Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole.

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP(C)) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP(C) interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP(C) with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP(C) and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

The study of surface properties of an IgE-sensitive aptasensor using an acoustic method.

We applied the acoustic transverse shear mode (TSM) method for study of the surface properties of a DNA aptasensor that specifically binds human immunoglobulin E (IgE). The biotinylated 45-mer DNA aptamers were immobilized on the surface of a self-assembled layer composed of a mixture of polyamidoamine dendrimers of the fourth generation with 1-hexadecanetiol covered by neutravidin. Using the TSM method, we studied the kinetics of changes of the series resonant frequency, f(s), and the motional resistance, R(m), of a quartz crystal transducer, used as a support for formation of the sensing layer. We have shown that attachment of the biotinylated DNA aptamers onto the surface covered by neutravidin results in a decrease of f(s), but in an increase of R(m). Similar changes of f(s) and R(m) were observed following addition of IgE. This suggests the contribution of friction forces to the crystal oscillation, which was taken into account in the calculation of the mass changes at the sensor surface following binding processes.

Properties of mixed alkanethiol-dendrimer layers and their applications in biosensing.

We studied the properties of mixed alkanethiol-dendrimer layers on a gold support and their application in biosensing. We showed that properties of glucose sensor can be modified using a different ratio of 1-hexadecanethiol (HDT) and poly(amidoamine) dendrimer of first generation (G1). The cyclic voltammetry in the presence of the redox couple, Fe(CN)(6)(3-)/Fe(CN)(6)(4-), was used for estimating how effectively the layer blocks the redox probe's access to the electrode surface. A scanning electrochemical microscope (SECM) was used to image the resulting distribution of the organic compounds. We found that with increasing content of dendrimers, the integrity of the layers was improved.

Properties of glucose biosensors based on dendrimer layers. Effect of enzyme immobilization.

The properties of glucose biosensors based on dendrimer layers on a gold support, which depend on the method of immobilization of glucose oxidase (GOX), were studied by amperometry. The kinetic parameters of enzymatic reactions, response time, sensitivity, detection limit, linear range, and enzyme turnover were determined. We showed that a more stable and sensitive sensor was obtained when GOX was immobilized on the dendrimer by crosslinking with glutaraldehyde in vacuum. This biosensor was stable for at least eight weeks. The response time was approximately 1.3 min, the detection limit of glucose was 25 micro M, and the apparent Michaelis-Menten constant was relative low ( K(m)=1.1+/-0.1 mM) in comparison with that for GOX in solution. The reason for these differences is discussed. The example of the application of the developed biosensors for the detection of mercury is also presented. The inhibitory effect of mercury on GOX activity was observed at mercury concentration of 100 nM.

Glucose biosensors based on dendrimer monolayers.

The peculiarities of glucose biosensors based on different generation of dendrimers (G0, G1 and G4) have been studied by amperometry and QCM techniques. It is shown that stable glucose biosensor can be obtained with low generation of dendrimers. The sensor sensitivity, however considerable, increased with increasing number of generation of dendrimers. This can be due to the increased volume of the dendrimer interior as well as with increased number of binding sites for glucose oxidase (GOX). QCM experiments showed that immobilization of GOX resulted in formation of enzyme multilayers on a dendrimer surface. The enzyme turnover for this system (0.1-0.01 s(-1)) was lower then that for immobilization of GOX onto a supported lipid films by means of avidin-biotin technology (1.1 s(-1)). However, dendrimer based biosensors are more stable in comparison with sBLM based sensors and could be stored in a refrigerator in dry conditions over 15 days without substantial loss of sensitivity.

Binding of avidin modified antibody to biotinylated metal supported membranes and liposomes changes the physical properties of lipid bilayer.

Various methods have been applied to study the physical properties of metal supported bilayer lipid membranes (s-BLM) and large unilamellar liposomes containing biotinylated phospholipids during the binding of IgG modified by avidin. Electrostriction method applied to s-BLM showed that addition of avidin-IgG (A-IgG) complex in a small concentration (0.2 mumol/l) resulted in an approximately twofold decrease of membrane capacitance, C, increase of elasticity modulus in direction perpendicular to the membrane plane, E perpendicular, by 5-20%, increase in intrinsic membrane potential, delta phi m, by approximately 30 mV, and an approximately 5-15% increase of the coefficient of dynamic viscosity, eta. The method of ultrasonic velocimetry showed that addition into the suspension of liposomes of unmodified IgG did not induce any changes in ultrasound velocity, while addition of A-IgG complex in a concentration range of 0-1 mumol/l led to an increase of the velocity number [u]. The plot of changes of [u] versus A-IgG concentration has a tendency to saturate at concentrations above 0.5 mumol/l, which suggests long-range interactions in the membrane induced by strong binding of A-IgG to the biotin sites on the membrane. Probably, this binding leads to a decrease of the coefficient of adiabatic compressibility of liposomes.

