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.     

Enzymatic methods for the determination of ethanol based on linear and cyclif flow-injection systems

Linear and cyclic systems are described for the determination of ethanol (ca. 0.17–30×10^?3 M). In the linear system, the solution passes either through a minicolumn of yeast alcohol dehydrogenase (YADH) immobilized on controlled-pore glass or through minicolumns of the immobilized YADH and of yeast aldehyde dehydrogenase immobilized on cyanogen bromide-activated Sepharose-4B. The NADH formed is monitored either spectrophotometrically or spectrofluorimetrically. In the cyclic system, the solution passes through the same enzyme columns, and the NADH produced is monitored similarly before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on cyanogen bromie-activated Sepharose-4B in the presence of α-ketoglutarate and ammonium ions also present in the flow system. the sample throughout for both systems is ca. 40 h^?1 and 50 h^?1 for spectrophotometric and spectrofluorimetric detection, respectively. An on-line double-injection technique is described as an alternative to the cyclic system for limiting the consumption of NAD⁺.     

Cyclic regeneration of nicotinamide adenine dinucleotide with immobilized enzymes: Flow-injection spectrofluorimetric determination of ethanol

Ethanol (0.05–0.5%) in water is determined by injection of a 20-μl sample into a solution of 1.5 × 10^?3 M NAD⁺ in pH 8.0 phosphate buffer flowing from a reservoir. The solution passes through a minicolumn of yeast alcohol dehydrogenase immobilized on controlled-pore glass (CPG). The NADH formed is monitored spectrofluorimetrically in the flow system, before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on CPG in the presence of glutarate and ammonium ions, also in the flowing solution. The solution then returns to the reservoir. The regeneration of NAD⁺ allows the same coenzyme solution to be used for 50 ethanol determinations daily for 4 days.     

Flow-injection determination of l-tyrosine in serum with an immobilized tyrosinase reactor and fluorescence detection

Tyrosinase is immobilized on controlled-pore glass beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum is deproteinized with tungstate and sulphuric acid. The carrier stream is 0.3 M phosphate buffer (pH 7.2), and is mixed with 5 M potassium hydroxide after the enzyme reactor. The fluorescent dihydroxyindole formed is detected at 490 nm (excitation at 375 nm). The calibration graph is linear for 1 × 10^?7 ?1 × 10^?4 M tyrosine; the detection limit is 2 × 10^?8 M.     

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.     

Spectrophotometric determination of copper by flow injection analysis with an on-line reduction column

A silver reductor minicolumn is used ina flow-injection system for reduction of copper(II) to copper(I), which is detected spectrophotometrically using bathocuproine disulphonic acid. The reductor functions very well at flow rates up to 4 ml min^?1; this allows sample injection ar rates up to 120 h^?1. Linear calibration is achieved for 5 × 10^?7– 1 × 10^?4 M copper. The detection limit is 3.4 ng and the midrange precision is 1%.     

Chemiluminescence Sensor with Mn(III)-Tetrakis (4-sulfonatophenyl)-porphyrin Immobilized on Dioctadecyldimethylammonium Chloride Bilayer Membranes Incorporated Into PVC Film

Abstract

An indicator phase for a flow-through chemiluminescence (CL) sensor composed of ordered surfactant assemblies, a polymer and a catalyst was evaluated by measuring adrenaline. The method is based on use of Mn (III)-porphyrin immobilized on a bilayer membrane contained in a blended film, prepared by incorporating dioctadecyl-dimethylammonium chloride into polyvinyl chloride. The sensor consisted of a Pyrex glass tube (30 mm × 5 mm i.d.) packed with silica glass wool, on which the indicator phase was coated, and a photomultiplier tube. The blend film functioned as a favorable reaction medium for the adrenaline CL, and further enhanced CL was observed with the immobilized catalyst. This indicator phase permitted adrenaline to be detected down to 3 × 10^?6 M with a 20 μl injection into a 0.3 M NaOH carrier solution. The relative standard deviation (n = 10) was 1.0% for 5 × 10^?5 M adrenaline. For 80 successive injections of 5 × 10^?5 M adrenaline, the variation of the CL signal was within the relative standard deviation. Almost the same sensitivity and precision were observed with the indicator phase stored in water for at least 3 days. The sensor was successfully applied to determine adrenaline in drug samples.     

