Digital SERS immunoassay of Interleukin-6 based on Au@Ag-Au nanotags

Interleukin-6 (IL-6) is a crucial cytokine involved in inflammation and immune regulation. However, the detection of IL-6 with ultrasensitivity and high specificity remains a significant challenge due to the inherent complexity of biofluids. Herein, we present a digital surface enhanced Raman scattering (SERS) immunoassay using core-shell Au@Ag-Au nanotags for IL-6 detection with ultrasensitivity and high reliability. A low-cost silicon chip was functionalized as capture substrates, employing novel SERS nanotags that exhibit strong, robust and reproducible signals at single-nanoparticle resolution as the amplification element. We proposed two analytical methods to validate single-molecule events follow a Poisson distribution and to quantify protein biomarkers over a broad linear dynamic range, respectively. The strong alignment between theoretical and experimental results enhances the method's reliability. Our assay provides two readouts: colorimetric analysis by naked eyes for high concentrations (>1 ng/mL) and digital SERS analysis for low concentrations. Following method optimization, we obtained a linear range from 100 fg/mL to 1 ng/mL (R² = 0.994) with a limit of detection (LOD) of 12.4 fg/mL, suitable for clinical applications. The method was tested for IL-6 quantification in healthy human serum and saliva, with recoveries from 92.4% to 105.3%. Finally, the immunoassay demonstrated strong consistency with the standard clinical laboratory method when tested with clinical serum samples. Thus, our proposed the digital SERS immunoassay is a promising tool for the precision clinical diagnosis of IL-6-related diseases or other conditions.