Nanosensor Chemical Cytometry for Characterizing the Efflux Heterogeneity of Nitric Oxide from Macrophages

Macrophages are a critical part of the human immune response, and their collective heterogeneity is implicated in disease progression and prevention. A nondestructive, label-free tool does not currently exist for profiling the dynamic, antigenic responses of single macrophages in a collection to correlate with specific molecular expression and correlated biophysical properties at the cellular level, despite the potential for diagnosis and therapeutics. Herein, we develop a nanosensor chemical cytometry (NCC) that can profile the heterogeneity of inducible nitric oxide synthase (iNOS) responses from macrophage populations. By integrating a near-infrared (nIR) fluorescent nanosensor array and collagen layer with microfluidics, the cellular lensing effect of the macrophage was utilized to characterize both nitric oxide (NO) efflux and refractive index (RI) changes at a single-cell level. Using a parallel, multichannel approach, distinct iNOS heterogeneities of macrophages can be monitored at an attomolar (10^-18 mol) sensitivity in a nondestructive and real-time manner with a throughput of exceeding the 200 cells/frame. We demonstrate that estimated mean NO efflux rates of macrophage populations are elevated from 342 (σ = 199) to 464 (σ = 206) attomol/cell·hr with a 3% larger increase in the heterogeneity, and estimated RI of macrophage decrease from 1.366 (σ = 0.015) to 1.359 (σ = 0.009) with trimodal subpopulations under lipopolysaccharide (LPS) activation. These measured values are also in good agreement with Griess assay results and previously reported measurements. This work provides an efficient strategy for single-cell analysis of macrophage populations for cellular manufacturing and biopharmaceutical engineering.