Use of a ruthenium(III), iron(II), and nickel(II) hexacyanometallate-modified graphite electrode with immobilized oxalate oxidase for the determination of urinary oxalate.

This paper describes the performance of a biosensor with an Ru(III), Ni(II), and Fe(II) hexacyanometallate-modified graphite electrode and immobilized oxalate oxidase for the determination of urinary oxalate. The addition of ruthenium enhances the electrochemical reversibility and chemical stability of the electrocrystallized layer and improves the sensitivity of the biosensor. Hydrogen peroxide, produced by the enzyme-catalyzed oxidation of oxalate, was measured at -50 mV vs an Hg Hg2CI2 3M KCl electrode in a solution of pH 3.6 succinic buffer, 0.1 M KCl, and 5.4mM ethylenediaminetetraacetic acid. The linear concentration range for the determination of oxalate was 0.18-280 microM. The recoveries of added oxalate (10-35 microM) from aqueous solution ranged from 99.5 to 101.7%, whereas from urine samples without oxalate (or with a concentration of oxalate below the detection limit) the recoveries of added oxalate ranged from 91.4 to 106.6%. The oxalate in 24 h urine samples, taken during their daily routine from 35 infants and children, was measured and found to range from 0.6 to 121.7 mg/L. There were no interferences from uric acid, acetylsalicylic acid, and urea in the concentration range investigated, but paracetamol and ascorbic acid did interfere. A good correlation (R2 = 0.9242) was found between values obtained for oxalate in real urine samples by 2 laboratories, with the proposed biosensor and ion chromatography, respectively.     

A novel biamperometric biosensor for urinary oxalate determination using flow-injection analysis

A biosensor for determination of oxalate concentration in urine has been developed by immobilisation of oxalate oxidase and peroxidase on the surface of an interdigitated gold electrode. Enzyme immobilisation was performed using BSA and glutaraldehyde. Biamperometric measurements were made in flow conditions both in aqueous oxalate solutions (tested concentration range between 50 μM and 10 mM) and in real urine samples (tested measuring range between 5 and 100 μM). Optimal working conditions were examined for flow-injection analysis, and good correlation was achieved between added oxalate quantity and the one measured by biosensor in urine matrix (R² = 0.9983). The influence of some interferences (ascorbic acid, uric acid, paracetamol, acetylsalicylic acid) was also studied using biamperometric measurement mode.     

Comparative study of potentiometric and amperometric tissue-based electrodes for oxalate

Acetone-precipitated pulp from banana skins is physicall entrapped at the tip of a carbon dioxide gas-sensor and on a hydrogen peroxide sensor probe to determine oxalate potentiometrically and amperometrically in aqueous solution and inurine. The enzyme present in the tissue is oxalate oxidase. The potentiometric response has a slope of 47–50 mV/decade for 1 × 10^?4 M–2 × 10^?3 M oxalate with a detection limit of 2 × 10^?5 M. The amperometric response is linear for 2 × 10^t-5–3 × 10^?4 M oxalate with a dectection limit of 2 × 10^?6 M. Average recoveries of oxalate added to aqueous samples were 96.2% and 98.0%, and average relative standrd deviations were 3.8% and 3.6% for the potentiometric and amperometric systems, respectively. Oxalate was determined in six control urine samples, with relative errors of about 2.5%, by both electrode systems after a simple clean-up.     

Sensitive Flow-Injection Electrochemical Determination of Hydrogen Peroxide at a Palladium Nanoparticle-Modified Pencil Graphite Electrode

A novel flow-injection amperometric method was proposed for the sensitive and enzymeless determination of hydrogen peroxide based on its electrocatalytic reduction at a palladium nanoparticle-modified pretreated pencil graphite electrode in a laboratory-constructed electrochemical flow cell. Cyclic voltammograms of the unmodified and modified electrodes were recorded in pH 7.0 phosphate buffer containing 0.10 M KCl at a scan rate of 50?mV s^?1 for the investigation of electrocatalytic reduction of hydrogen peroxide at the palladium nanoparticle-modified pretreated pencil graphite electrode. Cyclic voltammograms of the pretreated pencil graphite electrode revealed an irreversible oxidation peak and a weak reduction peak of hydrogen peroxide at +1100?mV and –450?mV vs. an Ag/AgCl/KCl saturated reference electrode. However, the reduction of hydrogen peroxide was observed at –100?mV with an increase in current in the cyclic voltammograms of the palladium nanoparticle-modified pretreated pencil graphite electrode compared to the unmodified electrode. These results indicate that the palladium nanoparticle-modified pretreated pencil graphite electrode exhibits efficient electrocatalytic activity for the reduction of hydrogen peroxide. A linear concentration range was obtained between .01 and 10.0?mM hydrogen peroxide with a detection limit of 3.0 µM from flow injection amperometric current–time curves recorded in pH 7.0 phosphate buffer at –100?mV and a 2.0?mL min^?1 flow rate. The novelty of this work relies on its use of a laboratory-constructed flow cell constructed for the pencil graphite electrode using these inexpensive, disposable, and electrochemically reactive modified electrodes for the amperometric determination of hydrogen peroxide in a flow injection analysis system.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

