Fabrication and characterization of a fully integrated microdevice for in-vitro single cell assays

The aim of this work is the development of a microdevice able to provide in-vitro assays at single cell level. Two modules, integrated in a single platform, are presented: interdigitated electrode arrays (IDEs)-based microsystem for the cell addressed delivery of bio-functionalized nano/microparticles and a cell size microelectrode array (MEA) for single cell electroporation. Both the modules are characterized by two levels of metal structures (buried connection lines made of Al 1% Si + Ti/TiN and gold electrodes) in order to reduce the fabrication costs and the dimensions while improving the device electrical performances. Additional steps of bulk micromachining are developed in order to realize the inlet microfluidics of the MEA-based module. Biocompatible polymers and quartz are used for microchannels and cells confinement respectively. In order to demonstrate the feasibility of this approach, both modules are individually characterized. The dielectrophoretic (DEP) capability of the former is demonstrated by using polystyrene microbeads and the bioaffinity of the latter is evaluated by successful Chinese Hamster Ovary (CHO) cells culture on chip. Moreover, preliminary results of electrochemical impedance spectroscopy [100Hz–1MHz] and of a Randles-based electrical model show the stability of electrode/solution interface parameters (│Z(f)│dispersion < 3%) before and after the cell culture

Homologous versus heterologous interactions in the bicomponent staphylococcal γ-haemolysin pore(1)

Staphylococcal γ-haemolysin HlgA–HlgB forms a β-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues – placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor–acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA–HlgB or HlgB–HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA–HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB–HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers