Investigation of unexpected migration behavior of adenosine in capillary zone electrophoresis

Summary The capillary zone electrophoresis of two common nucleosides, adenosine and inosine, was investigated. Both compounds were resolved in a 0.1 M sodium phosphate buffer, pH 7.5. Contrary to expectations, adenosine behaved at this pH— 5 pH units lower than the literature pK_a— as a negative ion, migrating behind mesityloxide (neutral marker) when working in normal polarity mode. To confirm the migration order, peaks were identified from absorption maxima, by high-speed scanning detector. The change in electrophoretic mobility with pH was investigated for the nucleosides, and 10 other background electrolytes were tried to match the separation capabilities of the sodium phosphate buffer. Most inorganic buffers showed comparable separation, while organic, Good-type buffers lacked selectivity.     

Poly(brilliant green)/carbon nanotube-modified carbon film electrodes and application as sensors

Poly(brilliant green) (PBG) films were formed on carbon film electrodes (CFE) by electropolymerisation of brilliant green monomer using potential cycling or at fixed potential from different pH solutions. The modified electrodes, PBG/CFE, were characterised by cyclic voltammetry (CV) in electrolytes of different pH by electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). In order to increase the stability of the polymer film and enhance the response, multi-walled carbon nanotubes (MWCNTs) were first deposited on CFE and then PBG was formed on top, PBG/CNT/CFE. The modified electrodes were applied to the amperometric determination of ascorbic acid (AA) in phosphate buffer pH?7.0 at 0.0 V vs. saturated calomel electrode (SCE) and the results were compared, the presence of CNT leading to a significant increase in sensitivity. An interference study was carried out and good separation between AA and dopamine (DA) peaks was achieved that led to the successful determination of DA without interferences. Other interferents: aspirin, acetaminophen, salicylic acid and uric acid exhibited no response on the PBG/CNT/CFE. Determination of AA in pharmaceutical samples was successfully performed.     

Determination of the performance of glass electrodes in aqueous solutions in the physiological ph range and at the physiological sodium ion concentration

A method for testing glass electrodes in the physiological pH range (6.4–7.6) and at the physiological sodium ion concentration (0.15 mol l^-1), based on indirect comparison of potentials with the hydrogen gas electrode, is developed according to a scheme described earlier. The hydrogen ion sensitivity and the sodium ion error of a glass electrode can be determined with three different aqueous solutions of amine buffers and their hydrochlorides; two of these have different pH values and one also contains a sodium salt (at the higher pH value). A cell without a liquid/liquid junction, containing silver/silver chloride reference electrodes, is used at 37° C. The accuracy of both determinations is ±0.2 mV (±0.003 pH). The results for some commercial glass electrodes tested with this method are presented.     

Generation and detection of single metal nanoparticles using scanning electrochemical microscopy techniques

Different pathways towards the generation and detection of a single metal nanoparticle (MNP) on a conductive carbon support for testing as an electrocatalyst are described. Various approaches were investigated including interparticle distance enhancement, electrochemical and mechanical tip-substrate MNP transfer onto macroscopic surfaces, scanning electrochemical microscopy (SECM)-controlled electrodeposition, and the use of selective binding monolayers on carbon fiber electrodes (CFEs) for solution-phase-selective adsorption. A novel SECM technique for electrodepositing MNPs on CFE tips immersed 100-200 nm below the electrolyte level was developed and used to generate single Pt and Ni nanoparticles. Following their generation, we demonstrate electrocatalytic detection of Fe3+ on individual Pt particles with the CFE in a Fe3+/H2SO4 solution. We also describe an approach of attaching MNPs to CFEs by controlling the composition of monolayers bonded to the CFE. By employing a monolayer with a low ratio of binding (e.g., 4-aminopyridine) to nonbinding molecules (e.g., aniline) and controlling the position of the CFE in a colloidal Pt solution with a SECM, we attached a single 15 nm radius Pt nanoparticle to the CFE. Such chemisorbed Pt particles exhibited a stronger adhesion on surface-modified CFEs and better mechanical stability during proton reduction than MNPs electrodeposited directly on the CFE.     

Fluorescence Detection of Adenosine Triphosphate in an Aqueous Solution Using a Combination of Copper(II) Complexes

Fluorescent ligands have been designed to form ternary complexes with a Cu(II) cation and phosphates in a buffer solution at physiological pH 7.4. It has been shown that a combination of two different ligands and CuCl(2) allows one to achieve high adenosine triphosphate/adenosine diphosphate, adenosine 5'-monophosphate selectivity, and ratiometric fluorescence sensing, while separately each ligand complex does not have such properties.     