Amperometric biosensor for oxalate determination in urine using sequential injection analysis

An amperometric flow biosensor for oxalate determination in urine samples after enzymatic reaction with oxalate oxidase immobilized on a modified magnetic solid is described. The solid was magnetically retained on the electrode surface of an electrode modified with Fe (III)-tris-(2-thiopyridone) borate placed into a sequential injection system preceding the amperometric detector. The variables involved in the system such as flow rate, aspired volumes (modified magnetic suspension and sample) and reaction coil length were evaluated using a Taguchi parameter design. Under optimal conditions, the calibration curve of oxalate was linear between 3.0-50.0 mg·L^-1, with a limit of detection of 1.0 mg·L^-1. The repeatability for a 30.0 mg·L^-1 oxalate solution was 0.7%. The method was validated by comparing the obtained results to those provided by the spectrophotometric method; no significant differences were observed.     

Electroimmobilization of MAO into a Polypyrrole Film and Its Utilization for Amperometric Flow Detection of Antidepressant Drugs

The preparation of a biosensor based on the enzymatic immobilization in polypyrrole polymer for the detection of antidepressant drugs is described. The enzyme monoamine oxidase (MAO) was immobilized by electropolymerization of pyrrole around a platinum electrode, at a constant potential of +0.75 V (vs. Ag/AgCl) in such a way to obtain a membrane thickness, which was constant and equal to 100 mC/cm². The biosensor was obtained from a 0.1 M KCl saline solution containing pyrrole at a concentration equal to 0.4 M and 2.5 mU/mL of MAO. The biosensor was adapted to a continuous flow injection analysis system (FIA) with the amperometric detection of hydrogen peroxide produced by enzymatic reaction carried out at a potential of +0.7 V (vs. Ag/AgCl), pH 7.4 and temperature of 37 °C. In optimized flow conditions, the biosensor presented an analytical response for fluoxetine in the interval between 0.67 and 4.33 mM, with a detection limit of 0.10 mM. The analytical use of the biosensor developed was evaluated through analysis of commercial pharmaceutical products containing fluoxetine, available on the Portuguese market. The amperometric flow results obtained do not differ significantly from the values resulting from analysis of the same products by the method proposed by the US Pharmacopeia, with sampling rates of 20–25 samples/hour.     

Amperometric Biosensor for Hydrogen Peroxide,Using Supramolecularly Immobilized Horseradish Peroxidase on the β‐Cyclodextrin‐Coated Gold Electrode

Horseradish peroxidase, previously modified with 1‐adamantane moieties, was supramolecularly immobilized on gold electrodes coated with perthiolated β‐cyclodextrin. The functionalized electrode was employed for the construction of an amperometric biosensor device for hydrogen peroxide using 1 mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (6 s) and a good linear response toward H₂O₂ concentration between 12 μM and 450 μM. The biosensor showed a sensitivity of 1.02 mA/M cm², and a very low detection limit of 5 μM. The electrode retained 97% of its initial electrocatalytic activity after 30 days of storage at 4 ⁰C in 50 mM sodium phosphate buffer, pH 7.0.     

Enzyme electrode system for oxalate determination utilizing oxalate decarboxylase immobilized on a carbon dioxide sensor

A highly selective enzyme electrode system for oxalate is described in which the enzyme oxalate decarboxylase is immobilized on a carbon dioxide gas-sensing electrode. The response of the system is linear with the logarithm of the oxalate concentration between 2 × 10^-4 and I × 10^-2 M with a slope of 57–60 mV/decade. The oxalate detection limit is 4 × 10^-5 M. Electrodes used with chemically immobilized enzyme are not affected by phosphate and sulfate at levels normally found in urine and are very stable showing no decrease in response after one month of operation. The enzyme electrode system functions well in urine, requiring minimal sample pretreatment. The recovery of oxalate added to five aliquots of a human control urine sample averaged 97.7% with an average relative standard deviation of 4.5%.     

