Direct Electrochemistry of Hemoglobin Immobilized on Carbon‐Coated Iron Nanoparticles for Amperometric Detection of Hydrogen Peroxide

A novel method for preparation of hydrogen peroxide biosensor was presented based on immobilization of hemoglobin (Hb) on carbon‐coated iron nanoparticles (CIN). CIN was firstly dispersed in a chitosan solution and cast onto a glassy carbon electrode to form a CIN/chitosan composite film modified electrode. Hb was then immobilized onto the composite film with the cross‐linking of glutaraldehyde. The immobilized Hb displayed a pair of stable and quasireversible redox peaks and excellent electrocatalytic reduction of hydrogen peroxide (H₂O₂), which leading to an unmediated biosensor for H₂O₂. The electrocatalytic response exhibited a linear dependence on H₂O₂ concentration in a wide range from 3.1 μM to 4.0 mM with a detection limit of 1.2 μM (S/N=3). The designed biosensor exhibited acceptable stability, long‐term life and good reproducibility.     

Immobilization and Bioelectrochemistry of Hemoglobin Based on Carrageenan and Room Temperature Ionic Liquid Composite Film

A novel biopolymer/room‐temperature ionic liquid composite film based on carrageenan, room temperature ionic liquid (IL) [1‐butyl‐3‐methylimidazolium tetra?uoroborate ([BMIM]BF₄)] was explored for immobilization of hemoglobin (Hb) and construction of biosensor. Direct electrochemistry and electrocatalytic behaviors of Hb entrapped in the IL‐carrageenan composite ?lm on the surface of glassy carbon electrode (GCE) were investigated. UV‐vis spectroscopy demonstrated that Hb in the IL‐carrageenan composite ?lm could retain its native secondary structure. A pair of well‐de?ned redox peaks of Hb was obtained at the Hb‐IL‐carrageenan composite ?lm modi?ed electrode through direct electron transfer between the protein and the underlying electrode. The heterogeneous electron transfer rate constant (k_s) was 2.02 s^?1, indicating great facilitation of the electron transfer between Hb and IL‐carrageenan composite film modi?ed electrode. The modi?ed electrode showed excellent electrocatalytic activity toward reduction of hydrogen peroxide with a linear range of 5.0×10^?6 to 1.5×10^?4 mol/L and the detection limit was 2.12×10^?7 mol/L (S/N=3). The apparent Michaelis‐Menten constant K_M^app for hydrogen peroxide was estimated to be 0.02 mmol/L, indicating that the biosensor possessed high af?nity to hydrogen peroxide. In addition, the proposed biosensor showed good reproducibility and stability.     

Construction of hyaluronan-silver nanoparticle–hemoglobin multilayer composite film and investigations on its electrocatalytic properties

In this work, hyaluronan-silver nanoparticles (HSNPs) were prepared by UV-initiated photoreduction, and protein hemoglobin (Hb) was then alternately assembled with the prepared negatively charged HSNPs into layer-by-layer (LBL) films on solid surface. The electrochemical behavior and electrocatalytic activities toward oxygen and hydrogen peroxide of the resulting films were studied. It was found that the HSNPs greatly enhanced the electron transfer reactivity of Hb as a bridge. The assembly films showed a pair of nearly reversible redox peaks with a formal potential of −0.32 V (vs. Ag/AgCl) for the heme Fe(III)/Fe(II) redox couple. The immobilized Hb in the films maintained its biological activity, showing a surface-controlled process with a heterogeneous electron transfer rate constant (k _s) of 1.0 s⁻¹ and displaying the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide. This work provides a novel model to fabricate LBL films with protein, polysaccharide and nanoparticles, which may establish a foundation for fabricating new type of biosensors based on the direct electron transfer of redox proteins immobilized in nanocomposite multilayer films with underlying electrodes.     

