Myoglobin/arylhydroxylamine film modified electrode: Direct electrochemistry and electrochemical catalysis

The adsorption processes and electrochemical behavior of 4-nitroaniline (4-NA) adsorbed onto glassy carbon electrodes (GCE) have been investigated in aqueous 0.1 M nitric acid (HNO₃) electrolyte solutions using cyclic voltammetry (CV). 4-NA adsorbs onto GCE surfaces, and upon potential cycling past −0.2 V, is transformed into the arylhydroxylamine (ArHA) derivative which exhibits a well-behaved pH dependent redox couple centered at 0.32 V at pH 1.5. It is noted as arylhydroxylamine modified glassy carbon electrodes (HAGCE). This modified electrode can be readily used as an immobilization matrix to entrap proteins and enzymes. In our studies, myoglobin (Mb) was used as a model protein for investigation. A pair of well-defined reversible redox peaks of Mb (Fe(III)-Fe(II)) was obtained at the Mb/arylhydroxylamine modified glassy carbon electrode (Mb/HAGC) by direct electron transfer between the protein and the GCE. The formal potential (E^0′), the apparent coverage (Γ^*) and the electron-transfer rate constant (k_s) were calculated as −0.317 V, 8.26 × 10⁻¹² mol/cm² and 51 ± 5 s⁻¹, respectively. Dramatically enhanced biocatalytic activity was exemplified at the Mb/HAGC electrode by the reduction of hydrogen peroxide (H₂O₂), trichloroacetic acid (TCA) and oxygen (O₂). The Mb/arylhydroxylamine film was also characterized by UV-visible spectroscopy (UV-vis), scanning electron microscope (SEM) indicating excellent stability and good biocompatibility of the protein in the arylhydroxylamine modified electrode. This new Mb/HAGC electrode exhibited rapid electrochemical response (2 s) for H₂O₂ and had good stability in physiological condition, showing the potential applicability of the films in the preparation of third generation biosensors or bioreactors based on direct electrochemistry of the proteins.     

Exploiting multi-function Metal-Organic Framework nanocomposite Ag@Zn-TSA as highly efficient immobilization matrixes for sensitive electrochemical biosensing

A novel multi-function Metal-Organic Framework composite Ag@Zn-TSA (zinc thiosalicylate, Zn(C₇H₄O₂S), Zn-TSA) was synthesized as highly efficient immobilization matrixes of myoglobin (Mb)/glucose oxidase (GOx) for electrochemical biosensing. The electrochemical biosensors based on Ag@Zn-TSA composite and ionic liquid (IL) modified carbon paste electrode (CPE) were fabricated successfully. Furthermore, the properties of the sensors were discussed by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometric current-time curve, respectively. The results showed the proposed biosensors had wide linear response to hydrogen peroxide (H₂O₂) in the range of 0.3–20,000 μM, to nitrite (NO₂⁻) for 1.3 μM–1660 μM and 2262 μM–1,33,000 μM, to glucose for 2.0–1022 μM, with a low detection limit of 0.08 μM for H₂O₂, 0.5 μM for NO₂⁻, 0.8 μM for glucose. The values of the apparent heterogeneous electron transfer rate constant (k_s) for Mb and GOx were estimated as 2.05 s⁻¹ and 2.45 s⁻¹, respectively. Thus, Ag@Zn-TSA was a kind of ideal material as highly efficient immobilization matrixes for sensitive electrochemical biosensing. In addition, this work indicated that MOF nanocomposite had a great potential for constructing wide range of sensing interface.     

Prussian Blue electrodeposited on MWNTs-PANI hybrid composites for H2O2 detection

A Prussian Blue (PB)/polyaniline (PANI)/multi-walled carbon nanotubes (MWNTs) composite film was fabricated by step-by-step electrodeposition on glassy carbon electrode (GCE). The electrode prepared exhibits enhanced electrocatalytic behavior and good stability for detection of H₂O₂ at an applied potential of 0.0 V. The effects of MWNTs thickness, electrodeposition time of PANI and rotating rate on the current response of the composite modified electrode toward H₂O₂ were optimized to obtain the maximal sensitivity. A linear range from 8 × 10⁻⁹ to 5 × 10⁻⁶ M for H₂O₂ detection has been observed at the PB/PANI/MWNTs modified GCE with a correlation coefficient of 0.997. The detection limit is 5 × 10⁻⁹ M on signal-to-noise ratio of 3. To the best of our knowledge, this is the lowest detection limit for H₂O₂ detection. The electrode also shows high sensitivity (526.43 μA μM⁻¹ cm⁻²) for H₂O₂ detection which is more than three orders of magnitude higher than the reported.     

