Electrocatalytic oxidation of glucose on Ni and NiCu alloy modified glassy carbon electrode

Nickel and nickel–copper alloy modified glassy carbon electrodes (GC/Ni and GC/NiCu) prepared by galvanostatic deposition were examined for their redox processes and electro-catalytic activities towards the oxidation of glucose in alkaline solutions. The methods of cyclic voltammetry (CV) and chronoamperometry (CA) were employed. The cyclic voltammogram of NiCu alloy demonstrates the formation of β/β crystallographic forms of the nickel oxyhydroxide under prolonged repetitive potential cycling in alkaline solution. It is also observed that the overpotential for O₂ evolution increases for NiCu alloy modified electrode. In CV studies, NiCu alloy modified electrode yields significantly higher activity for glucose oxidation compared to Ni. The oxidation of glucose was concluded to be catalyzed through mediated electron transfer across the nickel hydroxide layer comprising of nickel ions of various valence states. The anodic peak currents show linear dependency with the square root of scan rate. This behavior is the characteristic of a diffusion-controlled process. Under the CA regime, the reaction followed a Cottrellian behavior, and the diffusion coefficient of glucose was found to be 1 × 10⁻⁵ cm² s⁻¹, in agreement with diffusion coefficient obtained in CV studies.     

Direct electron transfer of cytochrome C and its electrocatalytic properties on multiwalled carbon nanotubes/ciprofloxacin films

In this study, stable and homogenous thin films of multiwalled carbon nanotubes (MWCNTs) were obtained on conducting surface using ciprofloxacin (CF, fluoroquinolone antibiotic) as an effective-dispersing agent. Further, MWCNTs/CF film modified electrodes (glassy carbon and indium tin oxide-coated glass electrode) are used successfully to study the direct electrochemistry of proteins. Here, cytochrome C (Cyt-C) was used as a model protein for investigation. A MWCNTs/CF film modified electrode was used as a biocompatible material for immobilization of Cyt-C from a neutral buffer solution (pH 7.2) using cyclic voltammetry (CV). Interestingly, Cyt-C retained its native state on the MWCNTs/CF film. The Cyt-C adsorbed MWCNTs/CF film was characterized by scanning electron microscopy (SEM), UV–visible spectrophotometry (UV-vis) and CV. SEM images showed the evidence for the adsorption of Cyt-C on the MWCNTs/CF film, and UV–vis spectrum confirmed that Cyt-C was in its native state on MWCNTs/CF film. Using CV, it was found that the electrochemical signal of Cyt-C was highly stable in the neutral buffer solution and its redox peak potential was pH dependent. The formal potential (−0.27 V) and electron transfer rate constant (13 ± 1 s⁻¹) were calculated for Cyt-C on MWCNTs/CF film modified electrode. A potential application of the Cyt-C/MWCNTs/CF electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2 × 10⁻⁶ to 7.8 × 10⁻⁵ M. The detection limit for determination of H₂O₂ has been found to be 1.0 × 10⁻⁶ M (S/N = 3). Thus, Cyt-C/MWCNTs/CF film modified electrode can be used as a biosensing material for sensor applications.     

Voltammetric sensor for nitrite determination based on its electrocatalytic reduction at the surface of p-duroquinone modified carbon paste electrode

A p-duroquinone (tetramethyl-p-benzoquinone) modified carbon paste electrode (DMCPE) was employed to study the electrocatalytic reduction of nitrite in aqueous solutions using cyclic voltammetry (CV), double potential-step chronoamperometry, and differential pulse voltammetry (DPV). It has found that under an optimum condition (pH 1.00), the reduction of nitrite at the surface of DMCPE occurs at a potential of about 660 mV less negative than that of an unmodified carbon paste electrode (CPE). The catalytic rate constant, k′_h, based on Andrieux and Saveant theoretical model was calculated as for scan rate 10 mV s^-1. Also, the apparent diffusion coefficient, D _app, was found as 2.5 × 10^–10 and 3.61 × 10^–5 cm² s^-1 for p-duroquinone in carbon paste matrix and nitrite in aqueous buffered solution, respectively. The values for αn_α were estimated to be −0.65 and −0.19 for the reduction of nitrite at the surface of DMCPE and CPE, respectively. The electrocatalytic reduction peak currents showed a linear dependence on the nitrite concentration, and a linear analytical curve was obtained in the ranges of 5.0 × 10^–5 M to 8.0 × 10^–3 M and 6.0 × 10^–6 M to 8.0 × 10^–4 M of nitrite concentration with CV and DPV methods, respectively. The detection limits (2σ) were determined as 2.5 × 10^–5 M and 4.3 × 10^–6 M by CV and DPV methods. This method was also applied as a simple, selective and precise method for determination of nitrite in real samples (the weak liquor from the wood and paper factory of Mazandaran province in Iran) by using a standard addition method.     

