Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

The direct voltammetry and electrocatalytic properties of catalase, which was adsorbed on the surface of multiwall carbon nanotubes (MWCNTs), was investigated. A pair of well-defined and nearly reversible cyclic voltammetry peaks for Fe(III)/Fe(II) redox couple of catalase adsorbed on the surface of MWCNTs at approximately -0.05 V versus reference electrode in pH 6.5 buffer solution, indicating the direct electron transfer between catalase and electrode. The surface coverage of catalase immobilized on MWCNTs glassy carbon electrode was approximately 2.4x10(-10) molcm-2. The transfer coefficient (alpha) was calculated to be 0.4, and the heterogeneous electron transfer rate constant was 80 s-1 in pH 7, indicating great facilitation of the electron transfer between catalase and MWCNTs adsorbed on the electrode surface. The formal potential of catalase Fe(III)/Fe(II) couple in MWCNTs film had a linear relationship with pH values between 2 and 11 with a slope of 58 mV/pH, showing that the electron transfer is accompanied by single proton transportation. Catalase adsorbed on MWCNTs exhibits a remarkable electrocatalytic activity toward the reduction of oxygen and hydrogen peroxide. The value for calculated Michaelis-Menten constant (1.70 mM) was high, indicating the potential applicability of the films as a new type of reagentless biosensor based on the direct electrochemistry of the catalase enzyme.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Direct electrochemistry of horseradish peroxidase immobilized on DNA/electrodeposited zirconium dioxide modified,gold disk electrode

A novel H₂O₂ biosensor is described which is based on immobilization of horseradish peroxidase (HRP) on DNA/electrodeposited, ZrO₂/modified, gold electrode. The DNA is attached via its 5′ end to ZrO₂ and this provides a microenvironment for the immobilization of various biomolecules and promotes electron transfer between HRP and the electrode surface. Under optimized conditions, the biosensor reduced H₂O₂ linearly between 3.5 μM and 10 mM with a detection limit of 0.8 μM at a signal-to-noise ratio of 3. In addition, the developed biosensor shows an acceptable stability and repeatability. Importantly, the analytical methodology could be further developed for the immobilization of other proteins and biocompounds.     

Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

The direct voltammetry and electrocatalytic properties of catalase (Cat) in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Cat-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetry peaks for Cat Fe(III)/Fe(II) redox couples at approximately -0.46 V vs. SCE in pH 7.0 buffers. The electron transfer between catalase and PG electrodes was greatly facilitated in the microenvironment of PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting square wave voltammograms with non-linear regression analysis. The formal potential of Cat Fe(III)/Fe(II) couples in PAM films had a linear relationship with pH between pH 4.0 and 9.0 with a slope of -56 mV pH(-1), suggesting that one proton is coupled with single-electron transfer for each heme group of catalase in the electrode reaction. UV-Vis absorption spectroscopy demonstrated that catalase retained a near native conformation in PAM films at medium pH. The embedded catalase in PAM films showed the electrocatalytic activity toward dioxygen and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) at Cat-PAM film electrodes was proposed.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

Direct electrochemistry of novel affinity-tag immobilized recombinant horse heart cytochrome c

During the last decade protein electrochemistry at miniaturized electrodes has become important not only for functional studies of the charge transfer properties of redox proteins but also for fostering the development of sensitive biosensor and bioelectronic devices. One of the major challenges in this field is the directed coupling between electronic and biologically active components. A prerequisite for a fast and reversible electron transfer between electrode and protein is that the protein can be bound to the electrode in a favourable orientation. We examined electrostatic and bioaffinity-tag binding strategies for the directed immobilization of horse heart cytochrome c (cytc) on gold electrode surfaces to achieve this goal. Horse heart cytc was expressed in E. coli either as non-modified or genetically modified, i.e. histidine (his)-tag containing protein. The his-tags were introduced at defined positions at the N- or C-terminus of the polypeptide. It was our aim to generate tagged-versions of cytc that facilitate strong electronic coupling between protein and electrode and, at the same time, retain their catalytic and regulatory properties. The combination of different immobilization strategies, e.g. his-tag and electrostatic immobilization also opens new avenues for bivalent immobilization of proteins. This is of interest for molecular bioelectronic and biosensing applications where the proteins are immobilized between two crossing electrodes.     

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.     

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl₂ produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential (E^0′) on the solution pH (56 mV pH^− 1) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10^− 11 mol cm^− 2_, indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant (k_s) was 1.43 s^− 1, indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K_m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.     

