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.

On the purpose of designing a novel generation of luminescent bioprobes for imaging and inhibition of tumour cells, a series of lanthanide-ruthenium complexes has been synthesized and characterized by 1H NMR, 13C NMR, absorption/emission spectroscopy, high-performance liquid chromatography, and mass spectroscopy. Those complexes are qualified to be considered as photo-activatable anticancer prodrugs which consist of a ruthenium (II) complex linked to a lanthanide-based cyclen chelate via a π-conjugated bridge. Comprehensive studies have been performed to evaluate their efficacy as pro-drugs which requires in cellulo activity, inhibiting ability, instant monitoring possibility, and safety to normal cells. The resulting complexes are proved to be promising agents for controllable anticancer therapy because the prodrug remains inactive in dark and the release of the active drug is induced by visible light. Drug delivery process can be quantitatively monitored by either the long-lived red europium emission under one- or two-photon excitation or potentially by magnetic resonance imaging signals. Besides of these, the correlation among the drug releasing amount, signaling emission intensity, and mass spectroscopy response, has been proposed for quick and simple quantitative analysis.

This joint PhD program is part of a collaboration between Hong Kong Baptist University (Dr. Gary K-L Wong) and Laboratoire d'Ingénierie Moléculaire Appliquée à l'Analyse (LIMAA - Dr. Loïc Charbonnière) funded by the Alsace region to synthesize new nanoprobes for sensing, imaging, and inhibiting cancer diseases. The first work was to synthesize new hybrid ultrabright nanoparticles. They have been obtained from a La0.9Tb0.1F3 core and coated by different ligands. Thanks to a mechanism of antenna effect, the brightness of the nanoparticles has been significantly improved. The second work was to synthesize a new ligand to photosensitize water-soluble La0.90Eu0.1F3 nanoparticles in order to improve the emission of europium. A second ligand and new heterometallic nanoparticles have been synthesized with the aim to promote the energy transfer from Tb(III) ions on the surface of the NPs to Eu(III) ions in the core of the nanoparticles and to get a very long excited-state lifetime and an exceptional quantum yield in aqueous solution. The last work was to functionalize water-soluble graphitic-carbon nitride (g-C3N4) nanoparticles by porphyrins. The porphyrins have been synthesized to generate singlet oxygen (1O2), to host a Ga3+ ion inside their cavity and with two different linkers to be coupled to nanoparticles. This system aims to be a pH sensor, and a PDT and PET theranostic agent.

Transition metal complexes offer potential alternatives to fluorescent organic compounds in various sensing applications. They show several characteristic properties over organic dyes, such as strong luminescence emission, long emission lifetime and large Stoke shift. Among transition metal complexes, cyclometalated iridium(III) (Ir) complexes are most widely explored for sensing applications, due to their bright and tuneable phosphorescence emission. Up to now, Ir(III) complexes have been successfully applied to detect a range of analytes in environmental and biological systems, such as cations, anion, small molecules and proteins. In this thesis, we deeply explored the capability of Ir(III) complexes to the detection of a range of targets including metal ions, small molecules and biomarkers. Several strategies are used to improve the biocompatibility of Ir(III)-based probes while retaining their desirable characteristics. In chapter 2, we developed a novel Ir(III) complex for the detection of Al3+ with a detection limit of 1 μM. The long lifetime of the complex was harnessed to distinguish luminescence response to Al3+ from autofluorescence in biological samples by TRES experiment, while the probe was also successfully applied for imaging Al3+ in living cells. The results have been published as Chem. Commun., 2016, 52, 3611. In chapter 3, we reported a new reaction-based luminogenic probe for imaging both H2S and hypoxia in living zebrafish. This probe demonstrated their utility for the detection of H2S in solution, living cells and zebrafish model, while it was also capable of discriminating hypoxic from normoxic cells and zebrafish model. The results have been published as Sens. Actuator B-Chem., 2018, 255, 1953. In chapter 4, we conjugated a natural product oridonin to an Ir(III) scaffold for tracking intracellular NF-κB. This complex was successfully applied to track NF-κB translocation induced by TNF-α, without affecting the translocation process. The results have been published as Chem. Eur. J., 2017, 23, 4929. In chapter 5, an Ir(III) scaffold with galactose moiety was designed and synthesized for discriminating ovarian carcinoma cell lines from normal cell lines. This probe can selectively "light up" ovarian carcinoma cells with negligible luminescence in normal cells. The results have been published as Anal. Chem., 2017, 89, 11679. These works have further demonstrated the utility of Ir(III) complex in the monitoring environment and studying biomolecules in living systems. In particular, the conjugation of endogenous molecule galactose or a natural compound oridonin to Ir(III) scaffolds highlights an effective solution to develop biocompatible probes. However, it should be pointed out that there is a need for developing a general strategy to improve the biocompatibility of luminescent Ir(III) complex-based probes, while there is huge potential for incorporating luminescent Ir(III) complexes-based sensing platforms into portable devices, and exploring theranostic probes.

