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MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers. We introduce a kinetic fingerprinting approach called single-molecule recognition through equilibrium Poisson sampling (SiMREPS) for the amplification-free counting of single unlabeled miRNA molecules, which circumvents thermodynamic limits of specificity and virtually eliminates false positives. We demonstrate high-confidence, single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biofluids, as well as &gt;500-fold discrimination between single nucleotide polymorphisms.

Kinetic fingerprinting to identify and count single nucleic acids