Direct Electrochemistry of Hemoglobin on Single‐Walled Carbon Nanotubes Modified Carbon Ionic Liquid Electrode and Its Electrocatalysis

In this article we report on the fabrication of a carbon ionic liquid electrode (CILE) by using a room temperature ionic liquid of 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) as binder. It was further modified by single‐walled carbon nanotubes (SWCNTs) to get a SWCNTs modified CILE denoted as SWCNTs/CILE. The redox protein of hemoglobin (Hb) was further immobilized on the surface of SWCNTs/CILE with the help of Nafion film. UV‐vis and FT‐IR spectra indicated that the immobilized Hb retained its native conformation in the composite film. The direct electrochemistry of Hb on the SWCNTs/CILE was carefully studied in pH 7.0 phosphate buffer solution (PBS). Cyclic voltammetric results indicated that a pair of well‐defined and quasireversible voltammetric peaks of Hb heme Fe(III)/Fe(II) was obtained with the formal potential (E°') at ?0.306 V (vs. SCE). The electrochemical parameters such as the electron transfer coefficient (α), the electron transfer number (n) and the apparent heterogeneous electron transfer rate constant (k_s) were calculated as 0.34, 0.989 and 0.538 s^?1, respectively. The fabricated Hb modified electrode showed good electrocatalytic ability to the reduction of trichloroacetic acid (TCA) in the concentration range from 20.0 to 150.0 mmol/L with the detection limit of 10.0 mmol/L (3σ).     

Direct Electrochemistry of Hemoglobin and Its Electrocatalysis Based on Hyaluronic Acid and Room Temperature Ionic Liquid

A biocomposite material of hyaluronic acid (HA) and room temperature ionic liquid, 1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (BMIMBF₄) was fabricated and characterized by voltammetry and electrochemical impedance spectroscopy. Hemoglobin (Hb), as a model protein, was immobilized in this biocomposite on glassy carbon electrode to investigate its direct electrochemistry. A pair of well‐defined, quasireversible cyclic voltammetric peaks appeared with the formal potentials (E°′) at ?0.360 V (vs. SCE). The Hb modified electrode exhibited an excellent electrocatalytic activity to the reduction of H₂O₂ with a wide linear range, good selectivity, sensitivity and reproducibility, which had potential application to fabrication the third‐generation biosensor.     

Ionic Liquid‐Hemoglobin‐Carbon Paste Composite Bioelectrode and Its Electrocatalytic Activity

A new carbon ionic liquid paste bioelectrode was fabricated by mixing hemoglobin (Hb) with graphite powder, ionic liquid 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (EMIMBF₄) and liquid paraffin homogeneously. Nafion film was cast on the electrode surface to improve the stability of bioelectrode. Direct electrochemistry of Hb in the bioelectrode was carefully investigated. Cyclic voltammetric results indicated that a pair of well‐defined and quasi‐reversible electrochemical responses appeared in pH 7.0 phosphate buffer solution (PBS), indicating that direct electron transfer of Hb was realized in the modified electrode. The formal potential (E^0′) was calculated as ?0.316 V (vs. SCE), which was the typical characteristic of the electrochemical reaction of heme Fe(III)/Fe(II) redox couple. Based on the cyclic voltammetric results the electrochemical parameters of the electrode reaction were calculated. This bioelectrode showed high electrocatalytic activity towards the reduction of trichloroacetic acid (TCA) with good stability and reproducibility.     

Application of NiMoO4 Nanorods for the Direct Electrochemistry and Electrocatalysis of Hemoglobin with Carbon Ionic Liquid Electrode

In this paper NiMoO₄ nanorods were synthesized and used to accelerate the direct electron transfer of hemoglobin (Hb). By using an ionic liquid (IL) 1‐butylpyridinium hexafluorophosphate (BPPF₆) modified carbon paste electrode (CILE) as the basic electrode, NiMoO₄ nanorods and Hb composite biomaterial was further cast on the surface of CILE and fixed by chitosan (CTS) to establish a modified electrode denoted as CTS/NiMoO₄‐Hb/CILE. UV‐vis and FT‐IR spectroscopic results showed that Hb in the film retained its native structures without any conformational changes. Electrochemical behaviors of Hb entrapped in the film were carefully investigated by cyclic voltammetry with a pair of well‐defined and quasi‐reversible redox voltammetric peaks appearing in phosphate buffer solution (PBS, pH 3.0), which was attributed to the direct electrochemistry of the electroactive center of Hb heme Fe(III)/Fe(II). The results were ascribed to the specific characteristic of NiMoO₄ nanorods, which accelerated the direct electron transfer rate of Hb with the underlying CILE. The electrochemical parameters of Hb in the composite film were further carefully calculated with the results of the electron transfer number (n) as 1.08, the charge transfer coefficient (α) as 0.39 and the electron‐transfer rate constant (k_s) as 0.82 s^?1. The Hb modified electrode showed good electrocatalytic ability toward the reduction of trichloroacetic acid (TCA) in the concentration range from 0.2 to 26.0 mmol/L with a detection limit of 0.072 mmol/L (3σ), and H₂O₂ in the concentration range from 0.1 to 426.0 µmol/L with a detection limit of 3.16×10^?8 mol/L (3σ).     

