Nanofabrication towards biophotonics

This thesis explores methods for fabrication of nanohole arrays, and their integration into a benchtop system for use as sensors or anti-counterfeit labels. Chapter 1 gives an introduction to plasmonics and more specifically nanohole arrays and how they have potential as label free sensors compared to the current biosensors on the market. Various fabrication methods are explored, including Focused Ion Beam, Electron Beam Lithography, Nanoimprint lithography, Template stripping and Phase Shift Lithography. Focused Ion Beam was chosen to fabricate the nanohole arrays due to its suitability for rapid prototyping and it’s relatively low cost. In chapter 2 the fabrication of nanohole arrays using FIB is described, and the samples characterised. The fabricated nanohole arrays are tested as bulk refractive index sensors, before a bioassay using whole molecule human IgG antibodies and antigen is developed and performed on the senor. In chapter 3 the fabricated sensors are integrated into a custom built system, capable of real time, multiplexed detection of biomolecules. Here, scFv antibodies of two biomolecules relevant to the detection of pancreatic cancer (C1q and C3) are attached to the nanohole arrays, and detection of their complementary proteins is demonstrated both in buffer (10 nM detection of C1q Ag) and human serum. Chapter 4 explores arrays of anisotropic (elliptical) nanoholes and shows how the shape anisotropy induces polarisation sensitive transmission spectra, in both simulations and fabricated arrays. The potential use of such samples as visible and NIR tag for anti-counterfeiting applications is demonstrated. Finally, chapter 5 gives a summary of the work completed and discusses potential future work in this area.

Effects of synbiotics on cancer risk biomarkers

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world and second most common (behind lung cancer) in developed countries. In recent years there has been much interest in the potential use of prebiotics, probiotics and synbiotics in the prevention and treatment of CRC. We have previously shown that synbiotic consumption in Azoxymethane treated rats modulates the immune system, influences the genotoxic potential of caecal contents and reduces the number of colonic tumours compared to control rats who did not receive the synbiotic. The aim of the current study was to identify biomarkers suitable for use as cancer risk markers and as intervention markers. A second aim was to determine the influence of synbiotic consumption on cancer risk biomarkers such as in vivo colonic mucosal proliferation and genotoxic damage along with examining the genotoxic, cytotoxic and tumour promoting potential of faecal water (FW). Synbiotic consumption altered the composition of the gastrointestinal flora and reduced in vivo genotoxic damage and the genotoxic potential of FW in cancer and polyp subjects. Synbiotic consumption also reduced the proliferative activity in the colonic mucosa in polyp subjects. In both cancer and polyp subjects gene expression in the colonic mucosa was modulated in synbiotic consuming subjects. In this and other studies the activity of natural killer cells, the level of PGE2 in FW, IL-12 production by PBMCs, genotoxic damage in the colonic mucosa and the tumour promoting activities of FW have been identified as possible biomarkers of cancer risk. Future large scale studies investigating these parameters in healthy and diseased individuals are needed to confirm the suitability of these markers in assessing cancer risk and the role of synbiotics in modulating them.

Development and optimisation of photonic crystal based nanosensors

This thesis involved the development of two Biosensors and their associated assays for the detection of diseases, namely IBR and BVD for veterinary use and C1q protein as a biomarker to pancreatic cancer for medical application, using Surface Plasmon Resonance (SPR) and nanoplasmonics. SPR techniques have been used by a number of groups, both in research [1-3] and commercially [4, 5] , as a diagnostic tool for the detection of various biomolecules, especially antibodies [6-8]. The biosensor market is an ever expanding field, with new technology and new companies rapidly emerging on the market, for both human [8] and veterinary applications [9, 10]. In Chapter 2, we discuss the development of a simultaneous IBR and BVD virus assay for the detection of antibodies in bovine serum on an SPR-2 platform. Pancreatic cancer is the most lethal cancer by organ site, partially due to the lack of a reliable molecular signature for diagnostic testing. C1q protein has been recently proposed as a biomarker within a panel for the detection of pancreatic cancer. The third chapter discusses the fabrication, assays and characterisation of nanoplasmonic arrays. We will talk about developing C1q scFv antibody assays, clone screening of the antibodies and subsequently moving the assays onto the nanoplasmonic array platform for static assays, as well as a custom hybrid benchtop system as a diagnostic method for the detection of pancreatic cancer. Finally, in chapter 4, we move on to Guided Mode Resonance (GMR) sensors, as a low-cost option for potential use in Point-of Care diagnostics. C1q and BVD assays used in the prior formats are transferred to this platform, to ascertain its usability as a cost effective, reliable sensor for diagnostic testing. We discuss the fabrication, characterisation and assay development, as well as their use in the benchtop hybrid system.

Insulin-like growth factor receptor activity in cancer biology & therapy responses

The Insulin-like Growth Factor 1 Receptor (IGF-1R) has an essential function in normal cell growth and in cancer progression. However, anti-IGF-1R therapies have mostly been withdrawn from clinical trials owing to a lack of efficacy and predictive biomarkers. IGF-1R activity and signalling in cancer cells is regulated by its C-terminal tail, and in particular, by a motif that encompasses tyrosines 1250 and 1251 flanked by serines 1248 and 1252 (1248- SFYYS-1252). Mutation of Y1250/1251 greatly reduces IGF-1-promoted cell migration, interaction with the scaffolding protein RACK1 in the context Integrin signalling, and IGF- 1R kinase activity. Here we investigated the phosphorylation of the SFYYS motif and characterise the conditions under which this motif may be phosphorylated under. As phosphorylated residues, the SFYYS motif may also serve to recruit interacting proteins to the IGF-1R. To this end we identified a novel IGF-1R interacting partner which requires phosphorylated residues in the SFYYS motif to interact with the IGF-1R. This interaction was found to be IGF-1-dependent, and required the scaffold protein RACK1. The interaction of this binding protein with the IGF-1R likely functions to promote maximal phosphorylation of Shc and ERK in IGF-1-stimulated cell migration, and may be important for IGF-1 signalling in cancer cells. Lastly, we have investigated possible kinases that may confer resistance or sensitivity to the IGF-1R kinase inhibitor BMS-754807. In this screen we identified ATR as a mediator of resistance and showed that suppression or chemical inhibition of ATR synergised with BMS-754807 to reduce colony formation. This work has contributes to our understanding of IGF-1R kinase regulation and signalling and suggests that administration of anti-IGF-1R drugs with ATR inhibitors may have therapeutic benefit.