Light Free Iridium Luciferase Conjugates Enable Carbon Centered Radical Proximity Labeling and Deep Tissue Microenvironment Mapping

Carbon-centered radicals offer unmatched nanometer-scale precision for proximity labeling, yet existing light-driven platforms such as μMap are limited by shallow tissue penetration and phototoxicity. We present the first fully light-free, dual-luciferase BRET system that redefines in vivo proximity proteomics. A bespoke Ir(III) photocatalyst-[Ir(dF(CF₃)ppy)₂(bpy-N₃-maleimide)]PF₆ is site-specifically tethered to both EGFR-targeting antibodies and luciferases (NanoLuc or RLuc8.6-535), placing the catalyst and donor within 10 nm for on-demand single-electron transfer. This architecture generates high-efficiency carbon radicals deep within tissue (>3 mm) without any external light source, eliminating phototoxicity while preserving nanometer spatial resolution. By leveraging NanoLuc’s intense emission and RLuc8.6-535’s spectral isolation, we achieve unprecedented signal-to-noise ratios and proteome coverage—unlocking a truly in vivo-compatible route for spatially resolved microenvironment mapping.