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Gluconobacter oxydanswhole cells and membrane fractions in combination with ferrocene mediators were used to study oxidation of ethanol. The efficiency of mediatorenzyme interaction was assessed by the ratio of maximum current to the apparent Michaelis constant (Imax/KM) for saturating mediator concentrations. The bioelectrocatalytic processes were found to be more efficient with membrane fractions. The highest Imax/KMvalue of 120 and 3200 µA g/mol for, respectively, cells and fractions was obtained for ferrocene carboxylic acid. In test measurements of biological oxygen demand for rye distillers’ grains, the values obtained by a biosensor based on Gluconobactermembrane fractions and ferrocene were found to correlate with the reference data.

We report on the fabrication and characterisation of the very first direct electron transfer‐based glucose/oxygen biofuel cell (BFC) operating in neutral glucose‐containing buffer and human serum. Corynascus thermophiluscellobiose dehydrogenase and Myrothecium verrucariabilirubin oxidase were used as anodic and cathodic bioelements, respectively. The following characteristics of the mediator‐, separator‐ and membrane‐less, a priori, non‐toxic and simple miniature BFC, was obtained: an open‐circuit voltage of 0.62 and 0.58 V, a maximum power density of ca. 3 and 4 μW cm–2at 0.37 and 0.19 V of cell voltage, in phosphate buffer and human serum, respectively.

A membrane‐free biofuel cell (BFC) is reported based on enzymes wired to graphite electrodes by means of Os‐complex modified redox polymers. For the anode cellobiose dehydrogenase (CDH) is used as a biocatalyst whereas for the cathode a laccase was applied. This laccase is a high‐potential laccase and hence able to reduce O2to H2O at a formal potential higher than +500 mV versusAg/AgCl. In order to establish efficient electrochemical contact between the enzymes and graphite electrodes electrodeposition polymers containing Os‐complex with specifically designed monomer compositions and formal potentials of the coordinatively bound Os‐complex were synthesised and used to wire the enzymes to the electrodes. The newly designed CDH/Os‐redox polymer anode was characterised at different pH values and optimised with respect to the nature of the polymer and the enzyme‐to‐polymer ratio. The resulting BFC was evaluated running on β‐lactose as a fuel and air/O2as an oxidising agent. The power output, the maximum current density and the electromotor force (Eemf) were found to be affected by the pH value, resulting in a maximum power output of 1.9 μW cm–2reached at pH 4.3, a maximum current density of about 13 μA cm–2at pH 3.5, and the highest Eemfapproaching 600 mV at pH 4.0.

A method for the in situ modification of hollow fiber membranes used as sampling units for microdialysis probes is presented. The method consists of adsorption-coating, high molecular-weight poly(ethylene imine), PEI, onto membranes, already fitted on microdialysis probes. Modification of membranes was designed to specifically explore the so-called Andrade effects and thus enhance the interaction of membranes with enzyme. The performances of polysulfone, polyethersulfone, and polyamide membranes modified with PEI-enzyme complex were evaluated based on the membrane extraction fraction for maltose and maltotriose and membrane morphology as examined by scanning electron microscopy. Of the membranes tested, the PEI-enzyme complex least affected the performance of the polysulfone membranes. Conversion of maltoheptaose to maltotriose and maltose was increased reproducibly (within a 5 % relative standard deviation) by 50 % for modified membranes compared to the native hollow fiber membranes. The results demonstrate the potential to effectively modify membranes already fitted on a microdialysis probe. Such a procedure can be modified and employed to either promote or reduce membrane–protein interaction for hollow fibers used as microdialysis sampling units or other similar membrane applications.

The search for an enzyme as a reagent for selective bioanalytical detection of the neurotoxic amino acid, β-N-oxalyl L-alpha, β-diaminopropionic acid, β-ODAP (found in grass pea, Lathyrus sativus) led to its redox catalytic reaction by glutamate oxidase (GluOx). Homogeneous kinetic studies and an immobilized GluOx reactor-based flow-injection assay were initially made for beta-ODAP with small immobilized GluOx/catalase glutamate destroying prereactors. The method was applied to examine the toxin content in processed grass pea. The kinetics and the equilibrium of the thermal isomerization of β-ODAP to the nontoxic isomer α-ODAP established that GluOx is specific to the neurotoxin. The first ever GluOx-based amperometric biosensor for liquid chromatography (LC) detection was reported in 1997. This biosensor coupled with a refractive index detector improved LC performance. The most recent work with GluOx resulted in MnO2-based screen-printed amperometric biosensor, with offline elimination of glutamate interference by glutamate decarboxylase. A single-shot chemiluminescent sensor developed for hydrogen peroxide is also proposed for β-ODAP with GluOx application. This decade of progress resulted from studies that included four Ph.D. (Ethiopia, Sweden, Austria), four M.Sc. (Ethiopia, Sweden) and Licentiate (Sweden) theses projects, plus one collaborative project in Sweden. The advances in grass pea research may be regarded as a model north–south cooperation for research and education.

