Fluorescent sensor development through surface functionalisation

Abstract

The research undertaken in this thesis is concerned with the attachment of fluorophores to both optical fibres and nanodiamonds to provide sensing devices with application to biological sensing. Fluorescent sensors are commonly used to detect biological analytes through a change in fluorescence, a concept introduced in Chapter 1. However, the successful application of fluorescent sensors to biological systems is inhibited by a number of challenges, including delivery of the fluorophore to the measurement site, photobleaching, and fluorophore cell permeability. Chapter 1 introduces optical fibres and nanodiamonds as solid supports for attachment of fluorophores to overcome these challenges. Chapter 2 presents a new fibre functionalisation architecture for dual imaging and sensing within a single optical fibre probe. The fluorescent pH sensor, 5(6)carboxy-SNARF2, was reacted with the N-terminus of a silk-binding peptide to form an amide bond and provide the peptide SBP-SNARF. A fluorescence polarisation assay showed this peptide to bind with a Kd of 36 μM when added to aqueous silk fibroin protein. Fibre probes were prepared by dip-coating the fibre tip into the silk and peptide mixture, which provided a uniform silk coating (determined by scanning electron microscopy) that was stable to repeated washing in water and did not affect the imaging light emitted. This allows concurrent optical coherence tomography (OCT) imaging and pH sensing, which was demonstrated in an in vitro fertilisation (IVF) setting. Specifically, the probe detected a change in pH of 0.04 in cumulus-oocyte complexes after metabolic stimulation with CoCl2 to produce lactic acid, with the distance between the probe tip and the oocyte monitored by simultaneous OCT acquisitions. Notably, OCT imaging of an ovary revealed the presence/absence of an oocyte within an ovarian follicle, an important first step toward improving IVF patient outcomes by limiting the number of follicle punctures required to collect oocytes. Collectively, these results demonstrate the utility of the new fibre coating system to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems. Chapter 3 reports the detection of AlexaFluor-532 tagged streptavidin through its binding to D-biotin, which was reacted with the N-terminus of a silk-binding peptide to form the complex SBP-Biotin. This complex then adheres to a silk-coated fibre tip to provide an optical fibre probe for streptavidin, which is stable to repeated washing and long-term exposure to water. The probes were prepared by two methods that either distribute the SBP-Biotin throughout the silk fibroin matrix, or isolate the SBP-Biotin to the exterior of the silk layer. Only probes with surface bound SBP-Biotin successfully detected streptavidin, with a fluorescence-based detection limit of 15 mg/mL. Atomic absorption spectroscopy revealed that silk coating formation was inhibited by a lithium concentration of 200 ppm, however reduction to less than 20 ppm by dialysis re-enabled fibre coating. Together, Chapters 2 and 3 demonstrate that judicious preparation of optical fibre probes provides an opportunity for a wide array of fibre-based sensors using the silk fibroin and silk-binding peptide-based fibre functionalisation architecture described herein. Chapter 4 explores functionalisation of carboxylic acid laden nanodiamonds (NDs) through amide bond formation. This was first probed using an analytical high pressure liquid chromatography system to quantify the amount of 4-fluorobenzylamine attached to NDs. However, it was found that 4-fluorobenzylamine did not bind to NDs. Next, 1H nuclear magnetic resonance spectroscopy was employed to determine that the amount of diamino-PEG molecules attached to the NDs varied between 0 and 0.2 mmol/g. This indicated an inconsistent yield from the amidation reactions, which was investigated in more detail across ten separate amidation reactions between 4-cyanobenzylamine and NDs. However, none of these reactions resulted in an observable loading of 4-cyanobenzylamine, and it was concluded that amide bond formation is not an effective strategy for ND functionalisation. Chapter 5 presents four carbon-binding peptides (1-DLC, 2-CN, 3-DF and 4-GF) for ND functionalisation. Retention of each peptide on NDs was assessed by colourimetric assay and their presence confirmed through infrared spectroscopy and thermogravimetric analysis. 1-DLC was found to be the most well-retained peptide, at 87% and 35% on detonation and high-pressure high-temperature NDs respectively. This interaction was determined to be predominantly electrostatic, while 2-CN bound through non-polar interactions. Both 1-DLC and 2-CN coatings improved the colloidal stability of detonation NDs in aqueous solution, however neither peptide affected the colloidal stability of high-pressure, high-temperature NDs. This study provides a new, highly adaptable approach to functionalise NDs using carbon-binding peptides. Chapter 6 presents the synthesis of 4-aminobutanenitrile, an important synthetic intermediate for neurological disorder therapeutics, including Parkinson’s and Alzheimer’s diseases. Preparation of 4-aminobutanenitrile by Co(II) catalysed reduction, or a one-pot Staudinger reduction, of 4-azidobutanenitrile was low yielding. The reported Staudinger reaction was investigated through 1H-NMR analysis to reveal formation of the iminophosphorane intermediate after 22 h at rt, and increasing the temperature to 40 °C promoted hydrolysis of this intermediate to the desired amine. The Staudinger reduction was performed using pyridine solvent in place of THF, with water added 3 h after reaction initiation. These conditions gave rise to 4-aminobutanenitrile in 69% yield and 94% purity (calculated by qHNMR) without chromatography. However, 4-aminobutanenitrile was found to be unstable at rt, and cyclised to 2-aminopyrroline over several days. This was circumvented by preparation of the hydrochloride salt, which was shown to be stable at rt. Hence, 4-aminobutanitrile is best stored as the corresponding hydrochloride salt.