Xanthine Sensors Based on Anodic and Cathodic Detection of Enzymatically Generated Hydrogen Peroxide

A xanthine biosensor was fabricated by the covalent immobilization of xanthine oxidase (XO) onto a functionalized conducting polymer (Poly‐5, 2′: 5′, 2″‐terthiophine‐3‐carboxylic acid), poly‐TTCA through the formation of amide bond between carboxylic acid groups of poly‐TTCA and amine groups of enzyme. The immobilization of XO onto the conducting polymer (XO/poly‐TTCA) was characterized using cyclic voltammetry, quartz crystal microbalance (QCM), and X‐ray photoelectron spectroscopy (XPS) techniques. The direct electron transfer of the immobilized XO at poly‐TTCA was found to be quasireversible and the electron transfer rate constant was determined to be 0.73 s^?1. The biosensor efficiently detected xanthine through oxidation at +0.35 V and reduction at ?0.25 V (versus Ag/AgCl) of enzymatically generated hydrogen peroxide. Various experimental parameters, such as pH, temperature, and applied potential were optimized. The linear dynamic ranges of anodic and cathodic detections of xanthine were between 5.0×10^?6?1.0×10^?4 M and 5.0×10^?7 to 1.0×10^?4 M, respectively. The detection limits were determined to be of 1.0×10^?6 M and 9.0×10^?8 M with anodic and cathodic processes, respectively. The applicability of the biosensor was tested by detecting xanthine in blood serum and urine real samples.     

Flow-injection determination of sulphite and assay of sulphite oxidase

Sulphite (1–80 × 10^?5 M) in formaldehyde-stabilized solutions is determined by injection into a flowing stream of pH 8.5 phosphate buffer, passing through a mini-column of sulphite oxidase immobilized on controlled-pore glass, with amperometric detection of the hydrogen peroxide produced. Sulphite oxidase (5–100 U l^?) is determined by injection into a flowing stream of formaldehyde-stabilized 2 × 10^?3 M sodium sulphite in pH 8.0 phosphate buffer; hydrogen peroxide is again monitored.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

Spectrophotometric determination of hydrogen peroxide with leuco Patent Blue Violet

Hydrogen peroxide (1 × 10^?7?10^?5 mol dm^?3) was determined in aqueous solution using leuco Patent Blue Violet and peroxidase. The reagent can also be applied in the same concentration range to the ethyl acetate extraction method described previously. This allows hydrogen peroxide to be determined in the presence of a variety of species which interfere in the direct method. A reagent consisting of leuco Patent Blue Violet, peroxidase, glucose oxidase and mutarotase was also applied to the determination of glucose.     

Spectrofluorimetric determination of hydrogen peroxide based on the catalytic effect of peroxidase-like manganese tetrakis(sulphophenyl) porphyrin on the oxidation of homovanillic acid

Traces of hydrogen peroxide (8.5 × 10^?8–2.5 × 10^?6 mol/l) and, indirectly, glucose (3–44 × 10^?6 mol/l) can be determined by the fluorescence reaction between homovanillic acid and hydrogen peroxide. Mn-TPPS₄ is found to have very similar catalytic properties to horse peroxidase.     

Determination of Hydrogen Peroxide by Electrochemiluminescence Using a Chitosan–graphene Composite Film Doped Cadmium-Tellurium Quantum Dot Modified Glassy Carbon Electrode

Here is reported the novel determination of hydrogen peroxide by electrochemiluminescence using a chitosan–graphene composite film doped cadmium-tellurium quantum dot modified glassy carbon electrode. The cadmium-tellurium quantum dots were studied by absorption and fluorescence spectroscopy. Scanning electron microscopy and electrochemical impedance spectroscopy were used to characterize the structure morphology of the composite matrix. The electrochemiluminescence emission was linear with the concentration of hydrogen peroxide in the range of 3.5?×?10^?7 to 1.1?×?10^?5?M with a determination limit of 2.1?×?10^?7?M. Furthermore, the modified electrode showed excellent reproducibility and stability.     

