In-situ construction of fluorescent probes for lysosomes imaging and lysosomal pH ratiometric detection based on bioorthogonal fluorescence

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Based on a novel fluorescent bioorthogonal reaction between 4-(6-Methyl-1,2,4,5-tetrazin-3-yl) benzoic acid (TE) and norbornenes (NC), we reported a convenient strategy to in-situ construct fluorescent probes directly in live cells for lysosomal studies. Once the cells were treated with morpholine modified norbornene (MP-NC), the intracellular lysosomes were decorated fast as functionalizing substrate for bioorthogonal counterparts to react. With the introduction of TE, lysosomes-targeted probe LP1 was in-situ constructed directly in live cells within 10 min and emitted bioorthogonal fluorescence (515 nm). In a similar way, probe LP2 was assembled inside lysosomes rapidly. The probe emitted green bioorthogonal fluorescence (480 nm) and red rhodamine fluorescence (590 nm) in acidic lysosomal environment. A linear response (R = 0.9702) of the fluorescence intensity ratio (I590/I480) was found in pH range of 3.9–5.3. Ratiometric detection of lysosomal pH was successfully performed. Our modular assembling strategy to construct fluorescent probes presented excellent convenience and flexibility. We hoped our work to expand the research toolbox of bioorthogonal chemistry and to benefit more fluorescent applications in biological systems.