An analysis of the kinetics of oxidation of ascorbate at poly(aniline)-poly(styrene sulfonate) modified microelectrodes

A detailed kinetic study is provided for the oxidation of ascorbate at poly(aniline)-poly(styrene sulfonate) coated microelectrodes. Flat films with a low degree of polymer spillover and a thickness much lower than the microelectrode radius were produced by controlled potentiodynamic electrodeposition. The currents for ascorbate oxidation are found to be independent of the polymer thickness, indicating that the reaction occurs at the outer surface of the polymer film. At low ascorbate concentrations, below around 40 mM, the currents are found to be mass transport limited. At higher ascorbate concentrations the currents became kinetically limited. The experimental data for measurements at a range of potentials are fitted to a consistent kinetic model and the results summarized in a case diagram. The results obtained for the poly(aniline)-(polystyrene sulfonate) coated microelectrode are compared to those for a poly(aniline)-poly(vinyl sulfonate) coated microelectrode and to the results of an earlier study of the reaction at poly(aniline)-poly(vinyl sulfonate) coated rotating disc electrodes. For poly(aniline)-poly(styrene sulfonate) the oxidation of ascorbate is found to proceed by one electron reaction whereas for poly(aniline)-poly(vinyl sulfonate) the reaction is found to be a two electron oxidation.     

Determination of Paracetamol in Presence of Ascorbic Acid in Pharmaceutical Products by Scanning Electrochemical Microscopy

The selective amperometric determination of paracetamol in pharmaceutical formulations containing ascorbate was achieved by removing the interfering species in the diffusion layer created between a platinum substrate and a disc microelectrode in a Scanning Electrochemical Microscopy (SECM) configuration, while the target analyte was kept unconsumed. After complete depletion of ascorbate, paracetamol was detected at the SECM tip in a free‐interference solution zone. The influence of the substrate potential and the gap distance on the efficiency of ascorbate removal was systematically examined. The effectiveness of the device towards the determination of paracetamol in pharmaceutical samples was evaluated and under optimal conditions the results obtained agreed well with the labeled value.     

O2>-DEPENDENT and -INDEPENDENT PHOTOOXIDATION OF QUERCETIN IN THE PRESENCE and ABSENCE OF RIBOFLAVIN and EFFECTS OF ASCORBATE ON THE PHOTOOXIDATION

Abstract— The self-sensitized photooxidation of quercetin was not suppressed by superoxide dismutase (SOD), but suppressed by ascorbate. During the suppression of quercetin photooxidation, ascorbate was oxidized. These results suggest the reduction of oxidized quercetin to quercetin by ascorbate. Quercetin photooxidation in the presence of both riboflavin and EDTA was suppressed by SOD by about 90%. The result suggests the participation of O₂^- in the photooxidation of quercetin. The participation of O₂^- in the quercetin oxidation was confirmed by a xanthine-xanthine oxidase system. Based on these results the physiological and pharmacological functions of quercetin are discussed.     

Effects of glutamate on dehydroascorbate uptake and its enhanced vulnerability to the peroxidation in cerebral cortical slices

Pro-oxidant properties of ascorbate have been studied with uses of brain tissues and neuronal cells. Here we address potential mechanism of ascorbate coupling with glutamate to generate oxidative stress, and the role which oxidized ascorbate (dehydroascorbate) transport plays in oxidative neuronal injury. Ascorbate in neurones can be depleted by adding glutamate in culture medium since endogenous ascorbate can be exchanged with glutamate, which enhances ascorbate/dehydroascorbate transport by depleting ascorbate in the neurons with the glutamate- heteroexchange. However, ascorbate is known readily being oxidized to dehydroascorbate in the medium. Glutamate enhanced the dehydroascorbate uptake by cells via a glucose transporter (GLUT) from extracellular region, and cytosolic dehydroascorbate enhanced lipid peroxide production and reduced glutathione (GSH) concentrations. Iso-ascorbate, the epimer of ascorbate was ineffective in generating the oxidative stress. These observations support the current concept that the high rates of dehydroascorbate transport via a GLUT after the release of ascorbate by glutamate leads to peroxidation, the role of glutamate on ascorbate/dehydroascorbate recycling being critical to induce neuronal death via an oxidative stress in the brain injury.     

