Single-Pair Fluorescence Resonance Energy Transfer (spFRET) for the High Sensitivity Analysis of Low-Abundance Proteins Using Aptamers as Molecular Recognition Elements

We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.