Voltammetry of Osmium End‐Labeled Oligodeoxynucleotides at Carbon,Mercury, and Gold Electrodes

Voltammetric behavior of oligodeoxynucleotide (ODN) 5′‐T₄₀ (GAA)₇–3′ end‐labeled with osmium tetroxide,2,2‐bipyridine [Os(VIII)bipy] was compared with Os(VIII)bipy‐base‐ and with Os(VI)bipy‐sugar‐modified thymine ribosides. Cyclic voltammograms of Os(VIII)bipy‐modified ODN at mercury and carbon electrodes were similar but not identical to those of Os(VIII)bipy‐modified thymine riboside. Treatment of the ODN with Os(VI)bipy did not result in the ODN modification, in agreement with the known specificity of the reagent to the sugar cis‐diols. We show that in addition to mercury and carbon electrodes, the gold electrode can be used to detect Os(VIII)bipy‐labeled ODN. Comparison of voltammetric behavior of end‐labeled ODN using three types of electrodes most frequently used in DNA analysis may help to optimize electrochemical DNA sensors.     

Voltammetry of osmium-modified DNA at a mercury film electrode: application in detecting DNA hybridization

Mercury film electrodes (MFE) have recently been used in nucleic acid electrochemical analysis as alternatives to the classical mercury drop ones. DNA modified with osmium tetroxide, 2,2'-bipyridine (Os,bipy) can be detected with a high sensitivity at mercury electrodes via measurements of a catalytic osmium signal. In this paper we show that mercury film on a glassy carbon electrode can be used in voltammetric analysis of Os,bipy-modified DNA. Application of the MFE as a detection electrode in double-surface electrochemical DNA hybridization assay involving osmium labeling of target DNA is demonstrated.     

Determination of nanogram quantities of osmium-labeled single stranded DNA by differential pulse stripping voltammetry.

Earlier, we showed that using differential pulse cathodic stripping voltammetry with hanging mercury drop electrode (HMDE), single-stranded (ss) DNA modified with osmium tetroxide, pyridine reagent (Os,py) can be determined at concentrations down to about 10-5 ng/ml. Here, we show that by exchanging Os,py for osmium tetroxide, 2,2'-bipyridine (Os,bipy) and decreasing the pH of the background electrolyte from neutrality to about pH 4, ssDNA can be determined at concentrations lower by one order of magnitude. Determination of DNA at such low concentrations may find use in various areas of molecular biology and in biotechnologies, including the development of DNA sensors.     

Covalent Labeling of Nucleosides with VIII‐ and VI‐Valent Osmium Complexes

Osmium tetroxide complexes with nitrogen ligands [Os(VIII)L] have been widely applied as probes of the DNA structure and as electroactive labels of DNA. Here we describe the electrochemical behavior of Os(VIII)2,2‐bipyridine (Os, bipy)‐base‐labeled nucleosides. We show that electroactive label can be introduced also in the nucleoside ribose residues using six‐valent osmium complex. Cyclic voltammograms of sugar‐Os(VI)‐modified ribosides are similar but not identical to those of the base‐modified ribosides. Our results showing the electroactivity of sugar modified ribosides pave the way to facile end‐labeling of RNA.     

End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes

Peptide nucleic acid (PNA), the DNA mimic with electrically neutral pseudopeptide backbone, is intensively used in biotechnologies and particularly in single-base mismatch detection in DNA hybridization sensors. We propose a simple method of covalent end-labeling of PNA with osmium tetroxide, 2,2′-bipyridine (Os,bipy). Os,bipy-modified PNA (PNA–Os,bipy) produces voltammetric stripping peaks at carbon and mercury electrodes. Peak potential (E_p) of one of the anodic peaks of PNA–Os,bipy at the pyrolytic graphite electrode (PGE) differs from E_p of the reagent, allowing PNA–Os,bipy analysis directly in the reaction mixture. At the hanging mercury drop electrode (HMDE) the PNA–Os,bipy yields a catalytic peak Cat_p, in addition to the redox couples. Using Cat_p it is possible to detect purified PNA–Os,bipy down to 1 pM concentration at accumulation time 60 s. To our knowledge this is the highest sensitivity of the electrochemical detection of PNA.     

New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces

Up to now, the development of the electrochemical DNA hybridization sensors relied on solid electrodes, on which both the hybridization and detection steps have been performed. Here we propose a new method in which the DNA hybridization is performed at commercially available magnetic beads and electrochemical detection on detection electrodes (DE). Due to minimum nonspecific DNA adsorption at the magnetic beads, very high specificity of the DNA hybridization is achieved. Optimum DE can be chosen only with respect to the given electrode process. It is shown that high sensitivity and specificity in the detection of relatively long target DNAs can be obtained (a) by using cathodic stripping voltammetry at mercury or solid mercury amalgam DEs for the determination of purine bases, released from DNA by acid treatment, and (b) by enzyme-linked immunoassay of target DNA modified by osmium tetroxide,2,2'-bipyridine (Os,bipy) at carbon DEs. Direct determination of Os,bipy at mercury and carbon electrodes is also possible.     

