Luděk Havran

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)25 (DBT) with covalently bound (dT)25 probe are used for the hybridization with target DNA containing adenine stretches. Target DNA is modified with osmium tetroxide,2,2′-bipyridine (Os,bipy) and the immunogenic DNA-Os,bipy adduct is determined by the enzyme-linked immunoassay with electrochemical detection. Electroinactive 1-naphthyl phosphate is used as a substrate and the electroactive product (1-naphthol) is measured on the carbon electrodes. Alternatively Os,bipy-modified target DNA can be determined directly by measuring the osmium signal on the pyrolytic graphite electrode (PGE). A comparison between determinations of the 67-mer oligodeoxynucleotide on carbon electrodes using (a) the guanine oxidation signal, (b) direct determination of the DNA-Os,bipy adduct and (c) its electrochemical immunoassay showed immunoassay to be the most sensitive method. In combination with DBT, the DNA hybridization of long target deoxyoligonucleotides (such as 67- and 97-mers) and a DNA PCR product (226-base pairs) have been detected by immunoassay at high sensitivity and specificity.

Aminophenyl‐ and Nitrophenyl‐Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection

DNA biosensors and chips are broadly utilized in the life sciences. Electrochemical detection is a less expensive but comparatively sensitive alternative to common optical methods. Although nucleic acids are electroactive themselves, diverse electroactive tags are used to increase sensitivity and specificity. Besides widely used DNA tags based on metal complexes, quantum dots, or phenothiazine dyes, some simple organic derivatives (such as aromatic amines or nitro compounds) exhibit distinct electrochemical activity, thus making them candidates for nucleic acid labeling. In particular, the nitro group appears promising for sensitive detection because of the high number of electrons (four or six) collected per nitro group reduction. So far, neither amino nor nitro groups have been used as specific electroactive DNA markers. Some modified 2’-deoxyribonucleoside triphosphates (dNTPs) bearing substituents at the nucleobase can be enzymatically incorporated into DNA by polymerases. This approach has been used for the construction of functionalized nucleic acids bearing diverse functional groups. Recently, aqueous-phase cross-coupling reactions of unprotected halogenated nucleoside triphosphates with boronic acids or acetylenes were developed and used in combination with polymerase incorporation for the two-step construction of modified nucleic acids, including ferrocene-labeled oligonucleotides (ONs). Herein, we report the synthesis of nucleoside triphosphates bearing aminophenyl and nitrophenyl groups attached to a nucleobase, their enzymatic incorporation, and the preliminary electrochemical properties of the labeled ONs. We expected that the fully conjugated aromatic system would respond well to the electronic changes arising from incorporation into nucleic acids, and result in changes of the redox potential of the label. The modified dNTPs were prepared by the single-step aqueous-phase cross-coupling reactions of halogenated dNTPs, in analogy to our previously developed procedures. The Suzuki–Miyaura reaction of 7-iodo-7-deaza-2’-deoxyadenosine 5’-triphosphate (7-I-7-deaza-dATP), 5-iodo-2’deoxyuridine 5’-triphosphate (5-I-dUTP), or 5-iodo-2’-deoxycytidine 5’-triphosphate (5-I-dCTP) with either 3-aminophenylor 3-nitrophenylboronic acid (Scheme 1) gave the

DNA hybridization at microbeads with cathodic stripping voltammetric detection

In electrochemical DNA hybridization sensors generally a single-stranded probe DNA was immobilized at the electrode followed by hybridization with the target DNA and electrochemical detection of the hybridization event at the same electrode. In this type of experiments nonspecific adsorption of DNA at the electrode caused serious difficulties especially in the case of the analysis of long target DNAs. We propose a new technology in which DNA is hybridized at a surface H and the hybridization is detected at the detection electrode (DE). This technology significantly extends the choice of hybridization surfaces and DEs. Here we use paramagnetic Dynabeads Oligo(dT)(25) (DBT) as a transportable reactive surface H and a hanging mercury drop electrode as DE. We describe a label-free detection of DNA and RNA (selectively captured at DBT) based on the determination of adenines (at ppb levels, by cathodic stripping voltammetry) released from the nucleic acids by acid treatment. The DNA and RNA nonspecific adsorption at DBT is negligible, making thus possible to detect the hybridization event with a great specificity and sensitivity. Specific detection of the hybridization of polyribonucleotides, mRNA, oligodeoxynucleotides, and a DNA PCR product (226 base pairs) is demonstrated. New possibilities in the development of the DNA hybridization sensors opened by the proposed technology, including utilization of catalytic signals in nucleic acid determination at mercury (e.g. signals of osmium complexes covalently bound to DNA) and solid DEs (e.g. using enzyme-labeled antibodies against chemically modified DNAs) are discussed.

