Electrochemical and catalytic investigation of carbon paste modified with Toluidine Blue O covalently immobilised on silica gel

Toluidine Blue O (TBO) was covalently bound on silica gel and mixed with graphite powder and paraffin oil to produce modified carbon paste electrodes. The formal potential (E°′) of the covalently bound TBO was found to be −100 mV versus Ag|AgCl (KCl sat.) at pH 7.0 and the E°′ varied less than anticipated for a 2-electron-proton type mediator with pH. The bound TBO was found to act as an efficient electron acceptor for NADH as well as electron donor for oxidised horseradish peroxidase (HRP). The kinetics and the mechanism of the reaction between NADH and TBO were investigated with cyclic voltammetry and using a rotating disc electrode. Further experiments were done in the flow injection mode injecting different concentrations of NADH. Similar studies were done in the presence of hydrogen peroxide when HRP was adsorbed onto the TBO modified silica gel carbon paste electrodes.     

NADH Oxidation Catalyzed by Electropolymerized Azines on Carbon Nanotube Modified Electrodes

Electropolymerizing azines on a carbon nanotube (CNT) modified electrode yields a high‐surface area interface with excellent electrocatalytic activity towards NADH oxidation. Electrodeposition of poly(methylene green) (PMG) and poly(toluidine blue) (PTBO) on the carboxylated CNT‐modified electrodes was achieved by cyclic voltammetry. The PMG‐CNT interface demonstrates 5.0 mA cm^?2 current density for NADH oxidation at 50 mV vs. Ag|AgCl in 20 mM NADH solution. The kinetics of NADH electrocatalysis were analyzed using a quantitative mass‐transport‐corrected model with NADH bulk concentration and applied potential as independent variables. This high‐rate poly(azine)‐CNT interface is potentially applicable to high‐performance bioconversion, bioenergy and biosensors involving NADH‐dependent dehydrogenases.     

Improvement of direct bioelectrocatalysis by cellobiose dehydrogenase on screen printed graphite electrodes using polyaniline modification

Modification of graphite based screen printed electrodes (SPEs) by electrosynthesised polyaniline (PANI) has been applied to improve the electron exchange between cellobiose dehydrogenase (CDH, EC 1.1.99.18) from the ascomycete Myriococcum thermophilum and the surface of the SPE. The redox intermediate layer of the conducting polymer promotes the bioelectrocatalysis providing a higher current for lactose oxidation at a lower potential compared to CDH immobilised on a plain SPE. The current of the SPE|PANI|CDH electrode was more than 5 times higher as compared to that of a SPE|CDH electrode at a potential of 0 mV vs. Ag|AgCl. When comparing the response obtained through direct electron transfer with that obtained through mediated electron transfer, it was clearly observed that the improved current of the SPE|PANI|CDH electrode is due to the specific role of PANI, rather than caused by a rise of enzyme loading. The operational stability of the enzyme electrode based on PANI modified SPE was 5 times higher compared with that based on plain SPE.     

Myoglobin/sol-gel film modified electrode: direct electrochemistry and electrochemical catalysis

Direct electrochemical and electrocatalytic behavior of myoglobin (Mb) immobilized on carbon paste electrode (CPE) by a silica sol-gel film derived from tetraethyl orthosilicate was investigated for the first time. Mb/sol-gel film modified electrodes show a pair of well-defined and nearly reversible cyclic voltammetric peaks for the Mb Fe(III)/Fe(II) redox couple at about -0.298 V (vs Ag/AgCl) in a pH 7.0 phosphate buffer solution. The formal potential of the Mb heme Fe(III)/Fe(II) couple shifted linearly with pH with a slope of 52.4 mV/pH, denoting that an electron transfer accompanies single-proton transportation. An FTIR and UV-vis spectroscopy study confirms that the secondary structure of Mb immobilized on an electrode by a sol-gel film still maintains the original arrangement. The immobilized Mb displays the features of a peroxidase and acts in an electrocatalytic manner in the reduction of oxygen, trichloroacetic acid (TCA), and nitrite. In comparison to other electrodes, the chemically modified electrodes used in this study for direct electrochemistry and electrocatalysis of Mb are easy to fabricate and fairly inexpensive. Consequently, the Mb/sol-gel film modified electrode provides a convenient way to perform electrochemical research on this kind of protein. It also has potential use in the fabrication of bioreactors and third-generation biosensors.     

