Phase-polarisation contrast for surface plasmon resonance biosensors

A technique of phase-polarisation contrast (PPC) for the enhancement of the contrast of a surface plasmon resonance (SPR) intensity profile is proposed and experimentally realised. The technique exploits the peculiarities of light phase and polarisation behaviour under SPR. It applies to non-optimum SPR coupling conditions and enables one to lower the resonant minimum of reflected intensity nearly to zero, and hence to increase substantially the ratio of the intensity from the resonance to that at the minimum. We observed the contrast enhancement by more than one order of magnitude when we applied the PPC scheme. The PPC can be efficiently employed in commercial SPR sensors, as it significantly reduces restrictions on allowable parameters of SPR-supporting metal films and biomolecular layers immobilised on them, facilitates SPR observation, and increases the accuracy of SPR shift measurements.     

Fucoxanthin metabolites in egg yolks of laying hens

Feeding experiments were conducted with White leghorn laying hens fed a carotenoid depleted control diet (containing some zeaxanthin and lutein) or this diet supplemented with 15% seaweed meal of established carotenoid composition. Egg yolk colour was estimated by use of a Roche Yolk Colour Fan and by detailed quantitative and qualitative carotenoid analysis of individual eggs of three laying hens during 4 weeks. Identification of the carotenoids included HPLC, VIS, MS, ¹H NMR data and partial synthesis. The results confirmed that fucoxanthin, the major carotenoid in seaweed meal, is not transferred to the yolk. However, fucoxanthin gave rise to the metabolites fucoxanthinol, fucoxanthinol 3'-sulphate and paracentrone, that are ascribed to enzymatic modifications occurring in the hens. The difuranoid auroxanthin encountered in the egg yolk was ascribed to violaxanthin and/or its furanoid derivatives present in the seaweed meal. Colour of individual yolks varied considerably. The pigmentation level is discussed.     

Volume conduction models for surface EMG; confrontation with measurements

Volume conduction models are used to describe and explain recorded motor unit potentials (MUPs). So far it has remained unclear which factors have to be taken into account in a volume conduction model. In the present study, five different models are confronted with measured MUP distributions over the skin surface above the m. biceps brachii generated by MUs at different depths and recorded by small surface electrodes. All model simulations include fibres of finite length. The models differ in the size of the volume conductor (finite/infinite), the number of different layers (1, 2 or 3) and the conductivities of these layers (representing muscle, subcutaneous fat and skin). All measured and simulated MUPs contain a mainly negative propagating wave followed by a positive wave simultaneously present at all electrode positions. The magnitude of the different MUP components relative to each other and as a function of motor unit (MU) and electrode position differ between the models studied and the measurements. All simulated MUPs changed faster with observation distance than the measured MUPs. The three-layer model, in which muscle tissue was surrounded by a subcutaneous fat layer and by a layer of skin resulted in MUPs closest to the measured MUPs.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

A new interference-free lysine biosensor using a non-conducting polymer film

An electrochemical biosensor for the determination of lysine to be used for rapid evaluation of food quality has been developed. Platinum electrodes have been coated by electropolymerisation with 1,2-diaminobenzene (1.2-DAB) using cyclic voltammetry. The reduction in the oxidation of interferents compared with the bare platinum electrode was 100% for ascorbic acid, 99% for acetaminophen and 99% for cysteine. The enzyme L-lysine-α-oxidase was then immobilised onto the polymer layer by passive adsorption and a calibration curve for lysine constructed. This gave a linear range of 1×10⁻⁵ mol/l to 1×10⁻³ mol/l and a limit of detection of 2×10⁻⁷ mol/l.     

Determination and pharmacokinetics of a furosemide–amiloride drug combination

The study presents an accurate and precise HPLC assay for the determination of furosemide and amiloride in human specimens. Both drugs were extracted from human plasma with ethyl acetate; furosemide was extracted at pH 1 and amiloride at pH 12. While chromatographic separation conditions, i.e., column, mobile phase and flow-rate were the same for both investigated drugs, furosemide was detected using a UV absorbance detector, whereas amiloride, because of its very low therapeutic range, was detected with a spectrofluorimetric detector. The linearity of the furosemide and amiloride assays were confirmed over the range of 30–3000 ng/ml and 0.5–30 ng/ml, respectively. These concentrations correspond well with the therapeutic ranges of both drugs. The extraction recoveries, depending on concentration, exceed 80% for furosemide and 74% for amiloride. The reported methods were applied to pharmacokinetic investigations of the two compounds taken in form of a drug combination.     

