Highly Accurate Multiplexed Nanoplasmonic Detection of Micrornas Using Splinted Ligation

Abstract

MicroRNAs (miRNAs) are short single-stranded RNAs that regulate gene expression at the post-transcriptional level. Abnormal expression of miRNAs is widely observed in cancer; hence, miRNAs have been recognized as important cancer biomarkers. However, the accurate detection of miRNAs is challenging owing to their short length and high sequence homology. In this study, we developed an accurate multiplexed miRNA detection method based on localized surface plasmonic resonance (LSPR) of metallic nanostructures. MiRNAs were labeled with DNA probes by splinted ligation and immobilized on a gold nanodot array. After further intensification via enzyme-assisted precipitation, the shift in LSPR absorption was measured to estimate the concentration of miRNAs. Three miRNAs (miR-34c, miR-99a, and miR-125b) were detected with very high sensitivity (limit of detection: 18–261 zmol). The clear discrimination of single-nucleotide variations and precise estimation of miRNA concentrations demonstrated the high quantitative accuracy of this method. Application to real samples revealed that miR-125b, an important cancer-related miRNA, is upregulated in gastric cancer cells.

Keywords: LSPR biosensing, MicroRNA, Gold nanodot array, Splinted ligation, Single nucleotide variation, Multiplexing