Nanometer-scale mapping and single-molecule detection with color-coded nanoparticle probes
- *Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, 101 Woodruff Circle, Suite 2001, Atlanta, GA 30322;
- †Department of Physics and Astronomy, Georgia State University, Atlanta, GA 30303; and
- ‡Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, VA Whitaker Building 4106, Atlanta, GA 30332
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Communicated by Mostafa A. El-Sayed, Georgia Institute of Technology, Atlanta, GA, December 29, 2007 (received for review April 26, 2007)
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Fig. 1.Schematic diagrams showing the design of color-coded nanoparticle probes for detecting single biomolecules in two different binding modes. (a) Direct binding between two bioconjugated nanoparticles leading to a separation distance of d 1. In this work, this mode of binding was used to construct rigid molecular structures (molecular rulers) for verification/validation studies of the precision in distance measurements. (b) Indirect sandwich-type binding in which two nanoparticles recognize the same target molecule at two different sites. This indirect mode of binding allows native biomolecules such as genes to be recognized and detected at the single-molecule level.
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Fig. 2.Automated processing of dual-color fluorescence images and nanoparticle colocalization at nanometer precision. (a) Dual-color fluorescence image of a red/green nanoparticle pair and separation into its individual color components (red and green). (b) Single-color fluorescence-intensity profiles after convolution and curve fitting with a two-dimensional Gaussian kernel function. (c) Determination of the distance between the red and green nanoparticles when mapped to the original color image. (d) Schematic diagram of the structure of a rigid DNA/nanoparticle construct and its expected contour length. Note that the experimentally determined distance (54 nm) is in close agreement with the expected length (55 nm).
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Fig. 3.Dual-color imaging and colocalization of red/green (R-G) nanoparticles attached to rigid DNA molecular rulers. (a and d) Wide-field fluorescence images obtained from a 24-bp DNA construct and a control (noncomplementary) sample under the same experimental conditions. (b and e) Expanded views of two selected areas in a and d (indicated by the boxes) showing colocalized signals (yellow) and isolated red and green signals. (c and f) Graphical rendering of colocalized red/green particle pairs and isolated particles corresponding to the signals observed in b and e. (Scale bars: a and d, 10 μm; b and e, 1 μm).
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Fig. 4.Dual-color imaging and colocalization of red/green nanoparticles attached to flexible DNA structures. (a and c) Wide-field fluorescence images obtained from sandwich DNA hybridization assays in the presence of a cancer gene (BCAR3) sequence (a) and in the absence of this target gene sequence under the same experimental conditions (c). (b and d) Magnified images of selected areas (boxes in a and c) for direct visualization of colocalized signals (yellow) in the complementary sample and the absence of colocalized signals in the noncomplementary sample. The expanded images were processed by separating and leveling the red and green signals. (Scale bars: a and c, 10 μm; b and d, 1 μm.)
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Fig. 5.Schematic drawings of two flexible structures in the head-to-tail configuration (a) and two constrained structures in the head-to-head configuration (b).
Footnotes
- §To whom correspondence may be addressed. E-mail: maywang{at}bme.gatech.edu or snie{at}emory.edu
- © 2008 by The National Academy of Sciences of the USA
