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.     

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 electron transfer for hemoglobin in biomembrane-like dimyristoyl phosphatidylcholine films on pyrolytic graphite electrodes

Stable thin films made from dimyristoyl phosphatidylcholine (DMPC) with incorporated hemoglobin (Hb) on pyrolytic graphite (PG) electrodes were characterized by electrochemical and other techniques. Cyclic voltammetry (CV) of Hb-DMPC films showed a pair of well-defined and nearly reversible peaks at about -0.27 V vs. saturated calomel electrode (SCE) at pH 5.5, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The electron transfer between Hb and PG electrodes was greatly facilitated in DMPC films. Apparent heterogeneous rate constants (ks) were estimated by fitting square wave voltammograms of Hb-DMPC films to a model featuring thin layer behavior and dispersion of formal potentials for redox center. The formal potential of Hb heme Fe(III)/Fe(II) couple in DMPC films shifted linearly between pH 4.5 to 11 with a slope of -48 mV pH-1, suggesting that one proton is coupled to each electron transfer in the electrochemical reaction. Soret absorption band positions suggest that Hb retains a near native conformation in DMPC films at medium pH. Differential scanning calorimetry (DSC) showed the phase transition for DMPC and Hb-DMPC films, suggesting DMPC has an ordered multibilayer structure. Trichloroacetic acid (TCA) was catalytically reduced by Hb-DMPC films with significant decreases in the electrode potential required.     

Heme protein-gluten films: voltammetric studies and their electrocatalytic properties

Direct electrochemistry and electrocatalysis of heme proteins, such as hemoglobin (Hb), myoglobin (Mb), and horseradish peroxidase (HRP), incorporated in gluten biopolymer films cast on pyrolytic graphite (PG) electrodes, were studied by voltammetry and amperometry. All the three protein-gluten films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about −0.28 V versus saturated calomel electrode (SCE) in pH 5.5 buffers, respectively, characteristic of the heme Fe(III)/Fe(II) redox couples, indicating enhanced electron transfer between the proteins and PG electrodes in a gluten film environment. The protein-gluten hydrogel films showed excellent stability. Positions of Soret absorption band of protein-gluten films suggested that the heme proteins kept their secondary structure similar to their native state in the films in the medium pH range. The heme proteins in gluten films were act as a biologic catalyst to catalyze reduction of oxygen or hydrogen peroxide. The voltammetric or amperometric responses of H₂O₂ at the protein-gluten film electrodes could be used to determine the concentration of 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.     

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.     

Direct electrochemistry and electrocatalysis of hemoglobin entrapped in semi-interpenetrating polymer network hydrogel based on polyacrylamide and chitosan

Semi-interpenetrating polymer network (semi-IPN) hydrogel based on polyacrylamide (PAM) and chitosan was prepared to immobilize redox protein hemoglobin (Hb). The Hb-PAM-chitosan hydrogel film obtained has been investigated by scanning electron microscopy (SEM) and UV-VIS spectroscopy. UV-VIS spectroscopy showed that Hb kept its secondary structure similar to its native state in the Hb-PAM-chitosan hydrogel film. Cyclic voltammogram of Hb-PAM-chitosan film-modified glass carbon (GC) electrode showed a pair of well-defined and quasi-reversible redox peaks for Hb Fe(III)/Fe(II), indicating that direct electron transfer between Hb and GC electrode occurred. The electron-transfer rate constant was about 5.51 s(-1) in pH 7.0 buffers, and the formal potential (E degrees ') was -0.324 V (vs. SCE). The dependence of E degrees ' on solution pH indicated that one-proton transfer was coupled to each electron transfer in the direct electron-transfer reaction. Additionally, Hb in the semi-IPN hydrogel film retained its bioactivity and showed excellent electrocatalytic activity toward H(2)O(2). The electrocatalytic current values were linear with increasing concentration of H(2)O(2) in a wide range of 5-420 microM. The unique semi-IPN hydrogel would have wide potential applications in direct electrochemistry, biosensors and biocatalysis.     

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 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.     

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.     

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.     

pH-dependent behaviors of electroactive myoglobin loaded into layer-by-layer films assembled with alginate and hydroxyethyl cellulose ethoxylate

In the present work, strong polybase quaternized hydroxyethyl cellulose ethoxylate (HECE) and weak polyacid alginate (AA) were assembled into {HECE/AA} n layer-by-layer (LBL) films on electrodes by electrostatic interaction between them, and the films were then immersed in myoglobin (Mb) solution to load Mb into the films, designated as {HECE/AA}n-Mb. The {HECE/AA}n-Mb films showed a nearly reversible cyclic voltammetric (CV) peak pair at about -0.34 V vs SCE in pH 7.0 buffers for Mb heme Fe(III)/Fe(II) redox couple, and the surface concentration of electroactive Mb in the films (Gamma*) was affected significantly by the pH of Mb loading solution and testing solution. The amount of Mb loaded from pH 5.0 solution was much larger than that from pH 9.0 solution, which is mainly attributed to the higher degree of swelling, porosity, and permeability of {HECE/AA}n films at pH 5.0 than at pH 9.0. In addition, the electrostatic interaction between Mb and the AA component in the films might also play an important role in Mb loading. The pH of the testing solution where {HECE/AA}n-Mb films were tested by CV also influenced the Gamma* value, showing that the fraction of electroactive Mb among the total Mb loaded into the films increased remarkably as the pH of the testing solution decreased. This result is rationalized in terms of the pH-dependent film permeability toward counterions and the electron-hopping mechanism in electron transfer of redox proteins in the film phase. This model system may provide a general and effective approach to control the electroactivity of immobilized redox proteins in the multilayer assembly containing weak polyions by adjusting pH and may guide us to develop the new kind of controllable electrochemical biosensors based on the direct electrochemistry of enzymes.     

