Multiplexed Nucleic Acid Hybridization Assays Using Single‐FRET‐Pair Distance‐Tuning

Abstract

Multiplexed photoluminescence (PL) detection plays an important role in chemical and biological sensing. Here, it is shown that time-gated (TG) detection of a single terbium-donor-based Förster resonance energy transfer (FRET) pair can be used to selectively quantify low nanomolar concentrations of multiple DNAs or microRNAs in a single sample. This study demonstrates the applicability of single-TG-FRET-pair multiplexing for molecular (Tb-to-dye) and nanoparticle (Tb-to-quantum-dot) biosensing. Both systems use acceptor-sensitization and donor-quenching for quantifying biomolecular recognition and modification of the donor-acceptor distance for tuning the PL decays. TG intensity detection provides extremely low background noise and a quick and simple one-step assay format. Single-TG-FRET-pair multiplexing can be combined with spectral and spatial resolution, paving the way for biosensing with unprecedented high-order multiplexing capabilities.

Advanced microRNA-based cancer diagnostics using amplified time-gated FRET

“…7 nm when using 0.34 nm per base pair and 0.4 ± 0.1 nm for both Tb and Cy5.5 and was shown to be in excellent agreement with time-resolved FRET measurements on double-stranded (ds) DNA. 35 When comparing the PL decays of the Tb-Cy5.5 FRET pair inside the RCP and inside dsDNA (ESI Fig. S2 † ), the average FRET-sensitized PL lifetime is significantly shorter for the RCP FRET-pair (0.4 ms vs. 2 ms), which means that the average FRET efficiency is higher (0.86 vs. 0.28).…”

Nanomaterials for Cancer Precision Medicine

“…Multiplexed detection of microRNA can provide complementary information for clinical decision‐making and evaluation of therapeutic outcome. Many methodologies have been developed for multiplex or single microRNA detection based on various mechanisms, including dye‐quencher cyclic enzymatic amplification method, microfluidic‐paper‐based analytical devices, donor–acceptor Förster resonance energy transfer (FRET), FISH fluorescence imaging, cascade hybridization reaction, and solid‐state biosensors . Most of these methods are developed for early diagnosis or prognosis of diseases according to their sensitivity, specificity, type of tissue or cellular samples.…”

“…Nanomaterials have been utilized for highly sensitive and specific microRNA detection. Quantum dots and graphene oxide have widely been used in the donor–acceptor Förster resonance energy transfer method . The detection selectivity of these methods is, however, heavily dependent on the ratio of signal amplification to background fluorescence.…”

Nanoparticles as energy donors and acceptors in bionanohybrid systems

“…The monitoring of fluorescence emission of the same wavelength with the microsecond time gate allowed distinguishing these two pathways which was utilized in the duplex assays for protease activity and oligonucleotides hybridization. Another approach was taken in miRNA hybridization sensor consisting of two oligonucleotide probes conjugated to a terbium complex and coupled with QD using different QD-Tb separation distances (Qiu et al, 2017a). Thus, the rate of FRET varied between the two probes which was reflected in different decay times.…”