Development of Functionalised Carbon Based Substrates for Neuronal Cell Culture and Production of Carbon Nanoparticles for Bioimaging Applications Andrew Hopper Thesis submitted to the University of Sheffield for the degree of Doctor of Philosophy Department of Materials Science and Engineering September 2014 0 Table of Contents Table of Contents ..................................................................................................................... 0 Abstract ..................................................................................................................................... 7 Acknowledgements .................................................................................................................. 9 Publications ............................................................................................................................ 10 Abbreviations ......................................................................................................................... 11 Chapter One: Literature Review .......................................................................................... 15 1. Introduction ...................................................................................................................... 15 1.1 Diamond Functionalisation Routes ................................................................................ 19 1.1.1 Non-Covalent Functionalisation .............................................................................. 20 1.1.2 Covalent Functionalisation ...................................................................................... 22 1.2 Nanodiamond Films ....................................................................................................... 25 1.3 Nanodiamond Films as Biocoatings ............................................................................... 28 1.3.1 Surfaces Which Influence the Proliferation or Differentiation of Cells .................. 28 1.3.2 Biosensors ................................................................................................................ 30 1.3.3 Retinal and Neural Prostheses ................................................................................. 32 1.4 Nanodiamond Particles .................................................................................................. 34 1.4.1 Nanodiamond Functionalisation Routes .................................................................. 38 1.4.2 Biocompatibility of Nanodiamond .......................................................................... 39 1.4.3 Applications of Nanodiamond Particles .................................................................. 40 1.4.3.1 Nanocomposites ................................................................................................ 40 1.4.3.2 Protein Replicas ................................................................................................ 41 1.4.3.3 Biolabelling ....................................................................................................... 42 1.4.3.4 Drug Delivery ................................................................................................... 44 1.4.3.5 Gene Delivery ................................................................................................... 47 1.4.3.6 Intracellular Antioxidant System ...................................................................... 48 1.4.3.7 Influence upon cell attachment ......................................................................... 48 1.5 Diamond-Like Carbon (DLC) as Biocoatings ............................................................... 49 1.5.1 Different Forms of DLC .......................................................................................... 49 1.5.1.1 Polymeric (Highly Hydrogenated) Amorphous Carbon (a-C:H) ...................... 50 1 1.5.1.2 Soft [(Hydrogenated a-C:H) and (Non-Hydrogenated a-C)] Amorphous Carbon ....................................................................................................................................... 50 1.5.1.3 Hard (Hydrogenated a-C:H) Amorphous Carbon and Tetrahedral (ta-C) Carbon ....................................................................................................................................... 50 1.5.2 DLC Coating Methods............................................................................................. 51 1.5.3 Properties of DLC .................................................................................................... 53 1.5.4 DLC Applications and its use as a Biocoating ........................................................ 54 1.5.4.1 Orthodontic Wires ............................................................................................. 55 1.5.4.2 Vascular Stents.................................................................................................. 56 1.5.4.3 Artificial Joint Implants .................................................................................... 56 1.5.4.4 Biosensors and Bioarrays .................................................................................. 58 1.5.4.5 Cell Growth Patterning ..................................................................................... 59 1.6 Carbon Nanoparticles ..................................................................................................... 62 1.6.1 Applications of Carbon Nanoparticles .................................................................... 64 1.6.1.1 In Vitro Bioimaging .......................................................................................... 65 1.6.1.2 In Vivo Biolabelling .......................................................................................... 67 1.7 Conclusion ...................................................................................................................... 68 1.8 Thesis Aims and Objectives ........................................................................................... 69 Chapter Two: Materials and Methods ................................................................................. 71 2.1 Diamond-Like Carbon (DLC) Study Methods............................................................... 71 2.1.1 Preparation of Diamond-Like Carbon Substrates - Pulsed Laser Deposition of DLC .......................................................................................................................................... 71 2.1.2 SEM Electron Beam Deposition of DLC ................................................................ 71 2.1.3 Synthesis of Trifluoroacetic acid protected-10-Aminodec-1-ene (TFAAD) .......... 72 2.1.4 Determination of optimal UV radiation exposure for TFAAD grafting ................. 72 2.1.5 Preparation of 10-amino-dec-1-ene Functionalised DLC ....................................... 73 2.1.6 Preparation of 2-(10-Undecen-1-yl)-1,3-dioxolane ................................................. 73 2.1.7 Preparation of 10-undecenal Functionalised DLC .................................................. 74 2.1.8 NG108-15 Cell Culture Medium ............................................................................. 74 2.1.9 NG108-15 Neuroblastoma Cell Culture .................................................................. 74 2.1.10 NG108-15 Cell Passaging ..................................................................................... 75 2.1.11 Resurrecting Cells.................................................................................................. 76 2.1.12 Primary Schwann Cell Isolation and Culture ........................................................ 76 2.1.13 Fluorescence Cell Staining .................................................................................... 77 2 2.1.14 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Viability Assay ................................................................................................................. 78 2.1.15 Contact Angle Measurements ................................................................................ 79 2.1.16 X-Ray Photoelectron Spectroscopy (XPS) ............................................................ 80 2.1.17 Scanning Electron Microscopy (SEM) .................................................................. 81 2.2 Nanodiamond (ND) Study Methods .............................................................................. 81 2.2.l Fenton Treated Nanodiamond Synthesis .................................................................. 81 2.2.2 Non-Fenton Treated Nanodiamond Synthesis ......................................................... 82 2.2.3 Synthesis of Trifluoroacetic acid protected-10-Aminodec-1-ene (TFAAD) .......... 82 2.2.4 Preparation of 10-Amino dec-1-ene functionalised Nanodiamond ......................... 83 2.2.5 Plasma Polymerisation of Acrylic Acid .................................................................. 83 2.2.6 Production of AAND Substrates ............................................................................. 84 2.2.7 NG108-15 Cell Culture Medium ............................................................................. 85 2.2.8 NG108-15 Neuroblastoma Cell Culture .................................................................. 85 2.2.9 Primary Schwann Cell Isolation and Culture .......................................................... 85 2.2.10 NG108-15 Neuroblastoma and Primary Schwann cell Co-Culture....................... 86 2.2.11 Primary