A Review on Direct Electrochemistry of Catalase for Electrochemical Sensors
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paper_title: Catalase immobilization on electrospun nanofibers : Effects of porphyrin pendants and carbon nanotubes

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Carbon nanotube (CNT) has been generally recognized as an electron acceptor, whereas porphyrin can act as an electron donor. In this work, acrylonitrile-based copolymers bearing porphyrin pendants were therefore blended with or without CNTs and then electrospun into nanofibers on which a redox enzyme, catalase, was covalently immobilized. The uniform nanofibers diameters are around 180 nm. Field emission scanning electron microscopy and transmission electron microscopy confirmed the morphologies of the nanofibers and the distribution of CNTs. Rougher surfaces as well as obvious protruded parts induced by the blending of CNTs were observed from the studied nanofibers. Catalase was immobilized onto the nanofiber surface through the activation of carboxyl groups by N-(3-dimethylaminopropyl)-N‘-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). Results indicate that both the introduction of porphyrin pendants and CNTs obviously improve the activity and stabilities of the immobilized catalases...
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paper_title: Novel method for the investigation of the electrochemistry of metalloproteins: cytochrome c

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The d.c. and a.c. cyclic voltammetries of horse heart ferricytochrome c have been investigated and it is shown that in the presence of 4,4′-bipyridyl, the electrochemistry corresponds to a quasi-reversible one-electron process, from which an E° value of +0·25 V vs. normal hydrogen electrode can be derived.
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paper_title: Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films.

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Abstract Protein–chitosan (CS) films were made by casting a solution of proteins and CS on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) incorporated in CS films gave a pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks at about −0.33 V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about −0.46 V which was not stable. All these peaks are located at the potentials characteristic of heme Fe III /Fe II redox couples of the proteins. The electrochemical parameters such as formal potentials (E° ′ ) and apparent heterogeneous electron-transfer rate constants ( k s ) were estimated by square-wave voltammetry with nonlinear regression analysis. Chitosan films contained considerable water and formed hydrogel in aqueous solution. Positions of the Soret absorbance band suggest that Mb and Hb in CS films keep their secondary structure similar to the native states in the medium pH range, while HRP and Ct retain their native conformation at least in the dry CS films. Scanning electron microscopy of the films demonstrated that interaction between the proteins and CS would make the morphology of dry protein–CS films very different from the CS films alone. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all four proteins in CS films.
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paper_title: Direct and indirect electrochemical investigations of metalloenzymes.

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Publisher Summary All redox metalloproteins can be investigated by electrochemical methods. There is no difficulty in achieving the electrochemistry of small redox proteins such as cytochromes, ferredoxins, blue copper proteins, and flavodoxins. Direct electrochemistry proceeds without the need for an electron transfer shuttle, or mediator, between the redox center of the protein and the electrode. However, at most metal electrodes the presence of a promoter is required. Such a compound binds to the electrode surface and, while not itself taking part in the electron transfer process, encourages electron transfer with the protein to proceed. Redox enzymes may be classified as being intrinsic or extrinsic in nature. To enable communication between an enzyme and the electrode, the surface of the latter must bind the enzyme and prevent denaturation. An intrinsic redox enzyme is one in which electron transfer associated with the catalyzed event is contained within the confines of the active site—that is, the enzyme lacks a natural long-range electron transfer pathway. Thus, achieving heterogeneous electron transfer with an electrode may require that (1) the site of the catalytic reaction be close to the enzyme surface, (2) the enzyme be able to deform without losing its activity, (3) the electrode surface (with or without a promoter) project into the enzyme, or (4) electron transfer pathways be introduced by modification. Indirect electrochemical communication between an electrode and a metalloenzyme may be achieved using an associated redox protein as a mediator, in other words, as an electron transfer shuttle between the enzyme and the working electrode.
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paper_title: Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis

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Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite (PG) electrodes. Hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP), and catalase (Cat) incorporated in PAMAM films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. While Hb-, Mb-, and HRP-PAMAM films showed the cyclic voltammetry (CV) peaks at about −0.34 V vs. saturated calomel electrode (SCE) in pH 7.0 buffers, Cat-PAMAM films displayed the peak pair at a more negative potential of −0.47 V. The protein-PAMAM films demonstrated a surface-confined or thin-layer voltammetric behavior. The electrochemical parameters such as apparent heterogeneous electron transfer rate constants (ks) and formal potentials (E°′) were estimated by square wave voltammetry with nonlinear regression analysis. UV–vis and IR spectroscopy showed that the proteins retained their near-native secondary structures in PAMAM films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the protein-PAMAM film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the proteins.
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paper_title: Enzyme-catalyzed direct electron transfer: Fundamentals and analytical applications

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This article overviews the fundamentals of the phenomenon of direct electron transfer in enzyme-catalyzed electrode reactions and the development of electroanalytical applications of the bioelectrocatalytic systems. A brief description of the enzymes capable of catalyzing electrochemical reactions by means of direct electron transfer is given. The physico-chemical background of bioelectrocatalysis is discussed in terms of the different concepts underlying the mechanism of electron transfer. The concept of a “molecular transducer” is introduced to designate a complex, formed by the electrocatalytically active enzyme on the electrode-electrolyte interface, which is directly responsible for the transduction of the chemical signal to an electric one. The role of this “molecular transducer” in enzyme electrodes and immunoelectrodes is discussed. The analytical applications of bioelectrocatalysis are categorized as systems employing either amperometric or potentiometric detection. Discussion focuses on the advantages of systems based on enzyme-catalyzed direct electron transfer as opposed to other enzyme-catalyzed electroanalytical devices. In conclusion, the trends towards upcoming practical applications are suggested as well as some directions in fundamental studies of bioelectrocatalysis as a phenomenon.
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paper_title: Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide.

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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.
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paper_title: Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors

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Direct electron transfer (DET) between redox enzymes and electrodes found the basis for third generation biosensors. Recent investigations in the authors' laboratories on the bioelectrochemistry of ...
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paper_title: Direct voltammetry of catalase immobilized on silica sol-gel and cysteine modified gold electrode and its application.

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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.
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paper_title: REVERSIBLE ELECTRODE REACTION OF CYTOCHROME C

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The heme protein, cytochrome c, was found to exhibit reversible electron transfer characteristics at an indium oxide electrode. The electrode reaction at this electrode was evaluated using cyclic voltammetry and differential pulse method.
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paper_title: Direct Electrochemistry of Catalase at a Gold Electrode Modified with Single-Wall Carbon Nanotubes

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The direct electrochemistry of catalase (Ct) was accomplished at a gold electrode modified with single-wall carbon nanotubes (SWNTs). A pair of well-defined redox peaks was obtained for Ct with the reduction peak potential at 0.414 V and a peak potential separation of 32 mV at pH 5.9. Both reflectance FT-IR spectra and the dependence of the reduction peak current on the scan rate revealed that Ct adsorbed onto the SWNT surfaces. The redox wave corresponds to the Fe(III)/Fe(II) redox center of the heme group of the Ct adsorbate. Compared to other types of carbonaceous electrode materials (e.g., graphite and carbon soot), the electron transfer rate of Ct redox reaction was greatly enhanced at the SWNT-modified electrode. The peak current was found to increase linearly with the Ct concentration in the range of 8 10 6 ±8 10 5 M used for the electrode preparation and the peak potential was shown to be pH dependent. The catalytic activity of Ct adsorbates at the SWNTs appears to be retained, as the addition of H2O2 produced a characteristic catalytic redox wave. This work demonstrates that direct electrochemistry of redox-active biomacromolecules such as metalloenzymes can be improved through the use of carbon nanotubes.
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paper_title: Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

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Abstract 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 (ks) and formal potential (E°′) 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 H2O2 at Cat-PAM film electrodes was proposed.
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paper_title: Interaction of sodium bis(2-ethylhexyljt) sulfosuccinate (AOT) with catalase and horseradish peroxidase in an aqueous solution and in the reverse micelles of AOT/n-heptane

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Some properties of catalase and horseradish peroxidase entrapped into reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane with those in an aqueous solution of AOT are compared. Secondary structure of catalase significantly changes and activity is completely lost in the presence of aqueous micellar solution of AOT. In AOT/n-heptane reverse micelles secondary structure of catalase does not change. AOT has no effect on horseradish peroxidase in aqueous solution. On the other hand slight changes in secondary structure of horseradish peroxidase in AOT/n-heptane reverse micelles appear. It is concluded that electrostatic interactions between catalase or horseradish peroxidase with AOT molecule at neutral pH are not the main factor determining reactivity of investigated enzymes in reverse micelles.
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paper_title: Direct Electron Transfer of Heme- and Molybdopterin Cofactor-Containing Chicken Liver Sulfite Oxidase on Alkanethiol-Modified Gold Electrodes

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Direct heterogeneous electron transfer (ET) of sulfite oxidase (SOx), a heme- and molybdopterin cofactor-containing intermembrane enzyme, was studied on alkanethiol-modified Au electrodes both with SOx entrapped between the modified Au electrode and a permselective membrane and with SOx adsorbed at the electrode surface, in the absence of any membrane. SOx in direct electronic communication with the electrode surface gave a quasi-reversible electrochemical signal with a midpoint potential of −120 mV vs Ag | AgCl corresponding to the redox transformations of the heme domain of SOx and with a heterogeneous ET constant in the order of 15 s-1. The efficiency of the bioelectrocatalytic 2e- oxidation of sulfite catalyzed by SOx in direct ET exchange with the electrode was shown to depend essentially on the nature of the alkanethiol layer. Adsorption and orientation of SOx on an 11-mercapto-1-undecanol (MuD-OH) self-assembled monolayer, i.e., terminally functionalized with OH groups, provided efficient catalytic...
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paper_title: Protein electrodes with direct electrochemical communication.

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Electrochemistry using direct electron transfer between an electrode and a protein or an enzyme has developed into a means for studying biological redox reactions and for bioanalytics, biosynthesis and bioenergetics. This review summarizes recent work on direct protein electrochemistry with special emphasis on our results in bioelectrocatalysis using isolated enzymes and enzyme-protein couples.
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paper_title: Electrospun nanofibrous membranes filled with carbon nanotubes for redox enzyme immobilization

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Abstract Catalases decompose hydrogen peroxide (H 2 O 2 ) into water and oxygen in an effective and environmentally friend way. The decomposition of H 2 O 2 by nanofibrous membrane-supported catalases is reported in this work. Poly(acrylonitrile- co - N -vinyl-2-pyrrolidone) (PANCNVP) and polyacrylonitrile (PAN) blending with or without multi-walled carbon nanotubes (MWCNTs) were electrospun into nanofibrous membranes as enzyme immobilization matrixes and were carefully characterized. To immobilize catalase covalently, the nanofibrous membranes were treated with alkali solution to generate carboxyl groups followed by activation procedure with EDC/NHS. Results indicate that the nanofibrous membranes can bind large amount of catalases due to their superior surface area to volume ratio. The decomposition behavior of H 2 O 2 with these immobilized catalases was explored. The increment of activity retention induced by the NVP moieties and/or MWCNTs is remarkable. Stabilities of the immobilized catalases including the effects of substrate pH and reaction temperature, thermal stability, operational stability and storage stability were also carefully studied. Results elucidate the improved stabilities of the nanofibrous membrane-supported catalases. In conclusion, the nanofibrous membranes electrospun from this biocompatible polymer, especially those filled with MWCNTs, are suitable matrices for catalase immobilization.
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paper_title: Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film.

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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.
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paper_title: An electrochemical investigation of hemoglobin and catalase incorporated in collagen films.

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Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around -0.35 V and -0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV-vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.
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paper_title: Effects of hydrophilic room-temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate on direct electrochemistry and bioelectrocatalysis of heme proteins entrapped in agarose hydrogel films

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Abstract The effects of 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF 4 ]) on direct electrochemistry and bioelectrocatalysis of hemoglobin (Hb), myoglobin (Mb), and catalase (Cat) entrapped in agarose hydrogel films were investigated. A small amount of water in [bmim][BF 4 ] is necessary to maintain the electrochemical activities of these heme proteins. The direct electron transfer between heme proteins and glassy carbon electrode (GC) is a surface-confined quasi-reversible process, corresponding to heme Fe(III) + e → heme Fe(II). Several electrochemical parameters of these processes, such as the charge-transfer coefficient ( a ) and the apparent electron transfer rate constant ( k s ), were calculated by nonlinear regression analysis of square wave voltammetry (SWV) experimental data. Furthermore, highly electrocatalytic activities of the heme protein–agarose films toward hydrogen peroxide (H 2 O 2 ) were observed, indicating that heme proteins entrapped in agarose films retained their bioelectrocatalytic activities in [bmim][BF 4 ] solution. The apparent Michaelis-Menten constant ( K m app ) of Mb for H 2 O 2 was evaluated to be 5.93 × 10 −4 mol L −1 . The catalytic reduction peak current is directly proportional to the concentration of H 2 O 2 between 1.28 × 10 −6 mol L −1 and 7.08 × 10 −4 mol L −1 . Experiments for Hb and Cat received similar results.
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paper_title: Recent developments in faradaic bioelectrochemistry

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Progress in the area of faradaic bioelectrochemistry over the last decade has been reviewed. Electrochemical and spectroscopic studies of the protein–electrode interface are discussed along with the use of electron transfer promoters, self-assembled monolayers (SAMs), and surfactant films. Voltammetric techniques are described that permit rapid, in-situ measurement of formal potentials while also providing kinetic resolution on the sub-millisecond time scale. Some enzymes, especially those of analytical interest, must be coupled or ‘wired’ to the electrode using mediators in order to achieve rapid electron transfer. Supramolecular structures designed to facilitate electron transfer and to immobilize co-factors are described. The use of electrochemically-based biosensors for in-vivo measurements is documented.
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paper_title: Direct electrochemistry of catalase on glassy carbon electrodes.

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Abstract Catalase was investigated as a possible catalyst of the electrochemical reduction of oxygen on glassy carbon electrodes. The presence of catalase dissolved in solution only provoked a moderate current increase, which was fully explained by the catalase-catalysed disproportionation of hydrogen peroxide (Scheme I). When catalase was adsorbed from dimethylsulfoxide on the surface of electrodes that did not undergo any electrochemical pre-treatment (EP), catalase efficiently catalysed oxygen reduction via direct electron transfer from the electrode (Scheme II). The results are discussed with respect to the electrode surface properties and the enzyme structure.
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paper_title: Direct voltammetry and catalysis of hemoenzymes in methyl cellulose film

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Horseradish peroxidase (HRP) and catalase (Cat) immobilized on edge-plane pyrolytic graphite (EPG) electrodes by methyl cellulose (MC). Both the hemoenzymes entrapped in the MC film undergo fast direct electron-transfer reaction, corresponding to hemeFe(III)+e→hemeFe(II). The formal potential (E0′), the apparent coverage (Γ), the electron transfer coefficient (α) and the apparent electron transfer rate constant (ks) were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. E0′ are linearly dependent on pH, indicating the electron transfer of Fe(III)/Fe(II) redox couple companied with the transfer of proton. The processes of catalytically reducing oxygen, hydrogen peroxide and nitric oxide by HRP and Cat entrapped in MC film are also explored.
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paper_title: Chemical and electrochemical approaches to the investigation of redox reactions of simple electron transfer metalloproteins

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Abstract The redox chemistry of simple metalloproteins such as cytochrome, plastocyanin and ferredoxin has been studied extensively by both chemically and electrochemically based techniques. However, despite the fact that complementary information can be obtained, in practice the results of the two methods generally have been reported and interpreted in an independent manner. In this paper an overview of the information that can be gained from the two approaches is presented. In both areas, a complex range of problems exists that are often considerably more challenging than those commonly encountered in studies on smaller redox active molecules. Consequently, interpretation of data obtained from either method when based on standard approaches and concepts is often fraught with danger. Nevertheless, knowledge related to important thermodynamic relationships, rates of electron transfer and structural changes associated with electron transfer processes are slowly being unravelled from both fields of endeavour. It is therefore proposed that the inherently difficult challenge of using both forms of methodology could now be attempted more frequently than currently is the case and that the dual approach provides an opportunity to synergistically enhance knowledge related to biologically important electron transfer processes of redox active metalloproteins.
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paper_title: Direct electrochemistry and electrocatalysis of heme-proteins in regenerated silk fibroin film

