Compounds with a nitrobenzoxadiazole (NBD) skeleton exhibit prominent useful properties including environmental sensitivity, high reactivity toward amines and biothiols (including H₂S) accompanied by distinct colorimetric and fluorescent changes, fluorescence-quenching ability, and small size, all of which facilitate biomolecular sensing and self-assembly. Amines are important biological nucleophiles, and the unique activity of NBD ethers with amines has allowed for site-specific protein labelling and for the detection of enzyme activities. Both H₂S and biothiols are involved in a wide range of physiological processes in mammals, and misregulation of these small molecules is associated with numerous diseases including cancers. In this review, we focus on NBD-based synthetic probes as advanced chemical tools for biomolecular sensing. Specifically, we discuss the sensing mechanisms and selectivity of the probes, the design strategies for multi-reactable multi-quenching probes, and the associated biological applications of these important constructs. We also highlight self-assembled NBD-based probes and outline future directions for NBD-based chemosensors. We hope that this comprehensive review will facilitate the development of future probes for investigating and understanding different biological processes and aid the development of potential theranostic agents.

A highly sensitive H₂S-specific near-infrared fluorescence-enhanced probe was developed for real-time imaging of endogenous H₂S in colorectal cancer cells (HCT116 and HT29) in mice.

Hydrogen sulfide (H₂S) is an important endogenous signalling molecule and also an important environmental target for detection. New reaction-based colorimetric and fluorescent turn-on probes based on selective thiolyling of NBD (7-nitro-1,2,3-benzoxadiazole) ether were explored for sensing of H₂S in aqueous buffer. The syntheses of both probes are simple and quite straightforward. The probes are highly sensitive and selective toward H₂S over other biologically relevant species. Probe 1 can be used to directly visualize H₂S by the naked eye and shows more than 1000-fold fluorescence increase upon reaction with H₂S. Probe 2 is a near-infrared fluorescent sensor for H₂S at physiological pH.

A FRET–ICT dual-quenching fluorescent probe with significant off–on response toward H₂S is rationally designed and prepared for imaging of H₂S in living cells.

A fluorescent probe installed with dual-reactive and dual-quenching groups was rationally designed and synthesized for highly selective and sensitive sensing of biothiols.

H₂S-specific fluorescent/colorimetric probes based on the thiolysis of NBD dyes are summarized.