Transition metal complex, especially iridium(III) complex, possesses substantial advantages in sensing applications. To date, a series of iridium(III) complex-based chemosensors and biosensors have been constructed for the detection of a wide range of biologically important analytes, including ions, small molecules, amino acids, peptides and proteins. Chapter 1 provides an overview of the general synthetic routes and properties of iridium(III) complexes. General strategies for the development of iridium(III) complex-based chemosensors and the utilization of iridium(III) complex as a DNA probe in biosensors are also reviewed. Chapter 2 describes the application of an iridium(III) complex as a switch "on-off-on" chemosensor for the detection of both exogenously supplied and endogenously generated sulfide ion in vitro, in cellulo and in vivo. The optimized probe (1-Fe 3+), which coordinates Fe 3+ ion to an iridium(III) complex, could achieve a limit of detection (LOD) of sulfide ion down to 2.9 µM and establish a linear detection range from 10 to 1500 µM. While 1-Fe 3+ did not show any luminescence response in vitro under a high concentration of thiols, it exhibited a significant luminescence enhancement when the concentration of thiols was perturbed in cellulo and in vivo. This phenomenon can be explained by the presence of cystathionine gamma-lyase (CGL) and cystathionine beta synthase (CBS) in cellulo that could catalyze the conversion of the hydrogen sulfide (H 2 S) precursors, including cysteine and glutathione (GSH), into H 2 S. The results of this work have been published in a peer-reviewed scientific journal (Biosens. Bioelectron, 2017, 94, 575). Chapter 3 discusses the adaptation of an oligonucleotide-based Vascular endothelial growth factor 165 (VEGF 165) biosensor on a portable microfluidic device. An iridium(III) complex with an extensive conjugation system was used as a long- lived and red-emitting G-quadruplex probe. The polypropylene (PP)-based suspended-droplet microfluidic chip allows easy sample introduction, flexible sample volume range and valve-free manipulation of a stepwise reaction. We successfully assembled all the required components, including a ultraviolet (UV) lamp, a filter, a rotatable sample holder and a detector, into a portable box. The device could achieve a LOD of VEGF 165 down to the picomolar level, which is comparable to the results of a conventional fluorometer. The results of this work have been published in a peer-reviewed scientific journal (Dalton Trans., 2019, 48, 9824) The integration of graphene oxide nanomaterial to an oligonucleotide- based isothermal signal amplification system is presented in Chapter 4. Strand displacement amplification (SDA) could substantially amplify the signal from the target Hepatitis B virus (HBV) gene, while the electron accepting graphene oxide could effectively quench the emission of iridium(III) complex and enlarged the luminescence fold-enhancement of the system. The system could achieve a LOD for the HBV gene down to the picomolar level and was selective for the wild-type HBV gene over the single-base mutated HBV gene. The operation mechanism and the important rules for the formation of a stable split G-quadruple are detailed in this chapter. The results of this work have been submitted to a peer-reviewed scientific journal. In Chapter 5, the adaptation of SDA on an exonuclease III-assisted amplification (EXO) as a quadruple-cycle phosphorescence amplification system is reported. A systematic three-round structural optimization campaign was performed for the first time to iteratively improve the G-quadruplex selectivity of a large pool of iridium(III) complex. The proof-of-principle application of a self-assembly tetrahedron nanostructure (TNS)-based aptasensor was demonstrated using a cancer biomarker mucin 1 as the target analyte. This TNS-based aptasensor revealed a 57% higher luminescence enhancement compared to the conventional dsDNA aptasensing approach. The results of this work will be submitted to a peer-reviewed scientific journal upon completion of mechanism studies. Chapter 6 summarizes the properties, advantages, improvements and potentials for the developed iridium(III) complex-based sensors