[Conductivity fluctuation of lipid bilayer membranes formed on a substrate]. / Fluktuatsii provodimosti visloinykh lipidnykh membran, sformirovannykhna podlozhkakh.

In the range of voltages -0.9-(+)0.9 V the fluctuations of conductivity of bilayer lipid membranes formed on the support (s-BLM) were measured. The new cut of the wire from stainless steel SS-10T served as the support. The similarity of current fluctuations for s-BLM as well as for support-electrolyte interface was observed, some quantitative distinctions are revealed. Three types of fluctuations are observed; 1) large current jumps observed as current steps at DC channel; 2) small current fluctuations near zero line with the spectrum of 1/f alpha type (flicker noise); 3) short current jumps, whose amplitudes are many folds higher than mean noise level, and the density of distribution function falls off as fluctuation magnitude raised to power 2.5 for s-BLM.

Stability of bilayer lipid membranes on different metallic supports.

In order to arrive at an optimized s-BLM protocol, the influence of various metallic supports (silver, iridium and stainless steel) of different diameters (from 0.14 to 0.33 mm) on the process of forming and stability of supported phospholipid bilayer membranes was studied. In addition, experiments were made using different membrane-forming solutions, ionic strength and pH. The transmembrane resistance and the current ratio between bare and lipid-coated wire at +670 mV were measured. Teflon-coated stainless steel support (0.33 mm in diameter) covered with 2% crude ox brain fraction of phospholipids was most convenient for the formation of supported bilayer lipid membranes on solid support. The well-known stabilizing effect of cholesterol was confirmed for the s-BLM system. Both the pH in the range 5.0-8.0 (silver and stainless steel supports) and the ionic strength (for all types of supports) in the range from 0.025 to 0.1 mol/l at pH 7.0 were found not to be crucial.

Application of biotin-streptavidin technology in developing a xanthine biosensor based on a self-assembled phospholipid membrane.

We have designed an amperometric xanthine minibiosensor based on a supported biotinylated phospholipid membrane. To this membrane streptavidin-modified xanthine oxidase was coupled. The fabricated biosensor corresponds in shape and size to a needle of 0.3 mm in diameter. The assay is based on the electrochemical detection of enzymatically generated hydrogen peroxide. The response to xanthine was linear up to 1 mmol.l-1 with a detection limit of 0.02 mmol.l-1. The response time was less than one minute and the biosensor was stable for at least five days.

Design of a glucose minisensor based on streptavidin-glucose oxidase complex coupling with self-assembled biotinylated phospholipid membrane on solid support.

A simple and fast procedure to prepare a glucose-sensitive minielectrode is presented. It is based on a biotin-modified phospholipid bilayer to which a streptavidin-glucose oxidase complex is coupled. The assay is based on the electrochemical detection of enzymatically generated hydrogen peroxide at the potential +670 mV. In the case of an air-saturated buffering solution the response to glucose was measured up to 50 mmol.L-1, with a linear portion up to 7 mmol.L-1. The influence of oxygen tension, pH, and temperature as well as possibly interfering substances was investigated. The prospect usage for the measurement of blood and urine was tested.

Influence of hypergravity on the pH profile and proteolytic activity of the avian gastrointestinal tract.

The present study deals with the effect of hypergravity (2xg) on the pH and on the proteolytic activity in the digesta of the gastrointestinal tract of Japanese quails during intense growth. The birds were raised on a semisynthetic diet containing free amino acids (A) and a commercial diet (B). During days 35 till 40 post-hatching the quails were exposed to hypergravity (2xg) using a specially designed centrifuge. On days 40 (experimental group, 2xg) and 41 (control group, 1xg) the animals were sacrificed. The pH of the digesta in various segments of the gastrointestinal tract was measured by means of a semi-microelectrode. Total proteolytic activity was determined by means of azo-dye-modified proteins serving as general proteolytic substrates. Hypergravity leads in general to an alkalization of digesta in various parts of the gastrointestinal tract. In case of the gizzard and duodenum (diet A) and also in the distal jejunum (diet B) the differences are significant. With both diets, hypergravity leads to a considerable decrease in the total proteolytic activity. The reduction is most expressed in the duodenum and jejunum. Changes in the pH of digesta compensate for the decrease in the proteolytic activity. This may explain why hypergravity per se does not seem to impair growth of the Japanese quails.

