Multiplexed and continuous single-molecule biosensing by spectral demixing of plasmon-enhanced fluorescence

Continuous monitoring of the concentration of biomarkers is a crucial step forward to capture time-dependent changes in early stage diagnostics. A novel class of continuous monitoring sensors relies on low-affinity interactions that are probed at the single-molecule level. Nevertheless, integration of multixplexing capabilities remains a tremendous challenge, despite its promise to provide more accurate diagnostics that will allow for early intervention. Here we demonstrate a multiplexed biosensor capable of continuous monitoring using single-molecule plasmon-enhanced fluorescence. Unfortunately, plasmon-enhancement of fluorescence not only leads to increased signal brightness but also causes strong reshaping of the dye’s emission spectrum. This has prohibited multiplexing that is most commonly implemented using multiple fluorophores of different colors. Here we overcome this barrier by experimentally quantifying the spectral reshaping as a function of the plasmon wavelength and linewidth. Using this knowledge we present a new spectral demixing approach that recovers the intrinsic dye spectrum from the spectrally distorted plasmon-enhanced spectrum. This enables the recovery of the intrinsic emission peak with a 2-fold improved precision despite the spectral distortion. We then demonstrate the use of spectral demixing for single-molecule biosensing where we show the multiplexed and continuous monitoring of two nucleic acids simultaneously. The approach enables the use of a single laser line for multicolor sensing while providing a high signal-to-noise ratio to enable cost-effective and multiplexed monitoring of biomarkers. • Continuous and multiplexed single-molecule biosensing is reported with nanomolar sensitivity and 10 min response time. • Multiplexing is achieved by two-color plasmon-enhanced fluorescence imaging at the single-molecule level. • Spectral reshaping of single-molecule fluorescence is quantified and a novel spectral demixing approach is presented to recover the intrinsic dye spectrum. • Despite strong spectral reshaping, plasmon-enhanced biosensing provides a 96% accuracy in analyte classification in a multiplexed sandwich assay format.