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

Haiyan Zhang,Dejian Zhou,Yuan Guo,Guoqiang Feng

doi:10.1039/c3nr02897f

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.

Highlights

Scheme 1 shows our approach to the quantum dot (QD)–DNA conjugate via the copper-free “click chemistry” (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 Forster resonance energy transfer (FRET) signals
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 nonspeci c adsorption and the structural compactness.[2]
A hydrophobic CdSe– ZnS core–shell QD 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

Summary

Introduction

The unique, size-dependent, highly stable and bright uorescence of quantum dots (QDs) make them powerful tools in broad ranges of bio-related applications.[1,2,3,4] In particular, their broad absorption and narrow and symmetric emission are extremely well-suited for Forster resonance energy transfer (FRET) based sensing, because these spectral characteristics enable a wide selection of excitation wavelengths to minimise direct excitation of the acceptor, reducing the background and improving the sensitivity.[1,2] numerous QD-FRET based biosensors have been reported.[3,4] Despite these, the sensitivity and speci city of the QD-FRET based biosensors have largely been limited by challenges in preparing compact and functional QD-bioconjugates that are stable and effectively resist non-speci c adsorption.[2,3,4] For example, water-soluble QDs prepared by ligand exchange are compact, but they o en show low stability in biologically relevant buffers and resistance to non-speci c adsorption, limiting their sensing speci city and robustness.[2]. The CFCC clicked QD–DNA conjugate is found to retain the native uorescence quantum yield (QY) of the parent QD, and well-suited for robust 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 even in complex media, e.g. 10% human serum, on a conventional uorimeter.

Results

Conclusion