A glass capillary microelectrode based on capillarity and its application to the detection of L-glutamate release from mouse brain slices.

A new glass capillary microelectrode for L-glutamate is described using pulled glass capillaries (tip size, approximately 12.5 microm) with a very small volume (approximately 2 microl) of inner solution containing glutamate oxidase (GluOx) and ascorbate oxidase. The operation of the electrode is based on capillary action that samples L-glutamate into the inner solution. The enzyme reaction by GluOx generates hydrogen peroxide that is detected at an Os-gel-HRP polymer modified Pt electrode in a three-electrode configuration. The amperometric response behavior of the electrode was characterized in terms of the capillarity, response time, sensitivity and selectivity for measurements of L-glutamate. The currents at 0 V vs. Ag/AgCl increased linearly with the L-glutamate concentration from 10 to 150 microM for in vitro and in situ calibrations. The response was highly selective to L-glutamate over ascorbate, dopamine, serotonin and other amino acids. The detection of L-glutamate in the extracellular fluids of different regions of mouse hippocampal slices under stimulation of KCl was demonstrated.     

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [(14)C]-ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [(14)C]-ascorbate uptake by GLUT following oxidation to [(14)C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.     

Studies on the kinetics of ascorbate oxidation at a ruthenium oxide hexacyanoferrate modified electrode towards the detection at microenvironments

The electrocatalytic oxidation of ascorbate on a ruthenium oxide hexacyanoferrate (RuOHCF) glassy carbon (GC) modified electrode was investigated at pH 6.9 by using rotating disc electrode (RDE) voltammetry. The influence of the systematic variation of rotation rate, film thickness, ascorbate concentration and the electrode potential indicated that the rate of cross-chemical reaction between Ru(III) centres immobilized into the film and ascorbate controls the overall process. The kinetic regime may be classified as a Sk″ mechanism and the second order rate constant for the surface electrocatalytic reaction was found to be 1.56 × 10⁻³ mol⁻¹ L¹ s⁻¹ cm. A carbon fibre microelectrode modified with the RuOHCF film was successfully used as an amperometric sensor to monitor the ascorbate diffusion in a simulated microenvironment experiment.     

Increased Sensitivity of Ascorbate Detection by Mediated Oxidation in Confined Electrochemical Cells

In this work, the EC’ mechanism involving ascorbate (AA⁻) and the oxidized form of ferrocenemethanol (FcMeOH) was explored as an analytical strategy to monitor AA⁻ at low concentration levels. The feasibility of this approach was investigated at different mass transport regimes utilizing macro- (glassy carbon) and ultramicroelectrodes (5 μm radius carbon disk). Cyclic voltammograms (CV) were recorded in acetate buffer solution (pH 3.7) using a glassy carbon electrode, and an expressive increase in the anodic peak current (and decrease in the cathodic peak) was noticed in the CV in a solution containing both FcMeOH and ascorbate, confirming the presence of an electrocatalytic process (EC’). The current increase was more pronounced when the reactants and products were confined in a thin solution layer, which was created by approaching an ultramicroelectrode close to an insulator surface. At optimized experimental conditions, a correlation between ascorbate concentration and the steady-state current measured at the ultramicroelectrode was established, allowing the development of an analytical method for ascorbate detection in the micromolar range. The proposed approach was used to quantify ascorbate in a commercial juice sample.     

Role of pyruvate and ascorbate production in regulation of antioxidant enzymes and membrane LPO levels in Fusarium Acuminatum

The role of pyruvate and ascorbate in the regulation of superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase enzymes; and, therefore, membrane lipid peroxidation (LPO) levels in Fusarium acuminatum was investigated in media containing either glycerin or glucose as a carbon source, depending on the incubation period, in the range of 5–25 g/L. Increasing SOD activity between d 9 and 16 of the incubation period showed a positive correlation with a significant increase in pyruvate production up to 15 g/L of glycerin and glucose. In addition, maximum ascorbate production was observed at 15 g/L of glycerin as 82.5 ± 2.1 and 20 g/L of glucose as 54±1.51, whereas CAT activity decreased with an increased concentration of both carbon sources. When compared with the LPO levels determined in media supplemented with glycerin and glucose, the minimum LPO level was 1.88±0.028 nmol of malondialdehyde/g wet wt at 15 g/L of glycerin on d 16, at which it was also observed to have a maximum pyruvate and ascorbate production and SOD, CAT, and GSH-Px activities of 75±1.42 μg/mL, 82.5±2.1 μg/mL, 32.5±0.634 μg/mL, 86.8±2.58 IU/mg, and 1.867 IU/mg, respectively. These results indicate that the biosynthesis of pyruvate and ascorbate may be involved in the regulation of antioxidant enzymes, depending on the glycerin and glucose concentrations, and also this defense network was effective in preventing membrane damage from oxidative stress.     

