František Jelen

Electrochemistry of Nucleic Acids and Development of DNA Sensors

In recent years our knowledge regarding the nucleotide sequences of a number of genomes has increased tremendously. With the completion of an increasing number of genomic sequences attention is currently focused on how the sequence data might be interpreted in terms of the structure, function, and control of biological systems. It now appears that methods of electrochemical analysis may find application in this exciting research area.

Biopolymer-modified electrodes in the voltammetric determination of nucleic acids and proteins at the submicrogram level

Abstract Nucleic acid- and protein-modified mercury and carbon electrodes can be prepared by immersing the electrode in a 5- μl drop of nucleic acid or protein solution. The biomacromolecule is strongly and irreversibly adsorbed at the electrode, resisting subsequent washing. The electrode is then transferred into a background electrolyte (not containing any biomacromolecule) followed by voltage scanning. This procedure is called adsorptive transfer stripping voltammetry (AdTSV). Nanogram amounts of nucleic acid can be determined with graphite electrodes, including the highly oriented pyrolytic graphite frequently used as a support in scanning probe microscopes, at relatively short waiting times without stirring of the solution. Even smaller amounts of nucleic acids are sufficient for analysis with the mercury electrode. Adsorption of DNA from different denaturing media produces DNA cyclic voltammetric peaks of different heights, suggesting that the arrangement of the DNA molecules at the electrode is influenced by the adsorption event and persists even after the electrode transfer to a non-denaturing medium. AdTSV with the mercury and graphite electrodes can be used for studies of virus suspensions producing DNA and protein signals, providing information about the virus particle degradation.

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.

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.

Cyclic voltammetry of echinomycin and its interaction with double-stranded and single-stranded DNA adsorbed at the electrode

Interactions of echinomycin (Echi) with DNA was studied by cyclic voltammetry (CV) with hanging mercury drop electrode (HMDE). Echinomycin was electrochemically active, yielding several signals. Interaction of Echi with dsDNA attached to a hanging mercury drop electrode resulted in high Echi signals, suggesting a strong binding of Echi to dsDNA by bis-intercalation at the electrode surface. Under the same conditions, interaction of Echi with ssDNA produced almost no Echi signal. This behavior is in agreement with a strong binding of Echi to dsDNA and a very weak binding of Echi to ssDNA observed earlier in solution. Echi, thus, appears to be a good candidate for redox indicator in electrochemical DNA hybridization sensors.

Adsorptive stripping square-wave voltammetry of DNA

Abstract Faradaic currents produced by DNA at neutral pH are studied by square-wave voltammetry (SWV) in connection with an HMDE. Under these conditions single-stranded DNA produces a cathodic peak (due to the reduction of adenine and cytosine residues) which is better developed than the peak obtained by CV and DPV; the SWV peak can thus be measured at substantially lower DNA concentration (down to tens of nanograms per milliliter). The anodic peak G , which is due to oxidation of the guanine reduction product (formed at potentials around −1.85 V against an Ag|AgCl|3 M KCl electrode), is studied in greater detail. At potentials more negative than −1.85 V a deeper reduction of the guanine residue takes place which results in a decrease of peak G and in changes of the peak potential. The dependences of the peak G on the pulse amplitude and square-wave frequency are unusual. Using adsorptive stripping transfer voltammetry it is possible to determine DNA in the presence of an excess of guanosine. Methylation of the DNA guanine residues by dimethyl sulfate results in a decrease of the peak G . A DNA-modified electrode can be used as a sensor for the DNA damage by methylating agents.

Voltammetry of native double-stranded, denatured and degraded DNAs

Abstract Degradation of calf thymus and plasmid DNAs was studied by means of conventional as well as adsorptive transfer stripping voltammetry in cyclic and a.c. modes in combination with an HMDE. These techniques detected sensitively small damages to the DNA double helix induced by relatively low doses of ionizing radiation as well as by partial depurination of DNA at a weakly acidic pH in the presence of HNO 2 . Electrochemical analysis did not require any special pretreatment of the sample, and the amount of DNA necessary for this analysis was below 1 μg. It was shown that dissolving DNA in concentrated perchloric acid resulted in a deep DNA degradation including depurination of DNA.

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.

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.

Interaction of DNA with echinomycin at the mercury electrode surface as detected by impedance and chronopotentiometric measurements.

The capacitance measurement (dependence of the differential capacitance C of the electrode double layer on potential E, C-E curves), electrochemical impedance spectroscopy (frequency response of the impedance Z of the electrode double layer-EIS) and constant current chronopotentiometry (dependence of dt/dE on potential at constant current, chronopotentiometric stripping analysis-CPSA) have been used for electrochemical study of echinomycin and its interaction with single-stranded (ss) and double-stranded (ds) DNA at the hanging mercury drop electrode (HMDE). The capacitance measurement showed that echinomycin gives a pseudocapacitance redox peak strongly dependent on the a.c. voltage frequency at the potential of -0.53 V. This peak is observed with dsDNA-echinomycin complex as well, but not with ssDNA treated by echinomycin. Similar results were obtained using CPSA measurements. Thus capacitance measurements and CPSA can distinguish with the aid of the bis-intercalator echinomycin the single-stranded and double helical form of DNA adsorbed at the mercury electrode surface. Impedance measurement in connection with adsorptive transfer technique can find the differences between ssDNA and dsDNA, which promise to use this technique for detection of dsDNA in hybridisation reactions.