Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research.
Typical microfluidic systems take advantage of multiple storage reservoirs, pumps and valves for the storage, driving and release of buffers and other reagents. However, the fabrication, integration, and operation of such components can be difficult. In particular, the reliance of such components on external off-chip equipment limits their utility for creating self-sufficient, stand-alone microfluidic systems. Here, we demonstrate a porous sponge made of polydimethylsiloxane (PDMS), which is fabricated by templating microscale water droplets using a T-junction microfluidic structure. High-resolution microscopy reveals that this sponge contains a network of pores, interconnected by small holes. This unique structure enables the sponge to passively release stored solutions very slowly. Proof-of-concept experiments demonstrate that the sponge can be used for the passive release of stored solutions into narrow channels and circular well plates, with the latter used for inducing intracellular calcium signalling of immobilised endothelial cells. The release rate of stored solutions can be controlled by varying the size of interconnecting holes, which can be easily achieved by changing the flow rate of the water injected into the T-junction. We also demonstrate the active release of stored liquids into a fluidic channel upon the manual compression of the sponge. The developed PDMS sponge can be easily integrated into complex micro/macro fluidic systems and prepared with a wide array of reagents, representing a new building block for self-sufficient microfluidic systems.
Background Supraphysiological hemodynamics are a recognized driver of platelet activation and thrombosis at high-grade stenosis and in blood contacting circulatory support devices. However, whether platelets mechano-sense hemodynamic parameters directly in free flow (in the absence of adhesion receptor engagement), the specific hemodynamic parameters at play, the precise timing of activation, and the signaling mechanism(s) involved remain poorly elucidated. Results Using a generalized Newtonian computational model in combination with microfluidic models of flow acceleration and quasi-homogenous extensional strain, we demonstrate that platelets directly mechano-sense acute changes in free-flow extensional strain independent of shear strain, platelet amplification loops, von Willebrand factor, and canonical adhesion receptor engagement. We define an extensional strain sensing “mechanosome” in platelets involving cooperative Ca2+ signaling driven by the mechanosensitive channel Piezo1 (as the primary strain sensor) and the fast ATP gated channel P2X1 (as the secondary signal amplifier). We demonstrate that type II PI3 kinase C2α activity (acting as a “clutch”) couples extensional strain to the mechanosome. Conclusions Our findings suggest that platelets are adapted to rapidly respond to supraphysiological extensional strain dynamics, rather than the peak magnitude of imposed wall shear stress. In the context of overall platelet activation and thrombosis, we posit that “extensional strain sensing” acts as a priming mechanism in response to threshold levels of extensional strain allowing platelets to form downstream adhesive interactions more rapidly under the limiting effects of supraphysiological hemodynamics.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.