G-quadruplexes represent a versatile sensing platform for the construction of label-free molecular detection assays due to their diverse structures that can be selectively recognized by G-quadruplex-specific luminescent probes. In this thesis, we have explored the applications of the label-free G-quadruplex-based luminescent detection platforms for the detection of biomolecules and metal ions. Chapter 1 provides an overview of the principles and recent developments of the field of luminescent oligonucleotide-based probes, and highlighting in particular the use of the “label-free” strategy for the construction of simple and inexpensive sensing platforms. Chapter 2 introduces the basic experiments performed during the course of this thesis, including UV/Vis absorption spectroscopy, luminescence spectroscopy, nuclear magnetic resonance, mass spectrometry circular dichroism spectroscopy and G-quadruplex fluorescent intercalator displacement assay. Chapter 3 describes a G-quadruplex-based switch-on luminescence assay for the detection of gene deletion using iridium(III) complex 1 as a G-quadruplex-selective probe. Our method is based on the formation of a split G-quadruplex upon hybridization of two critically designed quadruplex-forming sequences with the mutant DNA sequence, resulting in a “switch-on” luminescence response. Chapter 4 describes a label-free, oligonucleotide-based, switch-on luminescence detection method for T4 polynucleotide kinase activity using a G-quadruplex-selective luminescent iridium(III) complex 2. The application of the assay for screening potential T4 PNK inhibitors is also demonstrated. To our knowledge, this is the first metal-based assay for PNK activity that has been reported in the literature. Chapter 5 describes a label-free oligonucleotide-based luminescence switch-on assay for the selective detection of sub-nanomolar Pb2+ ions in aqueous solution and real water samples. Iridium(III) complex 1 was employed as a G-quadruplex-specific luminescent probe and a guanine-rich DNA sequence (PS2.M, 5.-GTG3TAG3CG3T2G2-3.) was employed as recognition unit for Pb2+ ions. The assay could detect Pb2+ ions in aqueous media with a limit of detection of 600 pM, and also exhibited good selectivity for Pb2+ ions over other heavy metal ions. Furthermore, the application of the assay for the detection of Pb2+ ions in spiked river water samples was demonstrated. Chapter 6 describes a label-free G-quadruplex-based luminescent switch-on assay for the selective detection of micromolar histidine in aqueous solution. Iridium(III) complex 8 was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5.-TG4AG3TG4AG3TG4A2G2-3.)/cupric ion (Cu2+) ensemble was employed as a recognition unit for histidine. The assay could detect down to 1 µM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine-masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated. Chapter 7 summarizes the work that was conducted in this thesis, and the future outlook of G-quadruplex-based sensing is presented.
| Identifer | oai:union.ndltd.org:hkbu.edu.hk/oai:repository.hkbu.edu.hk:etd_oa-1034 |
| Date | 26 August 2014 |
| Creators | He, Hongzhang |
| Publisher | HKBU Institutional Repository |
| Source Sets | Hong Kong Baptist University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Theses and Dissertations |
| Rights | The author retains all rights to this work. The author has signed an agreement granting HKBU a non-exclusive license to archive and distribute their thesis. |
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