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The hybridization chain reaction (HCR) is a simple and sensitive method for quantifying nucleic acids. Current approaches cannot combine a washing-free sensing format with multiplexed target quantification at low concentrations, which would be highly desirable for detection both in solution and in situ. Here, we demonstrate the implementation of time-gated Förster resonance energy transfer (TG-FRET) between terbium donors and dye acceptors into HCR for multiplexed quantification of microRNAs (miR-20a and miR-21) and their DNA analogues. HCR-TG-FRET provided washing-free nucleic acid quantification with very low limits of detection down to 240 amol (1.7 pM) of microRNA and 123 amol (0.88 pM) of DNA. Efficient distinction from very homologous microRNAs demonstrated high target specificity. Multiplexing with a single measurement, a single excitation wavelength, and a single FRET pair allowed for a simultaneous quantification of miR-20a and miR-21 at concentrations between 30 and 300 pM from the same sample. HCR-TG-FRET showed similar performance for serum-free and serum-containing samples without the use of RNase inhibitors. Our results present a significant improvement in current HCR approaches regarding simplicity, sensitivity, and multiplexing. The versatile diagnostic performance of HCR-TG-FRET even in challenging biological environments presents an important advantage for advanced nucleic acid biosensing.

Simple, Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction