Immobilization of horseradish peroxidase and nile blue into the ormosil nanocomposite for the fabrication of hydrogen peroxide biosensor based on MWCNT modified glassy carbon electrode

A novel approach to construct a second-generation amperometric biosensor is described. The classical redox dye nile blue (NB) as mediator and horseradish peroxidase as a base enzyme were coimmobilized into the multiwalled carbon nanotubes (MWCNTs) modified ormosil matrix. Nafion was dispersed into the matrix to enhance the rate of the electron transfer and prevent the cracking of the ormosil film. The surface morphology of MWCNT/NB/NAF/HRP nanocomposite was characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Cyclic voltammetry and amperometry measurements were used to study and optimize the performance of the resulting peroxide biosensor. The apparent Michaelis–Menten constant was determined to be 1.1 mM. The effect of pH, applied potential and amount of the HRP enzyme on the electrochemical biosensor has been systematically studied. The fabricated biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with wide linear range from 2 × 10⁻⁷ to 3.8 × 10⁻⁴ M, and low detection limit 1 × 10⁻⁷ M (S/N = 3) with fast response time <3 s. The facile procedure of immobilizing HRP and MWCNTs into the ormosil used in the present work can promote the development of electrochemical research for enzymes, proteins, biosensors, biofuel cells and other bioelectrochemical devices.     

Bean sprout peroxidase biosensor based on l-cysteine self-assembled monolayer for the determination of dopamine

A new bean sprout peroxidase was immobilized on a gold electrode modified with self-assembled monolayers (SAM) of l-cysteine for the determination of dopamine in pharmaceutical samples using square wave voltammetry. In the bean sprout–(SAM)–Au electrode, the peroxidase, in the presence of hydrogen peroxide, catalyzes the oxidation of dopamine to the corresponding quinone, which is electrochemically reduced back to dopamine at +0.15 V vs. Ag/AgCl. The performance and the factors influencing the response of this biosensor were studied in detail. The best performance was obtained using 0.1 mol L⁻¹ phosphate buffer solution (pH 6.0), 6.0 × 10⁻⁵ mol L⁻¹ hydrogen peroxide, frequency of 100 Hz, pulse amplitude of 80 mV and scan increment of 4.0 mV. The analytical curve was linear for dopamine concentrations from 9.91 × 10⁻⁶ to 2.21 × 10⁻⁴ mol L⁻¹ and the detection limit was 4.78 × 10⁻⁷ mol L⁻¹. The recovery of dopamine ranged from 98.0 to 111.8% and the relative standard deviation was 3.1% for a solution containing 1.30 × 10⁻⁵ mol L⁻¹ dopamine (n = 6). The lifetime of this biosensor was 15 days (at least 300 determinations). The results obtained for dopamine determination in pharmaceutical formulations using the proposed bean sprout–SAM–Au electrode were in agreement with those obtained with the standard method at the 95% confidence level.     

A sensitive nonenzymatic hydrogen peroxide sensor based on DNA–Cu complex electrodeposition onto glassy carbon electrode

In this paper, DNA–Cu²⁺ complex was electrodeposited onto the surface of glassy carbon (GC) electrode, which fabricated a DNA–Cu²⁺/GC electrode sensor to detect H₂O₂ with nonenzyme. Cyclic voltammogram of DNA–Cu²⁺/GC electrode showed a pair of well-defined redox peaks for Cu²⁺/Cu⁺. Moreover, the electrodeposited DNA–Cu²⁺ complex exhibited excellent electrocatalytic behavior and good stability for the detection of H₂O₂. The effects of Cu²⁺ concentration, electrodeposition time and determination conditions such as pH value, applied potential on the current response of the DNA–Cu²⁺/GC electrode toward H₂O₂ were optimized to obtain the maximal sensitivity. The linear range for the detection of H₂O₂ is 8.0 × 10⁻⁷ M to 4.5 × 10⁻³ M with a high sensitivity of −40.25 μA mM⁻¹, a low detection limit of 2.5 × 10⁻⁷ M and a fast response time of within 4 s. In addition, the sensor has good reproducibility and long-term stability and is interference free.     