The development of transition metal complex-based luminescent probes and theranostic have recently aroused tremendous interest for labelling and detecting environmental contaminants and cellular biomarkers, particularly in the use of real- time diagnosis and treatment of disease. Reasons behind include the unique photophysical properties of transition metal complexes, particularly in the properties of their long-lived and environmentally sensitive emission, which can be easily fine-tuned via the modification of the metal center and auxiliary ligand of the metal complex to achieve the desired emissive characteristics. In Chapter 2, a series of luminescent iridium(III) complexes were introduced and their synthesis and evaluation on their ability to interact with hydroxide (OH- ) ion in semi-aqueous media at ambient temperature were discussed. Upon addition of OH- ion, a nucleophilic aromatic substitution reaction takes place at the bromine groups of the N^N ligand of iridium(III) complex 2.1, resulting in the generation of product with a yellow-green luminescence. Complex 2.1 showed a 35-fold enhancement in emission signal at pH 14 when compared to neutral pH, and the detection limit for OH- ions was found to be 4.96 μM. Complex 2.1 exhibited high sensitivity and selectivity, long-lived luminescence and impressive stability. Additionally, practical application of complex 2.1 was demonstrated to be able to detect OH- ions in simulated wastewater. In Chapter 3, a series of luminescent iridium(III) complexes were introduced and their design and evaluation on their affinity to detect oxalyl chloride ((COCl)2) at ambient temperature were discussed. In the presence of (COCl)2, a double amidation reaction takes place at the diamino functionality of complex 3.1, leading to the switching-on of a long-lived red luminescence with 9-fold enhancement in emission signal. Complex 3.1 exhibited high sensitivity and selectivity and the detection limit for (COCl)2 was found to be 32 nM. Additionally, complex 3.1 can be used to detect (COCl)2 using a simple smartphone, which allows the detection to be a real-time one. iii In Chapter 4, a dual-functional luminescent probe and inhibitor was designed for the in-situ monitoring of neuraminidase (NA) using a structure-based molecular design strategy. The candidate iridium(III) complexes 4.1a-4.1d were synthesized by grafting an oseltamivir moiety as a binding unit onto signaling iridium(III) precursors, generating probes that allowed for the simultaneous inhibition and sensing of NA. Complexes 4.1a-4.1d showed strong yellow or red luminescence in aqueous buffer containing 0.5% acetonitrile in response to NA. In particular, complex 4.1d exhibited enhanced inhibition against NA compared to the FDA-approved antiviral drug, oseltamivir. Moreover, complex 4.1d also displayed a long-lived lifetime, large Stokes shift, and high quantum yield, allowing its luminescence output to be distinguished in the presence of an interfering auto- fluorescent background. We have successfully developed the first dual-functional molecule 4.1d for the in-situ inhibition and detection of NA, which provides the possibility for the in-field simultaneous therapy and monitoring of influenza infection. In Chapter 5, a general strategy was introduced for the development of a long- lifetime iridium(III) theranostic by grafting a well-known inhibitor as a "binding unit" onto an iridium(III) complex precursor as a "signaling unit". To further optimize their emissive properties, the effect of imaging behavior was explored by incorporating different substituents onto the parental "signaling unit". This design concept was validated by a series of tailored iridium(III) theranostic 5.2a-5.2h for the visualization and inhibition of EGFR in living cancer cells. By comprehensively assessing the theranostic potency of 5.2a-5.2h in both in vitro and in cellulo contexts, probe 5.2f containing electron-donating methoxy groups on the "signaling unit" was discovered to be the most promising candidate theranostic with desirable photophysical/chemical properties. Probe 5.2f selectively bound to EGFR in vitro and in cellulo, enabling it to selectively discriminate living EGFR- overexpressing cancer cells from normal cells that express low levels of EGFR with an "always-on" luminescence signal output. In particular, its long-lived lifetime enabled its luminescence signal to be readily distinguished from the interfering iv fluorescence of organic dyes by using time-resolved technique. Complex 5.2f simultaneously visualized and inhibited EGFR in a dose-dependent manner, leading to a reduction in the phosphorylation of downstream proteins ERK and MEK, and inhibition of the activity of downstream transcription factor AP1. Notably, complex 5.2f is comparable to the parental EGFR inhibitor 5.1b, in terms of both inhibitory activity against EGFR and cytotoxicity against EGFR- overexpressing cancer cells. This tailored dual-functional iridium(III) theranostic toolkit provides an alternative strategy for the real-time and personalized diagnosis and treatment of cancers. Chapter 6 discusses the challenges and future inspirations for the development of iridium(III) complex-based luminescent probes and theranostic

