FRET Enhancement in Aluminum Zero‐Mode Waveguides

Abstract

Zero-mode waveguides (ZMWs) are confining light into attoliter volumes, enabling single molecule fluorescence experiments at physiological micromolar concentrations. Among the fluorescence spectroscopy techniques that can be enhanced by ZMWs, Förster resonance energy transfer (FRET) is one of the most widely used in life sciences. Combining zero-mode waveguides with FRET provides new opportunities to investigate biochemical structures or follow interaction dynamics at micromolar concentration with single molecule resolution. However, prior to any quantitative FRET analysis on biological samples, it is crucial to establish first the influence of the ZMW on the FRET process. Here, we quantify the FRET rates and efficiencies between individual donor-acceptor fluorophore pairs diffusing in aluminum zero-mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large literature describing their use for single molecule fluorescence spectroscopy. We also compare the results between ZMWs milled in gold and aluminum, and find that while gold has a stronger influence on the decay rates, the lower losses of aluminum in the green spectral region provide larger fluorescence brightness enhancement factors. For both aluminum and gold ZMWs, we observe that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states (LDOS). Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single molecule FRET at physiological concentrations.

Matching Nanoantenna Field Confinement to FRET Distances Enhances Förster Energy Transfer Rates

“…Additionally, it is worth mentioning that the fluorescence decays found with gold structures are affected by a fast sub-5 ps decay originating from the gold photoluminescence. A positive element is that aluminum antennas are free of this effect; 35 …”

Förster resonance energy transfer rate in any dielectric nanophotonic medium with weak dispersion

“…FRET is the dominant energy transfer mechanism between emitters in nanometer proximity, since the rate has a characteristic ( ) r r Modern nanofabrication techniques have stimulated the relevant question whether Förster transfer can be controlled purely by means of the nanophotonic environment, while leaving the FRET pair geometrically and chemically unchanged. Indeed, theory and experiments have revealed both enhanced and inhibited FRET rates for many different nanophotonic systems, ranging from dielectric systems via plasmonic systems to graphene [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. At the same time, it is well known that the spontaneous-emission rate of a single emitter is controlled by the nanophotonic environment [40][41][42][43].…”

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

“…Major advantages of SMRT sequencing over second-generation sequencing methods include long average read lengths of more than 10,000 bases and lack of GC% bias 3, 5, 6 , critical for gap-free sequencing, and the ability to directly detect DNA base modifications by monitoring polymerase kinetics 2 . Apart from DNA sequencing, ZMWs have been exploited for single molecule RNA sequencing/epigenetics 7 and a variety of other single-molecule studies 8–13 . A critical limiting step of SMRT sequencing is the loading of long DNA templates into ZMW confinements.…”