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.

基于 4-(6-Methyl-1,2,4,5-tetrazin-3-yl) 苯甲酸 (TE) 和降冰片烯 (NC) 之间的新型荧光生物正交反应，我们报道了一种方便的原位构建策略荧光探针直接在活细胞中进行溶酶体研究。一旦用吗啉修饰的降冰片烯 (MP-NC) 处理细胞，细胞内溶酶体就被快速修饰为功能化底物，以供生物正交对应物反应。随着 TE 的引入，溶酶体靶向探针LP1在 10 分钟内直接在活细胞中原位构建， 并发出生物正交荧光 (515  nm)。以类似的方式，探针LP2在溶酶体内快速组装。探针发出绿色生物正交荧光（480 nm) 和红色罗丹明荧光 (590  nm) 在酸性溶酶体环境中。在 3.9-5.3的 pH 范围内发现荧光强度比 (I ₅₉₀ /I _{480 ) 的线性响应 (R = 0.9702)。}成功地进行了溶酶体pH的比率检测。我们构建荧光探针的模块化组装策略具有出色的便利性和灵活性。我们希望我们的工作能够扩展生物正交化学的研究工具箱，并有利于生物系统中更多的荧光应用。