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Robust and specific ratiometric biosensing using a copper-free clicked quantum dot-DNA aptamer sensor.

Zhang H, Feng G, Guo Y, Zhou D - Nanoscale (2013)

Bottom Line: We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free "click chemistry" (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA.We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor.Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. y.guo@leeds.ac.uk d.zhou@leeds.ac.uk.

ABSTRACT
We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free "click chemistry" (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.

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(A) Typical fluorescence spectra of QD–TBA20 (2 nM) pre-hybridized with DNA12-SM (60 nM) after addition of different [DNA29-NF] for 2 h (B) A plot of the corresponding fluorescence intensity ratio at 605 and 665 nm (I605/I665) as a function of [DNA29-NF], the inset shows I605/I665 responses in the sub-nM range.
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fig3: (A) Typical fluorescence spectra of QD–TBA20 (2 nM) pre-hybridized with DNA12-SM (60 nM) after addition of different [DNA29-NF] for 2 h (B) A plot of the corresponding fluorescence intensity ratio at 605 and 665 nm (I605/I665) as a function of [DNA29-NF], the inset shows I605/I665 responses in the sub-nM range.

Mentions: Fig. 3 reveals that this is indeed feasible, where the Atto647N FRET signal at ∼665 nm was almost diminished accompanied by a concurrent significant recovery of the QD fluorescence at 605 nm as the [DNA29-NL] was increased, suggesting a successful displacement of the DNA12-SM reporter strand by the DNA29-NL, leading to a significant increase (∼21 fold) of the I605/I665 ratio (from 1.32 ± 0.06 to 28.6 ± 2.2 as the [DNA29-NL] increased from 0 at 100 nM, see Fig. 3B). Interestingly, replacing the DNA12-SM with DNA12 as the FRET reporter strand led to a much smaller increase of the I605/I665 ratio under identical conditions (from 0.66 ± 0.02 to 3.48 ± 0.09, an increase of ∼5.3 fold, see ESI, Fig. S10† for details), suggesting that a high stability difference between the reporter and the probe DNAs for the common target is key to achieve efficient reporter strand displacement and hence the greatly increased I605/I665 ratio. The I605/I665 response curve as a function of [DNA29-NL] was found to be non-linear (Fig. 3B), where 500 pM [DNA29-NL] produced a signal consistently above the background (Fig. 3B, inset), suggesting that this sensor can readily detect 500 pM DNA29-NL without probe amplification. Therefore this signal-on DNA sensing approach developed here can readily detect ∼500 pM unlabelled DNA probes together with a maximum ratiometric signal enhancement of ∼21 fold, which is already competitive against some other more established DNA sensing approaches, such as molecular beacons (ca. 10–20 fold signal enhancement with single-quenchers, nM sensitivity)11b,c and a recently optimised electrochemical DNA sensor (ca. 8-fold).12d An advantage of our approach here is its ratiometric signal, which is insensitive to instrument noise and signal fluctuation, allowing more reliable target detection. In addition, the DNA displacement assay was found to work equally efficiently in complex media, such as PBS with a large excess of BSA (10 μM) and in 10% human serum, suggesting that it may have good potential for clinical applications.


Robust and specific ratiometric biosensing using a copper-free clicked quantum dot-DNA aptamer sensor.

Zhang H, Feng G, Guo Y, Zhou D - Nanoscale (2013)

(A) Typical fluorescence spectra of QD–TBA20 (2 nM) pre-hybridized with DNA12-SM (60 nM) after addition of different [DNA29-NF] for 2 h (B) A plot of the corresponding fluorescence intensity ratio at 605 and 665 nm (I605/I665) as a function of [DNA29-NF], the inset shows I605/I665 responses in the sub-nM range.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814187&req=5

fig3: (A) Typical fluorescence spectra of QD–TBA20 (2 nM) pre-hybridized with DNA12-SM (60 nM) after addition of different [DNA29-NF] for 2 h (B) A plot of the corresponding fluorescence intensity ratio at 605 and 665 nm (I605/I665) as a function of [DNA29-NF], the inset shows I605/I665 responses in the sub-nM range.
Mentions: Fig. 3 reveals that this is indeed feasible, where the Atto647N FRET signal at ∼665 nm was almost diminished accompanied by a concurrent significant recovery of the QD fluorescence at 605 nm as the [DNA29-NL] was increased, suggesting a successful displacement of the DNA12-SM reporter strand by the DNA29-NL, leading to a significant increase (∼21 fold) of the I605/I665 ratio (from 1.32 ± 0.06 to 28.6 ± 2.2 as the [DNA29-NL] increased from 0 at 100 nM, see Fig. 3B). Interestingly, replacing the DNA12-SM with DNA12 as the FRET reporter strand led to a much smaller increase of the I605/I665 ratio under identical conditions (from 0.66 ± 0.02 to 3.48 ± 0.09, an increase of ∼5.3 fold, see ESI, Fig. S10† for details), suggesting that a high stability difference between the reporter and the probe DNAs for the common target is key to achieve efficient reporter strand displacement and hence the greatly increased I605/I665 ratio. The I605/I665 response curve as a function of [DNA29-NL] was found to be non-linear (Fig. 3B), where 500 pM [DNA29-NL] produced a signal consistently above the background (Fig. 3B, inset), suggesting that this sensor can readily detect 500 pM DNA29-NL without probe amplification. Therefore this signal-on DNA sensing approach developed here can readily detect ∼500 pM unlabelled DNA probes together with a maximum ratiometric signal enhancement of ∼21 fold, which is already competitive against some other more established DNA sensing approaches, such as molecular beacons (ca. 10–20 fold signal enhancement with single-quenchers, nM sensitivity)11b,c and a recently optimised electrochemical DNA sensor (ca. 8-fold).12d An advantage of our approach here is its ratiometric signal, which is insensitive to instrument noise and signal fluctuation, allowing more reliable target detection. In addition, the DNA displacement assay was found to work equally efficiently in complex media, such as PBS with a large excess of BSA (10 μM) and in 10% human serum, suggesting that it may have good potential for clinical applications.

Bottom Line: We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free "click chemistry" (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA.We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor.Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. y.guo@leeds.ac.uk d.zhou@leeds.ac.uk.

ABSTRACT
We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free "click chemistry" (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.

Show MeSH