Chemical Modifications of Graphene for Biotechnology Applications
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Chemical modifications of graphene for biotechnology applications A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering 2016 Andrea Francesco Verre Schools of Materials 1 Index Figure captions………………………………………………………………………… 6 List of Abbreviations…...……………………………………………………………... 11 Abstract.………………………………………………………………………………...14 Declaration……………………………………………………………………………...15 Copyright statement…………………………………………………………………….16 Acknowledgments……………………………………………………………………...17 Chapter 1: Introduction… …………………………………………………………….. 20 1.1 Carbon nanomaterials………………………………….………………………….. 20 1.2 Graphene and related nanomaterials production………………………………….. 22 1.3 Characterization of Graphene and graphene related nanomaterials by microscopy…………………………………………………………..…………… .26 1.4 Characterization of Graphene and graphene related nanomaterials by spectroscopy…………………………………………………………..………… 28 1.5 Graphene Oxide Functionalization………………………………………………... 32 1.6 Lipid-Dip Pen Functionalization of Graphene… ………………………………….33 1.7 Graphene and related nanomaterials based substrates for stem cell culture and differentiation……………………………………………..… …………………….36 1.8 Conclusions………………………………………………………………………...47 References….…………………………………………………………………………..49 Chapter 2: Methodology………………………………………………………………. 62 2.1 GO synthesis and purification…………………………………………………….. 62 2.2 Biological functionalization of GO……………………………………………….. 62 2.3 Preparation of GO-based coatings… ………………………………………………64 2.4 Substrate Characterization… ………………………………………………………65 2.4.1 Atomic Force Microscopy… …………………………………………………… 65 2.4.2 Raman Spectroscopy……………………………………………………………. 66 2.4.3 X-Ray Photoelectron and Fourier Transform Infrared Spectroscopies………….,67 2.5 Biological Characterization of the Substrates…………………………………….. 69 2.5.1 Human Adipose Stem Cells Harvesting and Differentiation into dASC…...……69 2 2.5.2 MTS Assay………………………………………………………… 70 2.5.3 Live/Dead Assay…………………………………………………… 70 2.5.4 RNA extraction and gene expression studies…………………….... 71 2.5.5 Dorsal Root Ganglia (DRG) harvesting and immunohistochemistry 73 2.6 CVD graphene array fabrication……………………………………….74 2.6.1 CVD graphene transfers on silicon dioxide substrates……………....74 2.6.2 Photolithography on CVD graphene substrates……………………...74 2.7 Dip Pen Nanolithogrpahy Patterning and Membrane Protein Insertion.78 References………………………………………………………………….81 Introduction to Chapter 3… ……………………………………………….85 Chapter 3: Biochemical functionalization of graphene oxide for directing stem cell differentiation……………………………………………………………... 86 Abstract…………………………………………………………………… 86 3.1 Introduction.….………………………………………………………...87 3.2 Materials and methods………………………………………………... 88 3.2.1 Graphene based materials synthesis, substrates preparation and characterization… ……………………………………………………………………………...88 3.2.2 Human Adipose stem cells harvesting and culture…………………..89 3.2.3 Cell Proliferation and Live/Dead assays…………………………… 89 3.2.4 Quantitative real-time polymerase chain reaction (qRT-PCR)...……90 3.2.5 Rat DRG neurons harvesting, culture and immunohistochemistry… 90 3.2.6 Statistical analysis………………………………………………….. 91 3.3 Results and Discussion……………………………………………….. 91 3.3.1 Substrates Characterization… ………………………………………91 3.3.2 Effect on IKVAV functionalization on improving neuronal attachment and neurite outgrowth…………………………………………………………………. 93 3.3.3 Proliferation of ASCs on graphene substrates… ……………………94 3.3.4 Gene Expression Studies… …………………………………………95 3.4 Conclusions… ………………………………………………………...98 References……………………………………………………………….. 99 Supporting information………………………………………………….. 103 Introduction to Chapter 4…………………………………………………107 3 Chapter 4: Reduced GO substrates increase gene expression of neurotrophins and filament proteins by Schwann-like differentiated adipose stem cells… …. 108 Abstract……………………………………………………………………………….109 4.1 Introduction....…………………………………………………………………….109 4.2 Experimental… …………………………………………………………………..110 4.2.1 Graphene based materials synthesis, substrates preparation and characterization… ………………………………………………………………………………………..110 4.2.2 Human Adipose stem cells harvesting and differentiation……………………..110 4.2.3 Cell Proliferation and Live/Dead assays……………………………………….110 4.2.4 Quantitative real-time polymerase chain reaction (qRT-PCR)………………...110 4.2.5 Statistical analysis……………………………………………………………...111 4.3 Results and discussion…………………………………………………………...111 4.3.1 Substrates Characterization…………………………………………………….111 4.3.2 Proliferation of dASCs on graphene substrates………………………………...112 4.3.3 Gene Expression Studies……………………………………………………….112 4.4 Conclusions………………………………………………………………………115 References…………………………………………………………………………... 