Direct electrochemistry and electrocatalysis of heme proteins on SWCNTs-CTAB modified electrodes

Direct electrochemistry and electrocatalysis of heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) were studied with the protein incorporated single walled carbon nanotubes (SWCNTs)-cetylramethylammonium bromide (CTAB) nanocomposite film modified glassy carbon electrodes (GCEs). The incorporated heme proteins were characterized with Fourier transform infrared spectroscopy (FTIR), ultraviolet visible (UV) spectroscopy, atomic force microscopy (AFM) and electrochemistry, indicating the heme proteins in SWCNTs-CTAB nanocomposite films keep their secondary structure similar to their native states. The direct electron transfer between the heme proteins in SWCNTs-CTAB films and GCE was investigated. The electrochemical parameters such as formal potentials and apparent heterogeneous electrontransfer rate constants (k_s) were estimated by square wave voltammetry with nonlinear regression analysis. The heme protein-SWCNT-CTAB electrodes show excellent electrocatalytic activities for the reduction of H₂O₂ and NO₂⁻, which have been utilized to determine the concentrations of H₂O₂ and NO₂⁻.     

Electroactive films of heme protein-coated multiwalled carbon nanotubes

A novel method for fabricating protein-MWNT films on pyrolytic graphite (PG) electrodes was described. Positively charged hemoglobin (Hb) or myoglobin (Mb) in buffers at pH 5.5 or 5.0 was first adsorbed on the surface of acid-pretreated, negatively charged multiwalled carbon nanotubes (MWNTs) mainly by electrostatic interaction, forming a core-shell structure. The aqueous dispersion of protein-coated MWNTs was then cast on PG electrodes, forming protein-MWNT films after evaporation of solvent. The protein-MWNT films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, characteristic of heme Fe(III)/Fe(II) redox couples. The protein films were characterized by voltammetry, UV-vis spectroscopy, and scanning electron microscopy (SEM). This approach for assembly of protein-MWNT films showed higher surface concentration of electroactive proteins than the simple cast method, and the amount of proteins in the films could be controlled more precisely compared with the dipping method. Furthermore, the film assembly using this method was more stable than that using simple cast method. The proteins in MWNT films retained their near-native structure, and electrochemically catalyzed reduction of oxygen and hydrogen peroxide, suggesting the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of enzymes.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol–gel-derived tin oxide/gelatin composite films

Horseradish peroxidase (HRP) was immobilized into a new type of sol–gel-derived nano-sized tin oxide/gelatin composite film (SnO₂ composite film) using a sol–gel film/enzyme/sol–gel film “sandwich” configuration. Direct electrochemistry and electrocatalysis of HRP incorporated into the composite films were investigated. HRP/SnO₂ composite film exhibited a pair of stable and quasi-reversible cyclic voltammetric peaks for the HRP Fe(III)/HRP Fe(II) redox couple with a formal potential of about −0.25 V (vs. SCE) in a pH 6.0 phosphate buffer solution. The electron transfer between the enzyme and the underlying electrode was greatly enhanced in the microenvironment with nano-SnO₂ particles and nanoporous structures. Morphologies and microstructures of the composite films and HRP/composite films were characterized with TEM, AFM. Electrochemical impedance spectroscopy (EIS) was also used to feature the HRP incorporated into composite films. FTIR and UV–Vis spectroscopy demonstrated that HRP in the composite film could retain its native secondary structure. With the advantages of organic–inorganic hybrid materials, the HRP/SnO₂ composite film modified electrode displayed good stability and electrocatalytic activity to the reduction of H₂O₂, The apparent Michaelis-Menten constant was estimated to be 0.345 mM, indicating a high affinity of HRP entrapped into the composite film toward H₂O₂.     