A novel microbial biosensor based on Circinella sp. modified carbon paste electrode and its voltammetric application

Microbial biosensors have been developed for voltammetric determination of various substances. This paper describes the development of a new biosorption based microbial biosensor for determination of Cu²⁺. The developed biosensor is based on carbon paste electrode consisting of whole cells of Circinella sp. Cu²⁺ was preconcentrated on the electrode surface at open circuit and then cathodically detected with the reduction of Cu²⁺. The voltammetric responses were evaluated with respect to percentage cell loading in the carbon paste, preconcentration time, pH of preconcentration solution, scan rate and interferences. The optimum response was realized by biosensor constructed using 5 mg of dry cell weight per 100 mg of carbon paste in pH 5.5 preconcentration solution. Under the optimum experimental conditions, the developed microbial biosensor exhibited an excellent current response to Cu²⁺ over a linear range from 5.0 × 10⁻⁷ to 1.0 × 10⁻⁵ M (r² = 0.9938) with a detection limit of 5.4 × 10⁻⁸ M (S/N = 3). The microbial biosensor had good sensitivity and reproducibility (R.S.D. 4.3%, n = 6). Finally, the applicability of the proposed microbial biosensor to voltammetric determination of Cu²⁺ in real sample was also demonstrated and validated with atomic absorption spectrophotometric (AAS) method.     

Direct electrochemistry of glucose oxidase on screen-printed electrodes through one-step enzyme immobilization process with silica sol–gel/polyvinyl alcohol hybrid film

A one-step enzyme immobilization process with silica sol–gel/polyvinyl alcohol was described to achieve direct electrochemistry of glucose oxidase on screen-printed electrodes. The immobilized glucose oxidase displays a couple of stable and well-defined redox peaks with an electron transfer rate constant of 8.38 s⁻¹ and a formal potential of −460 mV (versus SCE) in phosphate buffer (0.05 M, pH 7.0) at a scan rate of 300 mV s⁻¹. The results suggested that conformation and bioactivity of glucose oxidase could be retained efficiently using the proposed immobilization method and the porous structure of screen-printed electrode surface was helpful for electron communication of glucose oxidase and the electrode. Furthermore, the modified electrode was used as a glucose biosensor, exhibiting a linear response to glucose concentration ranging from 0 to 4.13 mM and a sensitivity of 3.47 μA mM⁻¹ cm⁻² at an applied potential of −0.5 V. The detection limit of the biosensor is 9.8 μM, based on a signal-to-noise ratio of 3. The present work provided a promising strategy for fabricating a novel and disposable mediatorless glucose biosensor, which could be mass-produced through further development.     

Direct electrochemistry and electrocatalysis with horseradish peroxidase immobilized in polyquaternium-manganese oxide nanosheet nanocomposite films

A novel biocompatible polyquaternium (QY)-manganese oxide nanosheet (MNS) nanocomposite has been prepared and shown to be a promising matrix for horseradish peroxidase (HRP) immobilization. The resulting HRP-QY-MNS film was characterized by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy, which indicated that HRP retained its native structure in the nanocomposite film. An HRP-QY-MNS film-modified glassy carbon electrode exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks centered at −0.272 V (vs. Ag/AgCl) in pH 7.0 phosphate buffer solution. The direct electrochemical behavior of HRP was greatly enhanced in the QY-MNS nanocomposite film compared with that in single-component QY or MNS films. The immobilized HRP showed excellent electrocatalysis in the reduction of hydrogen peroxide (H₂O₂), which was exploited in the construction of an H₂O₂ biosensor. The linear range of the biosensor for H₂O₂ was found to be from 1.0 × 10⁻⁷ to 3.2 × 10⁻⁵ M with a correlation coefficient of 0.998. The detection limit was 7.8 × 10⁻⁸ M at a signal-to-noise ratio of 3. The biosensor exhibited rapid response and good long-term stability.     