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Abstract A biocompatible silk fibroin (SF) film provided a feasible microenvironment for heme-proteins to direct electron transfer on graphite electrodes (GE). Myoglobin (Mb), hemoglobin (Hb), horseradish peroxidase (HRP), and catalase (Cat) in corporated in SF films exhibited a pair of well-defined, nearly reversible cyclic voltammetric peaks, corresponding to the reaction of hemeFe (III) + e → hemeFe (II). The formal potential ( E 0 ), the apparent coverage ( Γ ) and the electron transfer rate constant ( k s ) of four proteins in SF films were evaluated by analyzing the cyclic voltammograms (CVs) of heme-proteins. The formal potential was pH dependent, suggesting that proton ion was involved in the reaction. Ultraviolet visible (UV–vis) spectra and reflectance absorbance infrared (RAIR) spectra indicated that heme-proteins in SF films were not grossly denatured. The structure of heme-proteins–SF films was investigated using scanning electron microscopy (SEM) and RAIR. It indicated that there existed intermolecular interaction between heme-proteins and SF and this governed their different morphology in SF films. Hydrogen peroxide and nitric oxide were catalytically reduced by the heme-proteins in SF films, showing the potential applicability of the heme-proteins–SF films as the new type of biosensors based on the protein film voltammetry.
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paper_title: Characterization for didodecyldimethylammonium bromide liquid crystal film entrapping catalase with enhanced direct electron transfer rate.

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Direct electrochemistry for catalase (CAT) embedded in the liquid crystal film of didodecyldimethylammonium bromide (DDAB) was investigated at pyrolytic graphite (PG) electrode by voltammetric methods. The reduction reaction involved the redox couple in CAT, i.e. FeIII/FeII couple. The electron transfer between the incorporated CAT and PG electrode was found to be greatly enhanced by DDAB. The heterogeneous electron transfer rate constant k(s) was fitted as 3.0 +/- 0.4 s(-1) using the nonlinear regression analysis of the square wave voltammograms at a series of pulse heights. The pH dependence of the formal potential for CAT in DDAB film was 57 mV/pH, which suggested one-proton-transfer together with a one-electron reaction. Visible absorption and reflectance-absorbance infrared (RAIR) spectra inferred the similar heme environment of CAT in DDAB film to its native status. Circular dichroism (CD) results indicated DDAB affected slightly on the second structure of CAT.
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paper_title: Enzyme encapsulation in layer-by-layer engineered polymer multilayer capsules.

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We report on the encapsulation of enzyme (catalase) by the controlled polymer multilayer coating of biocrystals, achieved by the sequential adsorption of oppositely charged polyelectrolytes onto enzyme crystal templates. An extremely high enzyme loading in each polymer capsule is obtained, and the activity of the encapsulated enzyme is preserved. The polymer-encapsulated enzyme is stable against protease degradation: The polymer-coated enzyme retains 100% of its activity after incubation for 100 min with protease, whereas uncoated, solubilized catalase loses more than 90% of its initial activity within 100 min under the same conditions. This simple, general, and versatile approach can potentially be applied for the encapsulation of various crystallized substances for catalysis and drug delivery applications.
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paper_title: Thin films of polyelectrolyte-encapsulated catalase microcrystals for biosensing.

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Polyelectrolyte (PE)-encapsulated catalase microcrystals were assembled onto gold electrodes by their sequential deposition with oppositely charged PEs, utilizing electrostatic interactions to form enzyme thin films for biosensing. The PE coating around the microcrystals provided a regular surface charge, thus facilitating the stepwise film growth, and it effectively prevented catalase leakage from the assembled films. The encapsulated catalase was shown to retain both its biological and its electrochemical activity. Direct electron transfer between catalase molecules and the gold electrode was achieved without the aid of any electron mediator. In pH 5.0 phosphate buffer solution, the apparent formal potential (E(o)') of catalase was -0.131 V (vs Ag/AgCl). As a H2O2 biosensor, films consisting of one layer of the encapsulated catalase displayed considerably higher (approximately 5-fold) and more stable electrocatalytic responses to the reduction of H2O2 than did corresponding films made of one layer of nonencapsulated catalase or solubilized catalase. An increase in either the number of "precursor" PE layers between the gold electrodes and the catalase microcrystal layers in the film or the number of PE layers encapsulating the catalase microcrystals was found to decrease the electrocatalytic activity of the electrode. At low precursor PE layer numbers (approximately 2) and PE encapsulating layers (approximately 4), the current response was proportional to the H2O2 concentration in the range 3.0 x 10(-6) to 1.0 x 10(-2) M. The overall electroactivity of the multilayer film increased for the first two layers of encapsulated catalase, after which a plateau was observed. This was attributed to the increasing difficulty of electron transfer and substrate diffusion limitations. The current approach of using immobilized PE-encapsulated enzyme microcrystals for biosensing provides a versatile method to prepare high enzyme content films with high and tailored enzyme activities.
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paper_title: Characterization for didodecyldimethylammonium bromide liquid crystal film entrapping catalase with enhanced direct electron transfer rate.

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Direct electrochemistry for catalase (CAT) embedded in the liquid crystal film of didodecyldimethylammonium bromide (DDAB) was investigated at pyrolytic graphite (PG) electrode by voltammetric methods. The reduction reaction involved the redox couple in CAT, i.e. FeIII/FeII couple. The electron transfer between the incorporated CAT and PG electrode was found to be greatly enhanced by DDAB. The heterogeneous electron transfer rate constant k(s) was fitted as 3.0 +/- 0.4 s(-1) using the nonlinear regression analysis of the square wave voltammograms at a series of pulse heights. The pH dependence of the formal potential for CAT in DDAB film was 57 mV/pH, which suggested one-proton-transfer together with a one-electron reaction. Visible absorption and reflectance-absorbance infrared (RAIR) spectra inferred the similar heme environment of CAT in DDAB film to its native status. Circular dichroism (CD) results indicated DDAB affected slightly on the second structure of CAT.
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paper_title: Interaction of sodium bis(2-ethylhexyljt) sulfosuccinate (AOT) with catalase and horseradish peroxidase in an aqueous solution and in the reverse micelles of AOT/n-heptane

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Some properties of catalase and horseradish peroxidase entrapped into reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane with those in an aqueous solution of AOT are compared. Secondary structure of catalase significantly changes and activity is completely lost in the presence of aqueous micellar solution of AOT. In AOT/n-heptane reverse micelles secondary structure of catalase does not change. AOT has no effect on horseradish peroxidase in aqueous solution. On the other hand slight changes in secondary structure of horseradish peroxidase in AOT/n-heptane reverse micelles appear. It is concluded that electrostatic interactions between catalase or horseradish peroxidase with AOT molecule at neutral pH are not the main factor determining reactivity of investigated enzymes in reverse micelles.
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paper_title: Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films.

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Abstract Protein–chitosan (CS) films were made by casting a solution of proteins and CS on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) incorporated in CS films gave a pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks at about −0.33 V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about −0.46 V which was not stable. All these peaks are located at the potentials characteristic of heme Fe III /Fe II redox couples of the proteins. The electrochemical parameters such as formal potentials (E° ′ ) and apparent heterogeneous electron-transfer rate constants ( k s ) were estimated by square-wave voltammetry with nonlinear regression analysis. Chitosan films contained considerable water and formed hydrogel in aqueous solution. Positions of the Soret absorbance band suggest that Mb and Hb in CS films keep their secondary structure similar to the native states in the medium pH range, while HRP and Ct retain their native conformation at least in the dry CS films. Scanning electron microscopy of the films demonstrated that interaction between the proteins and CS would make the morphology of dry protein–CS films very different from the CS films alone. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all four proteins in CS films.
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paper_title: Direct voltammetry of catalase immobilized on silica sol-gel and cysteine modified gold electrode and its application.

