Modular Orthogonal Dual-Channel Gold Nanosensor Platform for Ratiometric Imaging of Tumor-Associated Macrophage M1/M2 Polarization.

The phenotypic balance between M1 and M2 tumor-associated macrophages (MΦs) governs tumor immune homeostasis and immunotherapeutic outcomes. However, in situ visualization of this polarization equilibrium remains challenging. Conventional histological and cytological approaches only afford static ex vivo phenotyping, while single-biomarker-responsive fluorescent probes lack the capacity to simultaneously detect M1 and M2. Herein, we fabricate a modular dual-channel ratiometric nanosensor for simultaneous imaging and assessment of M1/M2 MΦ polarization states. Two spectrally orthogonal fluorophores responsive to nitric oxide (M1) and arginase-1 (M2) are conjugated onto ultrasmall gold nanoparticles (<3 nm) via orthogonal surface functionalization. This nanosensor maintains an "always-off" baseline through proximity-induced quenching and selectively recovers fluorescence upon biomarker activation, enabling cross-interference-free ratiometric detection. Capable of resolving spatial heterogeneity in MΦ polarization, the nanosensor reveals phenotypic switching in 2D cell cultures and 3D tumor spheroids, and imaging outputs show excellent consistency with flow cytometry (R2 = 0.96). Moving further to murine tumor models, it captures the progressive reduction in intratumoral M1/M2 ratiometric signals across tumor progression and non-invasively visualizes an elevation from ∼1.2 to ∼3.3 following immunotherapy, confirming efficient M2-to-M1 reprogramming. This work delivers a reliable optical tool for unraveling tumor immune microenvironment remodeling and provides a generalized strategy to design multi-analyte ratiometric nanosensors.