Amperometric biosensor for hydrogen peroxide based on the co-electrodeposition of horseradish peroxidase and methylene blue on an ITO electrode modified with an anodic aluminum oxide template

We report on a nano-array sensor for hydrogen peroxide (H₂O₂) that is based on a nanoporous anodic aluminum oxide template. This was used as a matrix for the co-immobilization of horseradish peroxidase (HRP) and methylene blue (MB) on the surface of an indium tin oxide electrode. The immobilized HRP retained its natural activity and MB is capable of efficiently shuttle electrons between HRP and the electrode. The new electrode was characterized by SEM and electrochemical methods. It exhibits fast response, long-term stability, high sensitivity and good selectivity to H₂O₂. Under optimized conditions, it linearly responds to H₂O₂ in the concentration range from 1.0?μM to 26?mM, with a detection limit of 0.21?μM (at S/N?=?3).

Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

Amperometric hydrogen peroxide biosensor based on a glassy carbon electrode modified with polythionine and gold nanoparticles

We report on a novel hydrogen peroxide biosensor that was fabricated by the layer-by-layer deposition method. Thionine was first deposited on a glassy carbon electrode by two-step electropolymerization to form a positively charged surface. The negatively charged gold nanoparticles and positively charged horseradish peroxidase were then immobilized onto the electrode via electrostatic adsorption. The sequential deposition process was characterized using electrochemical impedance spectroscopy by monitoring the impedance change of the electrode surface during the construction process. The electrochemical behaviour of the modified electrode and its response to hydrogen peroxide were studied by cyclic voltammetry. The effects of the experimental variables on the amperometric determination of H₂O₂ such as solution pH and applied potential were investigated for optimum analytical performance. Under the optimized conditions, the biosensor exhibited linear response to H₂O₂ in the concentration ranges from 0.20 to 1.6?mM and 1.6 to 4.0?mM, with a detection limit of 0.067?mM (at an S/N of 3). In addition, the stability and reproducibility of this biosensor was also evaluated and gave satisfactory results.

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H₂O₂) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM^?1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.

A silica-dextran nanocomposite as a novel matrix for immobilization of horseradish peroxidase, and its application to sensing hydrogen peroxide

We report on a novel matrix of solgel organic–inorganic nanocomposite that was fabricated from silica sol gel and dextran. It was used for the immobilization of horseradish peroxidase (HRP) to give a biosensor for hydrogen peroxide (H₂O₂). The sensor film was characterized by Fourier transform infrared and UV–vis spectroscopy with respect to structural features and the conformation of the enzyme. The topographies of the surface of the electrode were investigated by field emission scanning electron microscopy. The biosensor was used to determine H₂O₂ quantitatively in the presence of Methylene blue as a mediator with high electron transfer efficiency. A pair of stable and well defined quasi-reversible redox peaks of the HRP [Fe (III)]/HRP [Fe (II)] redox couple was observed at pH 7.0. The biosensor responds to H₂O₂ in the 0.5 mM to 16.5 mM concentration range, and the limit of detection is 0.5 mM.

A hydrogen peroxide biosensor based on direct electron transfer from hemoglobin to an electrode modified with Nafion and activated nanocarbon

A biosensor for hydrogen peroxide (HP) was developed by immobilizing hemoglobin on a glassy carbon electrode modified with activated carbon nanoparticles/Nafion. The characteristics of the sensor were studied by UV?Cvis spectroscopy and electrochemical methods. The immobilized Hb retained its native secondary structure, undergoes direct electron transfer (with a heterogeneous rate constant of 3.37?±?0.5?s^?1), and displays excellent bioelectrocatalytic activity to the reduction of HP. Under the optimal conditions, its amperometric response varies linearly with the concentration of HP in the range from 0.9???M to 17???M. The detection limit is 0.4???M (at S/N?=?3). Due to the commercial availability and low cost of activated carbon nanoparticles, it can be considered as a useful supporting material for construction of other third-generation biosensors.

Synthesis of multi-branched gold nanoparticles by reduction of tetrachloroauric acid with Tris base, and their application to SERS and cellular imaging

A biosensor for hydrogen peroxide was constructed by immobilizing horseradish peroxidase on chitosan-wrapped NiFe₂O₄ nanoparticles on a glassy carbon electrode (GCE). The electron mediator carboxyferrocene was also immobilized on the surface of the GCE. UV?Cvis spectra, Fourier transform IR spectra, scanning electron microscopy, and electrochemical impedance spectra were acquired to characterize the biosensor. The experimental conditions were studied and optimized. The biosensor responds linearly to H₂O₂ in the range from 1.0?×?10^?5 to 2.0?×?10^?3?M and with a detection limit of 2.0?×?10^?6?M (at S/N?=?3).

