Flow profiling of a surface-acoustic-wave nanopump.

The flow profile in a capillary gap and the pumping efficiency of an acoustic micropump employing surface acoustic waves is investigated both experimentally and theoretically. Ultrasonic surface waves on a piezoelectric substrate strongly couple to a thin liquid layer and generate a quadrupolar streaming pattern within the fluid. We use fluorescence correlation spectroscopy and fluorescence microscopy as complementary tools to investigate the resulting flow profile. The velocity was found to depend on the applied power approximately linearly and to decrease with the inverse third power of the distance from the ultrasound generator on the chip. The found properties reveal acoustic streaming as a promising tool for the controlled agitation during microarray hybridization.

Acoustic manipulation of small droplets.

Surface acoustic waves are used to actuate and process smallest amounts of fluids on the planar surface of a piezoelectric chip. Chemical modification of the chip surface is employed to create virtual wells and tubes to confine the liquids. Lithographically modulated wetting properties of the surface define a fluidic network, in analogy to the wiring of an electronic circuit. Acoustic radiation pressure exerted by the surface wave leads to internal streaming in the fluid and eventually to actuation of small droplets along predetermined trajectories. This way, in analogy to microelectronic circuitry, programmable biochips for a variety of assays on a chip have been realized.

Kinetics of membrane adhesion mediated by ligand-receptor interaction studied with a biomimetic system.

We report the first measurement of the kinetics of adhesion of a single giant vesicle controlled by the competition between membrane-substrate interaction mediated by ligand-receptor interaction, gravitation, and Helfrich repulsion. To model the cell-tissue interaction, we doped the vesicles with lipid-coupled polymers (mimicking the glycocalix) and the reconstituted ligands selectively recognized by alpha(IIb)beta(3) integrin-mediating specific attraction forces. The integrin was grafted on glass substrates to act as a target cell. The adhesion of the vesicle membrane to the integrin-covered surface starts with the spontaneous formation of a small (approximately 200 nm) domain of tight adhesion, which then gradually grows until the whole adhesion area is in the state of tight adhesion. The time of adhesion varies from few tens of seconds to about one hour depending on the ligand and lipopolymer concentration. At small ligand concentrations, we observed the displacement xi of the front of tight adhesion following the square root law xi approximately t(1/2), whereas, at high concentrations, we found a linear law xi approximately t. We show both experimentally and theoretically that the t(1/2)-regime is dominated by diffusion of ligands, and the xi approximately t-regime by the kinetics of ligands-receptors association.

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.

Analysis of the F-actin binding fragments of vinculin using stopped-flow and dynamic light-scattering measurements.

Using amino acids 884-1066 and 884-1012 expressed from chicken vinculin as fusion proteins with schistosomal glutathione S-transferase, we determined the binding kinetics of the protein fragments with F-actin. We established by the stopped-flow method a two-step binding process: an initial rapid reaction followed by a slower process. The latter is attributed to F-actin cross-linking and/or bundling, which was previously detected by viscometry and electron microscopy [Johnson, R. P. & Craig, S. W. (1995) Nature 373, 261-264]. This is also supported by dynamic light-scattering measurements, indicating dramatic changes in the internal actin filament dynamics, i.e. in bending undulations due to thermal noise. The similar size of the binding reaction for both fusion proteins with F-actin indicates that the F-actin binding site(s) on vinculin are located between residues 884-1012. No binding of pure glutathione S-transferase or its fusion protein with vinculin peptide 1012-1066 with F-actin was detected by either method.

Examination of temperature-induced 'gel-sol' transformation of alpha-actinin/cross-linked actin networks by static light scattering.

We studied the gel-sol transformation of F-actin/alpha-actinin solutions. Cross-linking of actin filaments by alpha-actinin shows a temperature-dependent increase in light scatter signal, (I)T. Higher F-actin/alpha-actinin molar ratios, r(A alpha) as well as increases in F-actin concentration, [A], and reduction of actin filament lengths, rAG, augment the maximal light intensity, I and shift the gel-sol transition point, Tg to higher temperatures. This behavior is interpreted in terms of the model developed by Tempel, M., Isenberg, G. and Sackmann, E. (1996) (Physical Review E 54, 1802-1810) based on the percolation theory. Using the temperature-dependent binding model of this theory allows instant prediction of the equilibrium constant, K for F-actin/alpha-actinin solutions at temperatures T < Tg.

Examining F-actin interaction with intact talin and talin head and tail fragment using static and dynamic light scattering.

We examined the binding kinetics of intact talin and talin head and tail fragment with F-actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact talin and talin tail fragment with filamentous actin. The latter can be attributed to F-actin cross-linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530-537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross-linking/bundling events with intact talin and talin tail fragment. No binding of the talin head fragment with F-actin was detected by either method.