Attachment of gold nanoparticles to glassy carbon electrode and its application for the direct electrochemistry and electrocatalytic behavior of hemoglobin

Gold nanoparticles have been attached onto glassy carbon electrode surface through sulfhydryl-terminated monolayer and characterized by X-ray photoelectron spectroscopy, atomic force microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The gold nanoparticles-attached glassy carbon electrodes have been applied to the immobilization/adsorption of hemoglobin, with a monolayer surface coverage of about 2.1 x 10(-10) mol cm(-2), and consequently obtained the direct electrochemistry of hemoglobin. Gold nanoparticles, acting as a bridge of electron transfer, can greatly promote the direct electron transfer between hemoglobin and the modified glassy carbon electrode without the aid of any electron mediator. In phosphate buffer solution with pH 6.8, hemoglobin shows a pair of well-defined redox waves with formal potential (E0') of about -0.085 V (versus Ag/AgCl/saturated KCl). The immobilized hemoglobin maintained its biological activity, showing a surface controlled electrode process with the apparent heterogeneous electron transfer rate constant (ks) of 1.05 s(-1) and charge-transfer coefficient (a) of 0.46, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide. A potential application of the hemoglobin-immobilized gold nanoparticles modified glassy carbon electrode as a biosensor to monitor hydrogen peroxide has been investigated. The steady-state current response increases linearly with hydrogen peroxide concentration from 2.0 x 10(-6) to 2.4 x 10(-4) M. The detection limit (3sigma) for hydrogen peroxide is 9.1 x 10(-7) M.     

Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition

Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K'(M)). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. (c) 1992 John Wiley & Sons, Inc.     

Direct electrochemistry and electrocatalysis of myoglobin on redox-active self-assembling monolayers derived from nitroaniline modified electrode

The adsorption processes and electrochemical behavior of 4-nitroaniline (4-NA) and 2-nitroaniline (2-NA) adsorbed onto glassy carbon electrodes (GCE) have been investigated in aqueous 0.1M nitric acid (HNO(3)) electrolyte solutions using cyclic voltammetry (CV). Nitroaniline adsorbs onto GCE surfaces and upon potential cycling past -0.55 V is transformed into the arylhydroxylamine (ArHA), which exhibits a well-behaved pH dependent redox couple centered at 0.32 V (pH 1.5). This modified electrode can be readily used as an immobilization matrix to entrap proteins and enzymes. In our studies, myoglobin (Mb) was chosen as a model protein for investigation. A pair of well-defined reversible redox peaks for Mb(Fe(III)-Fe(II)) was obtained at the Mb/arylhydroxylamine modified glassy carbon electrode (Mb/HAGCE) by direct electron transfer between the protein and the GCE. The formal potential (E(0')), the surface coverage (Gamma) and the electron transfer rate constant (k(s)) were calculated as -0.317 V, 4.15+/-0.5 x 10(-11)mol/cm(2) and 51+/-5s(-1), respectively. Dramatically enhanced biocatalytic activity was exemplified at the Mb/HAGCE for the reduction of hydrogen peroxide (H(2)O(2)), trichloroacetic acid (TCA) and oxygen (O(2)). The Mb/ArHA film was also characterized by UV-vis spectra, scanning electron microscope (SEM) indicating excellent stability and good biocompatibility for protein in the film. The applicability of the method to the determination of H(2)O(2) ( approximately 3%) in a commercial antiseptic solution and soft-contact lenses cleaning solutions were demonstrated. This new Mb/HAGCE exhibited rapid electrochemical response (with in 2s) with good stability in physiological condition.     

Electrochemiluminescence of luminol at the titanate nanotubes modified glassy carbon electrode

A new strategy for the construction of a sensitive and stable electrochemiluminescent platform based on titanate nanotubes (TNTs) and Nafion composite modified electrode for luminol is described, TNTs contained composite modified electrodes that showed some photocatalytic activity toward luminol electrochemiluminescence emission, and thus could dramatically enhance luminol light emission. This extremely sensitive and stable platform allowed a decrease of the experiment electrochemiluminescence luminol reagent. In addition, in luminol solution at low concentrations, we compared the capabilities of a bare glassy carbon electrode with the TNT composite modified electrode for hydrogen peroxide detection. The results indicated that compared with glassy carbon electrode this platform was extraordinarily sensitive to hydrogen peroxide. Therefore, by combining with an appropriate enzymatic reaction, this platform would be a sensitive matrix for many biomolecules. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrocatalytic reduction of protons and oxygen at glassy carbon electrodes coated with a cobalt(II)/platinum(II) porphyrin