Electrochemical synthesis of Fe3O4-PB nanoparticles with core-shell structure and its electrocatalytic reduction toward H2O2

The Fe₃O₄-Prussian blue (PB) nanoparticles with core-shell structure have been in situ prepared directly on a nano-Fe₃O₄-modified glassy carbon electrode by cyclic voltammetry (CV). First, the magnetic nano-Fe₃O₄ particles were synthesized and characterized by X-ray diffraction. Then, the properties of the Fe₃O₄-PB nanoparticles were characterized by CV, electrochemical impedance spectroscopy, and superconducting quantum interference device. The resulting core-shell Fe₃O₄-PB-modified electrode displays a dramatic electrocatalytic ability toward H₂O₂ reduction, and the catalytic current was a linear function with the concentration of H₂O₂ in the range of 1 × 10⁻⁷~5 × 10⁻⁴ mol/l. A detection limit of 2 × 10⁻⁸ (s/n = 3) was determined. Moreover, it showed good reproducibility, enhanced long-term stability, and potential applications in fields of magnetite biosensors.     

Electrochemical Behavior of Uric Acid and Epinephrine at a Meso-2,3-Dimercaptosuccinic Acid Self-Assembled Gold Electrode

A self-assembled electrode with a meso-2,3-dimercaptosuccinic acid (DMSA) monolayer has been characterized by electrochemical quartz crystal microbalance and complex impedance analysis, surface enhanced Raman spectroscopy and cyclic voltammetry. The self-assembled electrode was used for the simultaneous electrochemical detection of epinephrine (EP) and uric acid (UA) in phosphate buffer of pH 7.7. The simultaneous oxidation of EP and UA was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV), and the signals for each method were well separated with a potential difference of over 330 mV and without interference by each other. The detection limit of EP is 5.4 × 10⁻⁸ mol L⁻¹ by CV and 5.3 × 10⁻⁸ mol L⁻¹ by DPV and that of UA is 8.4 × 10⁻⁸ mol L⁻¹ by CV and 4.2 × 10⁻⁸ mol L⁻¹ by DPV. The DMSA self-assembled electrode can be applied to the simultaneous determination of EP and UA.     

Comparative study of electrolessly deposited Pd/Ag films onto p-silicon (100)-activated seed layers of Ag and Pd

Pd/Ag films were electrolessly deposited onto p-silicon (100)-activated seed layers of Ag and Pd, respectively, in the solution of 0.005 mol l⁻¹ AgNO₃ + 0.005 mol l⁻¹ PdCl₂ + 4.5 mol l⁻¹ NH₃ + 0.16 mol l⁻¹ Na₂EDTA+0.1 mol l⁻¹ NH₂NH₂ (pH 10.5) at room temperature. The morphology and composition of the films were studied comparatively by using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Cathodic polarization curves for hydrogen evolution were recorded in 0.5-mol l⁻¹ H₂SO₄ without illumination, in which the obtained films served as working electrodes. The experimental results show that the film obtained on the Ag seed layer was rather a pure Ag film and not a Pd/Ag film, and the Ag deposition rate on Pd sites was much faster than that on Ag sites.     

