Glucose biosensor based on disposable electrochemical paper-based transducers fully fabricated by screen-printing.

This paper describes a new approach for the massive production of electrochemical paper-based analytical devices (ePADs). These devices are fully fabricated by screen-printing technology and consist of a lineal microfluidic channel delimited by hydrophobic walls (patterned with diluted ultraviolet screen-printing ink in chromatographic paper grade 4) and a three-electrode system (printed with carbon and/or Ag/AgCl conductive inks). The printing process was characterised and optimized for pattern each layer with only one squeeze sweep. These ePADs were used as transducers to develop a glucose biosensor. Ionic strength/pH buffering salts, electrochemical mediator (ferricyanide) and enzyme (glucose dehydrogenase FAD-dependent) were separately stored along the microfluidic channel in order to be successively dissolved and mixed after the sample dropping at the entrance. The analyses required only 10 µl and the biosensors showed good reproducibility (RSD = 6.2%, n = 10) and sensitivity (0.426 C/M cm2), wide linear range (0.5-50 mM; r2 = 0.999) and low limit of detection (0.33 mM). Furthermore, the new biosensor was applied for glucose determination in five commercial soft-drinks without any sample treatment before the analysis. These samples were also analysed with a commercial enzymatic-kit assay. The results indicated that both methods provide accurate results.

Random walk in degree space and the time-dependent Watts-Strogatz model.

In this work, we propose a scheme that provides an analytical estimate for the time-dependent degree distribution of some networks. This scheme maps the problem into a random walk in degree space, and then we choose the paths that are responsible for the dominant contributions. The method is illustrated on the dynamical versions of the Erdos-Rényi and Watts-Strogatz graphs, which were introduced as static models in the original formulation. We have succeeded in obtaining an analytical form for the dynamics Watts-Strogatz model, which is asymptotically exact for some regimes.

Synthesis and functionalization of dextran-based single-chain nanoparticles in aqueous media.

Water-dispersible dextran-based single-chain polymer nanoparticles (SCPNs) were prepared in aqueous media and under mild conditions. Radiolabeling of the resulting biocompatible materials allowed the study of lung deposition of aqueous aerosols after intratracheal nebulization by means of single-photon emission computed tomography (SPECT), demonstrating their potential use as imaging contrast agents.

Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing.

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd(1) (cyt-cd(1)) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c(552) (cyt-c(552)), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 µM cyt-cd(1)/100 µM cyt-c(552) and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c(552) and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254±2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c(552) and cd(1) is 9.9×10(3)M(-1)s(-1). The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 µM; detection and quantification limits of 7 and 24 µM, respectively; sensitivity of 2.49±0.08 Amol(-1)cm(2) µM(-1). Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Resistance to benzimidazole and macrocyclic lactone anthelmintics in cattle nematodes in Argentina.

In April 2003, persistent scouring and ill-thrift that was reported in calves form an intensive beef rearing operation in central Argentina despite treatments with benzimidazole and ivermectin. In order to conduct a controlled faecal egg count reduction test on this herd, 40 calves 5-8-months-old were selected on the basis that they had a nematode eggs per gram (epg) of faeces count greater than 150. Animals were divided into four groups (1-4) of 10 calves. Calves of groups 1-3 were treated, respectively, with subcutaneous injection of ivermectin (200 mcg/kg), ricobendazole (4 mg/kg) and levamisole (7.5 mg/kg), while calves of group 4 remained as untreated controls. The egg count reductions carried out 10 days later were lower than 15% in calves treated with ivermectin and ricobendazole, but 100% in animals receiving levamisole. Pooled post-treatment faecal cultures showed larval percentages of 92 and 95 for Haemonchus and 8 and 5 for Cooperia in the faeces of calves treated with ivermectin and ricobendazole, respectively. This is the first reported case of Haemonchus parasiting cattle showing simultaneous resistance to avermectins and benzimidazole type anthelmintics.

GMR effect in CuCo annealed melt-spun ribbons.

A thorough microstructural and magnetic analysis has been performed on as-quenched and annealed (475 and 525 degrees C, 1 hour) melt-spun Cu100-xCox (x = 10 and 15) granular alloys, presenting a giant magnetoresistance (GMR) effect. The annealed samples are inhomogeneous with respect to the Co-particle sizes and interparticles distances and, therefore, these particles present superparamagnetic and ferromagnetic behaviours, which determine the GMR response. The samples x = 15, treated at 525 degrees C during 1 hour, presented the best GMR ratio (approximately 5% at room temperature to be the highest value approaching roughly to the saturation under an applied magnetic field of 15 KOe), with the coexistence of Co-particles with both kinds of magnetic behaviour.

Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A.

Immunisation against pathogens remains one of the most effective ways of preventing or reducing losses due to infectious diseases in animal husbandry. When inactivated vaccines are used, adjuvants are most often required to obtain satisfactory immune responses. One such type of adjuvant is saponin derived from the bark of Quillaja saponaria Molina, a tree of the rose family. A few different commercial sources exist, but due to the structural complexity and heterogeneity of these saponin preparations, it has been difficult to establish exactly which components are responsible for the adjuvant activity. By carefully selecting the bark source, we have succeeded in preparing a much less heterogeneous preparation of quillaja saponin. In this report we describe the preparation, in terms of structural complexity, hemolytic activity, adjuvant activity, and its ability to form ISCOM matrix. This new preparation could have implications for use per se, or as starting material for more effective preparation of pure substances.

Redox properties of the iron-sulfur clusters in activated Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough).

The periplasmic Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) contains three iron-sulfur prosthetic groups: two putative electron transferring [4Fe-4S] ferredoxin-like cubanes (two F-clusters), and one putative Fe/S supercluster redox catalyst (one H-cluster). Combined elemental analysis by proton-induced X-ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12-15 Fe) are present in 0.001-0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing reveals the existence of multiple charge conformers with pI in the range 5.7-6.4. Repeated re-chromatography results in small amounts of enzyme of very high H2-production activity determined under standardized conditions (approximately 7000 U/mg). The enzyme exists in two different catalytic forms: as isolated the protein is 'resting' and O2-insensitive; upon reduction the protein becomes active and O2-sensitive. EPR-monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equilibrium potentials are undefined, and previously reported apparent Em and n values [Patil, D. S., Moura, J. J. G., He, S. H., Teixeira, M, Prickril, B. C., DerVartanian, D. V., Peck, H. D. Jr, LeGall, J. & Huynh, B.-H. (1988) J. Biol. Chem. 263, 18,732-18,738] are not thermodynamic quantities. In the activated enzyme an S = 1/2 signal (g = 2.11, 2.05, 2.00; 0.4 spin/protein molecule), attributed to the oxidized H cluster, exhibits a single reduction potential, Em,7 = -307 mV, just above the onset potential of H2 production. The midpoint potential of the two F clusters (2.0 spins/protein molecule) has been determined either by titrating active enzyme with the H2/H+ couple (E,m = -330 mV) or by dithionite-titrating a recombinant protein that lacks the H-cluster active site (Em,7.5 = -340 mV). There is no significant redox interaction between the two F clusters (n approximately 1).

Application of hydrogenase in biotechnological conversions.

Evidence will be presented in this review article that the application of hydrogenase has large biotechnological possibilities. Our investigations show: Fast reaction of hydrogenase at an electrode surface to reduce H+; Photochemical production of H2 by hydrogenase by photosensitized Ru-complexes dissolved in reversed micellar membranes and vectorial H+ transport through the membrane to the water phase; The production of fine chemicals in reversed micelles by a system containing specific enzymes, hydrogenase and H2. The rules to obtain maximal conversion rates with this system will be presented.

A study of one of the iron-sulphur clusters in oxidized hydrogenase from Megasphaera elsdenii by magnetic-circular-dichroism spectroscopy.

The m.c.d. spectrum of the oxidized state of hydrogenase from Megasphaera elsdenii has been measured at liquid-helium temperatures. This oxidation state of the enzyme displays a characteristic rhombic e.p.r. signal with g-values of 2.101, 2.052 and 2.005 assigned previously to a [4Fe-4S]3+ cluster as in oxidized HiPIP (high-potential iron-sulphur protein) [Van Dijk, Grande, Mayhew & Veeger (1980) Eur. J. Biochem. 107, 251-261]. The low-temperature m.c.d. spectrum shows no features attributable to an oxidized four-iron cluster of the HiPIP type, but does reveal broad, positive peaks at 460 and 730 nm, which magnetize in a manner untypical of a spin S = 1/2 cluster with g-values close to 2. The m.c.d. spectrum is most closely similar to that of dye-oxidized P-clusters known in the enzyme nitrogenase. It is therefore proposed that the rhombic e.p.r. spectrum at a g-value close to 2 arises from an m.c.d.-silent radical species that may be related chemically to the cysteine persulphide species, RS-S., recently found in the hexacyanoferrate-oxidized seven-iron ferredoxin of Azotobacter vinelandii [Morgan, Stephens, Devlin, Stout, Melis & Burgess (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1931-1935].

