One Bead Three Targets: An Enzyme-Free Platform Enabling Simultaneous Detection of Multiplex MicroRNAs on a Single Microbead

Abstract Multiplexed microRNA analysis is of great significance in early diagnosis and prognosis of cancers. Based on the thermal cycling-assisted click nucleic acid ligation reaction, we wish to report an enzyme-free and mix-and-read fluorescence sensing strategy for the simultaneous detection of several species of miRNA on a single microbead (MB). In this study, the target miRNAs act as the templates to initiate the copper-free click DNA ligation between the bi-functional linker DNA probes (5′-biotin/3′-Aza-DBCO) and reporter DNA probe (3′-N3 and 5′-fluorophore coded for a specific target). Assisted with thermal cycling, each miRNA molecule can be recycled for many rounds to generate a large number of ligation products. Consequently, all of the fluorophore-encoded ligation products are accumulated on only one MB, leading to the highly concentrated fluorescence gathering. Through multi-channel fluorescence imaging of the fluorophore-anchored single MB, simultaneous detection of multiplex miRNAs can be realized with the limit of detection (LOD) lowered down to 16 fM. Benefiting from high sensitivity, excellent specificity, and multiplexed detection capability, the simple and enzyme-free method is promising in miRNA-related biomedical applications.