A new amperometric H<Subscript>2</Subscript>O<Subscript>2</Subscript> biosensor based on nanocomposite films of chitosan–MWNTs,hemoglobin, and silver nanoparticles

A new H₂O₂ biosensor was fabricated on the basis of nanocomposite films of hemoglobin (Hb), silver nanoparticles (AgNPs), and multiwalled carbon nanotubes (MWNTs)–chitosan (Chit) dispersed solution immobilized on glassy carbon electrode (GCE). The immobilized Hb displayed a pair of well-defined and reversible redox peaks with a formal potential (E ^θ′) of −22.5 mV in 0.1 M pH 7.0 phosphate buffer solution. The apparent heterogeneous electron transfer rate constants (k _s) in the Chit–MWNTs film was evaluated as 2.58 s⁻¹ according to Laviron’s equation. The surface concentration (Γ*) of the electroactive Hb in the Chit–MWNTs film was estimated to be (2.48 ± 0.25) × 10⁻⁹ mol cm⁻². Meanwhile, the Chit–MWNTs/Hb/AgNPs/GCE demonstrated excellently electrocatalytical ability to H₂O₂. Its apparent Michaelis–Menten constant (K _M^app) for H₂O₂ was 0.0032 mM, showing a good affinity. Under optimal conditions, the biosensors could be used for the determination of H₂O₂ ranging from 6.25 × 10⁻⁶ to 9.30 × 10⁻⁵ mol L⁻¹ with a detection limit of 3.47 × 10⁻⁷ mol L⁻¹ (S/N = 3). Furthermore, the biosensor possessed rapid response to H₂O₂ and good stability, selectivity, and reproducibility.     

New electrochemiluminescent biosensors combining polyluminol and an enzymatic matrix

Performant reagentless electrochemiluminescent (ECL) (bio)sensors have been developed using polymeric luminol as the luminophore. The polyluminol film is obtained by cyclic voltammetry (CV) on a screen-printed electrode either in a commonly used H₂SO₄ medium or under more original near-neutral buffered conditions. ECL responses obtained after performing polymerization either at acidic pH or at pH 6 have been compared. It appears that polyluminol formed in near-neutral medium gives the best responses for hydrogen peroxide detection. Polymerization at pH 6 by cyclic voltammetry gives a linear range extending from 8 × 10⁻⁸ to 1.3 × 10⁻⁴ M H₂O₂ concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H₂O₂-producing oxidase. Here, choline oxidase (ChOD) has been chosen as a model enzyme. To develop the biosensor, luminol has been polymerized at pH 6 by CV, and then an enzyme-entrapping matrix has been formed on the above modified working electrode. Different biological (chitosan, agarose, and alginate) and chemical (silica gels, photopolymers, or reticulated matrices) gels have been tested. Best performances have been obtained by associating a ChOD-immobilizing photopolymer with the polyluminol film. In this case, choline can be detected with a linear range extending from 8 × 10⁻⁸ to 1.3 × 10⁻⁴ M. This paper is based on the results presented in a poster that received a Poster Award on the occasion of XIII International Symposium on Luminescence Spectrometry in Bologna, Italy, on September, 7th-11th, 2008.     

Properties of ITO films prepared by reactive magnetron sputtering

Indium tin oxide (ITO) thin films were deposited by mid frequency pulsed dual magnetron sputtering using a metallic alloy target with 10 wt.% tin in an atmosphere of argon and oxygen. The aim of the work was to study the interdependence of structural, electrical and optical properties of ITO films deposited in the reactive and transition target mode, respectively. The deposition rate in the transition mode exceeds the deposition rate in the reactive mode by a factor of six, a maximum value of 100 nm·m min⁻¹ could be achieved. This corresponds to a static deposition rate of 200 nm min⁻¹. The lowest electrical resistivity of 1.1·10⁻³ Ω cm was measured at samples deposited in the high oxygen flow range in the transition mode. The samples show a good transparency in the visible range corresponding to extinction coefficients being below 10⁻². X-ray diffraction was used to characterise crystalline structure as well as film stress. ITO films prepared in the transition mode show a slightly preferred orientation in (211) direction, whereas films deposited in the reactive mode are strongly (222) oriented. Compared to undoped In₂O₃ all samples have an enlarged lattice. The lattice strain perpendicular to the surface is about 0.8% and 2.0% for films grown in the transition and the reactive mode, respectively. Deposition in the transition mode introduces a biaxial film stress in the range of −300 MPa, while stress in reactive mode samples is −1500 MPa.     