Fabrication of multilayer-film-modified gold electrode composed of myoglobin,chitosan, and polyelectrolyte-wrapped multi-wall carbon nanotubes by layer-by-layer assembled technique and electrochemical catalysis for hydrogen peroxide and trichloroacetic acid

Electroactive multilayer film of myoglobin (Mb)-, chitosan (CS)-, and poly(dimethyldiallylammonium chloride) (PDDA)-wrapped multi-wall carbon nanotubes (MWNTs) is fabricated on a gold electrode via layer-by-layer (LBL) technique. The assembled multilayer films is characterized by scanning electron microscopy (SEM), UV-vis spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). UV-vis spectroscopy showed that Mb in the films retained its near-native structure. The stable multilayerfilm-modified gold electrodes showed good electroactivity in protein-free buffer solution, which is originated from protein heme Fe(III)/Fe(II) redox couple. The modified electrode exhibited good electrocatalytic property toward reduction of H₂O₂ and trichloroacetic acid, indicating the potential application as amperometric biosensor. Published in Russian in Elektrokhimiya, 2008, Vol. 44, No. 11, pp. 1366–1376. The text was submitted by the authors in English.     

Electrochemical property and analysis application of biosensors in miscible nonaqueous media—Room-temperature ionic liquid

Stable heme proteins entrapped in dimethylformamide (DMF)–chitosan organohydrogel films modified electrodes were operated in neat hydrophilic room-temperature ionic liquid (IL) [bmim][BF₄] for the first time. The modified electrodes possess outstanding electrochemical response in [bmim][BF₄] without adding water. The morphology studies of films were demonstrated by atomic force microscopy (AFM). UV–Vis and FTIR spectroscopy showed that the heme proteins retained their native structure in organohydrogel films. Direct electrochemistry and bioelectrocatalysis of heme protein–organohydrogel films were investigated. Several electrochemical parameters such as the charge transfer coefficients (α) and the apparent electron transfer rate constant (k_s) of these processes were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental dates. Furthermore, high electrocatalytic activity to hydrogen peroxide (H₂O₂) was observed, indicating that heme proteins entrapped in organohydrogel films retained their bioelectrocatalytic activities in [bmim][BF₄]. Kinetic analysis of the cyclic voltammetry dates shows that heme protein–organohydrogel films operated in IL bring up to an enhancement of the biosensor sensitivity and a high affinity for H₂O_2.     

肌红蛋白在双十二烷基二甲基溴化铵-粘土多双层复合薄膜电极上的电化学与电催化

将双十二烷基二甲基溴化铵(DDAB)-粘土(Clay)复合物的水分散系与肌红蛋白(Mb)水溶液的混合物涂布到热解石墨(PG)电极表面,可制得Mb-DDAB-Clay薄膜电极.在pH5.5的缓冲溶液中,该薄膜电极在-0.25V(vs.SCE)处有一对可逆的循环伏安还原氧化峰,为Mb血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对的特征峰.在DDAB-Clay薄膜的微环境中,Mb与PG电极之间的电子传递得到极大促进,并显示了很好的稳定性.Soret吸收带的位置表明,在适中的pH范围内,Mb在薄膜中保持了其原始构象.X射线衍射实验结果表明,Mb的嵌入并未对薄膜的有序多层结构有很大影响.在DDAB-Clay薄膜环境中,Mb血红素Fe(Ⅲ)/Fe(Ⅱ)电对的式量电位在pH4.5～11.0范围内与溶液pH值成线性关系,表明Mb的电化学还原很可能是一个质子伴随一个电子的电极过程.Mb-DDAB-Clay薄膜可以用于催化还原溶解氧和三氯乙酸.     

