Urchinlike MnO2 nanoparticles for the direct electrochemistry of hemoglobin with carbon ionic liquid electrode

In this paper an urchinlike MnO(2) nanoparticle was synthesized by hydrothermal method and applied to the protein electrochemistry for the first time. By using a carbon ionic liquid electrode (CILE) as the basal electrode, hemoglobin (Hb) was immobilized on the surface of CILE with chitosan (CTS) and MnO(2) nanoparticle composite materials. Spectroscopic results indicated that Hb molecules retained its native structure in the composite film. A pair of well-defined redox peaks appeared on the cyclic voltammogram with the formal peak potential as -0.180 V (vs. SCE), which indicated that direct electron transfer of Hb was realized on the modified electrode. The result can be attributed to the specific characteristic of MnO(2) nanoparticle and the advantages of CILE, which facilitated the electron transfer rate. The fabricated CTS-MnO(2)-Hb/CILE showed good electrocatalytic ability to the reduction of trichloroacetic acid (TCA). Under the optimal conditions the catalytic current was in linear to TCA concentration in the range from 0.5 to 16.0 mmol L(-1) with the detection limit calculated as 0.167 mmol L(-1) (3σ). The result indicated that urchinlike MnO(2) nanoparticle had the potential application in the third generation electrochemical biosensors.     

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.     

MgFe-layered double hydroxide modified electrodes for direct electron transfer of heme proteins

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.     

A novel nitrite biosensor based on the direct electron transfer of hemoglobin immobilized on CdS hollow nanospheres

A novel nitrite biosensor based on the direct electron transfer of hemoglobin (Hb) immobilized on CdS hollow nanospheres (HS-CdS) modified glassy carbon electrode was constructed. The direct electron transfer of Hb showed a pair of redox peaks with a formal potential of -286 mV (vs. SCE) in 0.1M pH 7.0 phosphate buffer solution. It was a surface-controlled electrode process involving a single proton transfer coupled with a reversible one-electron transfer for each heme group of Hb. HS-CdS had a large specific surface area and good biocompatibility and had a better electrochemical response than that of solid spherical CdS. The immobilized Hb on HS-CdS displayed an excellent response to NO(2)(-) with one irreversible electrode process for NO reduction. Under optimal conditions, the biosensor could be used for the determination of NO(2)(-) with a linear range from 0.3 to 182 microM and a detection limit of 0.08 microM at 3 sigma based on the irreversible reduction of NO. HS-CdS provided a good matrix for protein immobilization and had a promising application in constructing sensors.     

A novel hydrogen peroxide biosensor based on the immobilization of hemoglobin on three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) film

The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface. Hemoglobin (Hb) has been successfully immobilized on the 3DOM GTD film and the fabrication process was characterized by Raman and UV-vis spectra. The results indicated that the Hb immobilized on the film retained its biological activity and the secondary structure of Hb was not destroyed. The direct electrochemistry and electrocatalysis of Hb immobilized on this film have been investigated. The Hb/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the Hb intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.20 and -0.48 V in 0.1M PBS (pH7.0), respectively. The Hb/3DOM GTD/ITO electrode exhibits an excellent eletrocatalytic activity, a wide linear range for H(2)O(2) from 5.0 μM to 1.0mM with a limit of detection of 0.6μM, high sensitivity (144.5 μA mM(-1)), good stability and reproducibility. Compared with the TiO(2) nanoneedles modified electrode, the GTD modified electrode has higher sensitivity and response peak current. The 3DOM GTD provided a good matrix for bioactive molecules immobilization, suggesting it has the potential use in the fields of H(2)O(2) biosensors.     

Immobilization of hemoglobin on zirconium dioxide nanoparticles for preparation of a novel hydrogen peroxide biosensor

Direct electrochemistry and thermal stability of hemoglobin (Hb) immobilized on a nanometer-sized zirconium dioxide (ZrO2) modified pyrolytic graphite (PG) electrode were studied. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of (7.90 +/- 0.93)s(-1) and a formal potential of -0.361 V (-0.12 V versus NHE) in 0.1M pH 7.0 PBS. Both nanometer-sized ZrO2 and dimethyl sulfoxide (DMSO) could accelerate the electron transfer between Hb and the electrode. Spectroscopy analysis of the Hb/ZrO2/DMSO film showed that the immobilized Hb could retain its natural structure. This modified electrode showed a high thermal stability up to 74 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the aid of an electron mediator. The electrocatalytic response showed a linear dependence on the H2O2 concentration ranging from 1.5 to 30.2 microM with a detection limit of 0.14 microM at 3sigma. The apparent Michaelis-Menten constant KMapp for H2O2 sensor was estimated to be (0.31 +/- 0.02) mM, showing a high affinity.     

