Ilja Czolkos

Fractal avalanche ruptures in biological membranes

Bilayer membranes envelope cells as well as organelles, and constitute the most ubiquitous biological material found in all branches of the phylogenetic tree. Cell membrane rupture is an important biological process, and substantial rupture rates are found in skeletal and cardiac muscle cells under a mechanical load. Rupture can also be induced by processes such as cell death, and active cell membrane repair mechanisms are essential to preserve cell integrity. Pore formation in cell membranes is also at the heart of many biomedical applications such as in drug, gene and short interfering RNA delivery. Membrane rupture dynamics has been studied in bilayer vesicles under tensile stress, which consistently produce circular pores. We observed very different rupture mechanics in bilayer membranes spreading on solid supports: in one instance fingering instabilities were seen resulting in floral-like pores and in another, the rupture proceeded in a series of rapid avalanches causing fractal membrane fragmentation. The intermittent character of rupture evolution and the broad distribution in avalanche sizes is consistent with crackling-noise dynamics. Such noisy dynamics appear in fracture of solid disordered materials, in dislocation avalanches in plastic deformations and domain wall magnetization avalanches. We also observed similar fractal rupture mechanics in spreading cell membranes.

Platform for controlled supramolecular nanoassembly.

We here present a two-dimensional (2D) micro/nano-fluidic technique where reactant-doped liquid-crystal films spread and mix on micro- and nanopatterned substrates. Surface-supported phospholipid monolayers are individually doped with complementary DNA molecules which hybridize when these lipid films mix. Using lipid films to convey reactants reduces the dimensionality of traditional 3D chemistry to 2D, and possibly to 1D by confining the lipid film to nanometer-sized lanes. The hybridization event was observed by FRET using single-molecule-sensitive confocal fluorescence detection. We could successfully detect hybridization in lipid streams on 250 nm wide lanes. Our results show that the number and density of reactants as well as sequence of reactant addition can be controlled within confined liquid crystal films, providing a platform for nanochemistry with potential for kinetic control.

High-Resolution Micropatterned Teflon AF Substrates for Biocompatible Nanofluidic Devices

We describe a general photolithography-based process for the microfabrication of surface-supported Teflon AF structures. Teflon AF patterns primarily benefit from superior optical properties such as very low autofluorescence and a low refractive index. The process ensures that the Teflon AF patterns remain strongly hydrophobic in order to allow rapid lipid monolayer spreading and generates a characteristic edge morphology which assists directed cell growth along the structured surfaces. We provide application examples, demonstrating the well-controlled mixing of lipid films on Teflon AF structures and showing how the patterned surfaces can be used as biocompatible growth-directing substrates for cell culture. Chinese hamster ovary (CHO) cells develop in a guided fashion along the sides of the microstructures, selectively avoiding to grow over the patterned areas.

Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger.

A Platform for Supramolecular Nanochemistry

There is increased need for analytical tools to probe (bio)molecular recognition and chemical interactions. This can be provided by nanochemistry, where the focus is on individual molecules, or molecular assemblies that, unlike bulk canonical ensembles, exhibit some degree of order. Nanochemical systems rely on self-assembly and (bio)molecular recognition, and one way to introduce order into a system is to link these processes to surfaces. We here present a 2D micro-/nano-fluidic technique where reactant-doped liquid crystal (LC) films spread and mix on micropatterned amphiphilic substrates. These phospholipid monolayer films are individually doped with complementary DNA strands modified in one end by a lipophilic anchor and with a fluorescent dye in the other end. By using fluorescence resonance energy transfer we monitor the hybridization of the complementary strands. Our results show that the density of reactants, number of different reactants as well as the sequence of reactant addition, can be controlled within LC films confined to micromachined substrates. The technology introduced here provides a platform for nanochemistry with potential for kinetic control where molecular assemblies with 2D orientational order can be established, controlled, and probed.View Large Image | View Hi-Res Image | Download PowerPoint Slide