[Utilization of free and protein-bound lysine in the Japanese quail]. / Vyuzitie vol'ného a v bielkovinách viazaného lyzínu u prepelice japonskej.

The utilization of dietary lysine for protein synthesis is affected by the digestibility of protein-bound lysine, by its intestinal resorption and by its oxidative catabolism. The approach chosen in this paper enables a comparison of the cumulative effect of these processes on the utilization of free and protein-bound lysine, respectively. The principle of the approach is based on a quantification of the expiration of 14C-labelled carbon dioxide after an oral administration of a diet, which contains L-(U-14C)-lysine either as a free amino acid or bound to yeast proteins. During an adaptation phase cockerels of the Japanese quail received a diet based mainly on ground wheat and wheat gluten. This diet was supplemented either with yeast proteins or with a mixture of L-amino acids which simulates the composition of the yeast proteins. In the main experiment the expiration of labelled carbon dioxide was measured during 240 minutes after the administration of the corresponding labelled diets. Just before treatment the animals were either in the postprandial phase or in a state of slight hunger. The maximum of expiration of labelled carbon dioxide occurred around the 60th minute after administration of the corresponding labelled diets. The cumulative expiration of labelled carbon dioxide, expressed in per cent of the radioactive dose used, amounts to 15.5% and 14.3% for free and protein-bound lysine, respectively. The utilization of both forms of lysine in the Japanese quail is lower than in broilers.

Utilisation of free and protein dietary lysine in chicks estimated with isotopes.

1. Utilisation of supplementary free L-lysine hydrochloride was estimated in growing chicks and compared to that of protein-bound lysine. The technique used is based on the oxidative catabolism of either free (U-14C)-L-lysine or the labelled lysine incorporated into yeast proteins. 2. The animals received a wheat-wheat gluten diet which was L-lysine-supplemented with either unlabelled yeast proteins or a mixture of synthetic amino acids simulating the yeast proteins or L-lysine hydrochloride alone. 3. At 13 and 15 days after hatching, expiry of (14C)--carbon dioxide was followed 8 h after dosing with the appropriate radiolabelled diet. After 4 h, 0.66% of the protein-bound and 5.3 to 5.7% of the free lysine radioactivity appeared as 14C--carbon dioxide. 4. It is concluded that under these conditions lysine from both sources was utilised more efficiently than had been assumed hitherto, protein-bound lysine being slightly better utilised than free lysine.

[Methoxydextran as an indicator of gastrointestinal transit in Japanese quail]. / Metoxydextrán ako indikátor gastrointestinálneho tranzitu u prepelice japonskej.

The recovery of polyethylene glycol, a substance frequently used as an indicator of the dynamics in the liquid phase of intestinal chyme, is considerably influenced by the presence of trichloroacetic acid in certain applications. The suitability of (methoxy-14C)methoxydextrane as an intestinal indicator in the Japanese quail was tested in the study. After the administration of this indicator, the bird expires less than 1% of the substance in form of 14CO2. The recovery of labelled methoxydextrane is 98 +/- 7%. The distribution of the indicator within the segments of the gastrointestinal tract does not differ significantly from the distribution of (14C)polyethylene glycol 4000, measured in the absence of trichloroacetic acid. The transit of the labelled methoxydextrane into the jejunum did not reduce the radio-chemical purity of the isotope, as compared with its original purity before administration. A slight decrease was only recorded during the measurement of the cumulative excretion of the indicator for 72 hours, but this decrease has no influence upon the suitability of methoxydextrane as an intestinal indicator. (Methoxy-14C)methoxydextrane does not interact with trichloroacetic acid, whereas when polyethylene glycol is used as an indicator, the substance precipitates from the liquid phase just at the concentration commonly used for the removal of protein from biological samples.

Quantification of lysine dynamics in the Japanese quail.

To cockerels of the Japanese quail four graduated flooding doses of 14C-labelled L-lysine (20-160 mg) in starch gel were administered into the crop. The animals were slaughtered between 40 and 210 min thereafter. The TCA soluble fraction of the gastrointestinal tract, of blood plasma, liver and muscle were analyzed for their lysine content and its specific radioactivity. Incorporation of radioactivity into the TCA insoluble fraction of the same organs was also followed. After quantification of lysine losses by catabolism and excretion a multicompartment model of lysine dynamics in the quail was set up. Model parameter values in general agree with independently obtained data. The necessity of model validation and extension as well as possibilities for model applications are pointed out.