title: A facile protocol for the immobilisation of vesicles, virus particles, bacteria, and yeast cells.
abstract: Immobilisation of liposomes and cells is often a prerequisite for long-term observations. The most common immobilisation approaches rely on surface modifications, encapsulation in porous materials or trapping in microfluidic channels by means of hurdle-like structures. While these approaches are useful for larger mammalian cells, the immobilisation of smaller organisms like bacteria and yeast or membrane model systems such as liposomes typically requires modification of their outer membrane to ensure that they are stably arrested at a defined position. Here, we present a protocol to immobilise biological objects, which can interact with hydrophobic cholesterol. A water-soluble molecule (cholesterol-PEG-biotin) is used as a linker, which can bind via avidin to biotinylated BSA (bBSA) previously absorbed on a glass surface. For better visualization, bBSA is arranged in a dot pattern by means of microcontact printing, and a microfluidic channel is used for sample supply. We show that our approach can be used to successfully immobilise artificial liposomes of different sizes, native (cell-derived) vesicles, vaccinia virions, Saccharomyces cerevisiae and Escherichia coli, simply by flushing the objects through the channel. Under these conditions, small liposomes and biological objects are stably arrested at high flow rates, while larger cells and liposomes can be released again by application of high shear stress. This protocol can be applied for long-term studies where fluids must be changed repeatedly, for measuring fast kinetics where rapid fluid exchange is essential, and to study the effects of shear stress.
authors.affiliation: ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, Zurich, Switzerland.
authors.forename: Phillip
authors.initials: P
authors.lastname: Kuhn
authors.forename: Klaus
authors.initials: K
authors.lastname: Eyer
authors.forename: Tom
authors.initials: T
authors.lastname: Robinson
authors.forename: Florian I
authors.initials: FI
authors.lastname: Schmidt
authors.forename: Jason
authors.initials: J
authors.lastname: Mercer
authors.forename: Petra S
authors.initials: PS
authors.lastname: Dittrich
mesh_terms: D000818:Animals; D002460:Cell Line; D018914:Cells, Immobilized; D022162:Cytoplasmic Vesicles; D004926:Escherichia coli / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D008081:Liposomes / Q000302:isolation & purification; D008828:Microbiological Techniques; D046210:Microfluidic Analytical Techniques; D008856:Microscopy, Fluorescence; D012441:Saccharomyces cerevisiae / Q000302:isolation & purification; D013314:Stress, Mechanical; D049490:Systems Biology; D014616:Vaccinia virus / Q000302:isolation & purification; D014771:Virion / Q000302:isolation & purification
chemical_list: D008081:Liposomes