Burst-Mode Near-Infrared Chemiluminescent Probes for In Vivo Imaging

Chemiluminescence (CL) offers excitation-free optical readout with minimal background signal, making it highly attractive for diagnostics and imaging. However, most reported 1,2-dioxetane-based chemiluminophores exhibit long-lasting CL but low instantaneous intensity, limiting their imaging applications. Here, we report a series of burst-mode near-infrared (NIR) chemiluminophores with high instantaneous brightness via a substituent-driven electronic-tuning approach to reduce the activation barrier of bond scission within chemiluminophores. After replacing the 3-methoxy group of 1,2-dioxetane in the dicyanomethylene-phenoxy-dioxetane (DPD) with different substituents, the 2,2,2-trifluoroethyloxy-modified analogue DPD4 is identified to show the activation barrier energy required for the transition state of O-O dissociation, approximately 3-fold lower than that of the methoxy-substituted DPD1. This reduction corresponds to a 15.0-fold increase in the relative chemiexcitation rate versus DPD1. Consequently, DPD4 displays pronounced burst-mode NIR emission, featuring a remarkable ∼79,978-fold intensity enhancement and a markedly shortened CL half-life (t_1/2,CL = 10 s) while retaining ultrahigh chemical stability with a half-life of 6.8 days in buffer at room temperature. Then, the optimized chemiluminophore DPD4 is constructed into an activatable probe DPD4_g to selectively trigger its strong CL responses by β-galactosidase (β-gal) in live cells. In vivo, DPD4_g distinguishes β-gal-overexpressing tumors, affording a 15.2-fold signal enhancement relative to β-gal-negative tumors. This work establishes trifluoroethyl substitution as a generic route to construct burst-mode chemiluminophores for sensitive CL molecular imaging in living systems.