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.