116 Introduction to Chapter 5… …………………………………………………………120 Chapter 5: Selective Immobilization of Membrane Proteins carried in Nanodiscs on Functionalized Graphene Oxide… ………………………………………………….121 Abstract…………………………………………………………………………….. 121 5.1 Introduction……………………………………………………………………...122 5.2 Methodology… …………………………………………………………………123 5.2.1 Preparation and functionalization of GO flakes……………………………….123 5.2.2 XPS and AFM Characterization………………………………………….........123 5.3 Results and Discussion……...…………………………………………………...123 5.3.1 XPS characterization of the carboxylation of graphene oxide………………...123 5.3.2 AFM studies of the protein binding on functionalized GO……………………126 5.4 Conclusions……………………………………………………………………...128 References…………………………………………………………………………...130 Introduction to Chapter 6…………………………………………………………....133 4 Chapter 6: Tail-anchoring proteins insertion into phospholipid biomimetic membranes on graphene………………………………………………………………………….134 Abstract…………………………………………………………………134 6.1 Introduction…………………………………………………………135 6.2 Materials and methods……………………………………………...136 6.2.1 Graphene-Array Fabrication……………………………………...137 6.2.2 Lipid Deposition by L-DPN……………………………………...137 6.2.3 Protein Insertion and Immunostaining…………………………...137 6.3 Results and Discussion……………………………………………. 138 6.4 Conclusions………………………………………………………...141 References……………………………………………………………...143 Chapter 7: Conclusions and future works……………………………...146 Intoduction to Appendix………………………………………………..149 Appendix 1: Graphene oxide selectively targets cancer stem cells, across multiple tumor types: Implications for non-toxic cancer treatment, via “differentiation-based nano- therapy”. Appendix 2: Molecular Dynamics Simulations of Biomimetic Phospholipid Membrane Organization from Dip Pen Nanolithography 5 Figure captions Chapter 1 Figure 1.1: a) Optical microscopy image of micromechanical exfoliated graphene flakes ; b) and c) AFM topography image of graphene flakes, d) SEM image of graphene field effect transistor, e) Schematic representation of graphene FET device. Image taken from [7] Figure 1.2: (a, b) SEM image of CVD-graphene films , (c, d) CVD-graphene films transferred on Si/SiO2 and glass substrates respectively. Image taken from reference [8] Figure 1.3: (a-b) SEM of sieded graphite and from the sediment after centrifugation respectively; c-g) bright field TEM image of exfoliated graphene obtained after the liquid exfoliation process.; h) Histogram of visual observation of flakes as function of number of layers. Image taken from [17] Figure 1.4: Structural models of GO proposed by Lerf-Klinowski. Image taken from [25] Figure 1.5: AFM topography image of reduced graphene oxide flakes. On the right hand side height profile measurement of the flakes on the spot highlighted from the black lines. Image taken from [30] Figure 1.6: Optical microscopy image of micromechanically exfoliated graphene flakes on arbitrary substrates. Image taken from [31] Figure 1.7: A) GO and rGO films; B) GO and rGO dispersions; C) Optical microscopy image of GO flakes; D)Optical microsocpy image of rGO flakes. Image taken from [34] Figure 1.8: AFM topography image of graphene flakes with height profile measurement. Image taken from [31] Figure 1.9: Raman Spectrum of mechanically exfoliated graphene flakes (a), Variation of 2D peak as function of different number of layers in micromechanical exfoliated graphene flake. Image taken from [33] Figure 1.10: Raman spectrum of functionalized graphene sheet (FGS), graphene oxide (GO) and graphite . Taken from reference [36] Figure 1.11 : XPS C1s deconvolution in 6 components. XPS C1s spectrum of GO (a), Graphite oxide (b), diamond (c), HOPG (d), sodium terephthalate (e), sodium dodecanedioate (f), hydroqunone (g). Taken from [39]. 6 Figure 1.12: Functionalization of GO by exploiting carboxylic functional groups. Image taken from [25] Figure 1.13: Schematics of DPN patterning. Images taken from [72] Figure 1.14: a) Relative expression of pluripotent stem cells markers; b) Fluorescence microscopy image after immunohistochemical analysis of Sox2 and Oct4. Image taken from [89] Figure 1.15: Immunostaining for desmin and osteocalcin (OCN) in green and nuclear marker DAPI In blue to evaluate the osteogenic differentiation of hMSCs grown on PET , PDMS and Si/SiO2 substrates with and without CVD graphene. Image taken from [101] Figure 1.16: Evaluation of osteogenic differentiation on PDMS, CVD graphene (G) and graphene oxide (GO) substrates by Alzarin Red staining. Image taken from [103]. Figure 1.17: Immunohistochemistry on hippocampal neurons grown on functionalized graphene oxide substrates. Image taken from [107] Figure 1.18: Neurite lenght outgrowth, number of branches, number of neurites and cell body area measurements of hippocampal neurons on GO, GO-MPC, and GO-DMAEMA substrates. Image taken from