Direct electrochemistry and electrocatalysis of cytochrome c immobilized on gold nanoparticles-chitosan-carbon nanotubes-modified electrode

A robust and effective nanohybrid film based on gold nanoparticles (GNPs)/chitosan (Chit)/multi-walled carbon nanotubes (MWNTs) was prepared by a layer-by-layer self-assembly technique. Cytochrome c (Cyt c) was successfully immobilized on the nanohybrid film modified glassy carbon (GC) electrode by cyclic voltammetry. The direct electron transfer between Cyt c and the modified electrode was investigated in detail. Cyt c shows a couple of quasi-reversible and well-defined cyclic voltammetry peaks with a formal potential (E^0′) of −0.16 V (versus Ag/AgCl) in pH 7.0 phosphate buffer solution (PBS). The Cyt c/GNPs/Chit/MWNTs modified GC electrode gives an improved electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂). The sensitivity is 92.21 μA mM⁻¹ cm⁻² and the calculated apparent Michaelis-Menten constant () is 0.791 mM, indicating a high-catalytic activity of Cyt c. The catalysis currents increase linearly to the H₂O₂ concentration in a wide range of 1.5 × 10⁻⁶ to 5.1 × 10⁻⁴ M with a correlation coefficient 0.999. The detection limit is 9.0 × 10⁻⁷ M (at the ratio of signal to noise, S/N = 3). Moreover, the modified electrode displays rapid response (5 s) to H₂O₂, and possesses good stability and reproducibility.     

Direct Electron Transfer and Electrocatalysis of Myoglobin Based on its Direct Immobilization on Carbon Ionic Liquid Electrode

Direct electron transfer of myoglobin (Mb) was achieved by its direct immobilization on carbon ionic liquid electrode (CILE) with a conductive hydrophobic ionic liquid, 1‐butyl pyridinium hexaflourophosphate ([BuPy][PF₆]) as binder for the first time. A pair of well‐defined, quasi‐reversible redox peaks was observed for Mb/CILE resulting from Mb redox of heme Fe(III)/Fe(II) redox couple in 0.1 M phosphate buffer solution (pH 7.0) with oxidation potential of ?0.277 V, reduction potential of ?0.388 V, the formal potential E°′ (E°′=(E_pa+E_pc)/2) at ?0.332 V and the peak‐to‐peak potential separation of 0.111 V at 0.5 V/s. The average surface coverage of the electroactive Mb immobilized on the electrode surface was calculated as 1.06±0.03×10^?9 mol cm^?2. Mb retained its bioactivity on modified electrode and showed excellent electrocatalytic activity towards the reduction of H₂O₂. The cathodic peak current of Mb was linear to H₂O₂ concentration in the range from 6.0 μM to 160 μM with a detection limit of 2.0 μM (S/N=3). The apparent Michaelis–Menten constant (K and the electron transfer rate constant (k_s) were estimated to be 140±1 μM and 2.8±0.1 s^?1, respectively. The biosensor achieved the direct electrochemistry of Mb on CILE without the help of any supporting film or any electron mediator.     

Direct electrocatalytic oxidation of nitric oxide and reduction of hydrogen peroxide based on α-Fe2O3 nanoparticles-chitosan composite

α-Fe₂O₃ nanoparticles prepared using a simple solution-combusting method have been dispersed in chitosan (CH) solution to fabricate nanocomposite film on glass carbon electrode (GCE). The as-prepared α-Fe₂O₃ nanoparticles were characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM). The nanocomposite film exhibits high electrocatalytic oxidation for nitric oxide (NO) and reduction for hydrogen peroxide (H₂O₂). The electrocatalytic oxidation peak is observed at +0.82 V (vs. Ag/AgCl) and controlled by diffusion process. The electrocatalytic reduction peak is observed at −0.45 V (vs. Ag/AgCl) and controlled by diffusion process. This α-Fe₂O₃-CH/GCE nanocomposite bioelectrode has response time of 5 s, linearity as 5.0 × 10⁻⁷ to 15.0 × 10⁻⁶ M of NO with a detection limit of 8.0 × 10⁻⁸ M and a sensitivity of −283.6 μA/mM. This α-Fe₂O₃-CH/GCE nanocomposite bioelectrode was further utilized in detection of H₂O₂ with a detection limit of 4.0 × 10⁻⁷ M, linearity as 1.0 × 10⁻⁶ to 44.0 × 10⁻⁶ M and with a sensitivity of 21.62 μA/mM. The shelf life of this bioelectrode is about 6 weeks under room temperature conditions.     