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.     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Sensitive biosensor for choline and acetylcholine involving fast immobilization of a bienzyme system on a disposable membrane

A biosensor was prepared for the determination of choline or acetylcholine by co-immobilizing choline oxidase and cholinesterase on a chemically preactivated membrane ready for use. This rapid procedure allows the coupling to be performed in a few minutes. The determination is based on the electrochemical detection of enzymatically generated hydrogen peroxide. This sensor has a detection limit of 5 × 10^?8 M. The response was obtained in 2 min and was linear up to 2 × 10^?5 M.     

Peroxide optrode based on micellar-mediated chemiluminescence reaction of luminol

Cetyltrimethylammonium bromide (CTAB) was used to overcome the pH mismatch of the luminol (5-amino-2,3-dihydrophthalazine-1,4-dione ) chemiluminescence reaction when coupled to the glucose/glucose oxidase reaction at neutral pH. The results demonstrate the feasibility of conducting both reactions simultaneously and efficiently at pH 7.5–8.5. The incorporation of the CTAB micellar system in the coupled luminol/enzymatic reaction allows quantification of glucose in the 3 × 10^?7?3 × 10^?4 M range. The relative standard deviation (RSD) for 5 replicates of 5 × 10^?5 M glucose was 3.8%. Also, hydrogen peroxide was quantified in the 1.2 × 10^?4?2.4 × 10^?8 M range with RSD 2.6%. The micellar-mediated luminol reaction was applied successfully to the determination of glucose in blood serum. Excellent agreement with reported results by standard assays was obtained.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.     

A Cholesterol Biosensor Based on Entrapment of Cholesterol Oxidase in a Silicic Sol‐Gel Matrix at a Prussian Blue Modified Electrode

A cholesterol biosensors fabricated by immobilization of cholesterol oxidase (ChOx) in a layer of silicic sol‐gel matrix on the top of a Prussian Blue‐modified glassy carbon electrode was prepared. It is based on the detection of hydrogen peroxide produced by ChOx at ?0.05 V. The half‐lifetime of the biosensor is about 35 days. Cholesterol can be determined in the concentration range of 1×10^?6?8×10^?5 mol/L with a detection limit of 1.2×10^?7 mol/L. Normal interfering compounds, such as ascorbic acid and uric acid do not affect the determination. The high sensitivity and outstanding selectivity are attributed to the Prussian Blue film modified on the sensor.     

Amperometric aspartate electrode

An amperometric enzyme electrode for L-aspartate determination was developed. The probe consisted of a platinum electrode which senses hydrogen peroxide produced from the reactions catalyzed by two enzymes co-immobilized on a preactivated polymeric membrane, α-Ketoglutarate in the presence of L-aspartate was transaminated to L-glutamate by aspartate aminotransferase and the glutamate produced was oxidized by glutamate oxidase, with concomitant production of hydrogen peroxide. Additional protective membranes eliminated interferences from glutamate and most electroactive compounds. The response curve of the probe was linear over the concentration range 1.0 × 10^?6 M to 2.0 × 10^?4 M aspartate and was useful for at least two months. Aspartic acid in some pharmaceutical products was determined and the results correlated well with a liquid chromatographic reference method and the manufacturer's specification.     

Flow-injection amperometric determination of oxalate with immobilized oxalate oxidase

Oxalate is immobilized on controlled-pore glass and is used on-line in a glass minicolumn (2.5×25 mm). The hydrogen peroxide formed is detected amperometrically. Oxalate (6×10^?6?9×10^?4 M) is determined in a flowing stream of pH 3.5 citrate (or succinate) buffer. As little as 20 ng (in 40 μl; 5.7×10^?6 M) of oxalate can be detected. Copper inhibition can be removed either by adding EDTA to the carrier stream or incorporating a chelating-resin minicolumn into the flow system prior to the enzyme column.     

An enzyme reactor electrode for determination of amino acids

L-Leucine can be determined with an enzyme reactor electrode containing L-amino acid oxidase immobilized with glutaraldehyde to glass. The reactor also contains immobilized catalase which splits the hydrogen peroxide formed. Oxygen for the reaction is also supplied by adding hydrogen peroxide to the samples. The electrode is an ammonia gas sensor. The calibration curve is strictly linear with Nernstian slope between 3·10^-5 and 10^-3 M leucine.     