Electrodeposited Graphene and Silver Nanoparticles Modified Electrode for Direct Electrochemistry and Electrocatalysis of Hemoglobin

By using a 1‐butylpyridinium hexafluorophosphate based carbon ionic liquid electrode (CILE) as the working electrode, graphene (GR) nanosheets and silver nanoparticles (Ag NPs) were step by step electrodeposited on the surface of the CILE with potentiostatic method. The fabricated Ag/GR/CILE was used as a new platform for protein electrochemistry and hemoglobin (Hb) was immobilized on its surface with chitosan (CTS) as film forming material. In 0.1 mol/L phosphate buffer solution, a pair of well‐defined and quasi‐reversible redox peaks appeared on the CTS/Hb/Ag/GR/CILE with a formal peak potential of ?0.202 V (vs. SCE) and a peak‐to‐peak separation (ΔE_p) of 68 mV, which indicated that direct electrochemistry of Hb was realized on the modified electrode. The results could be attributed to the synergistic effects of Ag NPs and GR nanosheets on the electrode surface, which provided a specific three‐dimensional structure with high conductivity and good biocompatibility. The Hb modified electrode showed excellent electrocatalysis to the reduction of trichloroacetic acid in the concentration range from 0.8 to 22.0 mmol/L with a detection limit of 0.42 mmol/L (3σ). Moreover, the modified electrode exhibited favorable reproducibility, long term stability and accuracy, with potential applications in the third‐generation electrochemical biosensor.     

Direct Electrochemistry and Electrocatalysis of Hemoglobin Based on Nafion‐Room Temperature Ionic Liquids‐Multiwalled Carbon Nanotubes Composite Film

A robust and effective composite film combined the benefits of Nafion, room temperature ionic liquid (RTIL) and multi‐wall carbon nanotubes (MWNTs) was prepared. Hemoglobin (Hb) was successfully immobilized on glassy carbon electrode surface by entrapping in the composite film. Direct electrochemistry and electrocatalysis of immobilized Hb were investigated in detail. A pair of well‐defined and quasi‐reversible redox peaks of Hb was obtained in 0.10 mol·L^?1 pH 7.0 phosphate buffer solution (PBS), indicating that the Nafion‐RTIL‐MWNTs film showed an obvious promotion for the direct electron transfer between Hb and the underlying electrode. The immobilized Hb exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis current was linear to H₂O₂ concentration in the range of 2.0×10^?6 to 2.5×10^?4 mol·L^?1, with a detection limit of 8.0×10^?7 mol·L^?1 (S/N=3). The apparent Michaelis‐Menten constant (K_m^app) was calculated to be 0.34 mmol·L^?1. Moreover, the modified electrode displayed a good stability and reproducibility. Based on the composite film, a third‐generation reagentless biosensor could be constructed for the determination of H₂O₂.     

Direct Electrochemistry and Electrocatalysis of Hemoglobin Immobilized in a Polymeric Ionic Liquid Film

A polymer film based on polymeric ionic liquid, which was poly(1‐vinyl‐3‐butylimidazolium chloride) (poly(ViBuIm⁺Cl^?)for short), was firstly used as matrix to immobilize hemoglobin (Hb). FTIR and UV‐vis spectra demonstrated that the native structure of Hb was well preserved after entrapped into the polymer film. The Hb immobilized in the poly(ViBuIm⁺Cl^?) film showed a fast direct electron transfer for the Hb‐Fe^III/Fe^II redox couple. Based on the direct electron transfer of the immobilized Hb, polyvinyl alcohol (PVA)/Hb/poly(ViBuIm⁺Cl^?)/GC electrode displayed good sensitivity and wide linear range for the detection of H₂O₂. The linear range of the PVA/Hb/poly(ViBuIm⁺Cl^?)/GC electrode to H₂O₂ is from 3.5 to 224 μM with a limit of detection of 1.17 μM. Such an avenue, which integrated polymeric ionic liquid and redox protein via a simple method, may provide a novel and efficient platform for the fabrication of biosensors, biofuel cells and other bioelectrochemical devices.     