Simultaneous Detection of Copper,Lead and Zinc on Tin Film/Gold Nanoparticles/Gold Microelectrode by Square Wave Stripping Voltammetry

In this work, three heavy metals (Cu(II), Pb(II) and Zn(II)) in wide potential window were simultaneously detected on tin film/gold nanoparticles/gold microelectrode (Sn/GNPs/gold microelectrode) by the method of square wave stripping voltammetry. The Sn/GNPs/gold microelectrode was fabricated by in situ plating of a Sn film on a gold nanoparticles (GNPs) modified gold microelectrode. The influence of hydrogen overflow on stripping of Zn(II) on the gold microelectrode was reduced by modification of GNPs, which made the stripping potential of target metals shift positively. The interference of sulfhydryl groups was reduced and the selectivity of the microelectrode was improved due to the addition of Sn in the detection solution. After accumulation at ?1.4 V for 300 s in acetate buffer solution (0.1 mol L^?1, pH 4.5), the Sn/GNPs/gold microelectrode revealed a good linear behavior in the examined concentration ranges from 5 to 500 µg L^?1 for Cu(II) and Pb(II), and from 10 to 500 µg L^?1 for Zn(II), with a limit of detection of 2 µg L^?1 for Cu(II), 3 µg L^?1 for Pb(II) and 5 µg L^?1 for Zn(II) (S/N=3). When compared with a Sb/GNPs/gold microelectrode and a Bi/GNPs/gold microelectrode, the Sn/GNPs/gold microelectrode showed the best stripping performance to Cu(II), Pb(II) and Zn(II). As a new type of environment‐friendly electrode, the Sn/GNPs/gold microelectrode has potential applications for detection of heavy metals.     

Simultaneous determination of the binding of amantadine and its analogues to synthetic melanin by liquid chromatography after precolumn derivatization with dansyl chloride

For the determination of amantadine (1-ADA), 2-adamantanamine (2-ADA), memantine (MEM), and rimantadine (RIM) in melanin binding studies, the simultaneous determination of 1-ADA or 2-ADA, MEM, and RIM is investigated by high-performance liquid chromatographic assay with dansyl chloride as a fluorescent derivative reagent. Dansyl derivatives with fluorescent intensity are detected at an excitation wavelength of 370 nm and an emission wavelength of 506 nm. Retention times of 1-ADA, 2-ADA, MEM, and RIM derivatives are 12.2, 12.2, 15.2, and 16.6 min, respectively. The peak of 1-ADA derivative coelutes with the 2-ADA derivative. The limits of detection for 1-ADA, 2-ADA, MEM, and RIM are 0.014, 0.007, 0.012, and 0.020microM, respectively (signal-to-noise ratio of 3:1). In the intra- and interday assay, the range of standard deviation to the average of 1-ADA, 2-ADA, MEM, and RIM is 4.6-12.7%. Their recovery is also good. The ranking order for synthetic melanin binding among these compounds is RIM > MEM > 2-ADA = 1-ADA. The method is simple, sensitive, and reproducible for simultaneously measuring 1-ADA or 2-ADA, MEM, and RIM. Also, it is useful to investigate their binding kinetics to melanin.     