An electrochemiluminescent biosensor for uric acid based on the electrochemiluminescence of bis-[3,4,6-trichloro-2-(pentyloxycarbonyl)-phenyl] oxalate on an ITO electrode modified by an electropolymerized nickel phthalocyanine film

Nickel(ii) tetrasulfophthalo-cyanine (NiTSPc) could be electrodeposited onto an indium tin oxide (ITO) electrode to form an electropolymerization film of NiTSPc. The electrochemiluminescent (ECL) behavior of bis-[3,4,6-trichloro-2-(pentyloxy-carbonyl)-phenyl] oxalate (BTPPO) on this modified electrode was investigated in detail. The emission of ECL of BTPPO can be greatly enhanced by hydrogen peroxide at this modified electrode. Thus, a new ECL biosensor for uric acid was developed based on the enzymatic reaction of uric acid in the presence of uricase to produce H(2)O(2). The developed sensor has been used to detect uric acid in real serum samples, and the results compared well with those obtained by the routine method.     

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.     

基于等离子体聚合膜固定酶的H2O2生物传感器

以玻碳电极为基础电极,用微波等离子体技术聚合沉积聚乙二胺等离子体膜,使之形成带氨基功能团的表面,再通过戊二醛交联共价固定辣根过氧化物酶,制得H2O2生物传感器.探讨了等离子体聚合膜的形成条件(如放电功率、单体流速、单体气压和聚合时间),讨论了工作电位、介体浓度和pH值对传感器响应的影响.此外,用红外光谱对等离子体聚合膜进行了表征.该传感器在5×10-7～1.1×10-3mol/LH2O2浓度范围内有线性响应,最低检测限为0.3μmol/L.将此传感器用于实际试样回收率的测定,结果良好.     

Determination of oxalic acid in urine by co-electroosmotic capillary electrophoresis with amperometric detection

Co-electroosmotic capillary electrophoresis with amperometric detection at a Cobalt phthalocyanine (CoPC) modified carbon paste electrode was evaluated for the determination of oxalic acid in urine. The running buffer consisted of 10 mM phosphate (pH=5.70) and 0.25 mM Cetyltrimethylammonium bromide. Under the optimum conditions, a detection limit of 0.12 muM was achieved for oxalic acid. The response was linear between 0.5 and 1000 muM with a correlation coefficient of 0.9995. Applications of the method to real urine samples were described.     

Reagentless enzyme electrode based on phenothiazine mediation of horseradish peroxidase for subnanomolar hydrogen peroxide determination

The development and characterization of a highly sensitive enzyme immobilized carbon based electrode for the determination of subnanomolar concentrations of hydrogen peroxide in aqueous samples is described. The biosensor consists of horseradish peroxidase (HRP) immobilized in solid carbon paste along with a suitable redox mediator. The latter allows the acceleration of the electroreduction of HRP in the presence of hydrogen peroxide. Several phenothiazines as mediators are investigated in a comparative manner and with respect to dimethylferrocene using cyclic voltammetry and amperometry. Insolubilization of the HRP in the solid carbon paste is achieved by cross-linking the enzyme with glutaraldehyde and bovine serum albumin. Several experimental parameters such as pH, mediator and enzyme content are considered. The hydrogen peroxide determination is better carried out in 0.1 M acetate buffer, pH 4.5, by amperometry at an applied potential of 0.0 V versus Ag/AgCl, 3 M NaCl concentration and by using the phenothiazine base as redox mediator. The biosensor response is linear over the concentration range 2 nM-10 microM with a detection limit of 1 nM. The linear range of the hydrogen peroxide response without a mediator in the biosensor is found between 2 and 40 microM. The biosensor can be used for more than 180 measurements. Additional modification of the electrode by incorporation of Nafion SAC-13 microparticles in the solid carbon paste allows detection of concentrations of hydrogen peroxide as low as 0.1 nM.     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Covalently Immobilized on Electrografted Organic Film on Screen‐Printed Carbon Electrode

A new convenient strategy to fabricate a third‐generation hydrogen peroxide biosensor was described. The screen‐printed carbon electrode (SPCE) was first modified with a layer of 4‐nitrophenyl assembled from the 4‐nitroaniline diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were converted to amines followed by crosslinking to the horseradish peroxidase (HRP) by glutaraldehyde. The redox chemistry of the active center of the HRP was observed and the HRP‐modified electrode displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. H₂O₂ was determined in a linear range from 5.0 μM to 50.0 μM, with a detection limit of 1.0 μM. Furthermore, the biosensor exhibited fast amperometric response, good reproducibility and long‐term stability.     

