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

Abstract

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.

Mixed metal zero-mode guides (ZMWs) for tunable fluorescence enhancement

“…While confocal and total internal reection (TIRF) modalities are limited to a concentration barrier (<10 nM) to detect a single molecule event, 6 nanoapertures allow imaging of biological interactions at single molecule levels in micromolar concentrations. [7][8][9][10][11][12][13][14][15] As a result, ZMWs have been widely used for genomic sequencing, [16][17][18] protein-protein interaction, [19][20][21][22] ligand-receptor binding, [23][24][25][26][27][28] membrane bound diffusion events 29,30 and the study of membrane proteins at single molecule levels. 31,32 This capability is due to the aperture dimensions of the ZMWs, which result in cut-off wavelengths for the transmission of light smaller than the wavelength of excitation light.…”

Zero-mode waveguide nanophotonic structures for single molecule characterization

“…Because only a small area of the cell membrane is probed, difference in transport behavior between finely-spaced areas on the cell membrane can be observed [ 131 ]. For more dynamic studies, ZMWs can be combined with other biological analysis techniques; for example, single-ligand binding has been demonstrated with cells cultured on a ZMW array in an integrated microfluidic device [ 132 ].…”

Gold Ion Beam Milled Gold Zero-Mode Waveguides

“…The geometry and opacity of the ZMW enables measurements at physiological concentrations (∼1–100 μM) in SMS [ 30 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. As a result, ZMWs have found wide application in studies of genomic sequencing [ 47 , 48 , 49 , 50 ], protein–protein interaction [ 51 , 52 , 53 , 54 ], ligand–receptor binding [ 55 , 56 , 57 , 58 , 59 , 60 , 61 ], membrane-bound diffusion events [ 62 , 63 ] and the study of membrane proteins [ 64 , 65 ]. ZMWs have also been used to manipulate exciton behavior in quantum dots [ 66 ], and ZMWs have been shown to extend Förster Resonance Energy Transfer (FRET) distances from 10 to 13.6 nm [ 67 ].…”