Single-Molecule Topochemical Analyses for Large-Scale Multiplexing Tasks

Multitasking is the pivotal feature in next-generation chemo- or bioanalyses. However, simultaneous analyses rarely exceed over three different tasks, which is ascribed to the limited space to accommodate analyzing units and the compromised signal-to-noise (S/N) level as the number of tasks increases. Here, by leveraging superior S/N of single-molecule techniques, we analyzed five microRNA biomarkers by spatially encoding miRNA recognition units with nanometers resolution in a DNA template, while decoding the analyte binding temporally in seconds. The hairpin stem is interspersed by internal loops to encode recognition units for miRNA. By mechanical unfolding of the hairpin, individual internal loops are sequentially interrogated for the binding of each miRNA. Using this so-called topochemical spatiotemporal analysis, we were able to achieve subpicomolar detection limits of miRNAs. We anticipate that this new single-molecule topochemical analysis can massively analyze single-molecule targets.