Fluorescence Brightness, Photostability, and Energy Transfer Enhancement of Immobilized Single Molecules in Zero-Mode Waveguide Nanoapertures

Abstract

Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor single molecules beyond the range accessible to normal microscopes. However, several aspects of the ZMW influence on the photophysics of fluorophores remain inadequately documented and sometimes controversial. Here, we thoroughly investigate the ZMW influence on the fluorescence of single immobilized Cy3B and Alexa 647 molecules, detailing the interplays between brightness, lifetime, photobleaching time, total number of emitted photons and Förster resonance energy transfer (FRET). Despite the plasmonicenhanced excitation intensity in the ZMW, we find that the photostability is preserved with similar photobleaching times as on the glass reference. Both the fluorescence brightness and the total numbers of photons detected before photobleaching are increased, with an impressive gain near five times found for Alexa 647 dyes. Finally, the single-molecule data importantly allow a loophole-free characterization of the ZMW influence on the FRET process. We show that the FRET rate constant is enhanced by 50%, demonstrating that nanophotonics can mediate the energy transfer. These results deepen our understanding of the fluorescence enhancement in ZMWs and are of immediate relevance for single-molecule biophysical applications.

Single Effective Complex Loading into Zero-Mode Waveguides Optimized with Fluorescence Evaluation at Quenching and Accumulation Checkpoints

“…ZMWs enable the capture of singlemolecule fluorescence signals at concentrations ranging from micromoles to millimoles, 3−5 facilitating the exploration of biological macromolecule interactions under physiological concentrations. These advantages have facilitated the extensive use of ZMWs in areas such as single-molecule sequencing, 6,7 biological membrane research, 8 single-molecular dynamic detection, 9,10 and real-time interaction detection. 11−13 The single molecule or complex such as a double-stranded deoxyribonucleic acid (DNA), 10,14,15 single-stranded DNA (ssDNA), 16 protein−DNA complex, 6,12,17,18 or protein− ribonucleic acid complex 19 complexes into the ZMW pores is more decisive for effective detection as they play an important role in biological reactions and mechanism studies.…”

“…These advantages have facilitated the extensive use of ZMWs in areas such as single-molecule sequencing, 6,7 biological membrane research, 8 single-molecular dynamic detection, 9,10 and real-time interaction detection. 11−13 The single molecule or complex such as a double-stranded deoxyribonucleic acid (DNA), 10,14,15 single-stranded DNA (ssDNA), 16 protein−DNA complex, 6,12,17,18 or protein− ribonucleic acid complex 19 complexes into the ZMW pores is more decisive for effective detection as they play an important role in biological reactions and mechanism studies. 20−22 Efficient loading of biological complexes requires not only the loading of an individual molecule into the ZMW pore but also the functionality of the complex.…”

On‐Chip Fluorescent Sensor for Chemical Vapor Detection

“…Waveguide evanescent field based fluorescence sensors (WFS) have been exploited for cancer detection, [ 18 ] deoxyribonucleic acid (DNA) sequencing, [ 19 ] single‐molecule detection, [ 19b,20 ] and food safety applications. [ 21 ] However, these reports were all focused on the detection of solutions.…”

Revealing the Binding Events of Single Proteins on Exosomes Using Nanocavity Antennas beyond Zero-Mode Waveguides

“…With a spatial resolution greater than the diffraction limit, the excitation volume in the hole is reduced to 10 –21 L. − In such a small excitation volume, there is a better time distinction between the molecular diffusion phenomenon (typically at the microsecond level, μs) and the binding phenomenon (typically at the millisecond level, ms), thereby improving the time resolution . When ZMWs are combined with fluorescence correlation spectrum and fluorescence energy transfer technology, single-molecule detection with a high signal-to-noise ratio (SNR) can be realized. − Numerous advancements have been made in nucleic acid sequencing, , protein interaction dynamics, ,− biomembrane behavior, − and other research areas.…”