Determination of pesticides in vegetable samples using an acetylcholinesterase biosensor based on nanoparticles ZrO2/chitosan composite film

An amperometric pesticides inhibition biosensor has been developed and used for determination of pesticides in vegetable samples. To eliminate the interference of ascorbic acid, multilayer films of polyelectrolyte (chitosan/polystyrensulfonate) were coated on the glass carbon electrode. Then, acetylcholinesterase was immobilized on the electrode based on surface-treated nanoporous ZrO₂/chitosan composite film as immobilization matrix. As a modified substrate, acetylthiocholine was hydrolysed by acetylcholinesterase and produced thiocholine which can be oxidized at +700?mV vs. SCE. Pesticides inhibit the activity of enzyme with an effect of decreasing of oxidation current. The experimental conditions were optimized. The electrode has a linear response to acetylthiocholine within 9.90?×?10^?6 to 2.03?×?10^?3?M. The electrode provided a linear response over a concentration range of 6.6?×?10^?6 to 4.4?×?10^?4?M for phoxim with a detection limit of 1.3?×?10^?6?M, over a range of 1.0?×?10^?8 to 5.9?×?10^?7?M for malathion, and over a range of 8.6?×?10^?6 to 5.2?×?10^?4?M for dimethoate. This biosensor has been used to determine pesticides in a real vegetable sample.     

Determination of uranium using a flow system with reagent injection. Application to the determination of uranium in ore leachates

The determination of uranium by a flow system with reagent injection is based on the reaction of U(IV) with Arsenazo III in 3.6 M HCl; U(IV) is generated by reduction of uranyl ion in a lead reductor minicolumn installed in the sample channel of the manifold. The interference effect caused by several ions is studied. The calibration graph is linear up to 1.0 × 10^?5 M (2.4 mg l^?1) and the detection limit is 2.8 × 10^?8 M (6.6 μg l^?1). The modification of the manifold by including a second valve to by-pass the reducing column allows the measurement of the difference in peak heights, which makes the method specific for uranium.     

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.     

An amperometric monoamine oxidase biosensor for determining some antidepressants

An amperometric biosensor based on a platinum screen-printed electrode and immobilized monoamine oxidase is developed to determine antidepressants of different classes. Petylyl, pyrazidol, and flu-oxetine can be determined with determination limits of 8 × 10^?9, 8 × 10^?7, and 8 × 10^?10 M, respectively. A procedure is proposed for determining fluoxetine in tablets. It is shown that petylyl can be selectively determined by an immunochemical technique using the developed biosensor and immobilized antibodies in the concentration range from 1 × 10^?4 to 1 × 10^?8 M.     

Kinetic studies of the oxidation of transition metal(II) malate complexes by peroxomonosulphate

The kinetics of oxidation of malic acid by peroxomonosulphate (PMS) in the presence of Cu(II) (2.50 × 10^?4–5.00 × 10^?3 M), Co(II) (2.00 × 10^?6–1.00 × 10^?5 M) and Ni(II) (5.00 × 10^?4–6.00 × 10^?3 M) were studied in the pH range 4.05–5.89. The oxidation of Ni(II) malate follows simple first-order kinetics with respect to both [PMS] and [Ni(II)], while the oxidations of Cu(II) malate and Co(II) malate show autocatalysis. There is an appreciable induction period in the Cu(II) malate oxidation, while Co(II) malate oxidation follows a simple curve. The initial oxidation product for all three systems was identified as malonic semialdehyde. Alcohol quenching studies suggest that, even in the Co(II) malate-PMS system, no radical intermediates such as $ {\text{SO}}_{4}^{ - .} $ or $ {\text{OH}}{}^{.} $ are detected. The malonic semialdehyde intermediate may react with M(II) malates to give a hemiacetal, which may be more reactive.     

Flow-injection spectrophotometric determination of acetyl-coenzyme A with immobilized phosphotransacetylase

Phosphotransacetylase (PTA) is immobilized on AF-Tresyl TOYOPEARL 650 gel and used on-line in a stainless-steel column (10 × 4 mm i.d.). CoA-SH liberated enzymatically from acetyl-CoA is reacted with Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the carrier stream. The calibration graph for acetyl-CoA is linear from 4 × 10^?6 to 4 × 10^?4 M and the detection limits is 8 × 10^?7 M. The immobilized enzyme can be employed for over 4 months without any significant decrease in activity. The enzyme retains its activity in methanol even though the initial rate of reaction is decreased.     

二茂铁衍生物电子传递剂键合的溶胶-凝胶葡萄糖生物传感器

The sol-gel derived glucose biosensor was developed, and the sol-gel membrane was organically modified by N-（3-triethoxysilylpropyl）-ferrocenylmethylamine （FcSi） as sol-gel precursor to make electrochemical biosensor. The structure of biosensor was sol-gel/FcSi＋GOx/GC type （glucose oxidase, GOx）. The ferrocene mediator was chemically immobilized to the silane network, and GOx was entrapped to the sol-gel glass network. Therefore, these structures prevented mediator leakage and retained the enzyme activity. Additionally, pH of electrolyte, temperature effects, and interference of positive substances with biosensor were investigated. And the electrochemical performance of biosensor was studied by amperometry. The results indicated that the linear range, detection limit. and response slope of biosensor was 2.00×10^-4-1.57×10^-3 mol·L^-1, 2.0×10^-4 mol·L^-1 and 5.06×10^5 nA·mol^- 1·L, respectively.     

