Single molecules detected using dual-colored probes

Michael A. Greenwood

Accurately detecting and identifying single biomolecules for chemical analysis and medical diagnostics could potentially unlock important new discoveries, but promising technology continues to be hindered by technical challenges.

Current methods, for instance, can handle only nanoliter amounts or less of a sample, and it is often difficult to separate molecules tagged with a nanoparticle from free-ranging nanoparticles in a mixture swirling with many components.

The ability to differentiate and track such molecules could aid in the detection of disease biomarkers and of infectious agents such as bacteria and viruses.

To overcome these barriers, investigator May D. Wang of Georgia Institute of Technology and colleagues from Emory University and from Georgia State University, all in Atlanta, are experimenting with color-coded optical probes that allow for rapid and comprehensive single-molecule detection as well as for nanometer-scale mapping.

Shown is dual-color imaging and localization of color-coded nanoparticles linked to single DNA molecules. The superimposed diagram shows structural mapping of the DNA complexes at nanometer-scale resolution. Courtesy of Amit Agrawal and May D. Wang, Georgia Institute of Technology.

They used as imaging agents nanobeads and quantum dots, both of which afforded enhanced brightness as well as stability against photobleaching in comparison with organic dyes and fluorescent proteins. The materials also have the advantage of being able to achieve various emission colors with a single light source.

The team used two binding methods to mark single molecules with red and green nanoprobes. The first involved two bioconjugated nanoparticles designed to recognize each other — the so-called direct-binding mode. In the second method, known as the indirect-binding mode, the two nanoparticles each are designed to recognize a specific site on the target molecule.

By sandwiching a molecule with two nanoprobes, the investigators computed the distance between the red and green probes, thus calculating the size and binding geometry of a target molecule. This type of spatial correlation allows targeted molecules to be separated from extraneous probes.

They tested the technique on two rigid DNA molecular rulers and found that the localization precision was >1 nm. It was 0.4 nm for the red nanoprobes (which emit more photons) and 0.7 nm for the green probes. They also calculated the overall detection efficiency of the probes to be between 80 and 85 percent. The probes were imaged with either a Nikon or Olympus epifluorescence microscope, and a Nikon digital camera captured the images. The method has the potential to be used with volume samples as large as a milliliter.

The investigators report also that they used in their research a software program designed for astronomy. The DAOPHOT system allows light from a single twinkling star, even if it is located in a dense cluster of stars, to be pinpointed and analyzed. Under the microscope, the brightly colored nanoprobes bear a striking resemblance to a starry night. They applied the software to their miniature display and discovered that it could locate color-coded nanoparticle probes accurately, processing up to 10 million nanoparticle pairs per minute.

PNAS, March 4, 2008, pp. 3298-3303.