Direct electrochemistry and electrocatalysis with horseradish peroxidase in Eastman AQ films

Stable films made from ionomer poly(ester sulfonic acid) or Eastman AQ29 on pyrolytic graphite (PG) electrodes gave direct electrochemistry for incorporated enzyme horseradish peroxidase (HRP). Cyclic voltammetry of HRP-AQ films showed a pair of well-defined, nearly reversible peaks at about -0.33 V vs. SCE at pH 7.0 in blank buffers, characteristic of HRP heme Fe(III)/Fe(II) redox couple. The electron transfer between HRP and PG electrode was greatly facilitated in AQ films. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E(o')) were estimated by fitting the data of square-wave voltammetry (SWV) with nonlinear regression analysis. Reflectance absorption infrared (RAIR) and UV-Vis absorption spectra demonstrated that HRP retained a near native conformation in AQ films. The embedded HRP in AQ films retained the electrocatalytic activity for oxygen, nitrite and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) with HRP-AQ films was proposed.     

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 and photosensitized redox reactions of a prussian blue film layered on a WO3/[Ru(bpy)3]2+/polymer hybrid film.

A hybrid film of WO(3)/tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)(3)](2+))/poly(sodium 4-styrenesulfonate) (PSS) (denoted as a WRP hybrid film) was prepared as a base layer on an indium tin oxide electrode substrate by cathodic electrodeposition from a colloidal ternary solution containing peroxotungstic acid, [Ru(bpy)(3)](2+), and PSS. Prussian blue, Fe(III) (4)[Fe(II)(CN)(6)](3) (Fe(II)-Fe(III)) was cathodically electrodeposited on the WRP hybrid film from a Berlin brown (Fe(III)-Fe(III)) colloidal solution to give a WRP/Fe(II)-Fe(III) bilayer film. Spectrocyclic voltammetry measurement of the WRP/Fe(II)-Fe(III) bilayer film reveals that Prussian white (Fe(II)-Fe(II)) is oxidized to Fe(II)-Fe(III) by electrogenerated Ru(III), and Fe(II)-Fe(III) is re-reduced to Fe(II)-Fe(II) by electrogenerated H(x)WO(3). Visible-light irradiation of the WRP hybrid film generates a small photocurrent (approximately 8 nA cm(-2)) at 0.4 V of an applied potential, whereas irradiation of the WRP/Fe(II)-Fe(II) bilayer film (Fe(II)-Fe(III) is electrochemically reduced to the Fe(II)-Fe(II) state) significantly generates a steady photoanodic current of 2.0-1.1 microA cm(-2) under the same conditions, thus demonstrating that the photoanodic current is produced by the layered Fe(II)-Fe(II) film. The photoaction spectrum of the bilayer film reveals that the photoanodic current is based on the photoexcitation of [Ru(bpy)(3)](2+). The photogeneration of Fe(II)-Fe(III) from Fe(II)-Fe(II) is shown by the absorption spectral change of the bilayer film on irradiation. These results corroborate the notion that Fe(II)-Fe(II) is oxidized by photogenerated Ru(III) to generate Fe(II)-Fe(III). However, the rate of photogeneration of Fe(II)-Fe(III) is slow, which could be ascribed to the fast back electron transfer (ET) from WO(3) to Ru(III), comparable with the forward ET from Fe(II)-Fe(II) to Ru(III). The fast back ET could be a crucial problem for the [Ru(bpy)(3)](2+)-sensitized reaction in the hybrid film.     

Direct electrochemistry of hemoglobin immobilized in the sodium alginate and SiO2 nanoparticles bionanocomposite film on a carbon ionic liquid electrode

In this paper a carbon ionic liquid electrode (CILE) was fabricated by using a room temperature ionic liquid of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) as binder. By using the CILE as basal electrode, the hemoglobin (Hb) molecule was immobilized on the surface of CILE with a sodium alginate (SA) hydrogel and SiO₂ nanoparticles organic-inorganic composite material. The direct electrochemical behaviors of Hb in the bionanocomposite film were further studied in a pH 7.0 Britton-Robinson (B-R) buffer solution. A pair of well-defined quasi-reversible cyclic voltammetric peaks of Hb was obtained on SA/nano-SiO₂/Hb/CILE with the formal potential (E^0’) at -0.355 V (vs. SCE), which was the characteristic of heme Fe(III)/Fe(II) redox couples. The formal potential of Hb Fe(III)/Fe(II) couple shifted negatively with increasing pH of solution with a slope of -45.2 mV/pH, which indicated that a one electron transfer accompanied with one proton transportation. The immobilized Hb showed good electrocatalytic manner to the reduction of trichloroacetic acid (TCA).     

