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.     

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.     

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.     

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.     

Spectrophotometric reaction rate method for the determination of osmium by its catalytic effect on the oxidation of gallocyanine by bromate

A simple kinetic spectrophotometric method was developed for the determination of osmium. The method is based on the catalytic effect of osmium as osmium tetroxide on the oxidation of gallocyanine by bromate at pH 7. The reaction is monitored spectrophotometrically by measuring the decreasing absorbance of gallocyanine at 620 nm by the fixed-time method. A detection limit of 0.01 ng/ml and linear calibration curve from 0.1 to 100 and from 100 to 1200 ng/ml Os(VIII) is reported. The relative standard deviation for 0.0100 microg/ml Os(VIII) is 0.8% (N = 10). The method is free from most interferences. Osmium in synthetic samples is determined by this method, with satisfactory results.     

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.     

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.     

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.     

Photometric determination of osmium with thiourea after extraction of the tetroxide

A general method for the determination of 5–1000 γ of osmium involves extraction of osmium tetroxide with chloroform or carbon tetrachloride, followed by shaking the organic solvent with a sulfuric acid solution, of thiourea to form red Os(NH₂CSNH₂)₆⁺³, whose color intensity is measured photometrically. A sharp separation of osmium from ruthenium can be obtained by reducing Os(VIII) and Ru(VIII) with ferrous sulfate and then oxidizing Os(IV) to Os(VIII) with nitric acid; ruthenium remains reduced and is not extracted by chloroform or carbon tetrachloride.     

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.     

Oxidation of tertiary silanes by osmium tetroxide

In the presence of an excess of pyridine ligand L, osmium tetroxide oxidizes tertiary silanes (Et(3)SiH, (i)Pr(3)SiH, Ph(3)SiH, or PhMe(2)SiH) to the corresponding silanols. With L = 4-tert-butylpyridine ((t)Bupy), OsO(4)((t)Bupy) oxidizes Et(3)SiH and PhMe(2)SiH to yield 100 +/- 2% of silanol and the structurally characterized osmium(VI) mu-oxo dimer [OsO(2)((t)Bupy)(2)](2)(mu-O)(2) (1a). With L = pyridine (py), only 40-60% yields of R(3)SiOH are obtained, apparently because of coprecipitation of osmium(VIII) with [Os(O)(2)py(2)](2)(mu-O)(2) (1b). Excess silane in these reactions causes further reduction of the OsVI products, and similar osmium "over-reduction" is observed with PhSiH(3), Bu(3)SnH, and boranes. The pathway for OsO(4)(L) + R(3)SiH involves an intermediate, which forms rapidly at 200 K and decays more slowly to products. NMR and IR spectra indicate that the intermediate is a monomeric Os(VI)-hydroxo-siloxo complex, trans-cis-cis-Os(O)(2)L(2)(OH)(OSiR(3)). Mechanistic studies and density functional theory calculations indicate that the intermediate is formed by the [3 + 2] addition of an Si-H bond across an O=Os=O fragment. This is the first direct observation of a [3 + 2] intermediate in a sigma-bond oxidation, though such species have previously been implicated in reactions of H-H and C-H bonds with OsO(4)(L) and RuO(4).     

Voltammetric behavior of DNA modified with osmium tetroxide 2,2'-bipyridine at mercury electrodes

Osmium tetroxide complexes with nitrogen ligands (L) are probes of DNA structure and electroactive labels of DNA. Here adducts of single-stranded (ss) DNA with osmium tetroxide 2,2'-bipyridine (DNA-Os,bipy) were studied by cyclic voltammetry for the first time. It was found that at neutral pH DNA-Os,bipy produces three redox couples in the potential range between 0 and -1 V (peaks I-III) and a cathodic peak at about -1.3 V (peak IV). The latter peak decreased with increasing scan rate, and peaks arising from the forward and reverse scans exhibited the same direction, suggesting catalytic nature of the electrode process. We concluded that this peak corresponds to the known differential pulse voltammetric (polarographic) peak of DNA-Os,L adducts for which catalytic hydrogen evolution is responsible. In contrast, currents of cathodic peaks II and III increased almost linearly with increasing scan rate, suggesting involvement of adsorption in the electrode processes. Adsorptive stripping square-wave voltammetry was used to analyze the DNA-Os,bipy at low concentrations. It was shown that at neutral pH, peak III can offer sensitivity in the ppb range, which is only little lower than that reached by catalytic peak IV. The latter peak is, however, superior in sensitivity at acid pH values.     

