Colloidal silver nanoparticles modified electrode and its application to the electroanalysis of Cytochrome c

A colloidal silver nanoparticles (CSNs) chemically modified electrode was prepared and its application to the electroanalysis of Cytochrome c (Cyt. c) was studied. The CSNs were prepared by reduction of AgNO₃ with NaBH₄, and were stabilized by oleate. They could be efficiently immobilized on the surface of a silver electrode. The result showed that the CSNs could clearly enhance the electron transfer process between Cyt. c and the electrode compared with bulk silver electrode. Linear sweep voltammetric measurement of Cyt. c at the chemical modified electrode indicated that the oxidative peak current of Cyt. c was linear to its concentration ranging from 8.0 nmol L⁻¹ to 3.0 μmol L⁻¹ with the calculated detection limit was about 2.6 nmol L⁻¹. The direct electrochemistry of Cyt. c was also studied by cyclic voltammetry.     

Catalytic reduction of hydrogen peroxide at metal hexacyanoferrate composite electrodes and applications in enzymatic analysis

The electrocatalytic activity of various metal hexacyanoferrates (Mhcfs) (i) immobilized on graphite electrodes, and (ii) as components of a composite electrode was investigated with respect to the reduction of hydrogen peroxide. The flow-through working electrode was a thin layer consisting of a composite of Mhcf, graphite, and polymethylmetacrylate (PMMA) as a binder, sandwiched between two Plexiglas plates. Among the pure Mhcfs immobilized on a graphite electrode, iron(III) hexacyanoferrate (Prussian blue) exhibits the highest electrocatalytic effect, whereas in the composite electrodes chromium(III) hexacyanoferrate (Crhcf) shows the highest activity and best performance and reproducibility for the electrochemical reduction of H₂O₂. The Crhcf electrode provides a linear dependence on H₂O₂ concentration in the range 2.5 × 10⁻⁶ mol L⁻¹ (LOD) to 1 × 10⁻⁴ mol L⁻¹ (phosphate buffer, pH 7). The sensor was applied for the detection of H₂O₂ enzymatically produced by glucose oxidase. The optimal conditions for the peroxide injection were 2 min after the beginning of the reaction and 25 °C with a detection limit of 7.0 × 10⁻⁶ mol L⁻¹ for glucose.     

The ultrasonic electropolymerization of an 5-[o-(4-bromine amyloxy)phenyl]-10,15,20-triphenylporphrin (o-BrPETPP) film electrode and its electrocatalytic properties to dopamine oxidation in aqueous solution

5-[o-(4-Bromine amyloxy)phenyl]-10,15,20-triphenylporphrin (o-BrPETPP) was electropolymerized on a glassy carbon electrode (GCE), and the electrocatalytic properties of the prepared film electrode response to dopamine (DA) oxidation were investigated. A stable o-BrPETPP film was formed on the GCE under ultrasonic irradiation through a potentiodynamic process in 0.1 M H₂SO₄ between −1.1 V and 2.2 V versus a saturated calomel electrode (SCE) at a scan rate of 0.1 V s⁻¹. The film electrode showed high selectivity for DA in the presence of ascorbic acid (AA) and uric acid (UA), and a 6-fold greater sensitivity to DA than that of the bare GCE. In the 0.05 mol L⁻¹ phosphate buffer (pH 6.0), there was a linear relationship between the oxidation current and the concentration of DA solution in the range of 5 × 10⁻⁷ mol L⁻¹ to 3 × 10⁻⁵ mol L⁻¹. The electrode had a detection limit of 6.0 × 10⁻⁸ mol L⁻¹(S/N = 3) when the differential pulse voltammetric (DPV) method was used. In addition, the charge transfer rate constant k = 0.0703 cm s⁻¹, the transfer coefficient α = 0.709, the electron number involved in the rate determining step n_α = 0.952, and the diffusion coefficient D_o = 3.54  10⁻⁵ cm² s⁻¹ were determined. The o-BrPETPP film electrode provides high stability, sensitivity, and selectivity for DA oxidation.     

