Optokinetically Encoded Nanoprobe-Based Multiplexing Strategy for MicroRNA Profiling

Multiplexed real-time analysis on multiple interacting molecules and particles is needed to obtain information on binding patterns between multiple ligands and receptors, specificity of bond formations, and interacting pairs in a complex medium, often found in chemical and biological systems, and difference in binding affinity and kinetics for different binding pairs in one solution. In particular, multiplexed profiling of microRNA (miRNA) in a reliable, quantitative manner is of great demand for the use of miRNA in cell biology, biosensing, and clinical diagnostic applications, and accurate diagnosis of cancers with miRNA is not possible without detecting multiple miRNA sequences in a highly specific manner. Here, we report a multiplexed molecular detection strategy with optokinetically (OK) coded nanoprobes (NPs) that show high photostability, distinct optical signals, and dynamic behaviors on a supported lipid bilayer (SLB) (OK-NLB assay). Metal NPs with three distinct dark-field light scattering signals [red (R), green (G), and blue (B)] and three different target miRNA half-complements were tethered to a two dimensionally fluidic SLB with mobile (M) or immobile (I) state. In situ single-particle monitoring and normalized RGB analysis of the optokinetically combinatorial assemblies among three M-NPs and three I-NPs with dark-field microscopy (DFM) allow for differentiating and quantifying 9 different miRNA targets in one sample. The OK-NP-based assay enables simultaneous detection of multiple miRNA targets in a highly quantitative, specific manner within 1 h and can be potentially used for diagnosis of different cancer types. We validated the OK-NLB assay with single-base mismatched experiments and HeLa cell-extracted total RNA samples by comparing the assay results to the quantitative reverse transcription polymerase chain reaction (qRT-PCR) results.