Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis

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 (k(s)) and formal potentials (E (degrees ')) 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.     

Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis

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 (k_s) 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.     

An electrochemical investigation of hemoglobin and catalase incorporated in collagen films

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.     

Studies on direct electron transfer and biocatalytic properties of heme proteins in lecithin film

Myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP) were incorporated in lecithin (PC) film on glassy carbon (GC) electrode by the method of vesicle-fusion. A pair of well-defined and quasi-reversible cyclic voltammetric peaks was obtained, which reflected the direct electron transfer of heme proteins. UV-Vis and reflectance absorption infrared (RAIR) spectroscopy showed that proteins in PC films remained at their secondary structure similar to their native states. Scanning electron microscopy (SEM) demonstrated the interaction between the proteins and PC would make the morphology of protein-PC films very different from the PC films alone. The immobilized proteins retained their biocatalytic activity to the reduction of NO and hydrogen peroxide, which provide the perspective to be the third generation sensors.     

Direct voltammetry and electrochemical catalysis with horseradish peroxidase in polyacrylamide hydrogel films

This paper reports the direct voltammetry of horseradish peroxidase (HRP) incorporated in amphiphilic polyacrylamide (PAM) films modified on pyrolytic graphite (PG) electrodes. Cyclic voltammetry of HRP-PAM films showed a pair of well-defined, nearly reversible peaks at approximately -0.33 V vs. SCE in pH 7.0 buffers, characteristic of HRP heme Fe(III)/Fe(II) redox couple. The PAM films in solution contained large amounts of water and formed a hydrogel, and provided a favorable microenvironment for HRP and facilitated its direct electron transfer with underlying PG electrodes. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E*') were estimated by fitting the data of square wave voltammetry (SWV) with the non-linear regression analysis. UV-vis absorption spectra demonstrated that HRP in PAM films retained its secondary structure similar to its native state. The embedded HRP in PAM films showed the electrocatalytic activity to various substrates such as nitrite, oxygen and hydrogen peroxide. The possible mechanism of catalytic reaction of H(2)O(2) with HRP-PAM films was proposed.     

Assembly of electroactive layer-by-layer films of hemoglobin and polycationic poly(diallyldimethylammonium)

Layer-by-layer (PDDA/Hb)(n) films were assembled by alternate adsorption of positively charged poly(diallyldimethylammonium) (PDDA) and negatively charged hemoglobin (Hb) at pH 9.2 from their aqueous solutions on pyrolytic graphite electrodes and other substrates. The assembly process was monitored and confirmed by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). CVs of (PDDA/Hb)(n) films showed a pair of well-defined, nearly reversible peaks at about -0.34 V vs SCE at pH 7.0, characteristic of Hb heme Fe(III)/Fe(II) redox couple. Positions of Soret absorption band and infrared amide II band of Hb in (PDDA/Hb)(8) films suggest that Hb in the films keeps its secondary structure similar to its native state. The electrochemical parameters of (PDDA/Hb)(8) films were estimated by square wave voltammetry, and the thickness of the PDDA/Hb bilayer was estimated by QCM and scanning electron microscopy. Trichloroacetic acid and nitrite (NO(2)(-)) were catalytically reduced at (PDDA/Hb)(8) film electrodes. The electrochemical catalytic reactions of O(2) and H(2)O(2) on (PDDA/Hb)(8) films were also studied.     

Voltammetric studies of hemoglobin-coated polystyrene latex bead films on pyrolytic graphite electrodes

A novel hemoglobin (Hb)-coated polystyrene (PS) latex bead film was deposited on pyrolytic graphite (PG) electrode surface. In the first step, positively charged Hb molecules in pH 5.0 buffers were adsorbed on the surface of negatively charged, 500 nm diameter PS latex beads bearing sulfate groups by electrostatic interaction. The aqueous dispersion of Hb-coated PS particles was then deposited on the surface of PG electrodes and, after evaporation of the solvent, Hb-PS films were formed. The Hb-PS film electrodes exhibited a pair of well-defined, quasi-reversible cyclic voltammetric (CV) peaks at about −0.36 V vs. SCE in pH 7.0 buffers, characteristic of Hb heme Fe(III)/Fe(II) redox couples. Positions of Soret absorption band of Hb-PS films suggest that Hb retains its near-native structure in the films in its dry form and in solution at medium pH. The Hb in PS films was also acted as a catalyst to catalyze electrochemical reduction of various substrates such as trichloroacetic acid (TCA), nitrite, oxygen and hydrogen peroxide.     

