A versatile microfluidic-SERS barcoding system is developed herein for sensitive and multiplexed imaging of circulating microRNAs through interfacial probing of encoded nanorod aggregates at diverse patterned nanogaps. The use of a single-layer, vertically oriented nanorod array creates a plasmonic coupling-based electromagnetic field with enormously enhanced Raman outputs. The introduction of herringbone micromixer with a circulated microflow sampling accelerates the hybridization and capture of nanorod aggregates on the plasmonic substrate. The method is able to achieve ideal sensitivities at sub-femtomolar levels for four miRNAs, with multiplexed assay capability for an integrated liquid biopsy. The digital profiling of serum miRNAs in mapping and barcoding formats both enable clear discrimination of untreated cancer patients from the healthy cohort, and precise classification of tumor subtypes, stages, and metastatic conditions, with an overall accuracy of 94%. The SERS-based microfluidic barcoding system therefore holds great promise in early cancer screening, diagnosis, and prognosis.
