SURFACE FUNCTIONALIZED SUBSTRATES AND THEIR INTERACTIONS WITH BIOMOLECULES AND CELLS CEN LIAN NATIONAL UNIVERSITY OF SINGAPORE 2004 SURFACE FUNCTIONALIZED SUBSTRATES AND THEIR INTERACTIONS WITH BIOMOLECULES AND CELLS CEN LIAN (B.Eng., ECUST) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENT First of all, I would like to express my cordial gratitude to my supervisors, Prof. K. G. Neoh and Prof. E. T. Kang, for their heartfelt guidance, invaluable suggestions and profound discussion throughout this work, for sharing with me their enthusiasm and active research interests, which are the constant source for inspiration accompanying me throughout this project. The valued knowledge I learnt from them on how to research work and how to enjoy it paves my way for this study and for my life long study. I would like to thank Dr. Li Sheng for his help in XPS and SEM operation, training and sample analysis. I am also grateful to all my colleagues for their kind help and encouragement, especially to Dr. Ling Qidan, Dr. Lu Zhiyun, Dr. Zhang Yan, Dr. Yang Guanghui, Mr. Ying Lei, Mr. Yu Weihong, Mr. Wang Wencai, Mr. Zhao Luping and Mdm Liu Xin for sharing with me their invaluable experience on the research field. In addition, I also appreciate the assistance and cooperation from lab technologists and officers of Department of Chemical and Environmental Engineering. Finally, but not least, I would give my special thanks to my parents for their continuous love, support, and encouragement. i TABLE OF CONTENTS ACKNOWLEDGEMENT i TABLE OF CONTENTS ii SUMMARY v NOMENCLATURE vii LIST OF FIGURES viii LIST OF TABLES xv Chapter INTRODUCTION Chapter LITERATURE SURVEY 2.1 Surface Graft Copolymerization 2.2 Further Functionalization of Grafted Surface 13 2.2.1 Immobilization of Biomolecules 13 2.2.2 Biofilm Inhibition 23 2.3 Substrates for Enzyme Immobilization, Cell Culture and Biofilm Prevention 30 2.3.1 Electroconductive Polypyrrole 30 2.3.2 Polymeric Nonporous Film and Porous Materials 33 Chapter SURFACE FUNCTIONALIZATION OF POLYPYRROLE FILM WITH GLUCOSE OXIDASE AND VIOLOGEN 36 3.1 Introduction 37 3.2 Experimental 40 3.3 Results and Discussion 47 3.4 Conclusion 66 Chapter SURFACE FUNCTIONALIZATION OF ELECTRICALLY CONDUCTIVE POLYPYRROLE FILM WITH HYALURONIC ACID 67 ii 4.1 Introduction 68 4.2 Experimental 70 4.3 Results and Discussion 76 4.4 Conclusion 92 Chapter ASSESSMENT OF BIOLOGICAL RESPONSES OF HYALURONIC ACID FUNCTIONALIZED POLYPYRROLE FILM 93 5.1 Interactions of Hyaluronic Acid Functionalized Polypyrrole Film with PC12 Cells 94 5.1.1 Introduction 94 5.1.2 Experimental 96 5.1.3 Results and Discussion 99 5.1.4 Conclusion 107 5.2 Antibacterial Assay of Hyaluronic Acid Functionalized Electroactive Polymer 108 5.2.1 Introduction 108 5.2.2 Experimental 108 5.2.3 Results and Discussion 112 5.2.4 Conclusion 117 Chapter SURFACE FUNCTIONALIZATION OF NONPOROUS AND POROUS SUBSTRATES TO ACHIEVE ANTIBACTERIAL PROPERTIES 118 6.1 Surface Functionalization Technique for Conferring Antibacterial Properties to Polymeric Film Surface 119 6.1.1 Introduction 119 6.1.2 Experimental 121 6.1.3 Results and Discussion 127 6.1.4 Conclusion 147 6.2 Surface Functionalization Technique for Conferring 148 iii Antibacterial Properties to Porous Material Surfaces 6.2.1 Introduction 148 6.2.2 Experimental 149 6.2.3 Results and Discussion 153 6.2.4 Conclusion 179 Chapter CONCLUSION 180 Chapter RECOMMENDATIONS FOR FURTHER STUDY 184 REFERENCES 188 LIST OF PUBLICATION 207 iv SUMMARY Polymeric substrates can readily undergo surface modification via graft polymerization with monomers bearing functional groups for further coupling reactions. The grafted polymeric substrates still retain their intrinsic bulk properties with the improvements in their surface characteristics for specific applications. In this thesis, different approaches of surface grafting were developed depending on the system of interest. Further functionalization of the grafted surfaces was carried out either by biomolecular immobilization or post derivatization. The main focus of this thesis is on the subsequent biological assays of such material-based systems with desired functionalities. Surface modification techniques were developed for the functionalization of electrically conductive polypyrrole (PPY) film with glucose oxidase (GOD) and an electron mediator: viologen moieties. Acrylic acid (AAc) graft copolymerized PPY film was used to covalently immobilize GOD through the formation of amide linkages. Parallel linkage of viologen moieties on the GOD immobilized