Direct electrochemistry of cytochrome c and biosensing for hydrogen peroxide on polyaniline grafted multi-walled carbon nanotube electrode

Cytochrome c (cyt c) was immobilized into a matrix consisting of polyaniline (PANI) and multi-walled carbon nanotubes (MWNT) by a new strategy. First, PANI chains were grafted onto MWNT through electropolymerization. Second, the amine groups in PANI chains were oxidized at an applied potential of +0.80 V to acquire positive charges that would effectively immobilize negatively charged cyt C. The ITO/MWNT-g-PANI(O)/cyt c electrode exhibited a pair of redox peaks with a peak potential separation (anodic to cathodic) of 0.25 V (vs Ag/AgCl) in 0.1 M phosphate buffer (pH 7.0). The results demonstrated that ITO/MWNT-g-PANI(O)/cyt c promoted direct electron transfer between cyt c and electrode with a high electron transfer rate constant (17 s⁻¹). The ITO/MWNT-g-PANI(O)/cyt c electrode catalyzes the reduction of H₂O₂. The ITO/MWNT-g-PANI(O)/cyt c biosensor displays an amperometric response to H₂O₂ with a linear concentration range from 0.5 μM to 1.5 mM (r = 0.99, n = 12), a high sensitivity (32.2 μAm M⁻¹) and fast response (9 s) and detection limit of 0.3 μM (S/N = 3).     

Modified glassy carbon electrode with multiwall carbon nanotubes as a voltammetric sensor for determination of noscapine in biological and pharmaceutical samples

A simple and highly sensitive method is described for direct voltammetric determination of noscapine in blood and pharmaceutical sample. Glassy carbon electrode with effective method is modified with multiwall carbon nanotubes (MWNTs) to cause activation of multiwall carbon nanotubes structures for electrocatalyzes of noscapine oxidation. The cyclic voltammetric (CV) results indicated that MWNTs remarkably enhances electrocatalytic activity toward the oxidation of noscapine, which is leading to considerable improvement of anodic peak current for noscapine, and allows the development of a highly sensitive voltammetric sensor for detection of noscapine in pharmaceutical and clinical samples. Under the optimum condition, the calibration curve was linear in the concentration range 4.0 × 10⁻⁷–1.0 × 10⁻⁴ mol L⁻¹ with the detection limit of 8.0 × 10⁻⁸ mol L⁻¹ and relative standard deviation (R.S.D.%) lower than 1.0% (n = 5). Finally, some kinetic parameters were determined and multistep mechanism for oxidation of noscapine for first time was proposed.     

Anodic stripping voltammetric determination of copper(II) using a functionalized carbon nanotubes paste electrode modified with crosslinked chitosan

The development and application of a functionalized carbon nanotubes paste electrode (CNPE) modified with crosslinked chitosan for determination of Cu(II) in industrial wastewater, natural water and human urine samples by linear scan anodic stripping voltammetry (LSASV) are described. Different electrodes were constructed using chitosan and chitosan crosslinked with glutaraldehyde (CTS-GA) and epichlorohydrin (CTS-ECH). The best voltammetric response for Cu(II) was obtained with a paste composition of 65% (m/m) of functionalized carbon nanotubes, 15% (m/m) of CTS-ECH, and 20% (m/m) of mineral oil using a solution of 0.05 mol L⁻¹ KNO₃ with pH adjusted to 2.25 with HNO₃, an accumulation potential of −0.3 V vs. Ag/AgCl (3.0 mol L⁻¹ KCl) for 300 s and a scan rate of 100 mV s⁻¹. Under these optimal experimental conditions, the voltammetric response was linearly dependent on the Cu(II) concentration in the range from 7.90 × 10⁻⁸ to 1.60 × 10⁻⁵ mol L⁻¹ with a detection limit of 1.00 × 10⁻⁸ mol L⁻¹. The samples analyses were evaluated using the proposed sensor and a good recovery of Cu(II) was obtained with results in the range from 98.0% to 104%. The analysis of industrial wastewater, natural water and human urine samples obtained using the proposed CNPE modified with CTS-ECH electrode and those obtained using a comparative method are in agreement at the 95% confidence level.     

A novel biosensor using entangled carbon nanotubes layer grown on an alumina substrate by CCVD of methane on FeOx–MgO

Glucose oxidase (GOx) was immobilized on entangled and high surface area carbon nanotubes (CNTs) grown on an alumina substrate, and direct electron transfer reaction between GOx and the electrode was revealed. Fe/MgO catalyst layer was spin-coated on the insulating alumina substrate and the CNT layer was grown on the catalyst by chemical vapor deposition of methane at 950 °C for 15 min. About 20–30 nm bundles of about 1 nm single-wall as well as 10 nm multiwall CNTs are formed. The redox process was surface-controlled and electron transfer coefficient and the rate constant were estimated to be 0.35 and 0.64 s⁻¹, respectively. In addition GOx immobilized on CNT layer showed a linear response range between 12 and 62 μM of glucose concentration. A detection limit and sensitivity of 0.1 μM and 635 μA mM⁻¹ cm⁻², respectively, were obtained for the biosensor.     