Preparation and Crystal Structure of K2Ag12Te7

Summary.  Single crystals of K₂Ag₁₂Te₇ (a = 11.460(2), c = 4.660(1) ?; V = 530.01 ?³; space group: P6₃/m; Z = 1) were synthesized under hydrothermal conditions at 250°C in concentrated aqueous KOH solution from elementary silver and tellurium. The crystal structure is characterized by trigonal prismatic KTe₆ polyhedra, connected via two common faces to KTe₃ rods parallel to [001]. These rods are combined by two crystallographically independent Ag atoms, each coordinated to four Te and three Ag atoms (Ag–Te and Ag–Ag < 3.1 ?) to a framework of the formula (K₂Ag₁₂Te₆)^2 + and with channels parallel to the sixfold axis. These channels are statistically occupied by one further Te atom per unit cell, distributed over two independent positions. Received May 17, 2001. Accepted (revised) July 3, 2001     

Preparation and Crystal Structure of K2Ag12Te7

 Single crystals of K₂Ag₁₂Te₇ (a = 11.460(2), c = 4.660(1) ?; V = 530.01 ?³; space group: P6₃/m; Z = 1) were synthesized under hydrothermal conditions at 250°C in concentrated aqueous KOH solution from elementary silver and tellurium. The crystal structure is characterized by trigonal prismatic KTe₆ polyhedra, connected via two common faces to KTe₃ rods parallel to [001]. These rods are combined by two crystallographically independent Ag atoms, each coordinated to four Te and three Ag atoms (Ag–Te and Ag–Ag < 3.1 ?) to a framework of the formula (K₂Ag₁₂Te₆)^2 + and with channels parallel to the sixfold axis. These channels are statistically occupied by one further Te atom per unit cell, distributed over two independent positions.     

Immobilization of flavine adenine dinucleotide onto nickel oxide nanostructures modified glassy carbon electrode: fabrication of highly sensitive persulfate sensor

A simple method was used to fabricate flavin adenine dinucleotide (FAD)/NiOx nanocomposite on the surface of glassy carbon (GC) electrode. Cyclic voltammetry technique was applied for deposition nickel oxide nanostructures onto GC surface. Owing to its high biocompatibility and large surface area of nickel oxide nanomaterials with immersing the GC/NiOx-modified electrode into FAD solution for a short period of time, 10–140 s, a stable thin layer of the FAD molecules immobilized onto electrode surface. The FAD/NiOx films exhibited a pair of well-defined, stable, and nearly reversible CV peaks at wide pH range (2–10). The formal potential of adsorbed FAD onto nickel oxide nanoparticles film, E ^o′ vs. Ag/AgCl reference electrode is −0.44 V in pH 7 buffer solutions was similar to dissolved FAD and changed linearly with a slope of 58.6 mV/pH in the pH range 2–10. The surface coverage and heterogeneous electron transfer rate constant (k _s ) of FAD immobilized on NiOx film glassy carbon electrode are 4.66 × 10⁻¹¹ mol cm⁻² and 63 ± 0.1 s⁻¹, indicating the high loading ability of the nickel oxide nanoparticles and great facilitation of the electron transfer between FAD and nickel oxide nanoparticles. FAD/NiOx nanocomposite-modified GC electrode shows excellent electrocatalytic activity toward S₂O₈²⁻ reduction at reduced overpotential. Furthermore, rotated modified electrode illustrates good analytical performance for amperometric detection of S₂O₈²⁻. Under optimized condition, the concentration calibration range, detection limit, and sensitivity were 3 μM–1.5 mM, 0.38 μM and 16.6 nA/μM, respectively.     

Immobilization of Hemoglobin on the Gold Colloid Modified Pretreated Glassy Carbon Electrode for Preparing a Novel Hydrogen Peroxide Biosensor

A novel hydrogen peroxide (H₂O₂) biosensor was developed by immobilizing hemoglobin on the gold colloid modified electrochemical pretreated glassy carbon electrode (PGCE) via the bridging of an ethylenediamine monolayer. This biosensor was characterized by UV-vis reflection spectroscopy (UV-vis), electrochemical impendence spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized Hb exhibited excellent electrocatalytic activity for hydrogen peroxide. The Michaelis–Menten constant (K _m) was 3.6 mM. The currents were proportional to the H₂O₂ concentration from 2.6 × 10⁻⁷ to 7.0 × 10⁻³ M, and the detection limit was as low as 1.0 × 10⁻⁷ M (S/N = 3).     