PPY film was facilitated by the coupling reaction of 4,4’-bipyridine and α,α’-dichloro-p-xylene with the grafted poly(vinyl benzyl chloride) chains on the PPY film surface. The effect of AAc monomer concentration used for grafting on the amount of GOD immobilized as well as on the corresponding film properties was assessed. The investigation of the enzymatic activities of the immobilized GOD was carried out under different temperatures as well as under an extreme condition of oxygen depletion. The effect of the viologen moieties as an electron transfer mediator in the proximity of the GODPPY system was thus addressed. v A method was then developed to immobilize hyaluronic acid (HA) through the molecular recognition of the carboxyl groups of HA by the primary amine end groups of the linker molecules introduced on the PPY film via grafting techniques. Subsequent biological assays of the immobilized polysaccharide were carried out. Different systems were employed, from the assay of the specific binding between a protein and HA to a dynamic cellular response mediated by the interaction between HA and cellsurface receptors. A bacteria-based environment was also applied to assess the antifouling properties of the HA-PPY system. A reduction in Escherichia coli (E. coli) adhesion was observed. However, this did not eradicate the development of a subsequent biofilm from the initially adhered bacteria. Such an observation became the motivation for the subsequent research into antibacterial surface treatments. Capitalizing on the advantages of versatility and flexibility offered by surface grafting techniques, a promising method was developed for the functionalization of substrates with bactericidal polycationic groups. This method involves the graft copolymerization of polymeric substrates with 4-vinylpyridine (4VP), followed by quaternization of the grafted pyridine groups into pyridinium groups with hexylbromide. The applicability of this method was substantiated by several substrates: poly(ethylene terephthalate) (PET) film, carbohydrate-based cellulosic materials (filter paper and cotton cloth) and poly(vinylidene fluoride) (PVDF) membrane, which showed promising bactericidal activities. The inhibition of biofilm formation of E. coli cells was effectively achieved for all the substrates tested. 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Surface Functionalization of Electrically Conductive Polypyrrole Film with Hyaluronic Acid, Langmuir, 18, pp.8633-8640. 2002. Cen L., K.G. Neoh and E.T. Kang. Surface Functionalization of Polypyrrole Film with Glucose Oxidase and Viologen, Biosens. Bioelectron., 18, pp.363-374. 2003. Li Y.L., K.G. Neoh, L. Cen and E.T. Kang. Physicochemical and Blood Compatibility Characterization of Polypyrrole Surface Functionalized with Heparin, Biotechnol. Bioeng., 84, pp.305-313. 2003. Cen L., K.G. Neoh and E.T. Kang. Surface Functionalization Technique for Conferring Antibacterial Properties to Polymeric and Cellulosic Surfaces, Langmuir, 18, pp.10295-10303. 2003. Cen L., K.G. Neoh, L. Ying and E.T. Kang. Surface Modification of Polymeric Films and Membranes to Achieve Antibacterial Properties, Surf. Interface Anal, 36, pp.716719. 2004. Cen L., K.G. Neoh and E.T. Kang. Antibacterial Activity of Cloth Functionalized with N-alkylated Poly(4-vinylpyridine), J. Biomed. Mater. Res. A., 71(A), pp.70-80. 2004. Cen L., K.G. Neoh, Y.L. Li and E.T. Kang. Assessment of In Vitro Bioactivity of Hyaluronic Acid and Sulfated Hyaluronic Acid Functionalized Electroactive Polymer, Biomacromolecules, 5, pp.2238-2246. 2004. Liu X., K.G. Neoh, L. Cen and E.T. Kang. Enzymatic Activity of Glucose Oxidase Covalently Wired via Viologen to Electrically Conductive Polypyrrole Films, Biosens. Bioelectron., 19, pp.823-834. 2004. Conference Presentations Cen L., K.G. Neoh, L. Ying and E.T. Kang. Surface Modification of Polymeric Films and Membranes to Achieve Antibacterial Properties, 10th European Conference on Applications of Surface and Interface Analysis (ECASIA), Oct 5-10, 2003. Cen L., K.G. Neoh and E.T. Kang. Interactions of Hyaluronic Acid Functionalized Polypyrrole Film with PC 12 Cells, International Conference on Materials for Advanced Technologies, Dec 7-12, 2003. Cen L., K.G. Neoh, Y.L. Li and E.T. Kang. Assessment of In Vitro Bioactivity of Hyaluronic Acid and Sulfated Hyaluronic Acid Functionalized Electroactive Polymer, International Conference on Materials for Advanced Technologies, Dec 7-12, 2003. 207 [...]