A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized in a tetrathiafulvalene-tetracyanoquinodimethane/multiwalled carbon nanotubes film

A new third-generation biosensor for H(2)O(2) assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ)/multiwalled carbon nanotubes (MWCNTs) modified gold electrode. The prepared HRP/TTF-TCNQ/MWCNTs/Au electrode was used for the bioelectrocatalytic reduction of H(2)O(2), with a linear range from 0.005 to 1.05mM and a detection limit of 0.5muM for amperometric sensing of H(2)O(2). In addition, a novel method on the basis of electrochemical quartz crystal microbalance (EQCM) measurements was proposed to determine the effective enzymatic specific activity (ESA) of the immobilized HRP for the first time, and the ESA was found to be greater at the TTF-TCNQ/MWCNTs/Au electrode than that at the MWCNTs/Au or TTF-TCNQ/Au electrode, indicating that the TTF-TCNQ/MWCNTs film is a good HRP-immobilization matrix to achieve the direct electron transfer between the enzyme and the electrode.     

Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode

The direct electrochemistry of glucose oxidase (GOD) adsorbed on a colloidal gold modified carbon paste electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -(449+/-1) mV in 0.1 M pH 5.0 phosphate buffer solution. The response showed a surface-controlled electrode process with an electron transfer rate constant of (38.9+/-5.3)/s determined in the scan rate range from 10 to 100 mV/s. GOD adsorbed on gold colloid nanoparticles maintained its bioactivity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a high sensitivity (8.4 microA/mM), a linear range from 0.04 to 0.28 mM and a detection limit of 0.01 mM at a signal-to-noise ratio of 3sigma. The sensor could exclude the interference of commonly coexisted uric and ascorbic acid.     

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.     

Electrochemical detection of mismatched DNA using a MutS probe

A direct and label-free electrochemical biosensor for the detection of the protein–mismatched DNA interaction was designed using immobilized N-terminal histidine tagged Escherichia coli. MutS on a Ni-NTA coated Au electrode. General electrochemical methods, cyclic voltammetry (CV), electrochemical quartz crystal microbalance (EQCM) and impedance spectroscopy, were used to ascertain the binding affinity of mismatched DNAs to the MutS probe. The direct results of CV and impedance clearly reveal that the interaction of MutS with the CC heteroduplex was much stronger than that with AT homoduplex, which was not differentiated in previous results (GT > CT > CC ≈ AT) of a gel mobility shift assay. The EQCM technique was also able to quantitatively analyze MutS affinity to heteroduplexes.     

Direct electrochemistry and electrocatalytic activity of catalase incorporated onto multiwall carbon nanotubes-modified glassy carbon electrode

The direct voltammetry and electrocatalytic properties of catalase, which was adsorbed on the surface of multiwall carbon nanotubes (MWCNTs), was investigated. A pair of well-defined and nearly reversible cyclic voltammetry peaks for Fe(III)/Fe(II) redox couple of catalase adsorbed on the surface of MWCNTs at approximately -0.05 V versus reference electrode in pH 6.5 buffer solution, indicating the direct electron transfer between catalase and electrode. The surface coverage of catalase immobilized on MWCNTs glassy carbon electrode was approximately 2.4x10(-10) molcm-2. The transfer coefficient (alpha) was calculated to be 0.4, and the heterogeneous electron transfer rate constant was 80 s-1 in pH 7, indicating great facilitation of the electron transfer between catalase and MWCNTs adsorbed on the electrode surface. The formal potential of catalase Fe(III)/Fe(II) couple in MWCNTs film had a linear relationship with pH values between 2 and 11 with a slope of 58 mV/pH, showing that the electron transfer is accompanied by single proton transportation. Catalase adsorbed on MWCNTs exhibits a remarkable electrocatalytic activity toward the reduction of oxygen and hydrogen peroxide. The value for calculated Michaelis-Menten constant (1.70 mM) was high, indicating the potential applicability of the films as a new type of reagentless biosensor based on the direct electrochemistry of the catalase enzyme.     