Constant Current Chronopotentiometric Stripping Analysis of Bioactive Peptides at Mercury and Carbon Electrodes

Constant current derivative chronopotentiometric stripping analysis (CPSA) was used to study bioactive peptides [Lys8]-vasopressin and angiotensin II at carbon paste electrode (CPE) and hanging mercury drop electrode (HMDE). Both peptides contain a single tyrosine residue which is oxidized at CPE close to +0.6 V (against Ag/AgCl/3 M KCl electrode) producing peak Y; this peak was by about 15 mV more positive in vasopressin than in angiotensin II. At HMDE vasopressin yielded peak S due to the reduction of the disulfidic bond in the peptide. No peak S was observed with angiotensin II as it does not contain any cystine/cysteine residues. Vasopressin produced different catalytic hydrogen reduction signals in media with cobalt ion and in media not containing any metal ion. The signal produced in the latter media (peak H), which appeared close to −1.7 V, was well separated from the background. No catalytic hydrogen reduction signal was obtained with angiotensin II. The above mentioned CPSA signals can be applied for the determination of the respective peptide at nanomolar concentrations. The least sensitive peak S appears which requires at least 100 nM vasopressin concentration at moderate accumulation time. By two orders of magnitude, higher sensitivities can be obtained with peaks Y and H. In measuring these peaks CPSA shows significant advantages over the voltammetric stripping techniques.

Reduction and oxidation of peptide nucleic acid and DNA at mercury and carbon electrodes

Abstract Peptide nucleic acid (PNA) is a DNA mimic that binds strongly and specifically to complementary DNA or RNA oligomers, but in contrast to DNA its backbone does not carry any electric charge. We used voltammetry in cyclic and square-wave modes to study reduction and oxidation signals of single stranded (ss)PNA and DNA decamers and pentadecamers with the same base sequences at mercury and carbon electrodes. The signals produced by the ssDNA and ssPNA oligomers at the hanging mercury drop electrode (HMDE), i.e. the cathodic peak CA (due to reduction of cytosine and adenine) and the anodic peak G (due to oxidation of the guanine reduction product) corresponded roughly to those observed earlier with ssDNAs. ssPNA peak potentials were more negative compared to DNA. Differences in the signals of ssPNA and ssDNA were explained primarily by different adsorption properties of these compounds. At an accumulation time of 5 min the detection limit of ssPNA was below 5 ng ml −1 . Constant current derivative chronopotentiometric stripping analysis (CPSA) at a pyrolytic graphite electrode produced two well-separated oxidation peaks of guanine and adenine residues in ssDNA and ssPNA in contrast to the poorly developed signals obtained by linear sweep (LS) and square wave (SW) voltammetries. The voltammetric signals were improved greatly as a result of application of a suitable baseline correction method. Using the polynomic method for LSV and moving average baseline correction for SWV, the ssDNA detection limits were comparable to those of CPSA at carbon electrodes as well to those obtained with peak G measurements at the HMDE.

The “Presodium” Catalysis of Electroreduction of Hydrogen Ions on Mercury Electrodes by Metallothionein. An Investigation by Constant Current Derivative Stripping Chronopotentiometry

Metallothionein (MT) yields on mercury electrodes a “presodium” catalysis of the evolution of hydrogen, which is, as a peak-shaped signal, particularly well measurable in chronopotentiometry, and which is, unlike presodium catalytic effects of other biopolymers, strongly dependent on the presence of cobalt ions in the solution. The behavior of the “presodium” derivative chronopotentiometric peak under various experimental conditions was tested.