Preparation, electrochemical behavior and performance of gallium hexacyanoferrate as electrocatalyst of H2O2

The gallium hexacyanoferrate (GaHCF) was synthesized chemically and characterized by FTIR technique. Its electrochemical behavior was carefully investigated by fabricating a GaHCF modified carbon paste electrode in various supporting electrolyte. The experimental results showed that in KNO₃, K₂SO₄, KCl and other supporting electrolyte, GaHCF yielded one pair of ill-defined redox waves with a formal potential of 0.9 V (versus SCE). In 0.050 mol L⁻¹ phosphate buffer solution (PBS, pH 6.8), however, GaHCF yielded one pair of well-defined redox peaks with a formal potential of 0.222 V. Furthermore, this modified electrode exhibited a high electrocatalytic activity toward the reduction of H₂O₂ in pH 6.8 PBS, with over-potential dramatically lower than that of on the bare carbon paste electrode. Amperometry was used for the determination of H₂O₂, under the optimal conditions, a linear dependence of the catalytic current versus H₂O₂ concentration was obtained in the range of 4.9 × 10⁻⁶ to 4.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1 × 10⁻⁶ mol L⁻¹ when the signal-to-noise ratio was 3, and a sensitivity of 27.9 μA mM⁻¹ (correlation coefficient of 0.997). Chronoamperometry was used to conveniently determine the diffusion coefficient of H₂O₂ in the solution.     

Enhanced solid-state electrogenerated chemiluminescence of Au/CdS nanocomposite and its sensing to H2O2

Au nanoparticles (AuNPs) are good quenchers once they closely contact with luminophore. Here we reported a simple approach to obtain enhanced electrogenerated chemiluminescence (ECL) behavior based on Au/CdS nanocomposite films by adjusting the amount of AuNPs in the nanocomposite. The maximum enhancement factor of about 4 was obtained at an indium tin oxide (ITO) electrode in the presence of co-reactant H₂O₂. The mechanism of this enhancement was discussed in detail. The strong ECL emission from Au/CdS nanocomposites film was exploited to determine H₂O₂. The resulting ECL biosensors showed a linear response to the concentration of H₂O₂ ranging from 1.0 × 10⁻⁸ to 6.6 × 10⁻⁴ mol L⁻¹ with a detection limit of 5 nmol L⁻¹ (S/N = 3) and good stability and reproducibility.     

Electrochemical study on cobalt film modified glassy carbon electrode and its application

A novel electroactive material for ascorbic acid (AA) determination was successfully prepared by plating/potential cycling method. The cobalt film was first deposited on the surface of glassy carbon electrode (GCE) in CoSO₄ solution by potential cycling, and then a cobalt film on the surface of GCE was activated by potential cycling in 0.1 mol L⁻¹ NaOH. The electrochemical performance of the resulted film (Co/GCE) and factors affecting its electrochemical activity were investigated by cyclic voltammetry and amperometry. This film electrode exhibited good electrocatalytic activity to the oxidation of AA. This biosensor had a fast response of AA less than 3 s and excellent linear relationships were obtained in the concentration range of 3 × 10⁻⁷ to 1 × 10⁻⁴ mol L⁻¹ with a detection limit of 2 × 10⁻⁷ mol L⁻¹ (S/N = 3) under the optimum conditions. Moreover, the selectivity, stability and reproducibility of this biosensor were evaluated with satisfactory results.     

Electrochemical synthesis of belt-like polyaniline network on p-phenylenediamine functionalized glassy carbon electrode and its use for the direct electrochemistry of horse heart cytochrome c

Three-dimension (3D) belt-like polyaniline (PAN) network has been prepared via electrochemical polymerization of aniline on p-phenylenediamine (PDA) functionalized glassy carbon electrode (GCE) using a three-step electrochemical deposition procedure. PDA was covalently binded on GCE via the formation of carbon-nitrogen bond between amine cation radical and the aromatic moiety of GCE surface using electrochemical oxidation procedure. X-ray photo-electron spectroscopy (XPS) and cyclic voltammetry have been performed to characterize the attachment of PDA on GCE. The images of scanning electron microscope (SEM) show that the 3D belt-like PAN network is uniform. The width and thickness of the PAN belt varies in the range of 1.5-5.5 μm and 0.1-0.8 μm, respectively. The distance between the belt-contacts ranges from 2.5 to 15 μm. The 3D belt-like PAN network modified GCE (PAN-PDA/GCE) exhibits an improved electro-activity of PAN at an extended pH up to 7.0. The PAN-PDA/GCE not only immobilizes but also leads to a direct electrochemical behavior of cytochrome c (Cyt c). The immobilized Cyt c maintains its activity, showing a surface-controlled electrode process with the electron-transfer rate constant (k_s) of 14.8 s⁻¹ and electron-transfer coefficient (α) of 0.48, and could be used for the electrocatalytic reduction of hydrogen peroxide (H₂O₂).     

