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.     

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.     

Nanoplated bismuth titanate sub-microspheres for protein immobilization and their corresponding direct electrochemistry and electrocatalysis

A layered inorganic perovskite sub-micrometer-scale material, nanoplated bismuth titanate (Bi₄Ti₃O₁₂) sub-microspheres (NBTSMs) constructed with tens of Bi₄Ti₃O₁₂ nanoplates, was for the first time synthesized by a facile hydrothermal synthesis strategy. The NBTSMs were employed as a supporting matrix to explore a novel immobilization and biosensing platform of redox proteins through a combined hydrogen bond and electrostatic assembly process. Biocompatibility, stability, reproducibility, and electrochemical and electrocatalytic properties of the resulting NBTSMs-based composite were studied by UV–vis absorption, FTIR, and electrochemical methods. The research results revealed that the NBTSMs-based composite was a satisfying matrix for proteins to effectively retain their native structure and bioactivity. With advantages of the Bi₄Ti₃O₁₂ layered material, facilitated direct electron transfer of the metalloenzymes with an apparent heterogeneous electron transfer rate constant (k_s) of 20.0 ± 3.8 s⁻¹ was acquired on the NBTSMs-based enzyme electrode. The NBTSMs-based biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with an apparent Michaelis–Menten constant (204 μM), wide linear range (2–430 μM), and low detection limit (0.46 μM, S/N = 3). These indicated that the nanoplate-constructed Bi₄Ti₃O₁₂ sub-microspheres were one of ideal candidate materials for direct electrochemistry of redox proteins and the construction of the related enzyme biosensors, and may find potential applications in biomedical, food, and environmental analysis and detection.     

Direct electrochemistry and electrocatalysis of heme-proteins entrapped in agarose hydrogel films

Three heme-proteins, including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), were immobilized on edge-plane pyrolytic graphite (EPG) electrodes by agarose hydrogel. The proteins entrapped in the agarose film undergo fast direct electron transfer reactions, corresponding to FeIII = e- --> FeII. The formal potential (E degrees'), the apparent coverage (Gamma), the electron transfer coefficient (alpha) and the apparent electron transfer rate constant (ks) were calculated by integrating cyclic voltammograms or performing nonlinear regression analysis of square wave voltammetric (SWV) experimental data. The E degrees's are linearly dependent on solution pH (redox Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet visible (UV-Vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the agarose film are little different from that proteins alone, and the conformation changes reversibly in the range of pH 3.0-10.0. Atomic force microscopy (AFM) images of the agarose film indicate a stable and crystal-like structure formed possibly due to the synergistic interaction of hydrogen bonding between N,N-dimethylformamide (DMF), agarose hydrogel and heme-proteins. This suggests a strong interaction between the heme-proteins and the agarose hydrogel. DMF plays an important role in immobilizing proteins and enhancing electron transfer between proteins and electrodes. The mechanisms for catalytic reduction of hydrogen peroxide and nitric oxide (NO) by proteins entrapped in agarose hydrogel were also explored.     

Composite system based on chitosan and room-temperature ionic liquid: direct electrochemistry and electrocatalysis of hemoglobin

A novel polymer/room-temperature ionic liquid (RTIL) composite material based on chitosan (Chi) and 1-butyl-3-methyl-imidazolium tetrafluoroborate (BMIM.BF(4)) was explored. The composite system can be readily used as an immobilization matrix to entrap proteins and enzymes. Hemoglobin (Hb) was chosen as a model protein to investigate the composite system. A pair of well-defined quasireversible redox peaks of hemoglobin were obtained at the Chi-BMIM.BF(4)-Hb composite-film-modified glassy carbon (GC) electrode by direct electron transfer between the protein and the GC electrode. Dramatically enhanced biocatalytic activity was exemplified at the Chi-BMIM.BF(4)-Hb/GC electrode by the reduction of oxygen and trichloroacetic acid. Thermogravimetric analysis (TGA) suggests that the Chi-BMIM.BF(4)-Hb composite has higher thermal stability than Chi-Hb itself. The Chi-BMIM.BF(4)-Hb film was also characterized by UV-visible spectra, indicating excellent stability in solution and good biocompatibility for protein. The unique composite material based on polymer and ionic liquid can find wide potential applications in direct electrochemistry, biosensors, and biocatalysis.     

DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer

A cascading hyperbranched polyamidoamine dendrimer was synthesized on the surface of bacterial magnetite from Magnetospirillum magneticum AMB-1 to allow enhanced extraction of DNA from fluid suspensions. Characterization of the synthesis revealed linear doubling of the surface amine charge from generations one through five starting with an amino silane initiator. Furthermore, transmission electron microscopy revealed clear dispersion of the single domain magnetite in aqueous solution. The dendrimer modified magnetic particles have been used to carry out magnetic separation of DNA. Binding and release efficiencies increased with the number of generations and those of bacterial magnetite modified with six generation dendrimer were 7 and 11 times respectively as many as those of bacterial magnetite modified with only amino silane.     

Effects of structure of polyamidoamine dendrimer on gene transfer efficiency of the dendrimer conjugate with alpha-cyclodextrin

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.     

Manno-oligosaccharide-binding ability of mouse RegIV/GST-fusion protein evaluated by complex formation with the carbohydrate-containing polyamidoamine dendrimer

The carbohydrate-binding properties of the C-type lectin-like mouse RegIV and glutathione S-transferase-fusion protein (GST-mRegIV) were examined using carbohydrate-containing polyamidoamine dendrimers (PD). GST-mRegIV showed affinity for mannan- and manno-oligosaccharide containing PD. Binding was inhibited by manno-oligosaccharides but not by mannose or other tested carbohydrates, suggesting that the binding site may have an extended structure in contrast with typical C-type lectins.     

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.     

Spectroscopic and molecular modeling studies of the interaction between morin and polyamidoamine dendrimer

Interactions between the polyamidoamine (PAMAM) dendrimer and drug molecules are of interest for their potential biomedical applications. The goal of this work is to examine the interaction of PAMAM‐C₁₂ 25% dendrimer with morin. The ultraviolet–visible, fluorescence spectroscopic methods as well as molecular modeling were used to analyze drug‐binding mode, binding constants and binding sites, etc. The experimental data showed that the binding constant of morin‐PAMAM‐C₁₂ 25% is about 10⁵ L/mol. The interaction of morin with PAMAM‐C₁₂ 25% is mainly driven by the hydrophobic, electrostatic, hydrogen bonds and van der Waals forces. There are mainly three classes of binding site of morin at the interface of PAMAM‐C₁₂ 25%. These results provided some useful information for self‐assembling and disassembling the PAMAM dendrimer as well as efficient drug delivery and therapeutic applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Enhancement of gene expression by polyamidoamine dendrimer conjugates with alpha-, beta-, and gamma-cyclodextrins.

To improve the transfection efficiency of nonviral vector, we synthesized the starburst polyamidoamine dendrimer conjugates with alpha-, beta-, and gamma-cyclodextrins (CDE conjugates), expecting the synergistic effect of dendrimer and cyclodextrins (CyDs). The (1)H NMR spectroscopic data indicated that alpha-, beta-, and gamma-CyDs are covalently bound to dendrimer in a molar ratio of 1:1. The agarose gel electrophoretic studies revealed that CDE conjugates formed the complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I in the same manner as dendrimer. CDE conjugates showed a potent luciferase gene expression, especially in the dendrimer conjugate with alpha-CyD (alpha-CDE conjugate) which provided the greatest transfection activity (approximately 100 times higher than those of dendrimer alone and of the physical mixture of dendrimer and alpha-CyD) in NIH3T3 and RAW264.7 cells. In addition, the gene transfer activity of alpha-CDE conjugate was superior to that of Lipofectin. The enhancing gene transfer effect of alpha-CDE conjugate may be attributable to not only increasing the cellular association, but also changing the intracellular trafficking of pDNA. These findings suggest that alpha-CDE conjugate could be a new preferable nonviral vector of pDNA.     

