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Single-molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets

Gunnarsson, Anders LU ; Jönsson, Peter LU ; Marie, Rodolphe LU ; Tegenfeldt, Jonas LU and Höök, Fredrik LU (2008) In Nano Letters 8(1). p.183-188
Abstract
We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limit in the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediate the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (Ø approximately 100 nm) to a DNA-modified substrate. On top of rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of the binding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential of this feature is demonstrated by... (More)
We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limit in the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediate the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (Ø approximately 100 nm) to a DNA-modified substrate. On top of rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of the binding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential of this feature is demonstrated by discriminating a single mismatch from a fully complementary sequence. The success of the method is critically dependent on a surface modification that provides sufficiently low background. This was achieved through self-assembly of a biotinylated copolymer, Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) on a silicon dioxide surface, followed by subsequent addition of streptavidin and biotinylated DNA. The proposed detection scheme is particularly appealing due to the simplicity of the sensor, which relies on self-assembly principles and conventional TIRFM. Therefore, we foresee a great potential of the concept to serve as an important component in future multiplexed sensing schemes. This holds in particular true in cases when information about binding kinetics is valuable, such as in single nucleotide polymorphism diagnostics. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nano Letters
volume
8
issue
1
pages
183 - 188
publisher
The American Chemical Society
external identifiers
  • pmid:18088151
  • wos:000252257700033
  • scopus:38749120901
ISSN
1530-6992
DOI
10.1021/nl072401j
language
English
LU publication?
yes
id
8687b89d-baef-47cb-ba48-84cd8f0131f6 (old id 987903)
date added to LUP
2008-01-29 11:50:38
date last changed
2017-05-28 04:14:48
@article{8687b89d-baef-47cb-ba48-84cd8f0131f6,
  abstract     = {We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limit in the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediate the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (Ø approximately 100 nm) to a DNA-modified substrate. On top of rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of the binding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential of this feature is demonstrated by discriminating a single mismatch from a fully complementary sequence. The success of the method is critically dependent on a surface modification that provides sufficiently low background. This was achieved through self-assembly of a biotinylated copolymer, Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) on a silicon dioxide surface, followed by subsequent addition of streptavidin and biotinylated DNA. The proposed detection scheme is particularly appealing due to the simplicity of the sensor, which relies on self-assembly principles and conventional TIRFM. Therefore, we foresee a great potential of the concept to serve as an important component in future multiplexed sensing schemes. This holds in particular true in cases when information about binding kinetics is valuable, such as in single nucleotide polymorphism diagnostics.},
  author       = {Gunnarsson, Anders and Jönsson, Peter and Marie, Rodolphe and Tegenfeldt, Jonas and Höök, Fredrik},
  issn         = {1530-6992},
  language     = {eng},
  number       = {1},
  pages        = {183--188},
  publisher    = {The American Chemical Society},
  series       = {Nano Letters},
  title        = {Single-molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets},
  url          = {http://dx.doi.org/10.1021/nl072401j},
  volume       = {8},
  year         = {2008},
}