Direct Electrochemical Oxidation of DNA on Polycrystalline Gold Electrodes

Electrochemical CV and SWV studies were performed with double stranded DNA from salmon testes (dsDNA) and single stranded DNAs, containing 25 nucleotides (ssDNA) directly adsorbed at polycrystalline Au electrodes. A distinct oxidation peak at +730 mV (SWV, scan rate 0.248 V s^?1) or at +730 – +780 mV (CV, scan rate from 0.3 to 1 V s^?1) was obtained with DNA‐modified Au electrodes after a time‐dependent prepolarization step at a positive potential value, i.e., at +500 mV (vs. Ag|AgCl), performed with the DNA‐modified Au electrodes dipped in a blank buffer solution. No electrochemical activity was detected when ssDNA, containing no guanines, was used for adsorptive modification of the Au electrodes. Electrochemical impedance measurements registered a possible reorganization of the adsorbed DNA layer in the course of the prepolarization, accompanied by decreasing in‐phase impedance. The results enable us to relate the oxidation process observed at the DNA‐modified Au electrodes with the oxidation of guanine residues in DNA.     

Investigation of the Effect of Different Glassy Carbon Materials on the Performance of Prussian Blue Based Sensors for Hydrogen Peroxide

Three different kinds of glassy carbon (GC‐R, GC‐K, GC‐G) were equally pretreated, further modified with electrochemically deposited Prussian Blue and used as sensors for hydrogen peroxide at an applied potential of ?50 mV (vs. Ag|AgCl). Their performance was evaluated with respect to the following parameters: the coverage and electrochemistry of the electrodeposited Prussian Blue, the sensitivity and the lower limit of detection for hydrogen peroxide, and the operational stability of the sensors. GC‐R showed the best behavior concerning the surface coverage and the operational stability of the electrodeposited Prussian Blue. For this electrode the sensitivity for hydrogen peroxide (10 μM) was 0.25 A/M cm² and the detection limit was 0.1 μM. Scanning electron microscopy was used to study the surfaces of the three electrodes before and after the electrodeposition of Prussian Blue and to search for the reason for the three different behaviors between the different glassy carbon materials. The Prussian Blue modified GC‐R was also used for the construction of a glucose biosensor based on immobilizing glucose oxidase in Nafion membranes on top of electrodeposited Prussian Blue layer. The operational stability of the glucose biosensors was studied in the flow injection mode at an applied potential of ?50 mV (vs. Ag|AgCl) and alternatively injecting standard solutions of hydrogen peroxide (10 μM) and glucose (1 mM) for 3 h. For the GC‐R based biosensor a 2.8% decrease of the initial glucose response was observed.     

Direct electron transfer of PQQ-glucose dehydrogenase at modified carbon nanotubes electrodes

In order to establish efficient enzyme-electrode-contacts for the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) different immobilisation strategies are investigated. Multi-walled carbon nanotubes (MWCNT) on gold electrodes are modified by chemical treatment and by (poly)-aniline derivatives. The electropolymerisation of methoxy-m-anilinesulfonic acid and m-aminobenzoic acid on the MWCNTs allows the covalent coupling of the PQQ-GDH. Such a poly-[ASA-ABA]/MWCNT/Au electrode can achieve current densities of up to 500 μA/cm² at a potential of 100 mV vs. Ag/AgCl. Furthermore investigations with small amounts of free PQQ indicate direct electron transfer between enzyme and electrode.     

