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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) Schematic approach to the Cu-free “clicked” QD–DNA conjugate. (B) Hybridization of a complementary dye-labelled DNA probe with the QD–DNA conjugate leads to QD sensitized dye FRET signals as a readout for labelled DNA detection. (C) Incubation of the QD–double-stranded (ds) DNA conjugate formed in (B) with a longer, unlabeled DNA displaces the shorter labelled DNA reporter, reducing the QD to dye FRET for label-free DNA detection. (D) Incubation of the QD–dsDNA conjugate (B) with a target protein that binds to the encoded aptamer sequence in the QD–dsDNA conjugate displaces the reporter DNA, leading to reduction of QD to dye FRET for label-free protein detection. The block arrows give the FRET directions.
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sch1: (A) Schematic approach to the Cu-free “clicked” QD–DNA conjugate. (B) Hybridization of a complementary dye-labelled DNA probe with the QD–DNA conjugate leads to QD sensitized dye FRET signals as a readout for labelled DNA detection. (C) Incubation of the QD–double-stranded (ds) DNA conjugate formed in (B) with a longer, unlabeled DNA displaces the shorter labelled DNA reporter, reducing the QD to dye FRET for label-free DNA detection. (D) Incubation of the QD–dsDNA conjugate (B) with a target protein that binds to the encoded aptamer sequence in the QD–dsDNA conjugate displaces the reporter DNA, leading to reduction of QD to dye FRET for label-free protein detection. The block arrows give the FRET directions.

Mentions: Scheme 1 shows our approach to the QD–DNA conjugate via the CFCC and its use in label- and label-free-detection of DNA and protein targets via target binding induced changes in the QD sensitized dye FRET signals. First, a multi-functional ligand, containing a dihydrolipoic acid (DHLA, for strong QD binding) head group, a polyethylene glycol moiety of a molecular weight of 600 (PEG600, for providing good water-solubility and effective resistance to non-specific adsorption of biomolecules) and a terminal azide group (for efficient DNA conjugation via the CFCC), DHLA–PEG600–N3, was prepared (see the ESI† for details).9,10 A PEGylated DHLA ligand was used as the QD surface capping ligand here because it represented an excellent balance between the requirements of high stability and resistance to non-specific adsorption (for robust biosensing) and the structural compactness (for high sensitivity).2 Then a hydrophobic CdSe–ZnS core–shell QD (λEM ∼ 605 nm, QY ∼ 20%, capped with hydrophobic trioctyl-phosphine oxide/trioctylphosphine) was made water-soluble by ligand exchange with the DHLA–PEG600–N3 in a mixed solvent of CHCl3–ethanol using our previously established procedures,3l yielding the QD–DHLA–PEG600–N3 which was readily soluble in polar solvents. Its fluorescence QY was found to decrease to ∼6.0% (and hence a decrease of ca. 70%), which is in good agreement with most other reports in the literature where most hydrophobic CdSe–ZnS core–shell QDs typically showed a QY decrease of 50–80% following the ligand exchange and transfer to aqueous media.3,4 A single-stranded (ss) target DNA encoded with a 29 mer anti-thrombin (TB) aptamer sequence with strong affinity for TB (Kd ∼ 0.5 nM, modified with a C6-amine at 5′, H2N–TBA, see Table 1)11 was reacted with an N-hydroxysuccinimide (NHS) ester activated cyclooctyne to yield TBA–cyclooctyne, which was then reacted with the QD–DHLA–PEG600–N3 in a mixed solvent of ethanol–water at a molar ratio of 30 : 1. This led to QD–TBA covalent conjugation via the efficient CFCC approach. Approximately 20 strands of TBAs were found to be conjugated to each QD, denoted as QD–TBA20 hereafter, this gave a DNA conjugation efficiency of ∼67%. The detailed experimental procedures for the ligand synthesis and QD–DNA conjugation are given in the ESI.† The QY of the resulting QD–TBA20 was determined as ∼5.9% using rhodamine 6G in ethanol as the calibration standard (QY 95%),3b which is effectively the same as that of the QD–DHLA–PEG600–N3 (ca. 6.0%).