Two different ethyl(hydroxyethyl) cellulose (EHEC) samples were characterized by size-exclusion chromatography (SEC) with multiangle light scattering (MALS) detection and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The aim of the study was to investigate the molar mass distribution and the heterogeneity of the substituent distribution, factors that are thought to affect the functional properties of EHEC. The presence of blocks of unsubstituted glucose units was studied by enzymic degradation of EHEC by two different endoglucanases from Trichoderma reesei. The SEC-MALS analysis of the hydrolysis products showed that both enzymes were strongly inhibited by the large number of substituents along the cellulose chain. However, as the weight-average molar mass was reduced from approximately 360 000 to 80 000 g/mol in one of the polymers and from 770 000 to 60 000 g/mol in the other polymer, it was suggested that both samples were composed of some unsubstituted regions where the enzymes got access to the glucosidic bonds. The amount of glucose released upon endoglucanase hydrolysis was determined by HPAEC−PAD, which gave information on the homogeneity of the substituent distribution. The production of unsubstituted glucose units indicated that one of the polymers had a more uneven distribution compared with the other. It was demonstrated that chemical characterization of EHEC is a complex task, which requires an analytical approach involving numerous different methods and techniques.

Glassy carbon (GC) electrodes were modified with a layer of zirconium phosphate (ZrP), using either direct chemical synthesis onto the surface or a ZrP gel droplet evaporation procedure. The azine-type dyes nile blue A (NB) and toluidine blue O (TB) were immobilized onto the ZrP-modified GC electrodes either by adsorption onto just the formed layer of ZrP or by inclusion into the ZrP matrix during its chemical synthesis. The electrochemical behavior of the GC·ZrP·dye composite electrodes was studied. For GC·ZrP-modified electrodes prepared by chemical synthesis on the surface, coverage by NB or TB of one or a few monolayers was found, withEmvalues for these redox couples slightly shifting by ca. 0.05 V to the negative direction. For the GC·ZrP electrodes prepared by gel droplet evaporation, theEmvalues for NB and TB appear initially shifted by ca. 0.2 V to the positive direction; however, both cathodic and anodic peaks return to their usual positions on the potential scale during soaking these electrodes in a buffer solution.

The redox-active azine dyes Nile blue A (NB) and Toluidine blue O (TB) were electropolymerized after preadsorption onto the surface of graphite electrodes by potential cycling, using an anodic scan limit of 0.9 or 1.0 V vs SCE, where irreversible electrooxidation of the dye proceeds, yielding polymerizable species. Electropolymerization of both dyes is followed by the progressive disappearance of the characteristic current peaks in the cyclic voltammograms, and formation of new ones, shifted by ca. 0.21 V to the positive direction. Also, a decrease and gradual disappearance of the characteristic luminescence at a maximum of 671 nm was observed during the electropolymerization of NB. The resulting electrodes, modified by electropolymerized derivatives of NB and TB, were shown to be able to catalyze the electrooxidation of the coenzyme NADH. Thus, the modified electrodes prepared can be used for amperometric detection of the reduced form of the coenzyme.

An important aspect of the cytochrome c electrochemistry is the possibility of coupling the ‘heterogeneous reactions’ with other redox enzymes. Cellobiose dehydrogenase, a 89170 Da glycoprotein that contains both FAD and a b-type haem as prosthetic groups, donates electrons to a number of acceptors, including cytochrome c. While haem b is surrounded mainly by acidic amino acids, cytochrome c displays positive charged lysine groups around the haem site. Thus a fast reaction between both proteins is explicable. In the presence of cellobiose, a catalytic current was observed, owing to the interaction of cellobiose dehydrogenase with electrostatically adsorbed cytochrome c. Adsorption of cytochrome c provides a technological model surface for vectorial electron transfer.

