<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（carbon-centered radicals）在邻近标记（proximity labeling）中具备无可比拟的纳米级精度，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（phototoxicity）缺陷。我们首次报道了完全无光的双荧光素酶生物发光共振能量转移（BRET）系统，重新定义了体内邻近蛋白质组学（in vivo proximity proteomics）的研究范式。本研究将定制化铱(III)光催化剂（Ir(III) photocatalyst）——[Ir(dF(CF₃)ppy)₂(bpy-N₃-maleimide)]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），使催化剂与能量供体的间距控制在10 nm以内，可按需触发单电子转移（single-electron transfer）。该体系可在无需外部光源的情况下，在深度超过3毫米的组织内部生成高效碳中心自由基，在保留纳米级空间分辨率的同时彻底消除光毒性。通过利用NanoLuc的强发光特性与RLuc8.6-535的光谱分离优势，本研究实现了前所未有的信噪比（signal-to-noise ratios）与蛋白质组覆盖度（proteome coverage），为空间分辨的微环境图谱绘制开辟了真正兼容体内环境的全新路径。