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Cell communication via chemical signaling depends on spatial and temporal concentration changes. Nitric oxide (NO), a gaseous signaling molecule, is critical in physiological and pathological processes. However, current NO sensing methods lack the spatiotemporal resolution necessary to study subcellular NO efflux. This study introduces an innovative sensory platform utilizing single‐walled carbon nanotubes (SWNT) as an optical transducer for the spatial and temporal detection of extracellular NO. The platform quantifies NO diffusion gradients produced by human (THP‐1) and murine (RAW 264.7) macrophage cells. The uniform fluorescence distribution of the nanoarray enables precise analysis of NO efflux directionality, both under and surrounding the cell. It is demonstrated that cellular adhesion to the surface of the sensory platform does not affect its fluorescence functionality or sensing response rate. By combining the platform's high spatiotemporal resolution with the advanced analysis methods, the SWNT sensor platform offers a robust tool for studying extracellular NO dynamics within the cellular microenvironment. This work lays the foundation for advanced diagnostic and therapeutic tools elucidating NO cellular communication analysis.
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Microelectrophoretic single-cell measurements with microfluidic devices.
Here we describe in detail the design, fabrication and operation of our automated high-throughput single cell microchip electrophoresis device with laser induced fluorescence detection. Our device features on-board integrated peristaltic pumps that generate flow directly within the microfluidic channels. Additionally, we have incorporated an optical fiber bridge that enables simultaneous fluorescence detection at two points of interest within the device without the need for additional optical components or detectors. The second detection spot is used to detect the intact cell immediately prior to lysis giving a signal at t = 0 s for each single-cell electropherogram. We can also use this signal to measure the absolute migration time of the separated analytes to confidently determine the identity of each peak. Finally, we demonstrate the application of our device for the measurement of intracellular nitric oxide (NO) levels in T-lymphocytes. Changes in NO levels within cells is associated with a number of chronic diseases including neurodegenerative, cardiovascular and cancers. We show that our system is capable of measuring NO levels under the following conditions: native, lipopolysaccharide stimulation, and inhibition of inducible nitric oxide synthase. It is our hope that the information and procedures described in this chapter may enable others to use or adapt our system for other analyses at the single cell level.
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- Award ID(s):
- 1804416
- PAR ID:
- 10146446
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 628
- ISSN:
- 1557-7988
- Page Range / eLocation ID:
- 223-241
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/https://doi.org/10.1016/bs.mie.2019.07.011