Thermal Aggregation of Bovine Serum Albumin in Conventional Buffers: An Insight into Molecular Level Interactions

We have studied the effect of some conventional buffers, which are used frequently for biological research, on the thermal aggregation behavior of bovine serum albumin (BSA). The aggregation kinetics of BSA in buffer solutions were investigated by using UV–Vis spectroscopy. The buffers include sodium phosphate buffer, TRIS buffer and imidazole buffer at physiological pH (7.4). Dynamic light scattering and scanning electron microscopy have been employed to illustrate the size and morphology of protein aggregates. The molecular level interactions of buffer molecules with BSA was probed by various spectroscopic techniques, including UV–Vis, fluorescence, and circular dichroism. The results of this study reveal that the strong interactions of the buffers with protein’s folded/unfolded structures lead to stabilization/destabilization of BSA. We have also explored the possible binding sites of BSA for these buffers using a molecular docking technique.     

Determination of impurities in a novel analogue of adenosine 5′-triphosphate by capillary electrophoresis

A capillary electrophoretic method using an uncoated capillary was developed to resolve potential impurities in FPL 67085XX, a novel phosphonate analogue of adenosine 5′-triphosphate. The effects of buffers, buffer concentration, eleunt pH and methanol were investigated. Optimum resolution of the impurities was achieved using an eluent consisting of 7% (v/v) methanol in 25 mM phosphate buffer-2 mM EDTA adjusted to pH 6.6.     

Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine

Adenosine is a neuromodulator that regulates neurotransmission. Adenosine can be monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes and ATP is a possible interferent in vivo because the electroactive moiety, adenine, is the same for both molecules. In this study, we investigated carbon-fiber microelectrodes coated with Nafion and carbon nanotubes (CNTs) to enhance the sensitivity of adenosine and decrease interference by ATP. Electrodes coated in 0.05 mg mL(-1) CNTs in Nafion had a 4.2 ± 0.2 fold increase in current for adenosine, twice as large as for Nafion alone. Nafion-CNT electrodes were 6 times more sensitive to adenosine than ATP. The Nafion-CNT coating did not slow the temporal response of the electrode. Comparing different purine bases shows that the presence of an amine group enhances sensitivity and that purines with carbonyl groups, such as guanine and hypoxanthine, do not have as great an enhancement after Nafion-CNT coating. The ribose group provides additional sensitivity enhancement for adenosine over adenine. The Nafion-CNT modified electrodes exhibited significantly more current for adenosine than ATP in brain slices. Therefore, Nafion-CNT modified electrodes are useful for sensitive, selective detection of adenosine in biological samples.     

Microelectrode Sensing of Adenosine/Adenosine‐5′‐triphosphate with Fast‐Scan Cyclic Voltammetry

Adenosine and adenosine‐5′‐triphosphate (ATP) are important extracellular signaling molecules. Here, we studied adenosine and ATP using fast‐scan cyclic voltammetry at carbon‐fiber microelectrodes. Although ATP and adenosine have similar oxidation potentials, ATP oxidation current was highly dependent on buffer pH and divalent cation concentrations but adenosine current was not. Therefore, they can be distinguished by adding a divalent cation chelator or calibrating electrodes at different pH values. The enzymatic degradation of adenosine by adenosine deaminase was monitored in a mixture of adenosine and ATP in presence of EDTA (ethylenediaminetetraacetate). This sensing method is promising for enzyme kinetics or in vitro studies.     

Permselectivity of neurotransmitters at overoxidized polypyrrole-film-coated glassy carbon electrodes

The permselectivity of neurotransmitters such as dopamine, epinephrine, and norepinephrine at overoxidized polypyrrole (OPPY)-film-coated glassy carbon electrodes has been investigated. The chemically-modified electrodes exhibit attractive permselectivity and antifouling properties of rejecting anionic species, e.g. ascorbate, etc. Compared with the response of neurotransmitters at modified electrodes overoxidized in phosphate buffer solution (pH 7.4), higher sensitivity and reversibility response can be obtained at modified electrodes overoxidized in sodium hydroxide solution. The effect of film thickness on the permselective response was tested. Rotating disk electrode experiments were used to determine the apparent diffusion coefficients of several electroactive solutes in the OPPY films. The influence of the hydrophobicity of the organic ions on the permeability within the polymer films was discussed. Dopamine and epinephrine were determined at the 1 x 10(-6)-1 x 10(-4) M level by means of voltammetry after an exposure period of 2 min in 0.1 M phosphate buffer (pH 7.4) with detection limits of 8 x 10(-7) M and 6 x 10(-7) M respectively.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Ultrathin Cell‐Membrane‐Mimic Phosphorylcholine Polymer Film Coating Enables Large Improvements for In Vivo Electrochemical Detection