The interface behavior of hemoglobin at carbon nanotube and the detection for H(2)O(2)

Direct electrochemistry of hemoglobin (Hb) is observed at carbon nanotube (CNT) interface. The adsorbing Hb can transfer electron directly at CNT interface compared with common carbon material. The heterogeneous electron transfer rate constant k of Hb can be calculated as 0.062 s⁻¹, the transfer coefficient α is 0.21 and the average surface coverage of Hb on CNT surface is 3.58 × 10⁻⁹ ± 2.7 × 10⁻¹⁰ mol/cm². It is found that the adsorbing Hb still keeps its catalytic activity to H₂O₂. This sensor was used to detect H₂O₂. The apparent Michaelis-Menten constant is calculated as 6.75 × 10⁻⁴ mol L⁻¹.     

Analysis of hydrogen peroxide and an organic hydroperoxide via the electrocatalytic Fenton reaction

A new concept for the electrochemical detection of hydrogen peroxide, and organic hydroperoxides is presented. One advantage of the significance of this technique is that it does not require chemical modification of the electrode or addition of enzymes. Direct electro-reduction of the peroxides was not observed on the carbon disk electrode as it is a kinetically slow process. Redox cycling of the iron complex is apparent as Fe^IIEDTA rapidly reduces the O-O bond of the peroxides (Fenton Reaction) upon its production by the kinetically facile electro-reduction of Fe^IIIEDTA. This provides an enhanced and steady-state reductive current as observed by cyclic voltammetry. These features are indicative of the electrocatalytic (EC′) mechanism. A calibration curve was constructed based on the chronoamperometric response at 32 s and a detection limit for H₂O₂ and t-butyl hydroperoxide was calculated to be 0.4 μM and 20 µM, respectively. This difference is attributable to the rate in which the iron(II) complex reduces the O-O bond, H₂O₂ (2.3 × 10⁵ M^− 1 s^− 1) being faster than for the organic peroxide (5.1 × 10⁴ M^− 1 s^− 1). The Fe^IIEDTA complex was observed to be unreactive toward dialkyl peroxides. This method may find use in the detection of peroxide-based explosives or in enzymatic assays as it is rapid, simple, inexpensive and should prove to be robust.     

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.     

Hydrogen Peroxide Biosensor Based on the Electrochemistry of the Myoglobin‐TATP Composite Film

Abstract

Direct electrochemistry of the myoglobin‐triacetone triperoxide (Mb‐TATP) composite on carbon paste (CP) electrode is reported. This electrode gives a well‐defined and quasi‐reversible cyclic voltammogram for the Mb Fe^III/Fe^II redox coupled with the formal potential (E?′) of ?0.302 V (vs. Ag/AgCl) in pH 6.92 phosphate buffer. Electronic and vibrational spectroscopies show that the Mb in the composite retains a structure similar to its native form. The enzymatic reactivity to the reduction of H₂O₂ has been studied for the Mb‐TATP film. The analytical performances have been obtained with the linear range of 78.32–1135.64 µM, the detection limit of 55 µM (S/N=3), and the apparent Michaelis‐Menten constant (K _m) of 662.8 µM. This H₂O₂ biosensor based on the electrocatalysis of the immobilized Mb presents a higher stability within two weeks.     

Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator

An amperometric biosensor has been developed for the determination of H₂O₂ in plant samples. Horseradish peroxidase (HRP) is immobilized on a sandwiched nano-Au particle / m-phenylenediamine polymer film by glutaraldehyde cross-linking. The film is formulated on the carbon paste electrode (CPE) blended with ferrocene as an electron transfer mediator. On the low concentration range, the current response is related to the H₂O₂ concentration linearly from 0 to 8×10^-6 M with a detection limit of 1.3×10^-7 M. On a wider concentration range of 8×10^-6 to 1.4×10^-4 M, the reciprocal of current response is linearly related to the reciprocal of H₂O₂ concentration. The apparent Michaelis-Menten constant (K_m^app) was calculated to be 0.0334 mM. The sensor has been tested by determining H₂O₂ concentration in plant leaf samples.     

Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Bioimprinted protein exhibits glutathione peroxidase activity

A strategy for design of bioimprinted proteins with glutathione peroxidase (GPX) activity has been proposed. The proteins imprinted with a glutathione derivative were converted into selenium-containing proteins by chemical modifying the reactive hydroxyl groups of serines followed by sodium hydrogen selenide displacement. These selenium-containing proteins exhibited remarkable GPX activities and the GPX activities of reduction of H₂O₂ by glutathione (GSH) were found to be 101-817 U μmol⁻¹, which approaches the activity of a selenium-containing catalytic antibody elicited by a hapten similar to our template. The steady state kinetic study for imprinted protein catalysis revealed Michaelis-Menten kinetics for both H₂O₂ and GSH, e.g. the pesudo-first-order rate constant k_cat (H₂O₂) and the apparent Michaelis constant K_m (H₂O₂) at 1 mM GSH were calculated to be 784 min⁻¹ and 1.24×10⁻³ M, respectively, and the apparent second-order rate constant k_cat (H₂O₂)/K_m (H₂O₂) was determined to be 6.33×10⁵ (M min)⁻¹. The kinetics and the template inhibition showed that the strategy might be a remarkably efficient one for generating artificial enzyme with GPX activity.     

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10⁻⁷ M), short response time (within 10 s), high sensitivity (88.6 μA mM⁻¹ cm⁻²) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H₂O₂ by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083 ± 0.001) ×10⁻³ M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.     

Direct electrochemistry of glucose oxidase entrapped in nano gold particles-ionic liquid-N,N-dimethylformamide composite film on glassy carbon electrode and glucose sensing

The direct electrochemistry of glucose oxidase (GOD) entrapped in nano gold particles (NAs)-N,N-dimethylformamide (DMF)-1-butyl-3-methylimidazolium hexafluophosphate (BMIMPF₆) composite film on a glassy carbon electrode (NAs-DMF-GOD (BMIMPF₆)/GC) has been investigated for first time. The immobilized GOD exhibits a pair of well-defined reversible peaks in 0.050 M pH 5 phosphate solutions (PS), resulting from the redox of flavin adenine dinucleotide (FAD) in GOD. The peak currents are three times as large as those of GOD-NAs-DMF film coated GC electrode (i.e. NAs-DMF-GOD (water)/GC). In addition, the NAs-DMF-GOD (BMIMPF₆) composite material has higher thermal stability than NAs-DMF-GOD (water). Results show that ionic liquid BMIMPF₆, DMF and NAs are requisite for GOD to exhibit a pair of stable and reversible peaks. Without any of them, the peaks of GOD become small and unstable. Upon the addition of glucose, the peak currents of GOD decrease and a new cathodic peak occurs at −0.8 V (versus SCE), which corresponds to the reduction of hydrogen peroxide (H₂O₂) generated by the catalytic oxidation of glucose. The peak current of the new cathodic peak and the glucose concentration show a linear relationship in the ranges of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁶ M and 2.0 × 10⁻⁶ to 2.0 × 10⁻⁵ M. The kinetic parameter I_max of H₂O₂ is estimated to be 1.19 × 10⁻⁶ A and the apparent K_m (Michaelis-Menten constant) for the enzymatic reaction is 3.49 μM. This method has been successfully applied to the determination of glucose in human plasma and beer samples, and the average recoveries are 97.2% and 99%, respectively.     

Electrochemistry and scanning electron microscopy of polyaniline/peroxidase-based biosensor

An amperometric biosensor was prepared by in situ deposition of horseradish peroxidase (HRP) enzyme on a polyaniline (PANI)-doped platinum disk electrode. The PANI film was electrochemically deposited on the electrode at 100 mV s⁻¹/Ag-AgCl. Cyclic voltammetric characterization of the PANI film in 1 M HCl showed two distinct redox peaks, which prove that the PANI film was electroactive and exhibited fast reversible electrochemistry. The surface concentration and film thickness of the adsorbed electroactive species was estimated to be 1.85×10⁻⁷ mol cm⁻² and approximately 16 nm, respectively. HRP was electrostatically immobilized onto the surface of the PANI film, and voltammetry was used to monitor the electrocatalytic reduction of hydrogen peroxide under diffusion-controlled conditions. Linear responses over the concentration range 2.5×10⁻⁴ to 5×10⁻³ M were observed. Spectroelectrochemistry was used to monitor the changes in UV-vis properties of HRP, before and after the catalysis of H₂O₂. The biosensor surface morphology was characterized by scanning electron microscopy (SEM) using PANI-doped screen-printed carbon electrodes (SPCEs) in the presence and absence of (i) peroxidase and (ii) peroxide. The SEM images showed clear modifications of the conducting film surface structure when doped with HRP, as well as the effect of hydrogen peroxide on the morphology of biosensor.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized in a magnetic nanoparticles-chitosan film

