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Background: The cellular secretion of proteins as signaling molecules is known to be foundational to developmental biology, wound healing, immunology and many diseases such as cancer. While the types of proteins secreted by cells are now well cataloged, we are only just beginning to understand and appreciate how cells organize these secretions in space and time for multi-cellular functionality. State-of-the-art measurements produce just one data point every two hours with a spatial resolution of hundreds of microns: orders of magnitude coarser than what is needed to precisely map these signaling pathways. This lack of resolution has its origins in the dominant technique for detecting cell secretions, which is based upon fluorescent labeling. The introduction of these labels necessarily halts or ends the experiment and as a result, they are only introduced after a cell has been secreting for hours or days, severely limiting both spatial and temporal resolutions.
Invention: To address this roadblock, we have developed a label-free technique based upon nanoplasmonic imaging which enables the measurement of individual cell secretions with time resolutions below one second and spatial resolutions below 10 µm. This is accomplished by lithographically patterning gold plasmonic nanostructures into arrays atop standard glass coverslips. The nanostructures are functionalized for biomolecular detection using standard thiol chemistries and the detection of analyte binding is imaged by a CCD camera. As a result, the technique integrates seamlessly on to commercially available wide-field and confocal microscopes, allowing real-time transmitted light and fluorescence imaging of the cells, as well as the plasmonic imaging of secreted proteins. We anticipate this technique will be broadly applicable to the real-time characterization of both paracrine and autocrine signaling pathways with applications in immunology, developmental biology, wound healing and numerous diseases such as cancer.
Advantages: Employing nanoplasmonic imaging to the study of extracellular signaling has brought with it a number of advantages over current techniques:
- The protein secretions are measured in real-time with the frequency of time points limited only by the exposure time of the camera, typically 250-400 ms.
- The gold plasmonic nanostructures are lithographically patterned onto standard glass coverslips enabling more traditional imaging techniques such as fluorescence and bright field imagery to be readily integrated into the experiments. Thus, morphological changes and intracellular fluorescent tags can be monitored simultaneously in real time.
- The nanostructures are calibrated for the quantitative determination of secreted protein concentration as a function of time and space.
- Arrays of Au nanostructures positioned sufficiently far away from the cells can be utilized as negative control arrays used to distinguish global variations in signal, e.g. instrumental drift, from localized cell secretions.
- The platform is designed to accommodate a wide variety of cell types relevant to wound healing, including neurons, blood cells and epithelial cells.
The combination of these advantages enabled us to measure secreted antibodies at sub-nanomolar concentrations with unparalleled spatial and temporal resolutions, while also monitoring cell health using fluorescence and transmitted light microscopy.
Applications: Because the cellular secretion of proteins plays a central role in cellular communication, the applications for this technique span biology. Proteins secretions are utilized by cells in a diverse range of fields such as immunology, wound healing, developmental biology and diseases such as cancer. Our plasmonic imaging technique is designed to integrate seamlessly with existing commercially available wide field and confocal microscopes. As such, it is meant to enable investigators to extend their current light microscopy techniques to incorporate a measurement that was previously unattainable: real time spatio-temporal mapping of protein secretions from single cells.
Licensing Status: Licensing and collaborative research and development is available to companies with commercial interest.
Lead Inventor: Marc P. Raphael
Patents: US Published Patent Application No. US-20140095100 entitled “Calibrating Single Plasmonic Nanostructures for Quantitative Biosensing” filed on September 27, 2013 to Marc; Raphael, Christodoulides; Joseph, Byers; Jeff
- M. P. Raphael, J. A. Christodoulides, J. B. Delehanty, J. P. Long, J. M. Byers, “Quantitative Imaging of Protein Secretions from Single Cells in Real Time”, Biophysical Journal, 105, p.602 (2013).
- M. P. Raphael, J. A. Christodoulides, J. B. Delehanty, J. P. Long, P. E. Pehrsson, J. M. Byers, “Quantitative LSPR Imaging for Biosensing with Single Nanostructure Resolution”, currently being revised for publication at Biophysical Journal (2012).
- M. P. Raphael, J. A. Christodoulides, S. P. Mulvaney, M. M. Miller, J. P. Long, J. M. Byers, “A new methodology for quantitative LSPR biosensing and imaging” Analytical Chemistry, 84, p.1367 (2012).
Navy Case Numbers: 102,395; 102,043; 101,529