A Doubly-Quenched Fluorescent Probe for Low-Background Detection of Mitochondrial H₂O₂

Hydrogen peroxide (H₂O₂) is an important product of oxygen metabolism and plays a crucial role in regulating a variety of cellular functions. Fluorescent probes have made a great contribution to our understanding of the biological role of endogenous H₂O₂. However, fluorescent probes for H₂O₂ featuring aryl boronates can suffer from moderate turn-on fluorescence responses. Strategies that can reduce the background fluorescence of these boronate-masked probes would significantly improve the sensitivity of endogenous H₂O₂ detection. In this work, we propose a general and reliable double-quenching concept for the design of fluorescent probes with low background fluorescence. A new fluorescent probe was developed for the detection of endogenous H₂O₂ in mitochondria of live cancer cells. This probe exploits a boronate-driven lactam formation and an eliminable quenching moiety simultaneously (i.e., the double-quenching effect) to reduce the background fluorescence, which ultimately results in the achievement of a >50-fold fluorescence turn-on. A linear concentration range of response between 1 and 60 μM and a detection limit of 0.025 μM can be obtained. This study not only presents a highly sensitive fluorescent probe for the detection of H₂O₂ but also provides a new concept for the design of fluorescent probes with a previously unachievable fluorescence off-on response ratio for other types of ROS and many other biologically relevant analytes.