Voltammetric microanalysis of DNA adducts with osmium tetroxide,2,2'-bipyridine using a pyrolytic graphite electrode

DNA and synthetic polynucleotides modified with a complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) produce specific voltammetric signals at pyrolytic graphite electrodes. Based on a sufficient potential separation between the peaks of Os,bipy-modified DNA (DNA-Os,bipy) and of free Os,bipy, and using an adsorptive transfer stripping voltammetric procedure involving extraction of free Os,bipy from the electrode by chloroform, DNA-Os,bipy can be determined in an excess of the free reagent. Under certain conditions, 140 pg of DNA-Os,bipy can be detected after a 5 min accumulation period. This analysis displays a more favorable sensitivity and a better selectivity for DNA structure than oxidation of DNA guanine moieties, and offers detection of osmium DNA markers at carbon electrodes.     

Differential pulse adsorptive stripping voltammetry of osmium-modified peptides

Complexes of osmium tetroxide with nitrogen ligands were developed and used in our laboratory as probes of the DNA structure. Here, we show that the complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) can be used for modification and electrochemical detection of proteins at neutral pH. Salmon luteinizing hormone (SLH) containing two tryptophan (Trp) residues and human luteinizing hormone (HLH) containing one Trp were modified by Os,bipy and measured by differential pulse adsorptive stripping voltammetry (DPAdSV) at a hanging mercury drop electrode (HMDE). The intensity of the DPAdSV catalytic signals corresponded to the number of Trp residues in the peptide molecule. Decreasing pH of the background electrolyte from 6.6 to 3.8 led to the increase of DPAdSV signals, suggesting that at pH 3.8, the DPAdSV detection limit might be well below 1 ng/ml. Our results suggest that Os,bipy is potentially useful for chemical modification of proteins.     

Butylacrylate‐nucleobase Conjugates as Targets for Two‐step Redox Labeling of DNA with an Osmium Tetroxide Complex

Modification of nucleic acids with osmium tetroxide reagents (Os,L, such as OsO₄,2,2′‐bipyridine, Os,bpy) has been applied in redox DNA labeling, in probing DNA structure as well as in studies of DNA interactions with other molecules. In natural DNA, primarily thymine residues form adducts with the Os,bpy in a structure selective manner. In this paper we introduce a new two‐step technique of DNA modification with the electroactive Os,bpy, consisting in enzymatic construction of DNA bearing butyl acrylate (BA) moieties attached to uracil at C5 or to 7‐deaza adenine at C7, followed by chemical modification of a reactive C=C double bond in the acrylate residue. We demonstrate a facile modification of the BA conjugates in both single‐ and double‐stranded (ds) DNA under conditions when modification within the nucleobase rings in ds DNA is hindered. Various DNA−Os,bpy adducts can easily be analyzed electrochemically and distinguished by different redox potentials. The two‐step procedure appears to be applicable in osmium redox labelling of ds DNA.     

Copper Solid Amalgam Electrodes

This work describes the behavior of copper solid amalgam electrodes (CuSAE). The applied potential range has been compared with that of the silver solid amalgam electrode (AgSAE) and the hanging mercury drop electrode (HMDE). In 0.05 M tetraborate buffer the applicable potential range of CuSAE is+0.945 V to ?1.75 V excluding ?0.2 V to ?0.5 V, where the anodic oxidation of copper occurs. CuSAE does not need other than electrochemical pre‐treatment, which has been documented by the evaluated repeatability of eleven voltammetric curves of Cd²⁺ (0.1 ppm), Pb²⁺ (0.1 ppm) and Mn²⁺ (0.5 ppm). The obtained results showed that CuSAE could substitute the solid copper, amalgamed copper or liquid copper amalgam electrodes, and can be applied for the study of systems needing an addition of Cu²⁺ ions into the measured solution.     

Voltammetric Determination of Azidothymidine Using Silver Solid Amalgam Electrodes

A new simple and direct electroanalytical method was developed for the determination of azidothymidine in commercial pharmaceutical preparations. It is based on differential pulse voltammetry at silver solid amalgam electrode with polished surface (p‐AgSAE) or surface modified by mercury meniscus (m‐AgSAE). The electroreduction of azidothymidine in basic media at these electrodes gives rise to one irreversible cathodic peak. Its potential in 0.05 mol L^?1 borate buffer, pH 9.3 at ca. ?1050 mV is comparable to that using hanging mercury drop electrode (HMDE). Achieved limits of quantitation are in the 10^?7 mol L^?1 concentration range for both amalgam electrodes. According to the procedure based on the standard addition technique, the recoveries of known amounts of azidothymidine contained in pharmaceutical preparations available in capsules were 101.4±1.8% (m‐AgSAE), 100.3±3.5% (p‐AgSAE) and 102.0±1.0% (HMDE) (n=10). There was no significant difference between the values gained by proposed voltammetric methods and the HPLC‐UV recommended by the United States Pharmacopoeia regarding the mean values and standard deviations.     

