Label-Free Quantification of Bilirubin Using a Refractive Index-Insensitive Nanolaminate SERS Substrate

Bilirubin is an important biomarker, where a small unbound fraction dissociated from albumin can cross the blood–brain barrier and induce neurotoxicity, such as kernicterus, at low nanomolar levels. Accurate detection of this low-level fraction remains challenging. Surface-enhanced Raman spectroscopy (SERS) enables label-free molecular detection; however, variations in the local refractive index (RI) at plasmonic hotspots can detune the resonance from the excitation wavelength, leading to signal fluctuations and limited quantitative reliability. Here, we present a multi-resonant nanolaminate SERS substrate designed to achieve RI-insensitive and robust signal enhancement. The vertically stacked metal–insulator–metal architecture provides broadband spectral overlap with both excitation and Raman scattering under dielectric loading, maintaining consistent enhancement across varying RI conditions. We demonstrate label-free bilirubin detection with a highly linear response over 10−9 to 10−4 M, achieving an R2 value of 0.99. Compared with previously reported bilirubin SERS substrates relying mainly on single-resonant plasmonic enhancement, this RI-insensitive design offers improved quantitative reliability under dielectric environmental changes. These results highlight the importance of RI-insensitive SERS design for reliable quantification and provide a general strategy for robust SERS-based biosensing.