Electrochemical DNA sensing using osmium complexes as hybridization indicators

A surface-based method for the study of the interactions of DNA with redox-active osmium complexes is described. The study was carried out using gold electrodes modified with DNA by adsorption and [Os(bpy)3]3+/2+ (bpy=2,2'-bipyridyl) or [Os(phen)3]3+/2+ (phen=1,10-phenantroline) as electrochemical indicators. The method, which is simple and reagent saving, allows the accumulation of osmium complexes on the DNA layer. The amount of osmium complex bound by the layer of double-stranded (dsDNA) or single-stranded DNA (ssDNA) adsorbed at gold electrodes was estimated from the cyclic voltammetric (CV) peak charge of osmium complex reduction. The dissociation constants (K) for the oxidized and reduced forms of a bound species are also estimated. [Os(phen)3]3+/2+ was applied to a probe for electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 70 pmol/cm2 on a gold electrode. Following hybridization with the complementary DNA, the osmium complex was electrochemically accumulated on the double-stranded DNA layer and the differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of [Os(phen)3]3+/2+ that was bound to the double-stranded DNA on the electrode.     

Evaluation of reductive amperometric detection in the liquid chromatographic determination of antineoplastic platinum complexes

The usefulness of reductive electrochemical detection at mercury drop electrodes has been determined for platinum complexes separated by solvent-generated anion-exchange high-performance liquid chromatography. Both current-sampled dropping mercury and hanging mercury drop electrodes (DME and HMDE) provide significant advantages over UV absorbance and off-line non-flame atomic absorption detection. The effects of chromatographic and polarographic parameters on analytical system performance have been investigated. By raising the detector cell temperature, the detector response to cis-dichlorodiammineplatinum(II) (DDP) can be shifted anodically to 0.0 V vs. Ag/AgCl, thereby increasing detector selectivity for this compound. The noise-limited minimum detectable quantities of DDP with DME and HMDE are 1.8 ng and 70 pg injected, respectively. DDP can be determined in untreated urine at levels below 100 ng/ml.     

2'-anthraquinone-conjugated oligonucleotide as an electrochemical probe for DNA mismatch

We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be approximately 50 s (-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates ( approximately 50 s (-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G--A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.     

Electrode potential-modulated cleavage of surface-confined DNA by hydroxyl radicals detected by an electrochemical biosensor

Damage to DNA frequently involves interruption of DNA sugar-phosphate strands (strand breaks, sb). Under aerobic conditions, transition metal ions cause DNA damage through production of reactive oxygen species (frequently via Fenton-type reactions). Formation of sb in covalently closed supercoiled (sc) DNA can be detected using an electrochemical biosensor based on a scDNA-modified mercury electrode. By controlling the potential of the electrode, this technique can be employed in studies of redox reactions involved in formation of DNA strand breaks, and to detect species involved in these reactions. ScDNA anchored at HMDE was cleaved by catalytic amounts of iron/EDTA ions in the absence of chemical reductants when appropriate electrode potential (sufficiently negative to reduce [Fe(EDTA)]- to [Fe(EDTA)]2-) was applied. The process required oxygen or hydrogen peroxide. The extent of DNA damage increased with the shift of the electrode potential to negative values, displaying a sharp inflection point matching the potential of [Fe(EDTA)]2-/[Fe(EDTA)]- redox pair. In the absence of transition metal ions, significant DNA damage was observed at potentials sufficiently negative for reduction of dioxygen at the mercury electrode. This observation suggests cleavage of the surface-attached scDNA by radical intermediates of oxygen reduction at HMDE.     