The development of transition metal complex-based luminescent probes and theranostic have recently aroused tremendous interest for labelling and detecting environmental contaminants and cellular biomarkers, particularly in the use of real- time diagnosis and treatment of disease. Reasons behind include the unique photophysical properties of transition metal complexes, particularly in the properties of their long-lived and environmentally sensitive emission, which can be easily fine-tuned via the modification of the metal center and auxiliary ligand of the metal complex to achieve the desired emissive characteristics. In Chapter 2, a series of luminescent iridium(III) complexes were introduced and their synthesis and evaluation on their ability to interact with hydroxide (OH- ) ion in semi-aqueous media at ambient temperature were discussed. Upon addition of OH- ion, a nucleophilic aromatic substitution reaction takes place at the bromine groups of the N^N ligand of iridium(III) complex 2.1, resulting in the generation of product with a yellow-green luminescence. Complex 2.1 showed a 35-fold enhancement in emission signal at pH 14 when compared to neutral pH, and the detection limit for OH- ions was found to be 4.96 μM. Complex 2.1 exhibited high sensitivity and selectivity, long-lived luminescence and impressive stability. Additionally, practical application of complex 2.1 was demonstrated to be able to detect OH- ions in simulated wastewater. In Chapter 3, a series of luminescent iridium(III) complexes were introduced and their design and evaluation on their affinity to detect oxalyl chloride ((COCl)2) at ambient temperature were discussed. In the presence of (COCl)2, a double amidation reaction takes place at the diamino functionality of complex 3.1, leading to the switching-on of a long-lived red luminescence with 9-fold enhancement in emission signal. Complex 3.1 exhibited high sensitivity and selectivity and the detection limit for (COCl)2 was found to be 32 nM. Additionally, complex 3.1 can be used to detect (COCl)2 using a simple smartphone, which allows the detection to be a real-time one. iii In Chapter 4, a dual-functional luminescent probe and inhibitor was designed for the in-situ monitoring of neuraminidase (NA) using a structure-based molecular design strategy. The candidate iridium(III) complexes 4.1a-4.1d were synthesized by grafting an oseltamivir moiety as a binding unit onto signaling iridium(III) precursors, generating probes that allowed for the simultaneous inhibition and sensing of NA. Complexes 4.1a-4.1d showed strong yellow or red luminescence in aqueous buffer containing 0.5% acetonitrile in response to NA. In particular, complex 4.1d exhibited enhanced inhibition against NA compared to the FDA-approved antiviral drug, oseltamivir. Moreover, complex 4.1d also displayed a long-lived lifetime, large Stokes shift, and high quantum yield, allowing its luminescence output to be distinguished in the presence of an interfering auto- fluorescent background. We have successfully developed the first dual-functional molecule 4.1d for the in-situ inhibition and detection of NA, which provides the possibility for the in-field simultaneous therapy and monitoring of influenza infection. In Chapter 5, a general strategy was introduced for the development of a long- lifetime iridium(III) theranostic by grafting a well-known inhibitor as a "binding unit" onto an iridium(III) complex precursor as a "signaling unit". To further optimize their emissive properties, the effect of imaging behavior was explored by incorporating different substituents onto the parental "signaling unit". This design concept was validated by a series of tailored iridium(III) theranostic 5.2a-5.2h for the visualization and inhibition of EGFR in living cancer cells. By comprehensively assessing the theranostic potency of 5.2a-5.2h in both in vitro and in cellulo contexts, probe 5.2f containing electron-donating methoxy groups on the "signaling unit" was discovered to be the most promising candidate theranostic with desirable photophysical/chemical properties. Probe 5.2f selectively bound to EGFR in vitro and in cellulo, enabling it to selectively discriminate living EGFR- overexpressing cancer cells from normal cells that express low levels of EGFR with an "always-on" luminescence signal output. In particular, its long-lived lifetime enabled its luminescence signal to be readily distinguished from the interfering iv fluorescence of organic dyes by using time-resolved technique. Complex 5.2f simultaneously visualized and inhibited EGFR in a dose-dependent manner, leading to a reduction in the phosphorylation of downstream proteins ERK and MEK, and inhibition of the activity of downstream transcription factor AP1. Notably, complex 5.2f is comparable to the parental EGFR inhibitor 5.1b, in terms of both inhibitory activity against EGFR and cytotoxicity against EGFR- overexpressing cancer cells. This tailored dual-functional iridium(III) theranostic toolkit provides an alternative strategy for the real-time and personalized diagnosis and treatment of cancers. Chapter 6 discusses the challenges and future inspirations for the development of iridium(III) complex-based luminescent probes and theranostic

