Band-Pass Fluorescence Activation via Dual-Responsive Molecular Design in the NIR-II Window for In Vivo pH Sensing

Real-time monitoring of physiological pH in living systems is essential for accurate disease evaluation and therapeutic guidance. However, conventional pH-responsive probes typically operate through monotonic activation and lack window selectivity, limiting their capacity to report further microenvironmental changes after crossing the activation threshold. Here, we report a NIR-II molecularly engineered band-pass pH (BPpH) sensing strategy that exhibits a non-monotonic “off-on-off” fluorescence response. By integrating an acid-responsive spirolactam switch with an alkaline-sensitive phenolic regulator, the BPpH probes enable window-restricted signal output within a defined pH range. BPpH-3 displays fluorescence activation from pH 3.6 to 5.2 and deactivation from pH 5.2 to 8.0, affording dual apparent pKa values of 4.16 and 7.42 and establishing a response window. Unlike conventional monotonic turn-on probes, this band-pass response enables continuous tracking of pH changes across the therapeutic process, and BPpH-3 further enables dynamic evaluation of therapeutic efficacy in a skin inflammation model, allowing timely assessment of treatment progression and dosing sufficiency. Moreover, its window-constrained response mitigates signal over-amplification under extreme acidification, facilitating improved tumor margin delineation for precision resection under NIR-II imaging guidance. The band-pass sensing strategy presented here provides a transferable framework for window-regulated detection of diverse physiological parameters and holds promise for integration with multiple diagnostic imaging modalities.