Direct electrochemistry of Cytochrome c on natural nano-attapulgite clay modified electrode and its electrocatalytic reduction for H2O2

Natural nano-structural attapulgite clay was purified by mechanical stirring with the aid of ultrasonic wave and its structure and morphology was investigated by XRD and transmission electron microscopy (TEM). Cytochrome c was immobilized on attapulgite modified glassy carbon electrode. The interaction between Cytochrome c and attapulgite clay was examined by using UV-vis spectroscopy and electrochemical methods. The direct electron transfer of the immobilized Cytochrome c exhibited a pair of redox peaks with formal potential (E0′) of about 17 mV (versus SCE) in 0.1 mol/L, pH 7.0, PBS. The electrode reaction showed a surface-controlled process with the apparent heterogeneous electron transfer rate constant (k_s) of 7.05 s⁻¹ and charge-transfer coefficient (α) of 0.49. Cytochrome c immobilized on the attapulgite modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide (H₂O₂). The calculated apparent Michaelis-Menten constant was 470 μmol/L, indicating a high catalytic activity of Cytochrome c immobilized on attapulgite modified electrode to the reduction of H₂O₂. Based on these, a third generation of reagentless biosensor can be constructed for the determination of H₂O₂.     

Fabrication of carbon paste electrode containing [PFeW11O39] polyoxoanion supported on modified amorphous silica gel and its electrocatalytic activity for H2O2 reduction

[PFeW₁₁O₃₉]⁴⁻ (PFeW₁₁) supported on the surface of 3-aminopropyl(triethoxy)silane modified silica gel was synthesized and used as a bulk modifier to fabricate a renewable three-dimensional chemically modified electrode. The electrochemical behavior of the modified electrode was investigated. Cyclic voltammetry studies showed that the PFeW₁₁ on the electrode surface sustained the same electrochemical properties as that of the PFeW₁₁ in solution. The preparation of chemically modified electrode is simple and quiet reproducible using inexpensive material. The modified electrode had high electrocatalytic activity toward H₂O₂ reduction and it was successfully applied as an electrochemical detector to monitor H₂O₂ in flow injection analysis (FIA). The electrocatalytic peak current was found to be linear with the H₂O₂ concentration in the range 10-200 μmol L⁻¹ with a correlation coefficient of 0.998 and a detection limit (3σ) of 7.4 μmol L⁻¹ H₂O₂. The electrode has the remarkable advantage of surface renewal owing to bulk modification, as well as simple preparation, good mechanical and chemical stability and reproducibility.     

Changes on iron electrode surface during hydrogen permeation in borate buffer solution

Hydrogen interaction with oxide films grown on iron electrodes at open circuit potential (E_oc) and in the passive region (+0.30 V_ECS) was studied by chronopotentiometry, chronoamperometry and electrochemical impedance spectroscopy techniques. The results were obtained in deaerated 0.3 mol L⁻¹ H₃BO₃ + 0.075 mol L⁻¹ Na₂B₄O₇ (BB, pH 8.4) solution before, during and after hydrogen permeation. The iron oxide film modification was also investigated by means of in situ X-ray absorption near-edge spectroscopy (XANES) and scanning electrochemical microscopy (SECM) before and during hydrogen permeation. The main conclusion was that the passive film is reduced during the hydrogen diffusion. The hydrogen permeation stabilizes the iron surface at a potential close to the thermodynamic water stability line where hydrogen evolution can occur. The stationary condition required for the determination of the permeation parameters cannot be easily attained on iron surface during hydrogen permeation. Moreover, additional attention must be paid when obtaining the transport parameters using the classical permeation cell.     

A novel DNA biosensor based on ssDNA/Cyt c/l-Cys/GNPs/Chits/GCE

A novel DNA biosensor was fabricated by modified multilayer of ssDNA, cytochrome c, l-cysteine, metal gold nanoparticles and Chitosan (denoted as ssDNA/Cyt c/l-Cys/GNPs/Chits/GCE). The behavior of the DNA biosensor was then investigated by voltammetry, impedance spectrum and atomic force microscope (AFM), and the morphologic differences among each layer of the DNA biosensor were also observed. Results revealed that two well-defined redox peaks exhibited at 0.120 V and 0.362 V, and the amount of adsorbed DNA was 1.672 × 10⁻¹⁰ mol cm⁻². We concluded that the modified electrode could be used to detect DNA with the indicator daunomycin.     