Magnetic nanoparticles (Fe₃O₄) were synthesized by a chemical coprecipitation method. X-ray diffraction (XRD) and transmission electron microscope (TEM) were used to confirm the crystallite structure and the particle's radius. The Fe₃O₄ nanoparticles and chitosan (CS) were mixed to form a matrix in which haemoglobin (Hb) can be immobilized for the fabrication of H₂O₂ biosensor. The Fe₃O₄-CS-Hb film exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks due to the redox of Hb-heme Fe (III)/Fe (II) in a pH 7.0 phosphate buffer. The formal potential of Hb-heme Fe(III)/Fe(II) couple varied linearly with the increase of pH in the range of 4.0-10.0 with a slope of 46.5 mV pH⁻¹, indicating that electron transfer was accompanied with single proton transportation in the electrochemical reaction. The surface coverage of Hb immobilized on Fe₃O₄-CS film glassy carbon electrode was about 1.13 × 10⁻¹⁰ mol cm⁻². The heterogeneous electron transfer rate constant (k_s) was 1.04 s⁻¹, indicating great facilitation of the electron transfer between Hb and magnetic nanoparticles-chitosan modified electrode. The modified electrode showed excellent electrocatalytic activity toward oxygen and hydrogen peroxide reduction. The apparent Michaelis-Menten constant for H₂O₂ was estimated to be 38.1 μmol L⁻¹.     

Differential pulse voltammetric determination of P(V) following adsorptive accumulation of alpha-[PMo(12)O(40)](3-) on a polypyrrole-modified glassy carbon electrode

On the basis of the formation and pre-concentration of an α-Keggin-type [PMo₁₂O₄₀]³⁻ complex, a novel voltammetric method was developed for the determination of trace levels of P(V). The α-[PMo₁₂O₄₀]³⁻ complex was formed by heating a 5×10⁻⁴ M Mo(VI)-0.2 M HCl-40% (v/v) CH₃CN system containing a trace amount of P(V) at 70 °C for 30 min. During the electrochemical polymerization of pyrrole in the α-[PMo₁₂O₄₀]³⁻ solution, the α-[PMo₁₂O₄₀]³⁻ complex was accumulated into the polypyrrole film on a glassy carbon electrode. The differential pulse voltammetric peak current due to the α-[PMo₁₂O₄₀]³⁻ complex incorporated in the polypyrrole film was linearly dependent on the P(V) concentration in the range of 5×10⁻⁹-5×10⁻⁷ M; a detection limit of 2×10⁻⁹ M was achieved.     

Direct electrochemistry and electrocatalysis of heme proteins on SWCNTs-CTAB modified electrodes

Direct electrochemistry and electrocatalysis of heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) were studied with the protein incorporated single walled carbon nanotubes (SWCNTs)-cetylramethylammonium bromide (CTAB) nanocomposite film modified glassy carbon electrodes (GCEs). The incorporated heme proteins were characterized with Fourier transform infrared spectroscopy (FTIR), ultraviolet visible (UV) spectroscopy, atomic force microscopy (AFM) and electrochemistry, indicating the heme proteins in SWCNTs-CTAB nanocomposite films keep their secondary structure similar to their native states. The direct electron transfer between the heme proteins in SWCNTs-CTAB films and GCE was investigated. The electrochemical parameters such as formal potentials and apparent heterogeneous electrontransfer rate constants (k_s) were estimated by square wave voltammetry with nonlinear regression analysis. The heme protein-SWCNT-CTAB electrodes show excellent electrocatalytic activities for the reduction of H₂O₂ and NO₂⁻, which have been utilized to determine the concentrations of H₂O₂ and NO₂⁻.     

Amperometric biosensor for hydrogen peroxide based on hemoglobin entrapped in titania sol-gel film

Hemoglobin (Hb) was entrapped in a titania sol-gel matrix and used as a mimetic peroxidase to construct a novel amperometric biosensor for hydrogen peroxide. The Hb entrapped titania sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process for protein immobilization. The morphologies of both titania sol-gel and the Hb films were characterized using scanning electron microscopy (SEM) and proved to be chemically clean, porous, homogeneous. This matrix provided a biocompatible microenvironment for retaining the native structure and activity of the entrapped Hb and a very low mass transport barrier to the substrates. H₂O₂ could be reduced by the catalysis of the entrapped hemoglobin at −300 mV without any mediator. The reagentless H₂O₂ sensor exhibited a fast response (less than 5 s) and sensitivity as high as 1.29 mA mM⁻¹ cm⁻². The linear range for H₂O₂ determination was from 5.0×10⁻⁷ to 5.4×10⁻⁵ M with a detection limit of 1.2×10⁻⁷ M. The apparent Michaelis-Menten constant of the encapsulated hemoglobin was calculated to be 0.18±0.02 mM. The stability of the biosensor was also evaluated.