Direct Electrochemistry of Glucose Oxidase at Carbon Nanotube‐gold Colloid Modified Electrode with Poly(diallyldimethylammonium chloride) Coating

Glucose oxidase showed direct electrochemical transfer at glassy carbon electrodes immobilized with carbon nanotube‐gold colloid (CNT‐Au) composites with poly(diallydimethylammonium chloride) (PDDA) coatings. The modified electrode (GC/CNT/Au/PDDA‐GOD) was employed for the amperometric determination of glucose. Under optimal conditions, the biosensor displayed linear response to glucose from 0.5 to 5 mM with a sensitivity of 2.50 mA M^?1 at an applied potential of ?0.3 V (vs. Ag|AgCl reference).     

Sensing the Lactic Acid in Probiotic Yogurts Using an L-Lactate Biosensor Coupled with a Microdialysis Fiber Inserted in a Flow Analysis System

An amperometric biosensor for the determination of L-lactic acid in probiotic yogurts has been assembled using L-lactate dehydrogenase (EC 1.1.1.27, LDH) entrapped in 1% (v/v) neutralized Nafion® solution deposited on Variamine blue redox mediator modified screen-printed electrodes. The Variamine blue was previously covalently linked to oxidized single-walled carbon nanotubes and used for modifying screen-printed electrodes. The electrochemical cell, containing the L-lactate biosensor operating at an applied working potential of +200 mV vs. Ag|AgCl, was coupled with a microdialysis fiber and connected with a flow system, thus obtaining a microdialysis based sampling experimental set-up. Various analytical parameters, such as the cofactor concentration (2 mM, NAD⁺), the flow rate (10.5 μL/min), the applied working potential (+200 mV vs. Ag|AgCl), the working buffer (50 mM phosphate buffer +0.1 M KCl), and pH (7.5), were optimized in batch amperometric experiments. The dynamic linear working range was comprised between 2·10^?4 and 1·10^?3 M. The proposed biosensor was challenged with real samples of yogurt, properly diluted in working buffer, and the performances of the L-lactate biosensor were compared with a commercially available kit for the determination of L-lactic acid in foodstuffs from R-Biopharm GmbH, Germany, showing a good agreement.     

Carbon Ceramic Electrodes Modified with Laccase from Trametes hirsuta: Fabrication,Characterization and Their Use for Phenolic Compounds Detection

Fungal laccase (Lc) from the basidiomycete Trametes hirsuta was immobilized on top of a carbon ceramic electrode using physical absorption. Direct, unmediated heterogeneous electron transfer between Lc and the carbon ceramic electrode (CCE) under aerobic conditions was shown. The bioelectrocatalytic reduction of oxygen on Lc‐CCE started at about 430 mV vs. Ag|AgCl|KCl_sat at pH 3.5 and moved with about 57 mV in the cathodic region per pH unit. The Lc‐modified CCE was then used as a biosensing detection element in a single line flow injection system for the amperometric determination of a variety of phenolic substrates of the enzyme. The experimental conditions were studied and optimized for catechol serving as a model compound. Statistical aspects were applied and the sensor characteristics and Michaelis‐Menten constants of the investigated phenolic compounds were calculated and compared with those obtained for solid graphite electrodes modified with Trametes hirsuta laccase. The results showed that the CCE based biosensor in comparison with the solid graphite based biosensor offers a lower detection limit, a wider linear dynamic range, and excellent operational stability with no sensor passivation, indicating that the sol–gel lattice improves the electrochemical behavior of the biosensor.     

