Optimized fluorescent probes for heparan sulfate sensing in live cells and human blood.

Heparan sulfate (HS) proteoglycans on the cell surface play pivotal roles in cell signaling, angiogenesis, endocytosis, infectious disease progression, and tumor invasion. Real-time detection and quantification of HS in live cells remain challenging due to its structural heterogeneity and probe internalization issues. Here, we report rationally designed fluorescent probes (1-3) incorporating a branched peptide receptor with fatty-acid and/or PEG conjugation for selective targeting of HS proteoglycans. Among them, the PEGylated probe 3 showed stable and selective ratiometric fluorescence responses to HS in RPMI medium, with exclusive cell-surface localization for 120 min. In contrast, the fatty-acid-conjugated probe 2 exhibited partial accumulation at the plasma membrane for approximately 20 min, but was subsequently internalized into the cytoplasm, limiting its cell-surface retention. Notably, probe 3 enabled quantitative monitoring of HS depletion in live cells following inhibition of HS sulfation by siRNA or chemical treatment, as well as after heparinase digestion, while simultaneously attenuating fibroblast growth factor signaling and suppressing cancer cell migration. Furthermore, PEG conjugation reduced nonspecific serum protein binding, thereby enhancing sensitivity and selectivity for HS detection in human blood. These findings demonstrate that PEG conjugation is an effective strategy to optimize fluorescent HS probes by enhancing cell-surface anchoring, prolonging retention, and improving ratiometric responses in both cellular and plasma environments.