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Zeptomole detection of DNA nanoparticles by single-molecule fluorescence with magnetic field-directed localization.
- Source :
-
Analytical biochemistry [Anal Biochem] 2012 Dec 01; Vol. 431 (1), pp. 40-7. Date of Electronic Publication: 2012 Aug 26. - Publication Year :
- 2012
-
Abstract
- Single-molecule fluorescence methods offer the promise of ultrasensitive detection of biomolecules, but the passive immobilization methods commonly employed require analyte concentrations in the picomolar range. Here, we demonstrate that superparamagnetic Fe(3)O(4) nanoparticles (NPs) can be used with an external magnetic field as a simple strategy to enhance the immobilization efficiency and thereby decrease the detection limit. Inorganic NPs functionalized with streptavidin were bound to biotinylated single-stranded DNA oligonucleotides, which were in turn annealed to complementary oligonucleotides labeled with a Cy3 fluorescent dye. Using an external magnetic field, the superparamagnetic nanoparticles were localized to a specific region within the flow chamber surface. From the single-molecule fluorescence time traces, single-step photobleaching indicated that the surface-immobilized NPs were primarily bound with a single Cy3-labeled oligonucleotide. This strategy gave a concentration detection limit for the Cy3-labeled oligonucleotide of 100aM, 3000-fold lower than that from an analogous strategy with passive immobilization. With a sample volume of 25μl, this method achieved a mole detection limit of approximately 2.5zmol (∼1500 molecules). Together, the results support that idea that single-molecule fluorescence methods could be used for biological applications such as detection and measurements of nucleic acids from biological or clinical samples without polymerase chain reaction amplification.<br /> (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Subjects :
- Ferrosoferric Oxide chemistry
Immobilized Nucleic Acids
Magnetic Fields
Nucleic Acid Hybridization
Oligonucleotides chemistry
Photobleaching
Streptavidin chemistry
Streptavidin metabolism
Surface Properties
Carbocyanines chemistry
DNA analysis
Fluorescent Dyes chemistry
Magnetite Nanoparticles chemistry
Microscopy, Fluorescence
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0309
- Volume :
- 431
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 22929698
- Full Text :
- https://doi.org/10.1016/j.ab.2012.08.017