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.