Direct electrochemistry and electrocatalysis of hemoglobin in poly-3-hydroxybutyrate membrane

Hemoglobin (Hb) can take direct electron-transfer reactions after being entrapped in poly-3-hydroxybutyrate (PHB) film. A pair of well-defined, quasi-reversible cyclic voltammetric peaks is thus obtained at an Hb-PHB modified pyrolytic graphite electrode. The anodic and cathodic peaks are located at -224 and -284 mV for a pH 5.0 acetate buffer solution. Meanwhile, the peroxidase activity of the protein in the membrane has been greatly enhanced, with the apparent Michaelis-Menten constant calculated to be 1076 microM. According to the direct electron transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb-PHB based hydrogen peroxide biosensor is prepared, with a linear range 6.0 x 10(-7) to 8.0 x 10(-4) M. The pathway of reductive dehalogenation of trichloroacetic acid is also discussed in detail. The highly reduced form of Hb produced in PHB film can be used to dechlorinate di- and monochloroacetic acid. The catalytic ability of Hb toward the reduction of nitric oxide has been investigated as well. Due to its biodegradability, low cost, chemical inertness, and especially its biocompatibility and non-toxicity, PHB would be a desirable film in the sensor field.     

Comparison of the macromolecular MR contrast agents with ethylenediamine-core versus ammonia-core generation-6 polyamidoamine dendrimer

Two novel macromolecular MRI contrast agents based upon generation-6 polyamidoamine dendrimers (G6) of presumed similar molecular size, but of different molecular weight, were compared in terms of their blood retention, tissue distribution, and renal excretion. Two G6s with either ammonia core (G6A) or with ethylenediamine core (G6E), which possessed 192 and 256 exterior primary amino groups, respectively, were used. These dendrimers were reacted with 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M). The G6--1B4M conjugates were reacted with (153)Gd for studying biodistribution and blood clearance or Gd(III) for the MRI study. 3D-micro-MR angiography of the mice were taken with injection of 0.033 mmol of Gd/kg of G6A--(1B4M-Gd)(192) or G6E--(1B4M-Gd)(256) using a 1.5-T superconductive MRI unit. Although numerous fine vessels of approximately 100 microm diameter were visualized on subtracted 3D-MR-angiography with both G6A--(1B4M-Gd)(192) and G6E--(1B4M-Gd)(256), (153)Gd-labeled saturated G6E-(1B4M)(256) remained in the blood significantly more than (153)Gd-labeled saturated G6A--(1B4M)(192) at later than 15 min postinjection (p < 0.01). In addition, G6E--(1B4M-Gd)(256) visualized these finer vessels longer than G6A--(1B4M-Gd)(192). The G6A--(1B4M-Gd)(192) showed higher signal intensity in the kidney on the dynamic MR images and brighter kidney images than G6E--(1B4M-Gd)(256). In conclusion, the G6A--(1B4M-Gd)(192) was observed to go through glomerular filtration more efficiently than G6E--(1B4M-Gd)(256) resulting faster clearance from the blood and higher renal accumulation, even though both of G6--1B4M conjugates have almost similar molecular size and same chemical structure. In terms of the ability of intravascular contrast agents, G6E--(1B4M-Gd)(256) was better due to more Gd(III) atoms per molecule and longer retention in the circulation than G6A--(1B4M-Gd)(192).     

Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films

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.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay-chitosan-gold nanoparticle nanocomposite

Gold nanoparticles stabilized by chitosan (AuCS) were hybridized with exfoliated clay nanoplates through electrostatic interaction. The resulting clay-chitosan-gold nanoparticle nanocomposite (Clay/AuCS) was used to modify glassy carbon electrode (GCE). HRP, a model peroxidase, was entrapped between the Clay/AuCS film and another clay layer. UV-vis spectrum suggested HRP retained its native conformation in the modified film. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among HRP, AuCS and clay during the modified film drying. The immobilized HRP showed a pair of quasi-reversible redox peaks at -0.195 V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), and the biosensor displayed a fast amperometric response to H(2)O(2) with a wide linear range of 39 microM to 3.1 mM. The detection limit was 9.0 microM based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K(m) (Michaelis-Menten constant) were evaluated to be 0.53, 2.95+/-0.20s(-1) and 23.15 mM, respectively.     

