Biofluids contain cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) that have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that increase the risk of sample loss and cross-contamination, respectively. To achieve the direct, rapid detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein describe an accelerated amplification-free single-molecule kinetic fingerprinting. This approach, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS), exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures individual target molecules onto the surface, while the other transiently interrogates them to generate kinetic fingerprints by intramolecular sin-gle-molecule Forster resonance energy transfer (smFRET). Formamide is used to further accelerate the kinetics of probe-target interactions and fingerprinting, while background signals are reduced by removing non-target-bound probes from the surface using toehold-mediated strand displacement. We show that iSiMREPS can detect in as little as 10 seconds two distinct, promising cancer biomarkers, miR-141 and a common EGFR exon 19 deletion, reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.
Exponential fluorescent amplification of individual RNAs using clampFISH probes