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Magnetically assisted DNA assays: high selectivity using conjugated polymers for amplified fluorescent transduction.

Xu H, Wu H, Huang F, Song S, Li W, Cao Y, Fan C - Nucleic Acids Res. (2005)

Bottom Line: We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors.Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum.We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiology and Nanomedicine, Shanghai Institute of Applied Physics, Chinese Academy of Sciences Shanghai 201800, China.

ABSTRACT
We report a strategy for conjugated polymer (CP)-based optical DNA detection with improved selectivity. The high sensitivity of CP-based biosensors arises from light harvesting by the CP and the related amplified fluorescent signal transduction. We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors. Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum. We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

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Signal amplification is offered by PF amplification, which is demonstrated by the comparison between the FRET signal sensitized by PF excitation and the signal generated from direct excitation of the fluorescein-tagged ssDNA signaling probe. Upper curve: the excitation wavelength was 380 nm, which excited PF (2.7 × 10−8 M). In the presence of fluorescein-tagged ssDNA probe (1 × 10−10 M), the energy was efficiently transferred from PF to the proximal fluorescein via FRET. Lower curve: the excitation wavelength was 480 nm, which directly excited the fluorescein of the ssDNA probe (1 × 10−10 M). Note that the emission of fluorescein at 520 nm was much more intense in the case of PF amplification than in the case of direct excitation.
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fig1: Signal amplification is offered by PF amplification, which is demonstrated by the comparison between the FRET signal sensitized by PF excitation and the signal generated from direct excitation of the fluorescein-tagged ssDNA signaling probe. Upper curve: the excitation wavelength was 380 nm, which excited PF (2.7 × 10−8 M). In the presence of fluorescein-tagged ssDNA probe (1 × 10−10 M), the energy was efficiently transferred from PF to the proximal fluorescein via FRET. Lower curve: the excitation wavelength was 480 nm, which directly excited the fluorescein of the ssDNA probe (1 × 10−10 M). Note that the emission of fluorescein at 520 nm was much more intense in the case of PF amplification than in the case of direct excitation.

Mentions: Previous spectroscopic studies have proven that the emission of PF has sufficient spectral overlap with the fluorescein absorption that they form an excellent FRET pair (38). Consistent with this, we observed well-defined FRET signals upon addition of PF to the test solution containing probe 2 (Figures 1 and 2). The light-harvesting PF sensitized the emission of the fluorescein (the energy-transfer acceptor), leading to fluorescent signal amplification. In fact, we could still observe well-defined PF-sensitized fluorescein emission at probe 2 concentrations that were sufficiently dilute (100 pM) that fluorescence emission was negligible by direct excitation of fluorescein (Figure 1). These results verify that light-harvesting polymers do provide optical signal amplification and improve the detection sensitivity.


Magnetically assisted DNA assays: high selectivity using conjugated polymers for amplified fluorescent transduction.

Xu H, Wu H, Huang F, Song S, Li W, Cao Y, Fan C - Nucleic Acids Res. (2005)

Signal amplification is offered by PF amplification, which is demonstrated by the comparison between the FRET signal sensitized by PF excitation and the signal generated from direct excitation of the fluorescein-tagged ssDNA signaling probe. Upper curve: the excitation wavelength was 380 nm, which excited PF (2.7 × 10−8 M). In the presence of fluorescein-tagged ssDNA probe (1 × 10−10 M), the energy was efficiently transferred from PF to the proximal fluorescein via FRET. Lower curve: the excitation wavelength was 480 nm, which directly excited the fluorescein of the ssDNA probe (1 × 10−10 M). Note that the emission of fluorescein at 520 nm was much more intense in the case of PF amplification than in the case of direct excitation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1131937&req=5

fig1: Signal amplification is offered by PF amplification, which is demonstrated by the comparison between the FRET signal sensitized by PF excitation and the signal generated from direct excitation of the fluorescein-tagged ssDNA signaling probe. Upper curve: the excitation wavelength was 380 nm, which excited PF (2.7 × 10−8 M). In the presence of fluorescein-tagged ssDNA probe (1 × 10−10 M), the energy was efficiently transferred from PF to the proximal fluorescein via FRET. Lower curve: the excitation wavelength was 480 nm, which directly excited the fluorescein of the ssDNA probe (1 × 10−10 M). Note that the emission of fluorescein at 520 nm was much more intense in the case of PF amplification than in the case of direct excitation.
Mentions: Previous spectroscopic studies have proven that the emission of PF has sufficient spectral overlap with the fluorescein absorption that they form an excellent FRET pair (38). Consistent with this, we observed well-defined FRET signals upon addition of PF to the test solution containing probe 2 (Figures 1 and 2). The light-harvesting PF sensitized the emission of the fluorescein (the energy-transfer acceptor), leading to fluorescent signal amplification. In fact, we could still observe well-defined PF-sensitized fluorescein emission at probe 2 concentrations that were sufficiently dilute (100 pM) that fluorescence emission was negligible by direct excitation of fluorescein (Figure 1). These results verify that light-harvesting polymers do provide optical signal amplification and improve the detection sensitivity.

Bottom Line: We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors.Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum.We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiology and Nanomedicine, Shanghai Institute of Applied Physics, Chinese Academy of Sciences Shanghai 201800, China.

ABSTRACT
We report a strategy for conjugated polymer (CP)-based optical DNA detection with improved selectivity. The high sensitivity of CP-based biosensors arises from light harvesting by the CP and the related amplified fluorescent signal transduction. We demonstrate that the use of magnetic microparticles significantly improves the selectivity of this class of DNA sensors. Compared with previously reported DNA sensors with CP amplification, this novel sensing strategy displays excellent discrimination against non-cognate DNA in the presence of a protein mixture or even human serum. We also demonstrate that the magnetically assisted DNA sensor can conveniently identify even a single-nucleotide mismatch in the target sequence.

Show MeSH