Spiropyran-based fluorescence sensors are an ideal target for intracellular metal ion sensing, due to their biocompatibility, red emission frequency and photo-controlled reversible analyte binding for continuous signal monitoring. However, increasing the brightness of spiropyran-based sensors would extend their sensing capability for live-cell imaging. In this work we look to enhance the fluorescence of spiropyran-based sensors, by incorporating an additional fluorophore into the sensor design. We report a 5-membered monoazacrown bearing spiropyran with metal ion specificity, modified to incorporate the pyrene fluorophore. The effect of N-indole pyrene modification on the behavior of the spiropyran molecule is explored, with absorbance and fluorescence emission characterization. This first generation sensor provides an insight into fluorescence-enhancement of spiropyran molecules.
Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H<sub>2</sub>O<sub>2</sub> using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H<sub>2</sub>O<sub>2</sub> to non-invasively detect H<sub>2</sub>O<sub>2</sub> near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H<sub>2</sub>O<sub>2</sub> and pH that is functional at biologically concentrations of H<sub>2</sub>O<sub>2</sub> and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.
We designed and fabricated a smart and stimuli responsive membrane to cater on demand molecular transporting applications. A novel photoswitchable peptide (PSP) was synthesized and attached inside nanoporous anodic alumina membranes (NAAMs) pores. The PSP specifically switched between its cis and trans photostationary states on exposure to 364 nm and 440 nm wavelength lights respectively, which not only provided the ability to control its pore diameter but also the surface chemistry. The switchable molecular transport properties of the PSP-NAAMs have been shown as a function of the light exposure. Most importantly, the molecular transport across PSP-NAAMs could be repeatedly switched between on and off state, which is highly significant for on-demand triggered drug release systems.
The photoisomerisation of azobenzenes between trans and cis results in well-defined changes in geometry and a considerable change of polarity. Thus, incorporating an azobenezene into a bioactive compound provides an opportunity to control biological activity, with ideally one configuration being active and the other inactive. This can allow the role of a specific biomolecule to be probed in its native environment by controlling activity both spatially and temporally using light. Incorporating such a photoswitchable moiety into the structure of a known GRK2 inhibitor can generate photoswitchable inhibitors, which can be used to reversibly regulate the activity of GRK2, and hence GPRCs.
The production of reactive oxygen species (ROS) is known to affect the developmental competence of embryos. Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) an important reactive oxygen species, is also known to causes DNA damage and defective sperm function. Current techniques require incubating a developing embryo with an organic fluorophore which is potentially hazardous for the embryo. What we need is a localised ROS sensor which does not require fluorophores in solution and hence will allow continuous monitoring of H<sub>2</sub>O<sub>2</sub> production without adversely affect the development of the embryo. Here we report studies on such a fibre-based sensor for the detection of H<sub>2</sub>O<sub>2</sub> that uses a surface-bound aryl boronate fluorophore carboxyperoxyfluor-1(CPF1). Optical fibres present a unique platform due to desirable characteristics as dip sensors in biological solutions. Attempts to functionalise the fibre tips using polyelectrolyte layers and (3-aminopropyl)triethoxysilane (APTES) coatings resulted in a limited signal and poor fluorescent response to H<sub>2</sub>O<sub>2</sub> due to a low tip surface density of the fluorophore. To increase the surface density, CPF1 was integrated into a polymer matrix formed on the fibre tip by a UV-catalysed polymerisation process of acrylamide onto a methacrylate silane layer. The polyacrylamide containing CPF1 gave a much higher surface density than previous surface attachment methods and the sensor was found to effectively detect H<sub>2</sub>O<sub>2</sub>. Using this method, biologically relevant concentrations of H<sub>2</sub>O<sub>2</sub> were detected, enabling remote sensing studies into ROS releases from embryos throughout early development.
The results of functionalizing exposed-core optical fiber with multiligand binding sensor molecules for ion detection is presented. We show that the capacity of the sensor molecules to bind multiple ligands is negated when the sensor molecules are covalently bound, making the method ineffective where multiligand binding fluoroionophores are needed. An alternate functionalization method using thin film polymer doped with multiligand binding fluoroionophores is shown, demonstrating the ability for ion detection in a case where multiligand binding is needed. This one step functionalizing process for optical fiber sensing applications does not require surface attachment functional groups and has the potential to be inline with fiber drawing so that long lengths of functionalized fiber can be fabricated.
The first nanoliter-scale regenerable ion sensor based on microstructured optical fiber (MOF) is reported. The air holes of the MOF are functionalized with a monoazacrown bearing spiropyran to give a switchable sensor that detects lithium ions down to 100 nM in nanoliter-scale volumes. Ion binding is turned on and off on upon irradiation with light, with the sensor being unaffected by multiple rounds of photoswitching. Unbound ions are flushed from the fiber in the ‘off’ state to allow the sensor to be reused. The integration of an ionophore into the sensor paves the way for the development of highly specific light-based sensing platforms that are readily adaptable to sense a particular ion simply by altering the ionophore design.