Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

Baibakov, Mikhail; Patra, Satyajit; Claude, Jean-Benoît; Moreau, Antonin; Lumeau, Julien; Wenger, Jérôme

doi:10.1021/acsnano.9b04378

Cited by 58 publications

(108 citation statements)

References 84 publications

(298 reference statements)

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“…Here, we focus on a fixed ZMW diameter of 110 nm as this size was shown to provide near-optimal fluorescence enhancement performance for both green and red dyes. 35 We observed similar adsorption effects for ZMW of diameters in the 85-200 nm range. So, our findings and conclusions reported here are quite general to ZMWs and can be applied other aluminum plasmonic nanostructures as well.…”

Section: Resultssupporting

confidence: 75%

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

Patra

Baibakov

Claude

et al. 2020

Sci Rep

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Zero mode waveguide (ZMW) nanoapertures efficiently confine the light down to the nanometer scale and overcome the diffraction limit in single molecule fluorescence analysis. However, unwanted adhesion of the fluorescent molecules on the ZMW surface can severely hamper the experiments. Therefore a proper surface passivation is required for ZMWs, but information is currently lacking on both the nature of the adhesion phenomenon and the optimization of the different passivation protocols. Here we monitor the influence of the fluorescent dye (Alexa Fluor 546 and 647, Atto 550 and 647N) on the non-specific adhesion of double stranded DNA molecule. We show that the nonspecific adhesion of DNA double strands onto the ZMW surface is directly mediated by the organic fluorescent dye being used, as Atto 550 and Atto 647N show a pronounced tendency to adhere to the ZMW while the Alexa Fluor 546 and 647 are remarkably free of this effect. Despite the small size of the fluorescent label, the surface charge and hydrophobicity of the dye appear to play a key role in promoting the DNA affinity for the ZMW surface. Next, different surface passivation methods (bovine serum albumin BSA, polyethylene glycol PEG, polyvinylphosphonic acid PVPA) are quantitatively benchmarked by fluorescence correlation spectroscopy to determine the most efficient approaches to prevent the adsorption of Atto 647N labeled DNA. Protocols using PVPA and PEG-silane of 1000 Da molar mass are found to drastically avoid the non-specific adsorption into ZMWs. Optimizing both the choice of the fluorescent dye and the surface passivation protocol are highly significant to expand the use of ZMWs for single molecule fluorescence applications.

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Section: Resultssupporting

confidence: 75%

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

Patra

Baibakov

Claude

et al. 2020

Sci Rep

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“…where N mol is the total number of detected molecules, B the background noise intensity, F the total fluorescence intensity, τ d the mean diffusion time and κ the aspect ratio of the axial to transversal dimensions of the detection volume. Obviously the nanoaperture geometry is more complicated than an open 3D volume, yet this model was found to correctly describe the FCS data inside the nanoapertures, 58,73 when the aspect ratio constant κ is set equal to 1. The fluorescence brightness per molecule is then computed as (F-B)/ N mol .…”

Section: Fcs Analysismentioning

confidence: 99%

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

et al. 2019

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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.

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“…Plasmon-enhanced FRET sensors have been extensively studied, including plasmonic nanoantennas [91] and ZMWs. [92,93] Maybe these sensors can be combined with solid-state nanopores in the future.…”

Section: Detection Of Plasmon Resonance Shift Sersmentioning

confidence: 99%

Electro‐Optical Detection of Single Molecules Based on Solid‐State Nanopores

Tong

et al. 2020

Small Structures

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Solid-state nanopore-based biosensors have received considerable attention in the past two decades and are promising for the next-generation single-molecule detection and sequencing platforms. Typically, the sensor is comprised of a thin inorganic membrane that separates two chambers containing an ion conductive solution. A nanoscale pore is drilled in the membrane as the only channel connecting the two chambers (cis chamber and trans chamber). The charged molecules are driven through the nanopore by an electric field applied across the membrane. At the same time, as the molecules block a fraction of the ionic current that flows through the pore, there will be a set of current pulses. The characteristics of the pulses, such as dwell time (duration of the pulses) and current blockages, give us a wealth of information about the passing analytes. Typically, excluded volumes, charges, capture rates, configurations, dynamics, and possible interactions of the analyte molecules can be concluded from electrical signals. Biological nanopores have been commercially used for DNA sequencing. [1] However, for solid-state nanopores, there are still challenges that need to be addressed for commercial use: specifically, spatial resolution, temporal resolution, and reproducibility. To achieve the identification of nucleotides during a stepwise displacement of DNA through the nanopores, our sensors must have a spatial resolution of 0.34 nm, considering the distance between adjacent base pairs of a double-stranded DNA chain. [2] Such a spatial resolution is a real challenge for nanofabrication of solid-state pores. Temporal resolution depends on the maximum bandwidth of the platforms and translocation speed of the analyte molecules. Limited temporal resolution leads to anomalously low event rates and distorted signals of proteins. [3] Signal reproducibility is another problem for solid-state nanopores compared with their biological counterparts. Biological nanopores, such as α-hemolysin [4] and mycobacterium smegmatis porin A, [5] serve as reliable sensors with excellent signal reproducibility because of the same genetically encoded structure. In comparison, due to the nonuniformity of the fabrication process or other unknown reasons, each solid-state nanopore is slightly different from one to another. In addition, there are other limitations for conventional solid-state nanopore sensors, such as low throughput, background noise, and lack of external handles to manipulate the analytes. Several strategies have been proposed to address these challenges, among which the combination of solid-state nanopores and optical detection has obvious advantages. Introducing a synchronized optical detection would help to improve the spatial resolution of solid-state nanopore platforms. Optical detection, in particular fluorescence [6] and surface-enhanced Raman spectroscopy (SERS), [7,8] has been widely used for

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Extending Single-Molecule Förster Resonance Energy Transfer (FRET) Range beyond 10 Nanometers in Zero-Mode Waveguides

Cited by 58 publications

References 84 publications

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

Surface passivation of zero-mode waveguide nanostructures: benchmarking protocols and fluorescent labels

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

Electro‐Optical Detection of Single Molecules Based on Solid‐State Nanopores

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