Metal ions are critical to a range of mammalian cell functions, including cell signalling, proliferation, differentiation and death. As such, changes in metal ion homeostasis can have a significant effect on cell health. Fluorescent chemosensors, in combination with specialised imaging technologies, provide a useful tool to study the role of metal ions in cellular processes as they enable detection in and around cells with spatial and temporal resolution. Commercially available and literature fluorescent probes, with core structures based on traditional fluorophores (e.g. fluorescein, coumarin and rhodamine) have been used extensively to study the relationship between cellular metal ion dynamics and disease. However, one drawback of current sensors is the lack of reusability. Photoswitchable molecules present as an alternative core unit for the development of fluorescent metal ion sensors, with the potential for reversible analyte binding. The photochromic spiropyran molecule is of particular interest in our research group, as the core structure can be readily functionalised for analyte selectivity, surface attachment, and tuning of the photochromic properties. Photo-controlled switching between the weakly fluorescent spiropyran (SP) and highly fluorescent merocyanine (MC) isomers occurs on irradiation with UV light or in the presence of the target metal ion, while visible light reverses the isomerisation (see Figure 1). This thesis describes the functionalisation of the spiropyran molecule in order to improve fluorescence intensity (Chapter 2), develop selective metal ion sensors for Mg²⁺ (Chapter 3) and investigate structure-metal ion selectivity relationships (Chapter 4). Chapter 1: Chapter 1 gives a broad introduction to fluorescent sensors and presents a literature review highlighting the use of photoswitchable spiropyrans as effective sensors for biologically relevant metal ions relevant such as calcium (Ca²⁺) and zinc (Zn²⁺), as well as the alkali metals sodium (Na⁺), potassium (K⁺) and lithium (Li⁺). Chapter 2: In Chapter 2, we took a unique approach to Ca²⁺ sensing with a rationally designed sensor which possess all the desirable characteristics (brightness, photostability and red fluorescence emission) of both the traditional and spiropyran-type sensor. This was accomplished by combining a traditional fluorophore (pyrene) with a photoswitch (spiropyran) with a Ca²⁺-selective ionophore. The rationally designed, pyrene-spiropyran hybrid Ca²⁺ sensor (Py-1) displays enhanced fluorescence intensity compared to a standalone spiropyran analogue. Importantly, Py-1 retains the characteristic red emission profile of the spiropyran, while fibre-based photostability studies show the sensor is stable after multiple cycles of photoswitching, without any sign of photodegradation. Such properties are of real advantage for cell-based sensing applications. An interesting observation is that, Py-1 presents with two excitation options; direct green excitation (532 nm) of the photoswitch for a red emission, and UV excitation (344 nm) of the component pyrene, which gives rise to distinct blue and red emissions. This proof-of-concept hybrid sensing system presents as a general approach to brighter spiropyran-based sensors. Chapter 3: Magnesium ions (Mg²⁺) play an important role in mammalian cell function; however, relatively little is known about the mechanisms of Mg²⁺ regulation in disease states. An advance in this field would come from the development of selective, reversible fluorescent sensors, capable of repeated measurements. To this end, Chapter 3 details the development of the first rationally designed, spiropyran-based fluorescent Mg²⁺ sensors. The most promising analogue, sensor 1, exhibits 2-fold fluorescence enhancement factor and 3-fold higher binding affinity for Mg²⁺ (Kd 6.0 µM) [d subscript] over Ca²⁺ (Kd 18.7 µM) [d subscript]. Incorporation of spiropyran-based sensors into optical fibre sensing platforms has been shown to yield significant signal-to-background changes with minimal sample volumes, a real advance in biological sensing that enables measurement on subcellular-scale samples. In order to demonstrate sensor compatibility within the light intense microenvironment of an optical fibre, photoswitching and photostability of 1 within a suspended core optical fibre (SCF) was subsequently explored, revealing reversible Mg²⁺ binding with improved photostability compared to the non- photoswitchable Rhodamine B fluorophore. The spiropyran-based sensors reported here highlight untapped opportunities for a new class of photoswitchable Mg²⁺ probe and present a first step in the development of a light-controlled, reversible dip-sensor for Mg²⁺. Chapter 4: In Chapter 4, the influence of multiple chelating groups on calcium ion (Ca²⁺) selectivity are explored with a series of spiropyran-based sensors incorporating both C8' and N1- indole metal ion binding domains. The sensors possess N1-indole functionalisation in the form of hydroxyethyl (SP-1), ethoxycarbonylbutyl (SP-2) and carboxybutyl (SP-3) groups, while all three sensors incorporate a 1-aza-15-crown-5 ionophore at the C8' position. Absorbance and fluorescence characterisation of metal ion binding revealed that in particular, sensor SP-3 gave excellent Ca²⁺-selectivity, improved dissociation constant (K𝖽 MC(SP-3)-Ca²⁺ = 22 µM) [d subscript] and quantum yield of fluorescence (Φ MC(SP-3)-Ca²⁺ = 0.37), compared to the other sensors. These results suggest the carboxybutyl N1-indole functionality of SP-3 may play a role in stabilizing Ca²⁺ in the 1-aza-15-crown-5 ionophore, promoting metal-induced isomerisation to the MC(SP-3)-Ca²⁺ complex and thus a bright, Ca²⁺-selective, red fluorescence signal. Appendix A: One sensor from the selectivity study in Chapter 4 (labelled ‘SHL’) was subsequently utilized with collaborators in a biological application, to study lithium ‘hot-spots’ in living colon cancer cells. The results show ion binding to the sensor intracellularly is dependent on exogenous Li⁺ transport into the cell, and repeated cycles of photoswitching gave reproducible changes in fluorescence, demonstrating the ability of the sensor to reversibly photoswitch in living cells. Furthermore, ‘hot-spots’ of Li⁺-SHL binding induced fluorescence were observed at the leading edges of migrating cells, which correlates with ion movement through aquaporin transmembrane channels. These results suggest that the aquaporin-1 (AQP1) ion channel could be a novel candidate for therapeutic interventions to manage metastasis in AQP1-dependent cancers. Appendix B: The themes of tailored selectivity and signal enhancement developed in this thesis are further explored in Appendix B, where we report on improved sensitivity in a nanoporous anodic alumina (NAA) sensing platform, targeted towards the detection of analytes in biological media. Sensing on an NAA platform utilises reflectometric interference spectroscopy (RIFS), where the amount of light-based signal is proportional to the degree of conformational change of the surface. This work describes a range of Au³⁺ selective binding molecules, in a series of combined surface attachment strategies in order to improve fundamental knowledge of surface-engineering in these nanoporous materials. The most sensitive functional molecules from sensing approaches (i) and (ii) were combined into a third sensing strategy whereby the nanoporous platforms are functionalised on both the top and inner surfaces concurrently. Engineering of the surface according to this approach resulted in an additive enhancement in sensitivity of up to 5-fold compared to previously reported systems.
Spiropyran, metal ion sensing, chemosensor, biosensor, photoswitch, fluorophore
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