Molecular valve consisting of poly(acrylic acid) gel

A molecular valve, consisting of poly(acrylic acid) gel-coated Au mesh, was developed based on volume change of the gel in response to cation concentration. The valve closed when concentration of cations such as H⁺, Na⁺, K⁺, Ca²⁺, Cu²⁺, or Al³⁺ was low, whereas opened upon increase in its concentration. The valve re-closed when water was flowed. The concentration where the valve opens was found to increase in the order of Al³⁺, Ca²⁺, and Na⁺ (2 × 10⁻⁴, 5 × 10⁻⁴, and 6 × 10⁻³ M, respectively). The response to Cu²⁺ ion showed similar behaviour, but the opening concentration was ca. 2 × 10⁻⁴ M, which is lower than that of Ca²⁺ ion. The valve appeared to close over the pH range from 3 to 12, whereas to open below and above it. The fastest response time to open the valve (less than 1 min) was obtained for a solution of pH 1–2. The valve showed repeatability at least 25 cycles upon successive loading of a solution of pH 2 and water. Effects of anions and pressure were also studied.     

Potentiometric sensor based on molecularly imprinted polymer for determination of melamine in milk

A polymeric membrane ion-selective electrode for determination of melamine is described in this paper. It is based on a molecularly imprinted polymer (MIP) for selective recognition, which can be synthesized by using melamine as a template molecule, methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a cross-linking agent. The membrane electrode shows near-Nernstian response (54 mV/decade) to the protonated melamine over the concentration range of 5.0 × 10⁻⁶ to 1.0 × 10⁻² mol L⁻¹. The electrode exhibits a short response time of 16 s and can be stable for more than 2 months. Combined with flow analysis system, the potentiometric sensor has been successfully applied to the determination of melamine in milk samples. Interference from high concentrations of ions co-existing in milk samples such as K⁺ and Na⁺ can be effectively eliminated by on-line introduction of anion- and cation-exchanger tandem columns placed upstream, while melamine existing as neutral molecules in milk of pH 6.7 can flow through the ion-exchanger columns and be measured downstream by the proposed electrode in an acetate buffer solution of pH 3.7.     

Electrochemical sensor for hydroperoxides determination based on Prussian blue film modified electrode

An electrochemical sensor for hydroperoxides determination was investigated. The sensor was based on the electrocatalytic reduction of hydroperoxides on Prussian blue (PB)-modified glassy carbon electrode. The modified electrode possesses a high electrocatalytic effect towards all studied peroxides with the highest effect obtained with H₂O₂ followed by tert-butyl hydroperoxide (TBH), cumene hydroperoxide (CH) and linoleic acid hydroperoxide (LAH). In addition, the modified electrode showed a good stability and a fast response time (<20 s). The lower detection limits of H₂O₂, TBH, CH and LAH were found to be 10⁻⁷ mol L⁻¹, 2 × 10⁻⁷ mol L⁻¹, 3.5 × 10⁻⁷ mol L⁻¹ and 4 × 10⁻⁷ mol L⁻¹, respectively. The electrochemical sensor was then applied for amperometric determination of peroxide value (PV) in edible oil at an applied potential of 50 mV (vs. Ag/AgCl (1 M KCl)). A good linearity has been found in the range 0.02–1.0 mequiv. O₂/kg, with a detection limit (S/N = 3) of 0.001 mequiv. O₂/kg. The precision of the method (R.S.D., n = 9) for within and between-days is better than 1.9% and 2.7%, respectively at 0.1 mequiv. O₂/kg. The method was successfully applied to the determination of PV in real edible oil samples with an excellent agreement with results obtained with the official standard procedure. The proposed method is accurate, simple, cheap and could be used to control edible oil rancidity with a high sample throughputs (more than 120 samples/h).     

