Unimolecular Tandem Nano-Amplifiers for Imaging Signaling Molecules during Wound Healing in Zebrafish

Real-time detection and efficient imaging of low-abundance active substances and signaling molecules in living system demand highly sensitive probes. Multiple amplification is the principal method for achieving such sensitivity. However, current multiple amplification probes operate on multimolecular mechanisms, which are vulnerable to interference from complex biological environments. Herein, we introduce a unimolecular tandem nanoamplifier (UTNA). The UTNA is designed by covalently linking a hyperbranched self-immolative polymer (hSIP) and squarylium (SQ), a dye known for its fluorescence enhancement upon binding to proteins. Initially, the SQ molecules are self-quenched and unable to bind to proteins. Upon active substance stimulation, the hSIP degrades and releases multiple SQ molecules, thus achieving the first level of amplification. Subsequently, the released SQ molecules bind to cytosolic proteins, resulting in a second level of amplification. This UTNA was employed to image signaling molecules (H₂O₂) fluctuations during the healing process following tail resection in zebrafish larvae.