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

paper_content:

Abstract 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 (ks) and formal potential (E°′) 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 H2O2 at Cat-PAM film electrodes was proposed.
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paper_title: Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film.

paper_content:

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.
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paper_title: An electrochemical investigation of hemoglobin and catalase incorporated in collagen films.

paper_content:

Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around -0.35 V and -0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV-vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.
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paper_title: Direct voltammetry and catalysis of hemoenzymes in methyl cellulose film

paper_content:

Horseradish peroxidase (HRP) and catalase (Cat) immobilized on edge-plane pyrolytic graphite (EPG) electrodes by methyl cellulose (MC). Both the hemoenzymes entrapped in the MC film undergo fast direct electron-transfer reaction, corresponding to hemeFe(III)+e→hemeFe(II). The formal potential (E0′), the apparent coverage (Γ), the electron transfer coefficient (α) and the apparent electron transfer rate constant (ks) were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. E0′ are linearly dependent on pH, indicating the electron transfer of Fe(III)/Fe(II) redox couple companied with the transfer of proton. The processes of catalytically reducing oxygen, hydrogen peroxide and nitric oxide by HRP and Cat entrapped in MC film are also explored.
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paper_title: Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis

paper_content:

Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite (PG) electrodes. Hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP), and catalase (Cat) incorporated in PAMAM films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. While Hb-, Mb-, and HRP-PAMAM films showed the cyclic voltammetry (CV) peaks at about −0.34 V vs. saturated calomel electrode (SCE) in pH 7.0 buffers, Cat-PAMAM films displayed the peak pair at a more negative potential of −0.47 V. The protein-PAMAM films demonstrated a surface-confined or thin-layer voltammetric behavior. The electrochemical parameters such as apparent heterogeneous electron transfer rate constants (ks) and formal potentials (E°′) were estimated by square wave voltammetry with nonlinear regression analysis. UV–vis and IR spectroscopy showed that the proteins retained their near-native secondary structures in PAMAM films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the protein-PAMAM film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the proteins.
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paper_title: Hydrogen peroxide biosensor based on direct electrochemistry of soybean peroxidase immobilized on single-walled carbon nanohorn modified electrode.

paper_content:

Single-walled carbon nanohorns (SWCNHs) were used as a novel and biocompatible matrix for fabricating biosensing devices. The direct immobilization of acid-stable and thermostable soybean peroxidase (SBP) on SWCNH modified electrode surface can realize the direct electrochemistry of enzyme. Cyclic voltammogram of the adsorbed SBP displays a pair of redox peaks with a formal potential of -0.24 V in pH 5 phosphate buffer solution. The formal potential has a linear relationship with pH from 3 to 9 with a slope of -48.7 mV/pH, close to the value of -55.7 mV/pH expected at 18 degrees C for the reversible transfer of one proton and one electron. Bioactivity of SBP remains good in SWCNH microenvironment, along with effective catalysis of the reduction of H(2)O(2). In the absence of a mediator, this H(2)O(2) biosensor exhibited a high sensitivity (16.625 microAL/mmol), a linear range from 0.02 to 1.2 mmolL(-1), and a detection limit of 5.0 x 10(-7) mmolL(-1), as well as acceptable preparation reproducibility and excellent stability.
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paper_title: Aqueous dispersions of SWCNTs using pyrrolic surfactants for the electro-generation of homogeneous nanotube composites. Application to the design of an amperometric biosensor

paper_content:

A new approach for the fabrication of homogeneous single-walled carbon nanotube/polypyrrole composites is presented. Stable SWCNT dispersions were obtained using the electropolymerizable (11-pyrrolyl-1-ylundecyl)triethylammonium tetrafluoroborate as surfactant. For these dispersions, we used commercially available HiPco® SWCNTs (purified) as received without any further treatment. Highly homogeneous composite materials could be electrogenerated, which were analyzed by scanning electron microscopy showing thin SWCNT bundles embedded in the polymer matrix. The quality of the SWCNT composite was tested with regard to its performance for biosensor applications. Enzymes like glucose oxidase and tyrosinase were entrapped in this copolymer during electropolymerization. The resulting glucose and catechol biosensors exhibit a drastic increase of sensitivities and maximum currents compared with the same sensor setup without nanotubes.
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paper_title: Direct Electrochemistry of Catalase at a Gold Electrode Modified with Single-Wall Carbon Nanotubes

paper_content:

The direct electrochemistry of catalase (Ct) was accomplished at a gold electrode modified with single-wall carbon nanotubes (SWNTs). A pair of well-defined redox peaks was obtained for Ct with the reduction peak potential at 0.414 V and a peak potential separation of 32 mV at pH 5.9. Both reflectance FT-IR spectra and the dependence of the reduction peak current on the scan rate revealed that Ct adsorbed onto the SWNT surfaces. The redox wave corresponds to the Fe(III)/Fe(II) redox center of the heme group of the Ct adsorbate. Compared to other types of carbonaceous electrode materials (e.g., graphite and carbon soot), the electron transfer rate of Ct redox reaction was greatly enhanced at the SWNT-modified electrode. The peak current was found to increase linearly with the Ct concentration in the range of 8 10 6 ±8 10 5 M used for the electrode preparation and the peak potential was shown to be pH dependent. The catalytic activity of Ct adsorbates at the SWNTs appears to be retained, as the addition of H2O2 produced a characteristic catalytic redox wave. This work demonstrates that direct electrochemistry of redox-active biomacromolecules such as metalloenzymes can be improved through the use of carbon nanotubes.
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paper_title: Picomolar Detection of Hydrogen Peroxide at Glassy Carbon Electrode Modified with NAD+ and Single Walled Carbon Nanotubes

paper_content:

Potential cycling was used for oxidation of NAD+ and producing an electroactive redox couple which strongly adsorbed on the electrode surface modified with single walled carbon nanotubes (SWCNTs). Modified electrode shows a pair of well defined and nearly reversible redox peaks at pH range 1–13 and the response showed a surface-controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (ks) of adsorbed redox couple onto CNTs films were about 6.32×10−10 mol cm−2 and 2.0 (±0.20) s−1, respectively, indicating the high loading ability of CNTs toward the oxidation product of NAD+ (2,8-dihydroxy adenine dinucleotide) and great facilitation of the electron transfer between redox couple and CNTs immobilized onto electrode surface. The modified electrode exhibited excellent electrocatalytic activity for H2O2 reduction at reduced overpotential. The catalytic rate constant for H2O2 reduction was found to be 2.22(±0.20)×104 M−1 s−1. The catalytic reduction current allows the amperometric detection of H2O2 at an applied potential of −0.25 V vs. Ag/AgCl with a detection limit of 10 pM and linear response up to 100 nM and resulting analytical sensitivity 747.6 nA/pM. The remarkably low detection limit (10 pM) is the lowest value ever reported for direct H2O2 determination on the electrodes at pH 7. The modified electrode can be used for monitoring H2O2 without the need for an enzyme or enzyme mimic. The proposed method for rapid amperometric detection of H2O2 is low cost and high throughput. Furthermore, the sensor can be used to any detection scheme that uses enzymatically generated H2O2 as a reactive product in biological systems.
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paper_title: Amperometric Glucose Biosensor Based on Immobilization of Glucose Oxidase in Chitosan Matrix Cross-Linked with Glutaraldehyde

paper_content:

A glucose biosensor was fabricated using chitosan film for the immobilization of glucose oxidase (GOX) and mediator in the following “sandwich” configuration:chitosan-ferrocene:GOX:chitosan using a carbon paste electrode as the basic electrode. To prevent loss of immobilized GOX under optimized conditions, the biosensor surface was cross-linked with glutaraldehyde (GA). Despite the multilayer configuration, the biosensor exhibited a relatively fast response (less than 10 s) and a linear response with glucose in the range of 8×10−4 to 1.7×10−2 M. The biosensor retained about 65 % of its original activity after 30 days of storage in a phosphate buffer at 4 °C. Applicability of the biosensor was demonstrated in real samples and the results obtained by this biosensor corroborate well with a classical UV spectrophotometric technique.
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paper_title: Direct electrochemistry and electrocatalysis of catalase immobilized on a SWNT-nanocomposite film

paper_content:

