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

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 (&gt;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.碳中心自由基在邻近标记领域具备无可比拟的纳米级精准度，但现有光驱动平台（如μMap）受限于组织穿透深度不足与光毒性问题。本研究首次提出完全无光照的双荧光素酶生物发光共振能量转移（Bioluminescence Resonance Energy Transfer, BRET）系统，重新定义了体内邻近蛋白质组学研究范式。本研究将定制化铱(III)光催化剂——[Ir(dF(CF₃)ppy)₂(bpy-N₃-马来酰亚胺)]PF₆——位点特异性偶联至靶向表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）的抗体与荧光素酶（NanoLuc或RLuc8.6-535）上，使催化剂与供体间距维持在10 nm以内，以实现按需触发单电子转移。该系统无需外接光源即可在组织深层（＞3 mm）高效生成碳中心自由基，在消除光毒性的同时保留纳米级空间分辨率。依托NanoLuc的强发光特性与RLuc8.6-535的光谱分离能力，本研究实现了前所未有的信噪比与蛋白质组覆盖度，为空间分辨微环境图谱绘制开辟了真正兼容体内环境的全新研究路径。