Hairpin formation in Friedreich's ataxia triplet repeat expansion

Triplet repeat tracts occur throughout the human genome. Expansions of a (GAA)(n)/(TTC)(n) repeat tract during its transmission from parent to child are tightly associated with the occurrence of Friedreich's ataxia. Evidence supports DNA slippage during DNA replication as the cause of the expansions. DNA slippage results in single-stranded expansion intermediates. Evidence has accumulated that predicts that hairpin structures protect from DNA repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia. How the latter repeat expansions avoid repair remains a mystery because (GAA)(n) and (TTC)(n) repeats are reported not to self-anneal. To characterize the Friedreich's ataxia intermediates, we generated massive expansions of (GAA)(n) and (TTC)(n) during DNA replication in vitro using human polymerase beta and the Klenow fragment of Escherichia coli polymerase I. Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA)(n) and (TTC)(n) hairpins during DNA synthesis. The results provide unifying evidence that predicts that hairpin formation during DNA synthesis mediates all of the disease-associated, triplet repeat expansions.     

Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution

We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3′ and/or 5′ extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5′ position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.     

Hybridization biosensor using di(2,2'-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3(')-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy)(2)Cl(2)](+) as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy)(2)Cl(2)](+) in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy)(2)Cl(2)](+) at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.     

Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Construction of DNA biosensor at glassy carbon surface modified with 4-aminoethylbenzenediazonium salt

A simple, label-free electrochemical impedance-spectroscopy method for sequence-specific detection of DNA using a 4-aminoethylbenzenediazonium (AEBD) salt as a binder for amino-modified probe DNA is reported. This novel method simplifies the anchoring of DNA at the GC surface and opens new ways for the detection of hybridization. The hybridization of target DNA, without and with mismatches, with the probe DNA anchored at the GC surface modified with AEBD, greatly increases the interfacial electron transfer resistance at the double-stranded DNA modified electrodes for the redox couple Fe(CN)(6)(3-/4-). The resistance was measured using electrochemical impedance spectroscopy. The sensor response increased linearly with logarithm of concentration of target DNA in the range 2×10(-12)÷2×10(-6) M. The obtained quantification limit was circa 6.5×10(-17) mole in a 7 μL droplet and corresponded to a concentration of 9.2×10(-12) M of target DNA in the sample. This limit is equivalent to the detection of circa 4×10(7) copies of DNA in a 7 μL droplet or circa 5.7×10(12) DNA copies in one litre of sample.     

Immunostimulatory PyNTTTTGT oligodeoxynucleotides: structural properties and refinement of the active motif

It is well known that synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within a given context stimulate B lymphocytes and plasmacytoid dendritic cells (PDCs) of the vertebrate immune system. We have reported that B lymphocyte and PDC stimulation in humans could also be efficiently achieved by using non-CpG ODNs bearing the immunostimulatory sequence (motif) PyNTTTTGT, wherein Py is C or T and N is any deoxyribonucleotide. We are now reporting a series of studies that gives further precision regarding the composition of this immunostimulatory motif. The analysis of hundreds of ODNs led us to the conclusion that the motif for optimal CpG-independent immune stimulation can be represented by a sequence of the following general formula: PyN(T/A)(T/C/G)(T/C/G)(T/G)GT, wherein Py is C or T and N is any deoxyribonucleotide and wherein at least two of the positions represented within parentheses are Ts. Requirements for optimal ODN activity are as follows: (1) at least one of the versions of the general motif must be located near the central portion of the ODN; and (2) the ODN must be 20 or more nucleotides long. PyN(T/A)(T/C/G)(T/C/G)(T/G)GT ODNs are active in a phosphorothioate or in a phosphodiester backbone. In a phosphodiester backbone a canonical motif is strongly required whereas in a phosphorothioate backbone specificity is, within certain limits, less strict. On the other hand, PyN(T/A)(T/C/G)(T/C/G)(T/G)GT oligonucleotides are inactive as a double chain or as a modified (phosphorothioate or hydroxyl-methyl modified) or unmodified RNA backbone.     