Electrocatalytic oxidation of deoxyguanosine on a glassy carbon electrode modified with a ruthenium oxide hexacyanoferrate film

The electrocatalytic oxidation of deoxyguanosine on a ruthenium hexacyanoferrate (RuOHCF) glassy carbon (GC) modified electrode was investigated in acid medium by using rotating disc electrode (RDE) voltammetry. Chronoamperometric experiments allowed information on the charge transport rate through the RuOHCF film and at a very short time window a diffusion-like behavior was observed with a D_ct value of 2.7 × 10⁻¹¹ cm² s⁻¹ for a film with Γ = 4.47 × 10⁻⁹ mol cm⁻². The influence of systematic variation of rotation rate, film thickness and the electrode potential indicates that the rate of cross-chemical reaction between Ru(IV) centers immobilized into the film and deoxyguanosine controls the overall electrodic process and the value of the rate constant was found to be 3.2 × 10⁶ mol⁻¹ L¹ s⁻¹. The relatively high rate constant of the cross-reaction, the facile penetration of the substrate through the film and the fast transport of electrons suggest that the electrocatalytic process occurs throughout the film layer.     

Kinetics and mechanism of oxygen reduction at hydrous oxide film-covered platinum electrode in alkaline solution

This study uses rotating ring-disk electrode (RRDE) and linear sweep voltammetry (LSV) to characterize oxygen reduction kinetics in alkaline solution on platinum electrodes with various thickness of hydrous oxide (oxyhydroxy) film. Oxyhydroxy films are created on Pt electrodes by pretreatment in 1.0 mol dm⁻³ KOH at a constant voltage. The pretreatment voltage ranges from −1.2 to 1.0 V and is increased stepwise before each new experimental run to produce seven discreet films. LSV plots show oxyhydroxy film thickness strongly inhibits oxygen reduction and is inversely proportional to RRDE oxygen reduction current I_D for LSV voltages E_D from −0.1 to −0.46 V, but this trend reverses at E_D more negative than −0.46 V so that the worst-performing electrode becomes the best. However, this improvement disappears at around −0.8 V, suggesting this change involves a negatively charged ion, possibly embedded into the metal in the top few atomic layers either interstitially or substitutionally. The 1.0 V-pretreated electrode in the E_D range from −0.46 to −0.9 V of highest oxygen reduction current also exhibits the lowest hydrogen peroxide production, with zero H₂O₂ produced at −0.6 V, indicating the brief presence of the oxyhydroxy film on the Pt surface has strong lingering effects. The post-oxyhydroxy Pt surface is very different than the native Pt for oxygen reduction pathway and efficiency. Reaction order with respect to oxygen is close to 1. The rate constants of the direct O₂ to H₂O electroreduction reaction are increased with decreasing the potential from −0.2 to −0.6 V, but the O₂ to H₂O₂ electroreduction is contrary to this expectation. The rate constants of H₂O₂ decomposition on the oxyhydroxy film-covered Pt electrode are near constant around 1 × 10⁻⁴ cm s⁻¹ at E_D > −0.5 V.     

Electrochemiluminescent behavior of luminol on the glassy carbon electrode modified with CoTPP/MWNT composite film

A glassy carbon electrode (GCE) modified with cobalt(II) meso-tetraphenylporphrine/multiwall-carbon nanotube (CoTPP/MWNT) was applied to investigate the electrochemiluminescent (ECL) behavior of luminol. The ECL intensity of luminol was found to be increased greatly on this modified electrode. The presence of cobalt(II) meso-tetraphenylporphrine (CoTPP) can catalyze the reduction of oxygen on the electrode surface to produce HOO⁻, which can increase the ECL intensity of luminol. Moreover, MWNT can provide the more effective area of the electrode, and can act as a promoter to enhance the electrochemical reaction. The proposed method enables a detection limit for luminol of 1.0 × 10⁻⁸ mol/L in the neutral solution. Under the optimum condition, the enhanced ECL intensity of luminol by H₂O₂ had a linear relationship with the concentration of H₂O₂ in the range of 1.0 × 10⁻⁷ to 8.0 × 10⁻⁸ mol/L with the detection limit of 5.0 × 10⁻⁹ mol/L.     