A novel nanohybrid of daunomycin and single-walled carbon nanotubes: photophysical properties and enhanced electrochemical activity

A nanohybrid adduct of the widely used, functional dye, daunomycin (DM), with single-walled carbon nanotubes (SWNTs) was prepared. Ultraviolet-visible-near infrared and fluorescence spectroscopy and electrochemistry of DM-functionalized SWNTs reveal that DM interacts with SWNTs through strong π–π stacking and there is a significant photo-induced charge-transfer interaction between the two components. Importantly, the novel adduct modified the glassy carbon (GC) electrode to give a much enhanced electrochemical activity than those of DM adsorbed onto not only the bare GC electrode but also the SWNTs-modified GC electrode.     

Electrochemical quartz crystal impedance and fluorescence quenching studies on the binding of carbon nanotubes (CNTs)-adsorbed and solution rutin with hemoglobin

Electrochemical quartz crystal impedance (QCI) technique was utilized to monitor in situ the adsorption of rutin (RT) onto a carbon nanotubes (CNTs)-modified gold electrode and to study the binding process of solution hemoglobin (Hb) to RT immobilized on the electrode. Time courses of the QCI parameters including crystal resonant frequency were simultaneously obtained during the RT adsorption and Hb-RT binding. In contrast to the negligible RT adsorption at a bare gold electrode, the modification by CNTs notably enhanced the amount of adsorption, and almost all of the adsorbed RT molecules were found to be electroactive. On the basis of the frequency response from the binding of adsorbed RT to solution Hb and the diminished electroactivity of adsorbed RT after the formation of the electrochemically inactive RT-Hb adduct, the average binding molar ratio of adsorbed RT to Hb was estimated to be 23.9:1, and the association constant (Ka) for the binding was estimated to be 2.87 x 106 (frequency) and 3.92 x 106 (charge) L mol-1, respectively. Comparable results were obtained from fluorescence quenching measurements in mixed solutions containing RT of fixed concentration and Hb of varying concentrations, demonstrating that the interfacial RT here behaved equivalently in the RT-Hb binding activity compared to that in solution. This work may have presented a new and general protocol involving CNTs to study many other electroactive natural antioxidants or drugs that are at the interface or in solution, their binding with proteins or other biomolecules, and changes of their antioxidant activity after the binding.     

Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified by Nafion and ordered mesoporous silica-SBA-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified by ordered mesoporous silica-SBA-15 and Nafion. The sorption behavior of GOD immobilized on SBA-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet–visible (UV–vis), FTIR, respectively, which demonstrated that SBA-15 can facilitate the electron exchange between the electroactive center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and SBA-15 matrices displays direct, nearly reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 3.89 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the absence of O₂, GOD immobilized on Nafion and SBA-15 matrices can produce a wide linear response to glucose in the positive potential range. Thus, Nafion/GOD-SBA-15/GC electrode is hopeful to be used in the third non-mediator's glucose biosensor. In addition, GOD immobilized on SBA-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. The Nafion/GOD-SBA-15/GC electrode can be utilized as the cathode in biofuel cell.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Nanocomposite of functionalized multiwall carbon nanotubes with nafion, nano platinum, and nano gold biosensing film for simultaneous determination of ascorbic acid, epinephrine, and uric acid

