Peter Rybár

High sensitive calixarene-based sensor for detection of dopamine by electrochemical and acoustic methods.

We synthesized 25,26,27,28-tetrakis(11-sulfanylundecyloxy)calix[4]arene (CALIX) sensitive to dopamine and confirmed its structure by (1)H NMR and mass spectrometry. Chemisorption of CALIX molecules or their mixtures with 1-dodecanethiols (DDT) or hexadecanethiols (HDT) resulted in formation of compact low permeable monolayers as revealed by cyclic voltammetry at presence of redox probe [Fe(CN)(6)](3-/4-). These self-assembled monolayers (SAMs) served as sensor for dopamine. Thickness shear mode acoustic method (TSM) has been used for study the interaction of dopamine with calixarene SAM. The admittance spectra of TSM transducer have been measured and used for simultaneous determination of the changes in series resonant frequency, f(S), and motional resistance, R(m), respectively. Addition of dopamine resulted in substantial decrease of f(S) and increase of R(m), which is evidence on increased viscoelastic contribution into the acoustic properties of the sensing layer. Limit of detection (LOD) for dopamine was 50 pM, which is much better in comparison with so far reported lowest LOD for dopamine-sensitive electrochemical sensors (20 nM). The sensor allowed discrimination between dopamine and epinephrine.

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl^phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.

Interaction of crystalline bacterial cell surface proteins with lipid bilayers in liposomes. A sound velocity study

Abstract Sound velocity and density measurements have been used to study physical properties of plain unilamellar liposomes and covered by a crystalline cell surface layer (S-layer) of Bacillus coagulans, strain E38-66. The liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and hexadecylamine. The measured parameters — velocity number [ u l and density ρ — decrease with increasing temperature for both suspensions of liposomes with S-layers (S-DPPC) and without S-layers. The values of [ u ] at T T >29°C larger for S-DPPC than those of plain liposomes. The density of the S-DPPC system was slightly greater than those of plain bilayer suspensions. The specific adiabatic compressibility, ϕ k / β 0 , calculated from the values of [ u ] and of the specific volume, ϕ k , increases with temperature for both liposome systems. The comparison of the parameter ϕ k / β 0 for S-DPPC and DPPC indicates that the adiabatic compressibility of S-DPPC at T T >20°C was lower in comparison with that of plain DPPC. This is evidence of an interesting phenomenon of softening and condensing effects of S-proteins on the lipid bilayer depending on the temperature.

Molecular acoustic as a new tool for the study of biophysical properties of lipoproteins.

The method of measurement of velocity and absorption of ultrasound at a fixed frequency (7.2 MHz) and measurement of density were used to study the physical properties of high- (HDL3) and low- (LDL) density lipoproteins. We found substantial changes in velocity number [u] and absorption number [alpha lambda] on temperature, which reflect structural changes in the hydrophobic core of LDL at the thermotropic-phase transition. The absorption number revealed broad changes in temperature for both classes of lipoproteins (LP). The density of LP also depends on temperature but in considerably less degree than the acoustic parameters. The values of acoustic parameters were determined, showing that LDL and HDL3 greatly differ with respect to adiabatic compressibility.

Affinity interactions on a liposome surface detected by ultrasound velocimetry

In this work, we performed targeted immobilization of immunoglobulins by means of bacterial S-layer proteins from Bacillus coagulans E38-66/V1 recrystallized on liposomes, which were exploited as immobilization matrix for antibody (Ab)-human IgG. The study of interaction of rabbit or swine anti-human IgG as antigens (Ag) was performed by means of measuring changes of ultrasound velocity. We showed that at a temperature of 25 degrees C, the increment of ultrasound velocity [u] linearly decreased following an increase of concentration of Ag. The decrease of [u] was presumably due to changes of hydration of the membrane due to the binding process. Approximately 10 times lower changes of [u] were observed at 45 degrees C for Ag-Ab interaction as well as for nonspecific interaction of Ag with liposomes covered by S-layer without Ab. No substantial differences in the behaviour of [u] were observed for interactions of human IgG with rabbit or swine anti-human IgG.