Silica‐Polyaniline Based Bienzyme Cholesterol Biosensor: Fabrication and Characterization

In the present investigation, silica‐polyaniline based bienzyme cholesterol biosensor is fabricated through a simple one‐step electrochemical method. The one‐step fabrication process involves electrochemical polymerization of N[3‐(trimethoxysilyl)propyl]aniline to result poly(N[3‐(trimethoxysilyl)propyl]aniline) (PTMSPA) and simultaneous immobilization of two enzymes, horseradish peroxidase (HRP) and cholesterol oxidase (ChOx) into PTMSPA matrix. The modified electrode is designated as PTMSPA‐HRP/ChOx‐ME. PTMSPA facilitates direct electron transfer between the electrode surface and the active redox centers of HRP. This enables the operation of a biosensor at a low working potential of about ?150 mV (vs. Ag/AgCl) for the detection of hydrogen peroxide. The PTMSPA‐HRP/ChOx‐ME demonstrates excellent analytical performance for the detection of cholesterol between 1 and 25 mM with high sensitivity and selectivity. PTMSPA possesses features suited for the fabrication of third‐generation biosensors.     

Study of the Electrochemical Behavior of Disperse Blue 1‐Modified Graphite Electrodes. Application to the Flow Determination of NADH

The preparation and the electrochemical study of Disperse Blue 1‐chemically modified electrodes (DB1‐CME), as well as their efficiency for the electrocatalytic oxidation of NADH is described. The proposed mediator was immobilized by physical adsorption onto graphite electrodes. The electrochemical behavior of DB1‐CME was studied with cyclic voltammetry. The electrochemical redox reaction of DB1 was found to be reversible, revealing two well‐shaped pair of peaks with formal potentials 152 and ?42 mV, respectively, (vs. Ag/AgCl/3M KCl) at pH 6.5. The current I_p has a linear relationship with the scan rate up to 800 mV s^?1, which is indicative for a fast electron transfer kinetics. The dissociation constants of the immobilized DB1 redox couple were calculated pK₁=4 and pK₂=5. The electrochemical rate constants of the immobilized DB1 were calculated k₁°=18 s^?1 and k₂°=23 s^?1 (Γ=2.36 nmol cm^?2). The modified electrodes were mounted in a flow injection manifold, poised at +150 mV (vs. Ag/AgCl/3M KCl) and a catalytic current due to the oxidation of NADH was measured. The reproducibility was 1.4% RSD (n=11 for 30 μM NADH) The behavior of the sensor towards different reducing compounds was investigated. The sensor exhibited good operational and storage stability.     

A cobalt(II) phthalocyanine with indole substituents: formation,characterization and electrocatalytic studies

Herein, we report the formation of a new cobalt(II) phthalocyanine (CoPc) containing peripheral tetra-substituted indole (CoPc-ind, 2) moieties. The derivatized phthalonitrile, 4-(indole-4-oxy)phthalonitrile (1) as well its corresponding metal complex was characterized by NMR (for 1), IR– and UV–Vis spectroscopy as well as TOF mass spectrometry and elemental analysis (for 2). The electrochemical properties of the N₄-macrocyclic metal complex were investigated using cyclic- and square-wave voltammetry as well as corroborated by UV–Vis spectroelectrochemistry. The CoPc was electrodeposited onto the surface of a Pt working electrode followed by the immobilization of multiwalled carbon nanotubes (MWCNTs) onto the modified working electrode surface. The electrocatalytic activity of the resultant modified electrode toward dopamine revealed a lower ΔE value of 80?mV versus Ag|AgCl for the modified (2-MWCNTs) Pt electrode compared to the bare Pt electrode (ΔE?=?280?mV vs. Ag|AgCl). The diffusion- and convection-controlled electron-transfer kinetics of the chemically modified electrode were evaluated by chronoamperometry and rotating disk electrode techniques. Electrochemical impedance spectroscopic studies revealed that the 2-MWCNTs Pt electrode had a lower charge-transfer resistance and a higher apparent electron-transfer rate constant.     