Hydrogen peroxide biosensor based on the direct electrochemistry of myoglobin immobilized on ceria nanoparticles coated with multiwalled carbon nanotubesby a hydrothermal synthetic method

A novel myoglobin-based electrochemical biosensor was developed. It is based on a nanocomposite prepared from multiwalled carbon nanotubes that were coated with ceria nanoparticles. UV-vis and electrochemical measurements displayed that the nanocomposite provides a biocompatible matrix for the immobilization of myoglobin (Mb) and also facilitates direct electron transfer between its active center and the surface of the electrode. Immobilized Mb exhibits excellent electrocatalytic activity toward the reduction of hydrogen peroxide (HP). The low apparent Michaelis-Menten constant of 63.3 μM indicates high bioactivity and enhanced affinity to HP. This study also shows that the nanocomposite is a promising support for immobilization of proteins and for the preparation of third-generation biosensors.     

Immobilization of hemoglobin on SBA-15 applied to the electrocatalytic reduction of H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The direct electron transfer between hemoglobin (Hb) and an electrode was realized by first immobilizing the protein onto SBA-15.The results of the immobilization showed that the adsorption was pH-dependent with a maximum adsorption near the isoelectric point of the protein, and SBA-15 with a larger pore diameter showed greater adsorption capacity for Hb. UV–vis spectroscopy and nitrogen adsorption analysis indicated that Hb was adsorbed within the channel of SBA-15 and no significant denaturation occurred to the protein. The Hb/SBA-15 composite obtained was used for the fabrication of a Hb biosensor to detect hydrogen peroxide. A pair of well-defined redox peaks at −0.337 and −0.370 V on the Hb/SBA-15 composite modified glassy carbon electrode was observed, and the electrode reactions showed a surface-controlled process with a single proton transfer at a scan rate range from 20 to 1,000 mV/s. The sensor showed a fast amperometric response, a low detection limit (2.3 × 10⁻⁹ M) and good stability for the detection of H₂O₂. The electrochemical results indicated that the immobilized Hb still retained its biological activity.     

Cellular biosensor based on red blood cells immobilized on Fe3O4 Core/Au Shell nanoparticles for hydrogen peroxide electroanalysis

A core/shell Fe₃O₄/gold nanocomposite was prepared for immobilizing of red blood cells on a gold electrode via conjugation to a cysteamine monolayer. The hemoglobin in the film undergoes direct electron transfer at a formal potential of ?330 mV and displays excellent electrocatalytic response to hydrogen peroxide, with a linear range from 9.6 µM to 2.6 mM. The limit of detection is 4.4 µM (S/N?=?3). The Michaelis–Menten constant is 120 µM. Owing to its good biocompatibility, the biosensor exhibits good stability and acceptable reproducibility. The nanocomposite film provided a good matrix for the immobilization of cells and for the preparation of cellular biosensors.     

Reagentless Biosensor for Hydrogen Peroxide Based on the Immobilization of Hemoglobin in Platinum Nanoparticles Enhanced Poly(chloromethyl thiirane) Cross‐linked Chitosan Hybrid Film

An unmediated hydrogen peroxide (H₂O₂) biosensor was prepared by co‐immobilizing hemoglobin (Hb) with platinum nanoparticles enhanced poly(chloromethyl thiirane) cross‐linked chitosan (CCCS‐PNs) hybrid film. CCCS could provide a biocompatible microenvironment for Hb and PNs could accelerate the electron transfer between Hb and the electrode. Spectroscopic analysis indicated that the immobilized Hb could maintain its native structure in the CCCS‐PNs hybrid film. Entrapped Hb exhibited direct electrochemistry for its heme Fe(III)/Fe(II) redox couples at ?0.396 V in the CCCS‐PNs hybrid film, as well as peroxidase‐like activity to the reduction of hydrogen peroxide without the aid of an electron mediator.     

