Highly accurate multiplexed nanoplasmonic detection of microRNAs using splinted ligation

• We developed a chip-based multiplexed nanoplasmonic detection method for microRNAs. • Single nucleotide variation between miRNAs could be clearly discriminated. • High quantitative accuracy was demonstrated in multiplexed detection. • Application to total RNAs showed excellent analytical performance of this method. 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 accuracy of this method. The relative abundance of miRNAs in total RNA was also successfully assessed using this method.