Single-microbead space-confined digital quantification strategy (SMSDQ) for counting microRNAs at the single-molecule level.

Quantification of microRNAs (miRNAs) at the single-molecule level is of great significance for clinical diagnostics and biomedical research. The challenges lie in the limits to transforming single-molecule measurements into quantitative signals. To address these limits, here, we report a new approach called a Single Microbead-based Space-confined Digital Quantification (SMSDQ) to measure individual miRNA molecules by counting gold nanoparticles (AuNPs) with localized surface plasmon resonance (LSPR) light-scattering imaging. One miRNA target hybridizes with the alkynyl-modified capture DNA probe immobilized on a microbead (60 μm) and the azide-modified report DNA probe anchored on AuNP (50 nm), respectively. Through the click reaction between the alkynyl and azide group, a single microbead can covalently link the AuNPs in the confined space within the view of the microscope. By digitally counting the light-scattering spots of AuNPs, we demonstrated the proposed approach with single-molecule detection sensitivity and high specificity of single-base discrimination. Taking the advantages of ultrahigh sensitivity, specificity, and the digital detection manner, the approach is suitable for evaluating cell heterogeneity and small variations of miRNA expression and has been successfully applied to direct quantification of miRNAs in one-tenth single-cell lysates and serum samples without RNA-isolated and nucleic acid amplification steps.