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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) Fluorescence spectra of the QD–TBA20 (CQD = 2 nM) after hybridization with different amounts of DNA29 for 2 h in PBS excited at 450 nm, the λabs minimum of Atto647N. (B) A plot of the integrated donor/acceptor fluorescence ratio, IDye/IQD, as a function of [DNA29]. The data were fitted to a two-stage linear relationship with fitting parameters of y = –0.539 + 0.1121x, R2 = 0.9978 over 10–40 nM, and y = –0.000395 + 0.0354x, R2 = 0.9947 over 0–5 nM (shown in the inset), in which the detection limit is based on. (C) Plot of the IDye/IQD ratios as a function of concentration of different length complementary DNA probes. (D) The IDye/IQD ratios for different length DNA probes at 40 nM.
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fig2: (A) Fluorescence spectra of the QD–TBA20 (CQD = 2 nM) after hybridization with different amounts of DNA29 for 2 h in PBS excited at 450 nm, the λabs minimum of Atto647N. (B) A plot of the integrated donor/acceptor fluorescence ratio, IDye/IQD, as a function of [DNA29]. The data were fitted to a two-stage linear relationship with fitting parameters of y = –0.539 + 0.1121x, R2 = 0.9978 over 10–40 nM, and y = –0.000395 + 0.0354x, R2 = 0.9947 over 0–5 nM (shown in the inset), in which the detection limit is based on. (C) Plot of the IDye/IQD ratios as a function of concentration of different length complementary DNA probes. (D) The IDye/IQD ratios for different length DNA probes at 40 nM.

Mentions: Fig. 2A shows that in general the QD fluorescence (peaking at ∼605 nm) was quenched progressively together with a concurrent simultaneous significant increase of the Atto647N FRET signal (peaking at ∼665 nm) with the increasing DNA29 concentration, [DNA29], suggesting efficient QD-sensitised dye FRET from hybridisation of the DNA29 with QD–TBA20. A careful examination of the Atto647N emission spectra over the 640–700 nm range revealed that the Atto647N emission obtained from direct excitation of 60 nM DNA29 was actually weaker than that of the QD-sensitized FRET signal for 0.25 nM DNA29 (see ESI, Fig. S5†), suggesting that the QD sensitized FRET is at least 240 times as efficient as direct excitation. To our knowledge, this has been the highest ratio of FRET-sensitized signals over that of direct excitation for the QD-FRET systems reported so far (most reported ratios in the literature typically ranged from ∼2.5 to 40).2,3 This is presumably because λEX = 450 nm, corresponding to the λabs minimum of the Atto647N, was used here to minimise the direct excitation of the Atto647N acceptor. Moreover, the CFCC conjugated QD–TBA20 here retained a much higher QY of the QD than those prepared via EDC–NHS coupling (see ESI, Fig. S1†), as a result, the sensing experiments were able to be performed at 2 nM QD, ∼10 to 500 fold lower than those reported previously (see Table 2).2,3 Such a high FRET-sensitised signal over the direct excitation background is highly advantageous for biosensing, which can effectively eliminate the need for background correction from direct acceptor excitation, making data analysis easy and straightforward.


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) Fluorescence spectra of the QD–TBA20 (CQD = 2 nM) after hybridization with different amounts of DNA29 for 2 h in PBS excited at 450 nm, the λabs minimum of Atto647N. (B) A plot of the integrated donor/acceptor fluorescence ratio, IDye/IQD, as a function of [DNA29]. The data were fitted to a two-stage linear relationship with fitting parameters of y = –0.539 + 0.1121x, R2 = 0.9978 over 10–40 nM, and y = –0.000395 + 0.0354x, R2 = 0.9947 over 0–5 nM (shown in the inset), in which the detection limit is based on. (C) Plot of the IDye/IQD ratios as a function of concentration of different length complementary DNA probes. (D) The IDye/IQD ratios for different length DNA probes at 40 nM.
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Related In: Results  -  Collection

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fig2: (A) Fluorescence spectra of the QD–TBA20 (CQD = 2 nM) after hybridization with different amounts of DNA29 for 2 h in PBS excited at 450 nm, the λabs minimum of Atto647N. (B) A plot of the integrated donor/acceptor fluorescence ratio, IDye/IQD, as a function of [DNA29]. The data were fitted to a two-stage linear relationship with fitting parameters of y = –0.539 + 0.1121x, R2 = 0.9978 over 10–40 nM, and y = –0.000395 + 0.0354x, R2 = 0.9947 over 0–5 nM (shown in the inset), in which the detection limit is based on. (C) Plot of the IDye/IQD ratios as a function of concentration of different length complementary DNA probes. (D) The IDye/IQD ratios for different length DNA probes at 40 nM.
Mentions: Fig. 2A shows that in general the QD fluorescence (peaking at ∼605 nm) was quenched progressively together with a concurrent simultaneous significant increase of the Atto647N FRET signal (peaking at ∼665 nm) with the increasing DNA29 concentration, [DNA29], suggesting efficient QD-sensitised dye FRET from hybridisation of the DNA29 with QD–TBA20. A careful examination of the Atto647N emission spectra over the 640–700 nm range revealed that the Atto647N emission obtained from direct excitation of 60 nM DNA29 was actually weaker than that of the QD-sensitized FRET signal for 0.25 nM DNA29 (see ESI, Fig. S5†), suggesting that the QD sensitized FRET is at least 240 times as efficient as direct excitation. To our knowledge, this has been the highest ratio of FRET-sensitized signals over that of direct excitation for the QD-FRET systems reported so far (most reported ratios in the literature typically ranged from ∼2.5 to 40).2,3 This is presumably because λEX = 450 nm, corresponding to the λabs minimum of the Atto647N, was used here to minimise the direct excitation of the Atto647N acceptor. Moreover, the CFCC conjugated QD–TBA20 here retained a much higher QY of the QD than those prepared via EDC–NHS coupling (see ESI, Fig. S1†), as a result, the sensing experiments were able to be performed at 2 nM QD, ∼10 to 500 fold lower than those reported previously (see Table 2).2,3 Such a high FRET-sensitised signal over the direct excitation background is highly advantageous for biosensing, which can effectively eliminate the need for background correction from direct acceptor excitation, making data analysis easy and straightforward.

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
Related in: MedlinePlus