PeT‐based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence‐based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT‐based turn‐on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT‐based turn‐on mechanism. The probe consists of a phenoxy‐1,2‐dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole‐dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal‐to‐noise ratio (over 5000‐fold). The PeT‐dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc‐DBCO and Max‐DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT‐based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT‐quenching pathways.
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