Polymer Platform for Peroxynitrite Detection Utilizing Benzopyrylium Chemistry

Continuous detection of peroxynitrite, a highly reactive oxygen and nitrogen species linked to the onset and severity of oxidative stress, is essential for real-time monitoring of disease progression. While benzopyrylium (BZP)-based two-photon fluorogenic probes offer promising selectivity, small molecule designs are restricted by short detection lifetimes, poor solubility, narrow pH stability, limited modularity and cytocompatibility. Here, we report the first polymeric BZP-based peroxynitrite probe, enabled by ring-opening metathesis polymerization (ROMP) of norbornene-functionalized BZP monomers (M2). These macromolecular platforms exhibit broad pH stability, excellent selectivity, and tunability in their response kinetics. Upon self-assembly into nanoparticles, the resulting system displays bathochromic shifts toward higher near-infrared two-photon emission (770 nm) for high-resolution ratiometric sensing. Exposure to peroxynitrite triggers progressive nanoparticle (23 nm) aggregation and swelling into complex globular morphologies (>200 nm), resulting in prolonged functional lifetimes (up to 60 hours) and distinct visualization. Compared to their small molecule counterparts, these macromolecular probes demonstrate substantially improved cytocompatibility in healthy CHO and SKOV-3 cancer cells up to 100 µM and enable dynamic imaging of continuous endogenous peroxynitrite generation in-vitro. This polymeric platform sets the stage for high-performance oxidative stress sensors capable of continuous, disease-relevant monitoring in biological environments.