Direct electrochemistry and electrocatalysis of hemoglobin in composite film based on ionic liquid and NiO microspheres with different morphologies

Flowerlike, spherical, and walnutlike NiO microspheres were respectively mixed with ionic liquid (IL) to form three stable composite films, which were used to immobilize hemoglobin (Hb) on carbon paste electrodes. Spectroscopic and electrochemical examinations revealed that the three NiO/IL composites were biocompatible matrix for immobilizing Hb, which showed good stability and bioactivity. However, electrochemical studies demonstrated that flowerlike NiO microspheres were far more effective than the other two in adsorbing Hb and facilitating the electron transfer between Hb and underlying electrode, which resulted from its unique flower architecture and large surface area. With advantages of flowerlike NiO and ionic liquid, a pair of stable and well-defined quasi-reversible redox peaks of Hb were obtained with a formal potential of -0.275 V (vs. Ag/AgCl) in pH 7.0 buffer. Meantime, flowerlike NiO modified electrode showed better electrocatalytic activity toward hydrogen peroxide reduction with a high sensitivity (15.7μAmM(-1)), low detection limit (0.68 μM) and small apparent Michaelis-Menten constant K(M) (0.18 mM). Flowerlike NiO could be a promising matrix for the fabrication of direct electrochemical biosensors in biomedical analysis.     

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 of hemoglobin in the hyaluronic acid films

Hemoglobin (Hb) in the hyaluronic acid (HA) was cast at pyrolytic graphite (PG) electrodes for researching its electrochemical and electrocatalytic properties. The formal potential and electron transfer rate constant of Hb on HA films were determined, and the stability of the films, the pH effect, and the influence of supporting electrolyte concentrations upon Hb electrochemistry on the films were investigated by cyclic voltammetry and square wave voltammetry. UV-Vis absorption and reflectance absorption infrared (RAIR) spectra showed that the protein on HA film retained near-native secondary structure. The stable Hb-HA/PG gave analytically useful electrochemical catalytic responses to hydrogen peroxide. Thus, the property of the HA film for sorption and retention of water maybe utilized to develop some new biosensors.     

Direct electrochemistry of hemoglobin in the hyaluronic acid films

Hemoglobin (Hb) in the hyaluronic acid (HA) was cast at pyrolytic graphite (PG) electrodes for researching its electrochemical and electrocatalytic properties. The formal potential and electron transfer rate constant of Hb on HA films were determined, and the stability of the films, the pH effect, and the influence of supporting electrolyte concentrations upon Hb electrochemistry on the films were investigated by cyclic voltammetry and square wave voltammetry. UV–Vis absorption and reflectance absorption infrared (RAIR) spectra showed that the protein on HA film retained near-native secondary structure. The stable Hb–HA/PG gave analytically useful electrochemical catalytic responses to hydrogen peroxide. Thus, the property of the HA film for sorption and retention of water maybe utilized to develop some new biosensors.     

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.     

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.     

Direct electrochemistry of horseradish peroxidase based on biocompatible carboxymethyl chitosan-gold nanoparticle nanocomposite

The nanocomposite composed of carboxymethyl chitosan (CMCS) and gold nanoparticles was successfully prepared by a novel and in situ process. It was characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectrophotometer (FTIR). The nanocomposite was hydrophilic even in neutral solutions, stable and inherited the properties of the AuNPs and CMCS, which make it biocompatible for enzymes immobilization. HRP, as a model enzyme, was immobilized on the silica sol-gel matrix containing the nanocomposite to construct a novel H(2)O(2) biosensor. The direct electron transfer of HRP was achieved and investigated. The biosensor exhibited a fast amperometric response (5s), a good linear response over a wide range of concentrations from 5.0 x 10(-6) to 1.4 x 10(-3)M, and a low detection limit of 4.01 x 10(-7)M. The apparent Michaelis-Menten constant (K(M)(app)) for the biosensor was 5.7 x 10(-4)M. Good stability and sensitivity were assessed for the biosensor.     

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.     

Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film

The composite film based on Nafion and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of ionic liquid (IL)-Mb interaction by ultraviolet-visible (UV-vis) spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peaks with a formal potential (E_o^’) of −0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0 to 180 μM with a detection limit of 0.14 μM at a signal/noise ratio of 3. The apparent Michaelis constant () for the electrocatalytic reaction was 22.6 μM. The stability, repeatability, and selectivity of the sensor were evaluated. The proposed biosensor has a lower detection limit than many other IL-heme protein-based biosensors and is free from common interference in hydrogen peroxide biosensors.     

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.     

Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry

Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility.     

Preparation of chitin/cellulose composite gels and films with ionic liquids

In this study, we performed preparation and characterizations of the chitin/cellulose composite gels and films using the two ionic liquids, 1-allyl-3-methylimidazolium bromide and 1-butyl-3-methylimidazolium chloride. First, chitin and cellulose were dissolved in each appropriate ionic liquid. Then, the two liquids were mixed in the desired ratios at 100 °C to give the homogeneous mixtures. The gels were obtained by standing the mixtures for 4 days. On the other hand, the films were obtained by casting the mixtures on glass plates, followed by soaking in water and drying. The obtained gels and films were characterized by XRD and TGA measurements. The mechanical properties of the gels and films were evaluated under compressive and tensile modes, respectively.     