Adsorptive Transfer Stripping AC Voltammetry of DNA Complexes with Intercalators

Using alternating current adsorptive transfer stripping voltammetry at hanging mercury drop electrode (HMDE), conformational changes of DNA due to binding of DNA intercalators were studied by means of the measurements of tensammetric DNA signals of peak 2 and peak 3. Untwisting of DNA by the intercalators in solution resulted in an altered DNA adsorption. After medium exchange and intercalator removal, the surface-confined DNA probably adopted a restrained structure, with untwisted segments attached to the electrode surface (yielding peak 2) and superhelical loops extending to the bulk of solution. This structure was more resistant to DNA surface denaturation than dsDNA adsorbed at HMDE in the absence of intercalators, which resulted in a decreased intensity of DNA peak 3. Similar effects were also observed when DNA was adsorbed at the electrode from a solution of low salt concentration. Upon introduction of DNA single-strand breaks, the specific behavior of DNA-intercalator complexes was eliminated.

Effect of spin-orbit coupling on reduction potentials of octahedral ruthenium(II/III) and osmium(II/III) complexes.

Reduction potentials of several M(2+/3+) (M = Ru, Os) octahedral complexes, namely, [M(H2O)6](2+/3+), [MCl6](4-/3-), [M(NH3)6](2+/3+), [M(en)3](2+/3+) [M(bipy)3](2+/3+), and [M(CN)6](4-/3-), were calculated using the CASSCF/CASPT2/CASSI and MRCI methods including spin-orbit coupling (SOC) by means of first-order quasi-degenerate perturbation theory. It was shown that the effect of SOC accounts for a systematic shift of approximately -70 mV in the reduction potentials of the studied ruthenium (II/III) complexes and an approximately -300 mV shift for the osmium(II/III) complexes. SOC splits the sixfold-degenerate (2)T(2g) ground electronic state (in ideal octahedral symmetry) of the M(3+) ions into the E((5/2)g) Kramers doublet and G((3/2)g) quartet, which were calculated to split by 1354-1573 cm(-1) in the Ru(3+) complexes and 4155-5061 cm(-1) in the Os(3+) complexes. It was demonstrated that this splitting represents the main contribution to the stabilization of the M(3+) ground state with respect to the closed-shell (1)A(1g) ground state in M(2+) systems. Moreover, it was shown that the accuracy of the calculated reduction potentials depends on the calculated solvation energies of both the oxidized and reduced forms. For smaller ligands, it involves explicit inclusion of the second solvation sphere into the calculations, whereas implicit solvation models yield results of sufficient accuracy for complexes with larger ligands. In such cases (e.g., [M(bipy)3](2+/3+) and its derivatives), very good agreement between the calculated (SOC-corrected) values of the reduction potentials and the available experimental values was obtained. These results led us to the conclusion that especially for Os(2+/3+) complexes, inclusion of SOC is necessary to avoid systematic errors of approximately 300 mV in the calculated reduction potentials.

Alkylsulfanylphenyl Derivatives of Cytosine and 7‐Deazaadenine Nucleosides, Nucleotides and Nucleoside Triphosphates: Synthesis, Polymerase Incorporation to DNA and Electrochemical Study

Aqueous Suzuki-Miyaura cross-coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5-iodocytosine and 7-iodo-7-deazaadenine with methyl-, benzyl- and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo-) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl- or tritylsulfanylphenyl moieties produced signals in [Co(NH(3))(6)](3+) ammonium buffer, attributed to the Brdička catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdička catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.

Benzofurazane as a New Redox Label for Electrochemical Detection of DNA: Towards Multipotential Redox Coding of DNA Bases

Benzofurazane has been attached to nucleosides and dNTPs, either directly or through an acetylene linker, as a new redox label for electrochemical analysis of nucleotide sequences. Primer extension incorporation of the benzofurazane-modified dNTPs by polymerases has been developed for the construction of labeled oligonucleotide probes. In combination with nitrophenyl and aminophenyl labels, we have successfully developed a three-potential coding of DNA bases and have explored the relevant electrochemical potentials. The combination of benzofurazane and nitrophenyl reducible labels has proved to be excellent for ratiometric analysis of nucleotide sequences and is suitable for bioanalytical applications.