Sensing glucose concentrations at GHz frequencies with a fully embedded Biomicro-electromechanical system (BioMEMS).

The progressive scaling in semiconductor technology allows for advanced miniaturization of intelligent systems like implantable biosensors for low-molecular weight analytes. A most relevant application would be the monitoring of glucose in diabetic patients, since no commercial solution is available yet for the continuous and drift-free monitoring of blood sugar levels. We report on a biosensor chip that operates via the binding competition of glucose and dextran to concanavalin A. The sensor is prepared as a fully embedded micro-electromechanical system and operates at GHz frequencies. Glucose concentrations derive from the assay viscosity as determined by the deflection of a 50 nm TiN actuator beam excited by quasi-electrostatic attraction. The GHz detection scheme does not rely on the resonant oscillation of the actuator and safely operates in fluidic environments. This property favorably combines with additional characteristics-(i) measurement times of less than a second, (ii) usage of biocompatible TiN for bio-milieu exposed parts, and (iii) small volume of less than 1 mm3-to qualify the sensor chip as key component in a continuous glucose monitor for the interstitial tissue.

[Permeability of cyanobacterial mucous surface structures for macromolecules].

The space of diffusive distribution of neutral hydrophilic macromolecules (dextrans with molecular sizes of 1.5 to 9 nm in the Stokes radius values) in the mucous surface structures (MSS) of intact bacterial cells has been studied for the first time on cyanobacteria. Cyanobacterial species and strains under study belong to different taxonomic groups, the members of which form MSS of various morphology and ultrastructure and can grow in association with plants and animals, inter alia as mucous microcolonies. The range of permeability has been determined by the fractionation of polydisperse dextrans method, previously applied for plants, in combination with electron microscopy. Dextrans are supposedly distributed in the MSS polysaccharide matrix in accordance with their sizes, in much the same way as in a macroporous unitary gel. The similarity of the chemical composition and macromolecular organization of cyanobacterial MSS with pectins of plant cell walls and the role of MSS and the intercellular matrix as permeability barriers in associative interactions of microorganisms are under consideration.

A long-term stable and adjustable osmotic pump for small volume flow based on principles of phloem loading.

An adjustable pump for microfluidics employing principles of osmoregulation analogous to those of phloem loading in plant leaves has been constructed and tested. Volume flow arises in a hollow fibre with vapour-permeable hydrophobic membrane. The fibre is connected to a source chamber filled with salt crystals and saturated salt solution. The source chamber takes up water through a relatively small membrane area and delivers saturated salt solution to one end of the capillary flow path within the hollow fibre. A stationary osmotic gradient is sustained in the hollow fibre lumen by constant input of saturated salt solution and radial osmotic water absorption. The strong temperature dependence of isothermal membrane distillation enables adjustment of the flow rate up to 20 nL/s. The pump provides pulse-free flow of any liquid with constant rate for at least 26 days without recharging the source chamber. Backpressures up to 1 bar decrease the flow rate by less than 4%. The volume delivered at a constant rate is more than 40 times larger than the volume of the source chamber. Osmoregulatory pumps of the described type may be useful for microinfusion, microdialysis and analytical microsystems.

Calibration of the viscometric glucose sensor before its use in physiological liquids--compensation for the colloid-osmotic effect.

The function of the recently described viscometric affinity sensor (VAS), which measures glucose due to its strong effect on the viscosity of a sensitive liquid containing Concanavalin A (ConA) and dextran, was analysed for osmotic and colloid-osmotic effects on the glucose reading. The suction of low- and high-molecular weight osmotica on the membrane of the microdialysis fibre was measured using a membrane osmometer built from the microdialysis probe of the VAS. The reduction of the sensor read-out in blood plasma can be completely explained by a change in small osmotic volume fluxes through the dialysis membrane, which affect the ConA concentration and the viscosity after the flow of the sensitive liquid through the dialysis probe. The measuring error could be prevented by the presence of the polyethylene glycol 6000 at an isotonic concentration in the glucose standard solutions used for sensor calibration.

Diffusion barriers of tripartite sporopollenin microcapsules prepared from pine pollen.