Nature of the cherry-red transient formed during the interaction of osmium tetroxide with hydrogen peroxide

Summary A cherry-red transitory coloration is formed when OsO₄ or osmium(VI) is added to alkaline hydrogen peroxide solution in the pH range 9–13. The transient has an absorption maximum at 530 nm and its concentration depends on the pH of reaction mixture reaching a maximum at pH 10.5–11. The transient is designated as a peroxo-derivative of osmium(VIII) [or an osmium(VII) — Superoxide radical pair if the peroxoderivative undergoes a fast intramolecular one-electron transfer].Many decades ago it was observed¹ that a cherry-red coloration appears transiently when osmium tetroxide solution is added to alkaline hydrogen peroxide solution. However, to our knowledge, there are no literature data about the nature of this transient. Further, we observed recently that there is a close connection between the rate of decomposition of hydrogen peroxide catalysed by osmium tetroxide and the intensity of the coloration, and therefore an attempt was made to investigate the transient by using a fast spectrophotometer technique.     

Characterization of a glucose sensor prepared by electropolymerization of pyrroles containing a tris-bipyridine osmium complex.

A glucose sensor was developed by electrocopolymerization using pyrroles containing a tris-bipyridine (bpy) osmium complex (Os-py), pyrrole (py), pyrrole propanoic acid (PPA) and glucose oxidase (GOx) to improve the key performance characteristics, such as the sensitivity, selectivity, and long-term stability. Tris-bipyridine osmium pyrrole complexes with four different methylene moieties were utilized to correlate the methylene length with the glucose sensor performance. The electrocatalytic response of glucose was clearly observed at electrodes modified with Os-py, except for the electrode immobilized with the Os-py complex containing the shortest methylene moiety. The current response to glucose increased up to a concentration of 100 mmol dm(-3). The electrocatalytic response to glucose at the [Os(bpy)(2)(py(6)-bpy)](2+/3+)/py/PPA/GOx electrode was stable for more than 100 days. Dissolved oxygen and potential interference compounds (ascorbic acid, uric acid, and acetaminophen) minimally perturbed the current response to glucose at the [Os(DM-bpy)(2)(py(6)-bpy)](2+/3+)/py/PPA/GOx electrode. Based on these results, a longer methylene moiety appears to improve the performance characteristics of a glucose sensor fabricated via the electropolymerization of tris-bipyridine osmium pyrrole complexes.     

Derivatisation of peptides with osmium tetroxide, 2,2′-bipyridine: capillary electrophoretic and MALDI-TOF mass spectrometric study

Site-specific chemical modification is a useful technology in characterisation of proteins, but the number of chemical probes of the protein structure reacting with proteins under mild conditions in aqueous solutions is rather limited. Here we studied the reaction of osmium tetroxide, 2,2′-bipyridine (Os,bipy) with several peptides using capillary zone electrophoresis (CZE) and matrix-assisted laser desorption-ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques showed formation of a stable complex of Os,bipy with tryptophan residues. In CZE peaks with different migration times and UV-Vis spectra were observed. MALDI-TOF MS showed the formation of a product with characteristic isotopic pattern corresponding to the presence of osmium atom. Oxidation of cysteine and methionine side chains to cysteic acid and methionine sulfone by Os,bipy was detected by CZE and confirmed by MALDI-TOF and post-source decay (PSD) mass spectra. PSD showed specific shifts of molecular weights of the peptides and their fragments after the derivatisation. We believe that Os,bipy may become a useful agent in the characterisation of proteins.     

Voltammetric Behavior of Osmium‐Labeled DNA at Mercury Meniscus‐Modified Solid Amalgam Electrodes. Detecting DNA Hybridization

The complex of osmium tetroxide with 2,2′‐bipyridine has been utilized as a probe of DNA structure and an electroactive marker of DNA in DNA hybridization sensors. It produces several voltammetric signals, the most negative of them has been observed only at mercury electrodes. This signal is of catalytic nature affording a high sensitivity of DNA determination. The catalytic current due to evolution of hydrogen in voltammetry of DNA modified by complex of osmium tetroxide with 2,2′‐bipyridine (DNA‐Os,bipy) was studied. Solid amalgam electrodes (modified with mercury menisci) of silver (m‐AgSAE), copper (m‐CuSAE), gold, and of combined bismuth and silver, were used as possible substitutes for mercury electrodes. Besides the hanging mercury drop electrode (HMDE), the catalytic current was observed only on m‐AgSAE and m‐CuSAE. Electrodes of gold and bismuth amalgams did not give the catalytic current. The detection limit of DNA‐Os,bipy on HMDE was 0.1 ng mL^?1 (RSD=2.3 %, N=11), and on m‐AgSAE 0.2 ng mL^?1 (RSD=3.1%, N=11). The m‐AgSAE was successfully applied as a detection electrode in double‐surface DNA hybridization experiments offering highly specific discrimination between complementary (target) and nonspecific DNAs, as well as determination of the length of a repetitive DNA sequence. The m‐AgSAE has proved a convenient alternative to the HMDE or carbon electrodes used for similar purposes in previous work.     

Kinetics of the labeling reactions of thymine, cytosine and uracil with osmium tetroxide bipyridine

The electroactive complex osmium tetroxide bipyridine holds great promise as a covalent label for biosensor applications regarding nucleic acids and protein detection. Labeling can easily be performed in the laboratory. Until now, almost only DNA species have been investigated using this label. Thymine (which occurs exclusively in DNA) is known to react much faster than cytosine and uracil. In order to explore the possibilities to modify and detect also RNA species in a timely fashion, we have investigated the kinetics of reactions of osmium tetroxide bipyridine with the pyrimidine bases in the micromolar concentration range at different temperatures by means of spectrophotometry. Results were confirmed using voltammetric detection for the determination of labeled oligonucleotides. The modification reaction can be easily completed at room temperature within 7 h, even in case of cytosine and uracil. At 60 °C, 3 h are sufficient for complete modification of all pyrimidine bases that are found in natural nucleic acids. These findings will be important for future biosensor applications with RNA species as target molecules.     

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.     

Protonation and H/D exchange reactions promoted by a sulfur-rich osmium hydride complex: identification of a labile dihydrogen complex

The sulfur-rich osmium nitrosyl complexes Bu(4)N[Os(NO)((bu)S(2))(2)] (1) [(bu)S(2)(2-) = 3,5-tert-butyl-1,2-benzenedithiolate(2-)] and [Os(NO)(py(bu)S(4))]Br ()[py(bu)S(4)(2-) = 2,6-bis(2-sulfanyl-3,5-di-tert-butylphenylthio)dimethylpyridine(2-)] have been synthesized. The molecular structure of 1 exhibits a square-pyramidal geometry with the NO group at the apical position. A pseudo-octahedral geometry with two thiolate and two thioether donors in trans configuration is found for 2. Compound 2 shows two quasi-reversible one-electron redox waves at E(1/2) = 0.51 and -0.46 V vs. NHE for the redox couples [Os(NO)(py(bu)S(4))](+1/0) and [Os(NO)(py(bu)S(4))](0/-1), respectively. 2 reacts with NaAlH(4) to produce Na[Os(H)(py(bu)S(4))] (3), which exhibits a typical hydride resonance in the (1)H NMR spectrum at delta =-15.03 ppm. Protonation of 3 with HBF(4)/CD(3)OD at 20 degrees C rapidly releases H(2)/HD to afford the dinuclear complex [Os(py(bu)S(4))](2) (4). Low temperature (1)H and (2)H NMR spectra of in [D(8)]THF with CH(3)OH or CD(3)OD at -80 degree C allow the observation of the formation of [Os(H(2)/HD)(py(bu)S(4))]. A 1 : 1 : 1 triplet at delta = -7.84 ppm [J(HD) = 31.2 Hz] and a relaxation time of T(1)(min) = 6 ms (-65 degrees C, 270 MHz) firmly establish the presence of eta(2)-H(2)/HD ligand. At room temperature, 3 interacts with D(2) (1 atm) and undergoes heterolytic D(2) cleavage followed by H/D exchange to form Na[Os(D)(py(bu)S(4))] (3a). A plausible cyclic mechanism has been proposed.     

Spectrophotometric methods for the determination of osmium-I Extraction and ultraviolet spectrophotometric determination of osmium tetroxide

An ultraviolet spectrophotometric method is presented for the détermination of milligram quantities of osmium in solutions of uranyl sulphate. Osmium is first oxidised to the octovalent state and the osmium tetroxide which is formed, is selectively extracted with chloroform. The ultraviolet absorption spectrum'of osmium tetroxide in chloroform has a series of absorption bands with peak absorbancies at 282, 289, 297, 304 and 312 mμ and molar absorbancy indexes of 1870, 1760,1640, 1400 and 1000, respectively. For each wavelength, the optimum concentration range for the determination of osmium was evaluated. From 0.4 to 3.3 mg of osmium can be determined with a coefficient of variation of 3%. Of the elements tested only chloride and octovalent ruthenium interfere; however, both of these interferences can be eliminated.