Photocatalytic Activation of Alkyl Diazirine Probes for In Situ Drug Profiling and Extracellular Vesicle-Based Diagnostics

Elucidating drug-target interactions within native biological environments is critical for rational drug design and personalized medicine. While photoaffinity labeling (PAL) serves as a powerful tool for capturing these transient interactions, conventional photoactivation by UV light suffers from phototoxicity, limited penetration, and low yields. Advances in visible-light photocatalysis provide new opportunities, yet current strategies often rely on direct catalyst conjugation to the drug, introducing steric and physicochemical perturbations that can compromise native binding affinity. Moreover, the photocatalytic activation of alkyl diazirines, despite their widespread utility in PAL, has remained largely unexplored. Here we report a photocatalysis-enhanced photoaffinity labeling (PE-PAL) platform that activates bioorthogonal alkyl diazirine probes using separate iridium photocatalysts under blue light. By employing lipid- and peptide-modified iridium bioconjugates, PE-PAL achieves efficient labeling at submicromolar catalyst loading with enhanced biocompatibility, enabling in situ drug analysis via cellular imaging and proteome profiling. We further extended this platform to extracellular vesicle (EV) analysis by integrating probe-mediated enzymatic amplification with nanoplasmonic resonators to directly profile drug-target interactions in clinical blood samples. From microliter-scale samples, we achieved sensitive, multiparametric analysis of disease-associated EV labeling indices, demonstrating the translational potential of PE-PAL for EV-based liquid biopsy.