The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.

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Published in ACS Omega, v. 2, issue 7, p. 3858-3867.

Copyright © 2017 American Chemical Society

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

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This work was funded through the Human Frontiers Science Program (RGY0081/2014) and the National Institute of Health DA 038817.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00934.

ao7b00934_si_001.pdf (1511 kB)
Schematic comparing standard ZMW and mf-ZMW, fabrication schematic of mf-ZMWs, and control image of binding specificity