Fluorescence energy transfer enhancement in aluminum nanoapertures

TL;DR

This study investigates how aluminum zero-mode waveguides enhance fluorescence resonance energy transfer (FRET) efficiency and rates, comparing aluminum and gold structures to optimize single-molecule biochemical analysis at physiological concentrations.

Contribution

The paper provides the first quantitative analysis of FRET rates and efficiencies in aluminum ZMWs, highlighting their advantages over gold in fluorescence enhancement for single-molecule studies.

Findings

Aluminum ZMWs increase fluorescence brightness more than gold in the green spectral region.

FRET rate scales linearly with donor decay rate and local density of optical states.

Aluminum ZMWs are promising for enhanced single-molecule FRET at physiological concentrations.

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, F\"{o}rster 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.