Direct amperometric determination of l-ascorbic acid (Vitamin C) at octacyanomolybdate-doped-poly(4-vinylpyridine) modified electrode in fruit juice and pharmaceuticals

The glassy carbon electrode (GCE) modified with Mo(CN)₈⁴⁻-incorporated-poly(4-vinylpyridine) (PVP/Mo(CN)₈⁴⁻), which has been recently shown to possess several attractive attributes as an efficient electrocatalytic electrode for l-ascorbic acid oxidation and its estimation, is used for l-ascorbic acid estimation directly in orange fruit juice and Celin tablet in a 0.1 M H₂SO₄ acid solution without any special treatment. Constant potential amperometry at 570 mV (saturated calomel electrode, SCE) in stirred solutions is used for this purpose. A good correlation is attained with the official titrametric method. To understand the possible electrocatalytic reaction mechanism for the electro-oxidation of l-ascorbic acid, calibration graphs over the range 1 × 10⁻⁵ to 1 × 10⁻² mol dm⁻³ l-ascorbic acid are compared for the three electrodes, ca. PVP/Mo(CN)₈⁴⁻, undoped PVP, and GCE; the curvature at high ascorbic acid concentration for the PVP/Mo(CN)₈⁴⁻ electrode is explained in terms of Michaelis–Menten (MM) saturation kinetics. The apparent MM constant (K_M), the maximum catalytic current (i_M), the complex decomposition rate constant (k_c), and the heterogeneous modified electrode rate constant (k^′_ME) are calculated from three different approaches. A reasonably high value of ≈1 × 10⁻² cm s⁻¹ is obtained for k^′_ME, indicating efficient l-ascorbic acid mediation at the PVP/Mo(CN)₈⁴⁻ electrode, thus accounting for quite a high sensitivity of this modified film electrode compared to several other modified electrodes.     

Surface-modified silicon nano-channel for urea sensing

Silicon nano-channels have been surface functionalized with the enzyme urease for biosensor applications to detect and quantify urea concentration. The device is nanofabricated from a silicon-on-insulator (SOI) wafer with a top down lithography approach. The differential conductance of silicon nano-channels can be tuned for optimum performance using the source drain bias voltage, and is sensitive to urea at low concentration. The experimental results show a linear relationship between surface potential change and urea concentration in the range of 0.1–0.68 mM. The sensitivity of our devices shows high reproducibility with time and different measurement conditions. The nano-channel urea biosensor offers the possibility of high quality, reusable enzyme sensor array integration with silicon-based circuits.     

Amperometric phenol biosensor based on polyaniline

The catechol biosensor is constructed by cross-linking between polyphenol oxidase (PPO) and polyaniline (PANI) using glutaraldehyde as a cross-linking agent. The PANI, which is electrochemically synthesized in a solution containing ionic liquid, 1-ethyl-3-methylimidazolium ethyl sulfate, possesses good electroactivity and high conductivity above pH 6. In the presence of catechol as a substrate, the biosensor exhibits a linear range from 0.2 to 80 μmol dm⁻³. The maximum response current (I_max) and the Michaelis–Menten constant (k^′_m) are 9.44 μA and 117 μmol dm⁻³, respectively. The effects of pH and operating potential are also explored to optimize measurement conditions. The activation energy (E_a) of the PPO catalytic reaction is 30.23 kJ mol⁻¹ in the B–R buffer. Electrochemical impedance spectroscopy (EIS), UV–vis and SEM are used to characterize the PANI–PPO biosensor. The biosensor exhibits good long-term stability.     

Application of carbon ionic liquid electrode for the electrooxidative determination of catechol

A carbon ionic liquid electrode (CILE) was constructed using graphite powder mixed with N-butylpyridinium hexafluorophosphate (BPPF₆) in place of paraffin as the binder, which showed strong electrocatalytic activity to the direct oxidation of catechol. In pH 3.0 phosphate buffer solution (PBS) a pair of redox peaks appeared on the CILE with the anodic and the cathodic peak potential located at 387 and 330 mV (vs. SCE), respectively. The electrochemical behaviors of catechol on the CILE were carefully investigated, and the electrochemical parameters were calculated with the results of the electrode reaction standard rate constant k_s as 1.27 s⁻¹, the charge-transfer coefficient α as 0.58 and the electron transferred number n as 2. Under the selected conditions, the anodic peak current increased linearly with the catechol concentration over the range from 1.0 × 10⁻⁶ to 8.0 × 10⁻⁴ mol L⁻¹ by cyclic voltammetry at the scan rate of 100 mV s⁻¹. The detection limit was calculated as 6.0 × 10⁻⁷ mol L⁻¹ (3σ). The CILE showed good ability to separate the electrochemical responses of catechol and ascorbic acid (AA) with the anodic peak potential separation as 252 mV (vs. SCE). The proposed method was further applied to the synthetic samples determination with satisfactory results.     

