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Single-Molecule Detection by Philip Tinnefeld

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Biomedical Measurement Science, BioSimilars, Drug Delivery Platform Technology, Ecosystems & Industrial Concentration in the Medical Device Sector, Medical Devices R&D Investment, Nanotechnology for Drug Delivery, Technology Transfer: Biotech and Pharmaceutical, tagged fluorescence correlation spectroscopy (FCS), Nanoantenna, nanoantenna-in-box, nanophotonics, Single-molecule detection on September 24, 2013| 1 Comment »

Curator: Tilda Barliya PhD

Original article by: Philip Tinnefeld (2)

The ability to detect a single chemical at extremely low concentrations and high contamination is vital for earlier disease diagnosis (I). Detecting a single chemical does not only help to detect disease earlier, it is also vital for toxin/drug screening, athlete drug monitoring, environmental surveillance, and homeland security.

To detect a single molecule the observation volume must host only one molecule of interest during the measurement (2). It is impossible to carry out single-molecule detection at concentrations exceeding the picomolar to low-nanomolar range, because the observation volume becomes populated by more than one fluorescent molecule as concentration increases.

This concentration barrier is crucial when using a Single-Molecule Technology such as:

protein-protein interactions
Enzyme-substrate analysis
DNA sequencing
etc

Jérôme Wenger and colleagues at AixMarseille Université in France, alongside collaborators at the ICFO-Institut de Ciencies Fotoniques and ICREA-Institució Catalana de Recerca i Estudis Avançats in Spain, describe a system that brings two complementary nanophotonic approaches together to achieve single-molecule detection with a high signal-to-noise ratio at a micromolar concentration (1, 2).

In recent years, two alternative nanophotonic approaches were used:

Light is confined to the near-field using nanoapertures of subwavelength dimensions (typically holes in a thin aluminum or gold film on a silica substrate) that do not allow light to propagate through. Figure 1a
Enhance the excitation field locally using a nanoantenna that converts freely propagating optical radiation into localized energy in the form of surface plasmons. By reducing the volume of the hot spot, it is possible to obtain enormous fluorescence enhancements up to more than 1,000-fold when compared with the case of an isolated fluorophore in solution Figure 1b

The first approach (Figure 1a) limitations were that although this approach reduces the observation volume and enables single-molecule detection in the micromolar range, further reduction of nanoaperture size has detrimental effects on the fluorescence signal because of the decreased excitation intensity and fluorescence quenching caused by the metal film.

It did however, has some encouraging results: This method has been used, for instance, to monitor the activity of the
enzyme polymerase1 and follow DNA sequencing in real time; and for visualizing a ribosome in action (3).

The second approach (Figure 1b) limitations were that this approach suffers from the background fluorescence of molecules far away from the hot spot but still within the diffraction-limited excited area.

Nanophotonic approach to enhance single molecule detection

“nanoantenna-in-box”

Wenger and colleagues synergistically combine these two approaches and place a nanoantenna within the spatial confinement of a nanoaperture (Fig. 1c), to which they called “nanoantenna-in the-box.

To demonstrate the capability of their device, they cover it with a solution containing a fluorescent dye and analyse the fluorescence signal coming out from the hot spot using fluorescence correlation spectroscopy (FCS).

The FCS technique measures the number of molecules in the effective observation volume as these diffuse in and out of it, as well as the brightness per molecule.

Limitations:

1. There are, however, a few limitations that have to be considered. Further reduction of the gap size may result in gaps being too small for biomolecules to enter.

2. The antenna-in-box device exhibits a large surface-to-volume ratio and sticky molecules might adsorb onto the gold surface, especially in the gap, which might lead to rapid surface degradation, surfaceinduced artefacts and other background.

To overcome some of the limitations, the authors used an approach that takes advantage of a recently developed DNA-scaffolded, gap-nanoantenna might represent a viable solution. The two nanoparticles forming the gap-nanoantenna are attached to a self-assembled DNA nanostructure that provides handles to place the biomolecule of interest in the hot spot.

In summary:

“Whatever direction the research field might take, it seems as though the development of sophisticated coverslips in which nanophotonic structures such as the antenna-in-box set-up are incorporated may unlock more potential for single- molecule detection than developing more powerful microscopes”.

Ref:

I. By: Grace Rattue. Nanotechnology For Detecting Diseases Earlier. http://www.medicalnewstoday.com/articles/245820.php

1. Deep Punj, Mathieu Mivelle, Satish Babu Moparthi, Thomas S. van Zanten, Hervé Rigneault, Niek F. van Hulst, María F. García-Parajó & Jérôme Wenger. A plasmonic ‘antenna-in-box’ platform for enhanced single-molecule analysis at micromolar concentrations. Nature Nanotechnology 8, 512–516 (2013) doi:10.1038/nnano.2013.98. http://www.nature.com/nnano/journal/v8/n7/full/nnano.2013.98.html?WT.ec_id=NNANO-201307

2. By: Philip Tinnefeld. Single-Molecule Detection: Breaking the concentration barrier. http://www.nature.com/nnano/journal/v8/n7/full/nnano.2013.122.html

3. Sotaro Uemura., Colin Echeverría Aitken., , Jonas Korlach., Benjamin A. Flusberg., Stephen W. Turner & Joseph D. Puglisi. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010). http://www.nature.com/nature/journal/v464/n7291/full/nature08925.html

4. G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld. Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas. Science 26 October 2012: Vol. 338 no. 6106 pp. 506-510. http://www.sciencemag.org/content/338/6106/506.abstract