A Dual-Channel NIR Fluorescent Probe for Self-Calibrated Ratiometric Imaging and In Vivo Discrimination of Malignant...

Alkaline phosphatase (ALP) is a well-established biomarker of tumor malignancy, yet the precise quantification and spatial mapping of its activity in living systems remain challenging with conventional single-channel probes. Herein, we report a rationally designed dual-channel near-infrared (NIR) fluorescent probe, P-CyBr-NBD, for ratiometric imaging and in vivo discrimination of tumors based on ALP activity. The probe integrates a NIR-emitting hemicyanine unit (CyBr) with a green-emitting fluorophore (NBD), which serve as the ALP-responsive signal and internal reference, respectively. Upon ALP-mediated dephosphorylation, the NIR signal of CyBr at 730 nm shows a marked fluorescence increase, while the green emission of NBD at 545 nm remains essentially constant, enabling self-calibrated ratiometric quantification of ALP activity via the fluorescence ratio (FI_730nm/FI_545nm). Encapsulation within lipid nanomicelles facilitates efficient cellular uptake and affords excellent specificity with minimal cytotoxicity. The dual-channel platform successfully distinguished ALP-high tumor cells (HeLa and HepG2) from ALP-low melanoma cells (B16F10) and normal cells (NIH3T3) by confocal imaging. In xenograft mouse models bearing HeLa or B16F10 tumors, ratiometric fluorescence imaging revealed a significantly higher signal ratio in HeLa tumors, enabling clear discrimination based on endogenous ALP activity. The probe exhibited selective activation and prolonged retention in ALP-overexpressing HeLa tumors, underscoring its potential for precise in vivo tumor imaging and enzymatic activity profiling. This work establishes a generalizable strategy for high-fidelity ratiometric enzymatic sensing and provides a useful tool for cancer diagnostics and microenvironmental analysis.