Carbon Nanotube Sensor Platforms for the Detection of Cellular Nitric Oxide Efflux Gradients
Authors: Acosta Ramirez Ivon , Iverson Nicole M , Sadak Omer 
Published: August 2024

Cell communication is the ability to receive, process, and transmit signals from the environment and within cells to ensure functional coordination during physiological events. Cells communicate between themselves using waves of chemical concentration that change in both direction and time. Nitric oxide (NO) is a gaseous, ubiquitous, intra - and inter-cellular signaling molecule. NO’s small size and lipophilic nature allows it to diffuse through cell membranes and reach intracellular compartments of nearby cells. However, NO detection in biological systems represents a challenge due to NO’s short half-life (in the range of seconds) before breaking down into different subproducts. The lack of spatial NO detection, not an upstream or downstream indicator of NO, has hindered understanding of the extracellular NO dynamic signaling during both physiological and pathological events. However, single wall carbon nanotube (SWNT)-based fluorescent sensors allow for the detection of NO, and when immobilized onto a glass substrate, they can be used to quantify extracellular NO in a spatial-temporal manner. We detected changes in extracellular NO efflux from different macrophage cell lines known for their NO production under inflammatory responses, specifically human THP-1 monocytes and murine macrophage RAW 264.7. The cells were seeded on the SWNT platform and treated with Liposaccharides (LPS) 24 h to increase their NO release. The SWNT platform’s sensing response to NO was detected by changes in fluorescence intensity. Fluorescence data was collected on a custom-built hyperspectral microscope to give us near infrared (nIR) signal quantification with spatial resolution. Images were collected before and after the respective NO induction method. To analyze the extracellular NO mappings, bright field images of the cells were used to extract the cell coordinates and translate them to fluorescent images. Hyperspectral image analysis was performed with MATLAB to determine SWNT fluorescence below each cell and its surrounding area. To validate that the change in the fluorescence response was attributed to NO and not from cellular interaction with the SWNT sensor, we added 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a NO scavenger, after visualizing the SWNT quenching to recover the initial fluorescence signal, showing that the cells didn’t affect SWNT functionality. Through fluorescence intensity changes with pixel specificity on the image acquisition, we were able to spatially quantify NO released by cells with dynamic diffusion gradients. The newly developed platform provides the means to quantify extracellular NO. In this study, only two cell lines were studied, but the platforms will allow for the quantification of NO for other cell types as well. By comparing extracellular NO concentrations for different types of cells, we will be able to better understand physiological and pathological patterns of NO release, leading to a better understanding of cellular signaling.