Resisting biomolecule adsorption onto the surface of brain‐implanted microelectrodes is a key issue for in vivo monitoring of neurochemicals. Herein, we demonstrate that an ultrathin cell‐membrane‐mimic film of ethylenedioxythiophene tailored with zwitterionic phosphorylcholine (EDOT‐PC) electropolymerized onto the surface of a carbon fiber microelectrode (CFE) not only resists protein adsorption but also maintains the sensitivity and time response for in vivo monitoring of dopamine (DA). As a consequence, the as‐prepared PEDOT‐PC/CFEs could be used as a new reliable platform for tracking DA in vivo and would help understand the physiological and pathological functions of DA.     

The impact of physiological buffer solutions on zinc oxide nanostructures: zinc phosphate conversion

Zinc oxide (ZnO) nanostructures have been widely used in biosensor applications. However, little attention has been given to the interaction of ZnO structures with physiological buffer solutions. In the present work, it is shown that the use of buffers containing phosphate ions leads to the modification of the ZnO tetrapodal micro/nanostructures when immersed in such solutions for several hours, even at the physiological pH (7.4). ZnO samples designed to be used as transducers in biosensors were immersed in phosphate buffers for several durations at pH = 5.8 and pH = 7.4. Their detailed morphological, structural and optical characterization was carried out to demonstrate the effect of the ZnO interaction with the phosphate ions. The pH had an important role in the ZnO conversion into zinc phosphate, with lower pH promoting a more pronounced effect. After 72 h and at pH = 5.8, a significant amount of the ZnO structures were converted into crystalline zinc phosphate, while immersion during the same time at pH = 7.4 resulted predominantly in amorphous zinc phosphate particles mixed with the original ZnO tetrapods. Photoluminescence spectra show remarkable changes with prolonged immersion times, particularly when the luminescence of the sample was investigated at 14 K. These findings highlight the importance of a careful analysis of the sensing results when phosphate-based buffer solutions are in contact with the ZnO transducers, as the changes observed on the transduction signal during sensing experiments may also comprise a non-negligible contribution from a phosphate-induced transformation of ZnO, which can hamper an accurate assessment of the sensing behavior.     

An oxygen cathode operating in a physiological solution

We report the electroreduction of O(2) to water under physiological conditions (pH 7.4, 0.15 M NaCl, 37.5 degrees C) at a current density of 5 mA cm(-2) and at a potential only 0.18 V reducing versus that of the reversible O(2)/H(2)O electrode at pH 7.4. The immobilized electrocatalyst enabling the reduction is the electrostatic adduct of bilirubin oxidase from Myrothecium verrucaria, a polyanion at pH >4.1, and the polycationic redox copolymer of polyacrylamide and poly (N-vinylimidazole) complexed with [Os (4,4'-dichloro-2,2'-bipyridine)(2)Cl](+/2+), cross-linked on carbon cloth. The current density of the rotating electrodes was O(2) transport limited up to 8.8 mA cm(-2); their kinetic limit was reached at 9.1 mA cm(-2). The operational life of the electrodes depended on their angular velocity, which defined not only the current density but also the mechanical shear stress stripping the electrocatalyst. When the electrodes were rotated at 300 rpm and were poised at -256 mV versus the potential of the reversible O(2)/H(2)O electrode, their 2.4 mA cm(-2) initial current density decreased to 1.3 mA cm(-2) after 6 days of continuous operation at 37.5 degrees C.     

Separation of human serum lipoproteins into three major classes by hydroxyapatite chromatography.

The separation of normolipidemic male serum lipoprotein fraction, prepared by ultracentrifugal flotation, was studied on hydroxyapatite columns. Potassium phosphate buffers in the pH range 5.6-7.4 were evaluated as eluents. The three main classes of the lipoproteins (high density, low density and very low density) can be separated on the Tiselius-type hydroxyapatite (Bio-Gel HTP DNA grade) column by elution with 75, 250 and 300 mM potassium phosphate buffer (pH 7.4), respectively.     

