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Nanowires and molecular beacons for DNA analysis
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apid, highly effective detection of nucleic acids has become commonplace, and many approaches are available. However, routine applications, such as medical diagnostic screening, require not just sensitivity and selectivity but also ease of use, reduced contamination risk, and the ability to simultaneously detect a range of target sequences. To meet the need, Christine Keating, Sharron Penn, and co-workers at Pennsylvania State University and Oxonica, Inc., have developed a metal-surfacebound molecular beacon (MB) probe assay that provides simultaneous, “closed tube” detection of multiple human viral pathogen targets with minimal risk of sample contamination ( J. Am. Chem. Soc. 2006, 128, 16,892–16,903). MB probes are short stretches of nucleic acid that fold into a hairpin or stem-and-loop structure. A fluorophore and a corresponding quenching molecule are linked to opposing ends. When the MB is in the closed, hairpin configuration, the fluorophore and quenching molecule are held close together and emission is quenched. The opposing ends separate when a target sequence binds to the loop sequence, resulting in detectable fluorescence. MB probes offer many advantages for DNA detection, Keating notes, especially for reducing sample handling. “There’s no target labeling and just a single hybridization step, with no washing afterwards,” she says. “With threestrand sandwich assays, you have two hybridizations and a wash after each one, which requires more sample manipulation and can lead to cross-contamination.” But it has been difficult to develop multiplexed MB probe assays because each target nucleic acid requires its own spectrally distinct fluorescent dye and corresponding quencher. The practical limit to date for simultaneous detection with MB probes in homogenous solution has been four target nucleic acids. © 2007 AMERICAN CHEMICAL SOCIETY
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HIV probes
Fluorescence intensity
Innovative use of patterned metal nanoparticles leads to multiplexed, sealed-chamber bioassays.
HIV HCV SARS
Schematic of the metal-surface-bound MB probe assay shows how it can pick out HIV oligonucleotides from a mixture of HIV, HCV, and SARS oligonucleotides.
However, MB probes can be arrayed on metal surfaces. The metal acts as the quencher so there is no need for a quenching moiety. Keating and colleagues recently developed an MB-based assay in which 5´ thiolated MB probes were assembled onto striped metal nanowires (Nanobiotechnology 2005, 1, 327–335). The nanowires could be electroplated with >100 distinct patterns of alternating Au and Ag stripes, resulting in easily distinguishable light and dark bar-code patterns of scattering intensity. “Attaching molecular beacons to a surface has several advantages,” including improved fluorescence and quenching, says Todd Krauss of the University of Rochester. Using bar-coded nanowires as the surface, he says, “gives you much greater and easier ability to detect multiple targets with only one dye.” In the current work, Keating and colleagues investigated the effects of varying loop and stem length, hybridization buffer type, salt concentration, and other factors on MB probe assay performance. The design principles, says Keating, are “not as simple as for a standard linear probe, because the secondary structure is so important.” The investigators were able to optimize quenching and fluorescence to achieve a limit of detection of 3 months, she notes. “It looks very fast and very visual,” says Catherine Murphy of the University of South Carolina. “You wouldn’t need a lot of training to read the results, so it could be very useful for diagnostic testing.” Keating says that optimization work is continuing with the nanowire-immobilized MB probe assays while her team also seeks to understand the underlying chemistry. “We don’t know enough about the physical chemistry of molecular beacons on these surfaces to be able to make predictions yet,” Keating says. “Historically, people often tried to avoid metal surfaces because they complicate everything, for example, by quenching fluorescence. But we’re finding out that they can be very useful.” a —Thomas Hayden
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