Electrochemical investigations of the reaction mechanism and kinetics between NADH and riboflavin immobilised on amorphous zirconium phosphate

Electrochemical investigations of the reaction mechanism and kinetics between riboflavin immobilised on zirconium phosphate (ZPRib) in carbon paste and NADH showed results yielding reliable information about aspects on the mechanism of the electron transfer reaction between the flavin and NADH. The formal potential (E°′) of the adsorbed riboflavin was −220 mV versus SCE at pH 7.0. A shift about 250 mV towards a more positive potential compared with its value in solution was assigned to the interaction between the basic nitrogen of riboflavin and the acid groups of ZP. The invariance of the E°′ with the pH of the contacting solution and the effect of different buffer constituents were attributed to the protection effect of ZP over the riboflavin. The electrocatalytic oxidation of NADH at the electrode was investigated using cyclic voltammetry and rotating disk electrode methodology using a potential of −50 mV versus SCE. The heterogeneous electron transfer rate constant, k _obs, was 816 M⁻¹ s⁻¹ and the Michaelis-Menten constant, K _M, was 1.8 mM (confirming a charge transfer complex intermediate in the reaction) for an electrode with a riboflavin coverage of 6.8 × 10⁻¹⁰ mol cm⁻². This drastic increase in the reaction rate between NADH and the immobilised riboflavin was assigned to the shift of the E°′. A surprising effect with addition of calcium or magnesium ion to the solution was also observed. The E°′ was shifted to −150 mV versus SCE and the reaction rate for NADH oxidation increased drastically. Received: 22 February 1999 / Accepted: 10 March 1999     

溴酚蓝修饰玻碳电极的电化学研究

溴酚蓝(BPB)常用作酸碱指示剂和光度分析的显色剂,颜色深,对生物大分子亲和力强,近年来,对它的应用研究主要集中在生物大分子,特别是蛋白质和酶的分析测定上,     

Electrochemical Polymerization of Azure Blue H and Its Electro-catalytic Activity toward NADH Oxidation

Poly (azure blue II) (PABII) thin film modified electrode was successfully assembled on the surface of a glassy carbon electrode by means of electrochemical polymerization, which was carried out with cyclic voltammetric sweeping in the potential range of ‐ 0.6 to + 1.3 V (vs. SCE) in Britton‐Robinson buffer solution (pH = 9.8) containing 1.25 ± 10–⁴ mol/L azure blue II. The effect of pH on the polymerization process of azure blue II and the electrochemical characteristics of the polymer‐modified electrodes were studied in detail. The experimental results indicated that the electropolymerization of azure blue II could take place in basic or neutral media. The cyclic voltammograms of poly (azure blue II) thin film modified electrode showed the presence of two couples of redox peaks. The film modified electrode exhibited potent and persistent electrocatalysis for oxidation of dihydronicotiamide adenine dinucleotide (NADH) in phosphate buffer media with a diminution of the overpotential of about 410 mV and an increase in peak current. The presence of some divalent cations in an electrolyte can greatly enhance the electrocatalytic current for oxidation of NADH. The electrocatalytic current increased linearly with NADH concentration from 1.0 ± 10–⁵ to 8.0 ± 10–³ mol/L in the presence of 4.0 ± 10–² mol/L Mg²⁺ cation. The detection limit (3s_b1/S) was 5.0 ± 10–⁶ mol/L, and the relative standard deviation of determination results was 4.2% for six successive determinations of 5.0 ± 10–⁴ mol/L NADH in the presence of Mg²⁺ cation.     

Comparison of the Electrochemical Reactivity of Electrodes Modified with Carbon Nanotubes from Different Sources

The electrochemical activity of five different commercial carbon nanotubes (CNT), prepared by the ARC discharge and chemical vapor deposition (CVD) methods, has been assessed and compared. The various multi‐walled CNT were immobilized onto a glassy carbon electrode using three different dispersing agents (Nafion, concentrated nitric acid and dimethylformamide (DMF)) and their voltammetric response to ferricyanide, NADH and hydrogen peroxide examined. SEM was used to characterize the surface morphology. The corresponding cyclic voltammetry and amperometric data showed that the electrocatalytic activity, the background current and the electroanalytical performance are strongly depended on the preparation of the CNT and on the dispersing agent used. The most favorable amperometric detection of NADH and hydrogen peroxide is observed at the NanoLab CVD‐produced CNT in connection to a DMF‐surface dispersion. ARC‐produced CNT display a smaller capacitance, particularly in connection to the DMF dispersion. Such differences in the electrochemical reactivity are attributed to the different surface chemistries (primarily defect densities) of the corresponding CNT layers, associated with the different production and dispersion protocols.     