Electroactive core-shell nanocluster films of heme proteins, polyelectrolytes, and silica nanoparticles

Novel protein core-shell nanocluster films were assembled layer by layer on solid surfaces. In the first step, positively charged heme protein hemoglobin (Hb) or myoglobin (Mb) and negatively charged poly(styrenesulfonate) (PSS) were alternately adsorbed on the surface of SiO2 nanoparticles, forming core-shell SiO2-(protein/PSS)m nanoclusters. In the second step, the SiO2-(protein/PSS)m nanoclusters and polycationic poly(ethylenimine) (PEI) were assembled layer by layer on various solid substrates, forming [[SiO2-(protein/PSS)m]/PEI]n films. Various techniques were used to characterize the nanoclusters and monitor the film growth. [[SiO2-(protein/PSS)m]/PEI]n films at pyrolytic graphite (PG) electrodes exhibited well-defined, chemically reversible cyclic voltammetric reduction-oxidation peaks characteristic of the heme Fe(III)/Fe(II) redox couples. The proteins in the films retained near native conformations in the medium pH range, and the films catalyzed electrochemical reduction of oxygen and hydrogen peroxide. Advantages of the nanocluster films over the simple [SiO2/protein]n layer-by-layer films include a larger fraction of electroactive protein and higher specific biocatalytic activity. Using this approach, biocatalytic activity can be tailored and controlled by varying the number of bilayers deposited on the nanoparticle cores and the number of nanocluster layers on electrodes.     

Direct Electrochemistry and Catalytic Activity of Hemoglobin and Myoglobin Entrapped in PEG Film

Abstract

Direct electrochemistry and electrocatalysis of two heme proteins, hemoglobin (Hb) and myoglobin (Mb), incorporated in polyethylene glycol (PEG) films, were studied by cyclic voltammetry. The two proteins exhibited a pair of well‐defined, quasi‐reversible cyclic voltammetric peaks with the apparent formal potential at about ?0.21 V (Hb) and ?0.22 V (Mb), respectively, vs. saturated calomel electrode (SCE) in pH 5.0 acetate buffer solution, characteristic of the h eme Fe(III)/Fe(II) redox couples, indicating enhanced electron transfer between the proteins and the substrate electrode in the PEG film environment. The protein–PEG films could also exhibit excellent stability. Meanwhile, positions of Soret absorption band of the proteins in the PEG films suggested that the heme proteins kept their secondary structure similar to their native state in the medium pH range. Oxygen, trichloroacetic acid, nitric oxide, and hydrogen peroxide could all be catalytically reduced by Hb or Mb in PEG films.     

Vapor‐Surface Sol‐Gel Deposition of Titania Alternated with Protein Adsorption for Assembly of Electroactive,Enzyme‐Active Films

Combining vapor‐surface sol‐gel deposition of titania with alternate adsorption of oppositely charged iron heme proteins provided ultrathin {TiO₂/protein}_n films with reversible voltammetry extended to 15 TiO₂/protein bilayers, more than twice that of more conventional polyion‐protein or nanoparticle‐protein films made by alternate layer‐by‐layer adsorption. Catalytic activity toward O₂, H₂O₂, and NO was also improved significantly compared to the conventionally fabricated films. The method involves vaporization of titanium butoxide into thin films of water, forming porous TiO₂ sol‐gel layers. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) were assembled by adsorption alternated with the vapor‐deposited TiO₂ layers. Improved electrochemical and catalytic performance may be related to better film permeability leading to better mass transport within the films, as suggested by studies with soluble voltammetric probes, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The electrochemical and electrocatalytic activity of the films can be controlled by tailoring the amount of water with which the metal alkoxide precursor vapor reacts and the number of bilayers deposited in the assembly.     

Non‐covalent Immobilization of Iron‐triazole (Fe(Htrz)3) Molecular Mediator in Mesoporous Silica Films for the Electrochemical Detection of Hydrogen Peroxide

