As drug delivery, therapy, and medical imaging are becoming increasingly cell-specific, there is a critical need for high fidelity and high-throughput screening methods for cell surface interactions. Cell membrane-mimicking surfaces, i.e., supported lipid bilayers (SLBs), are currently not sufficiently robust to meet this need. Here we describe a method of forming fluidic and air-stable SLBs through tethered and dispersed cholesterol groups incorporated into the bottom leaflet. Achieving air stability allows us to easily fabricate SLB microarrays from direct robotic spotting of vesicle solutions. We demonstrate their application as cell membrane-mimicking microarrays by reconstituting peripheral as well as integral membrane components that can be recognized by their respective targets. These demonstrations establish the viability of the fluidic and air-stable SLB platform for generating content microarrays in high throughput studies, e.g., the screening of drugs and nanomedicine targeting cell surface receptors.
A hallmark of cell-surface processes involving glycans is their multivalent interaction with glycan binding proteins (GBPs). Such multivalent interaction depends critically on the mobility and density of signaling molecules on the membrane surface. While glycan microarrays have been used in exploring multivalent interactions, the lack of mobility and the difficulty in controlling surface density both limit their quantitative applications. Here we apply a fluidic glycan microarray, with glycan density varying for orders of magnitude, to profile cell surface interaction using a model system, the adhesion of Escherichia coli (E. coli) to mannose. We show the quantitative determination of monovalent and multivalent adhesion channels; the latter can be inhibited by nanopartices presenting a high density of mannosyl groups. These results reveal a new E. coli adhesion mechanism: the switching in the FimH adhesion protein avidity from monovalent to multivalent as the density of mobile mannosyl groups increases; such avidity switching enhances binding affinity and triggers multiple fimbriae anchoring. Affinity enhancement towards FimH has only been observed before for oligo-mannose due to the turn on of secondary interactions outside the mannose binding pocket. We suggest that the new mechanism revealed by the fluidic microarray is of general significance to cell surface interactions: the dynamic clustering of simple sugar groups (homogeneous or heterogeneous) on the fluidic membrane surface may simulate the functions of complex glycan molecules.
Keeping protein molecules in the active state on a solid surface is essential to protein microarrays and other protein-based biosensors. Here, we show that the 2-D chemical environment controls the refolding of the denatured green fluorescent proteins tethered to solid surfaces. Refolding occurs readily on the repulsive PEG functionalized surface but is inhibited on the attractive--NH(2) functionalized surface. This result shows the critical importance of the 2-D chemical environment in the maintenance and revival of protein activity on surfaces and opens the door to designing 2-D molecular chaperones for protein folding.
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