ZnO/Cu nanocomposite: a platform for direct electrochemistry of enzymes and biosensing applications

Unique structured nanomaterials can facilitate the direct electron transfer between redox proteins and the electrodes. Here, in situ directed growth on an electrode of a ZnO/Cu nanocomposite was prepared by a simple corrosion approach, which enables robust mechanical adhesion and electrical contact between the nanostructured ZnO and the electrodes. This is great help to realize the direct electron transfer between the electrode surface and the redox protein. SEM images demonstrate that the morphology of the ZnO/Cu nanocomposite has a large specific surface area, which is favorable to immobilize the biomolecules and construct biosensors. Using glucose oxidase (GOx) as a model, this ZnO/Cu nanocomposite is employed for immobilization of GOx and the construction of the glucose biosensor. Direct electron transfer of GOx is achieved at ZnO/Cu nanocomposite with a high heterogeneous electron transfer rate constant of 0.67 ± 0.06 s(-1). Such ZnO/Cu nanocomposite provides a good matrix for direct electrochemistry of enzymes and mediator-free enzymatic biosensors.     

Reduced Graphene Oxide|Carbon Ceramic Electrode Modified with CdS‐Hemoglobin as a Sensitive Hydrogen Peroxide Biosensor

A sensitive hydrogen peroxide (H₂O₂) biosensor was developed based on a reduced graphene oxide|carbon ceramic electrode (RGO|CCE) modified with cadmium sulfide‐hemoglobin (CdS‐Hb). The electron transfer kinetics of Hb were promoted due to the synergetic function of RGO and CdS nanoparticles. The transfer coefficient (α) and the heterogeneous electron transfer rate constant (k_s) were calculated to be 0.54 and 2.6 s^?1, respectively, indicating a great facilitation achieved in the electron transfer between Hb and the electrode surface. The biosensor showed a good linear response to the reduction of H₂O₂ over the concentration range of 2–240 µM with a detection limit of 0.24 µM (S/N=3) and a sensitivity of 1.056 µA µM^?1 cm^?2. The high surface coverage of the CdS‐Hb modified RGO|CCE (1.04×10^?8 mol cm^?2) and a smaller value of the apparent Michaelis? Menten constant (0.24 mM) confirmed excellent loading of Hb and high affinity of the biosensor for hydrogen peroxide.     

Multilayer Assembly of Hemoglobin and Colloidal Gold Nanoparticles on Multiwall Carbon Nanotubes/Chitosan Composite for Detecting Hydrogen Peroxide

Chitosan (CS) was chosen for dispersing multi‐wall carbon nanotubes (MWNTs) to form a stable CS‐MWNTs composite, which was first coated on the surface of a glassy carbon electrode to provide a containing amino groups interface for assembling colloidal gold nanoparticles (GNPs), followed by the adsorption of hemoglobin (Hb). Repeating the assembly step of GNPs and Hb resulted in {Hb/GNPs}_n multilayers. The assembly of GNPs onto CS‐MWNTs composites was confirmed by transmission electron microscopy. The consecutive growth of {Hb/GNPs}_n multilayers was confirmed by cyclic voltammetry and UV‐vis absorption spectroscopy. The resulting system brings a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of GNPs and MWNTs. The resulting biosensor displays an excellent electrocatalytic activity and rapid response for hydrogen peroxide. The linear range for the determination of H₂O₂ was from 5.0×10^?7 to 2.0×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelis–Menten constant K_M^app value of 0.19 mM.     

