AsymmetricD–A–D′ RatiometricMolecule for Highly Specific Hypochlorous Acid Detection

Hypochlorous acid exists as HClO in acidic conditions and as ClO^– in alkaline conditions, posing a significant challenge for differentiation due to their strong and closely similar oxidative reaction activities. Addressing this challenge, our study presents an asymmetric donor–acceptor–donor′ (D–A–D′) molecular architecture for the design of a fluorescent probe (<b>PMT</b> NPs) that demonstrates exceptionally high specificity toward HClO alongside an optimized ratiometric response. The incorporation of the strong electron acceptor 2-(diphenylmethylene)malononitrile (A) modulates the reducing ability of the phenothiazine recognition site, adjusting the probe’s oxidation potential to an intermediate level between HClO and ClO^–. This adjustment directly dictates the probe’s selectivity, enabling it to respond exclusively to HClO. By incorporating D′, the probe’s response to HClO shifts the intramolecular charge transfer (ICT) from the original D–A to D′–A, instead of the usual D_ox–A as presented in previous works. This adjustment controls the blue shift in fluorescence wavelength upon recognition, thereby improving the accuracy of ratiometric signals <i>in vivo</i>. The ability of <b>PMT</b> NPs to precisely recognize HClO in acidic environments was validated through live cell imaging and <i>in vivo</i> experiments using zebrafish and mouse models, enabling real-time monitoring of HClO surges. This dual-pronged molecular design strategy, which combines D–A interaction modulation with a D–A–D′ molecular architecture, promises to revolutionize probe designs for various biomolecules and is anticipated to advance the understanding of diseases linked to these analytes.