Victoria V. Shumyantseva

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-graphite electrodes. Multi-walled carbon nanotubes (MWCNT) were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto MWCNT-modified rhodium-graphite electrodes. Cytochrome P450scc was also deposited onto both gold nanoparticle-modified and bare rhodium-graphite 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-graphite 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 microA/(mM mm(2)) and the linearity of the biosensor response was improved with respect to the use of gold nanoparticles.

Au-nanoparticles as an electrochemical sensing platform for aptamer–thrombin interaction

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.

Electrochemical nanobiosensor for express diagnosis of acute myocardial infarction in undiluted plasma

The myocardial infarction biomarker myoglobin was quantified at the biological level in undiluted plasma using developed electrochemical nanosensors with immobilized anti-myoglobin. Method for cardiac myoglobin detection is based on direct electron transfer between Fe(III)-heme and electrode surface modified with gold nanoparticles/didodecyldimethylammonium bromide (DDAB/Au) and antibodies. The procedure of myoglobin detection was optimized (pH, incubation times and characteristics of electrodes) to express determination of the marker in serum or plasma. Plasma of healthy donors and patients with acute myocardial infarction (AMI) was analyzed using electrochemical immunosensors and RAMP immunoassay. Square wave voltammetry cathodic peak of cardiac myoglobin reduction was found to be proportional to myoglobin quantity in plasma as determined by RAMP. The method proposed does not require signal enhancement or amplification; nor does it require labeled secondary antibodies. Immunosensor has a detection limit of 10 ng/ml (0.56 nM) and a broad range of working concentrations (10-1780 ng/ml; 0.56-100 nM). The whole procedure takes 30 min and can be used for express diagnosis of acute myocardial infarction.

Construction and characterization of bioelectrocatalytic sensors based on cytochromes P450.

Semisynthetic flavocytochromes RfP450 1A2, RfP450 2B4 and RfP450scc--molecular conjugates of protein with riboflavin--could be reduced on rhodium-graphite screen-printed thick film electrodes as was confirmed by cyclic voltammograms of immobilized enzymes. Amperometric enzyme electrodes for direct measurement of organic pollutants were developed. The efficiency of controlled potential electrolysis for the reduction of flavocytochromes P450 was comparable with traditional reduction by pyridine nucleotides. The rate constants for substrates conversion obtained by electrochemical methods were close to those obtained using NAD(P)H as an electron source.

Electrochemical investigations of cytochrome P450

In this paper we summarized our experimental data on the electrochemical reduction of cytochrome P450. Electrode/cytochrome P450 systems were analyzed in terms of the mechanisms underlying P450-catalyzed reactions. Bioelectrocatalysis-based screening of potential substrates or inhibitors of cytochrome P450, stoichiometry of the electrocatalytic cycle, redox thermodynamics and the peroxide shunt pathway were described. Characteristics, performance and potential application of cytochrome P450-electrodes are discussed.

Light‐driven biocatalysis with cytochrome P450 peroxygenases

The cytochrome P450 peroxygenases P450Bsβ (CYP152A1) from Bacillus subtilis and P450Cla (CYP152A2) from Clostridium acetobutylicum belong to a unique group of P450s with high synthetic potential. They consume hydrogen peroxide via the peroxide shunt and therefore do not require additional electron transfer proteins for biocatalytic activity. Their high synthetic potential is, however, impaired by their rather poor operational stability in the presence of hydrogen peroxide. Herein, we report the use of a light‐driven approach utilizing light‐excited flavins (riboflavin, flavin mononucleotide, or flavin adenine dinucleotide) and the electron donor ethylenediaminetetraacetate as the electron source for the in situ generation of hydrogen peroxide. This approach represents a simple and easily applicable way to promote oxyfunctionalization reactions catalyzed by P450 peroxygenases and is useful for biocatalysis with these enzymes.

Electrochemical methods for detection of post-translational modifications of proteins

Post-translational modifications of proteins play a key role in the regulation of various cellular processes. The analysis and identification of post-translational modifications are probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This review focuses on the electroactivity of amino acids as a tool for analysis of post-translational modifications of proteins. The most attention is paid to the electrochemical detection of phosphorylation/dephosphorylation and glycosylation of proteins, to the best-studied and functionally-significant modifications, and, also, to the electrochemical analysis of activity of enzymes responsible for carrying out phosphorylation/dephosphorylation of proteins. Recent advances in electrochemistry with special references to proteomics are outlined and innovative technologies for protein detection are highlighted.

SOI nanowire for the high-sensitive detection of HBsAg and α-fetoprotein

Silicon-on-isolator-nanowires (SOI-NWs) were used for the label-free, real-time biospecific detection of the hepatitis B marker HBsAg and cancer marker α-fetoprotein (AFP). Specific protein-protein recognition was carried out using individual NWs that were functionalized with antibodies. To solve the problem of non-specific binding of target protein molecules to the sensor element the use of a reference NW with immobilized antibodies against non-target proteins was proposed. Using individual SOI-NW surface functionalization allowed the fabrication of a NW array, containing working NWs and reference NWs within one chip. It was shown that this approach allows us to reach a detection limit up to 10(-14) and 10(-15) M for HBsAg and AFP, respectively. Our investigations also allowed us to reveal the influence of the charged state of the target protein molecules and antibodies in solutions with various pH values on the target protein detection limit. A high sensitivity NW-detector is of interest for the creation of diagnosticums for hepatitis B and for the early stages of cancer diseases.

Electrochemical reduction of sterol-14α-demethylase from Mycobacterium tuberculosis (CYP51b1)

The electrochemical reduction of the heme protein sterol-14α-demethylase from Mycobacterium tuberculosis (CYP51b1, or further CYP51) was investigated. Direct electron transfer was demonstrated between CYP51 and graphite screen-printed electrodes modified with gold nanoparticles and with the membrane-like synthetic surfactant didodecyl dimethylammonium bromide. The formal potential of the Fe3+/Fe2+ pair, E1/2, is equal to −273 mV (vs. Ag/AgCl). The cathodic current corresponding to the reduction of oxygen by immobilized heme protein was registered in the presence of oxygen. Addition of lanosterol, one of the substrates of the CYP51 family, to the oxygenated solution caused a concentration-dependent increase in the reduction current in voltammetric and amperometric experiments. Ketoconazole, an inhibitor of CYP51, inhibited the catalytic cathodic current in the presence of lanosterol. Electrochemical reduction of CYP51 may serve as an adequate alternative to the reconstituted system, which requires additional redox partners for the exhibition of catalytic activity of heme proteins of the cytochrome P450 superfamily.

Electrochemistry of Cytochromes P450: Analysis of Current-Voltage Characteristics of Electrodes with Immobilized Cytochromes P450 for the Screening of Substrates and Inhibitors

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14α-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450.