Self-assembled films of hemoglobin/laponite/chitosan: application for the direct electrochemistry and catalysis to hydrogen peroxide

A highly stable biological film was formed on the functional glassy carbon electrode (GCE) via step-by-step self-assembly of chitosan (CHT), laponite, and hemoglobin (Hb). Cyclic voltammetry (CV) of the Hb/laponite/CHT/GCE showed a pair of stable and quasi-reversible peaks for the Hb-Fe(III)/Fe(II) redox couple at about -0.035 V versus a saturated calomel electrode in pH 6.0 phosphate buffer at a scan rate of 0.1 V s(-1). The electrochemical reaction of Hb entrapped on the laponite/CHT self-assembled film exhibited a surface-controlled electrode process. The formal potential of the Hb-heme-Fe(III)/Fe(II) couple varied linearly with the increase of pH over the range of 3.0-8.0 with a slope of -63 mV pH(-1), which implied that an electron transfer was accompanied by single-proton transfer in the electrochemical reaction. The position of the Soret absorption band of this self-assembled Hb/laponite/CHT film suggested that the entrapped Hb kept its secondary structure similar to its native state. The self-assembled film showed excellent long-term stability, the CV peak potentials kept in the same positions, and the cathodic peak currents retained 90% of their values after 60 days. The film was used as a biological catalyst to catalyze the reduction of hydrogen peroxide. The electrocatalytic response showed a linear dependence on the H2O2 concentration ranging widely from 6.2 x 10(-6) to 2.55 x 10(-3) M with a detection limit of 6.2 x 10(-6) M at 3 sigma.     

Direct electron transfer between hemoglobin and pyrolytic graphite electrodes enhanced by Fe(3)O(4) nanoparticles in their layer-by-layer self-assembly films

Alternate adsorption of negatively charged Fe(3)O(4) nanoparticles from their pH 8.0 aqueous dispersions and positively charged hemoglobin (Hb) from its pH 5.5 buffers on solid substrates resulted in the assembly of {Fe(3)O(4)/Hb}(n) layer-by-layer films. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the film growth. A pair of well-defined, nearly reversible CV peaks for HbFe(III)/Fe(II) redox couples was observed for {Fe(3)O(4)/Hb}(n) films on pyrolytic graphite (PG) electrodes. Although the multilayered films grew linearly with the number of Fe(3)O(4)/Hb bilayers (n) and the amount of Hb adsorbed in each bilayer was generally the same, the electroactive Hb could only extend to 6 bilayers. This indicates that only those Hb molecules in the first few bilayers closest to the electrode surface are electroactive. The electrochemical parameters such as the apparent heterogeneous electron transfer rate constant (k(s)) were estimated by square wave voltammetry (SWV) and nonlinear regression. The Soret absorption band position of Hb in {Fe(3)O(4)/Hb}(6) films showed that Hb in the films retained its near native structure in the medium pH range. The {Fe(3)O(4)/Hb}(6) film electrodes also showed good biocatalytic activity toward reduction of oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite. The electrochemical reduction overpotentials of these substrates were lowered significantly by {Fe(3)O(4)/Hb}(n) films.     

Electrostatic adsorption of heme proteins alternated with polyamidoamine dendrimers for layer-by-layer assembly of electroactive films

A novel thin film of heme proteins, including hemoglobin (Hb), myoglobin (Mb), and catalase (Cat), was successfully assembled layer by layer with polyamidoamine (PAMAM) dendrimers on different solid surfaces. At pH 7.0, protonated PAMAM possesses positive surface charges, whereas the proteins have net negative surface charges at pH above their isoelectric points. Thus, layer-by-layer {PAMAM/protein}(n)() films were assembled with alternate adsorption of oppositely charged PAMAM and proteins from their aqueous solutions mainly by electrostatic interaction. The assembly process was monitored by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). The growth of the protein multilayer films was regular and linear, whereas the electroactivity of the films was only extended to a few bilayers. CVs of {PAMAM/protein}(n)() films showed a pair of well-defined and nearly reversible peaks characteristic of the protein heme Fe(III)/Fe(II) redox couples. Although {PAMAM/Hb}(n)() and {PAMAM/Mb}(n)() films showed very similar properties, {PAMAM/Cat}(n)() films displayed different and unique characters. The substrates with biological or environmental significance, such as oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite, were catalytically reduced at {PAMAM/protein}(n)() film electrodes, showing the potential applicability of the films as new types of biosensors or bioreactors based on direct electrochemistry of the proteins. Both the electrochemical and electrocatalytic activity of {PAMAM/protein}(n)() films can be tailored precisely by controlling the number of bilayers or the film thickness.     

