Dark-Field Illumination on Zero-Mode Waveguide/Microfluidic Hybrid Chip Reveals T4 Replisomal Protein Interactions

Abstract

The ability of zero-mode waveguides (ZMWs) to guide light energy into subwavelength-diameter cylindrical nanoapertures has been exploited for single-molecule fluorescence studies of biomolecules at micromolar concentrations, the typical dissociation constants for biomolecular interactions. Although epi-fluorescence microscopy is now adopted for ZMW-based imaging as an alternative to the commercialized ZMW imaging platform, its suitability and performance awaits rigorous examination. Here, we present conical lens-based dark-field fluorescence microscopy in combination with a ZMW/microfluidic chip for single-molecule fluorescence imaging. We demonstrate that compared to epi-illumination, the dark-field configuration displayed diminished background and noise and enhanced signal-to-noise ratios. This signal-to-noise ratio for imaging using the dark-field setup remains essentially unperturbed by the presence of background fluorescent molecules at micromolar concentration. Our design allowed single-molecule FRET studies that revealed weak DNA–protein and protein–protein interactions found with T4 replisomal proteins.

FRET Enhancement in Aluminum Zero‐Mode Waveguides

“…Earlier experiments on aluminum C-shaped nanoapertures did not reveal noticeable changes of the FRET efficiency [32]. A similar trend was reported for circular aluminum ZMWs but without quantitative data [33], while another study quantified a reduction of the FRET efficiency by 15% induced by the ZMWs [34]. Recently, our group has investigated FRET in ZMWs milled in gold films, reported a linear dependence of the FRET rate on the LDOS and quantified a slight variation of the FRET efficiency with the aperture diameter [48].…”

“…We point out that an intrinsic limitation of our approach based on random diffusion is that it provides only the spatially averaged fluorescence lifetime, while lifetime variations are expected within the ZMW depending on the position and orientation of the emitter respective to the metal [43,44]. Nevertheless, our net results remain relevant to describe the average properties of molecules randomly positioned inside ZMWs or randomly diffusing [11,[32][33][34].…”

“…13 of the Supporting Information). We anticipate that this quantification of the FRET process in ZMWs will promote their application as new devices for enhanced single molecule FRET analysis at physiological micromolar concentrations [8,10,11,[32][33][34].…”

“…ZMWs milled in aluminum are by far the most widely used structures as compared to other metals such as gold or copper [8][9][10][11][12][13][14][15][16][17][18][19][20][32][33][34], thanks to their commercial availability, low chemical reactivity and the availability of surface passivation protocols [36]. Due to the large interest raised by aluminum ZMWs and FRET separately, we believe that a clear quantification of FRET in aluminum ZMWs is necessary.…”

Optofluidic wavelength division multiplexing for single-virus detection

“…Current gold-standard techniques for nucleic acid and protein detection such as PCR and ELISA use fluorescent organic dyes as a means of signal reporting (4). Optical detection, by fluorescence or "label-free," is extremely sensitive, allowing for single-molecule and even single-dye detection under appropriate conditions (5)(6)(7)(8)(9)(10)(11). The availability of over a dozen dyes across the visible spectrum opens the door to implementing the desired multiplexing capability with multiple dyes, i.e., spectral channels (12)(13)(14).…”