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Title: Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices

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. Lastly, these methods should be applicable to a wide range of membrane proteins.
Authors:
 [1] ;  [1] ; ORCiD logo [2] ;  [1] ; ORCiD logo [2] ;  [1] ; ORCiD logo [1]
  1. Univ. of Kentucky, Lexington, KY (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
ACS Omega
Additional Journal Information:
Journal Volume: 2; Journal Issue: 7; Journal ID: ISSN 2470-1343
Publisher:
American Chemical Society (ACS)
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Cell and Molecular biology; Fluorescence; Lab-on-a-chip; Luminescence spectroscopy; Microfluidic devices; Nanostructures; Optical materials; Polysiloxanes; Proteins
OSTI Identifier:
1468258

Martin, W. Elliott, Ge, Ning, Srijanto, Bernadeta R., Furnish, Emily, Collier, C. Patrick, Trinkle, Christine A., and Richards, Christopher I.. Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices. United States: N. p., Web. doi:10.1021/acsomega.7b00934.
Martin, W. Elliott, Ge, Ning, Srijanto, Bernadeta R., Furnish, Emily, Collier, C. Patrick, Trinkle, Christine A., & Richards, Christopher I.. Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices. United States. doi:10.1021/acsomega.7b00934.
Martin, W. Elliott, Ge, Ning, Srijanto, Bernadeta R., Furnish, Emily, Collier, C. Patrick, Trinkle, Christine A., and Richards, Christopher I.. 2017. "Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices". United States. doi:10.1021/acsomega.7b00934. https://www.osti.gov/servlets/purl/1468258.
@article{osti_1468258,
title = {Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices},
author = {Martin, W. Elliott and Ge, Ning and Srijanto, Bernadeta R. and Furnish, Emily and Collier, C. Patrick and Trinkle, Christine A. and Richards, Christopher I.},
abstractNote = {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. Lastly, these methods should be applicable to a wide range of membrane proteins.},
doi = {10.1021/acsomega.7b00934},
journal = {ACS Omega},
number = 7,
volume = 2,
place = {United States},
year = {2017},
month = {7}
}