Self-assembly of electroactive layer-by-layer films of heme proteins with anionic surfactant dihexadecyl phosphate

Anionic surfactant dihexadecyl phosphate (DHP) with two hydrocarbon chains can be self-assembled into a double-layer structure with tail-to-tail configuration and negatively charged head groups toward outside in its aqueous dispersion. Due to this unique biomembrane-like structure, the "charge reversal" in DHP adsorption on solid surface was realized, and the DHP was successfully assembled with positively charged myoglobin (Mb) or hemoglobin (Hb) into {DHP/protein}(n) layer-by-layer films. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor or confirm the film assembly process. The {DHP/protein}(n) films grown on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible CV peaks at about -0.35 V vs SCE in pH 7.0 buffers, characteristic of the protein heme Fe(III)/Fe(II) redox couples. Based on the direct electrochemistry of heme proteins, the {DHP/protein}(n) films could also be used to electrochemically catalyze reduction of oxygen, hydrogen peroxide and nitrite with significant lowering of reduction overpotentials. Scanning electron microscopy (SEM), UV-vis spectroscopy, and reflectance absorption infrared (RAIR) spectroscopy were employed to characterize the {DHP/protein}(n) films, suggesting that the proteins in the films retain their near-native structure.     

Direct electrochemistry of hemoglobin in dimethyldioctadecyl ammonium bromide film and its electrocatalysis to nitric oxide

Hemoglobin (Hb) was successfully immobilized in dimethyldioctadecyl ammonium bromide (DOAB) film at pyrolytic graphite (PG) electrode. Electrochemical experiments revealed that Hb in DOAB film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.160 V versus saturated calomel electrode (SCE) in pH 5.0 buffer, characteristic of the heme Fe(III)/Fe(II) redox couple of Hb. The electron transfer (eT) rate between Hb and the PG electrode was 0.10 s(-1). Positions of the Soret absorbance band indicated that the Hb retained its secondary structure and was similar to its native state. Furthermore, the Hb in DOAB film acted as a biological catalyst towards the reduction of nitric oxide (NO). The voltammetric response of NO at the Hb-DOAB modified electrode could be used to determine the concentration of NO in solution.     

Assembly of myoglobin layer-by-layer films with poly(propyleneimine) dendrimer-stabilized gold nanoparticles and its application in electrochemical biosensing

The small-sized Au nanoparticles (3 nm) were prepared by reduction of HAuCl(4) in the presence of poly(propyleneimine) (PPI) dendrimers, forming the stable PPI-Au nanoclusters in aqueous medium. The PPI-Au nanoclusters might take a kind of "core-shell" structure, in which several PPI molecules were attached on the surface of one gold nanoparticle. The PPI-Au nanoclusters in aqueous dispersions and myoglobin (Mb) in its buffers at pH 5.0 were then alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes and other solid substrates, forming {PPI-Au/Mb}(n) layer-by-layer films, which was confirmed by cyclic voltammetry (CV) and quartz crystal microbalance (QCM). {PPI-Au/Mb}(n) films on PG electrodes demonstrated a pair of well-defined and quasi-reversible CV reduction-oxidation peaks for Mb heme Fe(III)/Fe(II) couple and good electrocatalytic properties toward reduction of oxygen and hydrogen peroxide. Compared with {Au/Mb}(n) multilayer films containing no dendrimers and {PAMAM/Mb}(n) films assembled by polyamidoamine (PAMAM) dendrimers and Mb but in the absence of Au nanoparticles, {PPI-Au/Mb}(n) films showed better electrochemical behaviors and catalytic performances, which may be attributed to the unique structure of PPI-Au nanoclusters and good conductivity of gold nanoparticles. This novel kind of protein multilayer films assembled with dendrimer-stabilized gold nanoparticles may provide a new and general approach to fabricate the biosensors and bioreactors based on the direct electrochemistry of proteins or enzymes.     