Detection of NADH and Ethanol at Titanium Containing MCM‐41 with Low Overpotential

Titanium‐containing MCM‐41 (Ti‐MCM‐41) modified glassy carbon electrode (GCE) can exhibit an excellent electrocatalytic activity towards the oxidation of β‐Nicotinamide adenine dinucleotide (NADH). A dramatic decrease in the overvoltage of NADH oxidation reaction is observed at 0.28 V vs. SCE. The application in the amperometric biosensing of ethanol using alcohol dehydrogenase enzyme (ADH) also has been demonstrated with this material. The proposed sensor shows a highly sensitivity, an acceptable reproducibility and a good stability. The linear range of ethanol is 25–1000 μM and the detection limit is 8.0 μM. Ti‐MCM‐41 modified electrode not only can be used to detect the concentration of NADH in biochemical reaction, but also as the potential matrix for the construction of dehydrogenases sensor.     

Low Potential Detection of NADH at Titanium‐Containing MCM‐41 Modified Glassy Carbon Electrode

Titanium‐containing MCM‐41 (Ti‐MCM‐41) modified glassy carbon electrode (GCE) can exhibit an excellent electrocatalytic activity towards the oxidation of β‐Nicotinamide adenine dinucleotide (NADH). A dramatic decrease in the over‐voltage of NADH oxidation reaction is observed at 0.28 V (vs. SCE). The modified electrode is found to be stable and reproducible. The electrode shows a linear response for a wide range of 10–1200 μM NADH and the detection limit is 8.0 μM. Ti‐MCM‐41 mesoporous molecular sieves provide an efficient matrix for development of NADH biosensors and the prepared electrode not only can be used to detect the concentration of NADH in biochemical reaction, but also as the potential matrix of the construction of dehydrogenases biosensor.     

Study of the Nano-Size Silica Sol–Gel Film as the Matrix of Chemically Modified Electrode

This paper reports a new method to make the sol–gel film from silicate. It provided more profitable conditions than hydrolysis of alkoxyl silane to prepare the high-quality nanometers thin film. The thin film could be applied as the matrix to directly modify the molecules of methylene blue on electrode surface and kept their electrochemical activity excellently. It exhibited the remarkable catalytic activity for the oxidation of NADH or reduction of hemoglobin while severally embedding them in the sol–gel film together with methylene blue.     

Electrochemical study of Meldola's blue,methylene blue and toluidine blue immobilized on a SiO<Subscript>2</Subscript>/Sb<Subscript>2</Subscript>O3 binary oxide matrix obtained by the sol-gel processing method

SiO₂/Sb₂O₃ (SiSb), having a specific surface area, S _BET, of 788 m² g⁻¹, an average pore diameter of 1.9 nm and 4.7 wt% of Sb, was prepared by the sol-gel processing method. Meldola's blue (MeB), methylene blue (MB) and toluidine blue (TB) were immobilized on SiSb by an ion exchange reaction. The amounts of the dyes bonded to the substrate surface were 12.49, 14.26 and 22.78 μmol g⁻¹ for MeB, MB and TB, respectively. These materials were used to modify carbon paste electrodes. The midpoint potentials (E _m) of the immobilized dyes were −0.059, −0.17 and −0.18 V vs. SCE for SiSb/MeB, SiSb/MB and SiSb/TB modified carbon paste electrodes, respectively. A solution pH between 3 and 7 practically did not affect the midpoint potential of the immobilized dyes. The electrodes presented reproducible responses and were chemically stable under various oxidation-reduction cycles. Among the immobilized dyes, MeB was the most efficient to mediate the electron transfer for NADH oxidation in aqueous solution at pH 7. In this case, amperometric detection of NADH at an applied potential of 0 mV vs. SCE gives linear responses over the concentration range of 0.1–0.6 mmol L⁻¹, with a detection limit of 7 μmol L⁻¹.     

Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly(vinyl sulphonate) films for use in a lactate biosensor

The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD⁺ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.