Micrometer-Sized Supported Lipid Bilayer Arrays for Bacterial Toxin Binding Studies

 

Jose M. Moran-Mirabal, Grant D. Meyer

 

 

We report the use of micron-sized lipid domains to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM)1. Supported lipid bilayer (SLB) patterns containing either ganglioside GT1b or GM1 were formed on substrates by vesicle fusion followed by polymer lift-off. The ganglioside-populated arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed by TIRFM and constants were extracted from a logistic model. Patterning of SLBs inside microfluidic channels allowed preparation of lipid domains with different compositions on a single device, which were used to achieve segregation from a binary mixture of the toxin fragments.

 

Specific recognition and binding form the basis of many biological assays. A common feature of all arrayed assays is the requirement of a patterning method that reduces cross-contamination between species, while retaining the flexibility to incubate the array with complex mixtures of binding agents. Patterning biomolecules with polymer lift-off provides desired features for biosensing assays, and for the study of interactions between surface patterned receptors and target analytes2. Supported Lipid Bilayers (SLBs) have received considerable attention in recent years for their intrinsic properties related to biological systems. Our group has previously shown the possibility patterning lipid bilayers with polymer lift-off3-4. The technique obviates the need for etched barriers for domain formation, and allows the creation of individual micron-size domains. An important application of patterned SLBs is the possibility to detect proteins that interact with lipid-soluble receptors and to elucidate their binding characteristics.

 

We have recently shown the use of micron-sized lipid arrays to evaluate the binding constants of bacterial toxins on a solid support1. Reactive ion etching was used to define openings on a polymer coating deposited on planar and microfluidic substrates. Lipid vesicles containing ganglioside receptors GM1 or GT1b were incubated with the microfabricated substrates and allowed to fuse and form SLBs in the patterned openings. After incubation the polymer was removed, revealing arrays of patterned SLBs. Microfluidic substrates allowed the formation of arrays with different receptor populations.

 

Patterned arrays were used to capture Cholera toxin B subunit (CTB) and Tetanus toxin C fragment (TTC) from solution. A gradient of concentrations was used to determine the limit of detection and the binding characteristics. Concentrations of 1 nM for CTB and 100 nM for TTC could be detected optically by TIRFM as distinct patterns determined by the lipid domains1. The apparent dissociation constants obtained from a Hill-Waud model fit of the binding curves for the toxins were: 37030 nanomolar for CTB and 1.10.3 micromolar for TTC1. Also, by using the polymer lift-off technique to pattern lipid arrays within microfluidic channels, it was shown that two toxins can be segregated from a mixed solution and bound to their corresponding receptors with high specificity. The reported results suggest that this approach is suitable for use in biosensor applications or cell stimulation studies and can be extended to protein or DNA microarray patterning.

 

 

Figure 1. Fluorescent toxins bound to supported lipid bilayer arrays in microfluidic channels (outlined by horizontal lines).

 

 

 

Figure 2. Binding assays for CTB. Inset: Hill-Waud model fit parameters.

 

 

Figure 3. Binding assays for TTC. Inset: Hill-Waud model fit parameters.

 

 

References:

 

1.      "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, Biophysical Journal, 89, 296-305 (2005).

 

2.      "Topographical Patterning of Chemically Sensitive Biological Materials Using a Polymer-Based Dry Lift Off", B. Ilic and H. G. Craighead, Biomedical Microdevices, 2, 317-322 (2000).

 

3.      "Mast Cell Activation on Patterned Lipid Bilayers of Subcellular Dimensions", Reid N. Orth, Min Wu, David A. Holowka, Harold G. Craighead, and Barbara A. Baird, Langmuir, 19, 1599-1605 (2003).

 

4.      "Creating Biological Membranes on the Micron Scale: Forming Patterned Lipid Bilayers Using a Polymer Lift-Off Technique", R. N. Orth, J. Kameoka, W. R. Zipfel, B. Ilic, W. W. Webb, T. G. Clark and H. G. Craighead, Biophysical Journal, 85, 2066-3073 (2003).