Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H(2)O(2). The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H(2)O(2) in the linear range from 2.6 x 10(-6) mol/L to 8.8 x 10(-3) mol/L with a detection limit of 6.4 x 10(-7) mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

A mediator-free amperometric hydrogen peroxide biosensor based on HRP immobilized on a nano-Au/poly 2,6-pyridinediamine-coated electrode

A mediator-free amperometric hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) enzyme on colloidal Au modified platinum (Pt) wire electrode, which was modified by poly 2,6-pyridinediamine (pPA). The modified process was characterized by electrochemical impedance spectroscopy (EIS), and the electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The biosensor displayed an excellent electrocatalytical response to reduction of H₂O₂ without the aid of an electron mediator, the linear range was 4.2 × 10⁻⁷–1.5 × 10⁻³ mol/L (r = 0.9977), with a detection limit of 1.4 × 10⁻⁷ mol/L. Moreover, the performance and factors influencing the resulted biosensor were studied in detail. The studied biosensor exhibited permselectivity, good stability and good fabrication 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.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

Amperometric glucose biosensor based on single-walled carbon nanohorns

The biosensing application of single-walled carbon nanohorns (SWCNHs) was demonstrated through fabrication of an amperometric glucose biosensor. The biosensor was constructed by encapsulating glucose oxidase in the Nafion-SWCNHs composite film. The cyclic voltammograms for glucose oxidase immobilized on the composite film displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of -0.453 V. The biosensor had good electrocatalytic activity toward oxidation of glucose. To decrease detection potential, ferrocene monocarboxylic acid was used as a redox mediator. The mediated glucose biosensor shows a linear range from 0 to 6.0 mM. The biosensor shows high sensitivity (1.06 microA/mM) and stability, and can avoid the commonly coexisted interference. Because of impressive properties of SWCNHs, such as high purity and high surface area, SWCNHs and their composites are expected to be promising material for biomolecular immobilization and biosensing applications.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.     

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.     

Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film

The composite film based on Nafion and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of ionic liquid (IL)-Mb interaction by ultraviolet-visible (UV-vis) spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peaks with a formal potential (E_o^’) of −0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0 to 180 μM with a detection limit of 0.14 μM at a signal/noise ratio of 3. The apparent Michaelis constant () for the electrocatalytic reaction was 22.6 μM. The stability, repeatability, and selectivity of the sensor were evaluated. The proposed biosensor has a lower detection limit than many other IL-heme protein-based biosensors and is free from common interference in hydrogen peroxide biosensors.     

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.     

Amperometric sensor based on ferrocene-modified multiwalled carbon nanotube nanocomposites as electron mediator for the determination of glucose

A kind of nanocomposite with good dispersion in water was prepared through covalent adsorption of ferrocenecarboxaldehyde on multiwalled carbon nanotubes (MWNTs) for electrical communication between glucose oxidase (GOD) and electrode. The ferrocene-modified multiwalled carbon nanotube nanocomposites (MWNTs-Fc) could be conveniently cast on electrode surfaces. With the aid of chitosan, GOD was then immobilized on the nanostructure film to form a reagentless amperometric sensor for glucose determination. FTIR spectra and cyclic voltammetry were used to characterize the nanocomposites. The presence of both ferrocene as mediator of electron transfer and MWNTs as conductor enhanced greatly the enzymatic response to the oxidation of glucose. The novel biosensor exhibited a fast response toward glucose with a detection limit of 3.0 × 10⁻⁶ mol/L and the linear range extended up to 3.8 × 10⁻³ mol/L.     

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 third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode

A new amperometric biosensor, based on adsorption of glucose oxidase (GOD) at the platinum nanoparticle-modified carbon nanotube (CNT) electrode, is presented in this article. CNTs were grown directly on the graphite substrate. The resulting GOD/Pt/CNT electrode was covered by a thin layer of Nafion to avoid the loss of GOD in determination and to improve the anti-interferent ability. The morphologies and electrochemical performance of the CNT, Pt/CNT, and Nafion/GOD/Pt/CNT electrodes have been investigated by scanning electron microscopy, cyclic voltammetry, and amperometric methods. The excellent electrocatalytic activity and special three-dimensional structure of the enzyme electrode result in good characteristics such as a large determination range (0.1-13.5mM), a short response time (within 5s), a large current density (1.176 mA cm(-2)), and high sensitivity (91mA M(-1)cm(-2)) and stability (73.5% remains after 22 days). In addition, effects of pH value, applied potential, electrode construction, and electroactive interferents on the amperometric response of the sensor were investigated and discussed. The reproducibility and applicability to whole blood analysis of the enzyme electrode were also evaluated.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

A novel nitrite biosensor based on the direct electron transfer of hemoglobin immobilized on CdS hollow nanospheres

A novel nitrite biosensor based on the direct electron transfer of hemoglobin (Hb) immobilized on CdS hollow nanospheres (HS-CdS) modified glassy carbon electrode was constructed. The direct electron transfer of Hb showed a pair of redox peaks with a formal potential of -286 mV (vs. SCE) in 0.1M pH 7.0 phosphate buffer solution. It was a surface-controlled electrode process involving a single proton transfer coupled with a reversible one-electron transfer for each heme group of Hb. HS-CdS had a large specific surface area and good biocompatibility and had a better electrochemical response than that of solid spherical CdS. The immobilized Hb on HS-CdS displayed an excellent response to NO(2)(-) with one irreversible electrode process for NO reduction. Under optimal conditions, the biosensor could be used for the determination of NO(2)(-) with a linear range from 0.3 to 182 microM and a detection limit of 0.08 microM at 3 sigma based on the irreversible reduction of NO. HS-CdS provided a good matrix for protein immobilization and had a promising application in constructing sensors.     

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 based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

Electrochemical properties of heme-protein in lauric acid films and its application as a biosensor

In this research, the enhancement of electron-transfer activity of hemoglobin (Hb) in lauric acid film was investigated for the first time. This type of composite film was made on a glassy carbon electrode by a casting method. Cyclic voltammetric result of the modified electrode displays a well-defined redox peak, which was attributed to the direct electrochemical response of Hb. Our results illustrate that Hb exchange electrons directly with electrode and exhibits the characteristics of peroxidase. When we use this modified electrode as a biosensor, it gives excellent performance in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). The parameters such as pH and applied potential of the biosensor influencing in H₂O₂ detection were optimized carefully. Through the optimal conditions, the proposed biosensor shows the linear range for H₂O₂ determination was from 1×10⁻⁵ to 1.25×10⁻⁴ mol L⁻¹ with a detection limit of 1×10⁻⁷ mol L⁻¹. The biosensor retained more than 90% of the initial response after 14 d.