Direct electrochemistry of hemoglobin immobilized in the sodium alginate and SiO2 nanoparticles bionanocomposite film on a carbon ionic liquid electrode

In this paper a carbon ionic liquid electrode (CILE) was fabricated by using a room temperature ionic liquid of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) as binder. By using the CILE as basal electrode, the hemoglobin (Hb) molecule was immobilized on the surface of CILE with a sodium alginate (SA) hydrogel and SiO₂ nanoparticles organic-inorganic composite material. The direct electrochemical behaviors of Hb in the bionanocomposite film were further studied in a pH 7.0 Britton-Robinson (B-R) buffer solution. A pair of well-defined quasi-reversible cyclic voltammetric peaks of Hb was obtained on SA/nano-SiO₂/Hb/CILE with the formal potential (E^0’) at -0.355 V (vs. SCE), which was the characteristic of heme Fe(III)/Fe(II) redox couples. The formal potential of Hb Fe(III)/Fe(II) couple shifted negatively with increasing pH of solution with a slope of -45.2 mV/pH, which indicated that a one electron transfer accompanied with one proton transportation. The immobilized Hb showed good electrocatalytic manner to the reduction of trichloroacetic acid (TCA).     

Fabrication and Electrochemical Behavior of Hemoglobin Modified Carbon Ionic Liquid Electrode

A new hemoglobin (Hb) and room temperature ionic liquid modified carbon paste electrode was constructed by mixing Hb with 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) and graphite powder together. The Hb modified carbon ionic liquid electrode (Hb‐CILE) was further characterized by FT‐IR spectra, scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Hb in the carbon ionic liquid electrode remained its natural structure and showed good direct electrochemical behaviors. A pair of well‐defined quasireversible redox peaks appeared with the apparent standard potential (E′) as ?0.334 (vs. SCE) in pH 7.0 phosphate buffer solution (PBS). The electrochemical parameters such as the electron transfer number (n), the electron transfer coefficient (α) and the heterogeneous electron transfer kinetic constant (k_s) of the electrode reaction were calculated with the results as 1.2, 0.465 and 0.434 s^?1, respectively. The fabricated Hb‐CILE exhibited excellent electrocatalytic activity to the reduction of H₂O₂. The calibration range for H₂O₂ quantitation was between 8.0×10^?6 mol/L and 2.8×10^?4 mol/L with the linear regression equation as I_ss (μA)=0.12 C (μmol/L)+0.73 (n=18, γ=0.997) and the detection limit as 1.0×10^?6 mol/L (3σ). The apparent Michaelis–Menten constant (K_M^app) of Hb in the modified electrode was estimated to be 1.103 mmol/L. The surface of this electrochemical sensor can be renewed by a simple polishing step and showed good reproducibility.     

Voltammetric Determination of Hemoglobin Using a Pencil Lead Electrode

In this paper the electrochemical behavior of hemoglobin (Hb) immobilized on a pencil lead electrode (PLE) was investigated. Immobilization of Hb on the pencil lead electrode was performed by nonelectrochemical and electrochemical methods. In phosphate buffer solution with pH 7.0 Hb showed a pair of well‐defined and nearly reversible redox waves (the anodic and cathodic peak potentials are located at ?0.18 V and ?0.22 V, respectively). The dependence of the anodic peak potential (E_pa) on the pH of the buffer solution indicated that the conversion of Hb? Fe(III)/Hb? Fe(II) is a one‐electron‐transfer reaction process coupled with one‐proton‐transfer. In addition the effect of scan rate on peak currents and peak separation potential was investigated and electrochemical parameters such as α and k_s were calculated. In the second part of this work, the ability of the electrode for determination of Hb concentration was investigated. The results showed a linear dynamic range from 0.15 to 2 µM and a detection limit of 0.11 µM. The relative standard deviation is 4.1 % for 4 successive determinations of a 1 µM Hb solution.     

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.     

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.     

Application of Titanium Dioxide Nanowires for the Direct Electrochemistry of Hemoglobin and Electrocatalysis

Titanium dioxide (TiO₂) nanowires were synthesized and used for the realization of direct electrochemistry of hemoglobin (Hb) with carbon ionic liquid electrode (CILE) as the substrate electrode. TiO₂‐Hb composite was casted on the surface of CILE with a chitosan film and spectroscopic results confirmed that Hb retained its native structure in the composite. Direct electron transfer of Hb on the modified electrode was realized with a pair of quasi‐reversible redox waves appeared, indicating that the presence of TiO₂ nanowires could accelerate the electron transfer rate between the electroactive center of Hb and the substrate electrode. Electrochemical behaviors of Hb on the modified electrode were carefully investigated with the values of the electron transfer coefficient (α), the electron transfer number and the heterogeneous electron transfer rate constant (k_s) as 0.58, 0.98 and 1.62 s^‐1. The Hb modified electrode showed excellent electrocatalytic activity to the reduction of trichloroacetic acid and NaNO₂ with wider linear range and lower detection limit, indicating the successful fabrication of a new third‐generation biosensor.     

