Multiplex detection of biomolecules is important in bionanotechnology and clinical diagnostics. Multiplexing using engineered solutions such as microarrays, synthetic nanopores, and DNA barcodes is promising, but they require sophisticated design/engineering and typically yield semiquantitative information. Single-molecule fluorescence resonance energy transfer (smFRET) is an attractive tool in this regard as it enables both sensitive and quantitative detection. However, multiplexing with smFRET remains a great challenge as it requires either multiple excitation sources, an antenna system created by multiple FRET pairs, or multiple acceptors of the donor fluorophore, which complicates not only the labeling schemes but also data analysis, due to overlapping of FRET efficiencies ( EFRET). Here, we address these currently outstanding issues by designing interconvertible hairpin-based sensors (iHabSs) with nonoverlapping EFRET utilizing a single donor/acceptor pair and demonstrate a high-confidence multiplex detection of unlabeled nucleic acid sequences. We validated the reliability of our approach by systematically omitting one target at a time. Further, we demonstrate that these iHabSs are fully recyclable, sensitive with a limit of detection of ∼200 pM, and able to discriminate against single base mismatches. The multiplexed approach developed here has the potential to benefit the fields of biosensing and diagnostics by allowing simultaneous and quantitative detection of unlabeled nucleic acid biomarkers.
Keywords: TIRF microscopy; multiplexing; recyclable sensors; single-molecule FRET; single nucleotide polymorphism (SNP).