A biopolymer-based carbon nanotube interface integrated with a redox shuttle and a D-sorbitol dehydrogenase for robust monitoring of D-sorbitol

We describe the preparation and characterization of a glassy carbon electrode modified with a bionanocomposite consisting of a hyaluronic acid, dispersed carbon nanotubes, and electrostatically bound toluidine blue. The electrode was used to detect NADH in the batch and flow-injection mode of operation. The electrode was further modified by immobilizing sorbitol dehydrogenase to result in biosensor for D-sorbitol that displays good operational stability, a sensitivity of 10.6???A?mM^?1?cm^?2, a response time of 16?s, and detection limit in the low micromolar range. The biosensor was successfully applied to off-line monitoring of D-sorbitol during its bioconversion into L-sorbose (a precursor in the synthesis of vitamin C) by Gluconobacter oxydans. The sample assay precision is 2.5% (an average RSD) and the throughput is 65?h^?1 if operated in the flow-injection mode. The validation of this biosensor against a reference HPLC method resulted in a slope of correlation of 1.021?±?0.001 (R ²?=?0.99997).

Electrochemistry and biosensing activity of cytochrome c immobilized in macroporous materials

An amperometric biosensor for hydrogen peroxide (H₂O₂) has been constructed by immobilizing cytochrome c on an indium/tin oxide (ITO) electrode modified with a macroporous material. Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of cytochrome c proceeds without the need for an electron mediator. A surface-controlled electron transfer process can be observed with an apparent heterogeneous electron-transfer rate constant (k_s) of 29.2?s^?1. The biosensor displays excellent electrocatalytic responses to the reduction of H₂O₂ to give amperometric responses that increase steadily with the concentration of H₂O₂ in the range from 5???M to 2?mM. The detection limit is 0.61???M at pH?7.4. The apparent Michaelis-Menten constant (K_m) of the biosensor is 1.06?mM. This investigation not only provided a method for the direct electron transfer of cytochrome c on macroporous materials, but also established a feasible approach for durable and reliable detection of H₂O₂.

Reagentless amperometric glucose biosensor based on the immobilization of glucose oxidase on a ferrocene@NaY zeolite composite

Ferrocene (Fc) was encapsulated in the cavities of a NaY zeolite by vapor diffusion via sublimation at below 100?°C. The resulting Fc@NaY zeolite composite was investigated by power X-ray diffraction, diffuse reflectance UV?Cvis and FT-IR spectroscopy, and by cyclic voltammetry. The results indicated that Fc was encapsulated into the zeolite whose microporous structure had remained intact. The Fc in the silica matrix had retained its electroactivity and did not leach out. A glucose biosensor was obtained by immobilization of the modified zeolite and glucose oxidase on a carbon paste electrode. It displays a linear response to glucose (from 0.8???M to 4.0?mM), a detection limit of 0.2???M, and a response time of 4?s. The good performance of the biosensor is ascribed to the biocompatibility of the zeolite and presence of Fc which facilitates the electron transfer from the enzyme to the surface of the electrode.

Simple construction of an enzymatic glucose biosensor based on a nanocomposite film prepared in one step from iron oxide, gold nanoparticles, and chitosan

The one-step synthesis is reported of a nanofilm composed of iron oxide and gold nanoparticles in a chitosan matrix that can act as a novel matrix for the immobilization of glucose oxidase (GOx) to fabricate a glucose biosensor. The use for the composite film strongly increased the effective electrode surface for loading of GOx. The size and shape of the iron oxide nanoparticles were examined by transmission electron micrograph. Direct electron transfer and electrocatalysis by GOx was investigated via cyclic voltammetry and chronoamperometry. Under optimized conditions, the biosensor has a response time of 6?s and a linear response in the range between 3???M and 0.57?mM of glucose, with a detection limit of 1.2???M at a signal-to-noise ratio of 3. This novel and disposable mediatorless glucose biosensor may form the basis for a future mass-produced glucose biosensor.