Tripartite sporopollenin microcapsules prepared from pine pollen (Pinus sylvestris L. and Pinus nigra Arnold) were analysed with respect to the permeability of the different strata of the exine which surround the gametophyte and form the sacci. The sexine at the surface of the sacci is highly permeable for polymer molecules and latex particles with a diameter of up to 200 nm, whereas the nexine covering the gametophyte is impermeable for dextran molecules, with a Stokes' radius > or =4 nm (Dextran T 70), and for the tetravalent anionic dye Evans Blue (Stokes' radius = 1.3 nm). The central capsules obtained by dissolution of the sporoplasts showed strictly membrane-controlled exchange of non-electrolytes, with half-equilibration times in the range of minutes (monosaccharides, oligosaccharides) to hours (dextran molecules with Stokes' radii up to 2.5 nm). The dependence of the permeability coefficients of the nexine for non-electrolytes on Stokes' radius or molecular weight shows that the aqueous pores through the nexine are inhomogeneous with respect to their size, and that most pores are too narrow for free diffusion of sugar molecules. To explain the barrier function of the nexine for Evans Blue, it is assumed that at least the larger pores, which enable slow permeation of dextran molecules, contain negative charges.

Recording of subcutaneous glucose dynamics by a viscometric affinity sensor.

AIMS/HYPOTHESIS: To provide a nonenzymatic sensor for glucose monitoring in subcutaneous tissue. METHODS: A continuously working affinity sensor based on the glucose-dependent viscosity of a sensitive liquid containing dextran and concanavalin A has been designed by arranging a microdialysis probe, two flow-resisting capillaries and two pressure transducers in a linear flow system. It allows synchronous processing of the viscosity of the sensitive liquid at the standard glucose concentration and the glucose concentration to be measured. In preliminary human trials the sensor was implanted into the subcutaneous tissue of the forearm and its read-out was compared with capillary blood concentrations. RESULTS: In vitro, the viscometric sensor shows a linear and long-term stable dependence on the glucose concentration without detectable drift. At the applied flow rate of the sensitive liquid (about 5 microliters/h) the technical delay is 5 to 10 min. The read-out of the implanted sensor followed the dynamics of the capillary blood glucose concentrations with a time-shift of 10 to 15 min but showed a systematic error when based on precalibration with polymer-free glucose solutions. After appropriate in vivo calibration, the read-out was in good or acceptable coincidence with capillary blood concentrations according to the error grid method and did not show any detectable reduction of sensitivity during the periods of measurement (up to 44 h). CONCLUSION/INTERPRETION: The viscometric-affinity sensor is an efficient tool for current research on glucose monitoring in the subcutaneous tissue and can potentially be further developed for routine clinical use.

Compensation of temperature and concanavalin A concentratration effects for glucose determination by the viscometric affinity assay.

A viscometer suitable for rapid measurements in small volumes of highly viscous liquids is described. Using this device the viscometric affinity assay for glucose was studied under variable conditions in order to obtain basic information for the design of a viscometric glucose sensor. The viscosity of the dextran/Concanavalin A (ConA) solution is sensitive to glucose in a broad range of the shear stress. However, for measuring the glucose concentration with this sensitive liquid the strong dependence of the absolute viscosity on temperature and ConA concentration has to be taken into account. For the purpose of calibration a parameter more suitable than the absolute viscosity is the relative fluidity (F(r)) that is defined by the actual measured viscosity at a given glucose concentration, the reference viscosity at a standard glucose concentration, and a constant linearization coefficient. F(r) shows a linear dependence on the glucose concentration in the therapeutically interesting range up to 30 mM and is not significantly changed by moderate variations of the ConA concentration or temperature.

Additional phosphate stabilises uninterrupted growth of a Dioscorea deltoidea cell culture.

Suspension cells of Dioscorea deltoidea Wall (strain D-1) were maintained in a semicontinuous culture (SCC) in shake flasks at a high growth rate. It was shown that continuous propagation growth of this culture is unstable on Murashige's and Skoog's (MS) medium due to P starvation. On a P-enriched MS-medium the SCC was stable even at mean specific growth rates >0.3 day(-1). Highest volumetric concentrations of furostanol glycosides were obtained, when a P-enriched SCC was not further subcultivated but fed with sucrose. The investigated culture is able to control phosphate uptake and to prevent toxicity on media with excess P. High concentrations of cellular P(i) did not effect the ratio of furostanol to starch.

