Analyte-Induced pK_a-Tunable Hemicyanine Dual-Locked Molecular Platform for In Vivo Tumor Precision Imaging

Dual-locked probes that precisely visualize the tumor microenvironment (TME) are poised to close a critical detection gap in personalized medicine. However, traditional dual-locked systems are still constrained by sluggish response kinetics, low sensitivity, intricate design, and limited generality. To overcome these challenges, we introduce a general "analyte-induced pK_a-tunable" (AIPT) hemicyanine dual-locked platform (pK_a-RH) for in vivo tumor precision imaging. Interestingly, the pK_a of pK_a-RH can be readily modulated by adjusting the electron-donating ability of the optically tunable site. Leveraging this feature, the analyte response alters the electron-donating capacity of the recognition unit, shifting the probe's pK_a and enabling simultaneous detection of both acidity and the other biomarker. To validate the versatility of pK_a-RH, we devised two dual-locked probes pK_a-CE (pK_a = 5.4) and pK_a-Cys (pK_a = 4.9), bearing acetyl (for carboxylesterase) and acrylate moiety (for Cys) recognition units that are cleaved to release pK_a-RH3 (pK_a = 6.6) in situ, concomitantly activating a pH response that elicits robust fluorescence enhancement. These two probes demonstrated rapid, specific, and sensitive response and high tumor-to-normal tissue ratios for hepatocellular carcinoma screening and imaging. Thus, the AIPT strategy establishes a generalizable dual-locked platform for next-generation dual-locked probes, offering a highly promising tool for intraoperative navigation and precision tumor imaging.