The electrocatalytic reduction of protons in 1.0 M perchloric acid at glassy carbon electrodes anodically modified with a Co(II)/Pt(II) porphyrin show shifts of 400 mV versus Ag/AgCl when compared to the same electrodes which have not been anodically modified. Anodic cycling of glassy carbon electrodes coated with the Co(II)/Pt(II) porphyrin in this study form stable electroactive films capable of improving both electroreduction of protons to hydrogen and oxygen to both peroxide and water. Electrooxidation of glassy carbon electrodes coated with the free base porphyrin show no improvement in catalytic ability for the reduction of protons in acidic solution or the reduction of molecular oxygen in basic solution. Glassy carbon electrodes coated with the Co(II)/Pt(II) porphyrin indicate, by rotating disk electrochemistry, that the electrocatalysis of oxygen is a two electron process leading to the formation of hydrogen peroxide. Koutecky-Levich plots of the data obtained from the reduction of oxygen at electrode surfaces coated with the Co(II)/Pt(II) porphyrin after oxidation of the surface indicate that 25% of the oxygen is reduced by four electrons directly to water while 75% of the oxygen is reduced by two electrons to hydrogen peroxide.     

Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide

Cyclic voltammetry was used for simultaneous formation and immobilization of nickel oxide nano-scale islands and catalase on glassy carbon electrode. Electrodeposited nickel oxide may be a promising material for enzyme immobilization owing to its high biocompatibility and large surface. The catalase films assembled on nickel oxide exhibited a pair of well defined, stable and nearly reversible CV peaks at about -0.05 V vs. SCE at pH 7, characteristic of the heme Fe (III)/Fe (II) redox couple. The formal potential of catalase in nickel oxide film were linearly varied in the range 1-12 with slope of 58.426 mV/pH, indicating that the electron transfer is accompanied by single proton transportation. The electron transfer between catalase and electrode surface, (k(s)) of 3.7(+/-0.1) s(-1) was greatly facilitated in the microenvironment of nickel oxide film. The electrocatalytic reduction of hydrogen peroxide at glassy carbon electrode modified with nickel oxide nano-scale islands and catalase enzyme has been studied. The embedded catalase in NiO nanoparticles showed excellent electrocatalytic activity toward hydrogen peroxide reduction. Also the modified rotating disk electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant catalase/nickel oxide modified glassy carbon electrodes exhibited fast amperometric response (within 2 s) to hydrogen peroxide reduction (with a linear range from 1 microM to 1 mM), excellent stability, long term life and good reproducibility. The apparent Michaelis-Menten constant is calculated to be 0.96(+/-0.05)mM, which shows a large catalytic activity of catalase in the nickel oxide film toward hydrogen peroxide. The excellent electrochemical reversibility of redox couple, high stability, technical simplicity, lake of need for mediators and short preparations times are advantages of this electrode. Finally the activity of biosensor for nitrite reduction was also investigated.     

Amperometric sensors based on Ni/Al and Co/Al layered double hydroxides modified electrode and their application for hydrogen peroxide detection

Two different hydrogen peroxide sensors were constructed with Ni/Al and Co/Al layered double hydroxides (LDHs) modified glassy carbon electrodes (GCE). Ni (Co)/Al-LDHs were synthesized by electrochemical method and were characterized by scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS). The advantages and shortcoming of the two hydrogen peroxide sensors were described in detail. Compared to Co/Al-LDHs modified electrode, sensors fabricated by Ni/Al-LDHs showed quicker heterogeneous electron transfer rate constants (k(s)), lower detection and better reproducibility. But Co/Al-LDHs modified electrode held the advantages of wider linear range and higher sensitivity. Further more, the different catalytic redox mechanisms of hydrogen peroxide on the Ni/Al/GCE and Co/Al/GCE were firstly comparatively explored.     

Fabrication of bienzyme nanobiocomposite electrode using functionalized carbon nanotubes for biosensing applications

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wider applicability. This work presents a novel approach to fabricate nanobiocomposite bienzymatic biosensor based on functionalized multiwalled carbon nanotubes (MWNTs) with the aim of evaluating their ability as sensing elements in amperometric transducers. Electrochemical behavior of the bienzymatic nanobiocomposite biosensor is investigated by Faradaic impedance spectroscopy and cyclic voltammetry. The results indicate that glucose oxidase (GOD) and horseradish peroxidase (HRP) are strongly adsorbed on the surface of the thionin (TH) functionalized MWNTs and demonstrate a facile electron transfer between immobilized GOD/HRP and the electrode via the functionalized MWNTs in a Nafion film. The functionalized carbon nanotubes act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centres of enzymes through TH. Linear ranges for these electrodes are from 10 nM to 10 mM for glucose and 17 nM to 56 mM for hydrogen peroxide with the detection limit of 3 and 6 nM, respectively. A remarkable feature of the bienzyme electrode is the possibility to determine glucose and hydrogen peroxide at a very low applied potential where the noise level and interferences from other electroactive compounds are minimal. Performance of the biosensor is evaluated with respect to response time, detection limit, selectivity, temperature and pH as well as operating and storage stability.     

