Nanohole-in-microsphere array (NiMA): An ultrasensitive digital-SERS tool for serum diagnosis of rheumatoid arthritis

Rheumatoid arthritis (RA) is a serious chronic systemic autoimmune disease leading to joint destruction for progressive disability. Accurate non-invasive diagnosis of RA in the early stages is vital to timely precision treatment. The advancements of surface-enhanced Raman spectroscopy (SERS) in label-free liquid biopsy for noninvasive diagnosis have been witnessed in the past decade. The recently-developed digital SERS (dSERS) further overcomes the spectral intensity fluctuation, strengthening reliability and robustness for quantitative analysis. Here we propose a high-throughput dSERS substrate by fs-laser parallel fabrication utilizing microsphere-generated photonic nanojet (PNJ), by which the nanohole-in-microsphere array (NiMA) is obtained with the minimum diameter of 115 nm and depth of 600 nm. The Au-nanoparticle-coated NiMA possesses high precision in liquid sampling (10^-18 L) and ultralow limit of detection. The discrete nanohole distribution with high homogeneity guarantees the independent acquisition of Raman spectrum for accuracy of Poisson's statistics in a small spectral dataset volume. The NiMA substrate is employed to collect the sera and diagnose by quantitative recognition of three biomarkers (homocitrulline, L-tryptophan and L-citrulline) in sera by dSERS approach. The accuracy of 88.0 %, sensitivity of 92.9 % and specificity of 81.8 % in distinguishing RA patients manifests the feasibility of serum dSERS protocol for clinical early-stage diagnosis.