硝酸还原酶在巯基自组装膜上的电化学性质

应用自组装技术将硝酸还原酶 (NR)构筑在硫醇自组装单分子膜上 ,研究了Au/半胱胺 /NR和Au/半胱胺 /NR/卵磷脂两种酶电极在磷酸缓冲液中的直接电化学行为     

Piperazine-based buffers for liposome coating of capillaries for electrophoresis

Anionic liposomes can be coated on fused-silica capillaries for electrophoresis in the presence of N-(hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) as background electrolyte (BGE) solution. In this work, the interaction of various compounds with zwitterionic and anionic phospholipid coatings was studied with HEPES at pH 7.4 as BGE solution. The chromatographic and electrophoretic behavior of three test sample solutions (anionic, cationic, and neutral) was investigated for evaluation of the phospholipid coatings. Our results show that hydrophobic interactions between analytes and the phospholipid coating are important for the migration of charged analytes. In addition, the performances of other piperazine-based buffers, i.e., N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid), piperazine-N,N'-bis(2-ethanesulfonic acid), and piperazine-N,N'-bis(hydroxypropane sulfonic acid), at pH 7.4, as liposome solvent and BGE solution were evaluated and compared with the performance of HEPES at pH 7.4. The anionic liposome solution comprised 80/20 mol% phosphatidylcholine/phosphatidylserine. A simple test solution was selected and the chromatographic and electrophoretic migration behavior of the analytes was evaluated. The results show that, in addition to HEPES, other piperazine-based buffers at pH 7.4 are suitable for coating of fused-silica capillaries with anionic liposomes.     

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 x 10(-5) to 2 x 10(-4)M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4-7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.     

The effect of electrolyte on the encapsulation efficiency of vesicles formed by the nonionic surfactant, 2C18E12

Encapsulation efficiencies of vesicles formed by the nonionic surfactant 1,2-dioctadecyl-rac-glycerol-3-omega-methoxydodecylethylene glycol (abbreviated as 2C18E12) and its phospholipid counterpart, distearoylphosphatidylcholine (DSPC) at 298 K, were determined by the entrapment of the water-soluble dye, carboxyfluorescein (CF) to be 0.045+/-0.001 and 0.03+/-0.04 L mol(-1) for 2C18E12 vesicles prepared using low osmolarity (270 m Osm) Krebs-Henseleit (K-H) buffer and a modified 'high salt' (1600 m Osm) variant of K-H buffer, respectively, and 0.64+/-0.01 and 0.31+/-0.04 Lmol(-1) for DSPC vesicles prepared under the same conditions and in the same buffers. Freeze fracture electron microscopy studies confirmed the presence of vesicles when 2C18E12 and DSPC were dispersed in water and both buffer solutions. Small angle neutron scattering (SANS) studies, using D2O in place of H2O, showed that when 2C18E12 vesicles were prepared in the 'high salt' variant of K-H buffer as opposed to K-H buffer or water, a higher proportion of multilamellar vesicles (MLV) were formed. Furthermore when prepared in the 'high salt' variant of K-H buffer, the 2C18E12 bilayers were thinner, and when present in the form of MLV exhibited a smaller layer of water separating the bilayers. However, even in the absence of electrolyte, 2C18E12 formed surprisingly thin bilayers due to the penetration of the polyoxyethylene chains into the hydrophobic chain region of the bilayer. Due to the dehydrating effect of the high concentration of electrolyte present in the 'high salt' variant of K-H, the polyoxyethylene head groups penetrated further into the hydrophobic region of the bilayer making the bilayer even thinner. In the case of the DSPC vesicles, although the SANS study showed an increase in the relative proportion of multilamellar to unilamellar vesicles when samples were prepared in the 'high salt' variant of K-H buffer, no differences were observed in the thickness and the d-spacing of the vesicle bilayers. Variable temperature turbidity measurements of 2C18E12, and DSPC vesicles prepared in water indicated phase changes at 320+/-0.5 and 327+/-0.5 K, respectively, and were unchanged when the 'high salt' variant of K-H buffer was used as hydrating medium. Taken together, these results suggest that a low phase transition temperature was not the reason for the poor entrapment efficiency of 2C18E12 vesicles but rather the very 'thin' hydrophobic barrier formed by the penetration of the polyoxyethylene chains into the hydrophobic region of the bilayer.     

阿昔洛韦在硼掺杂金刚石电极上的电化学行为及其与DNA相互作用的研究

利用硼掺杂金刚石(BDD)电极通过循环伏安法和微分脉冲伏安法研究了阿昔洛韦在0.10 mol/L磷酸盐缓冲溶液(pH 7.4)中的电化学行为及其与DNA的相互作用.与玻碳电极相比,阿昔洛韦在BDD电极上的循环伏安曲线在1.17 V处的氧化峰电流更大,背景电流较低.根据峰电位随溶液pH值和扫描速率的变化趋势考察了阿昔洛韦...