The Pore Size of Non-Graminaceous Plant Cell Walls Is Rapidly Decreased by Borate Ester Cross-Linking of the Pectic Polysaccharide Rhamnogalacturonan II.

The walls of suspension-cultured Chenopodium album L. cells grown continually for more than 1 year on B-deficient medium contained monomeric rhamnogalacturonan II (mRG-II) but not the borate ester cross-linked RG II dimer (dRG-II-B). The walls of these cells had an increased size limit for dextran permeation, which is a measure of wall pore size. Adding boric acid to growing B-deficient cells resulted in B binding to the wall, the formation of dRG-II-B from mRG-II, and a reduction in wall pore size within 10 min. The wall pore size of denatured B-grown cells was increased by treatment at pH

Binding of water, oil, and bile acids to dietary fibers of the cellan type.

Dietary fibers (DF) of the "cellan" type (consisting mainly or exclusively of undestroyed cells) were prepared as ethanol-dried materials from apple, cabbage, sugar-beet, soybean hulls, wheat bran, and suspension cultures of Chenopodium album L. and investigated with respect to their interactions with water, water-oil dispersions, bile acids, and oil. Water binding and retention capacities were found to be especially high in cellans obtained from thin-walled raw material. Water damp sorption by dry cellans, when analyzed according to the GAB and BET equations, shows a considerable fraction of monolayer water. At a water activity of 0.98, the cell and capillary spaces outside the walls remained in the air-filled state but the cell wall pores are filled with water. When the water content of a concentrated aqueous cellan suspension was equal to or below the water binding capacity, its rheological behavior was found to be of pseudoplastic nature. At a given dry weight concentration, yield stress and viscosity of such concentrated suspensions were highest for cellans with the highest water binding capacity. Dry cellan particles absorbed fatty oils without swelling but swell in a detergent-stabilized oil/water emulsion with a similar liquid absorption capacity as in water. In contrast to the dry or alkane-saturated cell wall, the hydrated wall is not permeable to oils in the absence of a detergent. Oil droplets may be entrapped within the cells, yielding a stable dispersion of oil in water. As DF of the cellan type absorb bile acids, preferentially glycoconjugates, from diluted solutions, they may have a potential to decrease the serum cholesterol level.

The boron requirement and cell wall properties of growing and stationary suspension-cultured chenopodium album L. cells

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (>/=7 &mgr;M) to the boron-deficient cells. C. album cells grown in the presence of 100 &mgr;M boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 &mgr;M boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 &mgr;M boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.

Apoplasmic and Protoplasmic Water Transport through the Parenchyma of the Potato Storage Organ.

Stationary volume fluxes through living and denatured parenchyma slices of the potato (Solanum tuberosum L.) storage organ were studied to estimate the hydraulic conductivity of the cell wall and to evaluate the significance of water transport through protoplasts, cell walls, and intercellular spaces. Slices were placed between liquid compartments, steady-state fluxes induced by pressure or concentration gradients of low- and high-molecular-mass osmotica were measured, and water transport pathways were distinguished on the basis of their difference in limiting pore size. The protoplasts were the dominating route for osmotically driven water transport through living slices, even in the case of a polymer osmoticum that is excluded from cell walls. The specific hydraulic conductivity of the cell wall matrix is too small to allow a significant contribution of the narrow cell wall bypass to water flow through the living tissue. This conclusion is based on (a) ultrafilter coefficients of denatured parenchyma slices, (b) the absence of a significant difference between ultrafilter coefficients of the living tissue slices for osmotica with low and high cell wall reflection coefficients, and (c) the absence of a significant interaction (solvent drag) between apoplasmic permeation of mannitol and the water flux caused by a concentration difference of excluded polyethylene glycol. Liquid-filled intercellular spaces were the dominating pathways for pressure-driven volume fluxes through the parenchyma tissue.