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.     

Cathodic adsorptive stripping voltammetric detection of tRNA by labelling with osmium tetroxide

This communication reports about adsorptive stripping voltammetric determination of baker’s yeast tRNA that was modified with the complex osmium tetroxide bipyridine. The uracil and cytosine bases are able to react with [OsO₄(bipy)] within 2 h. We observed a 42-fold higher sensitivity for the catalytic Os-peak on the hanging mercury drop electrode (HMDE) compared with an [OsO₄(bipy)]-modified 20-base ssDNA. We found a 0.27 μg/l detection limit for [OsO₄(bipy)]-tRNA. A linear calibration function was observed up to 3 μg/l. The effect of accumulation potential was very small. These findings possibly indicate a strong adsorption on the HMDE. Such labelling of tRNA with [OsO₄(bipy)] holds great promise for future biosensor application regarding the detection of other RNA species.     

Electrodes of Nontoxic Solid Amalgams for Electrochemical Measurements

The preparation, activation and electrochemical pretreatment of electrodes based on nontoxic solid amalgams were described. Testing of metal solid amalgam electrodes (MeSAEs) proved their broad applicability in many respects, e.g., as to the range of working potentials and the level of background currents, well comparable with those of the hanging mercury drop electrode (HMDE). A regeneration of their surfaces before each measurements could be simply automatized using a PC‐controlled system providing a reasonable repeatability of voltammetric measurements down to 3% RSD. Combination with stripping techniques at accumulation times t_ac=300 s the detection limit amounted to the concentration level of 1 ppb Cu(II), Pb(II), Cd(II), Zn(II), etc. Best electrochemical properties were exhibited by the silver solid amalgam electrode (AgSAE). For example, polished AgSAE (p‐AgSAE), completely free of liquid mercury, proved satisfactory even at more negative potentials enabling the determination of Zn(II), Mn(II), IO , etc. Moreover, even better repeatability of mercury meniscus modified AgSAE (m‐AgSAE) was due to better quality and renewability of its surface. In many cases further testing confirmed that under appropriate conditions MeSAEs represent good, often cheaper and more users‐friendly alternatives to HMDE.     

Voltammetric Determination of N,N‐Dimethyl‐4‐amine‐carboxyazobenzene at a Silver Solid Amalgam Electrode

The voltammetric behavior of N,N‐dimethyl‐4‐amino‐2′‐carboxyazobenzene was investigated by differential pulse voltammetry (DPV) at a mercury meniscus‐modified silver solid amalgam electrode (m‐AgSAE). Conditions have been found for its determination by DPV at m‐AgSAE in the concentration range of 0.4 to 15 μmol L^?1.     

Modification of Poly‐ and Oligosaccharides with Os(VI)pyridine. Voltammetry of the Os(VI) Adducts Obtained by Ligand Exchange

Polysaccharides and oligosaccharides were modified with Os(VI)pyridine complex followed by ligand exchange with different ligands such as 2,2′‐bipyridine or N,N,N′,N′‐tetramethylethylenediamine. The time of the modification was much shorter (taking about 15 min) then direct modification with the given Os(VI) complex. The resulting saccharide adducts were analyzed by voltammetric methods at carbon and mercury electrodes. The results showed that the proposed technique gives promise for a new approach to analysis of glycoproteins.     

Three Novel Mixed‐ligand Cadmium(II) Sulfonates with Aza‐aromatic Skeltons as Co‐ligands

To survey the influence of aza‐aromatic co‐ligands on the structure of Cadmium(II) sulfonates, three Cd(II) complexes with mixed‐ligand, [Cd^II(ANS)₂(phen)₂] ( 1 ), [Cd^II(ANS)₂(2,2′‐bipy)₂] ( 2 ) and [Cd^II(ANS)₂(4,4′‐bipy)₂]_n ( 3 ) (ANS = 2‐aminonaphthalene‐1‐sulfonate; phen = 1,10‐phenanthroline; 2,2′‐bipy = 2,2′‐bipyridine; 4,4′‐bipy = 4,4′‐bipyridine) were synthesized by hydrothermal methods and structurally characterized by elemental analyses, IR spectra, and single crystal X‐ray diffraction. Of the three complexes, ANS consistently coordinates to Cd²⁺ ion as a monodentate ligand. While phen in 1 and 2,2′‐bipy in 2 act as N,N‐bidentate chelating ligands, leading to the formation of a discrete mononuclear unit; 4,4′‐bipy in 3 bridges two Cd^II atoms in bis‐monodentate fashion to produce a 2‐D layered network, suggesting that the conjugate skeleton and the binding site of the co‐ligands have a moderate effect on molecular structure, crystal stacking pattern, and intramolecular weak interactions. In addition, the three complexes exhibit similar luminescent emissions originate from the transitions between the energy levels of sulfonate anions.     