Aptamer-based detection of thrombin by acoustic method using DNA tetrahedrons as immobilisation platform

The thickness shear mode acoustic method was used to study the binding of thrombin to DNA aptamers immobilised on the gold surface covered by DNA tetrahedrons. The binding of thrombin to conventional aptamers sensitive to fibrinogen (FBT) and heparin (HPT) exosites as well as to HPT in a loop configuration (HPTloop) made it possible to determine the constant of dissociation (KD) and the limit of detection (LOD). The sensing system composed of a HPTloop was characterised by KD = (66.7 ± 22.7) nM, which was almost twice as low as that of FBT and HPT. For this biosensor, a lower LOD of 5.2 nM compared with 17 nM for conventional HPT aptamers was also obtained. Less sensitive sensors based on FBT aptamers revealed an LOD of 30 nM which is in agreement with the lower affinity of these aptamers to thrombin in comparison with that of HPT. The surface concentration of DNA tetrahedrons was determined by the electrochemical method using [Ru(NH3)6]3+ as a redox probe. These experiments confirmed that the “step by step” method of forming the sensing layer, consisting first in chemisorption of DNA tetrahedrons onto a gold surface and then in hybridisation of the aptamer-supporting part with complementary oligos at the top of the tetrahedron, is preferable. In addition, atomic force microscopy was applied to analyse the topography of the gold layers modified stepwise by DNA tetrahedrons, DNA aptamers and thrombin. The height profiles of the layers were in reasonable agreement with the dimensions of the adsorbed molecules. The results indicate that DNA tetrahedrons represent an efficient platform for immobilisation of aptamers.

Local anesthetic tetracaine influences the physical properties of globular proteins

Abstract The interaction of local anesthetic tetracaine (TTC) with globular protein-bovine serum albumin (BSA) was studied by the measurement of sound velocity and density. We showed, that in its native form, at pH 7, the TTC in a concentration of 0.1 mmol l –1 resulted in an increase of adiabatic compressibility of BSA, while practically no changes of adiabatic compressibility was observed, when native form has been lost or changed (at pH 3). The binding of TTC on BSA was shown by measurement fluorescence quenching. Addition of TTC resulted BSA tryptophan fluorescence quenching that saturates at TTC concentration 1–3 mmol l –1 . The dissociation constant, K d =1.18±0.54 mmol l –1 , related to the binding of TTC to BSA was determined from fluorescence quenching experiments. We assume that TTC incorporates into hydrophobic core of BSA. This incorporation caused increase of BSA specific volume and consequently the conformational freedom of polypeptid chain could be the main reason of increase of BSA adiabatic compressibility.

Specific volume and compressibility of bilayer lipid membranes with incorporated Na,K-ATPase

Ultrasound velocimetry and densitometry methods were used to study the interactions of the Na,K-ATPase with the lipid bilayer in large unilamellar liposomes composed of dioleoyl phosphatidylcholine (DOPC). The ultrasound velocity increased and the specific volume of the phospholipids decreased with increasing concentrations of protein. These experiments allowed us to determine the reduced specific apparent compressibility of the lipid bilayer, which decreased by approx. 11% with increasing concentrations of the Na,K-ATPase up to an ATPase/DOPC molar ratio = 2 × 10⁻⁴. Assuming that ATPase induces rigidization of the surrounding lipid molecules one can obtain from the compressibility data that 3.7 to 100 times more lipid molecules are affected by the protein in comparison with annular lipids. However, this is in contradiction with the current theories of the phase transitions in lipid bilayers. It is suggested that another physical mechanisms should be involved for explanation of observed effect.

Adiabatic compressibility of red blood cell membrane: influence of skeleton.

Measurements of ultrasound velocity and density were used for determination of the adiabatic compressibility of red blood cells (RBC) during detachment of the membrane skeleton. Skeleton detachment was induced by addition of nystatin into a low ionic strength RBC suspension resulting in an increase (10%) of the ultrasound velocity concentration increment, [u], while the specific volume of cells, phi(v) did not change significantly. Changes of the concentration increment had rather long kinetics and were not completed even after 60 min. Both [u] and phiV values were used for calculation of the specific apparent adiabatic compressibility of RBC, phiK/beta0. The value of the specific apparent compressibility decreases following addition of nystatin. This corresponds to an increase in the volume elastic modulus of RBC membranes during detachment of the membrane skeleton. Control experiments with large unilamellar liposomes at conditions similar to that performed with the RBC did not reveal significant changes of [u] after the addition of nystatin. Our results show that the role of the membrane skeleton probably consists in maintaining higher compressibility of the RBC membranes. This may partly provide conditions for conformational changes of RBC membrane proteins.