Abstract Investigations are reported regarding the electrochemical performance of catalase (CAT) immobilized on a film composed of chitosan-wrapped single-walled carbon nanotubes (SWNTs), and the use of such a system as a biosensor. The immobilized CAT displays a pair of well-defined and quasi-reversible redox peaks, with a formal potential ( E °′) of −0.476 V vs. SCE in 0.1 M, pH 7.0 phosphate buffer solution; also the electrochemical response indicates a surface-controlled electrode process. The dependence of formal potential on solution pH indicated that the direct electron transfer reaction of CAT is a one-electron transfer coupled with a one-proton transfer reaction. The heterogeneous electron transfer rate constant was measured as 118 s −1 , indicating electron transfer between catalase and the modified electrode surface is greatly improved over that which is typically reported in the literature. This result is likely attributed to close contacts between the electroactive centers and the SWNTs. Experimental results demonstrate that absorbed CAT exhibits remarkable electrocatalytic activities toward the reduction of oxygen, hydrogen peroxide and nitrite. Also, the modified electrode exhibited good analytical performance for the amperometric determination of hydrogen peroxide and nitrite, and might find use as a third-generation biosensor based on the direct electrochemistry of catalase.
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paper_title: A biosensing platform based on horseradish peroxidase immobilized onto chitosan-wrapped single-walled carbon nanotubes

paper_content:

One-step, diameter-selective dispersion of single-walled carbon nanotubes (SWCNTs) has been accomplished through noncovalent complexation of the nanotubes with a water-soluble, biocompatible polymer chitosan at room temperature. Such chitosan-wrapped individual SWCNTs can be used for the immobilization of horseradish peroxidase (HRP) and be used to construct an electrode for direct bioelectrochemical sensing without an electron mediator. The direct electron transfer between HRP and the electrode surface was observed with a formal potential of approximately −0.35 V (vs. saturated calomel electrode) in phosphate buffer solution and the calculated heterogeneous electron transfer rate constant is approximately 23.5 s−1. Experimental results indicate that the immobilized HRP retains its catalytic activity for the reduction of nitric oxide. Such an HRP–SWCNT–chitosan-based biosensor exhibited a rapid response time of less than 6 s and a good linear detection range for nitrite concentration, from 25 to 300 μM with a detection limit of 3 μM. The apparent Michaelis–Menten constant (Km) and the maximum electrode sensitivity (imax/Km) are found to be 7.0 mM and 0.16 µA mM−1, respectively. Both the unique electrical properties of SWCNTs and biocompatibility of chitosan enable the construction of an excellent biosensing platform for improved electrocatalysis of HRP, allowing, specifically, the detection of trace levels of nitric oxide.
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paper_title: Bioinorganic Composites for Enzyme Electrodes

paper_content:

Sparingly soluble redox salts were combined with a model enzyme, glucose oxidase, in a host matrix of a biopolymer chitosan to form bioinorganic composite films on the surface of glassy carbon electrodes. Four redox salts, each containing the Ru(NH3)63+ cation and a selected anion, such as Ru(CN)64-, Fe(CN)64-, Co(CN)63- or IrCl63-, were studied. The composition and catalytic properties of such composite materials toward glucose oxidation were investigated by spectroscopic and electrochemical methods. The composite films provided an oxygen-independent electrical communication between the enzyme's redox centers and a glassy carbon surface at a potential as low as −0.10 V vs Ag/AgCl(3 M Cl-). The nature of the electrical communication is discussed in terms of redox mediation by the Ru(NH3)63+-containing ion pairs formed inside the biocomposites. The kinetic significance of the mediator's charge is considered by postulating that neutral ion pairs are more efficient redox mediators of the enzymatic reaction t...
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paper_title: Flow system for fish freshness determination based on double multi-enzyme reactor electrodes.

paper_content:

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.
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paper_title: Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film.

paper_content:

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.
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paper_title: An electrochemical investigation of hemoglobin and catalase incorporated in collagen films.

paper_content:

Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around -0.35 V and -0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV-vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.
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paper_title: Effects of hydrophilic room-temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate on direct electrochemistry and bioelectrocatalysis of heme proteins entrapped in agarose hydrogel films

paper_content:

Abstract The effects of 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF 4 ]) on direct electrochemistry and bioelectrocatalysis of hemoglobin (Hb), myoglobin (Mb), and catalase (Cat) entrapped in agarose hydrogel films were investigated. A small amount of water in [bmim][BF 4 ] is necessary to maintain the electrochemical activities of these heme proteins. The direct electron transfer between heme proteins and glassy carbon electrode (GC) is a surface-confined quasi-reversible process, corresponding to heme Fe(III) + e → heme Fe(II). Several electrochemical parameters of these processes, such as the charge-transfer coefficient ( a ) and the apparent electron transfer rate constant ( k s ), were calculated by nonlinear regression analysis of square wave voltammetry (SWV) experimental data. Furthermore, highly electrocatalytic activities of the heme protein–agarose films toward hydrogen peroxide (H 2 O 2 ) were observed, indicating that heme proteins entrapped in agarose films retained their bioelectrocatalytic activities in [bmim][BF 4 ] solution. The apparent Michaelis-Menten constant ( K m app ) of Mb for H 2 O 2 was evaluated to be 5.93 × 10 −4 mol L −1 . The catalytic reduction peak current is directly proportional to the concentration of H 2 O 2 between 1.28 × 10 −6 mol L −1 and 7.08 × 10 −4 mol L −1 . Experiments for Hb and Cat received similar results.
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paper_title: Enzyme sensor for L-lactate with a chitosan-mercury film electrode

paper_content:

SummaryAn amperometric enzyme sensor composed of a mercury film electrode and an enzyme-immobilized chitosan membrane is developed. This biosensor is based on both a mercury film electrode detecting the consumption of dissolved dioxygen following enzymatic reaction, and a chitosan membrane. The latter provides an excellent permselectivity and excludes electroactive interferents. The detection range of this biosensor was 1.0×10−5–3.0×10−4 mol/l and the relative standard deviation, R.S.D. at 5.0×10−5 mol/l was 1.4% (n=3). This biosensor was applied to the direct determination of L-lactate in human serum without pretreatment.
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paper_title: Direct electrochemistry and electrocatalysis of heme-proteins in regenerated silk fibroin film

paper_content:

Abstract A biocompatible silk fibroin (SF) film provided a feasible microenvironment for heme-proteins to direct electron transfer on graphite electrodes (GE). Myoglobin (Mb), hemoglobin (Hb), horseradish peroxidase (HRP), and catalase (Cat) in corporated in SF films exhibited a pair of well-defined, nearly reversible cyclic voltammetric peaks, corresponding to the reaction of hemeFe (III) + e → hemeFe (II). The formal potential ( E 0 ), the apparent coverage ( Γ ) and the electron transfer rate constant ( k s ) of four proteins in SF films were evaluated by analyzing the cyclic voltammograms (CVs) of heme-proteins. The formal potential was pH dependent, suggesting that proton ion was involved in the reaction. Ultraviolet visible (UV–vis) spectra and reflectance absorbance infrared (RAIR) spectra indicated that heme-proteins in SF films were not grossly denatured. The structure of heme-proteins–SF films was investigated using scanning electron microscopy (SEM) and RAIR. It indicated that there existed intermolecular interaction between heme-proteins and SF and this governed their different morphology in SF films. Hydrogen peroxide and nitric oxide were catalytically reduced by the heme-proteins in SF films, showing the potential applicability of the heme-proteins–SF films as the new type of biosensors based on the protein film voltammetry.
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paper_title: Characterization for didodecyldimethylammonium bromide liquid crystal film entrapping catalase with enhanced direct electron transfer rate.

paper_content:

Direct electrochemistry for catalase (CAT) embedded in the liquid crystal film of didodecyldimethylammonium bromide (DDAB) was investigated at pyrolytic graphite (PG) electrode by voltammetric methods. The reduction reaction involved the redox couple in CAT, i.e. FeIII/FeII couple. The electron transfer between the incorporated CAT and PG electrode was found to be greatly enhanced by DDAB. The heterogeneous electron transfer rate constant k(s) was fitted as 3.0 +/- 0.4 s(-1) using the nonlinear regression analysis of the square wave voltammograms at a series of pulse heights. The pH dependence of the formal potential for CAT in DDAB film was 57 mV/pH, which suggested one-proton-transfer together with a one-electron reaction. Visible absorption and reflectance-absorbance infrared (RAIR) spectra inferred the similar heme environment of CAT in DDAB film to its native status. Circular dichroism (CD) results indicated DDAB affected slightly on the second structure of CAT.
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paper_title: Attachment of Nanoparticles to Pyrolytic Graphite Electrode and Its Application for the Direct Electrochemistry and Electrocatalytic Behavior of Catalase

paper_content:

