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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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Label-free detection of thrombin using the CFCC clicked QD–DNA aptamer sensor using 2 nM QD–TBA20 pre-hybridized with DNA12-SM (60 nM) in PBS containing 20 μM BSA. A typical calibration curve showing the I605/I665 ratio as a function of thrombin concentration [TB] (inset: the response over 0–2 nM of [TB]).
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fig4: Label-free detection of thrombin using the CFCC clicked QD–DNA aptamer sensor using 2 nM QD–TBA20 pre-hybridized with DNA12-SM (60 nM) in PBS containing 20 μM BSA. A typical calibration curve showing the I605/I665 ratio as a function of thrombin concentration [TB] (inset: the response over 0–2 nM of [TB]).

Mentions: The CFCC clicked QD–TBA20 can be readily extended for label-free protein sensing via the anti-thrombin DNA aptamer sequence encoded within the TBA sequence, where the formation of thrombin (TB)–TBA complex can effectively displace the pre-hybridised reporter DNA12-SM, leading to the FRET decrease (and hence an increase of the I605/I665 ratio, see Scheme 1D). Fig. S11 (ESI†) reveals that this was indeed true, where the Atto647N FRET signal gradually decreased while the QD fluorescence increased concurrently as the target [TB] was increased, leading to the increased I605/I665 ratio (see Fig. 4). The maximum I605/I665 ratio obtained at 100 nM TB here (∼3.1) was not as high as that obtained in DNA29-NF detection (∼29), suggesting that the TB binding here is less efficient in displacing the DNA12-SM reporter strands from the QD–TBA20 conjugate as compared to DNA29-NF. Given that the binding affinity between the 29 mer anti-TB aptamer and TB (Kd ∼ 0.5 nM)11a is as strong as that of the TBA/DNA29 duplex (most likely to be in the low nM range as described above) here, the relatively low efficiency in displacing the reporter strands observed for TB here is therefore attributed to the significantly greater size of the TB–aptamer complex as compared to the TBA/DNA29 duplex, leading to steric hindrance and reduced accessibility for TB binding on the QD–DNA conjugate, especially under high [TB] conditions. Similar to the DNA29-NL based displacement assay above, a non-linear response curve between the I605/I665 signal and [TB] was also observed (Fig. 4). Moreover, the amplified response curve over the 0–2 nM [TB] range revealed that 500 pM [TB] produced a signal consistently above the background (Fig. 4, inset), suggesting that the CFCC clicked QD–DNA aptamer sensor can detect 500 pM TB directly without target pre-amplification. This sensitivity achieved here is among those of the most sensitive QD-FRET based label-free TB sensors using direct target detection without pre-amplification (see ESI, Table S1†). Moreover, this sensitivity is also comparable to those of other more established electrochemical sensing methods for TB detection (∼1 nM detection limit).12g


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)

Label-free detection of thrombin using the CFCC clicked QD–DNA aptamer sensor using 2 nM QD–TBA20 pre-hybridized with DNA12-SM (60 nM) in PBS containing 20 μM BSA. A typical calibration curve showing the I605/I665 ratio as a function of thrombin concentration [TB] (inset: the response over 0–2 nM of [TB]).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Label-free detection of thrombin using the CFCC clicked QD–DNA aptamer sensor using 2 nM QD–TBA20 pre-hybridized with DNA12-SM (60 nM) in PBS containing 20 μM BSA. A typical calibration curve showing the I605/I665 ratio as a function of thrombin concentration [TB] (inset: the response over 0–2 nM of [TB]).
Mentions: The CFCC clicked QD–TBA20 can be readily extended for label-free protein sensing via the anti-thrombin DNA aptamer sequence encoded within the TBA sequence, where the formation of thrombin (TB)–TBA complex can effectively displace the pre-hybridised reporter DNA12-SM, leading to the FRET decrease (and hence an increase of the I605/I665 ratio, see Scheme 1D). Fig. S11 (ESI†) reveals that this was indeed true, where the Atto647N FRET signal gradually decreased while the QD fluorescence increased concurrently as the target [TB] was increased, leading to the increased I605/I665 ratio (see Fig. 4). The maximum I605/I665 ratio obtained at 100 nM TB here (∼3.1) was not as high as that obtained in DNA29-NF detection (∼29), suggesting that the TB binding here is less efficient in displacing the DNA12-SM reporter strands from the QD–TBA20 conjugate as compared to DNA29-NF. Given that the binding affinity between the 29 mer anti-TB aptamer and TB (Kd ∼ 0.5 nM)11a is as strong as that of the TBA/DNA29 duplex (most likely to be in the low nM range as described above) here, the relatively low efficiency in displacing the reporter strands observed for TB here is therefore attributed to the significantly greater size of the TB–aptamer complex as compared to the TBA/DNA29 duplex, leading to steric hindrance and reduced accessibility for TB binding on the QD–DNA conjugate, especially under high [TB] conditions. Similar to the DNA29-NL based displacement assay above, a non-linear response curve between the I605/I665 signal and [TB] was also observed (Fig. 4). Moreover, the amplified response curve over the 0–2 nM [TB] range revealed that 500 pM [TB] produced a signal consistently above the background (Fig. 4, inset), suggesting that the CFCC clicked QD–DNA aptamer sensor can detect 500 pM TB directly without target pre-amplification. This sensitivity achieved here is among those of the most sensitive QD-FRET based label-free TB sensors using direct target detection without pre-amplification (see ESI, Table S1†). Moreover, this sensitivity is also comparable to those of other more established electrochemical sensing methods for TB detection (∼1 nM detection limit).12g

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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