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Title: Development of a novel, functional quantum dot-DNA/aptamer sensing technology
Author: Zhang, Haiyan
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2013
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Aptamers are short single-stranded DNA or RNA oligonucleotides artificially selected against specific targets. They exhibit high target binding affinity and exquisite specificity, making them very useful in developing biosensors for a wide range of targets, such as proteins, peptides, amino acids, drugs, metal ions and even whole cells. While fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have unique size-dependent, bright and extremely photo-stable fluorescence that make them as excellent fluorescent labels for biological imaging, sensing, cell tracking/trafficking and diagnostics. Their unique optical properties (broad absorption and narrow symmetric emission) are well-suited for FRET (fluorescence resonance energy transfer) based sensing applications. By combining the advance properties of both aptamers and QDs, this project aims to develop a sensitive, specific and robust aD-DNAlaptamer FRET based biosensing technology that can be used for rapid biosensing, diagnostics and environmental monitoring. A major hurdle here is the preparation of a compact, stable and water-soluble QD-bioconjugate that can effectively resist non-specific adsorption because FRET efficiency (E) decreases dramatically with the increasing donor-acceptor distance. Hence for high sensitivity, a compact QD-bioconjugate structure is essential. In this regard, a series of highly fluorescent, water-soluble CdSe/ZnS aDs were prepared first by ligand exchange with hydrophilic thiolated ligands, such as dihydrolipoic acid (DHLA), glutathione (GSH), dihydrolipoic acid-polyethyleneglycol (DHLAPEG600) derivatives. These QDs exhibited high fluorescence quantum yields (QYs, 6-30%), comparable to commercial water-soluble aDs (ca. 30%), but having significantly smaller hydrodynamic diameters «10 v.s. > 25 nm). Building upon these, three different QD-DNA aptamer sensing systems have been developed successfully: (1) A simple self-assembled aD-DNA system: I have found that thiolated DNA can self-assemble onto DHLA capped QDs to form compact, functional QD-DNA conjugates with small donor-acceptor distances, producing efficient FRET (E > 70%) at a relatively low (target: QD) copy number of 6:1. The resulting self-assembled aD-DNA (aptamer) conjugate has been used to detect low nM levels of labelled DNA target via QD sensitised dye FRET signal. Moreover, it has been successfully used for detection of nM level of a protein target via the encoded DNA aptamer sequence, although its specificity and stability still need further improvement. (2) A more stable and sensitive aD-dual-donor FRET sensing system based on an amine-modified DNA covalently coupled to a glutathione capped aD combined with the use of specific ethidium bromide (EB) intercalation in hybridized DNA duplex. As a result, both the aD and intercalated EBs can FRET to the dye acceptor appended to the complementary DNA, leading to significantly improved the overall FRET efficiency E, and hence sensitivity in both DNA and protein target detection down to pM level. (3) A Cu-free "clicked", robust, and_specific QD-DNA aptamer sensor. A compact, functional aD-DNA conjugate was prepared via the Cu-free "click chemistry" (CFCC) between a dihydrolipoic acid -polyethylene glycol-azide (DHLA-PEG600-N3) capped aD and a cyclooctyne modified DNA. The resulting QD-DNA conjugate is highly stable in biological buffer, effectively resisting nonspecific absorption, displaying a relatively small size (hydrodynamic radius - 5 nm) and retaining almost the native ay of the parent aD. Moreover, the CFCC based DNA conjugation method is also highly efficient, leading to high DNA loading (- 15-30 DNA strands per aD is readily achieved). This system is well-suited for robust biosensing: it can quantitate pM level of complementary DNA targets with SNP (singlenucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum. It can also detect pM level of a specific protein via the encoded DNA aptamer sequence. Compared with these approaches, the self-assembled system is the most convenient to prepare, but it has the least stability and cannot resist nonspecific absorption. The dual-donor FRET sensing system shows some enhancement on the stability and resisting nonspecific absorption, but it still cannot work in complex media, such as serum. The CFCC clicked QD-DNA aptamer sensor shows the highest stability, specificity and assay robustness, and can effectively work in clinical media. It can be readily extended to design sensors against other targets by simply clicking on specific aptamer sequences against such targets. Because the CFCC clicked QD-DNAlaptamer sensor shows high stability, specificity, robustness· and sensitivity, it may have a wide range of biosensing and diagnostic applications
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available