Preparation of clear noodles with mixtures of tapioca and high-amylose starches

Ways to simulate the making of clear noodles from mung bran starch were investigated by studying the molecular structures of mung bean and tapioca starches. Scanning electron micrographs showed that tapioca starch granules were smaller than those of mung bean starch. X-ray diffraction patterns of mung bean and tapioca starch were A- and C_A-patterns, respectively. Iodine affinity studies indicated that mung bean starch contained 37% of apparent amylose and tapioca starch contained 24%. Gel permeation chromatograms showed that mung bean amylopectin had longer peak chain-length of long-branch chains (DP 40) than that of tapioca starch (DP 35) but shorter peak chain-length of short-branch chains (DP 16) than that of tapioca starch (DP 21). P-31 n.m.r. spectroscopy showed that both starches contained phosphate monoesters, but only mung bean starch contained phospholipids. Physical properties, including pasting viscosity, gel strength, and thermal properties (gelatinization), were determined. The results of the molecular structure study and physical properties were used to develop acceptable products using mixtures of cross-linked tapioca and high-amylose maize starches. Tapioca starch was cross-linked by sodium trimetaphosphate (STMP) with various reaction times, pH values, and temperatures. The correlation between those parameters and the pasting viscosity were studied using a visco/amylograph. Starches, cross-linked with 0.1% STMP, pH 11.0, 3.5 h reaction time at 25, 35, and 45°C (reaction temperature), were used for making noodles. High-amylose maize starch (70% amylose) was mixed at varying ratios (9, 13, 17, 28, 37, and 44%) with the cross-linked tapioca starches. Analysis of the noodles included: tensile strength, water absorption, and soluble loss. Noodle sensory properties were evaluated using trained panelists. Noodles made from a mixture of cross-linked tapioca starch and 17% of a high-amylose starch were comparable to the clear noodles made from mung bean starch.     

Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma

The oxidative susceptibility of plasma obtained from rats after intragastric administration of quercetin was studied to know whether or not quercetin acts as an in vivo antioxidant after metabolic conversion. Quercetin was raised in the rat blood plasma essentially as glucuronide and/or sulfate conjugates. The plasma obtained from rats after quercetin administration was more resistant against copper sulfate-induced lipid peroxidation than the control plasma on the basis of the accumulation of cholesteryl ester hydroperoxides and the consumption of α-tocopherol. The results strongly suggest that some conjugated metabolites of quercetin act as effective antioxidants when plasma is subject to metal ion-induced lipid peroxidation.     

Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal dna sequences

Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (34–45%) and second (50–53%) internal transcribed spacers among the species was greater than in the 5.8S gene (3–5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern (s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.     

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca²⁺-ATPases is discussed.     

Novel system for real-time ex vivo lactate monitoring in human whole blood

The objective of the study was to evaluate the performance of an amperometric enzyme based lactate sensor and to investigate the possibility of replacing a double lumen catheter based blood withdrawal system with a heparin coated single lumen system. The inner lumen of a double lumen catheter which was placed in a peripheral vein was perfused with heparin solution. The outer lumen was used to collect heparinized blood samples at a defined flow rate. The single lumen system was attached to a heparinized catheter which was also placed in a peripheral vein. The undiluted blood samples were collected at a specified flow rate. A sensor flow chamber incorporating an amperometric thin-film lactate microbiosensor was placed in the sampling line for real-time lactate monitoring. Plasma lactate concentrations were measured during frequently performed hyperlactatemia bicycle ergometer experiments in six healthy volunteers (age 25.8±2.8 years, BMI 22.7±1 kg/m²). Additionally, plasma lactate was measured in real-time using the lactate sensors. The first three experiments were performed with a double lumen based catheter system whereas the following three experiments were performed with a heparin coated catheter system. The correlation coefficients of sensor readings and laboratory analyzer results in all six experiments were between 0.93 and 0.99, respectively (P<0.001). The miniaturized lactate sensors showed a linear range up to 25 mmol/l lactate concentration and 95% response times <30 s in undiluted serum. During the experiments maximum lactate concentrations of 14 mmol/l were achieved. Improvements of system performance using heparin coated catheter systems could be shown. The overall SD of the sensor readings compared to laboratory results using three double lumen catheter based systems was 0.91 mmol/l whereas the SD using three heparin coated systems was 0.65 mmol/l. In summary, real-time monitoring of lactate in human whole blood is feasible with such a device and can be improved by using heparin coated catheter systems.