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. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

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 -0.9 V (phosphate buffer, pH 7.4). The reduction peak currents of dUTP-N derivatives were found to increase with their molar concentrations. The dUTP-N3 with a double bond in the linker had the lowest reduction potential (about 100 mV less negative) among the derivatives studied. Further, dUTP-N nucleotides were tested as substrates in PCR and RPA to incorporate the electroactive labels into 90, 210, or 206 base pair long dsDNA amplicons. However, only a dUTP-N1 derivative with a shorter linker without the double bond demonstrated satisfactory compatibility with both PCR and RPA, though with a low reaction output of modified dsDNA amplicons (at 100% substitution of dTTP). The dsDNA amplicons produced by PCR with 85% substitution of dTTP by the dUTP-N1 in the reaction mixture were successfully detected by square wave voltammetry at micromolar concentrations at high square wave frequency.

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 (Ep) of 0.6-0.7 V and 0.8-0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between -0.9 V and -1 V, but they did not depend on concentration. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peaks at 0.8-0.9 V. In addition, the distinct reduction peaks at Ep between -0.8 V and -0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50-90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon output and labeling was achieved at the dUTP-Fl : dTTP and dUTP-Rh : dTTP molar ratios of 70% : 30% and 20% : 80% in the PCR mixture, respectively, allowing the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein and rhodamine modified dUTP appear as promising electroactive labels to develop direct electrochemical DNA assays for detecting PCR and RPA products.

Electrochemical Analysis in Studying ß-Amyloid Aggregation.

ß-amyloid (Aß) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aß aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aß aggregation have intensively being investigated using synthetic Aß peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aß aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aß (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aß oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aß, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aß are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aß aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.

Smartphone-assisted electrochemical sensor for reliable detection of tyrosine in serum.

Point-of-care devices have attracted a huge interest by the scientific community because of the valuable potentiality for rapid diagnosis and precision medicine through cost-effective and easy-to-use devices for on-site measurement by unskilled personnel. Herein, we reported a smartphone-assisted electrochemical device consisted of a screen-printed electrode modified with carbon black nanomaterial and a commercially available smartphone potentiostat i.e. EmStat3 Blue, for sensitive detection of tyrosine. Once optimized the conditions, tyrosine was detected in standard solutions by square wave voltammetry, achieving a linear range comprised between 30 and 500 µM, with a detection limit equal to 4.4 µM. To detect tyrosine in serum, the interference of another amino acid i.e. tryptophan was hindered using a sample treatment with an extraction cartridge. The agreement of results analyzing serum samples with HPLC reference method and with the developed smart sensing system demonstrated the suitability of this smartphone-assisted sensing tool for cost-effective and rapid analyses of tyrosine in serum samples.

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 impedance spectroscopy (EIS) technique offered a linear detection range (0.1-90ngmL(-1)) of myoglobin with sensitivity of 0.74kΩngmL(-1) (correlation coefficient, R(2)=0.97) and detection limit of 0.08ngmL(-1) (S/N=3). The mean percentage recovery of Mb in serum samples using this working biosensor is 97.33%. Furthermore, the proposed strategy can be a promising alternative for detection of Mb related cardiovascular disorders.

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.

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. It was shown that the cathodic peak in voltammograms at about -250 mV vs. Ag/AgCl can be associated with AMI. In parallel tests, cardiac myoglobin and troponin I, the AMI biomarkers, were determined in each sample by RAMP immunoassay. The applicability of the electrochemical testing for AMI diagnostics was confirmed by statistical methods: generalized linear model (GLM), linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA), artificial neural net (multi-layer perception, MLP), and support vector machine (SVM), all of which were created to obtain the "True-False" distribution prediction where "True" and "False" are, respectively, positive and negative decision about an illness event.

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.

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.

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 4.8+/-0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3+/-0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by capital P450 2B4 was 19.4+/-0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8+/-0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.

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.