General Preparation of Heme Protein Functional Fe3O4@Au‐Nps Magnetic Nanocomposite for Sensitive Detection of Hydrogen Peroxide

Stable magnetic nanocomposite of gold nanoparticles (Au‐NPs) decorating Fe₃O₄ core was successfully synthesized by the linker of Boc‐L‐cysteine. Transmission electron microscope (TEM), energy dispersive X‐ray spectroscopy (EDX) and cyclic voltammograms (CV) were performed to characterize the as‐prepared Fe₃O₄@Au‐Nps. The results indicated that Au‐Nps dispersed homogeneously around Fe₃O₄ with the ratio of Au to Fe₃O₄ nanoparticles as 5–10/1 and the apparent electrochemical area as 0.121 cm². After self‐assembly of hemoglobin (Hb) on Fe₃O₄@Au‐Nps by electrostatic interaction, a hydrogen peroxide biosensor was developed. The Fe₃O₄@Au‐Nps/Hb modified GCE exhibited fast direct electron transfer between heme center and electrode surface with the heterogeneous electron transfer rate constant (Ks ) of 3.35 s⁻¹. Importantly, it showed excellent electrocatalytic activity towards hydrogen peroxide reduction with low detection limit of 0.133 μM (S /D =3) and high sensitivity of 0.163 μA μM⁻¹, respectively. At the concentration evaluated, the interfering species of glucose, dopamine, uric acid and ascorbic acid did not affect the determination of hydrogen peroxide. These results demonstrated that the introduction of Au‐Nps on Fe₃O₄ not only stabilized the immobilized enzyme but also provided large surface area, fast electron transfer and excellent biocompatibility. This facile nanoassembly protocol can be extended to immobilize various enzymes, proteins and biomolecules to develop robust biosensors.     

Direct electrochemistry and enzymatic activity of hemoglobin immobilized in ordered mesoporous titanium oxide matrix

The novel highly ordered mesoporous titanium oxide (mesoTiO₂) materials, prepared by the “acid–base pairs” route, were firstly used for the immobilization of hemoglobin (Hb) and its bioelectrochemical properties were studied. FTIR and UV–vis spectroscopy demonstrated that Hb in the mesoTiO₂ matrix could retain its native secondary structure. The CV results of Hb/mesoTiO₂-modified electrode showed a pair of well-defined and quasi-reversible redox peaks centered at approximate −0.158 V (vs. SCE) in pH 6.0 phosphate buffer solution. It reflects the characteristic of Hb heme Fe (III)/Fe(II) redox couple with fast heterogeneous electron transfer rate. The immobilized Hb also displayed its good electrocatalytic activity for the reduction of hydrogen peroxide. The results demonstrate that the mesoTiO₂ matrix may improve the protein loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its high specific surface area, uniform three-dimensional well-ordered porous structure, suitable pore size and biocompatibility.     

Polyvinyl alcohol–ionic liquid composition for promoting the direct electron transfer and electrocatalysis of hemoglobin

The direct electron transfer and electrocatalysis of hemoglobin (Hb) entrapped in polyvinyl alcohol (PVA)–room temperature ionic liquid (i.e., 1-octyl-3-methylimidazolium hexafluorophosphate [OMIM]PF₆) composition has been investigated by using cyclic voltammetry and chronocoulometry. It is found that the composition can promote the direct electron transfer of Hb and the heterogeneous electron transfer rate constant (k_s) of immobilized Hb is enhanced to 19.9 s⁻¹. The immobilized Hb also shows high electro-catalytic activity towards the redox of oxygen, hydrogen peroxide and nitrite. The Michaelis constants (K_m) decrease to 1.2 × 10⁻⁴ M (for hydrogen peroxide) and 9.4 × 10⁻³ M (for nitrite). The surface concentration of electroactive Hb is estimated and it is ca. 1.4 × 10⁻¹⁰ mol cm⁻², meaning that several layers of immobilized Hb take part in the electrochemical reaction. When gold nanoparticles (GNP) is introduced into the composition, the resulting PVA–GNP–[OMIM]PF₆ composition presents better performance. The electrochemical characteristic of immobilized Hb is improved further. Thus PVA–GNP–[OMIM]PF₆ composition is more suitable for the immobilization of Hb. Therefore, it is a good strategy to prepare novel composition for protein immobilization by using several materials with different function.     

