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.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

Electrodeposition of gold nanoparticles on indium/tin oxide electrode for fabrication of a disposable hydrogen peroxide biosensor

A novel disposable third-generation hydrogen peroxide (H₂O₂) biosensor based on horseradish peroxidase (HRP) immobilized on the gold nanoparticles (AuNPs) electrodeposited indium tin oxide (ITO) electrode is investigated. The AuNPs deposited on ITO electrode were characterized by UV-vis, SEM, and electrochemical methods. The AuNPs attached on the ITO electrode surface with quasi-spherical shape and the average size of diameters was about 25 nm with a quite symmetric distribution. The direct electron chemistry of HRP was realized, and the biosensor exhibited excellent performances for the reduction of H₂O₂. The amperometric response to H₂O₂ shows a linear relation in the range from 8.0 μmol L⁻¹ to 3.0 mmol L⁻¹ and a detection limit of 2 μmol L⁻¹ (S/N = 3). The value of HRP immobilized on the electrode surface was found to be 0.4 mmol L⁻¹. The biosensor indicates excellent reproducibility, high selectivity and long-term stability.     

Amperometric hydrogen peroxide biosensor based on the immobilization of heme proteins on gold nanoparticles-bacteria cellulose nanofibers nanocomposite

A novel matrix, gold nanoparticles-bacterial cellulose nanofibers (Au-BC) nanocomposite was developed for enzyme immobilization and biosensor fabrication due to its unique properties such as satisfying biocompatibility, good conductivity and extensive surface area, which were inherited from both gold nanoparticles (AuNPs) and bacterial cellulose nanofibers (BC). Heme proteins such as horseradish peroxidase (HRP), hemoglobin (Hb) and myoglobin (Mb) were successfully immobilized on the surface of Au-BC nanocomposite modified glassy carbon electrode (GCE). The immobilized heme proteins showed electrocatalytic activities to the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ), which might be due to the fact that heme proteins retained the near-native secondary structures in the Au-BC nanocomposite which was proved by UV-vis and IR spectra. The response of the developed biosensor to H₂O₂ was related to the amount of AuNPs in Au-BC nanocomposite, indicating that the AuNPs in BC network played an important role in the biosensor performance. Under the optimum conditions, the biosensor based on HRP exhibited a fast amperometric response (within 1 s) to H₂O₂, a good linear response over a wide range of concentration from 0.3 μM to 1.00 mM, and a low detection limit of 0.1 μM based on S/N = 3. The high performance of the biosensor made Au-BC nanocomposite superior to other materials as immobilization matrix.     

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.     

Mesoporous silica hollow sphere (MSHS) for the bioelectrochemistry of horseradish peroxidase

In this work, novel mesoporous silica hollow spheres (MSHS) were chosen as an immobilization matrix, to construct a mediator-free third-generation HRP biosensor. UV-vis spectroscopy revealed that horseradish peroxidase (HRP) entrapped in MSHS could retain its native structure. FTIR spectroscopy and nitrogen adsorption-desorption isotherms indicated that HRP are intercalated into the mesopores. The direct electron transfer of HRP entrapped in MSHS was observed. A pair of stable and well-defined redox peaks of HRP with a formal potential of about −0.150 V (vs. Ag/AgCl) in 0.1 M pH 7.0 phosphate-buffered solution (PBS) were obtained. The biosensor exhibited a fast amperometric response to H₂O₂ with a linear range of 3.9 × 10⁻⁶ to 1.4 × 10⁻⁴ M (R = 0.997, N = 20). The detection limit was 1.2 × 10⁻⁶ M based S/N = 3.     

One-step construction of reagentless biosensor based on chitosan-carbon nanotubes-nile blue-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of novel reagentless biosensors by in situ formation of chitosan-carbon nanotubes-nile blue-horseradish peroxidase (CS-CNTs-NB-HRP) biocomposite film on electrode surface. The mediator effect of NB, conducting performance of CNTs and the biocompatible microenvironment of CS were combined by such one-step non-manual process. NB could interact with CNTs and resulted in good dispersion of CNTs-NB nanocomposites in aqueous solution. Cyclic voltammetry measurements demonstrated that electrons were efficiently shuttled between HRP and the electrode mediated by NB. The developed reagentless biosensor exhibited a fast amperometric response for the determination of H₂O₂ and 95% of the steady-state current was obtained within 2 s. The linear response of the reagentless biosensor for the determination of H₂O₂ ranged from 1.0 × 10⁻⁶ to 2.4 × 10⁻⁴ mol l⁻¹ with a detection limit of 1.2 × 10⁻⁷ mol l⁻¹. The biosensor exhibited high reproducibility and long-time storage stability. The as-prepared biosensor also showed effective anti-interference capability. The ease of the one-step non-manual technique and the promising feature of the biocomposite could serve as a versatile platform for fabricating electrochemical biosensors.     

