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 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.