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

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

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

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

Core--shell nanocluster films of hemoglobin and clay nanoparticle: direct electrochemistry and electrocatalysis

A novel core-shell protein nanocluster film, designated as clay-(Hb/PSS)(n), was fabricated on pyrolytic graphite (PG) electrodes. Positively charged hemoglobin (Hb) at pH 5.5 and negatively charged poly(styrenesulfonate) (PSS) were first assembled layer by layer on surface of clay nanoparticles from their solutions mainly by electrostatic attraction, forming a core-shell nanocluster structure in which clay nanoparticles were the "cores" and (Hb/PSS)(n) multilayers were the "shells". The aqueous dispersion of clay-(Hb/PSS)(n) nanoclusters was then cast on surface of PG electrodes, forming clay-(Hb/PSS)(n) nanocluster films after evaporation of solvent. Hb in clay-(Hb/PSS)(n) films exhibited a pair of well-defined and reversible cyclic voltammetric (CV) peaks at about -0.36 V vs. SCE in pH 7.0 buffers, characteristic of Hb heme Fe(III)/Fe(II) redox couples. Compared with other Hb-containing clay films, clay-(Hb/PSS)(n) films displayed smaller CV peak separation (DeltaE(p)), indicating the better electrochemical reversibility of Hb in these nanocluster films. The partially ordered structure of the films was characterized by X-ray diffraction (XRD) experiments. UV-VIS and reflection absorption infrared (RAIR) spectroscopy suggests that Hb retains its near-native structure in clay-(Hb/PSS)(n) films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at clay-(Hb/PSS)(n) film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on protein direct electrochemistry. The electrochemical and electrocatalytic activity of the films could be tailored by controlling the number of bilayers of the (Hb/PSS)(n) shells on the surface of clay nanoparticle cores.     

Electrochemistry and electrocatalysis of myoglobin immobilized in sulfonated graphene oxide and Nafion films

In this study, sulfonated graphene oxide (SGO) was synthesized and characterized by Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). It was used to make Mb–SGO–Nafion composite films by coating myoglobin (Mb) on the glassy carbon electrodes (GCE). Positions of the Soret absorption bands suggested that Mb retained its native conformation in the films. Mb–SGO–Nafion film modified electrode showed a pair of well-defined and nearly reversible cyclic voltammetry peaks at around −0.39 V versus saturated calomel electrode (SCE) in pH 7.0 buffers, characteristic of heme Fe(III)/Fe(II) redox couples. Electrochemical parameters such as electron transfer rate constant (k_s) and formal potential (E^o′) were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. Experimental data demonstrated that the electron transfer between Mb and electrode was greatly facilitated and showed good electrocatalytic properties toward various substrates, such as H₂O₂ and NaNO₂, with significant lowering of reduction overpotential.     

Enhanced electron-transfer reactivity of horseradish peroxidase in phosphatidylcholine films and its catalysis to nitric oxide

Direct electrochemistry of horseradish peroxidase (HRP) embedded in film of phosphatidylcholine (PC) is investigated at a pyrolytic graphite electrode by voltammetric methods. The electron-transfer reactivity between incorporated HRP and the electrode is found to be greatly enhanced by phosphatidylcholine film. Cyclic voltammetry (CV) of this incorporated peroxidase shows a pair of well-defined and nearly reversible peaks, and the cathodic and anodic peak potentials are located at about -0.261 and -0.180 V, respectively versus saturated calomel electrode at pH 5.5. Ultraviolet-visible absorption spectra indicate that the heme microenvironment of HRP in phosphatidylcholine film is similar to that of its native status. It is also observed that HRP modified electrode is able to catalyze the electrochemical reduction of nitric oxide. Experimental results reveal that the peak current related to nitric oxide reduction is linearly proportional to its concentration in the ranges of 2.0 x 10(-7) -5.0 x 10(-6) mol (-1) and 2.0 x 10(-5) -1.0 x 10(-4) mol(-1), based on which an unmediated biosensor for nitric oxide is developed.     

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

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

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.