Bilayer lipid membrane biosensor with enhanced stability for amperometric determination of hydrogen peroxide

In this paper, a polydopamine (PDA) film is electropolymerized on the surface of bilayer lipid membrane (BLM) which is immobilized with horseradish peroxidase (HRP). The coverage of the PDA film on HRP/BLM electrode is monitored by electrochemical impedance spectroscopy (EIS). The electrocatalytic reduction of H₂O₂ at the PDA/HRP/BLM electrode is studied by means of cyclic voltammetry (CV). The biosensor has a fast response to H₂O₂ of less than 5 s and an excellent linear relationship is obtained in the concentration range from 2.5 × 10⁻⁷ to 3.1 × 10⁻³ mol L⁻¹, with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The response current of BLM/HRP/PDA biosensor retains 84% of its original response after being stored in 0.1 mol L⁻¹ pH 7.0 PBS at 4 °C for 3 weeks. The selectivity, repeatability, and storage stability of PDA/HRP/BLM biosensor are greatly enhanced by the coverage of polydopamine film on BLM.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

Hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on ��-Al2O3 nanoparticles/chitosan film-modified electrode

An amperometric biosensor based on horseradish peroxidase (HRP) and ??-Al₂O₃/chitosan composite film at a glassy carbon electrode has been developed. Hydrogen peroxide (H₂O₂) was detected with the aid of ferrocene monocarboxylic acid mediator to transfer electrons between the electrode and HRP. The morphology and composition of the modified electrode were characterized by scanning electron microscopy and electrochemical impedance spectroscopy. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and amperometry. The effects of HRP concentration, the applied potential, and the pH values of the buffer solution on the response of the sensor were investigated for optimum analytical performance. The proposed biosensor showed high sensitivity (0.249?A M^?1?cm^?2) and a fast response (<5?s) to H₂O₂ with the detection limit of 0.07???M. The linear response range of the enzyme electrode to H₂O₂ concentration was from 0.5 to 700???M with a correlation coefficient of 0.9998. The apparent Michaelis-Menten constant of the biosensor was calculated to be 0.818?mM, exhibiting a high enzymatic activity and affinity for H₂O₂.     

Hydrogen peroxide biosensor based on hemoglobin modified zirconia nanoparticles-grafted collagen matrix

A novel method for the immobilization of hemoglobin (Hb) and preparation of reagentless biosensor was proposed using a biocompatible non-toxic zirconia enhanced grafted collagen tri-helix scaffold. The formed membrane was characterized with UV-vis and FT-IR spectroscopy, scanning electron microscope and electrochemical methods. The Hb immobilized in the matrix showed excellent direct electrochemistry with an electron transfer rate constant of 6.46 s⁻¹ and electrocatalytic activity to the reduction of hydrogen peroxide. The apparent Michaelis-Menten constant for H₂O₂ was 0.026 mM, showing good affinity. Based on the direct electrochemistry, a new biosensor for H₂O₂ ranging from 0.8 to 132 μM was constructed. Owing to the porous structure and high enzyme loading of the matrix the biosensor exhibited low limit of detection of 0.12 μM at 3σ, fast response less than 5 s and high sensitivity of 45.6 mA M⁻¹ cm⁻². The biosensor exhibited acceptable stability and reproducibility. ZrO₂-grafted collagen provided a good matrix for protein immobilization and biosensing preparation. This method was useful for monitoring H₂O₂ in practical samples with the satisfactory results.     

A biosensor based on urate oxidase-peroxidase coupled enzyme system for uric acid determination in urine

A new amperometric biosensor based on urate oxidase-peroxidase coupled enzyme system for the specific and selective determination of uric acid in urine was developed. Commercially available urate oxidase and peroxidase were immobilized with gelatin by using glutaraldehyde and fixed on a pretreated teflon membrane. The method is based on generation of H₂O₂ from urine uric acid by urate oxidase and its consuming by peroxidase and then measurement of the decreasing of dissolved oxygen concentration by the biosensor. The biosensor response depends linearly on uric acid concentration between 0.1 and 0.5 μM. In the optimization studies of the biosensor, phosphate buffer (pH 7.5; 50 mM) and 35 °C were obtained as the optimum working conditions. In addition, the most suitable enzyme activities were found as 64.9×10⁻³ U cm⁻² for urate oxidase and 512.7 U cm⁻² for peroxidase. And also some characteristic studies of the biosensor such as reproducibility, substrate specificity and storage stability were carried out.     

Enhancement of a conducting polymer-based biosensor using carbon nanotube-doped polyaniline

A biosensor with improved performance was developed through the immobilization of horseradish peroxidase (HRP) onto electropolymerized polyaniline (PANI) films doped with carbon nanotubes (CNTs). The effects of electropolymerization cycle and CNT concentration on the response of the biosensor toward H₂O₂ were investigated. It was found that the application of CNTs in the biosensor system could increase the amount and stability of the immobilized enzyme, and greatly enhanced the biosensor response. Compared with the biosensor without CNTs, the proposed biosensor exhibited enhanced stability and approximately eight-fold sensitivity. A linear range from 0.2 to 19 μM for the detection of H₂O₂ was observed for the proposed biosensor, with a detection limit of 68 nM at a signal-to-noise ratio of 3 and a response time of less than 5 s.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

An amperometric biosensor using toluidine blue as an electron transfer mediator intercalated in α-zirconium phosphate-modified horseradish peroxidase immobilization matrix