Assembly of quantum dots-mesoporous silicate hybrid material for protein immobilization and direct electrochemistry

An organized multi-components hybrid material, constructed by mesopores cellular foam silicate (MCFs) and quantum dots (QDs), was designed for the immobilization and biosensing of protein. The negative CdTe QDs were assembled on the surface of mesopores in amino group functionalized MCFs through electrostatic interaction to form QDs-MCFs hybrid material, which was used as the matrix to immobilize myoglobin (Mb) and fabricate modified protein electrode (Mb-QDs-MCFs/GC). FT-IR, UV-vis and PL spectroscopies were used to monitor the assembly process and also demonstrated that Mb was immobilized into the hybrid matrix without denaturation. Compared with the Mb-MCFs/GC electrode, the Mb-QDs-MCFs/GC electrode could not only realize enhanced direct electrochemistry but also display better sensitivity and wider linear range to the detection of hydrogen peroxide. The experiment results demonstrate that the hybrid matrix provides a biocompatible microenvironment for protein and supplies a necessary pathway for its direct electron transfer.     

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 electrochemistry and electrocatalysis of myoglobin immobilized on a hexagonal mesoporous silica matrix

The direct electrochemistry of myoglobin (Mb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Mb and HMS was investigated by using Fourier transfer infrared spectroscopy, nitrogen adsorption isotherm, and cyclic voltammetry. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the Mb intercalated in the mesopores and adsorbed on the surface of the HMS were observed with the formal potentials of -0.167 and -0.029V in 0.1M, pH 7.0, phosphate buffer solution, respectively. The electrode reaction showed a surface-controlled process with one proton transfer. The immobilized Mb displayed good electrocatalytic responses to the reduction of both hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), which were used to develop novel sensors for H(2)O(2) and NO(2)(-). The apparent Michaelis-Menten constants of the immobilized Mb for H(2)O(2) and NO(2)(-) were 0.065 and 0.72mM, respectively, showing good affinity. Under optimal conditions, the sensors could be used for the determinations of H(2)O(2) ranging from 4.0 to 124microM and NO(2)(-) ranging from 8.0 to 216microM. The detection limits were 6.2x10(-8) and 8.0x10(-7)M at 3 sigma, respectively. The HMS provided a novel matrix for protein immobilization and the construction of biosensors via the direct electron transfer of immobilized protein.     

Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan

Three heme proteins, myoglobin, hemoglobin, and cytochrome c, have been adsorbed onto chitosan-stabilized gold nanoparticles (Chit-Aus) modified Au electrode via a molecule bridge like cysteine. UV-vis spectra indicated that the proteins on Chit-Aus films retained near-native secondary structures. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectra and cyclic voltammetric techniques. It was demonstrated that Chit-Aus film could not only offer a friendly environment to immobilize protein molecules but also enhance the electron transfer ability between protein molecules and underlying electrode. The effects of scan rate and pH on the electrochemical behaviors of each heme protein are discussed in detail. The resultant electrode displayed an excellent electrocatalytic response to the reduction of H(2)O(2), long-term stability, and good reproducibility.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol-gel-derived ceramic-carbon nanotube nanocomposite film

The sol-gel-derived ceramic-carbon nanotube (SGCCN) nanocomposite film fabricated by doping multiwall carbon nanotubes (MWNTs) into a silicate gel matrix was used to immobilize protein. The SGCCN film can provide a favorable microenvironment for horseradish peroxidase (HRP) to perform direct electron transfer (DET) at glassy carbon electrode. The HRP immobilized in the SGCCN film shows a pair of well-defined redox waves and retains its bioelectrocatalytic activity to the reduction of O2 and H2O2, which is superior to that immobilized in silica sol-gel film.