Preparation and characterization of methylene blue-SDS- multiwalled carbon nanotubes nanocomposite for the detection of hydrogen peroxide

A nanocomposite was prepared by physical adsorption of?(cationic) methylene blue (MB) on (anionic) sodium dodecylsulfate (SDS) that was wrapped on multiwalled carbon nanotubes (MWCNTs) on the surface of a glassy carbon electrode. This electrostatic interaction enables electrical communication between the electrode and analyte. Horseradish peroxidase was then immobilized in a film of gelatin on the nanocomposite to form a biosensor for hydrogen peroxide. Scanning electron microscopy, transmission electron microscopy, Fourier transform infrared and UV?Cvis spectrometry, and cyclic voltammetry were applied to characterize the electrode. The addition of both MWCNTs and MB causes a synergistic effect and leads to a large signal enhancement. The prepared nanocomposite material modified sensor shows better response in presence of several interferences. The biosensor has detection limit of 5 nM of hydrogen peroxide (at S/N?=?3) with a linear response between 0.2???M and 1.4?mM. Its lifetime is >4?months under dry conditions at 4?°C.

Sensitive detection of hydrogen peroxide in foodstuff using an organic�Cinorganic hybrid multilayer-functionalized graphene biosensing platform

We report on a new electrochemical biosensing strategy for the sensitive detection of hydrogen peroxide (H₂O₂) in foodstuff samples. It is based on a gold electrode modified with layer of graphene patterned with a multilayer made from an organic?Cinorganic hybrid nanomaterial. Initially, a layer of thionine (Th) was assembled on the surface of the graphene nanosheets, and these were then cast on the surface of the electrode for the alternate assembly of gold nanoparticles and horseradish peroxidase. The large surface-to-volume ratio and high conductivity of the nanosheets provides a benign microenvironment for the construction of the biosensor. The use of such a multilayer not only shortens the electron transfer pathway of the active center of the enzyme due to the presence of gold nanoparticles, but also enhances the electrocatalytic efficiency of the biosensor toward the reduction of H₂O₂. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The number of layers, the operating potential, and the pH of the supporting electrolyte were optimized. Linear response is obtained for the range from 0.5???M to 1.8?mM of H₂O₂, the detection limit is 10 nM (at S/N?=?3), and 95% of the steady-state current is reached within 2?s. The method was applied to sense H₂O₂ in spiked sterilized milk and correlated excellently with the permanganate titration method.

Centri-voltammetry for biosensing systems: biocentri-voltammetric xanthine detection

Centri-voltammetry is a method that combines centrifugation and voltammetry. This method, developed by our group in 2003, yielded promising results when applied to trace analysis of metal ios. We demonstrate here the first application of centri-voltammetry to biosensing systems. A xanthine biosensor was constructed by immobilizing xanthine oxidase on a planar platinum electrode which then was placed at the bottom of a centri-voltammetric cell. The experimental parameters were optimized to give two linear ranges. The first is from 0.1 to 1???M, and the second from 5 to 50???M. The RSD is 3.4 (n?=?5). The biosensor was applied to the determination of xanthine in wine and in urine. Calculated recoveries are 101?±?0.61% (n?=?3) for wine samples, and 102?±?0.556% (n?=?3) for urine samples.

Fluorescent sensing of nitrite at nanomolar level using functionalized mesoporous silica

Aminopyrene was covalently anchored onto mesoporous silica through serial post-grafting to obtain a fluorescent solid that can be used as a sensing material for the determination of nitrite. The latter, in acidic medium, reacts with the secondary amino groups on the material to form a non-fluorescent nitroso derivative. Based on the fluorescence quenching caused by this specific reaction, a method was developed for the determination of nitrite at nanomolar levels. The range for detection of nitrite in 1.5?mol.L^?1 HCl is linear between 1.50?nM to 0.45???M and 0.45???M to 2.22???M, the detection limit being 1.10?nM and 0.307???M respectively at an S/N of 3.

Optical choline sensor based on a water-soluble fluorescent conjugated polymer and an enzyme-coupled assay

We report on a simple and sensitive water-soluble fluorescent conjugated polymer for use in a choline biosensor. Choline is oxidized by the enzyme choline oxidase (ChOx), and the hydrogen peroxide (H₂O₂) formed is used to oxidize catechol via catalysis by horseradish peroxidase. The product of oxidation acts as a quencher of the photoluminescence of a fluorescent conjugated polymer. The ratio of the fluorescence intensity of the system in the presence and absence of the choline, respectively, serves as the analytical information. It is proportional to the concentration of choline in the 0.1 μM to 20 μM concentration range. The detection limit for choline is 50 nM. The biosensor was successfully applied to the determination of choline in milk samples with satisfactory reproducibility and accuracy. This is the first biosensor where a ChOx/HRP enzyme-coupled assay is used in combination with a water-soluble conjugated polymer for the fluorescent detection of choline. In our opinion, it provides a common platform for further development of enzymatic biosensors based on fluorescent conjugated polymers.