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) Schematic approach to the Cu-free “clicked” QD–DNA conjugate. (B) Hybridization of a complementary dye-labelled DNA probe with the QD–DNA conjugate leads to QD sensitized dye FRET signals as a readout for labelled DNA detection. (C) Incubation of the QD–double-stranded (ds) DNA conjugate formed in (B) with a longer, unlabeled DNA displaces the shorter labelled DNA reporter, reducing the QD to dye FRET for label-free DNA detection. (D) Incubation of the QD–dsDNA conjugate (B) with a target protein that binds to the encoded aptamer sequence in the QD–dsDNA conjugate displaces the reporter DNA, leading to reduction of QD to dye FRET for label-free protein detection. The block arrows give the FRET directions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

sch1: (A) Schematic approach to the Cu-free “clicked” QD–DNA conjugate. (B) Hybridization of a complementary dye-labelled DNA probe with the QD–DNA conjugate leads to QD sensitized dye FRET signals as a readout for labelled DNA detection. (C) Incubation of the QD–double-stranded (ds) DNA conjugate formed in (B) with a longer, unlabeled DNA displaces the shorter labelled DNA reporter, reducing the QD to dye FRET for label-free DNA detection. (D) Incubation of the QD–dsDNA conjugate (B) with a target protein that binds to the encoded aptamer sequence in the QD–dsDNA conjugate displaces the reporter DNA, leading to reduction of QD to dye FRET for label-free protein detection. The block arrows give the FRET directions.
Mentions: Scheme 1 shows our approach to the QD–DNA conjugate via the CFCC and its use in label- and label-free-detection of DNA and protein targets via target binding induced changes in the QD sensitized dye FRET signals. First, a multi-functional ligand, containing a dihydrolipoic acid (DHLA, for strong QD binding) head group, a polyethylene glycol moiety of a molecular weight of 600 (PEG600, for providing good water-solubility and effective resistance to non-specific adsorption of biomolecules) and a terminal azide group (for efficient DNA conjugation via the CFCC), DHLA–PEG600–N3, was prepared (see the ESI† for details).9,10 A PEGylated DHLA ligand was used as the QD surface capping ligand here because it represented an excellent balance between the requirements of high stability and resistance to non-specific adsorption (for robust biosensing) and the structural compactness (for high sensitivity).2 Then a hydrophobic CdSe–ZnS core–shell QD (λEM ∼ 605 nm, QY ∼ 20%, capped with hydrophobic trioctyl-phosphine oxide/trioctylphosphine) was made water-soluble by ligand exchange with the DHLA–PEG600–N3 in a mixed solvent of CHCl3–ethanol using our previously established procedures,3l yielding the QD–DHLA–PEG600–N3 which was readily soluble in polar solvents. Its fluorescence QY was found to decrease to ∼6.0% (and hence a decrease of ca. 70%), which is in good agreement with most other reports in the literature where most hydrophobic CdSe–ZnS core–shell QDs typically showed a QY decrease of 50–80% following the ligand exchange and transfer to aqueous media.3,4 A single-stranded (ss) target DNA encoded with a 29 mer anti-thrombin (TB) aptamer sequence with strong affinity for TB (Kd ∼ 0.5 nM, modified with a C6-amine at 5′, H2N–TBA, see Table 1)11 was reacted with an N-hydroxysuccinimide (NHS) ester activated cyclooctyne to yield TBA–cyclooctyne, which was then reacted with the QD–DHLA–PEG600–N3 in a mixed solvent of ethanol–water at a molar ratio of 30 : 1. This led to QD–TBA covalent conjugation via the efficient CFCC approach. Approximately 20 strands of TBAs were found to be conjugated to each QD, denoted as QD–TBA20 hereafter, this gave a DNA conjugation efficiency of ∼67%. The detailed experimental procedures for the ligand synthesis and QD–DNA conjugation are given in the ESI.† The QY of the resulting QD–TBA20 was determined as ∼5.9% using rhodamine 6G in ethanol as the calibration standard (QY 95%),3b which is effectively the same as that of the QD–DHLA–PEG600–N3 (ca. 6.0%).

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