Year of Award

2014

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Department of Chemistry.

Principal Supervisor

Ma, Edmond Dik Lung

Keywords

Biomolecules, Luminescent probes, Metal ions, Oligonucleotides

Language

English

Abstract

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.

Comments

Thesis (Doctor of Philosophy)--Hong Kong Baptist University, 2014.;Principal supervisor: Dr. Ma Edmond Dik Lung.;Includes bibliographical references (pages 193-196)

Copyright

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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