Multiplexed Single-Molecule Detection of Nucleic Acid Biomarkers with a Distance-Tuned Single FRET Pair

Biomarkers have gained tremendous attention in recent years, as they offer reliable detection of diseases such as cancers and other health conditions. However, with the recent realization that one biomarker can be associated with more than one disease (cross-talk), there is a significant shift toward simultaneous monitoring of more than one biomarker to increase the accuracy of diagnosis. Despite a sizable effort made over the last several years, multiplexing using the common techniques including surface-enhanced Raman spectroscopy (SERS), microarrays, RT-qPCR, nanostring, fluorescence, and others requires target amplification, target labeling, or the use of additional probes/actuators, and hence, these requirements complicate the experiments and data analyses. Using single-molecule fluorescence resonance energy transfer (smFRET) and 4-way DNA junction strategy, we introduced a relatively simple platform for simultaneous detection of multiple nucleic acid biomarkers. While the traditional way of smFRET-multiplexing requires multiple excitation sources and multiple FRET pairs (donor-acceptor dyes), our approach requires only one set of excitation sources and one FRET pair. Specifically, we presented four sensors that provide nonoverlapping FRET traces upon target binding, thus offering a clear multiplexed detection. Further, the 4-way DNA strategy enables detection down to low femtomolar (×10^-15 M) level without requiring target labeling and amplification. Therefore, this simple and sensitive detection platform can benefit clinical diagnosis by offering early detection of diseases, including cancers.