Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

Abstract

Single molecule detection provides detailed information about molecular structures and functions, but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labelling.However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguides nanoapertures enable observing the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. While the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet

“…47 From experiments on circular nanoapertures milled in aluminum, we know that the optimum size for experiments in the visible spectral range is about 110 nm, 20 while it amounts to 60-70 nm for the UV. 60 The other dimension is then made as small as 30 nm to maximize the plasmonic coupling between the two long edges of the NR aperture. 45 Please note that the milling depths in the substrate is optimized as well following earlier works, 19,27 see Fig.…”

“…These results nd application in improving the performance of ZMWs for single molecule analysis at high concentrations, and are especially relevant for monitoring fast biomolecular dynamics, 4,5 realizing ultrafast single-photon sources, 14 and for interrogating molecules with ultraviolet plasmonics. [57][58][59][60][61][62] Methods…”

“…All uorescence traces are analyzed using Symphotime 64 soware (Picoquant). As in our previous works, 12,20,60 we t the FCS correlation functions with a standard Brownian diffusion model taking into account the background uorescence level and the photoblinking: 69,70 GðsÞ…”

“…Achieving the brightest emission rate together with short uorescence lifetimes is important for applications on fast biomolecular dynamics, 4,5 single-photon sources, 14 and also for the newly-developing eld of ultraviolet plasmonics. [57][58][59][60][61][62] Here we rationally explore the use of rectangular-shaped nanoapertures milled in aluminum to enhance the uorescence emission rate of single molecules from the near infrared (excitation 635 nm, detection 655 to 755 nm) down to the deep ultraviolet (excitation 266 nm, detection 310 to 410 nm). We use aluminum layers with optimized deposition parameters, 63,64 as the response for gold lms falls below 600 nm and hence gold lms cannot be used efficiently for dyes with emission from the green to the ultraviolet.…”

Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins

“…3d, the data for the UV fluorescent dye p-terphenyl is taken from our previous work Ref. 46 ). For streptavidin and βgalactosidase, we use the hydrodynamic radii independently measured in Ref.…”