Hydrogen peroxide sensing for reproductive health

The research presented in this thesis details the synthesis, surface functionalisation and photochemical studies of fluorescent probes for the detection of hydrogen peroxide (H₂O₂) in reproductive health. Chapter 1: H₂O₂ is an important reactive oxygen species (ROS) that is detrimental to the health of spermatozoa and embryos. Fluorescent probes are commonly used for the detection of ROS and here examples with different mechanisms of detection are examined; such as turn-on probes, turn-off probes, Förster resonance energy transfer (FRET)-based and photoinduced electron transfer (PET)-based probes. Specific reference is given to the aryl boronate and benzil classes of probe, which show good selectivity for H₂O₂ over other ROS. The attachment of the fluorescent probe to an optical fibre as a non-invasive sensing platform is discussed. This then allows sensing in a sensitive biological environment, such as an embryo, without exposure to the probe in solution. Fibre tip sensors and microstructured optical fibre-based sensors are discussed for use in such biological environments. Finally, a summary is provided detailing the objectives of this thesis and the chapters in which these are addressed. Chapter 2: Three aryl boronate probes [peroxyfluor-1 (PF1), carboxy peroxyfluor-1 (CPF1) and a novel probe 2(2-ethoxyethoxy)ethoxy peroxyfluor-1 (EEPF1)] were synthesised for use in the detection of H₂O₂ in human spermatozoa. The activity and selectivity of these probes was then compared to three commonly used commercial probes, 2’,7’-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE) and MitoSOX red (MSR). PF1 and EEPF1 were found to be effective at detecting H₂O₂ and peroxynitrite (ONOO⁻) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. Flow cytometry was used to demonstrate that EEPF1 is more effective at detecting ROS in spermatozoa compared to DCFH, DHE and MSR. Furthermore, EEPF1 distinguished poorly motile sperm from motile sperm as revealed by an enhanced production of ROS. Chapter 3: A fibre-tip based probe constructed by encapsulating CPF1-NHS in a polyacrylamide matrix is reported for the detection of H₂O₂. This non-invasive platform avoids the need to introduce an organic fluorophore into a sensitive cell such as an embryo as discussed above. A number of derivatives of PF1 were investigated, with carboxylated fluorophore CPF1 proving to be the easiest to synthesise and characterise. CPF1 was functionalised to glass slides using layer-by-layer deposition of polyelectrolytes. This functionalised surface showed a fluorescent response to H₂O₂ comparable to solution-based measurements. Three surface functionalisation methods were then investigated for attachment to an optical fibre tip, specifically polyelectrolyte deposition, silane monolayer formation, and light-catalysed polymerisation of acrylamide. The most effective method of functionalisation was found to be light-catalysed formation of a polyacrylamide matrix with the CPF1 embedded. These polyacrylamide fibre tip probes were then guided into microdroplets of bovine in vitro fertilisation (IVF) media using a micromanipulator. This was visualised under an optical microscope to detect the controlled release of H₂O₂. This fibre probe is thus compatible with imaging techniques used in IVF research laboratories. Chapter 4: This chapter presents the development of a single optical fibre tip probe capable of detecting both the concentration of H₂O₂ and the pH of the associated solution. The sensor was constructed by embedding two fluorophores [CPF1 and seminaphtharhodafluor-2 (SNARF2) for H₂O₂ and pH detection respectively] on the tip of an optical fibre using the previous developed polyacrylamide matrix methodology. The functionalised fibre probes reproducibly sensed pH with a resolution of 0.1 pH units. The probe also accurately detected H₂O₂ over a biologically significant concentration range, of 50-100 μM. This study revealed the importance of simultaneous detection of H₂O₂ and pH, where changes in pH were shown to affect the fluorescent response of CPF1. This new fibre probe offers potential for noninvasive detection of pH and H₂O₂ in biological environments using a single optical fibre. Chapter 5: Two new cell-permeable boron-dipyrromethene (BODIPY) based fluorescent probes for the detection of H₂O₂ were designed and synthesised. The aryl boronate peroxyBODIPY-1 (PB1) gave rise to a decrease in fluorescence on reaction with H₂O₂, while the fluorescence of the benzil-based nitrobenzoylBODIPY (NbzB) probe increased on reaction with H₂O₂. The benzil probe NbzB exhibited a high degree of selectivity for H₂O₂ over other ROS. The aryl boronate PB1 showed a greater change in fluorescence on reaction with H₂O₂ compared to NbzB, and PB1 also detected H₂O₂ in bovine oocytes under oxidative stress. These results suggest that aryl boronates (i.e. PB1) and benzils (i.e. NbzB) have use in biological environments requiring higher sensitivity or selectivity to H₂O₂. Chapter 6: The research discussed here extends the solution-based and fibre tip experiments to the detection of H₂O₂ in biological environments. Detection of H₂O₂ within cells is often frustrated by autofluorescence in the green emission region. Contrastingly, the red emission region in biological systems shows a lower autofluorescence background signal. Therefore a redemitting fluorescent probe for H₂O₂, naphthoperoxyfluor-1 (NPF1), was synthesised. However, when incubated with H₂O₂ in cuvette, NPF1 showed a greater than 20-fold reduced fluorescent response to H₂O₂ compared with CPF1. This poor sensitivity suggests that NPF1 should not be used for the detection of H₂O₂, but rather fluorophores with a greater fluorescent response should be utilised (e.g. CPF1). A reversible optical fibre-based sensor for H₂O₂ was then explored by attaching a reversible fluorescent probe for ROS (nicotinamide coumarin redox sensor 3, NCR3) to an optical fibre tip. The sensor was constructed using light-catalysed polymerisation to give a polymer matrix on the tip containing NCR3. This allowed the fibre tip to be reversibly oxidised by H₂O₂ and reduced by NaCNBH₃. The sensor exhibited good reversibility over at least seven cycles of oxidation and reduction, with consistent fluorescent ratios of its maxima at 500 and 635 nm. However, its fluorescence intensity decreased over time, suggesting that NCR3 leached from the polymer into the buffer solution. This nevertheless represents the first example of a reversible fibre sensor for ROS and is as such an important first step towards a reusable optical fibre probe for H₂O₂.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Physical Sciences, 2016