Spectral Super-Resolution Colloidal SERS Spectroscopy for Multiplexed Detection of Protein Biomarkers

Surface-enhanced Raman spectroscopy (SERS) possesses molecular specificity and single-molecule sensitivity. Yet, intensity-based SERS assays are vulnerable to nontarget analyte-induced intensity fluctuations, while frequency-shift-based SERS assays are constrained by the instrument's spectral resolution, limiting the translational quantitative applications of SERS. Herein, we introduce a stochastic colloidal plasmon-enhanced spectral sampling (SCOPE) strategy for spectral super-resolution SERS spectroscopy. Through large-scale stochastic spectral sampling in a chemically homogeneous, spectrally dynamic colloidal solution containing plasmonic nanoparticles and analyte molecules, we obtain an empirical approximation of the probability density function of the sample's spectral response. This enables accurate estimation of the true peak center with subresolution precision through Gaussian histogram fitting. Building on SCOPE, we develop a spectrally super-resolved colloidal SERS immunoassay for multiplexed detection of a panel of protein biomarkers spanning endocrine, cardiovascular, and hemostatic conditions. We believe this study paves the way for spectrally super-resolved spectroscopic applications in a variety of analytical domains.