This tutorial review summarizes the recent advances of the in situ molecular immobilization strategies and the utilization of these strategies for in situ bioimaging and therapeutics.

Significance: Long-term in situ imaging of biomolecules on the cell membrane was significant for studying the physiological processes emitted from cell signaling and cell adhesion. Thus, to develop an efficient tool to realize in situ imaging membrane-associated biomolecules, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome (HYPQ) characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate. Then, benefiting from the antidiffusion property of the constructed probe HYPQG, long-term in situ imaging of membrane-associated enzyme GGT was successfully realized. This work may provide an efficient strategy for constructing universal antidiffusion fluorescent probes to realize long-term in situ bioimaging.

Carboxylesterase (CE) is crucial in metabolizing ester-containing biomolecules and is particularly significant in liver metabolic diseases. Herein, we present the first activatable NIRF/PA dual-mode imaging probe QHD-CE for detection of CE in vitro and in vivo. QHD-CE displays excellent sensitivity and selectivity for CE with a high reaction efficiency (∼90 min). By utilizing QHD-CE, the dynamic changes of CE in drug-induced liver injury and diabetic mice models were monitored.