Combining recombinase polymerase amplification with tyrosine modified 2'-deoxyuridine-5'-triphosphate for direct voltammetric detection of double-stranded DNA: Application to potato pathogen Dickeya solani.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture.

Polymerase incorporation of 4-nitrophenyl modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

Three novel 2'-deoxyuridine-5'-triphosphates modified with 4-nitrophenyl groups via various linkers (dUTP-N1, dUTP-N2, and dUTP-N3) were tested as bearers of reducible electroactive labels as well as substrates suitable for enzymes used in polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) with a potential application to direct electrochemical detection of double-stranded deoxyribonucleic acid (dsDNA). In cyclic and square wave voltammograms on carbon screen printed electrodes, the labeled dUTP have demonstrated distinct reduction peaks at potentials of -0.7 V to -0.

Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection.

The 2'-deoxyuridine-5'-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (E) of 0.

Electrochemical Analysis in Studying β-Amyloid Aggregation.

β-amyloid (Aβ) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aβ aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aβ aggregation have intensively being investigated using synthetic Aβ peptides by methods based on monitoring of aggregates, including determination of their size and structure.

Impedimetric immunosensor for detection of cardiovascular disorder risk biomarker.

We report the construction and characterization of a novel, level free impedimetric immunosensor for rapid, sensitive and selective detection of myoglobin (Mb). Monoclonal anti-myoglobin (anti-Mb-IgG) antibody was immobilized on screen-printed multiwalled carbon nanotubes electrode for signal amplification without the need of natural enzymes. The fabrication of resulting immunosensor was extensively characterized by using scanning electron microscopy (SEM), fourier transform infrared (FT-IR) spectroscopy, cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS).

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.

Electrochemical approach for acute myocardial infarction diagnosis based on direct antibodies-free analysis of human blood plasma.

A novel direct antibodies-free electrochemical approach for acute myocardial infarction (AMI) diagnosis has been developed. For this purpose, a combination of the electrochemical assay of plasma samples with chemometrics was proposed. Screen printed carbon electrodes modified with didodecyldimethylammonium bromide were used for plasma charactrerization by cyclic (CV) and square wave voltammetry and square wave (SWV) voltammetry.

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.

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.

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4.

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E(0')=-450 mV) and electrodes, modified with cytochrome c (E(0')=-50 mV) or Prussian Blue (E(0')=0), as measuring electrodes (for H(2)O(2)) and Clark-type electrode (for O(2)). Benzphetamine N-demethylation rate was 17+/-3 nmol/nmol of enzyme/min, peroxide production was 4.

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.