Effects of NO2 ions on localised corrosion of steel in NaHCO3 + NaCl electrolytes

Pitting corrosion of carbon steel electrodes in 0.1 mol L⁻¹ NaHCO₃ + 0.02 mol L⁻¹ NaCl solutions was induced by anodic polarisation. The evolution of the breakdown potential E_b with NO₂⁻ concentration was investigated by linear voltammetry. E_b increased from −15 ± 5 mV/SCE for [NO₂⁻] = 0 up to 400 ± 50 mV/SCE for [NO₂⁻] = 0.1 mol L⁻¹. During anodic polarisation at potentials comprised between E_b([NO₂⁻] = 0) and E_b([NO₂⁻] ≠ 0), the behaviour of the whole electrode surface, followed by chronoamperometry, was compared to the behaviour of one single pit, followed via scanning vibrating electrode technique (SVET). Addition of a NaNO₂ solution after the beginning of the polarisation led to a rapid repassivation of pre-existing well-grown pits. In situ micro-Raman spectroscopy was then used to identify the corrosion products forming inside the pits. The first species to be detected in the presence of NO₂⁻ were mainly dissolved Fe(III) species, more likely [Fe^III(H₂O)₆]³⁺ complexes. Iron(II) carbonate FeCO₃, siderite, and carbonated green rust GR(CO₃²⁻) were also detected in the active pits, as in the absence of nitrite. But they were accompanied by maghemite γ-Fe₂O₃, a phase structurally similar to the passive film, that forms from the Fe(III) complexes. The Raman analyses then correlate with the SVET observations and confirm that the main effect of nitrite ions is to oxidize iron(II) into iron(III). The passive film would then form from the Fe(III) species still bound to the steel surface.     

A novel graphite-polyurethane composite electrode modified with thiol-organofunctionalized silica for the determination of copper ions in ethanol fuel

A graphite-polyurethane composite modified with 2-benzothiazolethiol organofunctionalized silica was evaluated as an alternative electrode in the determination of Cu²⁺ ions in ethanol fuel samples, on the basis of a differential pulse anodic stripping voltammetry procedure. This metal can be quantified by mixing ethanol fuel with 0.10 mol L⁻¹ KNO₃ aqueous solution and subsequent voltammetric measurement after the accumulation step. A maximum limit of 70% (v/v) ethanol in potassium nitrate aqueous solution was obtained for voltammetric measurements without loss of sensitivity for metal species. Factors affecting the pre-concentration and stripping steps were investigated and optimum conditions were employed to develop the analytical procedure. Using 20 min of accumulation time, the linear range of 0.1-1.2 μmol L⁻¹ was obtained with the limit of detection of 3.9 × 10⁻⁸ mol L⁻¹. The developed electrode was successfully applied to determine Cu²⁺ in commercial ethanol fuel samples. The proposed method was compared with a traditional analytical technique, the flame atomic absorption spectrometry, and no significant differences between the results obtained by both methods were observed according to statistical evaluation.     

Nucleic acid biosensor for DNA hybridization detection using rutin-Cu as an electrochemical indicator

The complex of rutin-Cu (C₈₁H₈₆Cu₂O₄₈, abbreviated by Cu₂R₃, R = rutin) was synthesized and characterized by elemental analysis and IR spectra. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Cu₂R₃ with salmon sperm DNA. It was revealed that Cu₂R₃ could interact with double-stranded DNA (dsDNA) by a major intercalation role. Using Cu₂R₃ as a novel electroactive indicator, an electrochemical DNA biosensor for the detection of specific DNA fragment was developed and its selectivity for the recognition with different target DNA was assessed by differential pulse voltammetry (DPV). The target DNA related to coliform virus gene could be quantified ranged from 1.62 × 10⁻⁸ mol L⁻¹ to 8.10 × 10⁻⁷ mol L⁻¹ with a good linearity (r = 0.9989) and a detection limit of 2.3 × 10⁻⁹ mol L⁻¹ (3σ, n = 7) was achieved by the constructed electrochemical DNA biosensor.     