The importance of quantitative Mössbauer spectroscopy of MoFe-protein from Azotobacter vinelandii.

The Mössbauer spectra of MoFe-protein of Azotobacter vinelandii, as isolated under dithionite and taken at temperatures from 125 K to 175 K, are the sums of four resolved quadrupole doublets. Our results indicate that the currently accepted interpretation of these doublets can be questioned. Our data reduction method converts the Mössbauer transmission spectra to source lineshape deconvolved absorption spectra linear in iron. We used these absorption spectra to determine the stoichiometry of the Fe clusters in MoFe-protein and we obtained much better fits if we assumed that there are four iron atoms in the 'Fe2+, doublet, two iron atoms in the 'S' doublet, twelve iron atoms in the 'D' doublet and sixteen iron atoms in the 'M' doublet. Therefore we propose that the MoFe-cofactor contains one molybdenum and eight iron atoms ('M'). We also argue that none of the previous Mössbauer spectroscopic studies have been performed on the highest-activity preparation now obtainable, nor has there been any study to prove that the Mössbauer spectra are independent of activity. We consider that the Mössbauer spectroscopic studies of the MoFe-protein of nitrogenase are a re-opened and unsolved problem.

Electron paramagnetic resonance and other properties of hydrogenases isolated from Desulfovibrio vulgaris (strain Hildenborough) and Megasphaera elsdenii.

The hydrogenases of Desulfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe-4S clusters, while M. elsdenii hydrogenase only releases part of its Fe-S clusters. The resting D. vulgaris enzyme shows only a small 3 Fe-xS type of EPR signal (maximum 5% electron equivalent). This amount can be increased to approximately 25% by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high-potential iron-sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin-like spectra.

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough).

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.

A pulse-radiolysis study of cytochrome c3. Kinetics of the reduction of cytochrome c3 by methyl viologen radicals and the characterisation of the redox properties of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough).

1. Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c3. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c3 are studied, The reaction between cytochrome c3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c3 molecule was Z = + 4.7 +/- 0.7. 2. After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome c3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, alpha. With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0 = - 0.250 - 0.088 alpha (in V). 3. An estimation is made of the energy of interaction between the haems due to electrostatic interactions (delta epsilon less than 32 mV) and due to ionic strength effects (- 12 mV less than delta epsilon less than 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c3 molecule are dependent on the degree of reduction of the other haems in the molecule. 4. The reaction of cytochrome c3 with MV+ or with ethanol radicals (EtOH) has been compared with the reactions of horse-heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV+ or EtOH with horse-heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.

On the molecular and submolecular structure of the semiquinone cations of alloxazines and isoalloxazines as revealed by electron-paramagnetic-resonance spectroscopy.

The thermodynamically stable and, therefore, analytically most important alloxazine and isoalloxazine radical cations have been studied in detail by electron paramagnetic resonance (EPR) spectroscopy. Isotopic and chemical substitutions have been made as in earlier studies with the less stable neutral and anionic species. The experimental spectra have been calculated with the aid of a more sophisticated computer-simulation program than previously used. Excellent fits were obtained only when all of the following atoms were taken into account in the hyperfine coupling scheme: N-5 H, N-10 H or CH3, C-6 H, C-7 H, C-8 H or CH3 and C-9 H. An additional but small coupling constant was required for the fit. This latter coupling constant is assigned to the nitrogen atom(s) of the pyrimidine subnucleus of (iso)alloxazine radical cations. The EPR-active proton is attached to N-5 as we also found for the neutral flavosemiquinone. The alloxazine and isoalloxazine radical cations exhibit an identical hyperfine coupling scheme but differ especially in the pyrazine nucleus with respect to the spin density distribution. This suggests that the geometrical structure of the two kinds of radicals is somewhat different. The highest spin density is, however, located at N-5 of (iso)alloxazine as has been found for the other flavosemiquinone species. The hyperfine coupling constants are interpreted in terms of spin densities and comparison is made with the most recently available quantum chemical calculations. All monomeric flavosemiquinone species are compared with each other and their differences in the submolecular structure are discussed briefly.

Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.

Protein mobility inside pyruvate dehydrogenase complexes as reflected by laser-pulse fluorometry. A new approach to multi-enzyme catalysis.

The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme-free complex is compared with complex to which Mg2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 degrees C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipo-amide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.