A hydrogen peroxide biosensor based on the direct electron transfer of hemoglobin encapsulated in liquid-crystalline cubic phase on electrode

Liquid crystal cubic phase formed with monoolein has been used as immobilizing matrix to host redox protein hemoglobin on glassy carbon electrode surface. The promoted direct electron transfer between hemoglobin and electrode was observed and a large average kinetic electron transfer rate constant k(s) of 3.03(±0.02)s(-1) was estimated. The electrode modified with cubic phase containing hemoglobin retains the bioactivity of hemoglobin and shows excellent bioelectrocatalytic activity to the reduction of hydrogen peroxide with a small apparent Michaelis-Menten constant of 0.25(±0.03)mM. A novel reagentless hydrogen peroxide biosensor was constructed using the hemoglobin-containing cubic phase modified electrode and the proposed hydrogen peroxide biosensor shows a linear range of 7.0-239μM with a detection limit of 3.1(±0.2)μM and good stability and reproducibility.     

Direct electrochemistry and electrocatalysis of hydrogen peroxide using hemoglobin immobilized in hollow zirconium dioxide spheres and sodium alginate films

A biocompatible composite made from hollow zirconium dioxide microspheres (HZMS) and sodium alginate (SA) is presented and used for the construction of a biosensor for hydrogen peroxide using a gold electrode. The composition, morphology and size were studied by transmission electron microscopy. FT-IR and UV-vis spectroscopy revealed that hemoglobin (Hb) entrapped in the ZHMS retains its native structure. A pair of stable and well-defined quasi-reversible redox peaks of Hb is obtained, with a formal potential of ?0.15 V at pH 7.0. The apparent heterogeneous electron transfer rate constant is 1.15 s^?1, indicating facile electron transfer that may result from the unique nanostructures and larger surface area of the HZMS. The amperometric response of the sensor varied linearly with the concentration of hydrogen peroxide in the range from 1.75 µM to 4.9 mM, with a detection limit of 0.6 µM (at S/N?=?3). The apparent Michaelis-Menten constant $ \left( {K_{\rm{M}}^{\rm{app}}} \right) $ is 1.6 mM. The biosensor possesses high sensitivity, good reproducibility, and long-term stability.     

Amperometric biosensor for hydrogen peroxide based on direct electrocatalysis by hemoglobin immobilized on gold nanoparticles/1,6-diaminohexane modified glassy carbon electrode.

A facile strategy of an amperometric biosensor for hydrogen peroxide based on the direct electrocatalysis of hemoglobin (Hb) immobilized on gold nanoparticles (GNPs)/1,6-diaminohexane (DAH) modified glassy carbon electrode (GCE) has been described. A uniform monolayer film of DAH was initially covalently bound on a GCE surface by virtue of the electrooxidation of one amino group of DAH, and another amino group was modified with GNPs and Hb, successively. The fabrication process was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The proposed biosensor exhibited an effective and fast catalytic response to the reduction of H2O2 with good reproducibility and stability. A linear relationship existed between the catalytic current and the H2O2 concentration in the range of 1.5x10(-6) to 2.1x10(-3) M with a correlation coefficient of 0.998 (n=24). The detection limit (S/N=3) was 8.8x10(-7) M.     

An Organic Sol‐Gel Film as Modifier to Construct Biosensor

A new amperometric biosensor for hydrogen peroxide (H₂O₂) was developed by adsorbing hemoglobin (Hb) on an organic sol‐gel film. The organic sol‐gel was prepared using resorcinol and formaldehyde as monomers. This sol‐gel film shows a biocompatible microenvironment for retaining the native activity of the adsorbed Hb. The direct electron transfer between Hb and electrode is achieved. Hb adsorbed on the film shows an enzyme‐like catalytic activity for the reduction of H₂O₂. The reduction peak currents are proportional linearly to the concentration of hydrogen peroxide in the range of 6×10^?8 to 3.6×10^?6 M, with a detection limit of 2.4×10^?8 M (S/N=3). This research enlarges the applications of organic sol‐gel materials in biosensor field.     

