Covalent Organic Framework‐Based Ultrasensitive Bioorthogonal Raman Probes for In Vivo Detection of Bone Crack

Abstract Bioorthogonal Raman probes offer significant advantages for multiplexed, background‐free biological imaging due to their unique vibrational fingerprints and photostability. However, their widespread application is limited by the inherently weak Raman scattering signal, which creates a pressing need for high‐sensitivity Raman probes. Here, chemically stable diacetylene (─C≡C─C≡C─) functionalized β‐ketoenamine covalent organic frameworks (BDDA COFs) are synthesized as Raman probes for in vivo imaging of bone cracks. The BDDA COFs exhibited significantly enhanced Raman intensity at 2205 cm −1 (up to ≈10 5 fold vs 5‐ethynyl‐2′‐deoxyuridine) compared to conventional alkyne‐based probes. Moreover, polydopamine (PDA)‐coated COFs (BDDA@PDA COFs) are prepared by utilizing the high affinity of PDA for calcium ions exposed at bone injury sites, thereby enabling highly sensitive and specific ex vivo and in vivo Raman imaging of bone cracks. The development of COFs containing alkyne groups as strong Raman imaging probes in the silent region provides a new paradigm for in vivo Raman imaging materials and also establishes a technical foundation for future applications in disease diagnosis and precision medicine.