Quantitative detection of exosomal microRNA extracted from human blood based on surface-enhanced Raman scattering

Since the nature of the exosomal lipid bilayer can allow miRNAs to be protected from degradation by cellular RNAses in body fluids, exosomal microRNA (miRNA) has become an ideal source of non-invasive biomarkers for the early diagnosis and prognosis. In this paper, a new surface-enhanced Raman scattering (SERS) analysis strategy combining stable SERS reporter element and duplex-specific nuclease (DSN)-assisted signal amplification for quantitative detection of exosomal miRNA extracted from human blood is proposed. Firstly, we prepared SERS signal reporter of Au@R6G@AgAu nanoparticles (R6G attachment on the gold nanoparticles, then encapsulated in AgAu alloy shell nanoparticles named as ARANPs) with an inter small nanogap to generate stable SERS signal. Then, ARANPs and separating substrate of silicon microbead (SiMB) were then covalently attached to the 3'- and 5'- end of capture probe (CP) targeting exosomal miRNA. Upon target miRNA binding, DNA in heteroduplexes could be specifically cleaved by DSN and resulted in the release of ARANPs from the surface of SiMB. Meanwhile, target miRNA remained intact and subsequently involved in the next round of target-recycling amplification. The combination of stable SERS intensity and signal amplification significantly improved the sensitivity of the sensing systems, resulting in detection limits of 5 fM. More importantly, this method also could be used for the detection of exosomal miRNAs extracted from the blood collected from patients of recurrence in non-small-cell lung cancer (NSCLC), with a detection of 5.0 mu L of sample volume, which has potential for point-of-care testing (POCT) in clinical analysis.