Rapid and efficient enzyme encapsulation in a dendrimer silica nanocomposite

We report the entrapment of horseradish peroxidase and quantitative encapsulation of glucose oxidase within silica nanoparticles by utilizing an amine-terminated dendritic template. Our improved strategy employs a water-soluble biomimetic template which is able to catalyze the condensation of Si(OH)(4) to silica nanoparticles while trapping an enzyme inside the mesoporous material. Kinetic analysis shows enzyme functionality to be mostly unchanged. Also, the role of pI and ionic strength within the encapsulation environment was found to strongly influence encapsulation. These results suggest that the electrostatic manipulation of a strong supramolecular silica-precipitating complex of enzyme and dendrimer has the potential of adding a vast array of chemical and biological activity to hybrid materials. [image: see text] Enzyme immobilization within a silica nanocomposite.     

Fructose/dioxygen biofuel cell based on direct electron transfer-type bioelectrocatalysis

One-compartment biofuel cells without separators have been constructed, in which d-fructose dehydrogenase (FDH) from Gluconobacter sp. and laccase from Trametes sp. (TsLAC) work as catalysts of direct electron transfer (DET)-type bioelectrocatalysis in the two-electron oxidation of d-fructose and four-electron reduction of dioxygen as fuels, respectively. FDH adsorbs strongly and stably on Ketjen black (KB) particles that have been modified on carbon papers (CP) and produces the catalytic current with the maximum density of about 4 mA cm(-2) without mediators at pH 5. The catalytic wave of the d-fructose oxidation is controlled by the enzyme kinetics. The location and the shape of the catalytic waves suggest strongly that the electron is directly transferred to the KB particles from the heme c site in FDH, of which the formal potential has been determined to be 39 mV vs. Ag|AgCl|sat. KCl. Electrochemistry of three kinds of multi-copper oxidases has also been investigated and TsLAC has been selected as the best one of the DET-type bioelectrocatalyst for the four-electron reduction of dioxygen in view of the thermodynamics and kinetics at pH 5. In the DET-type bioelectrocatalysis, the electron from electrodes seems to be transferred to the type I copper site of multi-copper oxidases. TsLAC adsorbed on carbon aerogel (CG) particles with an average pore size of 22 nm, that have been modified on CP electrodes, produces the catalytic reduction current of dioxygen with a density of about 4 mA cm(-2), which is governed by the mass transfer of the dissolved dioxygen. The FDH-adsorbed KB-modified CP electrodes and the TsLAC-adsorbed CG-modified CP electrodes have been combined to construct one-compartment biofuel cells without separators. The open-circuit voltage was 790 mV. The maximum current density of 2.8 mA cm(-2) and the maximum power density of 850 microW cm(-2) have been achieved at 410 mV of the cell voltage under stirring.     

Air diffusion biocathode with CueO as electrocatalyst adsorbed on carbon particle-modified electrodes

The current density of biofuel cells which use dissolved O₂ as electron acceptor is limited by O₂ supply to the electrode surface due to the low solubility and small diffusion coefficient of O₂ in the electrolyte solution. In order to increase the current density, we constructed an air diffusion biocathode which uses O₂ directly from the air. As cathodic biocatalyst, we utilized CueO from Escherichia coli, which belongs to the family of multi-copper oxidases. O₂ reduction was catalyzed by CueO adsorbed on Ketjen black-modified carbon paper electrodes. The hydrophobic electrode surface was obtained by optimizing the weight ratio of polytetrafluoroethylene binder to Ketjen black. The current density of O₂ reduction reached values as high as 20 mA cm^− 2 at 0 V vs. Ag|AgCl, KCl(sat.) in a citrate buffer (1.0 M, pH 5.0, 25 °C).     