The design and applications of quantum dots (QDs) as fluorescent probes for analyte sensing is presented. Cadmium based thiol-capped QDs were employed as probe for the detection of analytes. Comparative studies between core CdTe and core-shell CdTe@ZnS QDs showed that the overall sensitivity and selectivity of the sensor was dependent on the nature of the capping agent and the QDs employed, hence making CdTe@ZnS QDs a more superior sensor than the core. To explore the luminescent sensing of QDs based on the fluorescence “turn ON” mode, L-glutathione-capped CdTe QDs was conjugated to 4-amino-2,2,6,6-tetramethylpiperidine-N-oxide (4AT) to form a QDs-4AT conjugate system. The QDs-4AT nanoprobe was highly selective and sensitive to the detection of bromide ion with a very low limit of detection. Subsequently, metallo-phthalocyanines (MPcs) were employed as host molecules on the surface of QDs based on the covalent linking of the QDs to the MPc. Elucidation of the reaction mechanism showed that the fluorescence “turn ON” effect of the QDs-MPc probe in the presence of the analyte was due to axial ligation of the analytes to the Pc ring. Studies showed that the type of substituent attached to the MPc ring influenced the overall sensitivity of the probe. Additionally, a comparative investigation using newly synthesized phthalocyanine and triaza-benzcorrole complexes was conducted when these complexes were conjugated to CdSe@ZnS QDs for analyte sensing. Results showed that the triaza-benzcorrole complex can be employed as a host-molecule sensor but displayed a lower sensitivity for analyte sensing in comparison to the phthalocyanine complex.

The high incidence rate of Nasopharyngeal Carcinoma (NPC) in southern China, including Hong Kong, has attracted worldwide attention. According to the Center for Health Protection in Hong Kong, there were 841 new cases of NPC, with 655 cases of males and 186 cases of females in 2013. The development of NPC is highly associated with the infection of one human herpes virus, the Epstein-Barr virus (EBV). Given that the homodimerization of one of the EBV endogenous protein-Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for both viral genome maintenance and infected-cell survival, thus the interference of EBNA1 homodimerization would be a novel strategy for the inhibition of EBV-positive tumours. In this thesis we devote to conjugate several kinds of organic fluorophores with various EBV-specific peptides in order to achieve the highly responsive and selective imaging, as well as the effective inhibition of EBV-positive tumours in vitro and in vivo. The first research focused on the conjugation of a styrene pyridine fluorophore with two EBNA1-specific peptides, aiming to develop a dual-probe for the imaging and inhibition of EBV-positive tumour cells. Then we tried to introduce a Nuclear Localization Sequence into the EBNA1-specific peptide, and used an Intra-molecular Charge Transfer characterized fluorophore for the following second research, it showed an impressively responsive signal when the probe binds with EBNA1 both in vitro and in vivo, more importantly, only 4 μg probe can inhibit 92.8% of growth inhibition of an EBV-positive tumour. Along this line, our last research centred on the further improvement of the imaging by taking advantage of lanthanide.

Luminescent transition metal complexes have arisen as viable alternatives to organic dyes for sensory applications due to their notable advantages. This thesis aimed to synthesize different kinds of iridium(III) complexes as chemosensors and G-quadruplex probes for the detection of metal ions, small molecules, proteins and DNA to demonstrate the versatility of iridium(III) complex in luminescence sensing. Iridium(III) complex chemosensors were synthesized and developed for the detection of Cu2+ and cysteine. The iridium(III) complex plays the role of the "signaling unit", which transduces the analyte binding event into an optical (luminescent) signal and the "receptor unit" attached to the metal complex selectively binds the analyte of interest. Meanwhile, a series of iridium(III) complexes incorporating a variety of C^N and N^N donor ligands were synthesized and were shown to exhibit G-quadruplex-selective binding properties via emission titration, fluorescence resonance energy transfer melting and G-quadruplex fluorescent intercalator displacement experiments. These G-quadruplex-selective Ir(III) complexes were utilized as signal transducers to monitor the conformational changes of oligonucleotides in label-free oligonucleotide-based luminescent detection platforms for metal ion (Ag+), small molecules (cocaine), protein (insulin and AGR2) and gene deletion.