Amperometric detection of hypoxanthine and xanthine by enzymatic amplification using a gold nanoparticles-carbon nanohorn hybrid as the carrier

A novel gold nanoparticles-single-walled carbon nanohorn (GNPs-SWCNH) hybrid was synthesized for the construction of an amperometric biosensing platform. The GNPs-SWCNH hybrid was stable in aqueous solution for at least two weeks, and was characterized with scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy. The average diameter of GNPs in situ synthesized on the SWCNH was 5-8 nm, and the good interaction between GNPs and SWCNH was confirmed by ultraviolet-visible absorption spectroscopy. The GNPs-SWCNH immobilized on a platinum electrode showed high electrochemical activity toward the oxidation of hydrogen peroxide and uric acid with low applied potentials. Combining with the enzymatic reaction of xanthine oxidase (XOx), a biosensor for hypoxanthine and xanthine was constructed. The XOx-GNPs-SWCNH-based biosensor exhibited good responses to hypoxanthine and xanthine with the linear ranges of 1.5 to 35.4 and 2.0 to 37.3 μM, and the detection limits of 0.61 and 0.72 μM, respectively. The recovery test showed acceptable results. The gold nanoparticles functionalized carbon nanohorns provided a promising way to construct an electrochemical platform for sensitive biosensing.     

Electrochemical Surface Structuring with Polyaniline Wrapped Hb for Hydrogen Peroxide Biosensing

Through the electrodeposition of aniline with hemoglobin (Hb) on zincoxide‐gold colloidal sols (ZnO‐AuNPs) modified indium oxide electrode, a hydrogen peroxide (H₂O₂) biosensor was constructed. Polyaniline (PANI) form a nano‐cage wrapped Hb, which provided a comfortable and stable site for the immobization of Hb. UV‐vis spectrum was employed to characterize Hb retained original structure in the resulting Hb‐PANI/ZnO‐AuNPs membrane. Electrochemical investigation of the biosensor showed a pair of well‐defined, quasi‐reversible redox peaks with E_pa= ‐0.139 V and E_pc = ‐0.238 V (vs. SCE) in 0.1 M pH 7.0 phosphate buffer solution at the scan rate of 100 mV/s. The biosensor displayed a fast response time (<3 s) and broad linear response to H₂O₂ in the range from 1.5 μM to 1.7 mM with a detection limit of 0.8 μM (S/N = 3).     

A Simple Layer‐by‐Layer Assembly Strategy for a Reagentless Biosensor Based on a Nanocomposite of Methylene Blue‐Multiwalled Carbon Nanotubes

A simple layer‐by‐layer (LBL) assembly strategy was established for constructing a novel reagentless biosensor based on a nanocomposite of methylene blue multiwalled carbon nanotubes (MB‐MWNTs). A nanocomposite of MB‐MWNTs was obtained by direct premixing and possessed good dispersion in barbital‐HCl buffer. Through electrostatic interactions, the nanocomposite of MB‐MWNTs could alternately be assembled with horseradish peroxidase (HRP) on the Au electrode modified with precursor films. UV/Vis spectra and scanning electron microscopy (SEM) were applied to reveal the formation of the nanocomposite of MB‐MWNTs. The LBL assembly process was also verified by electrochemical impedance spectroscopy (EIS). The MB is a well‐established mediator and efficiently facilitated the electron shuttle between the HRP and the electrode, as demonstrated by the cyclic voltammetry (CV) measurements. The as‐prepared reagentless biosensor exhibited a fast response for the determination of hydrogen peroxide (H₂O₂) and reached 95% of the steady‐state current within 3 s. It was found that the linear response range of the reagentless biosensor for H₂O₂ was from 4.0 μM to 3.78 mM with a detection limit of 1.0 μM and a sensitivity of 22.5 μA mM⁻¹. The biosensor exhibited a high reproducibility and stability.