Zero-mode waveguide (ZMW) nano-apertures milled in metal films were proposed to improve the Förster resonance energy transfer (FRET) efficiency and enable single-molecule FRET detection beyond the 10 nm barrier, overcoming the restrictions of diffraction-limited detection in a homogeneous medium. However, the earlier ZMW demonstrations were limited to the Atto 550-Atto 647N fluorophore pair, asking the question whether the FRET enhancement observation was an artifact related to this specific set of fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to investigate single-molecule FRET at large donor-acceptor separations exceeding 10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different chemical structure, surface charge, and hydrophobicity as compared to their Atto counterparts. Our single molecule data on Alexa 546-Alexa 647 demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm, extending the spatial range available for FRET and confirming the earlier conclusions. By showing that the FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, this report is an important step to establish the relevance of ZMWs to extend the sensitivity and detection range of FRET, while preserving its ability to work on regular fluorescent dye pairs.
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