A unique bimetallic, nano platinum (Pt) with nano gold (Au) on nafion (NF) incorporated with functionalized multiwall carbon nanotubes (f-MWCNTs) composite film (f-MWCNTs-NF-PtAu) was developed by the potentiostatic method. The composite film exhibits promising efficient catalytic activity towards the oxidation of mixture of biochemical compounds and simultaneous measurement of ascorbate anion, epinephrine and urate anion in aqueous buffer solution (pH 6.75). Both, the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used for the measurement of electroanalytical properties of neurotransmitters by means of composite film modified electrodes. Well-separated voltammetric peaks were obtained for ascorbate, epinephrine and urate anions with the peak separations of 0.222 and 0.131V. The composite film can also be produced on gold and transparent semiconductor indium tin oxide electrodes for different kinds of studies such as electrochemical quartz crystal microbalance (EQCM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The incorporation of Pt and Au onto the f-MWCNTs-NF was revealed by the EQCM technique and the morphology of the film was studied using SEM, AFM and scanning electrochemical microscopy (SECM) techniques. Further, extensive studies were carried out using SECM for obtaining the surface current topographic images of composite film modified electrodes, and these indicated the presence of f-MWCNTs-NF-PtAu composite film on the electrode.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Construction and direct electrochemistry of orientation controlled laccase electrode

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O₂ reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.     

Structure and biosensor characteristics of complex between glucose oxidase and plasma-polymerized nanothin film

The structure and biosensor characteristics of complex between glucose oxidase (GOD) and plasma-polymerized nanothin film (PPF), in which the thickness is several nanometers, were investigated by atomic force microscopy (AFM) and electrochemical measurement. The GOD molecules were densely adsorbed onto the PPF surface treated by nitrogen plasma and the individual GOD molecules were observed. Subsequently, the GOD densely packed array on the PPF surface was subsequently treated by plasma polymerization (overcoating). AFM image showed that the thicker film gave the smoother surface, indicating that the GOD adsorbed on the surface was embedded more deeply in PPF. The amperometric biosensor characteristics of the GOD-PPF complex on a platinum electrode showed the current increment due to the enzymatic reaction with glucose addition, indicating that enzyme activity was retained although the enzyme has been exposed to the plasma gas related to diffusion of the substrate. This means that under mild exposure to organic plasma, the enzyme does not become seriously dysfunctional. Amperometric biosensor characteristics were strongly affected by monomer and thickness of PPF overcoating related with the diffusion of the substrate (glucose). Considering that the film deposition was performed using microfabrication-compatible organic plasma, our new method for protein architecture has a great potential of enabling high throughput production of bioelectronic devices.     

Chemisorption of proteins and their thiol derivatives onto gold surfaces: characterization based on electrochemical nonlinearity

This study was conducted to monitor the electrochemical responses of two proteins (bovine serum albumin (BSA) and gelatin) and their thiol derivatives adsorbed onto gold (Au) electrodes, which were analyzed by a "nonlinear" impedance method. A sinusoidal voltage is applied to a protein-containing aqueous solution and the waveform of the output current is analyzed by fast Fourier transformation (FFT). The intensities of the higher harmonics in the FFT varied with the species of protein and their thiol derivatives, and with time. From the higher harmonics, voltage-dependent capacitance and conductance were quantitatively evaluated to differentiate the state of adsorbed protein. Adsorption and desorption characteristics of BSA and its thiol derivative on the Au surface were continuously measured by a quartz crystal microbalance (QCM) in situ. The microscopic state of thiol-derivatized BSA adsorbed onto the Au surface was imaged by atomic force microscopy (AFM). In general, thiol-derivatized proteins were tightly adsorbed on the Au surface and showed no desorption. The present electrochemical measurements clearly differentiated adsorption characteristics of physically adsorbed (physisorbed) and chemically adsorbed (chemisorbed) proteins on Au surfaces.     

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.     

