Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Here we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz) / trans-cyclooctene (TCO) mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 < 1 sec) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.
Keywords: bioorthogonal chemistry; cancer; click chemistry; diagnostic Wiley; energy transfer; fluorescence; fluorescent probes; quenching; tetrazine; tetrazines.