Abstract: Electrochemical investigations of the reaction mechanism and kinetics between riboflavin immobilised on zirconium phosphate (ZPRib) in carbon paste and NADH showed results yielding reliable information about aspects on the mechanism of the electron transfer reaction between the flavin and NADH. The formal potential (E°′) of the adsorbed riboflavin was −220 mV versus SCE at pH 7.0. A shift about 250 mV towards a more positive potential compared with its value in solution was assigned to the interaction between the basic nitrogen of riboflavin and the acid groups of ZP. The invariance of the E°′ with the pH of the contacting solution and the effect of different buffer constituents were attributed to the protection effect of ZP over the riboflavin. The electrocatalytic oxidation of NADH at the electrode was investigated using cyclic voltammetry and rotating disk electrode methodology using a potential of −50 mV versus SCE. The heterogeneous electron transfer rate constant, k obs, was 816 M−1 s−1 and the Michaelis-Menten constant, K M, was 1.8 mM (confirming a charge transfer complex intermediate in the reaction) for an electrode with a riboflavin coverage of 6.8 × 10−10 mol cm−2. This drastic increase in the reaction rate between NADH and the immobilised riboflavin was assigned to the shift of the E°′. A surprising effect with addition of calcium or magnesium ion to the solution was also observed. The E°′ was shifted to −150 mV versus SCE and the reaction rate for NADH oxidation increased drastically.

Adsorption of a number of flavins, phenazines, phenoxazines, and phenothiazines on zirconium phosphate (ZP) was carried out in aqueous solution. The adsorbed organic dyes on ZP were used to prepare modified carbon paste electrodes. The electrochemical properties of the immobilized dyes were investigated and also their possible use to electrocatalytically oxidize NADH. The formal potential (E0′) of most of the adsorbed flavins, phenoxazines, and phenothiazines shifted markedly towards more positive potentials compared with their values in solution. The pH of the contacting solution did not affect their E0′‐values between pH 1 and 9. The phenazines did neither present good electrochemical response nor electrocatalytic activity for NADH oxidation and their E0′‐values remained pH dependent. In contrast, adsorbed flavins, phenoxazines, and phenothiazines presented good electron transfer rates between the electrode and the adsorbed molecule dye at pH 7.0.

The on‐line coupling of high‐performance anion‐exchange chromatography (HPAEC) with ion spray mass spectrometry, for the analysis of neutral and acidic oligosaccharides, is described. On‐line desalting is performed by a cation‐exchange membrane system which replaces sodium for hydronium ions from the effluent stream by means of either electrolysis of water or a combination of electrolysis and pneumatically supplied sulphuric acid. In contrast to formerly described systems no booster pump between the cation‐exchange system and the ion spray interface is needed, due to the almost complete absence of back‐pressure. The on‐line removal of sodium ions prior to the interface by means of electrolysis, and use of ion spray as an ionization technique, enables the routine use of gradients of sodium acetate up to a total sodium concentration of 0.6 M. After optimization of the HPAEC/MS system molecular mass determination could be obtained at concentrations down to 3 μg/mL (20 ng of each compound) for α‐1, 4‐glucose oligomers up to a degree of polymerization of 7 (DP7). For maltodextrines the applicable mass range could be extended to over 3000 Da by the detection of at least DP20, as the doubly charged disodiated molecule. A series of galacturonic acid oligomers could be detected up to DP4 at a concentration of 10 μg/mL (67 ng per comLpound), using a sodium acetate gradient increasing to a total sodium concentration of 0.6 M. © 1998 John Wiley & Sons, Ltd.

A fully integrated screening system for phenolic compounds was developed incorporating on-line solid phase extraction, fractionation and biosensor detection. Two different types of biosensors, solid graphite and carbon paste electrodes incorporating the enzyme tyrosinase, were compared and used in the screening system. Interfacing of the solid phase extraction and fractionation with the biosensor detection was given special attention since the biosensors were not compatible with the organic modifier used for desorption of phenols from the solid phase extraction step. The system was validated with conventional analytical techniques. Surface water samples from the Ebro river were spiked with 1,10, and 25 µg L−1of catechol, phenol,p-cresol, respectively. Three out of seven samples were spiked and the correct samples were identified, containing phenols equivalent to the spiked concentrations.

A two-dimensional, column liquid chromatographic system is used for the determination of the D- and L-enantiomers of amino acids in biological samples. Separation of the amino acids is first on ion-exchange column by gradient elution with a sodium citrate-sodium chloride buffer. Enantioseparation is by subsequent injection of 3 µl heart-cuts of the individual amino acids onto a second column with a chiral crown ether stationary phase. Finally, fluorescence detection is after post-column labelling of the amino acids using ano-phthalaldehyde-2-mercaptoethanol reagent solution. The high separation power and selectivity of the system allow processing of complex samples with hardly any additional treatment and the determination of small quantities of D-amino acids in the presence of excess L-form. Applicability of the system is illustrated by the determination of D- and L-aspartate, serine, glutamate and alanine in various complex biological samples, such as protein hydrolysates, urine and biotechnological and food samples. Data are given on detectability, repeatability and linearity.