Voltammetric Detection of Thrombin by Labeling with Osmium Tetroxide Bipyridine and Binding with Aptamers on a Gold Electrode

This communication reports on electrochemical detection of thrombin based on labeling with osmium tetroxide bipyridine [OsO₄(bipy)]. Tryptophan amino acids can be labeled at the C−C‐double bond, and at least some tryptophan moieties are accessible for labeling in thrombin. Using the catalytic hydrogen signal from adsorptive stripping voltammetry performed on hanging mercury drop electrode, we could detect as little as 1.47 nM [OsO₄(bipy)]‐modified thrombin. We also tested the binding of [OsO₄(bipy)]‐modified thrombin with the classic thrombin binding aptamer (TBA) on gold electrodes. This preliminary study revealed that even after modification, a major part of the affinity was conserved, and that the aptamer self‐assembled monolayer (SAM) could be regenerated several times. Molecular simulations confirm that [OsO₄(bipy)]‐modified thrombin largely preserves the high binding affinity also of the alternative HD22 aptamer to thrombin, albeit at slightly reduced affinities due to steric hindrance when tryptophans 96 and 237 are labelled. Based on these simulations, compensatory modifications in the aptamer should result in significantly improved binding with labelled thrombin. This combined experimental‐computational approach lays the groundwork for the rational design of improved aptamer sensors for analytical applications.     

Voltammetric Determination of 4‐Nitrophenol and 5‐Nitrobenzimidazole Using Different Types of Silver Solid Amalgam Electrodes – A Comparative Study

The voltammetric behavior of two genotoxic nitro compounds (4‐nitrophenol and 5‐nitrobenzimidazole) has been investigated using direct current voltammetry (DCV) and differential pulse voltammetry (DPV) at a polished silver solid amalgam electrode (p‐AgSAE), a mercury meniscus modified silver solid amalgam electrode (m‐AgSAE), and a mercury film modified silver solid amalgam electrode (MF‐AgSAE). The optimum conditions have been evaluated for their determination in Britton‐Robinson buffer solutions. The limit of quantification (L_Q) for 5‐nitrobenzimidazole at p‐AgSAE was 0.77 µmol L^?1 (DCV) and 0.47 µmol L^?1 (DPV), at m‐AgSAE it was 0.32 µmol L^?1 (DCV) and 0.16 µmol L^?1 (DPV), and at MF‐AgSAE it was 0.97 µmol L^?1 (DCV) and 0.70 µmol L^?1 (DPV). For 4‐nitrophenol at p‐AgSAE, L_Q was 0.37 µmol L^?1 (DCV) and 0.32 µmol L^?1 (DPV), at m‐AgSAE it was 0.14 µmol L^?1 (DCV) and 0.1 µmol L^?1 (DPV), and at MF‐AgSAE, it was 0.87 µmol L^?1 (DCV) and 0.37 µmol L^?1 (DPV). Thorough comparative studies have shown that m‐AgSAE is the best sensor for voltammetric determination of the two model genotoxic compounds because it gives the lowest L_Q, is easier to prepare, and its surface can be easily renewed both chemically (by new amalgamation) and/or electrochemically (by imposition of cleaning pulses). The practical applicability of the newly developed methods was verified on model samples of drinking water.     

Electrochemical Detection of Asymmetric PCR Products by Labeling with Osmium Tetroxide

Single stranded DNA‐targets from asymmetric polymerase chain reaction (PCR) of a sequence of the gram positive, spore forming bacterium Clostridium acetobutylicum were detected by square‐wave voltammetry after labeling with osmium tetroxide bipyridine and hybridization with DNA capture probes immobilized on gold electrodes. The asymmetric PCR, performed with a 10‐fold excess of the forward‐primer, was used without any further purification for hybridization with protective strands and covalent labeling with osmium tetroxide bipyridine. Square‐wave voltammetric signals of 20 nmol/L targets were significantly higher at 50 °C compared with 23 °C hybridization temperature. A fully noncomplementary protective strand yielded thoroughly modified targets unable for further hybridization. Coupling this with thermal discrimination opens new opportunities for sequence specific DNA detection.