Direct electrochemistry and electrocatalysis based on film of horseradish peroxidase intercalated into layered titanate nano-sheets

Intercalation of horseradish peroxidase (HRP) into layered titanate by assembling it with titanate nano-sheets (TNS) was firstly used for fabrication of enzyme electrode (HRP-TNS electrode). XRD result revealed that HRP-TNS film featured layered structure with HRP monolayer intercalated between the titanate layers. UV-vis spectra result indicated the intercalated HRP in TNS film well retained its native structure. The HRP-TNS film was uniform with porous structures which were confirmed by SEM. The immobilized HRP in the TNS film exhibited fast direct electron transfer and showed a good electrocatalytic performance to H2O2 with high sensitivity, wide linear range and low detection. The excellent electrochemical performance of the HRP-TNS electrode was attributed to biocompatibility of the titanate sheets, porous architectures of the HRP-TNS film which retained activity of HRP to large extent, avoided aggregation of HRP, provided better mass transport and allowed more HRP loading per unit area. Thus, the simple method described here provides a novel and effective platform for immobilization of enzyme in realizing direct electrochemistry and has a promising application in fabrication of the third-generation electrochemical biosensors.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode

Due to their unique physicochemical properties, doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, selecting glucose oxidase (GOD) as a model enzyme, we investigated the direct electrochemistry of GOD based on the B-doped carbon nanotubes/glassy carbon (BCNTs/GC) electrode with cyclic voltammetry. A pair of well-defined, quasi-reversible redox peaks of the immobilized GOD was observed at the BCNTs based enzyme electrode in 0.1M phosphate buffer solution (pH 6.98) by direct electron transfer between the protein and the electrode. As a new platform in glucose analysis, the new glucose biosensor based on the BCNTs/GC electrode has a sensitivity of 111.57 microA mM(-1)cm(-2), a linear range from 0.05 to 0.3mM and a detection limit of 0.01mM (S/N=3). Furthermore, the BCNTs modified electrode exhibits good stability and excellent anti-interferent ability to the commonly co-existed uric acid and ascorbic acid. These indicate that boron-doped carbon nanotubes are the good candidate material for the direct electrochemistry of the redox-active enzyme and the construction of the related enzyme biosensors.     

Direct electrochemistry of hemoglobin in gold nanowire array

Gold nanowire array has been proven to be efficient support matrixes for the immobilization of hemoglobin (Hb). The vertically oriented nanowire array provides an ordered well-defined 3D structure with nanowire density approximately 5 x 10(8)cm(2). The adsorption of ferritin onto the nanowire surface was visualized by transmission electron microscopy. When Hb was adsorbed, UV-vis absorption and Fourier transform infrared (FT-IR) spectra show no obvious denaturation of Hb in the nanowire array. The Hb-modified nanowire array exerted direct electron transfer and gave a well-defined, nearly reversible redox couple with formal potential of -0.225 V. The quantity of electroactive Hb varied with the changing of the morphology of the electrode and found to increase with the increasing of the nanowire length. Comparisons of voltammetric and quartz crystal microbalance measurements show that 70% of the Hb molecules adsorbed are electroactive when the length of the nanowire was 2 microm. Both of the Hb-modified nanowire array and the unmodified nanowire array demonstrate good electrocatalytic reduction ability for hydrogen peroxide. With the adsorption of glucose oxidase onto the bare nanowire surface, sensitive and selective glucose biosensors can be fabricated.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

On-chip detection of myoglobin based on fluorescence

A disposable immunosensor cartridge was developed that allows antibodies to be immobilized on the surface for the detection of myoglobin, a marker for the early assessment of acute myocardial infarction (AMI) using fluorescence techniques. The anti-myoglobin antibody was immobilized on a polystyrene substrate based on covalent bonding via silanization. The immunosensor chip layers were fabricated from sheets by CO(2)-laser ablation. The functionalized polystyrene surfaces were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antigen-antibody reaction as a sandwich enzyme-linked immunosorbent assay (ELISA) with a horseradish peroxidase-conjugated secondary antibody (HRP-anti-myoglobin), addition of fluorogenic substrate produced a fluorescent dye which was quantified on-chip using fluorescent technique. The immunosensor response was linear for myoglobin concentrations between 20 and 230 ng/ml (r=0.991, n=3). The detection limit was found to be 16 ng/ml, which is lower than the clinical cut-off value for myoglobin in healthy patients. This protocol could be extended to the detection of other important cardiac markers simultaneously in microchannels.     

Green composite films prepared from cellulose,starch and lignin in room-temperature ionic liquid

A series of novel biobased composite films derived from cellulose, starch and lignin were prepared from an ionic liquid (IL), 1-allyl-3-methylimidazolium chloride (AmimCl) by coagulating in a nonsolvent condition. The ionic liquid can be recycled with a high yield and purity after the green film was prepared. The uniform design method was applied to investigate mechanical properties of the biobased composite films. The effect of each component and their associated interactive effects were investigated. The experimental results showed that contents of cellulose, lignin and starch had a significant influence on the mechanical properties of composite films. The composite films showed relatively excellent mechanical properties in dry and wet states owing to the mutual property supplement of different components. The composite films were characterized via FT-IR, X-ray diffraction (XRD) and scanning electron microscope (SEM). Their thermal stability and gas permeability were also investigated, and the results showed that the composite films had good thermal stability and high gas barrier capacity and give a CO₂:O₂ permeability ratio close to 1.     

Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase.

This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.