An official website of the United States government
Progress has been made studying cell-cell signaling communication processes. However, due to limitations of current sensors on time and spatial resolution, the role of many extracellular analytes is still unknown. A single walled carbon nanotube (SWNT) platform was previously developed based on the avidin-biotin immobilization of SWNT to a glass substrate. The SWNT platform provides real time feedback about analyte concentration and has a high concentration of evenly distributed sensors, both of which are essential for the study of extracellular analytes. Unfortunately, this initial SWNT platform is synthesized through unsterile conditions and cannot be sterilized post-production due to the delicate nature of the sensors, making it unsuitable for in vitro work. Herein the multiple-step process for SWNT immobilization is modified and the platform’s biocompatibility is assessed in terms of sterility, cytotoxicity, cell proliferation, and cell morphology through comparison with non-sensors controls. The results demonstrate the SWNT platform’s sterility and lack of toxicity over 72 h. The proliferation rate and morphology profiles for cells growing on the SWNT platform are similar to those grown on tissue culture substrates. This novel nano-sensor platform preserves cell health and cell functionality over time, offering opportunities to study extracellular analytes gradients in cellular communication.
more »
« less
Programming‐Assisted Imaging of Cellular Nitric Oxide Efflux Gradients and Directionality via Carbon Nanotube Sensors
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.
more »
« less
- Award ID(s):
- 2145494
- PAR ID:
- 10570191
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Small Science
- Volume:
- 5
- Issue:
- 4
- ISSN:
- 2688-4046
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Single-wall carbon nanotubes (SWNT) have a strong and stable near-infrared (nIR) signal and can interact with target analytes selectively, even at the single molecule level, to alter fluorescence intensity and/or emission peak wavelength. SWNT have been employed as nIR optical sensors for detecting a variety of analytes. However, high cost, long fabrication time, and poor fluorescence yield limit the current methods for immobilizing SWNT sensors on solid substrates. Recently, our group reported a protocol for SWNT immobilization resulting in high fluorescence yield, signal longevity, fluorescence distribution, and quick sensing response time. However, it takes 5 days to fabricate these sensor arrays. We have improved our previously reported protocol to immobilize SWNT sensors with a method that takes only 2 days, results in a platform with similar surface morphology, and has a higher fluorescence intensity than the previous platforms without sacrificing the sensing capabilities.more » « less
-
Abstract A plant's response to external and internal nitrogen signals/status relies on sensing and signaling mechanisms that operate across spatial and temporal dimensions. From a comprehensive systems biology perspective, this involves integrating nitrogen responses in different cell types and over long distances to ensure organ coordination in real time and yield practical applications. In this prospective review, we focus on novel aspects of nitrogen (N) sensing/signaling uncovered using temporal and spatial systems biology approaches, largely in the model Arabidopsis. The temporal aspects span: transcriptional responses to N-dose mediated by Michaelis-Menten kinetics, the role of the master NLP7 transcription factor as a nitrate sensor, its nitrate-dependent TF nuclear retention, its “hit-and-run” mode of target gene regulation, and temporal transcriptional cascade identified by “network walking.” Spatial aspects of N-sensing/signaling have been uncovered in cell type-specific studies in roots and in root-to-shoot communication. We explore new approaches using single-cell sequencing data, trajectory inference, and pseudotime analysis as well as machine learning and artificial intelligence approaches. Finally, unveiling the mechanisms underlying the spatial dynamics of nitrogen sensing/signaling networks across species from model to crop could pave the way for translational studies to improve nitrogen-use efficiency in crops. Such outcomes could potentially reduce the detrimental effects of excessive fertilizer usage on groundwater pollution and greenhouse gas emissions.more » « less
-
Brown, Sam Paul (Ed.)Bacteria commonly exist in multicellular, surface-attached communities called biofilms. Biofilms are central to ecology, medicine, and industry. TheVibrio choleraepathogen forms biofilms from single founder cells that, via cell division, mature into three-dimensional structures with distinct, yet reproducible, regional architectures. To define mechanisms underlying biofilm developmental transitions, we establish a single-molecule fluorescence in situ hybridization (smFISH) approach that enables accurate quantitation of spatiotemporal gene-expression patterns in biofilms at cell-scale resolution. smFISH analyses ofV. choleraebiofilm regulatory and structural genes demonstrate that, as biofilms mature, overall matrix gene expression decreases, and simultaneously, a pattern emerges in which matrix gene expression becomes largely confined to peripheral biofilm cells. Both quorum sensing and c-di-GMP-signaling are required to generate the proper temporal pattern of matrix gene expression. Quorum sensing signaling is uniform across the biofilm, and thus, c-di-GMP-signaling alone sets the regional matrix gene expression pattern. The smFISH strategy provides insight into mechanisms conferring particular fates to individual biofilm cells.more » « less
-
Recent technological advances have enabled spatially resolved measurements of expression profiles for hundreds to thousands of genes in fixed tissues at single-cell resolution. However, scalable computational analysis methods able to take into consideration the inherent 3D spatial organization of cell types and nonuniform cellular densities within tissues are still lacking. To address this, we developed MERINGUE, a computational framework based on spatial autocorrelation and cross-correlation analysis to identify genes with spatially heterogeneous expression patterns, infer putative cell–cell communication, and perform spatially informed cell clustering in 2D and 3D in a density-agnostic manner using spatially resolved transcriptomic data. We applied MERINGUE to a variety of spatially resolved transcriptomic data sets including multiplexed error-robust fluorescence in situ hybridization (MERFISH), spatial transcriptomics, Slide-seq, and aligned in situ hybridization (ISH) data. We anticipate that such statistical analysis of spatially resolved transcriptomic data will facilitate our understanding of the interplay between cell state and spatial organization in tissue development and disease.more » « less
