Miltiades I. Karayannis

Enzyme Based Amperometric Biosensors for Food Analysis

This review introduces the principles of amperometric detection e.g. oxygen electrodes, hydrogen peroxide electrodes, NADH detection, mediators-aid detection, conductive organic salts and wiring electrodes. A short categorization and description of the materials commonly used for the construction of electrodes, e.g., platinum, glassy carbon, different types of graphite, screen-printed electrodes, rigid carbon-polymer biocomposites, zeolites, clays, and polymeric membranes is given. Approaches to construction of biosensors with respect to various strategies of enzyme immobilization, e.g., physical binding, covalent binding, gel entrapment, electropolymerization, sol-gel techniques and self-assembled architectures are also presented. The requirements and problems for sensing in food industry, examples of enzyme electrodes, published in the literature during the last half-decade, commercial biosensors released into the market along with the current and modern instrumentation, are also presented.

An optimised single-reagent method for the speciation of chromium by flame atomic absorption spectrometry based on surfactant micelle-mediated methodology

The toxicity of chromium in the environment is dependent on the species in which it exists. This paper outlines a method for the analysis of the oxidation states of Cr employing a suitable chelating agent and the cloud point phenomenon for Cr(VI) and total Cr analysis. The method involves preconcentration of metal chelates followed by air-acetylene flame atomic absorption spectrometric analysis. The chelating agent chosen for this task is the ammonium pyrrolidinedithiocarbamate, which reacts with either Cr(VI) or total Cr under specific experimental conditions. The condensed surfactant phase with the metal chelate(s) is introduced into a flame atomic absorption spectrometer, whereby discrimination of Cr species is feasible by calculating the Cr(III) concentration from the difference between total Cr and Cr(VI). A multivariate design was employed to study the variables affecting the overall analytical performance for total Cr assay. The analytical curves are rectilinear up to 100 micrograms l-1 for both oxidation states of the metal. The limits of detection are 0.6 microgram l-1 and the relative standard deviation (n = 5) at a concentration of 30 micrograms l-1 for both species is around 2.0%. The method was validated by analysing BCR 544 reference material certified for both Cr species. High recoveries in the range 96-107% were attained for the environmental and biological samples tested.

Determination of hydrogen peroxide by using a flow injection system with immobilized peroxidase and long pathlength capillary spectrophotometry

The development of a highly sensitive method for the determination of nanomolar concentrations of hydrogen peroxide in the liquid phase is described. This paper demonstrates for the first time a flow injection analysis (FIA) system with immobilized enzyme reactor combined with a total internal reflective cell (a liquid waveguide capillary cell (LWCC)) and spectrophotometric detection, for the development of an improved procedure for the determination of hydrogen peroxide. Moreover, the newly synthesized 4-aminopyrazolone derivative, 4-amino-5-(p-aminophenyl)-1-methyl-2-phenyl-pyrazol-3-one (DAP), is used as a color coupler in its oxidative condensation with the sodium salt of N-ethyl-N-sulphopropylaniline sodium salt (ALPS) which acts as a hydrogen donor. Immobilization of peroxidase is achieved by coupling the periodate-treated enzyme to aminopropyl controlled-pore glass (CPG) beads. The determination of hydrogen peroxide is carried out in a 0.1 M phosphate buffer and the product is monitored at 590 nm with a charge-coupled device (CCD) detector equipped with fiber optics in a fully computerized system. The interference of different species, mainly ionic, was investigated. The method permits detection down to 4 nmol l −1 hydrogen peroxide (signal-to-noise ratio = 3). A linear calibration graph was obtained over the range 20–700 nmol l −1 . The relative standard deviation (R.S.D.) at 300 nmol l −1 H2O2 is 1.7% (n = 7). The method was successfully applied for the determination of hydrogen peroxide in samples from a vat-cleaning process.

