Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Polyaniline based catalase biosensor for the detection of hydrogen peroxide and azide

The CAT/PANi/ITO bioelectrode has been prepared as a catalase biosensor and shows response for monitoring not only of H₂O₂ but also azide. The sensor exhibited an excellent response to the H₂O₂ and azide. The linear range of H₂O₂ was 0.064∼1 mM and for azide 0.125∼4 mM, respectively. Catalase biosensor was based on the principle of the measurements as the decrease in the differentiation of oxygen level, which has been caused by the inhibition of catalase in the bioactive layer of the biosensor by azide. The repeatability experiments were done in triplicate. The logarithm response of the biosensor to H₂O₂ (r² = 0.99), as well as, for azide (r² = 0.90) were reported, respectively. The bioelectrode was characterized by CV and AFM. The proposed biosensor would be applied for the determination of H₂O₂ and azide in various biological samples.     

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H(2)O(2). The pH effect on amperometric response to H(2)O(2) was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.     

Gold-coated silica-fiber hybrid materials for application in a novel hydrogen peroxide biosensor

We describe the preparation and characterization of a novel type of core-shell hybrid material for application in a novel hydrogen peroxide biosensor, where the structure consists of a continuous gold shell that encapsulates the silica fiber. The SiO(2)@Au nanofibers had been synthesized by electrospinning silica sol, and then golden seeds were in situ grown on the fiber, lastly the gold-seeded silica fibers were further coated by continuous gold shells. The above nanocomposites had satisfactory chemical stability, excellent biocompatibility and efficient electron transfer property, which may have potential application for the highly sensitive chemical or biological sensors. Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of the SiO(2)@Au nanocomposites at indium tin oxide (ITO). The biosensor showed high sensitivity and fast response upon the addition of H(2)O(2) and the linear range to H(2)O(2) was from 5×10(-6) to 1.0×10(-3)M with a detection limit of 2 μM (S/N=3). The apparent Michaelis-Menten constant of the biosensor was 1.11 mmol L(-1). These results indicated that SiO(2)@Au nanocomposites have potential for constructing of a variety of electrochemical biosensors.     

A microbial biosensor for hydrogen sulfide monitoring based on potentiometry

In this study, a microbial biosensor for hydrogen sulfide (H₂S) detection based on Thiobacillus thioparus immobilized in a gelatin matrix was developed. The T. thioparus was immobilized via either surface adsorption on the gelatin matrix or entrapment in the matrix. The bacterial and gelatin concentration in the support were then varied in order to optimize the sensor response time and detection limit for both methods. The optimization was conducted by a statistical analysis of the measured biosensor response with various bacterial and polymer concentrations. According to our experiments with both immobilization methods, the optimized conditions for the entrapment method were found to be a gelatin concentration of 1% and an optical density of 82. For the surface adsorption method, 0.6% gelatin and an optical density of 88 were selected as the optimal conditions. A statistical model was developed based on the extent of the biosensor response in both methods of immobilization. The maximum change in the potential of the solution was 23.16 mV for the entrapment method and 34.34 mV for the surface absorption method. The response times for the entrapment and adsorption methods were 160 s and 105 s, respectively. The adsorption method is more advantageous for the development of a gas biosensor due to its shorter response time.     

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.     

Amperometric biosensor for the detection of hydrogen peroxide using catalase modified electrodes in polyacrylamide

A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.     

Fabrication of hydrogen peroxide biosensor based on Ni doped SnO2 nanoparticles

Ni doped SnO(2) nanoparticles (0-5 wt%) have been prepared by a simple microwave irradiation (2.45 GHz) method. Powder X-ray diffraction (XRD) and transmission electron microscopy (TEM) studies confirmed the formation of rutile structure with space group (P(42)/mnm) and nanocrystalline nature of the products with spherical morphology. Direct electrochemistry of horseradish peroxidase (HRP)/nano-SnO(2) composite has been studied. The immobilized enzyme retained its bioactivity, exhibited a surface confined, reversible one-proton and one-electron transfer reaction, and had good stability, activity and a fast heterogeneous electron transfer rate. A significant enzyme loading (3.374×10(-10) mol cm(-2)) has been obtained on nano-Ni doped SnO(2) as compared to the bare glassy carbon (GC) and nano-SnO(2) modified surfaces. This HRP/nano-Ni-SnO(2) film has been used for sensitive detection of H(2)O(2) by differential pulse voltammetry (DPV), which exhibited a wider linearity range from 1.0×10(-7) to 3.0×10(-4)M (R=0.9897) with a detection limit of 43 nM. The apparent Michaelis-Menten constant (K(M)(app)) of HRP on the nano-Ni-SnO(2) was estimated as 0.221 mM. This excellent performance of the fabricated biosensor is attributed to large surface-to-volume ratio and Ni doping into SnO(2) which facilitate the direct electron transfer between the redox enzyme and the surface of electrode.     

A novel enzymatic hydrogen peroxide biosensor based on Ag/C nanocables

A novel enzymatic hydrogen peroxide sensor was successfully fabricated based on the nanocomposites containing of Ag/C nanocables and gold nanoparticles (AuNPs). Ag/C nanocables have been synthesized by a hydrothermal method and then AuNPs were assembled on the surface of Ag/C nanocables. The nanocomposites were confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS). The above nanocomposites have satisfactory chemical stability and excellent biocompatibility. Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of the Ag/C/Au nanocomposites at glassy carbon electrode (GCE). The results indicated that the Ag/C/Au nanocomposites exhibited excellent electrocatalytic activity to the reduction of H(2)O(2). It offered a linear range of 6.7×10(-9) to 8.0×10(-6) M, with a detection limit of 2.2×10(-9) M. The apparent Michaelis-Menten constant of the biosensor was 51.7×10(-6) M. These results indicated that Ag/C/Au nanocomposites have potential for constructing of a variety of electrochemical biosensors.     

Amperometric hydrogen peroxide biosensor based on covalently immobilizing thionine as a mediator

A novel amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on the 2,6-pyridinedicarboxylic acid (PDC) polymer, thionine and nano-Au was successfully fabricated. In this strategy, PDC polymer acted as the matrices to covalently immobilize the thionine, and then hemoglobin was successfully adsorbed on the nano-Au which was electro-deposited on to thionine modified electrode surface. The preparation process of modified electrode was characterized with electrochemical impedance spectroscopy and atomic force microscope. The analytical performance of proposed biosensor toward H₂O₂ was investigated by cyclic voltammetry and chronoamperometry. The resulted biosensor exhibited fast amperometric response (within 6 s) to H₂O₂, and linear range was from 9.1 μM to 5.0 mM with the detection limit of 2.6 μM (S/N = 3). The apparent Michaelis–Menten constant (K _M^app) was evaluated to be 3.2 mM. Furthermore, the resulted biosensor showed good stability and reproducibility.     

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.     

Immobilization of hemoglobin on zirconium dioxide nanoparticles for preparation of a novel hydrogen peroxide biosensor

Direct electrochemistry and thermal stability of hemoglobin (Hb) immobilized on a nanometer-sized zirconium dioxide (ZrO2) modified pyrolytic graphite (PG) electrode were studied. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of (7.90 +/- 0.93)s(-1) and a formal potential of -0.361 V (-0.12 V versus NHE) in 0.1M pH 7.0 PBS. Both nanometer-sized ZrO2 and dimethyl sulfoxide (DMSO) could accelerate the electron transfer between Hb and the electrode. Spectroscopy analysis of the Hb/ZrO2/DMSO film showed that the immobilized Hb could retain its natural structure. This modified electrode showed a high thermal stability up to 74 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the aid of an electron mediator. The electrocatalytic response showed a linear dependence on the H2O2 concentration ranging from 1.5 to 30.2 microM with a detection limit of 0.14 microM at 3sigma. The apparent Michaelis-Menten constant KMapp for H2O2 sensor was estimated to be (0.31 +/- 0.02) mM, showing a high affinity.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

Amperometric biosensor for hydrogen peroxide based on horseradish peroxidase onto gold nanowires and TiO2 nanoparticles

An electrochemical biosensor for determination of hydrogen peroxide (H₂O₂) was fabricated, based on the electrostatic immobilization of horseradish peroxidase (HRP) with one-dimensional gold nanowires (Au NWs) and TiO₂ nanoparticles (nano-TiO₂) on a gold electrode. The nano-TiO₂ can give a biocompatible microenvironment and compact film, and the Au NWs can provide fast electron transferring rate and greatly add the amount of HRP molecules immobilized on the electrode surface. Au NWs were characterized by ultraviolet–visible spectra and transmission electron microscope. The electrode modification process was probed by cyclic voltammetry and electrochemical impedance spectroscopy. Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. Under optimal conditions, the linear range for the determination of H₂O₂ was from 2.3 × 10⁻⁶ to 2.4 × 10⁻³ M with a detection limit of 7.0 × 10⁻⁷ M (S/N = 3). Moreover, the proposed biosensor showed superior stability and high sensitivity.     

