Interfacial assembly of bilayer plasmonic films with dynamic hotspot regulation for ultrasensitive surface-enhanced Raman spectroscopy detection

Interfacial assembly enables bilayer plasmonic SERS films; • Dynamic hotspot regulation governs multilayer SERS performance; • Ultrasensitive quantitative detection demonstrated in serum. Interfacial assembly of plasmonic nanostructures provides a versatile strategy for engineering Surface-enhanced Raman Spectroscopy (SERS) substrates, yet the fundamental mechanism governing multilayer SERS enhancement remains poorly understood. Here, we report interfacially assembled bilayer plasmonic films with precisely controlled Au nanoparticle layer numbers and uncover a previously unrecognized nonmonotonic enhancement behavior, in which the bilayer configuration unexpectedly outperforms both monolayer and multilayer counterparts through a dynamic hotspot regulation mechanism. The bilayer architecture exhibits optimal SERS performance, achieving ultralow limits of detection of 1.05 × 10 −14  M for crystal violet (CV) and 1.0 × 10 −11  M for paclitaxel (PTX) with excellent reproducibility (RSD < 5%). Time-resolved SERS measurements and Raman mapping reveal that solvent-driven analyte enrichment and interlayer nanogap evolution generate temporally stabilized plasmonic hotspots, producing a robust signal plateau window and establishing a direct structure–dynamics–performance relationship fundamentally distinct from conventional static hotspot amplification models. Benefiting from this mechanism, the bilayer films enable quantitative PTX detection in human serum with a limit of detection of 6.9 × 10 −10  M and show strong agreement with LC–MS analysis in 50 clinical samples (R 2  = 98.78%). Furthermore, in an orthotopic breast cancer mouse model, SERS-guided dose adjustment maintains antitumor efficacy while reducing systemic toxicity. This work not only provides insights into a previously unexplored mechanism of hotspot regulation in multilayer plasmonic assemblies but also provides a mechanistically guided interfacial engineering strategy for designing robust SERS substrates for ultrasensitive quantitative detection in complex biological environments. Scheme 1. Dynamic hotspot regulation arising from interlayer nanogap evolution and analyte enrichment establishes a structure–dynamics–performance correlation in bilayer plasmonic films. This mechanism produces a stable signal plateau window and enables ultrasensitive and quantitative paclitaxel monitoring for precision chemotherapy. Download: Download high-res image (164KB) Download: Download full-size image