A novel H₂O₂ biosensor was constructed employing α-zirconium phosphate as a new support substrate to hold an electron shuttle toluidine blue between a glassy carbon electrode and horseradish peroxidase. Toluidine blue was intercalated into α-zirconium phosphate-modified horseradish peroxidase immobilization matrix cross-linked on a glassy carbon electrode surface via bovine serum albumin-glutaraldehyde. This co-immobilization matrix of the mediator and the enzyme was formed from the α-zirconium phosphate (α-ZrP)-toluidine blue (TB) inclusion colloid in which horseradish peroxidase (HRP) was dissolved. Intercalation of TB in layered α-ZrP was investigated by scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and electrochemical measurements. TB immobilized in this way underwent a quasi-reversible electrochemical redox reaction at the electrode. Cyclic voltammetry and amperometric measurements demonstrated good stability and efficiently-shuttled electrons between HRP and the electrode. The sensor responded rapidly to H₂O₂ with a detection limit of 3.0 × 10^–7 mol/L.     

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.     

An H2O2 Biosensor Based on Immobilization of Horseradish Peroxidase Labeled Nano‐Au in Silica Sol‐Gel/Alginate Composite Film

Abstract

A novel approach to assemble an H₂O₂ amperometric biosensor was introduced. The biosensor was constructed by entrapping horseradish peroxidase (HRP) labeled nano‐scaled particulate gold (nano‐Au) (HRP‐nano‐Au electrostatic composite) in a new silica sol‐gel/alginate hybrid film using glassy carbon electrode as based electrode. This suggested strategy fully merged the merits of sol‐gel derived inorganic‐organic composite film and the nano‐Au intermediator. The silica sol‐gel/alginate hybrid material can improve the properties of conventional sol‐gel material and effectively prevent cracking of film. The entrapment of HRP in the form of HRP‐nano‐Au can not only factually prevent the leaking of enzyme out of the film but also provide a favorable microenvironment for HRP. With hydroquinone as an electron mediator, the proposed HRP electrode exhibited good catalytic activity for the reduction of H₂O₂. The parameters affecting both the qualities of sol‐gel/alginate hybrid film and the biosensor response were optimized. The biosensor exhibited high sensitivity of 0.40 Al mol^?1 cm^?2 for H₂O₂ over a wide linear range of concentration from 1.22×10^?5 to 1.46×10^?3 mol L^?1, rapid response of <5 s and a detection limit of 0.61×10^?6 mol L^?1. The enzyme electrode has remarkable stability and retained 86% of its initial activity after 45 days of storage in 0.1 mol L^?1 Tris‐HCl buffer solutions at pH 7.     

Analytical Biosensing of Hydrogen Peroxide on Brilliant Cresyl Blue/Multiwalled Carbon Nanotubes Modified Glassy Carbon Electrode

A cationic quinine‐imide dye brilliant cresyl blue (BCB) and horseradish peroxidase (HRP) were co‐immobilized within ormosil on multiwalled carbon nanotubes modified glassy carbon electrode for the fabrication of highly sensitive and selective hydrogen peroxide biosensor. The presence of epoxy group in ormosil as organic moiety improves the mechanical strength and transparency of the film and amino group provides biocompatible microenvironment for the immobilization of enzyme. The presence of MWCNTs improved the conductivity of the nanocomposite film. The surface characterization of MWCNT modified ormosil nanocomposite film was performed with scanning electron microscopy (SEM) and atomic force microscopy (AFM). 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 1.5 mM. The proposed H₂O₂ biosensor exhibited wide linear range from 3×10^?7 to 1×10^?4 M, and low detection limit 1×10^?7 M (S/N=3) with fast response time <5 s. The probable interferences in bio‐matrix were selected to test the selectivity and no significant response was observed in the biosensor. This biosensor possessed good analytical performance and long term storage stability.     

An Amperometric Biosensor Based on the Coimmobilization of Horseradish Peroxidase and Methylene Blue on a Carbon Nanotubes Modified Electrode

A novel hydrogen peroxide biosensor has been constructed based on the characteristics of the carbon nanotube. The multiwall carbon nanotube (MWNT) was used as a coimmobilization matrix to incorporate horseradish peroxidase (HRP) and electron transfer mediator methylene blue (MB) onto a glassy carbon electrode surface. Cyclic voltammetry and amperometric measurements were employed to demonstrate the feasibility of methylene blue as an electron carrier between the immobilized peroxidase and the surface of glassy carbon electrode. The amperometric response of this resulting biosensor to H₂O₂ shows a linear relation in the range from 4 μM to 2 mM. The detection limit was 1 μM when the signal to noise ratio is 3. The presence of dopamine and ascorbic acid hardly affects the sensitive determination of H₂O₂. This biosensor also possesses very good stability and reproducibility.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a β-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the co-immobilization of horseradish peroxidase (HRP) and methylene blue on a β-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H₂O₂ in the linear range from 2.5 × 10^–6 to 4.0 × 10^–3 M with a detection limit of 0.3 μM. The sensor was stable in continuous operation.