndrome, shortening the duration of therapy and reducing the need for daily intake of diclofenac. Pharmacodynamic and pharmacokinetic data have been confirmed by electrochemical tests of electrocatalytic activity of cytochrome P450 3A4 (CYP3A4). Electrochemical approach to the study of catalytic activity of cytochrome P450 and the impact of vitamins and natural compounds on electrocatalysis is an accurate and effective touch-sensitive method allowing to use low concentrations of protein at an electrode (10-15 mol/electrode), to conduct the analysis without using protein pairs (cytochrome B5, NADPH-dependent reductase) and to identify the interaction of drugs in preclinical studies. When comparing the influence of B vitamins (B1, B2, B6) in the same concentration (300 μM) according to the electrochemical analysis, riboflavin (vitamin B2) is most effectively inhibits the interaction of diclofenac with cytochrome P450 3A4. Vitamin-like substance taurine with antioxidant properties and antioxidant vitamins stimulated electrochemical reduction of cytochromes P450 3A4 and P450 2C9. The obtained data confirm that it is possible that the influence of vitamins on cytochromes P450 3A4 (CYP3A4) и P450 2C9 (CYP2C9) allows to regulate pharmacokinetic parameters and the pharmacodynamic effect intensity