Nanozyme‐Coupled SERS Paper Platform With Alkaline‐Mediated Hotspot Densification for Small Biomolecule Detection

ABSTRACT Reliable quantification of low‐Raman‐activity small biomolecules remains a challenge for paper‐based surface‐enhanced Raman scattering (SERS) platforms due to sparse hotspot distributions within disordered fibrous networks. Here, we present a dual‐amplification SERS platform fabricated on a layer‐by‐layer silver nanoparticle (AgNP)‐chitosan paper substrate to overcome both molecular and substrate‐level limitations. To compensate for the low inherent Raman activity of the target analyte, a nanozyme‐coupled surrogate reporter strategy was developed. The peroxidase‐like activity of AgNPs transduces analyte concentration into quantifiable oxidized 3,3′,5,5′‐tetramethylbenzidine (ox‐TMB) SERS signals through a redox‐based signal‐OFF mechanism, where uric acid (UA) reduces ox‐TMB. Concurrently, substrate SERS performance is enhanced via alkaline‐mediated interfacial regulation. Sodium hydroxide treatment deprotonates the chitosan backbone, driving a conformational contraction that densifies the plasmonic hotspot network. Using UA as a model analyte at tear‐relevant concentrations, the optimized platform achieves quantitative detection within the clinical range of 25–300 µ m ( R 2 = 0.933) with high target selectivity. This dual‐amplification strategy provides a versatile framework for sensitive SERS diagnostics on flexible paper substrates.