Carbon paste electrode modified with a binuclear manganese complex as a sensitive voltammetric sensor for tryptophan

We report on a carbon paste electrode that was modified with a binuclear manganese(II) complex by the drop-coating method. A study on the mechanism of the electro-oxidation of tryptophan (Trp) at this electrode indicated that it enables Trp to be determined with good sensitivity and selectivity. Second-order derivative linear sweep voltammetry at pH 4.1 revealed that a sensitive anodic peak appears at 812?mV (vs. SCE) whose current is proportional to the concentration of Trp in the concentration range from 0.1 to 1.0???mol?L^?1 and 1.0 to 80???mol?L^?1, with a detection limit (S/N?=?3) of 0.08???mol?L^?1 (60?s of accumulation). The method was applied to the determination of Trp in amino acid injection solutions with satisfactory results.

Biosensor based on a glassy carbon electrode modified with tyrosinase immmobilized on multiwalled carbon nanotubes

We describe a biosensor for phenolic compounds that is based on a glassy carbon electrode modified with tyrosinase immobilized on multiwalled carbon nanotubes (MWNTs). The MWNTs possess excellent inherent electrical conductivity which enhances the electron transfer rate and results in good electrochemical catalytic activity towards the reduction of benzoquinone produced by enzymatic reaction. The biosensor was characterized by cyclic voltammetry, and the experimental conditions were optimized. The cathodíc current is linearly related to the concentration of the phenols between 0.4???M and 10???M, and the detection limit is 0.2???M. The method was applied to the determination of phenol in water samples.

Electrochemical oxidation of p-nitrophenol using graphene-modified electrodes, and a comparison to the performance of MWNT-based electrodes

The electrochemical oxidation of p-nitrophenol (p-NP) has been studied comparatively on a graphene modified electrode and a multiwall carbon nanotube (MWNT) electrode by using cyclic and differential pulse voltammetry. The sensors were fabricated by modifying screen-printed electrodes with graphene and MWNT nanomaterials, respectively, both dispersed in Nafion polymer. p-NP is irreversibly oxidized at +0.9?V (vs. the Ag/AgCl) in solutions of pH 7. The height and potential of the peaks depend on pH in the range from 5 to 11. In acidic media, p-NP yields a well-defined oxidation peak at +0.96?V which gradually increases in height with the concentration of the analyte. In case of differential pulse voltammetry in sulfuric acid solution, the sensitivity is practically the same for both electrodes. The modified electrodes display an unusually wide linear response (from 10???M to 0.62?mM of p-NP), with a detection limit of 0.6???M in case of the graphene electrode, and of 1.3???M in case of the MWNT electrode.

Direct electrochemistry of horseradish peroxidase based on hierarchical porous calcium phosphate microspheres

Biomorphic calcium phosphate (CaP) microspheres with hierarchical porous structure were synthesized using natural cole pollen grains as templates and were further employed for the immobilization of horseradish peroxidase (HRP). Scanning electron microscopy and Fourier transform infrared spectroscopy revealed (a) the porous structure of the CaP microspheres, (b) the effective immobilization, and (c) the retention of the conformation of HRP on CaP. The immobilized HRP was placed on a glassy carbon electrode where it underwent a direct, fully reversible, and surface-controlled redox reaction with an electron transfer rate constant of 1.96 s^?1. It also exhibits high sensitivity to the reduction of H₂O_2. The response to H₂O₂ is linear in the 5.00 nM to 1.27 μM concentration range, and the sensitivity is 30357 μA?mM^?1?cm^?2. The detection limit (at an SNR of 3) is as low as 1.30 nM. The apparent Michaelis–Menten constant (K _M ^app ) of the immobilized enzyme is 0.92 μM. This new CaP with hierarchical porous structure therefore represents a material that can significantly promote the direct electron transfer between HRP and an electrode, and is quite attractive with respect to the construction of biosensors.