Inkjet-Assisted Printing of Encapsulated Polymer/Biopolymer Arrays A Dissertation Presented to The Academic Faculty by Rattanon Suntivich In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Materials Science and Engineering Georgia Institute of Technology August 2014 Copyright © 2013 by Rattanon Suntivich i Inkjet-Assisted Printing of Encapsulated Polymer/Biopolymer Arrays Approved by: Dr. Vladimir V. Tsukruk, Advisor Dr. Johnna S. Temenoff School of Materials Science and Department of Biochemical Engineering Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Donggang Yao Dr. Valeria Milam School of Materials Science and School of Materials Science and Engineering Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Zhiqun Lin School of Materials Science and Engineering Georgia Institute of Technology Date Approved: May 13, 2014 ii Dedicated to my loving family iii ACKNOWLEDGEMENTS I would like to thank Prof. Vladimir V. Tsukruk for giving me the opportunity to join his research group at the Georgia Institute of Technology. Not only for the unforgettable opportunity, but also for the thinking process, knowledge, inspiring guidance and encouragement throughout my research during these past five years. I will appreciate this learning experience for the rest of my life and implement what I have learned to generate useful products for improving the quality of living of people in our society. I would also like to thank Prof. Donggang Yao, Prof. Zhiqun Lin, Prof. Valeria Milam, and Prof. Johnna S. Temenoff for their helpful feedback, suggestions, and kind willingness to be a part of my dissertation committee. A good support network is necessary for great outcomes, so for that I am thankful to former and current members of SEMA group. Particularly, I would like to thank Dr. Ikjun Choi, Dr. Olga Shchepelina, and Dr. Irina Drachuk for their constant support and guidance. I would also like to thank former lab members, Dr. Zachary Combs, Dr. Dhaval Deepak Kulkarni, Dr. Kyle Anderson, and Dr. Maneesh Gupta for their support, as well as current members Kesong Hu, Sidney Malak, and Weinan Xu for their help and support. Finally, I am very grateful to my family for their unconditional love and support. Sincere thanks goes to all my family, Rinrada Luechapanichkul, Guntarika Suntivich, Sukannika Suntivich, and Boondarik Suntivich for their moral support over the years. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS iv LIST OF TABLES viii LIST OF FIGURES ix SUMMARY xiii CHAPTER 1 INTRODUCTION 1 1.1 Background 1 1.2 Inkjet printing technique 7 1.3 Motivation 11 CHAPTER 2 RESEARCH GOALS, OBJECTIVES, AND OVERVIEW 14 2.1 Goals 14 2.2 Technical objectives 15 2.3 Organization and composition of dissertation 17 CHAPTER 3 EXPERIMENTAL DETAILS 20 3.1 Materials 20 3.1.1. Silk fibroin 20 3.1.2. Synthetic polymers 23 3.1.3. Substrates 24 3.2. Fabrication of inkjet-assisted LbL arrays 24 3.2.1. Multiple deposition of LbL films by inkjet printing 24 3.2.2. Patterning with inkjet printing 26 3.2.3. Encapsulation using inkjet printing and stamping technique 26 3.2.4 Preparing E-coli cells 27 v 3.3. Characterization of encapsulated arrays 28 3.3.1. Ellipsometry 28 3.3.2. Atomic Force Microscopy (AFM) 28 3.3.3. Confocal Laser Scanning Microscopy (CLSM) 29 3.3.4 Optical microscopy 30 3.3.5.Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) 30 3.3.6. Scanning Electron Microscope (SEM) 31 3.4. Experimental studying of inkjet-assisted LbL encapsulated arrays property 31 3.4.1. Printing and patterning performance 31 3.4.2. Surface morphology and film thickness 31 3.4.3. Film stability 32 3.4.4. Cell viability 32 3.4.5. pH responsive properties of inkjet-assisted LbL dot array 33 3.4.6. Biosensing properties of inkjet/stamping-assisted LbL encapsulated E-coli array 33 CHAPTER 4 INKJET-ASSISTED LAYER-BY-LAYER PRINTING OF ENCAPSULATED ARRAYS 35 4.1 Introduction 35 4.2 Experimental details 37 4.3 Results and discussion 39 vi CHAPTER 5 INKJET PRINTING OF SILK NEST ARRAYS FOR CELL HOSTING 61 5.1 Introduction 61 5.2 Experimental details 64 5.3 Results and discussion 67 CHAPTER 6 FREE STANDING SILK-BASED BIOSENSING WITH INKJET PRINTING AND STAMPING TECHNIQUE 77 6.1 Introduction 77 6.2 Experimental details 79 6.3 Results and discussion 82 CHAPTER 7 GENERAL CONCLUSIONS AND BROADER IMPACT 91 7.1 General conclusions an discussion 91 7.2 Significance and broader impact 94 APPENDIX 101 REFERENCES 104 VITA 118 vii LIST OF TABLES Table 7.2.1. The critical issues and possible solutions for improved LbL encapsulation 95 Table 7.2.1.