Dual-ratiometric fluorescence sensing and real-time detection of HOCl and NQO1 using a single fluorescent probe under one-wavelength excitation

Overexposure to hypochlorous acid (HOCl) can lead to cellular oxidative stress. In response to the stress, the body activates related factor that upregulates a potent antioxidant enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1). A method that can simultaneously detect HOCl and NQO1 can help to understand their interactions during homeostasis. However, the methods are still limited due to the complexity of the biological environment. In this work, we present for the first time a dual-ratiometric fluorescent probe ( NH ) that can distinguish between HOCl and NQO1. Probe NH had ratiometric fluorescence responses by changing from red (λ max em = 643 nm) to green (λ max em = 517 nm) in the presence of HOCl and from red to mixed blue (λ max em = 483 nm)/red in the presence of NQO1 under single-wavelength excitation. This probe had high selectivity and sensitivity towards HOCl and NQO1 in vitro. Moreover, probe NH could specifically image exogenous or endogenous HOCl and NQO1 in living cells through different emission channels. This probe provides a practical tool for studying the correlation of HOCl and NQO1 in biological systems. A dual-ratiometric fluorescent probe was developed for differential detection of HOCl and NQO1 under single-wavelength excitation, and had been successfully applied to detect exogenous/endogenous HOCl and NQO1 in living cells by exhibiting different fluorescent colors. • A dual-ratiometric fluorescent probe NH for differential detection of HOCl and NQO1 was developed for the first time. • Probe NH could detect HOCl and NQO1 under one-wavelength excitation. • Probe NH could be successfully applied for the detection of HOCl and NQO1 in living cells with different fluorescence colors. • Probe NH could be used for detecting HOCl and NQO1 in living cells by exhibiting different color combinations.