Superoxide Dismutase-Loaded PLGA Nanoparticles Protect Cultured Human Neurons Under Oxidative Stress

The objective of our study was to investigate the neuroprotective efficacy of superoxide dismutase (SOD), loaded in poly(D,L-lactide co-glycolide; PLGA) nanoparticles (NPs), in cultured human neurons challenged with hydrogen peroxide (H(2)O(2))-induced oxidative stress. We hypothesized that the protected and sustained intracellular delivery of SOD encapsulated in NPs would demonstrate better neuroprotection from oxidative stress than either SOD or pegylated SOD (PEG-SOD) in solution. SOD-NPs (approximately 81 +/- 4 nm in diameter, 0.9% w/w SOD loading) released the encapsulated SOD in an active form with 8.2% cumulative release during the first 24 h, followed by a slower release thereafter. The results demonstrated that PLGA-NPs are compatible with human neurons, and the neuroprotective effect of SOD-NPs is dose-dependent, with efficacy seen at >100 U SOD, and less significant effects at lower doses. Neither SOD (25-200 U) nor PEG-SOD (100 U) in solution demonstrated the neuroprotective effect under similar conditions. The neuroprotective effect of SOD-NPs was seen up to 6 h after H(2)O(2)-induced oxidative stress, but the effect diminished thereafter. Confocal microscopic studies demonstrated better intracellular neuronal uptake of the encapsulated model protein (fluorescein isothiocyanate-labeled BSA) than the protein in solution. Thus, the mechanism of efficacy of SOD-NPs appears to be due to the stability of the encapsulated enzyme and its better neuronal uptake after encapsulation.     

In vitro continuous amperometric monitoring of 5-hydroxytryptamine release from enterochromaffin cells of the guinea pig ileum

A diamond microelectrode was used to sensitively, reproducibly and stably record overflow of 5-hydroxytryptamine (5-HT, serotonin) from enterochromaffin cells (EC) of the intenstinal mucosal layer. 5-HT is an important neurotransmitter and paracrine signalling molecule in the gastrointestinal tract. The diamond microelectrode was formed by overcoating a sharpened 76 microm diameter Pt wire with a thin layer of conducting diamond. After insulation with polypropylene, the conically-shaped microelectrode had a diameter of about 10 microm at the tip and 80 microm at the cylindrical portion. The exposed length was 100-200 microm. Continuous amperometry with the microelectrode poised at a detection potential of 700 mV vs. Ag|AgCl was used to measure 5-HT overflow as an oxidation current. 5-HT overflow was elicited by both mechanical and electrical stimulation. Some minor electrode fouling, a common problem with the oxidative detection of 5-HT, was seen for diamond but the response stabilized enabling recording in vitro. Both 5-HT and the paracrine hormone, melatonin, were detected in the extracellular solution. The 5-HT oxidation current increased in the presence of the serotonin transporter (SERT) inhibitor, fluoxetine (1 microM), providing evidence that the oxidation current was associated with 5-HT.     

Enhancement of the sensitivity and selectivity of oxidation of H2O2 on platinum wire at low working potential by platinization and covering of heteropolypyrrole film for amperometric micro-biosensor construction

A microelectrode for glucose determination was constructed by immobilization of glucose oxidase (GOx) on a platinized platinum (Pt) by electrochemical polymerization of a solution containing GOx, pyrrole, and a substituted pyrrole, 4-(3-pyrrolyl)-4-oxobutyric acid. Due to platinization and covering with the polymerized heteropolypyrrole (hPPy) film, the electrode prepared showed high sensitivity to H₂O₂ at a low potential and significantly reduced the response to electroactive compounds, such as ascorbate, urate and 4-acetamidophenol. Working at 200 mV (vs. SCE) the electrode showed a linear response to glucose from 1.6 to 10 mM with a high sensitivity of 1 μA/mM, whereas the response to 1 mM ascorbate, urate, and 4-acetamidophenol was 0.53 μA, 18 nA and 4 nA, respectively, which was about 2.5%, 1.0% and 1.0% of that at a bare electrode. The stability of the electrode was tested at intervals of three or five days, and each test lasted about two hours. After 6 months examination, only 30% of its activity was lost.     

Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses

In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.     

Determination of diclofenac sodium in eagle's minimum essential medium with Earle's balanced salt solution

A validated method was developed for determination of diclofenac sodium, considered a model hydrophilic drug for in vitro permeation studies, in Eagle's Minimum Essential Medium (MEM) with Earle's balanced salt solution. Liquid chromatography was used to determine diclofenac sodium. This method was developed with a reversed-phase column Supercosil LC 18, DB 25 cm x 4.5 mm; the mobile phase was methanol with 3% (v/v) acetic acid-Milli-Q water (74 + 26), and detection was at 283 nm. The detection and quantitation limits were 2.41 x 10(-8) and 3.31 x 10(-5) microg/microL, respectively. The accuracy within-day (n = 3) and day-to-day (n = 7) was 98.83%; the mean variation coefficient for inter- (n = 7) and intraday precision (n = 3) was 12.20%, thus, not exceeding 15%. This method can be used as an analytical procedure for the determination of diclofenac sodium in MEM for in vitro permeation studies.     

