Monitoring Enzyme Activity on the Surface of a Self‐Referenced Near‐White Light Emitting Assembly of Atomic Clusters

Sensitive detection of protease activity in complex biological environments remains challenging due to signal instability, background interference, and the lack of internal calibration. Here, we report a self‐referenced, white‐light–emitting surface defect‐engineered nanocluster assembly for selective and ratiometric detection of proteinase K activity and inhibition. Manganese‐doped, zinc‐mediated gold nanocluster (Zn Au NC) assemblies functionalized with bovine serum albumin (BSA) generate tricolor emission from BSA, the nanocluster core, and Mn ²⁺ , producing near‐white‐light fluorescence that serves as an intrinsic internal reference. Proteinase K induces coordinated spectral changes, including enhanced and blueshifted BSA emission with simultaneous quenching of Zn Au NC and Mn ²⁺ emissions, resulting in systematic chromaticity shifts and a robust ratiometric response. Mechanistic studies attribute these changes to proteolytic cleavage of BSA and disruption of the nanocluster assembly, corroborated by circular dichroism measurements. The platform exhibits high selectivity for proteinase K , enables inhibitor detection, and performs reliably in fetal bovine serum. This self‐calibrated nanocluster platform provides a versatile strategy for enzyme‐responsive sensing with potential applications in biochemical diagnostics and inhibitor screening.