New degenerate metal-oxide electrodes for nearly reversible direct electron transfer to the redox proteins

Heavily doped cadmium tin oxide (CTO) film electrodes were developed for fast electron exchange with redox proteins. The metal oxide films showed nearly reversible electron transfer for the [2Fe–2S] proteins spinach ferredoxin (Sp fd) and putidaredoxin (Pdx), and the well-studied heme protein horse heart cytochrome c. These represent a family of proteins that are of comparable size, but vary significantly in overall charge, formal redox potential, and type of metal center. The unmediated electron exchange was achieved through variation of metal oxide film synthesis parameters that led to an increase of the charge carrier concentration up to the levels typical for degenerate semiconductors. In addition, the flat band potential of the films was shifted close to or more positive of the formal redox potentials of proteins such that the semiconductor electrodes would be utilized in an accumulation mode. The rates and sustainability of electron transfer for the two ferredoxins obtained on these cadmium tin oxide electrodes are as high or higher than previously reported.     

Electroreduction of some pyridine carboxamides on carbon electrodes in aqueous solutions

The electroreductions of the NAD⁺ model compounds nicotinamide (I), N₁-methyl nicotinamide (II), N′-methyl nicotinamide (III) and isonicotinamide (IV) on carbon electrodes have been studied in aqueous media in the pH range 0–12 by linear-sweep cyclic voltammetry (Scheme 1, I-IV). Logarithmic analyses of the reduction peaks were performed by computing the convolution of the current with time as a function of the potential. On the basis of the experimental results it was concluded that the irreversibility of the electron transfers increased when a glassy carbon electrode was used, and this irreversibility being more marked when a plastic formed carbon electrode was employed. The reduction processes occurred with more difficulty on carbon electrodes than on mercury electrodes. Both the reduction and the reoxidation (when occurred) processes changed with respect to those observed on mercury electrodes, being irreversible electron transfers the rate-determining steps in most cases. Thus, for compounds I, II and III at pH < 2 the reductions occurred by the uptake of two electrons and two H⁺ ions, and the rate determining step was found to be the first one-electron transfer, for I and III, and the irreversible second electron transfer, preceded by the uptake of an H+ ion, for II. At pH>3 the processes consisted of electrodimerization reactions, preceded by the protonation of the heterocyclic nitrogen in cases I and III. The second electron transfer of the electroreduction of IV always appeared irreversible, in contrast with that found for mercury electrodes.     

血红蛋白在离子液体[BMIM]PF6碳糊电极上的直接电化学

制备了离子液体[BMIM]PF6修饰碳糊电极(CILE), 并对其形貌和电化学行为进行了表征. 采用涂布法利用壳聚糖-皂土有机-无机复合膜将血红蛋白(Hb)固定于CILE电极表面, 利用紫外可见光谱、红外光谱和电化学方法等手段对包埋于膜内的Hb的性质进行了表征. 结果表明, Hb在薄膜内保持了其原始构象与生物活性, 循环伏安实验表明, 在pH=7.0的Britton-Robinson (B-R)缓冲液中, Hb表现出一对峰形良好的准可逆氧化还原峰, 为Hb Fe(III)/Fe(II)电对的特征峰, 对其直接电化学行为进行了研究, 求出式电位为-0.352 V(vs SCE), 电子转移数为0.885, 电荷传递系数为0.578, 表观异相电子转移速率常数为0.149 s-1.     

Layer-by-layer films of hemoglobin or myoglobin assembled with zeolite particles: electrochemistry and electrocatalysis

Positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 in solutions and negatively charged zeolite particles in dispersions were alternately adsorbed onto solid surfaces forming [zeolite/protein](n) layer-by-layer films, which was confirmed by quartz crystal microbalance (QCM) and cyclic voltammetry (CV). The protein films assembled on pyrolytic graphite (PG) electrodes exhibited a pair of well-defined, nearly reversible CV peaks at about -0.35 V vs. SCE at pH 7.0, characteristic of the heme Fe(III)/Fe(II) redox couples. Hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)) in solution were catalytically reduced at [zeolite/protein](7) film modified electrodes, and could be quantitatively determined by CV and amperometry. The shape and position of infrared amide I and II bands of Hb or Mb in [zeolite/protein](7) films suggest that the proteins retain their near-native structure in the films. The penetration experiments of Fe(CN)(6)(3-) as the electroactive probe into these films and scanning electron microscopy (SEM) results indicate that the films possess a great amount of pores or channels. The porous structure of ]zeolite/protein](n) films is beneficial to counterion transport, which is crucial for protein electrochemistry in films controlled by the charge-hopping mechanism, and is also helpful for the diffusion of catalysis substrates into the films. The proteins with negatively charged net surface charges at pH 9.0 were also successfully assembled with like-charged zeolite particles into layer-by-layer films, although the adsorption amount was less than that assembled at pH 5.0. The possible reasons for this were discussed, and the driving forces were explored.