酪氨酸铜催化抗坏血酸有氧氧化动力学研究

抗坏血酸(H_2A)是一个强还原剂,在人体内受微量过渡金属离子、其可溶性络合物及金属酶-抗坏血酸氧化酶的催化,经过两个单电子的转移,氧化成脱氢抗坏血酸(A).本文以Cu(Ⅱ)与酪氨酸组成的络合物为催化剂,测定抗坏血酸氧化过程的动力学参数及其可能存在的中间体,以便进一步了解H_2A在接近生理条件氧化时,电子转移的途径。实验部分本实验是在pH=7.44以纯氧饱和的磷酸缓冲溶液中进行。除Cu(Ⅱ)-酪氨酸按文献     

Bacteriorhodopsin photocycle kinetics analyzed by the maximum entropy method

A maximum entropy method (MEM) was developed for the study of the bacteriorhodopsin photocycle kinetics. The method can be applied directly to experimental kinetic absorption data without any assumption for the number of the intermediate states taking part in the photocycle. Though this method does not give a specific kinetics, its result is very useful for selection between possible photocycle kinetics. Using simulated data, it is shown that MEM gives correct results for the number of the intermediate states and the amplitude distributions around the characteristic lifetimes. Analyzing experimental absorption data at five different wavelengths, MEM gives seven or eight characteristic lifetimes, which means that at least so many distinct intermediate states exist during the photocycle. Many possible photocycle kinetic models were studied and compared with the MEM result. The best agreement was found with a branching photocycle model of eight intermediate states (K, L, M(1), M(2), M(3), M(4), N, O). The branching occurs at the L intermediate state (M(1) and M(2) being in one branch and M(3) and M(4) in the other branch), but at high pH it occurs already at the K state.     

Enhancement of the sensitivity and selectivity of oxidation of H2O2 on platinum wire at low working potential by platinization and covering of heteropolypyrrole film for amperometric micro-biosensor construction

A microelectrode for glucose determination was constructed by immobilization of glucose oxidase (GOx) on a platinized platinum (Pt) by electrochemical polymerization of a solution containing GOx, pyrrole, and a substituted pyrrole, 4-(3-pyrrolyl)-4-oxobutyric acid. Due to platinization and covering with the polymerized heteropolypyrrole (hPPy) film, the electrode prepared showed high sensitivity to H₂O₂ at a low potential and significantly reduced the response to electroactive compounds, such as ascorbate, urate and 4-acetamidophenol. Working at 200 mV (vs. SCE) the electrode showed a linear response to glucose from 1.6 to 10 mM with a high sensitivity of 1 μA/mM, whereas the response to 1 mM ascorbate, urate, and 4-acetamidophenol was 0.53 μA, 18 nA and 4 nA, respectively, which was about 2.5%, 1.0% and 1.0% of that at a bare electrode. The stability of the electrode was tested at intervals of three or five days, and each test lasted about two hours. After 6 months examination, only 30% of its activity was lost. Received: 16 March / Revised: 26 July 1998 / Accepted: 1 August 1998     

pH敏感纳米凝胶的制备及其在siRNA转染中的应用

通过分散聚合的方法,以改性了双键的葡聚糖(Dex-AA)作为交联剂,甲基丙烯酸二甲氨基乙酯(DMAEMA)作为单体,过硫酸铵(APS)和四甲基乙二胺(TEMED)分别作为引发剂和助引发剂,合成了不同交联度的、具有pH敏感内吞增强作用的葡聚糖纳米凝胶(DD-NGs),并测试了其复合siRNA进行转染的能力.实验结果表明,该纳米凝胶表面带有正电荷,具有较好的担载siRNA进入肿瘤细胞并沉默基因的能力,且具有pH响应粒径变化的性质.在pH=7.4的体液环境中,纳米凝胶与基因的复合物粒子较小;在肿瘤酸性(pH=6.8)条件下,纳米凝胶与基因的复合物粒子变大,显著地增强了肿瘤细胞对纳米凝胶与基因复合物的内吞.