Direct electron transfer and enzymatic activity of hemoglobin in a hexagonal mesoporous silica matrix

The direct electrochemistry of hemoglobin (Hb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Hb and the HMS was investigated using UV-Vis spectroscopy, FT-IR, and electrochemical methods. The direct electron transfer of the immobilized Hb exhibited two couples of redox peaks with the formal potentials of -0.037 and -0.232 V in 0.1 M (pH 7.0) PBS, respectively, which corresponded to its two immobilized states. The electrode reactions showed a surface-controlled process with a single proton transfer at the scan rate range from 20 to 200 mV/s. The immobilized Hb retained its biological activity well and displayed an excellent response to the reduction of both hydrogen peroxide (H2O2) and nitrate (NO2-). Its apparent Michaelis-Menten constants for H2O2 and NO2- were 12.3 and 49.3 microM, respectively, showing a good affinity. Based on the immobilization of Hb on the HMS and its direct electrochemistry, two novel biosensors for H2O2 and NO2- were presented. Under optimal conditions, the sensors could be used for the determination of H2O2 ranging from 0.4 to 6.0 microM and NO2- ranging from 0.2 to 3.8 microM. The detection limits were 1.86 x 10(-9) M and 6.11 x 10(-7) M at 3sigma, respectively. HMS provided a good matrix for protein immobilization and biosensor preparation.     

Direct electron transfer and electrocatalysis of hemoglobin adsorbed on mesoporous carbon through layer-by-layer assembly

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.     

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.     

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.     

The direct electron transfer of glucose oxidase and glucose biosensor based on carbon nanotubes/chitosan matrix

The glucose oxidase (GOD) is entrapped in the composite of carbon nanotubes/chitosan and direct electron transfer reaction between GOD and electrode takes place. The electron transfer rate of GOD is greatly enhanced to 7.73 s(-1) in the system, which is more than one-fold higher than that of flavin adenine dinucleotide adsorbed on the carbon nanotubes (3.1 s(-1)). This maybe results from the conformational change of GOD in the microenvironment enabling the accessibility of active site for GOD to the electrode. Additionally, the bioactivity of GOD modified in the composite on electrode surface is kept. So as-prepared electrode can be used as a glucose biosensor exhibiting higher sensitivity (0.5 microA mM(-1)) and better stability. The facile procedure of immobilizing GOD will promote the developments of electrochemical research for protein, biosensors and other bioelectrochemical devices.     

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.     

Highly sensitive and selective hydrogen peroxide biosensor based on hemoglobin immobilized at multiwalled carbon nanotubes-zinc oxide composite electrode

A highly sensitive and selective amperometric hydrogen peroxide (H(2)O(2)) biosensor based on immobilization of hemoglobin (Hb) at multiwalled carbon nanotubes-zinc oxide (MWCNT/ZnO) composite modified glassy carbon electrode (GCE) is reported. ZnO microsponges were electrochemically grown on MWCNT surface by the simple, cost-effective, green, electrochemical method at room temperature. The MWCNT/ZnO/Hb composite film showed a pair of well-defined, quasi-reversible redox peaks with a formal potential (E°') of -0.336V, characteristic features of heme redox couple of Hb. The electron transfer rate constant (k(s)) of immobilized Hb was 1.26s(-1). The developed biosensor showed a very fast response (>2s) toward H(2)O(2) with good sensitivity, wide linear range, and low detection limit of 0.02μM. The fabricated biosensor showed interesting features, including high selectivity, acceptable stability, good reproducibility, and repeatability along with excellent conductivity, facile electron mobility of MWCNT, and good biocompatibility of ZnO. The fabrication method of this biosensor is simple and effective for determination of H(2)O(2) in real samples with quick response, good sensitivity, high selectivity, and acceptable recovery.     

