Single-mismatch detection using gold-quenched fluorescent oligonucleotides

Dubertret, Benoît; Calame, Michel; Libchaber, Albert

doi:10.1038/86762

Cited by 1,201 publications

(1,008 citation statements)

References 28 publications

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“…This is somewhat surprising because the absorption bands of gold often result in fluorescence quenching [25][26][27][28]. However, SPCE occurs over distances from the metal surface of 20-200 nm [20,21,29] which is longer than the distances for Forster resonance energy transfer quenching.…”

Section: Discussionmentioning

confidence: 99%

First observation of surface plasmon-coupled electrochemiluminescence

Zhang

Gryczyński

Lakowicz

2004

Chemical Physics Letters

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Section: Discussionmentioning

confidence: 99%

First observation of surface plasmon-coupled electrochemiluminescence

Zhang

Gryczyński

Lakowicz

2004

Chemical Physics Letters

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“…16,21,22,24,25,29 Figure 6 shows assay response as a function of target oligonucleotide concentration for HCV beacon probes hybridized in 0.3 M PBS at 50 °C. The y-axis is fractional coverage, estimated on the basis of the fluorescence intensity as compared to the maximum intensity.…”

Section: Nanowire Mb Assay Sensitivitymentioning

confidence: 99%

“…We note that both surface-based immunoassays 54 and MB probes in solution also exhibit several orders of magnitude in dynamic range of fluorescence response to target concentration. 25 Factors that affect sensitivity include the number of wires in each assay (surface area), surface coverage of beacon probes, hybridization thermodynamics, and beacon probe quenching efficiency. The highest target concentration tested (1 × 10 −6 M) provided 3.0 × 10 13 target strands/sample, which decreased to 3.0 × 10 9 /sample for 1 × 10 −10 M target.…”

Section: Nanowire Mb Assay Sensitivitymentioning

confidence: 99%

“…Metals can provide extremely efficient quenching; Dubertret et al showed that 1.4 nm Au clusters gave better performance in molecular beacons than the common molecular quencher, DABCYL. 25 Krauss and co-workers bound MB-style probes to planar Au films via 5′ thiol groups. Quenching efficiencies were improved as compared to MBs immobilized on glass.…”

Section: Introductionmentioning

confidence: 99%

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Coupling Molecular Beacons to Barcoded Metal Nanowires for Multiplexed, Sealed Chamber DNA Bioassays

et al. 2006

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We have combined molecular beacon (MB) probes with barcoded metal nanowires to enable nowash, sealed chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based MB assays are discussed. Loop regions of 24 bases and 5 base pair stem regions in the beacon probes gave optimal performance. Our results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. Dengue virus-specific probes with predicted solution-phase ΔG of folding in 500 mM buffered NaCl of approximately −4 kcal/mol performed better than those with ΔG > −2 or < −6 kcal/mol. Buffered 300-500 mM NaCl was selected after comparison of several buffers previously reported for similar types of assays, and 200-500 mM NaCl was found to be the optimal ionic strength for the hybridization temperatures (25 and 50 °C) and probe designs used here. Target binding to the surface as a function of solution concentration fit a Sips isotherm with K d = 1.7 ± 0.3 nM. The detection limit was ∼100 pM, limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for human immunodeficiency, hepatitis C, and severe acute respiratory viruses were assayed simultaneously in a no-wash, sealed chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, we demonstrated that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over 3 months.

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“…Au NPs quench fluorescence with a greater efficiency 5 and over greater distances 16 than molecular quenchers. We designed nano-flares using 13 nm Au NPs, since this size particle is an efficient quencher, can be densely functionalized with oligonucleotides, 12a and does not efficiently scatter visible light, which is important for designing optical probes with minimal interference.…”

mentioning

confidence: 99%

Nano-Flares: Probes for Transfection and mRNA Detection in Living Cells

et al. 2007

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We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular "nanoflares" for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells.Probes to visualize and detect intracellular RNA including those used for in situ staining, 1 molecular beacons, 2 and fluorescent resonance energy transfer (FRET) pairs 3 are important tools to measure and quantify activity in living systems in response to external stimuli. 4 However, these probes are often difficult to transfect, require additional agents for cellular internalization, and can be unstable in cellular environments. These factors can lead to a high background signal and the inability to detect targets. Here we show how novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore complements can be used as both transfection agents and cellular "nano-flares" for visualizing and quantifying RNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, 5 cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, 6 thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Specifically, nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells. The discovery and subsequent development of the oligonucleotide-nanoparticle conjugate have led to a variety of new opportunities in molecular diagnostics 11 and materials design. 12 Recently, it has been demonstrated that oligonucleotide-functionalized nanoparticles enter cells and can act as antisense agents to control gene expression. 6 These "antisense particles" are not only delivery vehicles, 13 but also single entity regulation and transfection agents that undergo facile cellular internalization, resist enzymatic degradation, and bind intracellular targets with affinity constants that are as much as two orders of magnitude greater than free oligonucleotides. 14 Moreover, they can be easily modified with potent designer materials such as locked nucleic acids 15 and are nontoxic under conditions required for gene regulation.The nano-flares described herein are oligonucleotide functionalized nanoparticle conjugates designed to provide an intracellular fluorescence signal that correlates with the relative amount of a specific intracellular RNA. By utilizing nanopart...

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Single-mismatch detection using gold-quenched fluorescent oligonucleotides

Cited by 1,201 publications

References 28 publications

First observation of surface plasmon-coupled electrochemiluminescence

First observation of surface plasmon-coupled electrochemiluminescence

Coupling Molecular Beacons to Barcoded Metal Nanowires for Multiplexed, Sealed Chamber DNA Bioassays

Nano-Flares: Probes for Transfection and mRNA Detection in Living Cells

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