... subsequently immobilized with GOD Figure 3.5 Effect of AAc monomer concentration on the surface graft concentration of the AAc polymer and PPY film conductivity Figure 3.6 XPS C 1s and Cl 2p core-level spectra of the PPY film (a) and (b) after UV-induced surface graft copolymerization with 10 vol.% AAc and 10 vol.% VBC in dioxane; (c) and (d) after surface grafting with 10 vol.% AAc and 10 vol.% VBC in... micrographs of PC12 cells after culturing in the presence of NGF added at the same time as cell seeding, after 36 h on TCPS (a) and (b); after 36 h on P-5%H-Si-HA (c) and (d); after 96 h culture on TCPS (e) and (f) and after 96 h on P-5%H-Si-HA surface (g) and (h) Figure 5.4 Scanning electron micrographs of PC12 cells without NGF after 96 h culture, on TCPS (a) and (b); on P-5%H-Si-HA surface (c) and (d) Micrographs... subsequent silanization with 1 vol.% ATS in dioxane) (P-5%H-Si), the HA functionalized PPY film (after graftcopolymerized with HEA and subsequently silanized) (P-5%H-Si-HA), and tissue culture polystyrene (TCPS) The attachment was assessed 2 h after cell seeding both with and without 50 ng/ml NGF Figure 5.2 Adhesion kinetics of PC12 cells on the PPY, P-5%H-Si, P-5%H-Si-HA and TCPS substrates All the assessments... the surface graft concentration of the HEA polymer and on the amounts of chemisorbed silane Figure 4.5 XPS C 1s and N 1s core-level spectra of (a) and (b) PPY film after UVinduced graft copolymerization with HEA in 5 vol.% HEA monomer in dioxane solution and subsequently silanized with 1 vol.% ATS in dioxane; (c) and (d) the PPY film graft-copolymerized with 5 vol.% HEA and subsequently silanized with. .. cloth and (b) functionalized cotton cloth surfaces after immersion in a PBS suspension of 107 cells/ ml of wild type multi-microorganisms for 2 h Figure 6.21 Scanning electron micrographs of (a) and (b) pristine cotton cloth, (c) and (d) functionalized cotton cloth after deposition of 200-µl aliquots containing 107 cells/ ml and 109 cells/ ml of wild type microorganisms in sterile water, respectively, and. .. graft-copolymerized with 5 vol.% HEA and subsequently silanized with 1 vol.% ATS in dioxane) after storage in (a) and (b) air at room temperature for 2 days; (c) and (d) air at room temperature for 4 days; (e) and (f) water for 2 days; (g) and (h) water for 4 days Figure 5.1 PC12 cell attachment on pristine PPY, silanized and HEA graftcopolymerized PPY film (carried out with 5 vol.% HEA monomer in dioxane solution and. .. Number of viable E coli cells in PBS at 37oC as a function of time in contact with the different substrates The cell number was determined by surface- spread method Figure 6.1 Schematic representation of the process of surface functionalization substrates with pyridinium groups Figure 6.2 XPS wide scan, C 1s and N 1s core-level spectra of (a), (b) and (c) pristine PET film; (d), (e) and (f) of PET film... graft-copolymerization with 4VP using 10 vol.% monomer in isopropanol, and (c) P-10 film Figure 6.5 Optical micrographs of (a) and (d) pristine PET, (b) and (e) P-1, (c) and (f) P-10 surfaces after exposure to airborne and waterborne E coli respectively, and subsequent incubation in solid growth agar for 24 h Figure 6.6 Scanning electron micrographs of (a) PET and (b) P-10 films after exposure to airborne E coli and. .. copolymerization with 4VP in 10 vol.% monomer in isopropanol and subsequent derivatization with 10 vol.% hexylbromide in nitromethane Figure 6.15 Scanning electron micrographs of (a) and (b) FP after exposure to airborne and waterborne E coli respectively and subsequently incubated with solid growth agar for 24 h; (c) and (d) FP-10 after exposure to airborne and waterborne E coli, respectively, and subsequently... electron micrographs of (a) and (b) pristine cotton cloth after exposure to airborne and waterborne E coli, respectively, and subsequently incubated with solid growth agar for 24 h; (c) and (d) functionalized cotton cloth after exposure to airborne and waterborne E coli, respectively, and subsequently incubated with solid growth agar for 24 h xiii Figure 6.18 Number of viable E coli cells in PBS at 37oC as . SURFACE FUNCTIONALIZED SUBSTRATES AND THEIR INTERACTIONS WITH BIOMOLECULES AND CELLS CEN LIAN . NATIONAL UNIVERSITY OF SINGAPORE 2004 SURFACE FUNCTIONALIZED SUBSTRATES AND THEIR INTERACTIONS WITH BIOMOLECULES AND CELLS CEN LIAN (B.Eng., ECUST) . (f) and after 96 h on P-5%H-Si-HA surface (g) and (h). Figure 5.4 Scanning electron micrographs of PC12 cells without NGF after 96 h culture, on TCPS (a) and (b); on P-5%H-Si-HA surface (c) and