Multiscale molecular engineering co-modulated dual-mode ratiometric probe for monitoring tumor-associated macrophage repolarization.

Precise modulation of structure-function relationship of molecules is at the core of driving breakthroughs in biosensing technology through chemical strategy. Here, we present a near-infrared (NIR) ratiometric fluorescence/photoacoustic dual-mode probe TPcy through a multiscale chemical modulation strategy. On the molecular scale, the synergistic design of the six-membered ring-centered cyanine skeleton and the N-nitrosylation response site achieves a significant spectral signal redshift driven by the nitric oxide (NO) response, and the introduction of the sulfonic acid side chain brings about a dual enhancement of biocompatibility and response rate. Further, the targeting unit maleimide-mediated protein covalent coupling achieves in situ anchoring and microenvironment enrichment of tumors without disturbing the probe function. TPcy enables dynamic tracking of immunotherapy-triggered M2-to-M1 macrophage repolarization through spatiotemporally resolved dual-modal imaging, revealing correlations between immune activation and therapeutic outcomes. This work establishes a multiscale co-regulation paradigm, offering a universal chemical design strategy for high-precision molecular imaging tools in precision immunotherapy.