Miroslav Fojta

Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode

AbstractIn this paper, we present an electrochemical DNA–protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.n Figureᅟ

Enzyme-linked electrochemical DNA ligation assay using magnetic beads

AbstractDNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5 biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3 digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5′-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.n FigureEnzyme-linked electrochemical detection of a ligated DNA strand using magnetic beads. Anti-digoxigenin antibody conjugate with alkaline phosphatase (ALP) is bound to digoxigenin label of the ligated product immobilized at streptavidin-coated magnetic beads via biotin tag on its opposite end. Then substrate for ALP (napht-1-yl phosphate) is added and enzymatically converted to napht-1-ol, an electroactive indicator, which is subsequently detected electrochemically at a carbon electrode

Biophysical and electrochemical studies of protein–nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein–nucleic acid (NA) interactions. The importance of NA structure and protein–NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein–DNA and protein–RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein–NA interactions. Recent applications of electrochemical methods in studies of protein–NA interactions are discussed in detail.Graphical abstract

G-quadruplex-based structural transitions in 15-mer DNA oligonucleotides varying in lengths of internal oligo(dG) stretches detected by voltammetric techniques

Electrochemical methods, particularly when applied in connection with mercury-containing electrodes, are excellent tools for studying nucleic acids structure and monitoring structural transitions. We studied the effect of the length of the central (dG)n stretch (varying from 0 to 15 guanine residues) in 15-mer oligodeoxynucleotides (ODN, G0 to G15) on their electrochemical and interfacial behavior at mercury and carbon electrodes. The intensity of guanine oxidation signal at the carbon electrode (peakxa0Gox) was observed to increase continuously with number of guanines between 0 and 15, with only a slight positive shift for ODNs with seven or more guanines in the central segment. Very different effects were observed when the peakxa0GHMDE was measured at the mercury electrode. Intensity of the latter signal increased with number of guanines up to G5, and decreased sharply with further elongation of the (dG)n stretch. CD spectroscopy and electrophoresis experiments revealed formation of parallel intermolecular quadruplex structures for ODNs containing five or more G residues. Further measurements made by cyclic and alternating-current voltammetry revealed a strong influence of the ODN structure on their behavior at electrically charged surfaces.

Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

In this report, we present a simple electrochemical detection protocol for the detection of specific PCR-amplified DNA fragments, based on incorporation of biotin tags into DNA amplicons during PCR run in the presence of a biotinylated nucleoside triphosphate. For detection, an enzyme-linked electrochemical system involving streptavidin–alkaline phosphatase conjugate attached to the biotinylated DNA, adsorbed at the surface of a disposable pencil graphite electrode, is used. The enzyme converts an inactive indicator, 1-naphthyl phosphate, into electrochemically oxidizable indicator 1-naphthol that is subsequently detected. Excellent selectivity of this fast, facile, and inexpensive analysis not requiring any sophisticated electrode modification and its applicability for off-line monitoring of DNA amplification is demonstrated. Applications of the technique include detection of the presence of specific nucleotide sequences in biological samples, such as sequences related to pathogenic microorganism or transgenes.Graphical abstract

Electrochemical behavior of anthraquinone- and nitrophenyl-labeled deoxynucleoside triphosphates: a contribution to development of multipotential redox labeling of DNA

Electrochemical properties of base-modified cytosine or 7-deazaadenine nucleoside triphosphates (dNTPs) bearing electrochemically active anthraquinone or 3-nitrophenyl moieties were studied using cyclic voltammetry with the hanging mercury drop electrode. The anthraquinone moiety in the dNTPs gives well-pronounced reversible quinone/hydroquinone redox signals around −0.40xa0V (against Ag|AgCl|3M KCl reference electrode), while the nitro group in 3-nitrophenyl exhibits irreversible reduction to hydroxylamine around −0.45xa0V that can be reversibly oxidized to corresponding nitroso compound close to 0.0xa0V. Both anthraquinone and hydroxylamine redox groups can be selectively switched off by further electrochemical transformation, depending on negative potential applied and composition of the background electrolyte. Results of this study suggest that both nucleobase and the conjugate label moiety influence remarkably the adsorbability and/or intermolecular interactions taking part at the electrode surface. The potential analytical utilization of these phenomena is discussed.Graphical abstract

