Sequentially activated fluorescent probes based on a purine scaffold: enabling precise spatiotemporal monitoring of H2O2 and tyrosine hydroxylase in the brainstem NTS during spontaneous hypertension

Abstract

Spontaneous hypertension (SH) is a prevalent chronic cardiovascular disorder characterized by the synergistic elevation of hydrogen peroxide (H₂O₂) levels and tyrosine hydroxylase (TH) activity in the brainstem nucleus tractus solitarius (NTS). Traditional detection techniques lack the specificity and spatiotemporal resolution to monitor the dynamic interplay of these two core pathological biomarkers, hindering the in-depth exploration of SH pathogenesis. Herein, a series of novel cascade-activated fluorescent probes (PPTHs) were rationally designed and synthesized based on a purine core, which achieve specific fluorescence responses only upon sequential activation by H₂O₂ and TH. In vitro assays demonstrated that the probes exhibited high sensitivity toward H₂O₂ in SH-SY5Y cell lysates, with a reliable limit of detection (LOD) and ideal anti-interference capability. Live-cell imaging further confirmed that purine-based molecules not only successfully mitigated probe adsorption on the cell membrane but also effectively improved the imaging signal-to-noise (S/N) ratio. Notably, PPTH-2-assisted confocal imaging clearly distinguished the differential fluorescence signals between normotensive control and SHR groups, which correlated with endogenous H₂O₂ level and TH activity in the NTS region. Our study presents a robust fluorescent probe platform for the synchronous detection of H₂O₂ and TH, offering a promising molecular tool for the early diagnosis and elucidation of the pathological mechanisms of SH.