Flow-injection determination of d-mannitol with immobilized mannitol dehydrogenase

A flow-injection system is described for the determination of d-mannitol. Mannitol dehydrogenase is immobilized on poly(vinyl alcohol) beads and packed in a column (5 cm × 4 mm i.d.). The NADH formed is detected fluorimetrically. The response is linear between 5 × 10^?7 and 1 × 10^?4 M mannitol and the detection limit is 1 × 10^?7 M. The throughput is 30 samples per hour. The reactor is stable for at least 8 weeks.     

Electrocatalysis of Horseradish Peroxidase Immobilized on Cobalt Nanoparticles Modified ITO Electrode

Abstract

It is the first time that Horseradish peroxidase (HRP) was successively immobilized on the magnetic cobalt nanoparticles modified ITO (indium tin oxide) electrode. Morphologies of electrode surface were featured by the field emission‐scanning electron microscope (FSEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the modified process of electrode. Direct electrochemistry and electrocatalysis of HRP immobilized on nano‐Co/ITO were investigated. The biosensor exhibited high sensitivity, good stability, and excellent electrocatalytic activity to the reduction of H₂O₂. Under the optimized experimental conditions, a calibration curve over 2.0×10^?9～2.0×10^?8 mol l^?1 and 2.0×10^?7～2.0×10^?6 mol l^?1, with a limit of detection of 1.9×10^?9 mol l^?1 was obtained. The apparent Michaelis‐Menten constant (K _M ^app ) for HRP/nano‐Co/ITO electrode was calculated to be 0.79 mmol l^?1, indicating a higher affinity of HRP attached on the modified electrode.     

An optical-fibre sensor for fluoride

The optical sensor is based on the formation of a fluoride ternary complex from an immobilized fluorescent binary zirconium-calcein blue chelate at pH 2.2. The enhanced fluorescence enables fluoride to be determined in the range 2.63 × 10^?5 ?4.21 × 10^?4 M (0.5–8.00 mg l^?1) with a limit of detection of 2.63 × 10^?5 M. The response of the sensor to fluoride is affected by ionic strength and temperature. The response time depends on the particle size and the packing density of the polymer (XAD-4) substrate used.     

Electrocatalytic studies of covalently immobilized metal tetra-amino phthalocyanines onto derivatized screen-printed gold electrodes

Metal tetra-amino phthalocyanine complexes (MTAPc; where M is Co or Mn) were immobilized on screen-printed gold electrodes pre-modified with monolayers of benzylamino groups. The functionalized electrodes were then activated using benzene-1,4-dicarbaldehyde as a linker before MTAPc complexes were immobilized. The surface coverages for the modified electrodes confirmed the perpendicular orientation of the MTAPcs. The apparent electron transfer constant (k_app) for the electrodes is 2.2?×?10^?5 cm.s^?1 for both CoTAPc and MnTAPc modified electrodes as calculated with data from impedance measurements. The k_app values for the bare and benzylamino modified electrodes were found to be 1.2?×?10^?4 cm.s^?1 and 4.9?×?10^?6 cm.s^?1, respectively. The electrocatalysis of the modified electrodes towards detection of H₂O₂ gave significant peak current densities and electrocatalytic potentials at ?0.28 V and ?0.31 V for the MnTAPc and CoTAPc modified electrodes, respectively.     

Immobilized enzyme electrode for the determination of salicylate in blood serum

This determination of salicylate in blood serum is based on application of an immobilized enzyme electrode. Salicylate hydroxylase (E.C.1.14.13.1) is chemically immobilized onto a pig intestine mounted on an oxygen electrode. The signals are monitored amperometrically and the resulting output voltage is read using a simple adapter. The experimental parameters and possible interferences are discussed. Samples containing 1.0 × 10^?5?1.87 × 10^?3 M (1.6–300 μg ml^?1) salicylate were assayed with relative standard deviations between 1.3% and 6% and recoveries between 98.7 and 103%. Results obtained by the proposed method and by the established clinical method for randomly spiked pooled serum samples correlated well (r = 0.99).     

Spectrophotometric study of coverage and acid-base equilibrium of a chemically bonded base

An aniline azo dye is immobilized on glass slides by reacting an organosilane reagent with the glass surface. The immobilized dye absorption spectral characteristics closely resemble those of the analogous solution form of the dye. The coverage of the immobilized dye, 1.1·10^?10 mol cm^?2, is consistent with simple monolayer coverage. The immobilized dye is a weaker base than the soluble form by an equilibrium constant factor of about 10, consistent with an electrostatic effect.