Electrochemical behavior of 7-deazaguanine- and 7-deazaadenine-modified DNA at the hanging mercury drop electrode

DNA modification with synthetic analogs of natural nucleotides and/or their conjugates with external redox active groups is applied in the development of electrochemical DNA sensors or assay for DNA hybridization, SNP typing, DNA damage and so forth. 7-Deazapurines (Pu*) are analogs of natural purine bases in which N7 atom is replaced by CH group. The Pu* bases retain Watson–Crick base pairing of their parent purines (and the ability to form duplex DNA) but are incapable of Hoogsteen pairing (and thus cannot be involved in triplex or quadruples DNA structures). Previously, we studied electrochemical oxidation of Pu* residues in DNA fragments (prepared by PCR in the presence of Pu* deoxynucleoside triphosphates) at a carbon electrode and reported on significantly lower potentials of oxidation of both 7-deazaguanine (G*) and 7-deazaadenine (A*), compared to natural guanine (G) and adenine (A), respectively. In this work, we studied faradaic and tensammetric responses of G*- or A*-modified DNA on the hanging mercury drop electrode (HMDE). While A* was reduced at the HMDE, giving rise to a similar irreversible cathodic peak as the natural A, G* did not yield any peak analogous to the peak G due to guanine, in agreement with a loss of corresponding redox site in G*. Responses of DNA modified with A* were relatively similar to those of unmodified DNA (albeit we observed certain differences in tensammetric peak currents). Effects of G substitution by G* were more pronounced, being reflected in diminution of peak due to guanine, decrease of the peak CA (due to cytosine and adenine reduction) and in significantly changed shape of tensammetric DNA signals, indicating altered adsorption/desorption processes. While substitution of A by A* resulted in certain destabilization of the DNA duplex at the negatively charged HMDE surface (in qualitative agreement with significantly decreased melting temperature of the same DNA duplexes in solution), G*-modified duplex DNA displayed apparently lower susceptibility to surface denaturation.Graphical abstract

Carbon Electrodes in Electrochemical Analysis of Biomolecules and Bioactive Substances: Roles of Surface Structures and Chemical Groups

Abstract In this chapter we focus on the properties of graphitic carbon and boron-doped diamond (BDD) electrodes from the point of view of surface micro- and nanostructures, chemistry of surface termination, effects of these characteristics on electron transfer and adsorption characteristics of the electrodes. Their properties are discussed in relation to electrochemical responses measured for typical groups of analytes, such as nucleic acid and protein constituents or other electroactive compounds. In general, basal planes of graphitic materials are sites of preferential adsorption of organic compounds while the edge planes and defects are sites of fast electron transfer for a number of analytes. At the BDD, fast electron transfer kinetics is observed particularly at hydrogenated and polished surfaces. Surface termination with oxygenous groups causes the electron transfer rate to decrease and renders the carbon surface hydrophilic properties, manifested in decreased adsorption of hydrophobic molecules and involvement of coulombic forces and hydrogen bonds in interactions of relevant analytes with the electrode surfaces. Examples of applications in the area of electroanalysis of biomolecules and their components are briefly discussed.

Determination of 2-nitrophenol using carbon film electrode

This study is focused on the application of a carbon film electrode for the determination of micromolar concentrations of pesticide 2-nitrophenol using modern voltammetric methods. For the determination of 2-nitrophenol, direct current (DCV) and differential pulse (DPV) voltammetry were chosen. The following optimal conditions for the determination of 2-nitrophenol were found: Britton–Robinson buffer of pH 5.0 for DCV and pH 6.0 for DPV, and the regeneration potential cycles (Einxa0=xa00xa0mV, Efinxa0=xa00xa0mV). Under these conditions, limit of quantification was found to be 1.2xa0×xa010−6xa0molxa0dm−3 for DCV and 2.0xa0×xa010−6xa0molxa0dm−3 for DPV in deionized water. The limit of quantification for model samples of drinking water was 3.0xa0×xa010−7xa0molxa0dm−3 for DCV and 1.0xa0×xa010−6xa0molxa0dm−3 for DPV. The applicability of carbon film electrode for the determination of micromolar concentrations of 2-nitrophenol based on cathodic reduction of present nitro group and in model samples of drinking water was confirmed.Graphical abstract