Analyte‐Targeted Plasmonic Hotspots on Superlattice Mirror Enable Ultra‐Broad‐Range SERS Sensing of Acetylcholinesterase

Abstract Surface‐enhanced Raman spectroscopy (SERS) enables molecular fingerprinting, but its widespread application is limited by poor detection sensitivity and signal reproducibility due to inefficient analyte retention in hotspots, less than 1 in 100, 000 molecules. To address this, we present a dynamic sensing strategy that actively couples in‐situ analyte recognition with the real‐time formation of plasmonic hotspots. The system integrates a resonant plasmonic nanoparticle superlattice monolayer as a 2D optical cavity and gap‐enhanced Au‐Ag superparticles functionalized with cyclodextrin molecular spacers. Enzymatic recognition of acetylcholinesterase (AChE), a pivotal neurofunctional enzyme, induces the self‐assembly of high‐density nanoparticle‐on‐“superlattice mirror” (NPoSM) nanocavities through competitive host‐guest displacement during acetylthiocholine hydrolysis. This active guidance ensures spatiotemporal synchronization between nanocavity formation, hotspot activation, and target binding. The system achieves high specificity with background‐free, ultrasensitive AChE detection over a 9‐order dynamic range (10 −8 to 10 U/L), offering a restricted detection platform for SERS sensing in molecular diagnostics, neurotoxicity assessment, and environmental monitoring.