Chemical Cross-Linking of a Redox Mediator Thionin for Electrocatalytic Oxidation of Reduced β-Nicotinamide Adenine Dinucleotide

Abstract

Thionin, a redox mediator that has been used to study the electrochemical behavior of reduced β-nicotinamide adenine dinucleotide (NADH), was chemically cross-linked on the surface of a spectroscopic graphite electrode by using a triisocyanate cross-linking agent. The electrodes modified in this manner had a purple film with an additional reversible redox couple a t E° of +73 mV vs. Ag/AgCl compared to uncross-linked thionin. The thionin modified electrode mediated oxidation of NADH with response to NADH between 7.0 × 10–7 to 1.8 × 10–3 M, a sensitivity of 113 pAJcmWmM, and a detection limit of 0.5 μM.     

碳糊电极上无机膜固载血红蛋白的直接电化学

报道了用硅溶胶-凝胶(Sol-gel)膜将血红蛋白(Hb)固载于碳糊电极上的直接电化学行为.研究结果表明,Hb-Sol-gel修饰的碳糊电极在pH=7.0的缓冲溶液中于-0.275V(vs.Ag/AgCl)处有一对可逆的循环伏安氧化-还原峰,为Hb血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对的特征峰.HbFe(Ⅲ)/Fe(Ⅱ)电对的式量电位在pH5.0～11.0范围内与溶液pH值呈线性关系,表明Hb的电化学还原很可能是一个质子伴随着一个电子的电极过程.FTIR光谱证实,Sol-gel膜对Hb的固载没有破坏其天然结构.Hb-Sol-gel修饰的碳糊电极能够催化还原H₂O₂,可望将其用于制作第三代生物传感器.     

Characterization of graphite electrodes modified with laccase from Trametes versicolor and their use for bioelectrochemical monitoring of phenolic compounds in flow injection analysis

Spectrographic graphite electrodes were modified through adsorption with laccase from Trametes versicolor. The laccase-modified graphite electrode was used as the working electrode in an amperometric flow-through cell for monitoring phenolic compounds in a single line flow injection system. The experimental conditions for bioelectrochemical determination of catechol were studied and optimized. The relative standard deviation of the biosensor for catechol (10 μM, n=12) was 1.0% and the reproducibility for six laccase-modified graphite electrodes, prepared and used different days was about 11%. The optimal conditions for the biosensor operation were: 0.1 M citrate buffer solution ( at pH 5.0), flow rate of 0.51 ml min⁻¹ and a working potential of −50 mV versus Ag|AgCl. At these conditions the responses of the biosensor for various phenolic compounds were recorded and the sensor characteristics were calculated and compared with those known for biosensors based on laccase from Coriolus hirsutus, cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium and horseradish peroxidase (HRP).     

Enhanced NADH Oxidation Using Polytyramine/Carbon Nanotube Modified Electrodes for Ethanol Biosensing

Polytyramine (PT) has been electro‐deposited onto multi‐walled carbon nanotube (MWCNT) modified glassy carbon (GC) electrodes via oxidation of tyramine in 0.1 M H₃PO₄ by cycling the potential over the range of −400 mV to 1300 mV (versus Ag/AgCl). The reactivity of the resulting chemically‐modified electrodes was characterized using cyclic voltammetry in the presence and absence of reduced nicotinamide adenine dinucleotide (NADH). The modified electrodes displayed electrochemical activity due to the formation of quinone species and were catalytically active towards NADH oxidation by lowering the oxidation peak potential by 170 mV compared to the value of the MWCNT modified electrode with a peak potential of 180±10 mV (versus Ag/AgCl). The MWCNT/PT surface was further characterized using SEM and XPS methods, which indicated that a thin polymeric film had been formed on the electrode surface. The present work demonstrates the advantage of using PT as a platform that combines both the immobilization of alcohol dehydrogenase (ADH) and the mediation of NADH oxidation at a low overpotential essential to the design of high performance ethanol biosensors, all within an easily electropolymerizable film. The resulting biosensor displayed an ethanol sensitivity of 4.28±0.06 μA mM⁻¹ cm⁻², a linear range between 0.1 mM and 0.5 mM and a detection limit of 10 μM.