Palladium zero-mode waveguides for optical single-molecule detection with nanopores

Abstract

Holes in metal films do not allow the propagation of light if the wavelength is much larger than the hole diameter, establishing such nanopores as so-called zero-mode waveguides (ZMWs). Molecules, on the other hand, can still pass through these holes. We use this to detect individual fluorophore-labelled molecules as they travel through a ZMW and thereby traverse from the dark region to the illuminated side, upon which they emit fluorescent light. This is beneficial both for background suppression and to prevent premature bleaching. We use palladium as a novel metal-film material for ZMWs, which is advantageous compared to conventionally used metals. We demonstrate that it is possible to simultaneously detect translocations of individual free fluorophores of different colours. Labelled DNA and protein biomolecules can also be detected at the single-molecule level with a high signal-to-noise ratio and at high bandwidth, which opens the door to a variety of single-molecule biophysics studies.

Slowing down an analyte in a nanopore

“…Thus by labeling the analyte with a fluorophore its delayed passage through the pore with the bi-level voltage profile can be detected optically independent of the pore current value. For example, in [23] a fluorophore-labeled molecule passes from the unlit (dark) cis side of an e-cell to the lighted trans side where it emits light and is detected.…”

“…Blockade-based detection requires a considerable degree of measurement precision to be able to distinguish among the blockades due to the four nucleobases (A, T, C, and G). Optical methods based on fluorescent labels [13] require much less precision. Alternatively transverse electronic measurements of the current across the pore can be measured with 1 to 2 orders of magnitude higher than longitudinal blockade currents [14].…”

Transport receptor occupancy in Nuclear Pore Complex mimics

“…Other platforms that avoid the use of an applied voltage can also be envisioned, e.g. by using zeromode-waveguides (ZMW 55 ), where translocating molecules are freely diffusing (i.e. not driven by an electrical field) and optically detected.…”

The NEOtrap – en route with a new single-molecule technique

“…Such NEO-FRET would benefit from the best of both worlds: a broad temporal bandwidth via electrical detection and signal interpretation with sub-nm-precision from the correlated optical detection. The electro-optical combination of nanopores with zero-mode waveguides has been used already to further localize the confocal laser excitation ( Assad et al., 2017 ; Jadhav et al., 2019 ; Klughammer and Dekker, 2021 ). And, additional spectroscopic combinations such as vibrational spectroscopy, circular dichroism, etc.…”