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.     

Comparison of DNA Hybridization at Rotating and Heated Gold Disk Electrodes

This paper reports about the influence of temperature, hybridization time and convection upon the detection of osmium tetroxide bipyridine‐labeled target oligonucleotides at rotating gold disk (RDE) and heated low temperature co‐fired ceramics (LTCC) gold disk electrodes. We used mixed self‐assembled monolayers of hexathiol‐linked probe oligonucleotides and mercaptohexanol on the gold surface of the electrodes for the hybridization detection of the labeled targets by means of square‐wave voltammetry. Due to protective strands, the osmium tetroxide‐modified target strands were still able to hybridize with the immobilized probe strands. The hybridization of such osmium tetroxide bipyridine‐modified target strands with thiol‐linked probe strands immobilized on gold yielded large reversible square‐wave‐voltammetric signals. Rotation speed and, hence, mass transport due to convection has only marginal effects. On the other hand, temperature affects greatly the hybridization step as indicated by both heated LTCC electrode in cold and RDE in warm hybridization solution. Calculated detection limits of 3.6 and 3.1 nM targets at the RDE and the LTCC electrode, respectively, have been almost the same at both types of electrodes. Applying an appropriate temperature during hybridization is more important than mechanically enhanced mass transport.     

Electrochemical detection of osmium tetroxide-labeled PCR-products by means of protective strands

This communication reports about how single-stranded 136 base polymerase chain reaction (PCR) products labeled with electrochemically active osmium tetroxide bipyridine can be detected voltammetrically by hybridization with probe strands immobilized on gold electrodes. These electroactive ssDNA targets have been obtained by means of Lambda Exonuclease treatment of the double-stranded PCR products followed by hybridization of the remaining single strands with short protective strands and covalent labeling with osmium tetroxide bipyridine. Square-wave voltammetric signals of these osmium labels have been obtained only upon hybridization with the immobilized probe strands. An optimal 50 °C hybridization temperature has been found with a saturation of the probe layer at 30 min hybridization time and 7.5 nmol/l target concentration. The blank capture probe layer alone did not yield any signal. Unprotected strands produced almost no interference. Such double-selective switch-on electrochemical hybridization assays hold great promise for the specific detection of PCR products.     

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.     

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.     

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 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.     

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.     

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

This article reports about the detection of DNA melting curves at heated electrochemical biosensors. Osmium tetroxide‐bipyridine‐labeled target oligonucleotides are hybridized with probe oligonucleotides immobilized on gold electrodes. Then, the gold electrode is successively heated in order to measure a complete melting curve consisting of alternating current voltammetric signals. Melting temperatures ?_m, determined at various ionic strengths and in dependence on different numbers of base pair mismatches, have been compared with those obtained by means of UV spectrophotometry. The proposed method holds great promise for the fast and easy parallel detection of nucleic acids sequences on selectively heated electrode arrays. A stringent hybridization temperature can be easily adjusted in order to discriminate base pair mismatches.     

Facile end-labeling of RNA with electroactive Os(VI) complexes

Ribose at the 3′-end of oligonucleotides (oligos) selectively modified by Os(VI)2,2′-bipyridine (bipy) produced two CV redox couples at pyrolytic graphite electrode. Using square wave voltammetry (SWV) 22-mer oligos can be detected down to 250 nM. At mercury electrodes the Os(VI)bipy-oligo adducts produced an electrocatalytic peak at ~?1.2 V allowing their determination down to picomolar concentrations. High specificity of Os(VI)bipy for ribose in nucleic acids and high sensitivity of the determination at mercury and solid amalgam electrodes give promise for new efficient methods of microRNA determination.     

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.     

Ethylene addition to Ru(CH2)(O)3 – A theoretical study

Quantum chemical calculations using density functional theory (B3LYP) were carried out to elucidate the reaction pathways for ethylene addition to the ruthenium compound RuO₃CH₂. These investigations show that the parent compound is relatively unstable and its rearrangement gives access to very diverse isomers and addition products with comparable relative energies and reaction barriers. The results are compared to our previous study on the analogous osmium system OsO₃CH₂ and we show that reactivity of both compounds towards ethylene is quite similar. In both cases, the [3 + 2]_C,O cycloaddition pathway is preferred kinetically and thermodynamically. The exothermicity (–68.8 kcal/mol) of this reaction is higher for the ruthenium system than for the osmium homologue. While this pathway is unrivaled for the osmium system, the [3 + 2]_O,O cycloaddition pathway is able to compete kinetically for the ruthenium system.     

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.     

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.     

Efficient stripping voltammetric detection of organophosphate pesticides using NanoPt intercalated Ni/Al layered double hydroxides as solid-phase extraction

We developed a simple strategy for designing a sensitive electrochemical stripping voltammetric sensor for organophosphate pesticides (OPs) based on solid-phase extraction (SPE) using nanosized Pt intercalated Ni/Al layered double hydroxides (labeled as NanoPt-LDHs). By assembling NanoPt with LDHs together, the resulting NanoPt-LDHs are highly efficient to capture OPs. It dramatically facilitates the enrichment of OPs onto their surface and realizes the sensitive stripping voltammetric detection of methyl parathion (MP) as a model of OPs. The stripping analysis shows highly linear over MP concentration ranges of 0.001–0.15 and 0.3–1.0 μg mL^? 1 with a detection limit of 0.6 ng mL^–1 (S/N = 3). The combination of NanoPt, LDHs, SPE, and square-wave voltammetry (SWV) provides a fast, simple, and sensitive electrochemical method for OPs.     