Electrocatalysis of Hemoglobin in ZnO Nanoparticle/Ionic Liquid Composite Film Modified Glassy Carbon Electrode

A nanobiocompatible composite containing hemoglobin (Hb), ZnO nanoparticles (nano‐ZnO) and ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) was fabricated and further modified on the glassy carbon electrode (GCE). The electrochemical behaviours of Hb in the composite film were carefully studied and a pair of quasi‐reversible redox peaks appeared in pH 7.0 phosphate buffer solution, which was attributed to the electrode reaction of Hb heme Fe(III)/Fe(II) redox couple. The presences of nano‐ZnO and BMIMPF₆ in the film can retain the bioactivity of Hb and greatly enhance the direct electron transfer of Hb. The immobilized Hb showed high stability and good electrocatalytic ability to the reduction of hydrogen peroxide and O₂.     

Electrocatalytic Determination of Hydrazine and Phenol Using a Carbon Paste Electrode Modified with Ionic Liquids and Magnetic Core‐shell Fe3O4@SiO2/MWCNT Nanocomposite

A carbon paste electrode was modified with 2‐(4‐Oxo‐3‐phenyl‐3,4‐dihydroquinazolinyl)‐N′‐phenyl‐hydrazinecarbothioamide, magnetic core? shell Fe₃O₄@SiO₂/MWCNT nanocomposite and ionic liquid (n‐hexyl‐3‐methylimidazolium hexafluoro phosphate). The electro‐oxidation of hydrazine at the surface of the modified electrode was studied using electrochemical approaches. This modified electrode offers a considerable improvement in voltammetric sensitivity toward hydrazine, compared to the bare electrode. Square wave voltammetry (SWV) exhibits a linear dynamic range from 7.0×10^?8 to 5.0×10^?4 M and a detection limit of 40.0 nM for hydrazine. The diffusion coefficient and kinetic parameters (such as electron transfer coefficient and the heterogeneous rate constant) for hydrazine oxidation were also determined. The prepared modified electrode exhibits a very good resolution between the voltammetric peaks of hydrazine and phenol that makes it suitable for the detection of hydrazine in the presence of phenol in real samples.     

Electrochemistry and electrocatalysis of hemoglobin in Nafion/nano-CaCO3 film on a new ionic liquid BPPF6 modified carbon paste electrode

Room temperature ionic liquid N-butylpyridinium hexafluorophosphate (BPPF6) was used as a binder to construct a new carbon ionic liquid electrode (CILE), which exhibited enhanced electrochemical behavior as compared with the traditional carbon paste electrode with paraffin. By using the CILE as the basal electrode, hemoglobin (Hb) was immobilized on the surface of the CILE with nano-CaCO3 and Nafion film step by step. The Hb molecule in the film kept its native structure and showed good electrochemical behavior. In pH 7.0 Britton-Robinson (B-R) buffer solution, a pair of well-defined, quasi-reversible cyclic voltammetric peaks appeared with cathodic and anodic peak potentials located at -0.444 and -0.285 V (vs SCE), respectively, and the formal potential (E degrees') was at -0.365 V, which was the characteristic of Hb Fe(III)/Fe(II) redox couples. The formal potential of Hb shifted linearly to the increase of buffer pH with a slope of -50.6 mV pH-1, indicating that one electron transferred was accompanied with one proton transportation. Ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopy studies showed that Hb immobilized in the Nafion/nano-CaCO3 film still remained its native arrangement. The Hb modified electrode showed an excellent electrocatalytic behavior to the reduction of H2O2, trichloroacetic acid (TCA), and NaNO2.     

