Layer‐by‐Layer Chitosan– <scp>AuNPs</scp> @ <scp>PTS</scp> Multilayer Films for Noninvasive Lactate Sensing

ABSTRACT The study developed and characterized a noninvasive lactate biosensor based on layer‐by‐layer (LbL) films of chitosan (Chi) and AuNPs@PTS, a gold nanoparticle–polythiophene composite. LbL assemblies with varying bilayer numbers were fabricated and used to immobilize lactate oxidase (LOx), yielding Chi/AuNPs@PTS/LOx electrodes. Film growth and properties were probed by UV–vis absorption, fluorescence, and electrochemical measurements. Absorbance and fluorescence increased with the number of bilayers, consistent with exponential film buildup. Cyclic voltammetry (CV) revealed that two bilayers enabled faster charge transfer and lower resistance than thicker films, identifying this architecture as optimal for enzyme immobilization. LOx was deposited by drip casting and cross‐linked with glutaraldehyde. Lactate sensing by CV was verified in standards and human sweat samples. The platform exhibited a linear analytical response from 0.5 to 30 mM, covering typical sweat concentrations, and demonstrated noninvasive measurement capability. Together, these results show that a minimal (two‐bilayer) Chi/AuNPs@PTS film provides an efficient matrix for LOx, supporting sensitive, sweat‐relevant lactate detection. The straightforward LbL fabrication and enzyme cross‐linking are compatible with low‐cost processing. Overall, the study establishes functional design principles by correlating the number of LbL bilayers with charge‐transfer properties and electrochemical performance to balance sensitivity with stability for wearable monitoring applications.