Micelle mediated extraction of magnesium from water samples with trizma-chloranilate and determination by flame atomic absorption spectrometry

This article describes an analytical method for the determination of magnesium taking advantage of the cloud point phenomenon employing a suitable chelating agent (chloranilate) for Mg analysis. The method encompasses pre-concentration of the metal chelate followed by flame atomic absorption spectrometry (FAAS) analysis. The chelating agent chosen for this task is a newly synthesised salt of chloranilic acid, trizma-chloranilate, which reacts with Mg but at the same time has a very low affinity for other metallic cations like silicon, aluminium and sodium, which interfere with the determination of Mg in FAAS. The condensed surfactant phase with the metal chelate(s) is introduced into the flame of an atomic absorption spectrometer after its treatment with an acidified methanolic solution. In this way, complex and time-consuming steps for sample treatment are avoided while increased sensitivity is achieved by the presence of both methanol and surfactant in the aspirated sample. The analytical curve was rectilinear in the range of 5-220 mugl(-1) and the limit of detection was as low as 0.75 mugl(-1) with a standard deviation of 5.2%. The method was applied for the determination of Mg in natural and mineral waters with satisfactory results and recoveries in the range of 97-102%.

Bioelectrochemical Determination of Citric Acid in Real Samples Using a Fully Automated Flow Injection Manifold

An enzymic method for the determination of citric acid in fruits, juices and sport drinks is proposed. The method is based on the action of the enzymes citrate lyase, oxaloacetate decarboxylase and pyruvate oxidase, which convert citric acid into H2O2 with the latter being monitored amperometrically with a H2O2 probe. The enzymes pyruvate oxidase and oxaloacetate decarboxylase were immobilized. A multi-membrane system, consisting of a cellulose acetate membrane for the elimination of interferants, an enzymic membrane and a protective polycarbonate membrane were placed on a Pt electrode and used with a fully automated flow injection manifold. Several parameters were optimized, resulting in a readily constructed and reproducible biosensor. Interference from various compounds present in real samples was minimized. Calibration graphs were linear over the range 0.01-0.9 mM pyruvate, 0.015-0.6 mM oxaloacetate and 0.015-0.5 mM citrate. The throughput was 30 samples h-1 with an RSD of 1.0% (n = 8); the mean relative error was 2.4% compared with a standard method. The recovery was 96-104%. A 8-10% loss of the initial activity of the sensor was observed after 100-120 injections.

Copper fractionation with dissolved organic matter in natural waters and wastewater--a mixed micelle mediated methodology (cloud point extraction) employing flame atomic absorption spectrometry.

A cloud point extraction-preconcentration methodology for the speciation analysis of free and organically complexed metal species in natural waters is presented. The method is based on the neutralization of the electrostatic charge of the humate-metal complexes with a positively charged surfactant in a high ionic strength solution environment. The resulting complexes are conveniently solubilized in the micelles of a non-ionic surfactant medium and are thus separated from the bulk aqueous phase. Free metal species are also determined by complexation with a conventional chelating agent under mild conditions. The overall procedure is easy, rapid and allows for a high sample throughput in terms of massive analysis of many samples in the same time period. The method offers substantially low detection limits of 8.5 and 0.9 micrograms l-1 for bound and labile species respectively, with a calibration curve rectilinear in the wide range 40-150 micrograms l-1 for the humate associated and 4-40 micrograms l-1 for the free metal species. The method is free from interferences yielding recoveries in the range 97-102% for various samples of different matrixes.

Electrocatalytic Oxidation of NADH in Flow Analysis by Graphite Electrode Modified with 2,6-Dichlorophenolindophenol Salts

The preparation of a 2,6-dichlorophenolindophenol (DCPI) modified graphite electrode is described. DCPI was successfully immobilized by physical adsorption onto a plain graphite electrode (DCPI-CME) and onto graphite electrodes pretreated with lanthanum nitrate (DCPI-La-CME) or thorium nitrate (DCPI-Th-CME). The electrochemical behavior of DCPI-CME was extensively studied using cyclic voltammetry. The electrochemical redox reaction of DCPI was found to be fairly reversible at low coverage with Eo′ = +55 mV (vs. Ag/AgCl/3M KCl) at pH 6.5. A pKa value of 5.8 ± 0.1 for immobilized form of DCPI is determined from the intersection of the lines in the plot Eo′ vs. pH. The current Ip has a linear relationship with the scan rate up to 1200 mV s−1, which is indicative for very fast electron transfer kinetics. The calculated value of the standard rate constant is ko = 18 ± 4 s−1. No decrease of either the anodic or the cathodic current of the cyclic voltammogram was observed after 500 runs of successive sweeps. The influence of the morphology of the electrode surface on the electrochemical behavior of the DCPI-CME was studied and a mathematical model is proposed, which partly describes the dependence of the geometrical area of the electrode surface on the grid of the emery paper. The modified electrodes were mounted in a flow-injection manifold, poised at +60 mV (vs. Ag/AgCl/3M KCl) and a catalytic current due to the oxidation of NADH was observed reducing thus the oxidation overpotential of NADH for about 400 mV. Interference from various reductive species present in real samples was investigated. The repeatability was 1.2 % RSD (n = 10 for 0.1 mM NADH). The sensor showed good operational and storage stability.