A third-generation H2O2 biosensor based on horseradish peroxidase-labeled Au nanoparticles self-assembled to hollow porous polymeric nanopheres

A novel third-generation biosensor for hydrogen peroxide (H2O2) was developed by self-assembling gold nanoparticles to hollow porous thiol-functionalized poly(divinylbenzene-co-acrylic acid) (DVB-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in hollow porous thiol-functionalized poly(DVB-co-AA) nanosphere latex to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups of the nanospheres. Finally, horseradish peroxidase (HRP) was immobilized on the surface of the gold nanoparticles. The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The resulting biosensor showed a wide linear range of 1.0 microM-8.0mM and a detection limit of 0.5 microM estimated at a signal-to-noise ratio of 3. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.     

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.     

Myoglobin immobilization on electrodeposited nanometer-scale nickel oxide particles and direct voltammetry

Prosperity of information on the reactions of redox-active sites in proteins can be attained by voltammetric studies in which the protein sample is located on a suitable surface. This work reports the presentation of myoglobin/nickel oxide nanoparticles/glassy carbon (Mb/NiO NPs/GC) electrode, ready by electrochemical deposition of the NiO NPs on glassy carbon electrode and myoglobin immobilization on their surfaces by the potential cycling method. Images of electrodeposited NiO NPs on the surface of glassy carbon electrode were obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). A pair of well-defined redox peaks for Mb(Fe(III)-Fe(II)) was obtained at the prepared electrode by direct electron transfer between the protein and nanoparticles. Electrochemical parameters of immobilized myoglobin such as formal potential (E(0')), charge transfer coefficient (alpha) and apparent heterogeneous electron transfer rate constant (k(s)) were estimated by cyclic voltammetry and nonlinear regression analysis. Biocatalytic activity was exemplified at the prepared electrode for reduction of hydrogen peroxide.     

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at l00-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO₂, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated matix after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.     

P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes

Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H₂O₂ due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine--oxidase.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

Hydrogen peroxide sensitive amperometric biosensor based on horseradish peroxidase entrapped in a polypyrrole electrode

The enzyme horseradish peroxidase (HRP) has been entrapped in situ by electropolymerization of pyrrole onto a platinum electrode. The latter was previously coated by a polypyrrole layer for better adhesion of the biocatalyst film and in order to avoid the enzyme folding onto the Pt electrode. The biosensor allowed the determination of hydrogen peroxide in the concentration range comprised between 4.9 x 10(-7) and 6.3 x 10(-4) M. The biosensor retained more than 90% of its original activity after 35 days of use.     

An amperometric immunosensor based on an electrochemically pretreated carbon-paraffin electrode for complement III (C3) assay

An electrochemical immunosensor based on the adsorption of anti-complement III antibody onto an electrochemical pretreated carbon-paraffin electrode has been proposed for the detection of complement III (C(3)). The competitive immunoassay format was adopted with horseradish peroxide-C(3) (HRP-C(3)) as a tracer, 3,3'5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide as the enzyme substrates. In order to measure the amount of HRP-C(3) binding onto the electrode surface, the product of the enzyme catalytic reaction was detected at 100 mV (vs. Ag/AgCl reference electrode). The system was optimized to realize a reliable determination of C(3) in the range of 0.06-10 microg/ml. It exhibits some advantages, such as simplicity of fabrication, rapidity of measurement, and satisfactory sensitivity and reproducibility.     

Electrochemical Sensors for Direct Reagentless Measurement of Superoxide Production by Human Neutrophils

Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised.     

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Detection of basal acetylcholine in rat brain microdialysate

A liquid chromatography-electrochemistry (LC-EC) method is described for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. This method is based on the separation of ACh and choline (Ch) by microbore liquid chromatography followed by passage of the effluent through a post-column immobilized enzyme reactor (IMER), containing acetylcholinesterase (AChE) and choline oxidase (ChO), and then the electrochemical detection of the hydrogen peroxide produced. Instead of the conventional platinum electrode used for the anodic detection of hydrogen peroxide, a peroxidase-redox polymer modified glassy carbon electrode operated at + 100 mV vs. Ag/AgCl has been used to detect the reduction of hydrogen peroxide. With this method, a detection limit of 10 fmol (injected) for ACh (S/N = 3:1) was obtained and the basal ACh concentration in striatal microdialysate was determined without using esterase inhibitors.