Abstract A highly hydrophilic, nontoxic, and conductive effect of colloidal gold nanoparticles (GNP) and multi-walled carbon nanotubes (MWCNT) on pyrolytic graphite electrode has been demonstrated. The direct electron transfer of catalase (CAT) was achieved based on the immobilization of MWCNT/CAT-GNP on a pyrolytic graphite electrode by a Nafion film. The immobilized catalase displayed a pair of well-defined and nearly reversible redox peaks in 0.1 M phosphate buffer solution (PBS) (pH 6.98). The dependence of E°′on solution pH indicated that the direct electron transfer reaction of catalase was a single-electron-transfer coupled with single-proton-transfer reaction process. The immobilized catalase maintained its biological activity, showing a surface controlled electrode process with an apparent heterogeneous electron transfer rate constant (k s) of 1.387±0.1 s−1 and charge-transfer coefficient (α) of 0.49, and displayed electrocatalytic activity in the electrocatalytic reduction of hydrogen peroxide. ...
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paper_title: Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions

paper_content:

MANY properties of colloids and suspensions depend on the particle size. Series of monodisperse suspensions of the same chemical composition but of rather different particle sizes may be used to study particle size dependent phenomena, such as Brownian motion, light scattering, sedimentation and electrophoresis of small particles. We have used such series to demonstrate the increased tendency of metal suspensions to coagulate in the presence of electrolytes as the radius of the particles increases1.
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paper_title: Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film.

paper_content:

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.
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paper_title: An electrochemical investigation of hemoglobin and catalase incorporated in collagen films.

paper_content:

Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around -0.35 V and -0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV-vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.
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paper_title: Effects of hydrophilic room-temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate on direct electrochemistry and bioelectrocatalysis of heme proteins entrapped in agarose hydrogel films

paper_content:

Abstract The effects of 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF 4 ]) on direct electrochemistry and bioelectrocatalysis of hemoglobin (Hb), myoglobin (Mb), and catalase (Cat) entrapped in agarose hydrogel films were investigated. A small amount of water in [bmim][BF 4 ] is necessary to maintain the electrochemical activities of these heme proteins. The direct electron transfer between heme proteins and glassy carbon electrode (GC) is a surface-confined quasi-reversible process, corresponding to heme Fe(III) + e → heme Fe(II). Several electrochemical parameters of these processes, such as the charge-transfer coefficient ( a ) and the apparent electron transfer rate constant ( k s ), were calculated by nonlinear regression analysis of square wave voltammetry (SWV) experimental data. Furthermore, highly electrocatalytic activities of the heme protein–agarose films toward hydrogen peroxide (H 2 O 2 ) were observed, indicating that heme proteins entrapped in agarose films retained their bioelectrocatalytic activities in [bmim][BF 4 ] solution. The apparent Michaelis-Menten constant ( K m app ) of Mb for H 2 O 2 was evaluated to be 5.93 × 10 −4 mol L −1 . The catalytic reduction peak current is directly proportional to the concentration of H 2 O 2 between 1.28 × 10 −6 mol L −1 and 7.08 × 10 −4 mol L −1 . Experiments for Hb and Cat received similar results.
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paper_title: Direct electrochemistry and electrocatalysis of heme-proteins in regenerated silk fibroin film

paper_content:

Abstract A biocompatible silk fibroin (SF) film provided a feasible microenvironment for heme-proteins to direct electron transfer on graphite electrodes (GE). Myoglobin (Mb), hemoglobin (Hb), horseradish peroxidase (HRP), and catalase (Cat) in corporated in SF films exhibited a pair of well-defined, nearly reversible cyclic voltammetric peaks, corresponding to the reaction of hemeFe (III) + e → hemeFe (II). The formal potential ( E 0 ), the apparent coverage ( Γ ) and the electron transfer rate constant ( k s ) of four proteins in SF films were evaluated by analyzing the cyclic voltammograms (CVs) of heme-proteins. The formal potential was pH dependent, suggesting that proton ion was involved in the reaction. Ultraviolet visible (UV–vis) spectra and reflectance absorbance infrared (RAIR) spectra indicated that heme-proteins in SF films were not grossly denatured. The structure of heme-proteins–SF films was investigated using scanning electron microscopy (SEM) and RAIR. It indicated that there existed intermolecular interaction between heme-proteins and SF and this governed their different morphology in SF films. Hydrogen peroxide and nitric oxide were catalytically reduced by the heme-proteins in SF films, showing the potential applicability of the heme-proteins–SF films as the new type of biosensors based on the protein film voltammetry.
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paper_title: Direct electrochemistry and electrocatalysis of catalase immobilized on multi-wall carbon nanotubes modified glassy carbon electrode and its application

paper_content:

Abstract The direct electrochemical and electrocatalytic properties of catalase (CAT) immobilized on the multi-wall carbon nanotubes (MWCNT) modified glassy carbon (MWCNT/GC) electrode was investigated in 0.1 M Tris–HCl buffer solution. It was found that CAT immobilized on the MWCNT/GC electrode could undergo a direct and quasi-reversible reaction with the formal potential ( E 0 ′) of −0.456 V. The electrochemical reaction involves the transfer of one electron and one proton. Furthermore, La 3+ can affect the electrochemical behavior of the immobilized CAT depending on the concentration of La 3+ . The low concentration of La 3+ can promote, but the high concentration of La 3+ would inhibit the electrochemical reaction of the immobilized CAT. This is due to that the different concentrations of La 3+ could increase or decrease the exposure extent of the heme group in CAT. CAT immobilized on the MWCNT/GC electrode could keep its biocatalytic activity for the H 2 O 2 reduction. These results illustrated that MWCNT is a good carrier for immobilizing the biomicromolecules. This may be useful in the biosensors and biofuel cells.
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paper_title: Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films.

paper_content:

Abstract Protein–chitosan (CS) films were made by casting a solution of proteins and CS on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) incorporated in CS films gave a pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks at about −0.33 V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about −0.46 V which was not stable. All these peaks are located at the potentials characteristic of heme Fe III /Fe II redox couples of the proteins. The electrochemical parameters such as formal potentials (E° ′ ) and apparent heterogeneous electron-transfer rate constants ( k s ) were estimated by square-wave voltammetry with nonlinear regression analysis. Chitosan films contained considerable water and formed hydrogel in aqueous solution. Positions of the Soret absorbance band suggest that Mb and Hb in CS films keep their secondary structure similar to the native states in the medium pH range, while HRP and Ct retain their native conformation at least in the dry CS films. Scanning electron microscopy of the films demonstrated that interaction between the proteins and CS would make the morphology of dry protein–CS films very different from the CS films alone. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all four proteins in CS films.
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paper_title: Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

paper_content:

Abstract 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 2.4 × 10 −10 mol cm −2 . The transfer coefficient (α) 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.
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paper_title: Direct voltammetry and catalysis with Mycobacterium tuberculosis catalase-peroxidase, peroxidases, and catalase in lipid films.

paper_content:

Stable films of dimyristoylphosphatidylcholine and M. tuberculosis catalase-peroxidase (KatG), several peroxidases, myoglobin, and catalase showed reversible FeIII/FeII voltammetry on pyrolytic graphite electrodes and catalytic current for hydrogen peroxide and oxygen. Amperometric responses for these films to H2O2 at 0 V are likely to contain significant contributions from catalytic reduction of oxygen produced during the catalytic cycles. Relative apparent turnover rates at pH 6 based on steady-state currents at 0 V versus SCE in the presence of H2O2 were in the order horseradish peroxidase > cytochrome c peroxidase (CcP) > soybean peroxidase > myoglobin > KatG > catalase. Lower currents for the very efficient peroxide scavengers KatG and catalase may be related to the instability of their compounds I in the presence of H2O2. KatG catalyzed the electrochemical reduction of oxygen more efficiently than catalase and CcP but less efficiently than the other peroxidases. DMPC films incorporating glucose oxidase and peroxidases gave good analytical responses to glucose, demonstrating the feasibility of dual enzyme-lipid films for biosensor fabrication.
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paper_title: Attachment of Nanoparticles to Pyrolytic Graphite Electrode and Its Application for the Direct Electrochemistry and Electrocatalytic Behavior of Catalase

paper_content:

Abstract A highly hydrophilic, nontoxic, and conductive effect of colloidal gold nanoparticles (GNP) and multi-walled carbon nanotubes (MWCNT) on pyrolytic graphite electrode has been demonstrated. The direct electron transfer of catalase (CAT) was achieved based on the immobilization of MWCNT/CAT-GNP on a pyrolytic graphite electrode by a Nafion film. The immobilized catalase displayed a pair of well-defined and nearly reversible redox peaks in 0.1 M phosphate buffer solution (PBS) (pH 6.98). The dependence of E°′on solution pH indicated that the direct electron transfer reaction of catalase was a single-electron-transfer coupled with single-proton-transfer reaction process. The immobilized catalase maintained its biological activity, showing a surface controlled electrode process with an apparent heterogeneous electron transfer rate constant (k s) of 1.387±0.1 s−1 and charge-transfer coefficient (α) of 0.49, and displayed electrocatalytic activity in the electrocatalytic reduction of hydrogen peroxide. ...
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paper_title: Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

paper_content:

Abstract 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 (ks) and formal potential (E°′) 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 H2O2 at Cat-PAM film electrodes was proposed.
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paper_title: Direct electrochemistry of catalase at multiwalled carbon nanotubes-nafion in presence of needle shaped DDAB for H2O2 sensor.

paper_content:

The direct electrochemistry of catalase (CAT) at didodecyldimethylammonium bromide (DDAB) present on nafion dispersed multiwalled carbon nanotubes (MWCNTs-NF) modified glassy carbon electrode (GCE) has been reported. The presence of DDAB in MWCNTs-NF-CAT film enhances the surface coverage concentration of CAT (Fe(III/II)) to 48%. Similarly, in presence of DDAB, there is a 57% enhancement in electron transfer rate (ks) with 66% increase in CAT stability. (Fe(III/II)) redox couple exhibits linear dependence with the pH variation (-51 mV pH(-1)). The UV-vis absorption spectroscopy study reveals the entrapped CAT in DDAB film retains its native structure at MWCNTs-NF modified electrodes. Similarly, electrochemical impedance spectroscopy results confirm the co-existence of CAT and DDAB in the modified film. Further, scanning electron microscopy results reveal the structural morphological difference between various components in MWCNTs-NF-(DDAB/CAT) film. The cyclic voltammetry (CV) and amperometry (i-t curve) have been used for the measurement of electroanalytical properties of H2O2 by means of various film modified GCEs. The sensitivity values of MWCNTs-NF-(DDAB/CAT) film for H2O2 using CV (35.62 microA mM(-1)cm2) are higher than the values which are obtained for MWCNTs-NF-CAT film (2.74 mmicroA mM(-1)cm2). Similarly, the sensitivity values using i-t curve are 101.74 microA mM(-1)cm2 for MWCNTs-NF-(DDAB/CAT) and 74.69 microA mM(-1)cm2 for MWCNTs-NF-CAT film. Finally, the diffusion coefficient of H2O2 at MWCNTs-NF-(DDAB/CAT) film (3.4 x 10(-10) cm2 s(-1)) has been calculated using rotating disc electrode studies.
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paper_title: Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film.

paper_content:

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.
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paper_title: An electrochemical investigation of hemoglobin and catalase incorporated in collagen films.

paper_content:

Collagen, an electrochemically inert protein, formed films on pyrolytic graphite (PG) electrodes, which provided a suitable microenvironment for heme proteins to transfer electron directly with the underlying electrodes. Hemoglobin (Hb) and catalase (Cat) incorporated in collagen films exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks at around -0.35 V and -0.47 V (vs. SCE) in pH 7.0 buffers, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. UV-vis spectra showed that the heme proteins in collagen films retained their near-native conformations in the medium pH range. The results of scanning electron microscopy (SEM) demonstrated that the interaction between heme proteins and collagen made the morphology of dry protein-collagen films different from the collagen films alone. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') of the films were estimated by using square wave voltammograms (SWV) and nonlinear regression analysis. The heme protein-collagen film electrodes were also used to catalyze the reduction of nitrite, oxygen and hydrogen peroxide, indicating potential applications of the films for the fabrication of a new type of biosensor that does not use mediators.
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paper_title: Effects of hydrophilic room-temperature ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate on direct electrochemistry and bioelectrocatalysis of heme proteins entrapped in agarose hydrogel films

paper_content:

Abstract The effects of 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF 4 ]) on direct electrochemistry and bioelectrocatalysis of hemoglobin (Hb), myoglobin (Mb), and catalase (Cat) entrapped in agarose hydrogel films were investigated. A small amount of water in [bmim][BF 4 ] is necessary to maintain the electrochemical activities of these heme proteins. The direct electron transfer between heme proteins and glassy carbon electrode (GC) is a surface-confined quasi-reversible process, corresponding to heme Fe(III) + e → heme Fe(II). Several electrochemical parameters of these processes, such as the charge-transfer coefficient ( a ) and the apparent electron transfer rate constant ( k s ), were calculated by nonlinear regression analysis of square wave voltammetry (SWV) experimental data. Furthermore, highly electrocatalytic activities of the heme protein–agarose films toward hydrogen peroxide (H 2 O 2 ) were observed, indicating that heme proteins entrapped in agarose films retained their bioelectrocatalytic activities in [bmim][BF 4 ] solution. The apparent Michaelis-Menten constant ( K m app ) of Mb for H 2 O 2 was evaluated to be 5.93 × 10 −4 mol L −1 . The catalytic reduction peak current is directly proportional to the concentration of H 2 O 2 between 1.28 × 10 −6 mol L −1 and 7.08 × 10 −4 mol L −1 . Experiments for Hb and Cat received similar results.
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paper_title: Direct electrochemistry and electrocatalysis of heme-proteins in regenerated silk fibroin film

paper_content:

Abstract A biocompatible silk fibroin (SF) film provided a feasible microenvironment for heme-proteins to direct electron transfer on graphite electrodes (GE). Myoglobin (Mb), hemoglobin (Hb), horseradish peroxidase (HRP), and catalase (Cat) in corporated in SF films exhibited a pair of well-defined, nearly reversible cyclic voltammetric peaks, corresponding to the reaction of hemeFe (III) + e → hemeFe (II). The formal potential ( E 0 ), the apparent coverage ( Γ ) and the electron transfer rate constant ( k s ) of four proteins in SF films were evaluated by analyzing the cyclic voltammograms (CVs) of heme-proteins. The formal potential was pH dependent, suggesting that proton ion was involved in the reaction. Ultraviolet visible (UV–vis) spectra and reflectance absorbance infrared (RAIR) spectra indicated that heme-proteins in SF films were not grossly denatured. The structure of heme-proteins–SF films was investigated using scanning electron microscopy (SEM) and RAIR. It indicated that there existed intermolecular interaction between heme-proteins and SF and this governed their different morphology in SF films. Hydrogen peroxide and nitric oxide were catalytically reduced by the heme-proteins in SF films, showing the potential applicability of the heme-proteins–SF films as the new type of biosensors based on the protein film voltammetry.
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paper_title: Characterization for didodecyldimethylammonium bromide liquid crystal film entrapping catalase with enhanced direct electron transfer rate.

paper_content:

Direct electrochemistry for catalase (CAT) embedded in the liquid crystal film of didodecyldimethylammonium bromide (DDAB) was investigated at pyrolytic graphite (PG) electrode by voltammetric methods. The reduction reaction involved the redox couple in CAT, i.e. FeIII/FeII couple. The electron transfer between the incorporated CAT and PG electrode was found to be greatly enhanced by DDAB. The heterogeneous electron transfer rate constant k(s) was fitted as 3.0 +/- 0.4 s(-1) using the nonlinear regression analysis of the square wave voltammograms at a series of pulse heights. The pH dependence of the formal potential for CAT in DDAB film was 57 mV/pH, which suggested one-proton-transfer together with a one-electron reaction. Visible absorption and reflectance-absorbance infrared (RAIR) spectra inferred the similar heme environment of CAT in DDAB film to its native status. Circular dichroism (CD) results indicated DDAB affected slightly on the second structure of CAT.
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paper_title: Myoglobin on multi-walled carbon nanotubes modified electrode: direct electrochemistry and electrocatalysis

paper_content:

Abstract Electrochemical behavior of myoglobin (Myb) incorporated on multi-walled carbon nanotubes (MWNTs) modified GC electrode is investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The results indicate that Myb can be strongly adsorbed on the surface of the MWNT-modified electrode to form an approximate monolayer film. MWNTs can greatly promote the redox of horse heart Myb, on which a pair of well-defined and nearly reversible CV peaks for Myb Fe(III)/Fe(II) redox couple were obtained in pH 7.0 buffers. The formal potential of Myb on MWNT modified electrode shifts linearly with pH with a slope of −56.4 mV/pH, showing that the electron transfer is accompanied by single proton transportation. The high efficiency of the Myb/MWNT modified electrode towards the catalytic electro-reduction of nitric oxide has been observed. Potential application of Myb/MWNT modified electrode as biosensors to monitor NO is proposed.
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paper_title: Catalase immobilization on electrospun nanofibers : Effects of porphyrin pendants and carbon nanotubes

paper_content:

Carbon nanotube (CNT) has been generally recognized as an electron acceptor, whereas porphyrin can act as an electron donor. In this work, acrylonitrile-based copolymers bearing porphyrin pendants were therefore blended with or without CNTs and then electrospun into nanofibers on which a redox enzyme, catalase, was covalently immobilized. The uniform nanofibers diameters are around 180 nm. Field emission scanning electron microscopy and transmission electron microscopy confirmed the morphologies of the nanofibers and the distribution of CNTs. Rougher surfaces as well as obvious protruded parts induced by the blending of CNTs were observed from the studied nanofibers. Catalase was immobilized onto the nanofiber surface through the activation of carboxyl groups by N-(3-dimethylaminopropyl)-N‘-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS). Results indicate that both the introduction of porphyrin pendants and CNTs obviously improve the activity and stabilities of the immobilized catalases...
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paper_title: Covalent immobilization of redox enzyme on electrospun nonwoven poly(acrylonitrile-co-acrylic acid) nanofiber mesh filled with carbon nanotubes: a comprehensive study.

paper_content:

In this work, novel conductive composite nanofiber mesh possessing reactive groups was electrospun from solutions containing poly(acrylonitrile-co-acrylic acid) (PANCAA) and multi-walled carbon nanotubes (MWCNTs) for redoxase immobilization, assuming that the incorporated MWCNTs could behave as electrons transferor during enzyme catalysis. The covalent immobilization of catalase from bovine liver on the neat PANCAA nanofiber mesh or the composite one was processed in the presence of EDC/NHS. Results indicated that both the amount and activity retention of bound catalase on the composite nanofiber mesh were higher than those on the neat PANCAA nanofiber mesh, and the activity increased up to 42%. Kinetic parameters, K(m) and V(max), for the catalases immobilized on the composite nanofiber mesh were lower and higher than those on the neat one, respectively. This enhanced activity might be ascribed to either promoted electron transfer through charge-transfer complexes and the pi system of carbon nanotubes or rendered biocompatibility by modified MWCNTs. Furthermore, the immobilized catalases revealed much more stability after MWCNTs were incorporated into the polymer nanofiber mesh. However, there was no significant difference in optimum pH value and temperature, thermal stability and operational stability between these two immobilized preparations, while the two ones appeared more advantageous than the free in these properties. The effect of MWCNTs incorporation on another redox enzyme, peroxidase, was also studied and it was found that the activity increased by 68% in comparison of composite one with neat preparation.
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paper_title: Electrospun nanofibrous membranes filled with carbon nanotubes for redox enzyme immobilization

paper_content:

Abstract Catalases decompose hydrogen peroxide (H 2 O 2 ) into water and oxygen in an effective and environmentally friend way. The decomposition of H 2 O 2 by nanofibrous membrane-supported catalases is reported in this work. Poly(acrylonitrile- co - N -vinyl-2-pyrrolidone) (PANCNVP) and polyacrylonitrile (PAN) blending with or without multi-walled carbon nanotubes (MWCNTs) were electrospun into nanofibrous membranes as enzyme immobilization matrixes and were carefully characterized. To immobilize catalase covalently, the nanofibrous membranes were treated with alkali solution to generate carboxyl groups followed by activation procedure with EDC/NHS. Results indicate that the nanofibrous membranes can bind large amount of catalases due to their superior surface area to volume ratio. The decomposition behavior of H 2 O 2 with these immobilized catalases was explored. The increment of activity retention induced by the NVP moieties and/or MWCNTs is remarkable. Stabilities of the immobilized catalases including the effects of substrate pH and reaction temperature, thermal stability, operational stability and storage stability were also carefully studied. Results elucidate the improved stabilities of the nanofibrous membrane-supported catalases. In conclusion, the nanofibrous membranes electrospun from this biocompatible polymer, especially those filled with MWCNTs, are suitable matrices for catalase immobilization.
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paper_title: Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide.

paper_content:

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.
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paper_title: Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors

paper_content:

Direct electron transfer (DET) between redox enzymes and electrodes found the basis for third generation biosensors. Recent investigations in the authors' laboratories on the bioelectrochemistry of ...
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Title: A Review on Direct Electrochemistry of Catalase for Electrochemical Sensors

Section 1: Introduction
Description 1: Introduce the significance of studying direct electrochemistry (DET) of catalase (CAT) and its applications in enzyme biosensors, biofuel cells, and biomedical devices.

Section 2: Polyelectrolyte Encapsulated CAT
Description 2: Discuss the research on encapsulating CAT in polyelectrolyte films and its impact on enzyme activity and stability.

Section 3: Surfactant Modified Matrices for CAT Immobilization
Description 3: Examine the immobilization of CAT onto surfactant modified matrices and the resulting electrochemical properties and stability.

Section 4: Immobilization of CAT at Various Polymers and Gel Matrices
Description 4: Review the studies where CAT has been immobilized on different polymeric and gel matrices and analyze their electrochemical behaviors.

Section 5: Dendrimer Matrices for CAT Immobilization
Description 5: Discuss the use of dendrimer matrices for CAT immobilization and the resulting properties of the enzyme films.

Section 6: Nanomaterial Matrices used for CAT Immobilization
Description 6: Detail the types of nanomaterials used for CAT immobilization and their influence on the direct electrochemistry of CAT.

Section 7: CAT Immobilized in Single-wall Carbon Nanotubes (SWCNTs) Matrices
Description 7: Explore the immobilization of CAT within SWCNTs matrices and the enhancements in electron transfer and catalytic activity.

Section 8: CAT Immobilized in a SWCNTs-CS Matrix
Description 8: Discuss the use of SWCNTs with chitosan (CS) matrix for CAT immobilization and the resulting improvements in stability and sensitivity.

Section 9: CAT Immobilized in Nafion-nano Gold-MWCNT Matrices
Description 9: Review the incorporation of multiwalled carbon nanotubes (MWCNTs) and gold nanoparticles in Nafion for CAT immobilization and its electrochemical performance.

Section 10: CAT Immobilized on MWCNTs Incorporated Glassy Carbon Electrodes
Description 10: Analyze the direct electrochemistry of CAT when immobilized on MWCNTs modified glassy carbon electrodes.

Section 11: Covalent Immobilization of CAT on Conductive Composite Nanofiber Meshes
Description 11: Explore the covalent immobilization of CAT on conductive nanofiber meshes and their electrochemical properties.

Section 12: CAT Immobilized on Nickel Oxide Nanoparticles Modified Glassy Carbon Electrodes
Description 12: Discuss the immobilization of CAT on nickel oxide nanoparticles modified electrodes and the resulting electrocatalytic properties.

Section 13: Benefits of Nanomaterial Matrices above Nanomaterial Free Matrices for Efficient CAT Immobilization towards the Development of CAT Based Electrochemical Sensors
Description 13: Compare the advantages of nanomaterial matrices over nanomaterial free matrices in terms of electrochemical performance and stability of CAT-based sensors.

Section 14: Conclusions
Description 14: Provide a summary of the findings on the direct electrochemistry of CAT in various matrices and highlight the superiority of nanomaterial matrices.