Viscosimetric affinity assay.

Affinity ligands and/or affinity receptors may be quantified by a viscosimetric assay which can be carried out with a simple technique and has the potential of broad applications. The viscosimetric affinity assay is based on the high contribution of affinity bonds to the viscosity of an aqueous dispersion of a hydrocolloid that is bearing affinity ligands. In dispersions of such sensitive hydrocolloids at a concentration above the overlapping point, agglutination is not possible and the modulation of viscosity by the formation or dissociation of intercolloidal affinity bonds may be several orders of magnitude larger than the basic viscosity measurable in the absence of intercolloidal affinity bonds. If dispersions (30 g liter-1) of branched dextran with high molecular weight were used as reagent for concanavalin A (Con A), the Con A concentration necessary for a significant rise in viscosity was decreased with increasing colloid size. The viscosity of dispersions containing both a ligand-bearing high-molecular-weight dextran and an appropriate polyvalent receptor protein (lectin or antibody) showed a dependence on the concentration of free ligands (sugars or insulin) according to the law of mass action. In this competitive mode the viscosimetric affinity assay seems to be well adaptable to many analytical problems.

[Separation of recombinant proteins by chromatography on vesicular sorbents]. / Razdelenie rekombinantnykh belkov s pomoshch'iu khromatografii na vezikuliarnykh sorbentov.

Vesicle chromatography, a recently developed method for separation of biomolecules, uses the vesicular packing (VP) material (clusters of microcapsules derived from plant cells), which was tested with respect to its application for the recombinant protein separation. Since VP has a well-defined separation limit, biomolecules are distributed in two separate peaks: large molecules are excluded and small molecules permeate through cell walls into the empty cell lumen. Recombinant proteins frequently form oligomers, which differ from monomers not only in size but also chemically and biologically. In the present study, separations of the recombinant proinsulin fusion protein oligomer and monomer, the recombinant human gamma-interferon monomer and dimer and recombinant tumour necrosis factor-alpha were investigated. For peak identification, the fractions and starting samples of the recombinant proteins were analysed by HPLC. The separations occurred without any sorption effects and with high efficiency and resolution of the protein peaks at a short column (10 cm). The VP is characterised by a high load ability, which favours the scale-up purification of the recombinant proteins. The combination of VP and HPLC is a considerable advance in biotechnology separation.

Induction of betacyanin formation in Chenopodium album cell cultures by co-cultivation with the duckweed Wolffia arrhiza.

Cells of Chenopodium album and whole plants of the duckweed Wolffia arrhiza were cocultivated. In the presence of Wolffia arrhiza the synthesis of a red-violet pigment (betacyanin) was induced in several cells or cell clusters of Chenopodium album in the light. The exchange of solutes through the liquid phase was necessary for the induction of pigment formation. The red-violet cells could be selected and subcultivated resulting in a red callus. A reddish cell suspension was obtained in liquid culture in the presence of the duckweeds.

Entrapment of dextran in plant cell capsules by reversible change of cell wall permeability.

Vesicular packing material (VP) made of clusters of extracted higher plant cells with the intact framework of their cell wall was used so far for permeation chromatography (vesicle chromatography). The objective of this study was to devise a method to entrap dextran in the vesicles. This can provide a means to entrap biocatalysts and secondly, to create aqueous two-phase systems with a stationary dextran phase for liquid-liquid partition chromatography. Dextran of molecular sizes above the separation limit of the plant cell wall cannot permeate into the intracellular space in aqueous medium. However, in hydrophilic organic solvent/water mixtures, dextran molecules can diffuse into the capsules. The removal of the organic solvent leaves the dextran trapped inside. There was an inverse correlation between the percentage of dextran permeating through the cell wall (Pperm) and the concentration of solvent required for dextran precipitation. The increase of permeability is therefore considered to be caused, to a great extent, by the decrease of the effective size of dextran molecules due to decreased solvation. Pperm was inversely correlated to the dielectric constants and the polarities of the solvents and, in the case of protic solvents, the hydrogen-bond acidities. No correlation was found to the hydrogen-bond basicities.

