Analyte-Induced p <i>K</i> _a -Tunable Hemicyanine Dual-Locked Molecular Platform for <i>In Vivo</i> 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 p<i>K</i>_a-tunable" (<b>AIPT</b>) hemicyanine dual-locked platform (<b>p</b><i><b>K</b></i>_<b>a</b><b>-RH</b>) for <i>in vivo</i> tumor precision imaging. Interestingly, the p<i>K</i>_a of <b>p</b><i><b>K</b></i>_<b>a</b><b>-RH</b> 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 p<i>K</i>_a and enabling simultaneous detection of both acidity and the other biomarker. To validate the versatility of <b>p</b><i><b>K</b></i>_<b>a</b><b>-RH</b>, we devised two dual-locked probes <b>p</b><i><b>K</b></i>_<b>a</b><b>-CE</b> (p<i>K</i>_a = 5.4) and <b>p</b><i><b>K</b></i>_<b>a</b><b>-Cys</b> (p<i>K</i>_a = 4.9), bearing acetyl (for carboxylesterase) and acrylate moiety (for Cys) recognition units that are cleaved to release <b>p</b><i><b>K</b></i>_<b>a</b><b>-RH3</b> (p<i>K</i>_a = 6.6) <i>in situ</i>, 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 <b>AIPT</b> 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.