Sticky DNA: effect of the polypurine.polypyrimidine sequence

The polypurine.polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA.TC)(37), which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA.TCC)(n) inserts (where n = 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA.TTC)(150) (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA.TCCTTC)(65) of the same approximate length (390 bp). A sequence that is replete with polypurine.polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA.TTC)(n) (where n = 176 or 80) readily formed sticky DNA with (GAAGGA.TCCTTC)(65) cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.     

Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR

Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5′-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.     

Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes.

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Electrochemical characterization and DNA-binding property of dipyridophenazine complexes of osmium (II).

Novel dipyridophenazine (DPPZ) complexes of osmium (II), [Os(L)2(DPPZ)]2+ [L = 2,2'-bipyridyl (bpy)(1), 4,4'-diamino-2,2'-bipyridyl (DA-bpy)(2), 4,4'-dimethyl-2,2'-bipyridyl(DM-bpy)(3), and 4,4'-dicarboxyl-2,2'-bipyridyl (DC-bpy)(4)] have been synthesized and characterized. The DNA-binding properties of the complexes were studied by electrochemical methods. As the results, complex 2 shows higher affinity to DNA than other osmium complexes. The binding constant, K of complex 2 to calf thymus DNA has been determined to be 2.3 x 10(7) M-1 by normal pulse voltammetry (NPV).     

Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of guanine residues, whereas the more positive peak, A, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.     

Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes

The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigitated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the presence of K(3)[Fe(CN)(6)]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K(3)[Fe(CN)(6)], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN)(6)(3-) ions through defects in the ODN monolayer determines the impedance of Au-ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K(3)[Fe(CN)(6)] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms.     

Metallic oxide CdIn2O4 films for the label free electrochemical detection of DNA hybridization

DNA functionalised semiconductor metallic oxide electrodes have been developed for the direct electrochemical detection of DNA hybridization, without labelling or the introduction of a redox couple. Conductive CdIn(2)O(4) thin films with controlled properties were deposited on glass substrates using an aerosol pyrolysis technique. The films exhibit a polycrystalline microstructure with a surface roughness of 1.5 nm (r.m.s.) and an electrical resistivity ranging between 1 and 3 x 10(-3) Omega cm. These electrodes were functionalised using hydroxylation and silanisation steps, to allow the binding of DNA probe sequences (20 bases). The electrical detection of DNA hybridization with complementary sequences has been performed using electrochemical impedance spectrometry (EIS) measuring the variation of impedance before and after hybridization. Two set-ups were used, a standard set-up including three electrodes and a set-up including two symmetrical electrodes. In both configurations, a significant increase of the impedance modulus, more particularly of the real part of the impedance (160-225% according to the electrochemical cell used) has been obtained over a frequency range of 10-10(5)Hz. DNA hybridization has also been systematically confirmed using the fluorescence spectrometry. This study emphasizes the high sensitivity of the CdIn(2)O(4) as a working electrode for the detection of biological events occurring at the electrode surface.     

Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies

The polypurine/polypyrimidine (PuPy) tracts present in the human genome are known to be scattered among and within chromosomes. In PuPy tract sequences, triplex formation occurs readily under physiological conditions, leaving single-stranded DNAs capable of hybridization with complementary single-stranded DNAs and RNAs. The formation of single-strands and transmolecular triplexes is thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining fluorescence in situ "nondenaturing" hybridization employing PuPy tract probes and immunodetection by antitriplex antibodies. The nondenaturing hybridization technique, which has been used to detect RNA, may detect single-stranded DNAs in nondenatured nuclei, if present. Probes such as (GA/TC)(n) and (GAA/TTC)(n) sequences gave sequence-specific signals that overlapped with or were closely associated with triplexes immunolocalized by using known antitriplex antibodies. Pretreatment of nuclei with antitriplex antibodies blocked probe signal formation. Signal formation was resistant to pretreatment of nuclei with RNases but sensitive to single strand-specific nucleases. Triplexes visualized differentially with distinct PuPy tract probes were associated spatially with centromeric sequences in the interphase nucleus in a sequence-specific manner.