A Tetrazine Amplification System for Visual Detection of Trace Analytes via Click-Release Reactions

Achieving visual detection of analytes at ultra-low concentrations in complex mixtures remains a persistent challenge. While sophisticated techniques offer single-molecule sensitivity, practical hurdles remain, necessitating tailored signal amplification systems for direct visual detection. In this study, we develop a strategy for the visualized detection of tetrazine through a "click-release-oxidation-cycle" (CROC) cascade amplification process. We systematically describe the construction and synthesis of this system, the kinetic process of click release, the kinetics of oxidation to tetrazine and its cascade amplification effect in trace amounts of tetrazine. This system is capable of amplifying the signal of tetrazine at a concentration as low as 2 nM by 10⁵-fold, thereby providing a clearly visible purple signal. Finally, as proof of concept, we successfully apply this method to visually detect trace β-galactosidase (β-gal) and Pd²⁺.