In situ detection of proteases has been extensively exploited for diverse biomedical applications; however, spatially controlled amplified imaging of extracellular proteases at diseased sites remains a significant challenge. Here, we report the design of DNA-peptide-PNA hybrid probes for sensitive imaging of protease activity within the acidic extracellular milieu of lesions, achieving enhanced spatial selectivity. In the strategy, both a pH-sensitive peptide and a protease-cleavable peptide substrate are rationally integrated into a DNA-based signal amplification platform. Specifically, a pH (low) insertion peptide (pHLIP) enables controlled localization of a peptide-substrate-engineered DNA initiator on the cell membrane under acidic conditions. Subsequent protease-mediated cleavage of the peptide substrate triggers signal amplification via initiator-mediated hybridization chain reaction (HCR), resulting in markedly improved spatial controllability and detection sensitivity for extracellular protease profiling in diseased microenvironments. Furthermore, the molecular imaging capability of the DNA-Peptide-PNA hybrid system was validated for matrix metalloproteinase 2/9 (MMP2/9) in vivo using both 4T1 tumor and osteoarthritis models. These results highlight the potential of this strategy as a versatile platform for the specific imaging of diverse enzymes within extracellular environments characterized by pH dysregulation.

Angew. Chem. Int. Ed. 2026, e8169928 https://onlinelibrary.wiley.com/doi/10.1002/anie.8169928