Development of 1-(2-pyridylazo)-2-naphthol-modified polymeric membranes for the effective batch pre-concentration and determination of zinc traces with flame atomic absorption spectrometry

The development of 1-(2-pyridylazo)-2-naphthol (PAN)-modified polymeric membranes for the effective batch pre-concentration and determination of zinc traces with flame atomic absorption spectrometry (FAAS) is described. The method is based on the chemical bonding of the metal species with a suitable ligand, which has been immobilized into a water-insoluble cellulose acetate (CA) membrane followed by simple rinsing of the chelating agent-metal complex with an acidified methanolic solution. The latter is directly aspirated to the nebulizer of a FAA spectrophotometer without any other treatment. As an analytical demonstration, trace concentrations of Zn(II) were successfully detected in real samples, such as seawater, river and lake water, wastewater as well as in a reference material, without any laborious and time-consuming treatment. Several working parameters were investigated. A pre-concentration factor of 100 was achieved by simple immersing of a circular piece of the CA-PAN membrane (0.6 cm diameter) in the tested samples for 30 min at room temperature. The analytical curve was rectilinear up to 30 mug l(-1) zinc with detection limit of 0.7 mug l(-1), a quantitation limit of 2.0 mug l(-1) and a relative standard deviation lower than 2%.

The use of surfactant-based separation techniques for monitoring of orthophosphate in natural waters and wastewater

In an effort to monitor orthophosphate in natural waters and wastewater in the Ipirous region (Greece), an analytical methodology was established owing to the need for interference-free determination at the low mg/l levels. The method applied for the determination of orthophosphate is based on its reaction with molybdate towards a yellow heteropoly acid complex, which is electrically balanced with a cationic surfactant (cetylatrimethylammonium bromide) towards a complex non-polar derivative. This derivative is conveniently solubilized in the micelles of a non-ionic surfactant under mild conditions and measured spectrophotometrically at 370 nm after its uptake with a sulfuric acid-methanol solution. By simply preconcentrating 10 ml of sample volume a detection limit of 2.6 microM was obtained. Due to the required dilution step and signal enhancement caused by the presence of surfactant, interference from the presence of anionic species (mostly silicate and arsenate) was minor and could be conveniently alleviated by the use of tartaric acid and Na(2)SO(3), respectively. The method was successfully applied to real samples obtained for the natural sites subject to the monitoring survey ranging in their matrix complexity from clean river water to wastewater.

Electrocatalysis of sulphide with a cellulose acetate film bearing 2,6-dichlorophenolindophenol. Application to sewage using a fully automated flow injection manifold

The application of an electrochemical sensor, based on a glassy carbon electrode, modified with a cellulose acetate polymeric film bearing 2,6-dichlorophenolindophenol (CA/DCPI-CME), for flow injection analysis of sulphide, is described. The overall reaction was found to obey a catalytic regeneration mechanism (EC mechanism) and the electrochemical rate constant k(f) for the electrocatalytic oxidation of sulphide was evaluated. The modified electrodes were mounted in a flow-injection manifold, poised at +0.08 mV versus Ag/AgCl/3 M KCl at pH 7.25 and utilised for the determination of sulphide in urban waste samples. The proposed method correlates well with a colorimetric method. Parameters such as working pH, sample size, flow rate and temperature were studied. Interferants of various compounds normally present in real samples were also tested. Calibration graphs were linear over the range 0.02-1 mM sodium sulphide for CA/DCPI-CMEs hydrolysed in KOH for 6 min. The throughput was 25 samples per h and the R.S.D. was 1.2% (n=7) for 0.1 mM sodium sulphide. Recoveries for spiked urban waste samples ranged from 99 to 120%. The sensor remained active for more than 2 weeks under specified conditions.