Hemoglobin niobate composite based biosensor for efficient determination of hydrogen peroxide in a broad pH range

Inorganic layered niobates (HCa2Nb3O10) were used as immobilization matrices of hemoglobin (Hb) because of their tunable interlayer spaces, large surface areas and good biocompatibilities. A pair of well-defined, quasi-reversible cycle voltammertric peaks were obtained at the Hb-HCa2Nb3O10 modified pyrolytic graphite electrode, suggesting that the layered niobates facilitate the electron transfer between the proteins and the electrode. Hb-HCa2Nb3O10 modified electrode exhibited electrocatalytic response for monitoring H2O2 with a large linear detection range from 25 microM to 3.0 mM and a relatively high sensitivity of 172 microA mM-1 cm-2. Based on the stabilizing effect of the layered niobates, Hb-HCa2Nb3O10 modified electrode can detect H2O2 in strongly acidic and basic solutions with pH of 1-12, which greatly expands the application fields of 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.     

Direct electrochemistry and electrocatalysis based on a film of horseradish peroxidase intercalated into Ni-Al layered double hydroxide nanosheets

Positively charged Ni-Al layered double hydroxide nanosheets (Ni-Al LDHNS) have been used for the first time as matrices for immobilization of horseradish peroxidase (HRP) in order to fabricate enzyme electrodes for the purpose of studying direct electron transfer between the redox centers of proteins and underlying electrodes. X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM) revealed that the HRP-Ni-Al LDHNS film had an ordered structure and that HRP was intercalated into Ni-Al LDHNS with a monolayer arrangement. Field emission scanning electron microscopy (FESEM) showed that the HRP-Ni-Al LDHNS film had a uniform, porous morphology. UV-vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the Ni-Al LDHNS film. The immobilized HRP in Ni-Al LDHNS on the surface of a glassy carbon electrode (GCE) exhibited good direct electrochemical and electrocatalytic responses to the reduction of hydrogen peroxide (H(2)O(2)) and trichloroacetic acid (TCA). The resulting H(2)O(2) biosensor showed a wide linear range from 6.00x10(-7)M to 1.92x10(-4)M, low detection limit (4.00x10(-7)M) and good stability. The results show that Ni-Al LDHNS provide a novel and efficient platform for the immobilization of enzymes and realizing direct electrochemistry and that the materials have potential applications in the fabrication of third-generation biosensors.     

Reagentless biosensor for hydrogen peroxide based on immobilization of protein in zirconia nanoparticles enhanced grafted collagen matrix

A novel matrix, zirconia nanoparticles enhanced grafted collagen (ZrO2-grafted collagen) hybrid composite, for immobilization of protein and biosensing was developed. The scanning electron microscopy, UV-vis and Fourier transform infrared spectra, and electrochemical measurements showed that the matrix was well biocompatible and could retain the bioactivity of immobilized protein to a large extent. The direct electron transfer of the immobilized myoglobin (Mb) exhibited a couple of stable and well-defined redox peaks with the formal potential of -336 mV (versus SCE) in 0.1M pH 7.0 PBS. This matrix could accelerate the electron transfer between Mb and the electrode with a surface-controlled process and an electron transfer rate constant of 3.58+/-0.35s-1 at 10-500 mVs-1. The Mb immobilized in the matrix showed a high thermal stability up to 70 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the help of an electron mediator. The linear response range of the biosensor to H2O2 concentration was from 1.0 to 85.0 microM with the limit of detection of 0.63 microM at a signal-to-noise ratio of 3sigma. The biosensor exhibited high sensitivity, acceptable stability and reproducibility. This work opened a way for the further study on the direct electron transfer and biosensing application of the immobilized protein in collagen-related matrices.     

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.     

A novel nitrite biosensor based on single-layer graphene nanoplatelet-protein composite film

A novel nitrite biosensor was developed through a sensing platform consisted of single-layer graphene nanoplatelet (SLGnP)-protein composite film. SLGnP with the virtues of excellent biocompatibility, conductivity and high sensitivity to the local perturbations can provide a biocompatible microenvironment for protein immobilization and a suitable electron transfer distance between electroactive centers of heme protein and electrode surface. A pair of well-defined and quasi-reversible cyclic voltammetric peaks that reflected the direct electrochemistry for ferric/ferrous couple of myoglobin (Mb) was achieved at the composite film modified electrode. Field emission scanning electron microscopy (FESEM) and ultraviolet visible spectra (UV-vis) were utilized to characterize the composite film. The results demonstrated that the morphology of the composite film was unique and the protein in the composite film retained its secondary structure similar to the native state. The composite film also displayed excellent electrocatalytic ability for the reduction of nitric oxide, which was applied to determine nitrite indirectly. It exhibited good electrochemical response to nitrite with a linear range from 0.05 to 2.5 mM and a detection limit of 0.01 mM.     

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.