Direct electrochemistry and electrocatalysis of myoglobin immobilized on a hexagonal mesoporous silica matrix

The direct electrochemistry of myoglobin (Mb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Mb and HMS was investigated by using Fourier transfer infrared spectroscopy, nitrogen adsorption isotherm, and cyclic voltammetry. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the Mb intercalated in the mesopores and adsorbed on the surface of the HMS were observed with the formal potentials of -0.167 and -0.029V in 0.1M, pH 7.0, phosphate buffer solution, respectively. The electrode reaction showed a surface-controlled process with one proton transfer. The immobilized Mb displayed good electrocatalytic responses to the reduction of both hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), which were used to develop novel sensors for H(2)O(2) and NO(2)(-). The apparent Michaelis-Menten constants of the immobilized Mb for H(2)O(2) and NO(2)(-) were 0.065 and 0.72mM, respectively, showing good affinity. Under optimal conditions, the sensors could be used for the determinations of H(2)O(2) ranging from 4.0 to 124microM and NO(2)(-) ranging from 8.0 to 216microM. The detection limits were 6.2x10(-8) and 8.0x10(-7)M at 3 sigma, respectively. The HMS provided a novel matrix for protein immobilization and the construction of biosensors via the direct electron transfer of immobilized protein.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol-gel-derived ceramic-carbon nanotube nanocomposite film

The sol-gel-derived ceramic-carbon nanotube (SGCCN) nanocomposite film fabricated by doping multiwall carbon nanotubes (MWNTs) into a silicate gel matrix was used to immobilize protein. The SGCCN film can provide a favorable microenvironment for horseradish peroxidase (HRP) to perform direct electron transfer (DET) at glassy carbon electrode. The HRP immobilized in the SGCCN film shows a pair of well-defined redox waves and retains its bioelectrocatalytic activity to the reduction of O2 and H2O2, which is superior to that immobilized in silica sol-gel film.     

Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan

Three heme proteins, myoglobin, hemoglobin, and cytochrome c, have been adsorbed onto chitosan-stabilized gold nanoparticles (Chit-Aus) modified Au electrode via a molecule bridge like cysteine. UV-vis spectra indicated that the proteins on Chit-Aus films retained near-native secondary structures. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectra and cyclic voltammetric techniques. It was demonstrated that Chit-Aus film could not only offer a friendly environment to immobilize protein molecules but also enhance the electron transfer ability between protein molecules and underlying electrode. The effects of scan rate and pH on the electrochemical behaviors of each heme protein are discussed in detail. The resultant electrode displayed an excellent electrocatalytic response to the reduction of H(2)O(2), long-term stability, and good reproducibility.     

Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode

Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.     

Direct electrochemistry of lactate dehydrogenase immobilized on silica sol-gel modified gold electrode and its application

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol-gel film on gold electrode was investigated, and an obvious cathodic peak at about -200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (alpha) of 0.79 and the apparent heterogeneous electron transfer rate constant (K(s)) of 3.2 s(-1). The activated voltammetric response and decreased charge-transfer resistance of Ru(NH(3))(6)(2+/3+) on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol-gel and the LDH embedded in silica sol-gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 x 10(-6) to 3.0 x 10(-5) mol L(-1) and the detection limit was 8.0 x 10(-7) mol L(-1) at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized on carbon paste electrode by silica sol-gel film

Direct electrochemical and electrocatalytic behaviors of hemoglobin (Hb) immobilized on carbon paste electrode (CPE) by a silica sol-gel film derived from tetraethylorthosilicate (TEOS) were investigated for the first time. Hb/sol-gel film modified electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for Hb Fe(III)/Fe(II) redox couple at about -0.312 V (versus Ag/AgCl) in a pH 7.0 phosphate buffer. The formal potential of Hb heme Fe(III)/Fe(II) couple varied linearly with the increase of pH in the range of 5.0-10.0 with a slope of 49.44 mV pH(-1), which suggests that a proton transfer is accompanied with each electron transfer (ET) in the electrochemical reaction. The immobilized Hb displayed the features of peroxidase and gave excellent electrocatalytic performance to the reduction of O2, NO2(-) and H2O2. The calculated apparent Michaelis-Menten constant was 8.98 x 10(-4)M, which indicated that there was a large catalytic activity of Hb immobilized on CPE by sol-gel film toward H2O2. In comparison with other electrodes, the chemically modified electrodes, used in this direct electrochemical study of Hb, are easy to be fabricated and rather inexpensive. Consequently, the Hb/sol-gel film modified electrode provides a convenient approach to perform electrochemical research on this kind of proteins. It also has potential use in the fabrication of the third generation biosensors and bioreactors.     

