An Internal-Standard-Integrated Digital SERS Immunoassay for Accurate and Precise Trace-Level Cytokine Quantification in Plasma

Abstract Accurate and precise quantification of trace-level cytokines in complex biological matrices remains challenging, limiting their use as longitudinal biomarkers in clinical studies. Here, we present a nanotechnology-enabled surface-enhanced Raman scattering (SERS) platform incorporating an internal standard (IS) for digital, attomolar-level multiplex cytokine quantification. This platform enables precise digital trace-level detection of IL-1β, IL-6, IL-12, and TNF-α. Using an orthogonal recombinant protein IS to correct assay- and SERS-related variability, this platform addresses key barriers to reliable trace-protein measurements and broader clinical adoption. Incorporation of the IS reduced the inter-replicate coefficient of variation from ∼40–60% to 2–3%, and improved accuracy to ∼95–105%, enabling robust cytokine quantification in human plasma. Compared with a widely used clinical electrochemiluminescence (ECL) assay, our internal-standard-integrated chip enables accurate and precise quantification while maintaining multiplex capability, consistently detecting cytokine levels near or below ECL-defined quantification limits. As a proof-of-concept application, we evaluated the platform using plasma samples from individuals with MDD, a clinically relevant context in which aberrant immune activation and low-level inflammation have been implicated. These measurements support the analytical robustness of the internal-standard-integrated digital SERS immunoassay and its potential utility for future longitudinal cytokine monitoring studies in complex samples.