Determination of rutin using a CeO2 nanoparticle-modified electrode

CeO₂ nanoparticles approximately 12 nm in size were synthesized and subsequently characterized by XRD, TEM and UV-vis spectroscopy. Then, a gold electrode modified with CeO₂ nanoparticles was constructed and characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The modified electrode demonstrated strong catalytic effects with high stability towards electrochemical oxidation of rutin. The anodic peak currents (measured by differential pulse voltammetry) increased linearly with the concentration of rutin in the range of 5.0 × 10⁻⁷–5.0 × 10⁻⁴ mol · L⁻¹. The detection limit (S/N = 3) was 2.0 × 10⁻⁷ mol · L⁻¹. The relative standard deviation (RSD) of 8 successive scans was 3.7% for 5.0 × 10⁻⁶ mol · L⁻¹ rutin. The method showed excellent sensitivity and stability, and the determination of rutin in tablets was satisfactory.     

Probing traces of hydrogen peroxide by use of a biosensor based on mediator-free DNA and horseradish peroxidase immobilized on silver nanoparticles

A new electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) has been developed by immobilizing horseradish peroxidase (HRP) on silver colloids (nanosilver) and use of a DNA-functionalized interface. In the presence of the DNA and the nanosilver the immobilized HRP gives a pair of well-defined redox peaks with an electron-transfer rate constant of 3.27 ± 0.91 s⁻¹ in pH 7.0 PBS. The presence of DNA also provides a biocompatible microenvironment for enzyme molecules, greatly amplifies the amount of HRP molecules immobilized on the electrode surface, and improves the sensitivity of the biosensor. Under optimum conditions the biosensor has electrocatalytic activity in the reduction of hydrogen peroxide with linear dependence on H₂O₂ concentration in the range 1.5 × 10⁻⁶ to 2.0 × 10⁻³ mol L⁻¹; the detection limit is 5.0 × 10⁻⁷ mol L⁻¹ at a signal-to-noise ratio of 3. The value of HRP in the composite membrane was found to be 1.62 mmol L⁻¹. These results suggest that the properties of the complex film, with its bioelectrochemical catalytic activity, could make it useful for development of bioelectronic devices and for investigation of protein electrochemistry at functional interfaces.     

Slow salt-induced aggregation of citrate-covered silver particles in aqueous solutions of cellulose derivatives

In this work, the salt-induced aggregation of bare and polymer-covered silver particles has been studied with the aid of light scattering and UV-visible spectroscopy. Light scattering on a suspension of bare silver particles at a low salt concentration shows that the cluster fractal dimension d _f changes from 1.6 to 2 in the course of the aggregation process, whereas no restructuring of the clusters is observed at a higher salinity where d _f ≈ 1.6. The growth of the clusters over time can be described by a power law R _h ∝ t ^α , where R _h is the apparent hydrodynamic radius. The UV-visible experiments revealed that increasing the size of the bare silver particles lead to a significant broadening and red-shift of the absorbance band, whereas for salt-induced growth of Ag clusters, a blue-shift and broadening was observed. Addition of salt to a suspension of silver particles and hydroxyethylcellulose divulged a slower broadening of the surface plasmon peak than without polymer.     

DNA microarrays for visual detection of human pathogenic microorganisms based on tyramine signal amplification coupled with gold label silver stain

The utility of DNA microarrays is severely limited by their restricted sensitivity. Tyramine signal amplification (TSA) coupled with gold label silver stain (GLSS) was introduced in DNA microarrays for visual detection of human pathogenic microorganisms. First, a TSA system was introduced to the microarrays after the microarrays were prepared and hybridized with biotinylated targets. This procedure leads to large amounts of biotin-conjugated tyramine depositing at the site of enzyme reaction under HRP catalysis. Second, streptavidin–nanogold was introduced and accumulated by specific binding of biotin and streptavidin. Finally, silver staining was performed. The images of the spots were scanned with a visible light scanner and quantified with ArrayVision 7.0 software. Detection conditions were systematically optimized. Then the sensitivity among TSA coupled with GLSS, GLSS, and TSA coupled with Cy3 was compared. The optimized conditions were: streptavidin–HRP (1 mg mL⁻¹) dilution 1:1500, biotin–tyramine dilution 1:200 (+0.5% H₂O₂), streptavidin–nanogold dilution 1:100 (all diluted in 1 × PBS + 1% BSA) and silver stain time of 10 min. The sensitivity of TSA coupled with GLSS was 100-fold higher than that of GLSS, and was identical with that of TSA coupled with Cy3. Meanwhile, the specificity of the microarrays were not affected. This implied that TSA coupled with GLSS was a sensitive visual detection method and would be an ideal alternative to fluorescence-based detection for DNA microarrays.     

