Phenylamine‐1,2‐Dioxetanes: Promising Class of Chemiluminescent Luminophores for Aqueous Sensing and Bacterial Detection

Abstract Chemiluminescence offers distinct advantages for bioimaging and sensing, notably by eliminating the need for external light excitation and minimizing background interference. While the original phenoxy‐1,2‐dioxetanes have served as the cornerstone of chemiluminescent probe design, their efficiency is significantly compromised in aqueous environments. In this study, we report the development and evaluation of phenylamine‐substituted 1,2‐dioxetanes as a new class of luminophores with markedly enhanced performance under physiological conditions. By incorporating amino‐substituted benzoates, we achieved over 200‐fold higher chemiluminescent emission compared to a conventional phenoxy analog. Among these, the mono‐methylated probe NHMe‐Diox exhibited improved properties, including fast chemiexcitation kinetics, high quantum yield, and excellent signal‐to‐noise ratios, enabling sensitive detection of β‐galactosidase activity in vitro and in live bacterial cells. These phenylamine‐1,2‐dioxetane probes function as single‐component probes without requiring surfactants or enhancers, offering a significant advance in the design of chemiluminescent tools for biological and diagnostic applications. Significantly, probe NHMe‐Diox demonstrated a 130‐fold greater sensitivity compared to its phenoxy‐1,2‐dioxetane analog for the detection of E. coli bacterial cells.