Stripping voltammetric analysis of organophosphate pesticides based on solid-phase extraction at zirconia nanoparticles modified electrode

A sensitive electrochemical stripping voltammetric method for analyzing organophosphate (OP) compounds was developed based on solid-phase extraction (SPE) at zirconia (ZrO₂) nanoparticles modified electrode. ZrO₂ nanoparticles were proved as a new sorbent for SPE of OP pesticides. Because of the strong affinity of ZrO₂ for the phosphoric group, nitroaromatic OPs can strongly bind to the ZrO₂ nanoparticle surface. The combination of SPE with square-wave voltammetry (SWV) provided a fast, sensitive, and selective electrochemical method for nitroaromatic OP compounds using methyl parathion (MP) as a model. The stripping response was highly linear over the MP range of 0.003–2.0 μg/mL, with a detection limit of 0.001 μg/mL. The fast extraction ability of ZrO₂ nanoparticles makes it promising sorbent for various solid-phase extractions.     

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.     

Quantum dot-based electrochemical DNA biosensor using a screen-printed graphite surface with embedded bismuth precursor

This work reports the development of screen-printed quantum dots (QDs)-based DNA biosensors utilizing graphite electrodes with embedded bismuth citrate as a bismuth precursor. The sensor surface serves both as a support for the immobilization of the oligonucleotide and as an ultrasensitive voltammetric QDs transducer relying on bismuth nanoparticles. The utility of this biosensor is demonstrated for the detection of the C634R mutation through hybridization of the biotin-tagged target oligonucleotide with a surface-confined capture complementary probe and subsequent reaction with streptavidin-conjugated PbS QDs. The electrochemical transduction step involved anodic stripping voltammetric determination of the Pb(II) released after acidic dissolution of the QDs. Simultaneously with the electrolytic accumulation of Pb on the sensor surface, the embedded bismuth citrate was converted in situ to bismuth nanoparticles enabling ultra-trace Pb determination. The biosensor showed a linear relationship of the Pb(II) peak current with respect to the logarithm of the target DNA concentrations from 0.1 pmol L^− 1 to 10 nmol L^− 1, and the limit of detection was 0.03 pmol L^− 1. The biosensor exhibited effective discrimination between a single-base mismatched sequence and the fully complementary target DNA. These “green” biosensors are inexpensive, lend themselves to easy mass production, and hold promise for ultrasensitive bioassay formats.     

Demetallation of methemoglobin in cellulose nanofibril–TiO2 nanoparticle composite membrane electrodes

Porous composite films containing cellulose nanofibrils (from sisal) and TiO₂ nanoparticles (ca. 6 nm diameter) are obtained in a layer-by-layer assembly process. Each layer consists of ca. 0.18 μg cellulose nanofibrils and ca. 0.72 μg TiO₂ (determined by QCMB) and adds a thickness of ca. 16 nm (by AFM) to the uniform deposit. The TiO₂ nanophase is creating conducting pathways for electrons in a relatively open cellulose structure (ca. 82% open pores) potentially suitable for the immobilization of large redox proteins such as methemoglobin.Methemoglobin is shown to readily adsorb into the cellulose–TiO₂ film. However, electrochemical responses for the immobilized methemoglobin in aqueous 0.1 M phosphate buffer at pH 5.5 suggest that facile demetallation occurs. Experiments with Fe³⁺ in the absence of protein result in voltammetric responses indistinguishable from those observed for immobilized methemoglobin. In the presence of ethylenediamine tetraacetic acid (EDTA) the voltammetric signals for the Fe³⁺ immediately disappear. Complementary experiments conducted in 0.1 M acetate buffer at pH 5.5 demonstrate that methemoglobin can indeed be immobilized in electrochemically active form and without demetallation loss of the voltammetric signal in the presence of EDTA. Demetallation appears to occur (i) in the presence of phosphate, (ii) at pH 5.5, (iii) and in the presence of a charged surface.     

Selective determination of trichloroacetic acid using silver nanoparticle coated multi-walled carbon nanotubes

Silver nanoparticle coated multi-walled carbon nanotubes (Ag/MWCNT) were prepared and used to fabricate a modified electrode. The Ag/MWCNT composites were observed by a transmission electron microscope (TEM), and the electrochemical properties of the Ag/MWCNT composite modified glassy carbon electrode were characterized by electrochemical measurements. The results showed that these composites had a favorable catalytic ability for the reduction of trichloroacetic acid (TCAA). Square wave voltammetric (SWV) technique was applied to detect TCAA. Under optimum conditions, the voltammetric determination of TCAA was performed with a linear range of 5.0 × 10^? 6–1.2 × 10^? 4 mol L^? 1 and a detection limit of 1.9 × 10^? 6 mol L^? 1 (S/N = 3).