Transient changes in length and growth of wheat coleoptile segments following treatments with osmotica and auxin.

The dependence of elongation on the osmotic potential of the medium was investigated, using coleoptile segments (CS) of Triticim aestivum L. (cv. Hartri) and an optoelectronic device. The study aimed at separating the osmoelastic response from the irreversible growth response when an osmoticum (mannitol) was added, and to compare both processes in order to consider the possibility of growth-induced reduction in turgor pressure. The prompt inhibition of elongation registered just after addition of 50 mM mannitol as well as the subsequent resumption of the original elongation rate could be quantitatively explained by the extent and the kinetics of the osmoelastic relaxation. An initial reduction in the irreversible elongation component by mild osmotic stress could not be demonstrated. Above a critical value, the irrevesible growth was insensitive to a further increase in water potential. The minimum turgor pressure required to drive steady growth was not far from zero in both the presence and absence of auxin. The rate (r) of osmotically caused shortening per unit change of water potential [Formula: see text] was determined from the kinetics of CS shortening induced by addition of mannitol at nearly isotonic concentration (300 mM). This parameter relates a fractional change in length to the difference in water potential between inside and outside, and was assumed to depend largely on the hydraulic resistance of the tissue and cuticle. It was found to be independent of IAA. The relatively low value of Γ suggests significant reduction of turgor at high growth rates. In accordance with this conclusion, the extent of osmoelastic shortening after a transfer to 300 mM mannitol (dependent on wall strain) was significantly decreased in the presence of IAA. Addition of 100 µM IAA to CS growing at a constant rate induced pronounced oscillations in the rate of elongation, which may be connected with the change in elastic cell wall strain. Whereas the steady state growth rate before the addition of IAA was the same in the presence and in the absence of 50 mM mannitol, the maximum growth rate found after addition of IAA was substantially reduced in the mannitol variant.

Inclusion and fractionated release of nucleic acids using microcapsules made from plant cells.

The encapsulation and fractionated release of nucleic acids on vesicular packing (VP) materials have been investigated. The earlier described dependence of the permeation of nucleic acid molecules through the vesicle membranes on the salt concentration is a necessary precondition for both encapsulation and fractionation. Encapsulation is achieved by applying a suitable sample onto a VP column that has been equilibrated with a high-salt buffer. In that buffer the sample molecules are permeable. Immediately after sample application, elution is started with a low-salt buffer, from which the sample molecules are excluded. At the front between the two buffers the permeability changes, and some of the sample molecules distributed inside the vesicles cannot pass through the membranes. These encapsulated molecules can be released by increasing the salt concentration in the eluent. If the encapsulated nucleic acid sample is polydisperse, a stepwise or linear increase in the salt concentration leads to a fractionated release. The fractions obtained differ in their molecular size composition.

[Permselect--a new substance for leukocyte elimination]. / Permselect--ein neues Material für die Leukozytenelimination.

Permselect particles consisting of purified plant cell wall ghosts obtained from the biomass of a suspension culture of Chenopodium album L. show a very high efficiency for leukocyte binding. A package containing 1 g dry Permselect eliminates 99-100% of leukocytes from 80 ml of fresh blood, preserved whole blood, or red blood cell concentrates. At comparable conditions Permselect shows a higher efficiency of leukocyte binding than commercial filter material (cotton, cellulose acetate). The vitality parameter of filtered erythrocytes and their stability during storage were not altered. The data are discussed with respect to causality of leukocyte binding to the pectin-containing surface of the cell wall particles.

[Exclusion chromatography for the separation of cryoprotective agents from freeze-preserved blood cells]. / Ausschlusschromatographie zur Trennung kryoprotektiver Substanzen von gefrierkonservierten Blutzellen.

A method for separating low molecular cryoprotectiva from freeze-conserved erythrocyte- and thrombocyte-concentrates by exclusion chromatography has been described. A new vesicular packing material has been used. Only 25 to 30 minutes are necessary in order to separate glycerol respectively dimethylsulphoxide (DMSO) completely from the cells. 86.5% of the erythrocytes and 75.4% of the thrombocytes were recovered after the separation process.