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

Jose M. Moran-Mirabal ^*, Joshua B. Edel ^*, Grant D. Meyer ^*, Dan Throckmorton , Anup K. Singh  and Harold G. Craighead ^*

^* Applied and Engineering Physics, Cornell University, Ithaca, New York; and Microfluidics, Sandia National Laboratories, Livermore, California

Correspondence: Address reprint requests to Harold G. Craighead, Applied and Engineering Physics, Cornell University, Ithaca, NY 14853. Tel.: 607-255-8707; Fax: 607-255-7658; E-mail: hgc1{at}cornell.edu.

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.

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