Determination of acetaldehyde by a chemiluminescence method

The determination of acetaldehyde was achieved by monitoring the chemiluminescence emission from the luminol-potassium hexacyanoferrate (III) reaction in the presence of xanthine oxidase. The linear range was three orders of magnitude, the detection limit (2σ) was 4 × 10^?7 M and the relative standard deviation (n = 5) was 10.6% for 4.3 × 10^?7 M. No interference was observed from seven organic and inorganic species at a 1000-fold excess relative to a concentration of 1 × 10^?5 M acetaldehyde.     

Quantitation of enzymatically generated hydrogen peroxide based on aqueous peroxyoxalate chemiluminescence

The method involves the reaction of 4,4′-{oxalyl bis[(trifluoromethylsulfonyl)imino]-ethylene}-bis(4-methylmorpholinium trifluoromethanesulfonate) with hydrogen peroxide in the presence of rhodamine-B. Precise measurements, with 1–3% relative standard deviation, can be made in both static and flow systems. In the flow system, the response to hydrogen peroxide is linear from 10^?2 M hydrogen peroxide down to the limit of detection of 7 × 10^?5 M.     

Pure copper vs. mixed copper and palladium hexacyanoferrates for glucose biosensing applications

A glucose amperometric biosensor was developed. Glucose oxidase enzyme was immobilized by means of a Nafion membrane on glassy carbon modified with an electrochemically deposited mixed Cu and Pd hexacyanoferrate (CuPdHCF). According to the data provided by X-ray atomic spectroscopy measurements, this Cu- and Pd-based hexacyanoferrate is likely to be a mixture of single CuHCF and PdHCF pure phases. The biosensor performances were evaluated by recording the steady-state currents due to submillimolar additions of glucose to a potassium buffer solution (pH 5.5) and exploiting the electrocatalytic reduction of the enzymatically produced hydrogen peroxide. The CuPdHCF-based biosensor exhibited a sensitivity of 8.1?±?0.6 A M^?1 m^?2, a limit of detection of 1.4?×?10^?5 M, and a linear response range extending between 5?×?10^?5 and 4?×?10^?4 M, with a dynamic response range up to 4?×?10^?3 M glucose. Electrode sensitivity and signal stability resulted more satisfactory as compared to those of a CuHCF-based biosensor fabricated according to the same procedure. The selectivity was investigated through an interference study. The response to easily oxidizable species was found to be low enough to allow glucose determination in biological samples.     

Spectrophotometric Determination of Hydrogen Peroxide and Glucose Based on Hemin Peroxidase-Like Catalyzed Oxidation of Bromopyrogallol Red

In an ammonium buffer medium at pH 8.9–9.5, hemin exhibits mimetic peroxidase activity, and has a catalytic effect on the oxidative decoloration of bromopyrogallol red (BPR) with hydrogen peroxide. On this basis and in presence of ethanol as an effect-enhancing agent, a spectrophotometric determination of hydrogen peroxide is described with an apparent molar absorptivity of 4.00×10⁴?l?mol^?1?cm^?1 and a linear range from 3.2×10^?7 to 3.2×10^?5?mol?l^?1. BPR has advantages over some of widely used chromogenic substrates in aspects of sensitivity, simplicity and detection wavelength, while hemin has better stability than peroxidase. The system can be easily coupled with a glucose oxidase-catalyzed reaction, and glucose in the concentration range of 6.0×10^?7? 3.2×10^?5?mol?l^?1 is spectrophotometrically determined. The method has been applied to the analyses of synthetic water and human serum samples. The Michaelis parameters and the mechanism of the mimetic peroxidase reaction are also investigated.     

Study of the mimetic peroxidase-catalysed chemiluminescence reaction

The chemiluminescence behaviour of the reaction in which the Mn-TPPS₄ complex the mimetic enzyme of peroxidase [manganese tetrakis(sulphophenyl)porphine] acts as a catalyst for the oxidation of luminol by hydrogen peroxide was studied. The reaction product luminesces at 427 nm. Trace amounts of hydrogen peroxide and glucose can be determined with detection limits of 5.5 × 10^?9 and 2.7 × 10^?9 M, respectively. The characteristics of Mn-TPPS₄ were compared with those of horseradish peroxidase.