A conductive crosslinked graphene/cytochrome c networks for the electrochemical and biosensing study

The direct electrochemistry of catalytically active cytochrome C (Cyt c) adsorbed together with a 3-dimensional network of chemically synthesized graphene on glassy carbon electrode has been readily obtained in aqueous phosphate buffer. Direct electrical communication between the redox center of Cyt c and the modified graphene-based electrode was established. The modified electrode was employed as a high-performance hydrogen peroxide (H₂O₂) biosensor. The Cyt c present in modified electrode exhibited a pair of quasi-reversible redox peaks with a midpoint potential of ?0.380 and ?0.2 V, cathodic and anodic, respectively. Investigations into the electrocatalytic activity of the modified electrode upon hydrogen peroxide exposure revealed a rapid amperometric response (5 s). Under optimized conditions, the linear range of response to H₂O₂ concentration ranged from 5 × 10^?7 to 2 × 10^?4 M with a detection limit of 2 × 10^?7 M at a signal-to-noise ratio of 3. The stability, reproducibility, and selectivity of the proposed biosensor are discussed in relation to the morphology and composition of the modified electrode.     

Electrochemistry and biosensing activity of cytochrome c immobilized on a mesoporous interface assembled from carbon nanospheres

We report on an amperometric biosensor for hydrogen peroxide. It is obtained via layer-by-layer assembly of ordered mesoporous carbon nanospheres and poly(diallyldimethylammonium) on the surface of an indium tin oxide (ITO) glass electrode and subsequent adsorption of cytochrome c. UV–vis absorption spectroscopy was applied to characterize the process of forming the assembled layers. Cyclic voltammetry revealed a direct and quasi-reversible electron transfer between cytochrome c and the surface of the modified ITO electrode. The surface-controlled electron transfer has an apparent heterogeneous electron-transfer rate constant (k _s ) of 5.9?±?0.2?s^?1 in case of the 5-layer electrode. The biosensor displays good electrocatalytic response to the reduction of H₂O₂, and the amperometric signal increase steadily with the concentration of H₂O₂ in the range from 5?μM to 1.5?mM. The detection limit is 1?μM at pH 7.4. The apparent Michaelis-Menten constant (K _m ) of the sensor is 0.53?mM. We assume that the observation of a direct electron transfer of cytochrome c on mesoporous carbon nanospheres may form the basis for a feasible approach for durable and reliable detection of H₂O₂.

Dendritic Silver/Silicon Dioxide Nanocomposite Modified Electrodes for Electrochemical Sensing of Hydrogen Peroxide

A novel biosensor for hydrogen peroxide was prepared by immobilizing horseradish peroxidase (HPR) on newly synthesized dendritic silver/silicon dioxide nanocomposites, which were coated on a glassy carbon electrode. The modified electrode was characterized with XPS, SEM, and electrochemical methods. This biosensor showed a very fast amperometric response to hydrogen peroxide with a linear range from 0.7 to 120 μM, a limit of detection of 0.05 μM and a sensitivity of 1.02 mA mM^?1 cm^?2. The Michaelis‐Menten constant of the immobilized HRP was estimated to be 0.21 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity. The preparation of the proposed biosensor was convenient, and it showed high sensitivity and good stability.     

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.

Amperometric sensor for hydrogen peroxide based on electric wire composed of horseradish peroxidase and toluidine blue-multiwalled carbon nanotubes nanocomposite

A kind of nanocomposites with good dispersion in water was prepared through noncovalent adsorption of toluidine blue (Tb) on multiwalled carbon nanotubes (MWCNT) for electric communication between horseradish peroxidase (HRP) and electrode. The nanocomposites could be conveniently cast on electrode surface. With the aid of chitosan, HRP was then immobilized on the nanostructure to form a reagentless amperometric sensor for hydrogen peroxide. UV-vis spectroscopy and electrochemical impedance spectroscopy were used to characterize the adsorption of Tb on MWCNT. The presence of both Tb as mediator of electron transfer and MWCNT as conductor enhanced greatly the enzymatic response to the reduction of hydrogen peroxide. The novel biosensor exhibited fast response towards hydrogen peroxide with a detection limit of 1.7x10(-6)M and the linear range extended up to 4x10(-4)M without the interference of ascorbic acid and uric acid. The Michaelis-Menten constant (K'(m)) of the immobilized HRP was evaluated to be 0.16mM.