Abstract

Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. To surmount these barriers, we have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic Förster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also reveals transport properties of a trapped particle, which enables discrimination between FRETfluors attached to a target and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. Finally, we demonstrate detection of both simple and complex mixtures of mRNA, dsDNA, and proteins, providing proof-of- concept for applications to amplification-free sensing of low-abundance targets in highly heterogeneous samples. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis that is nearly orthogonal to changing the geometry of a FRET construct. As a result, the number of distinguishable FRET-based labels can be combinatorically expanded while maintaining chemical compatibility, opening up new possibilities for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical components.