(Continue) The critical issues and possible solutions for improved LbL encapsulation 96 viii LIST OF FIGURES Figure 1.1. Different techniques for encapsulating therapeutic molecules: (A) loading assembled LbL capsules, (B) Encapsulating crystallized therapeutic materials with LbL shells and (C) LbL encapsulation with porous cores. 3 Figure 1.2. PEI-(TA/PVPON-55) capsules in FITC-dextran solution at different FITC- 4 dextran molecular weight. A) MW = 4000, B) MW = 70000, and C) MW = 500000. 4 Figure 1.3. Fluorescent image of uncoated and coated furosemide microcrystals with (PSS/PDDA) + (PSS/ gelatin) multilayers. 5 2 6 Figure 1.4. TEM images with different magnification of mesoporous silica (a-c) and three LbL layer of PDDA/Si on the silica template (d). 6 NP Figure 1.5. CLSM images of FITC-POD-loaded in different porous silica template. (a) large pore size 2.5 µm and (b) small pore size 1.8 µm. 7 Figure 1.6. Formation of ink droplet using thermal DOD inkjet printing. 8 Figure 1.7. Deformation modes of piezoelectric inkjet printing for ink droplet formation. 8 Figure 1.8. Phase-contrast microscopic images of the printed endothelial cells on PET culture disk using inkjet printing. 9 Figure 1.9. Cell diversion of Human fibrosarcoma cells after printed using inkjet printing and incubation for 48 hrs. 10 Figure 2.1. Experimental concept of inkjet-assisted LbL formation of encapsulated polymer arrays: A) Multilayer LbL assembly by inkjet printing, B) Target loading, C) Creation of additional protective layers, D) Completed sandwiched dot array, and E) Completed dots on different substrates and released structures. 14 Figure. 2.2. Summary of proposed approaches of inkjet-assisted LbL encapsulation procedure. 16 Figure 3.1.1: Schematic demonstrates hierarchical spider silk structures. 21 Figure 3.1.2. Chemical structure of anionic silk and cationic silk; silk-poly(glutamic) acid and silk-poly(lysine). 23 Figure 3.2.1. Inkjet-assisted LbL printing and encapsulation of LBL arrays: A) Formation of LbL dots, B) Rhodamine dye loading, C) Formation of capping film, D) ix dye-encapsulated array, and E) An optical image of a PVPON droplet injected from a 50 µm nozzle. 25 Figure 4.3.1. Optical microscopic images demonstrate the LBL dot array with different exposure times in buffer with pH 3.5: (A) 1 bilayer, (B) 3 bilayers, and (C) 5 bilayers. 39 Figure 4.3.2. Optical (Left) and fluorescent (Right) images of a 10x10 array PVPON/PMAA LbL films with encapsulated Rhodamine dye on PS coated substrates: A) 2 bilayers, B) 6 Bilayers, and C) 10 bilayers. 40 Figure 4.3.3. Variations of diameter of LbL encapsulation films as a function of number of bilayers on different substrates. 41 Figure 4.3.4. AFM images of LBL dot with diffrent exposure times in buffer pH 3.5: (A) 1 bilayer, (B) 3 bilayers, and (C) 5 bilayers. The bottom right image in (C) is a 3-D image of 5 bilayer LbL dot after expose to buffer for 15 hrs. The scale size is 100 µm for all images. The height is 500 nm for all AFM images and 2 µm for 3-D AFM image. 42 Figure 4.3.5. Optical images display the nozzle and droplet size of (A) PVPON droplet from a 50 µm nozzle in diameter and (B) Rhodamine droplet from a 20 µm nozzle in diameter. 43 Figure 4.3.6. Higher resolution AFM images (Left; 20x20 µm and Right; 5x5 µm) displaying the surface morphology of LBL dots with (A) 1 bilayer, (B) 3 bilayers, and (C) 5 bilayers. The height is 200 nm for all images. 44 Figure 4.3.7. High resolution AFM images displaying the surface morphology of LBL films with different exposure times in buffer at pH 3.5: (A) 1 bilayer, (B) 3 bilayers, and (C) 5 bilayers. The scale size is 20 µm and the height is 200 nm for all images. 45 Figure 4.3.8. High resolution AFM images displaying the varied surface morphology of the LbL films with diffrent exposure times in buffer pH 3.5: (A) 1 bilayer, (B) 3 bilayers, and (C) 5 bilayers. The scan size is 5 µm and the height is 200 nm for all images. 46 Figure 4.3.9. Thickness of LbL dots (empty) and LbL dye-encapsulated dots (dashed) vs number of bilayers. The error bars represent the average micro roughness of each sample. 47 Figure 4.3.10.Optical and fluorescent images of 10x10 arrays of PVPON/PMAA LbL dots with encapsulated Rhodamine dye with different number of bilayers: A) 2 bilayers, B) 6 bilayers, and C) 10 bilayers. 48 x