Mesoporous silica thin films encapsulating a molecular iron‐triazole complex, Fe(Htrz)₃ (Htrz=1,2,4,‐1H‐triazole), have been generated by electrochemically assisted self‐assembly (EASA) on indium‐tin oxide (ITO) electrode. The obtained modified electrodes are characterized by well‐defined voltammetric signals corresponding to the Fe^II/III centers of the Fe(Htrz)₃ species immobilized into the films, indicating fast electron transfer processes and stable operational stability. This is due to the presence of a high density of redox probes in the material (1.6×10^?4 mol g^?1 Fe(Htrz)₃ in the mesoporous silica film) enabling efficient charge transport by electron hopping. The mesoporous films are uniformly deposited over the whole electrode surface and they are characterized by a thickness of 110 nm and a wormlike mesostructure directed by the template role played by Fe(Htrz)₃ species in the EASA process. These species are durably immobilized in the material (they are not removed by solvent extraction). The composite mesoporous material (denoted Fe(Htrz)₃@SiO₂) is then used for the electrocatalytic detection of hydrogen peroxide, which can be performed by amperometry at an applied potential of ?0.4 V versus Ag/AgCl and by flow injection analysis. The organic‐inorganic hybrid film electrode displays good sensitivity for H₂O₂ sensing over a dynamic range from 5 to 300 μM, with a detection limit estimated at 2 μM.     

Long Distance Electron Transfer Across >100 nm Thick Au Nanoparticle/Polyion Films to a Surface Redox Protein

Glutathione‐decorated 5 nm gold nanoparticles (AuNPs) and oppositely charged poly(allylamine hydrochloride) (PAH) were assembled into {PAH/AuNP}_n films fabricated layer‐by‐layer (LbL) on pyrolytic graphite (PG) electrodes. These AuNP/polyion films utilized the AuNPs as electron hopping relays to achieve direct electron transfer between underlying electrodes and redox proteins on the outer film surface across unprecedented distances >100 nm for the first time. As film thickness increased, voltammetric peak currents for surface myoglobin (Mb) on these films decreased but the electron transfer rate was relatively constant, consistent with a AuNP‐mediated electron hopping mechanism.     

Electropolymerization of iron tetra(<Emphasis Type="Italic">o</Emphasis>-aminophenyl)porphyrin from aqueous solution and the electrocatalytic behavior of modified electrode

Stable electroactive iron tetra(o-aminophenyl)porphyrin (FeTAPP) films are prepared by electropolymerization from aqueous solution by cycling the electrode potential between −0.4 and 1.0 V vs Ag/AgCl at 0.1 V s⁻¹. The cyclic voltammetric response indicates that polymerization takes place after the oxidation of amino groups, and the films could be produced on glassy carbon (GC) and gold electrodes. The film growth of poly(FeTAPP) was monitored by using cyclic voltammetry and electrochemical quartz crystal microbalance. The cyclic voltammetric features of Fe(III)/Fe(II) redox couple in the film resembles that of surface confined redox species. The electrochemical response of the modified electrode was found to be dependent on the pH of the contacting solution with a negative shift of 57 mV/pH. The electrocatalytic behavior of poly(FeTAPP) film-modified electrode was investigated towards reduction of hydrogen peroxide, molecular oxygen, and chloroacetic acids (mono-, di-, and tri-). The reduction of hydrogen peroxide, molecular oxygen, and dichloroacetic acid occurred at less negative potential on poly(FeTAPP) film compared to bare GC electrode. Particularly, the overpotential of hydrogen peroxide was reduced substantially. The O₂ reduction proceeds through direct four-electron reduction mechanism.     

Myoglobin in polyacrylamide hydrogel films: direct electrochemistry and electrochemical catalysis

Electrochemical behavior of myoglobin (Mb) incorporated in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Mb–PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for Mb Fe(III)/Fe(II) redox couple at about −0.27 (vs. SCE) in pH 5.5 buffers. The electron exchange of Mb with PG electrodes was greatly enhanced in PAM films. The apparent heterogeneous electron transfer rate constant (k_s) and formal potential (E°′) were estimated by fitting the data of square wave voltammetry (SWV) with non-linear regression analysis. The formal potential of Mb–PAM films shifted linearly with pH with a slope of −0.52 V, showing the electron transfer was accompanied by a single-proton transportation. Positions of Soret absorbance band of Mb–PAM films suggest that Mb maintains its secondary structure similar to its native state in the films in the medium pH range. Oxygen, trichloroacetic acid (TCA) and nitrite were catalytically reduced by Mb–PAM film electrodes with significant lowering of overpotential. Potential application of Mb–PAM films as biosensors to monitor some substrates was proposed.     

Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film

Heme proteins were immobilized on glass carbon electrodes by poly (N-isopropylac-yamide-co-3-methacryloxy-propyl-trimethoxysilane) (PNM) and exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.35 V versus a saturated calomel electrode in pH 7.0 buffer solution, corresponding to hemeFe(III)+e-->hemeFe(II). Some electrochemical parameters were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. The formal potential was linearly dependent on pH, indicating the electron transfer of Fe(III)/Fe(II) redox couple accompanied by the transfer of proton. Ultraviolet visible and Fourier transform infrared spectra suggested that the conformation of proteins in the PNM films retained the essential feature of its native secondary structure. Atomic force microscopy images demonstrated the existence of interaction between heme proteins and PNM. N,N-dimethylformamide (DMF) played an important role in immobilizing proteins and enhancing electron transfer between proteins and electrodes. Electrochemical catalytic reductions of hydrogen peroxide and trichloroacetic acid by proteins entrapped in PNM film were also discussed, showing the potential applicability of the film modified electrodes as a biosensor.     

Oscillations of pH at the Fe│H2SO4 interface during anodic dissolution

An unmodified Pt microelectrode and a Pt microelectrode coated with polyaniline were used in conjunction with a scanning electrochemical microscope (SECM) to study anodic dissolution in the Fe│H₂SO₄ system. The concentrations of Fe^2 + (c_Fe2 +) measured with the unmodified microelectrode and the pH values measured with the polyaniline-modified microelectrode were recorded in situ during current oscillations in the Fe│H₂SO₄ system and were found to change periodically at the Fe│H₂SO₄ interface. The changes in c_Fe2 + may be caused by the periodic formation and dissolution of surface film(s), which could be salt films and/or oxide films. If a salt film is formed, it is unlikely to affect the pH. Since the pH changes periodically during the current oscillations, it can be deduced that the surface film is mainly composed of oxide, and that the formation and dissolution of the oxide film play a key role in the current oscillations of the system.     

Electrochemistry and Electrocatalysis with Myoglobin in Biomembrane-Like DHP-PDDA Polyelectrolyte-Surfactant Complex Films

The polyelectrolyte-surfactant complex DHP-PDDA was prepared by reacting the anionic surfactant dihexadecylphosphate (DHP) with polycationic poly(diallyldimethylammonium) (PDDA). Thin films made from DHP-PDDA on solid substrates demonstrated an ordered multibilayer structure by XRD and DSC. Incorporated myoglobin (Mb) in DHP-PDDA films on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for the Mb Fe(III)/Fe(II) couple at about -0.3 V vs SCE in pH 7.0 buffers. Electron transfer between Mb and PG electrodes was greatly facilitated in the film microenvironment. The positions of the Soret absorption band suggest that Mb maintains its secondary structure similar to its native state in DHP-PDDA films in the medium pH range. Mb could act as an enzyme-like catalyst in DHP-PDDA films as demonstrated by catalytic reduction of trichloroacetic acid, nitrite, and oxygen with a decrease in the electrode potentials required. Mb-DHP-PDDA films may thus have potential application as biosensors. Copyright 2001 Academic Press.     

Electrochemical Fabrication of Prussian Blue Nanocube‐decorated Electroreduced Graphene Oxide for Amperometric Sensing of NADH

In this study, Prussian blue (PB) film on the electroreduced graphene oxide (ERGO)‐modified Au electrode surface (ERGO/PB) is easily prepared by means of cyclic voltammetric technique in the mixture of K₃Fe(CN)₆ and FeCl₃. Its electrochemical behaviors for NADH biosensor are studied. The structural and morphological characters of modified electrode material are analyzed with using of XPS, XRD, Raman, EDS, and SEM techniques. ERGO/PB hybrid nanocomposite for NADH biosensor is exhibited to the higher catalytic effect (linear range from 1.0 to 100 μM, detection limit of 0.23 μM at S/N=3) compared to naked Au, ERGO‐modified Au, and PB‐modified Au electrodes. In addition to, ERGO/PB electrode was used to voltammetric and amperometric detection of H₂O₂. ERGO/PB electrodes also showed the same behavior as the NADH sensor. This ERGO/PB‐modified electrode supplied a simple, new, and low‐cost route for amperometric sensing of both NADH and H₂O₂.     

Influence of Ionic Strength on Electrochemical Responses of Myoglobin Loaded in Polysaccharide Layer‐by‐Layer Assembly Films

Two polysaccharides hydroxyethyl cellulose ethoxylate (HECE) and hyaluronic acid (HA) were assembled into {HECE/HA}_n layer‐by‐layer films on electrodes. The films were then immersed in myoglobin (Mb) solutions to load Mb into the films. The Mb‐loaded films showed a nearly reversible cyclic voltammetric (CV) peak pair at ?0.34 V vs. SCE in pH 7.0 buffers. The effect of ionic strength in Mb loading solutions and CV testing solutions on the CV response of the films was investigated. The direct electrochemistry of Mb loaded in the films could also be used to electrocatalyze the reduction of oxygen and H₂O₂ in solution.     

Electrochemical and electrocatalytic properties of myoglobin and hemoglobin incorporated in carboxymethyl cellulose films

Protein-CMC films were made by casting a solution of myoglobin (Mb) or hemoglobin (Hb) and carboxymethyl cellulose (CMC) on pyrolytic graphite electrodes. In pH 7.0 buffers, Mb and Hb incorporated in CMC films gave a pair of well-defined and quasi-reversible cyclic voltammetric peaks at about -0.34 V vs. SCE, respectively, characteristic of heme Fe(III)/Fe(II) redox couples of the proteins. The electrochemical parameters such as apparent standard heterogeneous electron transfer rate constants (k(s)) and formal potentials (E degrees ') were estimated by square wave voltammetry with nonlinear regression analysis. In aqueous solution, stable CMC films absorbed large amounts of water and formed hydrogel. Scanning electron microscopy of the films showed that interaction between Mb or Hb and CMC would make the morphology of dry protein-CMC films different from the CMC films alone. Positions of Soret absorbance band suggest that Mb and Hb in CMC films retain their secondary structure similar to the native states in the medium pH range. Trichloroacetic acid, nitrite, oxygen, and hydrogen peroxide were catalytically reduced at protein-CMC film electrodes.     

A hydrogen peroxide biosensor based on Langmuir-Blodgett technique: Direct electron transfer of hemoglobin in octadecylamine layer

The present paper describes the modification of hemoglobin (Hb)-octadecylamine (ODA) Langmuir-Blodgett (LB) film on a gold electrode surface to develop a novel electrochemical biosensor for the detection of hydrogen peroxide. Atomic force microscopy (AFM) image of Hb-ODA LB film indicated Hb molecules existed in ODA layer in a well-ordered and compact form. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of 4.58 ± 0.95 s⁻¹ and a formal potential of −185 mV (versus Ag/AgCl) in phosphate buffer (1.0 mM, pH 5.0) contain 0.1 M KCl at a scan rate of 200 mV s⁻¹, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The formal potential of Hb heme Fe(III)/Fe(II) redox couple in ODA film shifted linearly between pH 5 and 8 with a slope of −23.8 mV pH⁻¹, suggesting that proton took part in electrochemical reaction. The ODA could accelerate the electron transfer between Hb and the electrode. This modified electrode showed an electrochemical activity to the reduction of hydrogen peroxide (H₂O₂) without the aid of any electron mediator.