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Real-time monitoring of the evolution of bacterial infection-associated multiple radical species is critical to accurately profile the pathogenesis and host-defense mechanisms. Here, we present a unique dual wavelength near-infrared (NIR) cyanine-dyad molecular probe (HCy5-Cy7) for simultaneous monitoring of reactive oxygen and nitrogen species (RONS) variations both in vitro and in vivo. HCy5-Cy7 specifically turns on its fluorescence at 660 nm via superoxide or hydroxyl radical (O 2 .- , . OH)-mediated oxidation of reduced HCy5 moiety to Cy5, while peroxynitrite or hypochlorous species (ONOO - , ClO - )-induced Cy7 structural degradation causes the emission turn-off at 800 nm. Such multispectral but reverse signal responses allow multiplex manifestation of in situ oxidative and nitrosative stress events during the pathogenic and defensive processes in both bacteria-infected macrophage cells and living mice. Most importantly, this study may also provide new perspectives for understanding the bacterial pathogenesis and advancing the precision medicine against infectious diseases., (© 2021 Wiley-VCH GmbH.)

Real‐time monitoring of the evolution of bacterial infection‐associated multiple radical species is critical to accurately profile the pathogenesis and host‐defense mechanisms. Here, we present a unique dual wavelength near‐infrared (NIR) cyanine‐dyad molecular probe (HCy5‐Cy7) for simultaneous monitoring of reactive oxygen and nitrogen species (RONS) variations both in vitro and in vivo. HCy5‐Cy7 specifically turns on its fluorescence at 660 nm via superoxide or hydroxyl radical (O2.−,.OH)‐mediated oxidation of reduced HCy5 moiety to Cy5, while peroxynitrite or hypochlorous species (ONOO−, ClO−)‐induced Cy7 structural degradation causes the emission turn‐off at 800 nm. Such multispectral but reverse signal responses allow multiplex manifestation of in situ oxidative and nitrosative stress events during the pathogenic and defensive processes in both bacteria‐infected macrophage cells and living mice. Most importantly, this study may also provide new perspectives for understanding the bacterial pathogenesis and advancing the precision medicine against infectious diseases. [ABSTRACT FROM AUTHOR]