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WIREs Nanomed Nanobiotechnol
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Plasmonic antennas and zero‐mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy toward physiological concentrations

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Single‐molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero‐mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano‐optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Ultrasmall detection volumes are needed to investigate enzymatic function at the single molecule level. Histogram of Michaelis constant KM for 118,000 enzymes taken from the Brenda database (http://www.brenda‐enzymes.org/) in November 2013. The top axis shows the detection volume required to isolate a single molecule. The vertical bars indicate the effective concentration regime and detection volume reached by different techniques (TIRF: total internal reflection fluorescence microscopy; ZMW: zero mode waveguides).
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Bottom‐up approaches to plasmonic antennas for enhanced single molecule fluorescence. (a) A single gold nanoparticle is used as optical antenna. (Reprinted with permission from Ref . Copyright 2013 OSA). (b) Fluorescence enhancement versus the near‐field detection volume obtained with single gold nanoparticles, the nanoparticle diameter is annotated close to the data point. (c) Cryo‐EM of a plasmonic dimer antenna made of two 40 nm gold particles linked with a 30 base pairs double stranded DNA. (Reprinted with permission from Ref .Copyright 2012 NPG). (d) DNA origami pillar with two gold nanoparticles forming a dimer antenna. Fluorescent labeled ssDNA sequences in solution can transiently hybridize with complimentary sequences in the origami structure at the hotspot between the particles. (Reprinted with permission from Ref . Copyright 2012 AAAS). (e) Numerical simulation of electric field intensity for single and dimer of 80 nm diameter gold particles. The incoming light is horizontally polarized at a wavelength of 640 nm, the gap distance in the dimer is 23 nm. (Reprinted with permission from Ref . Copyright 2012 AAAS). (f) Scatter plot of fluorescence intensity versus lifetime of the ATTO647N‐labeled DNA origami pillar with binding sites for one (monomer) and two (dimer) 80‐nm diameters particles. (Reprinted with permission from Ref . Copyright 2012 AAAS).
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Top‐down approaches to plasmonic antennas for enhanced single molecule fluorescence. (a) Gold bowtie antenna covered by fluorescent molecules (arrows) in PMMA resin. The lower image shows the computed local intensity enhancement. The scale bar is 100 nm. (Reprinted with permission from Ref . Copyright 2009 NPG). (b) Plasmonic bowtie antennas surrounded by a fluid supported lipid bilayer, where fluorescently labeled Ras proteins are anchored in the upper leaflet of the lipid membrane. Fluorophores tethered to the supported membrane can diffuse in the plane and thereby pass through the nanogaps. (Reprinted with permission from Ref . Copyright 2012 ACS). (c) Antenna‐in‐box platform for single‐molecule analysis at high concentrations. (Reprinted with permission from Ref . Copyright 2013 NPG). Scatter plots of the fluorescence enhancement factor (d) and measured detection volume (e) as a function of gap size for the antenna‐in‐box. (d and e: Reprinted with permission from Ref . Copyright 2013 NPG).
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(a) Optical antenna carved on top of a NSOM aperture probe. Topography, biochemical recognition and fluorescence images can be recorded simultaneously at nanometre resolution. (Reprinted with permission from Ref . Copyright 2008 NPG). (b) Scanning electron microscope image of a tip‐on‐aperture probe. (Reprinted with permission from Ref . Copyright 2010 Wiley‐VCH). (c) Zoomed‐in confocal microscopy image of LFA‐1 at the cell surface of monocytes visualized by confocal microscopy (left). The right panel shows the NSOM imaging of the highlighted region in the confocal image. (Reprinted with permission from Ref . Copyright 2010 Wiley‐VCH).
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Corrugated nanoapertures to control the fluorescence directivity with surface plasmon waves. (a) Scanning electron microscope image of a single aperture of 140 nm diameter milled in gold with two concentric grooves of period 440 nm and depth 65 nm. (b) Sketch of the experiment to illustrate the photon sorting ability: the central aperture is filled with a mixed solution of Alexa Fluor 647 and Rhodamine 6G. (c) Radiation patterns in the back focal plane of the objective for emission centered at 670 nm and 560 nm. (d) Fluorescence radiation pattern for the two different emission wavelengths. (Reprinted with permission from Ref . Copyright 2011 ACS).
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Zero‐mode waveguides and nanoapertures to enhance the detection of single fluorescent molecules at micromolar concentrations. (a) Nanoaperture for enhanced single molecule fluorescence detection at micromolar concentrations. (Reprinted with permission from Ref . Copyright 2003 AAAS). (b) Electron microscope images of 120 and 160 nm apertures milled in gold. (Reprinted with permission from Ref . Copyright 2010 ACS). (c) Field intensity distribution on a 120 nm water‐filled gold aperture illuminated at 633 nm. (Reprinted with permission from Ref . Copyright 2010 ACS). The spatially‐averaged excitation intensity over the nanoaperture detection volume as seen by FCS is enhanced about three times as compared to the diffraction‐limited confocal spot. (d) Comparison of normalized FCS correlation traces between confocal and nanoaperture configurations: to reach similar amplitudes, the concentration was increased by a factor 400 for the nanoaperture. Moreover, the nanoaperture enables observing short diffusion times with significantly improved signal‐to‐noise ratio. (Reprinted with permission from Ref . Copyright 2009 ACS). (e) Observation volumes measured for aluminum apertures. The right axis shows the corresponding concentration to ensure there is a single molecule in the observation volume. (Reprinted with permission from Ref . Copyright 2005 APS). (f) Fluorescence brightness enhancement factor for Alexa Fluor 647 molecules in apertures milled in gold (laser excitation 633 nm) and for Rhodamine 6G molecules in apertures milled in aluminum (laser excitation 488 nm) (Reprinted with permission from Ref . Copyright 2008 APS).
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Application of zero‐mode waveguides to investigate cell membranes below the diffraction limit. (a) Scanning electron microscope image of cross‐sectional cuts of nanoapertures. Cell membranes have been outlined (light gray), and aperture locations have been circled. Cell membrane spanning a nanoaperture dips down (arrow), suggesting membrane invagination. The scale bar is 500 nm. (Reprinted with permission from Ref . Copyright 2007 IOP). (b) Fluorescence micrographs of cells labeled with DiI‐C 12 membrane probe through 280 nm aluminum apertures. (Reprinted with permission from Ref . Copyright 2007 IOP). (c) Normalized FCS correlation functions and numerical fits (thick lines) obtained for the FL‐GM1 ganglioside lipid analog, demonstrating a significant diffusion time reduction in the nanoaperture. (Reprinted with permission from Ref . Copyright 2007 BS). (d) Molecular diffusion times versus aperture area for the FL‐GM1 ganglioside and FL‐PC phosphatidylcholine. (Reprinted with permission from Ref . Copyright 2007 BS).
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Application of zero‐mode waveguides to single‐molecule real‐time DNA sequencing. (a) Principle of the experiment: a single DNA polymerase is immobilized at the bottom of a ZMW, which enables detection of individual phospholinked nucleotide substrates against the bulk solution background as they are incorporated into the DNA strand by the polymerase. (b) Schematic event sequence of the phospholinked dNTP incorporation cycle, the lower trace displays the temporal evolution of the fluorescence intensity. (c) Section of a fluorescence time trace showing 28 incorporations events with four color detection. Pulses correspond to the least‐squares fitting decisions of the algorithm. (Reprinted with permission from Ref . Copyright 2009 AAAS).
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