Polyaniline protected gold nanoparticles based mediator and label free electrochemical cortisol biosensor

Polyaniline protected gold nanoparticles (PPAuNPs) were electrophoretically deposited onto a gold electrode, and utilized to fabricate an electrochemical cortisol biosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized onto the surface of a PPAuNP/Au electrode using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. BSA was employed for blocking nonspecific adsorption on the electrode surface. PPAuNP formation and BSA/C-Mab/PPAuNP/Au electrode fabrication were characterized using transmission electron microscopy, atomic force microscopy and electrochemical impedance techniques, respectively. Cyclic voltammetry and differential pulse voltammetric techniques were used to determine the cortisol concentration in a phosphate buffer saline (PBS) solution. Results confirmed that the PPAuNP based electrode was stable during repeated scans and exhibited repeatable redox peaks. Further, the BSA/C-Mab/PPAuNP/Au electrode in the PBS buffer accurately detected cortisol in the range of 1 pM-100 nM with a sensitivity of 1.63 μAM(-1). The biosensor was found to be selective against BSA and 17-α-hydroxy progesterone. This research establishes the feasibility of using a PPAuNP based matrix for a label and mediator free electrochemical biosensor for cortisol, a stress biomarker.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

Fabrication of polymeric ionic liquid/graphene nanocomposite for glucose oxidase immobilization and direct electrochemistry

A novel polymeric ionic liquid functionalized graphene, poly(1-vinyl-3-butylimidazolium bromide)-graphene (denoted as poly(ViBuIm(+)Br(-))-G), was synthesized. FTIR, UV-vis spectra and TEM were used to characterize the formation of as synthesized nanocomposites. Due to the modification of the polymeric ionic liquid, poly(ViBuIm(+)Br(-))-G can not only be dispersed well in aqueous solutions to form a homogeneous colloidal suspension of individual nanosheets, but also exhibit a strong positive charge. Based on self-assembly, the negatively charged glucose oxidase (GOD) was immobilized onto the poly(ViBuIm(+)Br(-))-G to form a GOD/poly(ViBuIm(+)Br(-))-G/glassy carbon (GC) electrode under mild conditions. With the advantage of both poly(ViBuIm(+)Br(-)) and graphene, poly(ViBuIm(+)Br(-))-G can provide a favorable and conductive microenvironment for the immobilized GOD and thus promote their direct electron transfer at the GC electrode. Furthermore, the GOD/poly(ViBuIm(+)Br(-))-G/GC electrode displayed an excellent sensitivity, together with a wide linear range and excellent stability for the detection of glucose. Accordingly, these unique properties of such novel nanocomposite generate a promising platform for the construction of mediator-free enzymatic biosensors.     

Electrochemical characteristics of a gold nanoparticle-modified controlled enzyme–electrode contact junction electrode

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5–40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.

     

Attachment of gold nanoparticles to glassy carbon electrode and its application for the direct electrochemistry and electrocatalytic behavior of hemoglobin

Gold nanoparticles have been attached onto glassy carbon electrode surface through sulfhydryl-terminated monolayer and characterized by X-ray photoelectron spectroscopy, atomic force microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The gold nanoparticles-attached glassy carbon electrodes have been applied to the immobilization/adsorption of hemoglobin, with a monolayer surface coverage of about 2.1 x 10(-10) mol cm(-2), and consequently obtained the direct electrochemistry of hemoglobin. Gold nanoparticles, acting as a bridge of electron transfer, can greatly promote the direct electron transfer between hemoglobin and the modified glassy carbon electrode without the aid of any electron mediator. In phosphate buffer solution with pH 6.8, hemoglobin shows a pair of well-defined redox waves with formal potential (E0') of about -0.085 V (versus Ag/AgCl/saturated KCl). The immobilized hemoglobin maintained its biological activity, showing a surface controlled electrode process with the apparent heterogeneous electron transfer rate constant (ks) of 1.05 s(-1) and charge-transfer coefficient (a) of 0.46, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide. A potential application of the hemoglobin-immobilized gold nanoparticles modified glassy carbon electrode as a biosensor to monitor hydrogen peroxide has been investigated. The steady-state current response increases linearly with hydrogen peroxide concentration from 2.0 x 10(-6) to 2.4 x 10(-4) M. The detection limit (3sigma) for hydrogen peroxide is 9.1 x 10(-7) M.