Electrochemistry and Electrocatalysis of a Nafion/Nano‐CaCO3/Hb Film Modified Carbon Ionic Liquid Electrode Using BMIMPF6 as Binder

In this paper a room temperature ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) was used as binder for the construction of carbon ionic liquid electrode (CILE) and a new electrochemical biosensor was developed for determination of H₂O₂ by immobilization of hemoglobin (Hb) in the composite film of Nafion/nano‐CaCO₃ on the surface of CILE. The Hb modified electrode showed a pair of well‐defined, quasi‐reversible redox peaks with E_pa and E_pc as ?0.265 V and ?0.470 V (vs. SCE). The formal potential (E°′) was got by the midpoint of E_pa and E_pc as ?0.368 V, which was the characteristic of Hb Fe(III)/Fe(II) redox couples. The peak to peak separation was 205 mV in pH 7.0 Britton–Robinson (B–R) buffer solution at the scan rate of 100 mV/s. The direct electrochemistry of Hb in the film was carefully investigated and the electrochemical parameters of Hb on the modified electrode were calculated as α=0.487 and k_s=0.128 s^?1. The Nafion/nano‐CaCO₃/Hb film electrode showed good electrocatalysis to the reduction of H₂O₂ in the linear range from 8.0 to 240.0 μmol/L and the detection limit as 5.0 μmol/L (3σ). The apparent Michaelis–Menten constant (K_M^app) was estimated to be 65.7 μmol/L. UV‐vis absorption spectroscopy and FT‐IR spectroscopy showed that Hb in the Nafion/nano‐CaCO₃ composite film could retain its native structure.     

Sensitive Electrochemical Determination of Catechol with a Graphene Modified Carbon Ionic Liquid Electrode

In this paper a graphene (GR) modified carbon ionic liquid electrode (CILE) was fabricated and used as the voltammetric sensor for the sensitive detection of catechol. Due to the specific physicochemical characteristics of GR such as high surface area, excellent conductivity and good electrochemical properties, the modified electrode exhibits rapid response and strong catalytic activity with high stability toward the electrochemical oxidation of catechol. A pair of well‐defined redox peaks appeared with the anodic and the cathodic peak potential located at 225 mV and 133 mV (vs.SCE) in pH 6.5 phosphate buffer solution, respectively. Electrochemical behaviors of catechol on the GR modified CILE were carefully investigated and the electrochemical parameters were calculated with the results of the electrode reaction standard rate constant (k_s) as 1.24 s^?1, the charge transfer coefficient (α) as 0.4 and the electron transfer number (n) as 2. Under the selected conditions the differential pulse voltammetric peak current increased linearly with the catechol concentrations in the range from 1.0 × 10^‐7 to 7.0 × 10^?4mol L‐1 with the detection limit as 3.0 × 10^?8mol L‐1 (3σ). The proposed method was further applied to the synthetic waste water samples determination with satisfactory results     

Electrochemistry and Voltammetric Determination of Adenosine with N‐Hexylpyridinium Hexafluorophosphate Modified Electrode

An ionic liquid N‐hexylpyridinium hexafluorophosphate (HPPF₆) modified carbon paste electrode was fabricated for the sensitive voltammetric determination of adenosine in this paper. Carbon ionic liquid electrode (CILE) was prepared by mixing graphite powder and HPPF₆ together and the CILE was characterized by scanning electron microscopy (SEM) and electrochemical methods. The electrochemical behaviors of adenosine on the CILE were studied carefully. Compared with the traditional carbon paste electrode (CPE), a small negative shift of the oxidation peak potential appeared with greatly increase of the oxidation peak current, which indicated the presence of ionic liquid in the carbon paste not only as the binder but also as the modifier and promoter. Under the optimal conditions the oxidation peak current increased with the adenosine concentration in the range from 1.0×10^?6 mol/L to 1.4×10^?4 mol/L with the detection limit of 9.1×10^?7 mol/L (S/N=3) by differential pulse voltammetry. The proposed method was applied to the human urine samples detection with satisfactory results.     

High Performance Oxide Functionalized Nitrogen‐Doped Mesocellular Carbon Foam for Biosensor Construction

Nitrogen‐doped mesocellular carbon foam (denoted as MCF? CN_x) with high surface area and large pore volume was prepared and characterized in detail. The MCF? CN_x was further functionalized by oxidation with HNO₃ (denoted as MCF? CN_x‐O) in order to effectively improve its hydrophilicity and biocompatibility. Both MCF? CN_x and MCF? CN_x‐O were used for immobilization of Hb and design of electrochemical biosensors. The activity of Hb immobilized on MCF? CN_x‐O is two times higher than that of Hb immobilized on MCF? CN_x. The Hb‐MCF? CN_x‐O‐Nafion modified electrode displays fast response, high sensitivity and low detection limit to the detection of hydrogen peroxide. The sensitivity of Hb‐MCF? CN_x‐O‐Nafion modified electrode (477 μA mM^?1 cm^?2) is twice that of Hb‐MCF? CN_x‐Nafion modified electrode.