Facile preparation of disposable immunosensor for Shigella flexneri based on multi-wall carbon nanotubes/chitosan composite

Based on multi-wall carbon nanotubes (MWCNT)/chitosan/horseradish peroxidase labeled antibodies to Shigella flexneri (HRP-anti-S. flexneri) biocomposite film on a screen-printed electrode (SPE) surface, a disposable immunosensor has been developed for the rapid detection of S. flexneri. The HRP-anti-S. flexneri can be entrapped into MWCNT/chitosan composite matrix without other cross-linking agent. Thionine and H₂O₂ were used as the mediator and substrate, respectively. The surface morphologies of modified films were characterized by atomic force microscope (AFM). Cyclic voltammery (CV) was carried out to characterize the electrochemical properties of the immobilization of materials on the electrode surface and quantified S. flexneri. Due to the strong electrocatalytic properties of MWCNT and HRP toward H₂O₂, the response signal was significantly amplified. S. flexneri could be detected by the decrease of the reduction peak current before and after immunoreaction. Under optimal conditions, S. flexneri could be detected in the range of 10⁴ to 10¹⁰ cfu mL⁻¹, with a detection limit of 2.3 × 10³ cfu mL⁻¹ (S/N = 3). Furthermore, the proposed immunosensor exhibited a satisfactory specificity, reproducibility, stability and accuracy, indicating that the proposed immunosensor has potential application for a facile, rapid and harmless immunoassay.     

Electrochemical sensor for sulfite determination based on a nanostructured copper-salen film modified electrode

The electrochemical preparation described herein involved the electrocatalytic oxidation of sulfite on a platinum electrode modified with nanostructured copper salen (salen = N,N′-ethylenebis(salicylideneiminato)) polymer films. The complex was prepared and electropolymerized at a platinum electrode in a 0.1 mol L⁻¹ solution of tetrabutylammonium perchlorate in acetonitrile by cyclic voltammetry between 0 and 1.4 V vs. SCE. After cycling the modified electrode in a 0.50 mol L⁻¹ KCl solution, the estimated surface concentration was found to be equal to 2.2 × 10⁻⁹ mol cm⁻². This is a typical behavior of an electrode surface immobilized with a redox couple that can usually be considered as a reversible single-electron reduction/oxidation of the copper(II)/copper(III) couple. The potential peaks of the modified electrode in the electrolyte solution (aqueous) containing the different anions increase with the decrease of the ionic radius, demonstrating that the counter-ions influence the voltammetric behavior of the sensor. The potential peak was found to be linearly dependent upon the ratio [ionic charge]/[ionic radius]. The oxidation of the sulfite anion was performed at the platinum electrode at +0.9 V vs. SCE. However, a significant decrease in the overpotential (+0.45 V) was obtained while using the sensor, which minimized the effect of oxidizable interferences. A plot of the anodic current vs. the sulfite concentration for chronoamperometry (potential fixed = +0.45 V) at the sensor was linear in the 4.0 × 10⁻⁶ to 6.9 × 10⁻⁵ mol L⁻¹ concentration range and the concentration limit was 1.2 × 10⁻⁶ mol L⁻¹. The reaction order with respect to sulfite was determined by the slope of the logarithm of the current vs. the logarithm of the sulfite concentration.     

Direct electron-transfer and electrochemical catalysis of hemoglobin immobilized on mesoporous Al2O3

The direct electrochemistry of hemoglobin (Hb) has been achieved by immobilizing Hb on mesoporous Al₂O₃ (meso-Al₂O₃) film modified glassy carbon (GC) electrode. Meso-Al₂O₃ shows significant promotion to the direct electron-transfer of Hb, thus it exhibits a pair of well defined and quasi-reversible peaks with a formal potential of −0.345 V (vs. SCE). The electron-transfer rate constant (k_s) is estimated to be 3.17 s⁻¹. The immobilized Hb retains its biological activity well and shows high catalytic activity to the reduction of hydrogen peroxide (H₂O₂) and nitrite (NO₂⁻). Under the optimized experimental conditions, the catalytic currents are linear to the concentrations of H₂O₂ and NO₂⁻ in the ranges of 0.195-20.5 μM and 0.2-10 mM, respectively. The corresponding detection limits are 1.95 × 10⁻⁸ M and 3 × 10⁻⁵ M (S/N = 3). The resulting protein electrode has high thermal stability and good reproducibility due to the protection effect of meso-Al₂O₃. Ultraviolet visible (UV-vis) absorption spectra and reflection-absorption infrared (RAIR) spectra display that Hb keeps almost natural structure in the meso-Al₂O₃ film. The N₂ adsorption-desorption experiments show that the pore size of meso-Al₂O₃ is about 14.4 nm, suiting for the encapsulation of Hb (average size: 5.5 nm) well. Therefore, meso-Al₂O₃ is an alternative matrix for protein immobilization and biosensor preparation.