Adsorption of glucose oxidase at platinum-multiwalled carbon nanotube-alumina-coated silica nanocomposite for amperometric glucose biosensor

Glucose oxidase (GOx) has been immobilized in platinum-multiwalled carbon nanotube-alumina-coated silica (Pt-MWCNT-ACS) nanocomposite modified glassy carbon electrode by adsorption to provide a novel amperometric glucose biosensor. The morphology, nature, and performance of the resulting GOx-Pt-MWCNT-ACS nanobiocomposite modified glassy carbon electrode were characterized by field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, cyclic voltammetry, and amperometry. The influence of various experimental conditions was examined for the determination of the optimum analytical performance. The optimized glucose biosensor displayed a wide linear range of up to 10.5 mM, a high sensitivity of 113.13 mA M⁻¹ cm⁻², and a response time of less than 5 s. The sensitivity for the determination of glucose at the GOx-Pt-MWCNT-ACS nanobiocomposite modified glassy carbon electrode is better than at common GOx-Pt-CNT nanobiocomposite modified electrodes. The proposed biosensor has good anti-interferent ability and long-term storage stability after coating with Nafion, and it can be used for the determination of glucose in synthetic serum.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-κ-NN′]dichlormanganous and DNA

A new Mn(II) complex of MnL₂Cl₂ (L = azino-di(5,6-azafluorene)-κ²-NN′) was synthesized and utilized as an electrochemical indicator for the determination of hepatitis B virus (HBV) based on its interaction with MnL₂Cl₂. The electrochemical behavior of interaction of MnL₂Cl₂ with salmon sperm DNA was investigated on glassy carbon electrode (GCE). In the presence of salmon sperm DNA, the peak current of [MnL₂]²⁺ was decreased and the peak potential was shifted positively without appearance of new peaks. The binding ratio between [MnL₂]²⁺ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 3.72 × 10⁸ mol² L⁻². The extent of hybridization was evaluated on the basis of the difference between signals of [MnL₂]²⁺ with probe DNA before and after hybridization with complementary sequence. Control experiments performed with non-complementary and mismatch sequence demonstrated the good selectivity of the biosensor. With this approach, a sequence of the HBV could be quantified over the range from 1.76 × 10⁻⁸ to 1.07 × 10⁻⁶ mol L⁻¹, with a linear correlation of r = 0.9904 and a detection limit of 6.80 × 10⁻⁹ mol L⁻¹. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between MnL₂Cl₂ and DNA was found to be primary intercalation binding.     

Direct electron transfer of myoglobin in mesoporous silica KIT-6 modified on screen-printed electrode

A disposable hydrogen peroxide biosensor was developed based on the direct electron transfer of myoglobin (Mb) on mesopores KIT-6 modified screen-printed electrode (SPE) which was manually performed to fabricate the planar carbon electrodes. KIT-6 is a new material which can absorb abundant of Mb molecules. A mixture of Mb and KIT-6 was immobilized with nafion on electrode. The cyclic voltammetry experiment indicated that a pair of stable and well-defined reduction peaks with a formal potentials of −0.35, and −0.28 V versus saturated calomel electrode (SCE) was obtained, using the present modified electrode in phosphate buffer saline (0.05 M, pH 7.0) at scan rate of 100 mV s⁻¹, characteristic of Mb heme Fe(III)/Fe(II) redox couple. The heterogeneous electron transfer rate constant k_s was estimated to be 16.93 s⁻¹. And the formal potential was pH-dependent, having two slopes of −54.7 and −49.3 mV/pH which illustrated one electron transfer. This modified electrode was applied to detect H₂O₂ with sensitivity of 55.68 mA M⁻¹ cm⁻². Infrared spectrum and UV–vis absorption spectra of immobilized Mb film were recorded. In conclusion, KIT-6 increases the electron transfer activity of Mb and this kind of H₂O₂ biosensor is low cost for using disposable.