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1.
He P  Li M  Hu N 《Biopolymers》2005,79(6):310-323
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.  相似文献   

2.
Shan W  Liu H  Shi J  Yang L  Hu N 《Biophysical chemistry》2008,134(1-2):101-109
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.  相似文献   

3.
Cao D  Hu N 《Biophysical chemistry》2006,121(3):209-217
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
Li Shen 《BBA》2004,1608(1):23-33
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.  相似文献   

7.
Liu Y  Liu H  Hu N 《Biophysical chemistry》2005,117(1):27-37
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.  相似文献   

8.
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.  相似文献   

9.
He P  Hu N  Zhou G 《Biomacromolecules》2002,3(1):139-146
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.  相似文献   

10.
Lu H  Li Z  Hu N 《Biophysical chemistry》2003,104(3):623-632
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.  相似文献   

11.
Sun H  Hu N 《Biophysical chemistry》2004,110(3):411-308
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.  相似文献   

12.
In this study, the Fe-based layered double hydroxides (Mg(3)Fe LDH) were used to immobilize heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) for fabrication of heme/Mg(3)Fe LDH film on glassy carbon electrode (Mg(3)Fe-heme/GCE). The possible role of iron in framework of LDH to promote direct electron transfer (DET) of heme proteins was investigated using an LDH containing non-iron as a reference. Hb was selected as a model protein for studying the electrocatalytic activity of immobilized heme in LDH film. The Mg(3)Fe-Hb/GCE displayed an enhanced electrocatalytic reduction towards H(2)O(2). The biosensor showed a very low detection limit (0.036μM) and apparent Michaelis-Menten constant (7.98μM). This work outlines that Fe-based LDH modified electrode provides a promising platform for immobilization of heme proteins and development of sensitive biosensors.  相似文献   

13.
In this study, magnetic core-shell Fe(3)O(4)@Al(2)O(3) nanoparticles (NPs) attached to the surface of a magnetic glassy carbon electrode (MGCE) were used as a functional interface to immobilize several heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) for fabricating protein/Fe(3)O(4)@Al(2)O(3) film. Transmission electron microscope, UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the films. With the advantages of the magnetism and the excellent biocompatibility of the Fe(3)O(4)@Al(2)O(3) NPs, the protein/Fe(3)O(4)@Al(2)O(3) film could be easily fabricated in the present of external magnetic field, and well retained the bioactivity of the immobilized proteins, hence dramatically facilitated direct electron transfer of heme proteins and excellent electrocatalytic behaviors towards H(2)O(2) were demonstrated. The presented system avoids the complex synthesis for protecting Fe(3)O(4) NPs, supplies a facile, low cost and universal way to immobilize proteins, and is promising for construction of third-generation biosensors and other bio-magnetic induction devices.  相似文献   

14.
A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.  相似文献   

15.
Guo W  Hu N 《Biophysical chemistry》2007,129(2-3):163-171
Myoglobin (Mb), with net positive surface charges at pH 5.0, was successfully assembled into layer-by-layer films on various solid surfaces with poly(methacrylic acid) (PMAA) at different pH, designated as {PMAA(pH 5.0)/Mb}n, {PMAA(pH 6.5)/Mb}n, and {PMAA(pH 8.0)/Mb}n, respectively. As a weak polycarboxylic acid with pKa=6 - 7, PMAA carried different negative charges at different pH due to different ionization degree of its carboxylic acid groups. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the assembly of {PMAA/Mb}n films. All the results showed that the adsorption amount of Mb in each bilayer had an "unexpected" sequence of {PMAA(pH 5.0)/Mb}n>{PMAA(pH 6.5)/Mb}n>{PMAA(pH 8.0)/Mb}n, which could be explained by the formation of soluble complex of PMAA-Mb at pH 8.0 and the cooperative effect of hydrogen bonding and induced electrostatic interaction between Mb and PMAA at pH 5.0. The influence of ionic strength in exposure solution and in Mb adsorbate solution was investigated, and the results supported the above explanations. The {PMAA/Mb}n films provided a suitable microenvironment for Mb to retain its near-native structure and transfer electron with underlying electrodes. The reversible CV peak pair for Mb Fe(III)/Fe(II) redox couple could be used to catalyze reduction of hydrogen peroxide electrochemically, showing the potential applicability of the films as the new type of biosensors or bioreactors based on the direct electrochemistry of Mb. The electrochemical and electrocatalytic behaviors of protein layer-by-layer films with weak polyelectrolytes could thus be controlled by adjusting the solution pH of weak polyelectrolytes.  相似文献   

16.
Denitrifying NO reductases are evolutionarily related to the superfamily of heme--copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N(2)O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe(B) site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV--vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu(I)- or Zn(II)-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}(7) species that can be observed at liquid nitrogen temperature, the Fe(II)-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using (15)NO and (15)N(18)O. The apo and Cu(I)-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe(II) at the Fe(B) site shifts the heme ν(FeNO) by 17 cm(-1) and the ν(NO) by -50 cm(-1) to 1549 cm(-1). This low ν(NO) is without precedent for a six-coordinate heme {FeNO}(7) species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe(II). Detection of a similarly low ν(NO) in the Zn(II)-loaded protein supports this interpretation.  相似文献   

17.
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 -0.33V vs saturated calomel electrode in pH 7 buffers, respectively, while catalase (Ct) in CS films showed a peak pair at about -0.46V 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 degrees (')) 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.  相似文献   

18.
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.  相似文献   

19.
Using chitosan as an effective linker between CMK-3 and glassy carbon electrode surface, {Hb/CMK-3}n multilayer film-modified electrodes were constructed through layer-by-layer assembly. The morphology of thus-formed {Hb/CMK-3}n film was characterized by scanning electron micrographs, and the interaction of hemoglobin (Hb) with CMK-3 was studied by UV-vis spectroscopy and electrochemical methods. Under optimal conditions, {Hb/CMK-3}6 film showed a couple of stable and well-defined redox peaks at about -377 and -296 mV in pH 7.0 buffers. Furthermore, the {Hb/CMK-3}6 film displayed excellent electrocatalysis to the reduction of both H2O2 and O2. Based on thus-formed film and its direct electron transfer behavior, a novel biosensor was presented for the determination of H2O2 ranging from 1.2 to 57 muM with the detection limit of 0.6microM at S/N=3. CMK-3 provided a desirable matrix for protein immobilization and biosensor preparation.  相似文献   

20.
Lu Q  Chen X  Wu Y  Hu S 《Biophysical chemistry》2005,117(1):55-63
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.  相似文献   

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