Co-aggregation as an Exceptionally Simple Platform to Prepare Fluorogenic Tetrazine Probes with On-Demand Fluorophore Selection

Life science has progressed with applications of fluorescence probes — fluorophores linked to functional units responsive to biological events. To meet the varied demands across experiments, simple organic reactions to connect fluorophores and functional units are developed, enabling the on-demand selection of fluorophore-functional unit combinations. However, organic synthesis requires professional equipment and skills, which hamper the life scientists’ research. In this study, we demonstrate that the co-aggregation of hydrophobic molecules in water can serve as a simple, synthesis-free strategy for probe preparation. We focused on tetrazine — a difficult-to-prepare yet useful functional unit that provides effective biorthogonal reactivity and strong fluorogenicity to connected fluorophores. Simply mixing the tetrazine molecules and aggregation-induced emission luminogens (AIEgens) in water, co-aggregation is induced, and the emission of AIEgens is quenched. Subsequent click reaction bioorthogonally turns on the emission, identifying those coaggregates as fluorogenic probes. We utilized this bioorthogonal fluorogenicity to distinguish overlapping emission signals in a time-dependent manner, enabling multi-organelle imaging with two same-color fluorophores. Our study showcases the potential of our co-aggregation method for the on-demand preparation of fluorescent probes as well as protocols and molecular design principles in this approach, offering an effective solution to evolving needs in life science research.