An electroanalytical study of the drug proflavine

A square wave adsorptive stripping voltammetric (SWAdSV) method was developed for the determination of proflavine. The electrochemical behaviour of proflavine was investigated by cyclic (CV) and square wave voltammetry (SWV) at the hanging mercury drop electrode (HMDE) and carbon paste electrode (CPE). Different parameters were tested to optimize the conditions of the determination. Better results were obtained by square wave voltammetry using CPE where two oxidation and a reduction peak, appeared, at 0.19, 0.94 and 0.20 V, respectively. The peak at 0.19 V is quasi-reversible and deposition dependent. Linearity was observed in the range of (0.2–23.4) × 10⁻⁸ M (r = 0.998) during the anodic scan and in the range of (1.17–117) × 10⁻⁸ M (r = 0.999) during the cathodic scan. The second peak at 0.94 V is irreversible and deposition independent. The linearity of this peak was observed in the range of (1.29–11.7) × 10⁻⁸ M (r = 0.998). The method was applied to the analysis of bovine serum and gave satisfactory results. Correspondence: S. Th. Girousi, Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University, Thessaloniki 54124, Greece     

The Potential Energies of Cohesion of a Crystalline Organic Enantiomer and the Racemate; on the Energy for the Liquid Racemate [1]

The cohesion potential energy of the crystal of one enantiomer of ethyl 3-cyano-3-(3,4-dimethyloxyphenyl)-2,2,4-trimethylpentanoate, −47.7 ± 0.1 kJ mol⁻¹ (0–90°C), was found out from the heat of sublimation (123.2 ± 5.1 kJ mol⁻¹, 78.6°C) and the kinetic energies for the gas phase and the crystal. It was found that the entropy function of Debye’s theory of solids mathematically agreed with the vibrational entropy of the gas (variationally obtained), allowing to disclose the vibrational energy using the Debye energy function (E _vib 835.0 kJ mol⁻¹ (78.6°C), E ⁰ included). E _kin for the crystal (771.1 kJ mol⁻¹ (78.6°C)) was obtained by Debye’s theory with the experimental heat capacity. The cohesion energy represented a moderate part of the sublimation energy. The cohesion energy of the racemic crystal, −44.2 kJ mol⁻¹, was obtained by the heat of formation of the crystal in the solid state (3.0 kJ mol⁻¹, 83.3°C) and E _kin for the crystal (by Debye’s theory). The decrease in cohesion on formation of the crystal accounted for the energy of formation. The change in potential energy on liquefaction of the racemate from the gas state was disclosed obtaining added-up E _{vib + rot} for the liquid in the way as to E _vib for the gas, the Debye entropy function being increasedly suited for the liquid (E _{vib + rot} 763.4 kJ mol⁻¹ (115.4°C)). Positive ΔE _pot, 13.0 kJ mol⁻¹, arised from the increase in electronic energy (Δ _l ν_mean − 154.3 cm⁻¹, by the dielectric nature of the liquid), added to the cohesion energy.     

A hydrogen peroxide biosensor based on direct electrochemistry of hemoglobin in palladium nanoparticles/graphene-chitosan nanocomposite film

Thermally two-dimensional lattice graphene (GR) and biocompatibility chitosan (CS) act as a suitable support for the deposition of palladium nanoparticles (PdNPs). A novel hydrogen peroxide (H₂O₂) biosensor based on immobilization of hemoglobin (Hb) in thin film of CS containing GR and PdNPs was developed. The surface morphologies of a set of representative membranes were characterized by means of scanning electron microscopy and showed that the PdNPs are of a sphere shape and an average diameter of 50 nm. Under the optimal conditions, the immobilized Hb showed fast and excellent electrocatalytic activity to H₂O₂ with a small Michaelis–Menten constant of 16 μmol L⁻¹, a linear range from 2.0 × 10⁻⁶ to 1.1 × 10⁻³ mol L⁻¹, and a detection limit of 6.6 × 10⁻⁷ mol L⁻¹. The biosensor also exhibited other advantages, good reproducibility, and long-term stability, and PdNPs/GR–CS nanocomposites film would be a promising material in the preparation of third generation biosensor.