Hemoglobin niobate composite based biosensor for efficient determination of hydrogen peroxide in a broad pH range

Inorganic layered niobates (HCa2Nb3O10) were used as immobilization matrices of hemoglobin (Hb) because of their tunable interlayer spaces, large surface areas and good biocompatibilities. A pair of well-defined, quasi-reversible cycle voltammertric peaks were obtained at the Hb-HCa2Nb3O10 modified pyrolytic graphite electrode, suggesting that the layered niobates facilitate the electron transfer between the proteins and the electrode. Hb-HCa2Nb3O10 modified electrode exhibited electrocatalytic response for monitoring H2O2 with a large linear detection range from 25 microM to 3.0 mM and a relatively high sensitivity of 172 microA mM-1 cm-2. Based on the stabilizing effect of the layered niobates, Hb-HCa2Nb3O10 modified electrode can detect H2O2 in strongly acidic and basic solutions with pH of 1-12, which greatly expands the application fields of biosensors.     

HRP-based biosensor for monitoring rifampicin

Pyrrole was electropolymerized onto a Pt electrode in the presence of LiClO(4) and horseradish peroxidase (HRP). This HRP-based biosensor has been used for the amperometric detection of rifampicin (RIF) in the presence of a constant concentration of H(2)O(2). The C(H(2)O(2)) as well as the applied potential (E(ap)) and the pH of the phosphate buffer have simultaneously been optimized through a central composite design. Under these conditions, repeatability, reproducibility, and stability of the modified electrode have been analyzed. The detection limit for RIF has been calculated taking into account the probability of false-positive (alpha) and -negative (beta), reaching a value of 5.06x10(-6) mol dm(-3). The biosensor was applied to the determination of RIF in pharmaceutical preparations and biological samples.     

A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.     

Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite

A novel amperometric biosensor for hydrogen peroxide (H(2)O(2)) was developed by entrapping horseradish peroxidase (HRP) in a new ormosil composite doped with ferrocene monocarboxylic acid-bovine serum albumin conjugate and multiwall carbon nanotubes (MWNTs). The ormosil was prepared using 3-(aminopropyl)triethoxysilane and 2-(3,4 epoxycyclohexyl)-ethyltrimethoxy silane as monomers. The encapsulated conjugate showed excellent electrochemistry and acted as an electron transfer mediator. The presence of MWNTs improved the conductivity of the composite film. This matrix showed a biocompatible microenvironment for retaining the native activity of the entrapped HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H(2)O(2). The proposed H(2)O(2) biosensor exhibited a linear range of 0.02-4.0 mM with a detection limit of 5.0 microM (S/N = 3) and a K(M)(app) value of 2.0 mM. It could be used for flow injection analysis of hydrogen peroxide with a liner range from 0.02 to 4.5 mM, sensitivity of 0.042 microA/mM and analytical time of 20 s per sample. This biosensor possessed good analytical performance and storage stability.     

Immobilizing Pt nanoparticles and chitosan hybrid film on polyaniline naofibers membrane for an amperometric hydrogen peroxide biosensor

A convenient and effective way for fabricating amperometric hydrogen peroxide (H₂O₂) biosensor was designed in this paper. First, the polyaniline (PANI) nanofibers membrane with good conductance and high surface area was electropolymerized on a gold electrode surface. Then, Pt nanoparticle (PtNP) was electrochemically deposited on the PANI nanofibers membrane. Finally, the hybrid film of gold nanoparticle, chitosan, and horseradish peroxidase (HRP) was cast onto the modified electrode to form a stable biofunctional film, which was also employed as a protective layer to PtNP. The proposed biosensor exhibited a rapid response to H₂O₂ with the linear range from 7.0 × 10⁻⁶ to 1.4 × 10⁻² M and a detection limit of 2.8 × 10⁻⁶ M (S/N = 3). The sensitivity of 558 μA mM⁻¹ cm⁻² was obtained. The Michaelis–Menten constant, K_\textM^\textapp K_{\text{M}}^{\text{app}} value was 1.90 mM suggesting a high affinity. Moreover, it displayed a good reproducibility and long-term stability.