Modulation of catalase peroxidatic and catalatic activity by nitric oxide

Previously, we found that catalase enhanced the protection afforded by superoxide dismutase to Escherichia coli against the simultaneous generation of superoxide and nitric oxide (Brunelli et al., Arch. Biochem. Biophys. 316:327-334, 1995). Hydrogen peroxide itself was not toxic in this system in the presence or absence of superoxide dismutase. We therefore investigated whether catalase might consume nitric oxide in addition to hydrogen peroxide. Catalase rapidly formed a reversible complex stoichiometrically with nitric oxide with the Soret band shifting from 406 to 426 nm and two new peaks appeared at 540 and at 575 nm, consistent with the formation of a ferrous-nitrosyl complex. Catalase consumed more nitric oxide upon the addition of hydrogen peroxide. Conversely, micromolar concentrations of nitric oxide slowed the catalase-mediated decomposition of hydrogen peroxide. Catalase pretreated with nitric oxide and hydrogen peroxide regained full activity after dialysis. Our results suggest that catalase can slowly consume nitric oxide while nitric oxide modestly inhibits catalase-dependent scavenging of hydrogen peroxide. The protective effects of catalase in combination with superoxide dismutase may result from two actions; reducing peroxynitrite formation by scavenging nitric oxide and by scavenging hydrogen peroxide before it reacts with superoxide dismutase to form additional superoxide.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

High biological activity of a recombinant protein immobilized onto polystyrene

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.     

Direct voltammetry of catalase immobilized on silica sol-gel and cysteine modified gold electrode and its application

The direct voltammetry of catalase (CAT) immobilized in silica sol-gel film in the presence of cysteine on gold electrode was investigated. The CAT electrode showed a pair of well-defined and quasi-reversible cyclic voltammetry peaks. It can be used as an electrochemical biosensor for the determination of hydrogen peroxide. The calibration range of H(2)O(2) was from 1 to 30 micromolL(-1) and the detection limit was 0.4 micromolL(-1) at a signal-to-noise ratio of 3. The interaction of CAT and aluminum ion was also investigated based on the CAT-modified electrode. The electrochemical activity of the CAT-modified electrode was increased with the addition of Al(3+). The experimental results of voltammetry and fluorescence spectroscopy indicated that the conformation of CAT molecule was altered by the formation of Al-CAT complex with Al(3+), which may influence the activity of CAT.     

Characterization and properties of catalase immobilized onto controlled pore glass and its application in batch and plug-flow type reactors

Bovine liver catalase was covalently immobilized onto controlled pore glass (CPG) beads modified with 3-aminopropyltriethoxysilane (3-APTES) followed by treatment with glutaraldehyde. Coupling of catalase onto CPG was optimized to improve the efficiency of the overall immobilization procedure. The optimum coupling conditions: pore diameter of CPG, pH, buffer concentration, temperature, coupling time and initial catalase amount per grams of carrier were determined as 70 nm, 6.0, 75 mM, 5 °C, 7 h and 6 mg catalase, respectively. Catalytic efficiencies (k_cat/K_m) and thermal inactivation rate constants (k_i) of ICPG1 were determined and compared with that of free catalase. Suitability of ICPG1 was also investigated by using it in batch and plug-flow type reactors. When the remaining activity of ICPG1 retained was about 50% of its initial activity the highest total productivity of ICPG1 was determined as 7.6 × 10⁶ U g immobilized catalase⁻¹ in plug-flow type reactor. However, the highest total productivity of ICPG1 was 6.2 × 10⁵ U g immobilized catalase⁻¹ in batch type reactor. ICPG1 may have great potentials as biocatalyst for the application in decomposition of hydrogen peroxide in plug-flow type reactor.     

Enzymatic activity under tangential flow conditions of photochemically grafted membranes containing immobilized catalase

Catalase has been immobilized within sandwich membranes prepared by the photoinduced grafting of an epoxy-diacrylate prepolymer onto commercial asymmetric cellulose membranes. The enzymatic activity of the membrane composite of hydrogen peroxide decomposition has been studied in a recirculation apparatus under tangential flow conditions without ultrafiltration. The enzymatic membranes were exposed to very low mechanical stresses and showed a very good catalytic performance and durability. Initial reaction rates, measured at 25 degrees C as a function of both substrate concentration and enzyme amount immobilized per unit membrane surface, indicate that the mechanism of action of catalase is not altered after immobilization, although substrate diffusion through the original thin layer of membranes may become rate controlling. (c) 1993 John Wiley & Sons, Inc.