Micrometer-Sized Supported Lipid Bilayer Arrays for Bacterial Toxin Binding Studies through Total Internal Reflection Fluorescence Microscopy

Abstract

In this article, we present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy. The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated distearoylphosphatidylcholine:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside G(T1b) or G(M1). The ganglioside-populated SLB arrays were then exposed to either cholera toxin B subunit or tetanus toxin C fragment. Binding was assayed on planar substrates by total internal reflection fluorescence microscopy down to 100 pM concentration for cholera toxin subunit B and 10 nM for tetanus toxin fragment C. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is influenced by the microenvironment of the SLB and the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions.

High-Density Arrays of Submicron Spherical Supported Lipid Bilayers

“…Some groups have reported K D as low as 4.55 pM, 37 while other groups have reported values as high as 370 nM. 38 Shi and coworkers formed supported lipid bilayers with 2 % GM1 in a microfluidic chip, analyzed CTX binding with total internal reflectance fluorescence (TIRF) microscopy and calculated a K D of 0.46 nM and a K H of 0.39 nM. 39 Winter et al determined a K D value of approximately 30 nM using colloidal phase transition analysis of lipid-coated beads.…”

Evidence for Leaflet-Dependent Redistribution of Charged Molecules in Fluid Supported Phospholipid Bilayers

“…Such control is crucial for many applications. For instance, asymmetric partitioning of charged lipid fluorophores or ligands may influence widely used fluorescence microscopy measurements7 or skew analyses based on estimated densities of ligands in outer leaflets8-10. Further, better control over asymmetries in model substrate-supported systems may allow designing more complex model membranes that mimic interleaflet asymmetry and processes (e.g., flip-flop) in natural biological membranes11, 12.…”

Reusable, reversibly sealable parylene membranes for cell and protein patterning

“…1). For micropatterning applications, parylene-C has been used to pattern antibodies, 23 lipid bilayers, 24,25 proteins, 26 and cells. 23,26 In these studies, a 1 lm thick layer of parylene is vapor deposited, etched into a stencil, used for patterning, lifted-off, and discarded.…”