`      DEVELOPMENT OF POROUS POLYELECTROLYTE MULTILAYERS FOR FUNCTIONAL COMPOUND DELIVERY AND SURFACE SUPERWETTABILITY  By Jing Yu                A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements  for the degree of  Chemical Engineering-Doctor of Philosophy 2016    ` ABSTRACT DEVELOPMENT OF POROUS POLYELECTROLYTE MULTILAYERS FOR FUNCTIONAL COMPOUND DELIVERY AND SURFACE SUPERWETTABILITY  By Jing Yu  Layer-by-layer (LbL) assembled polyelectrolyte multilayers (PEMs), followed by simple post treatment at acidic pHs provide one of the most promising methods to generate porous polymeric thin films. The primary aim of this work is to obtain a precise control on the porous structures, design the porous multilayers according to the specific applications, and meanwhile shorten the processing time that goes into the LbL assembly.  Firstly, we studied the preliminary step to fabricate the porous multilayers, that is, the LbL assembly of the poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayers. More specifically, we investigated the growth behavior and surface topography of the multilayers. It was found that the effect of molecular weights of both PAA and PAH on the film growth is highly dependent on the charge density and deposition time in both linear and exponential growth regimes. Unique surface patterns were obtained by tuning the molecular weight of polyelectrolytes, deposition time, and the number of bilayers. Secondly, we developed the porous multilayers for the application of functional compound delivery. We first studied the formation of porous multilayers. Both the LbL assembly and the post treatment would affect the porous structures in a synergetic manner. The application of polyelectrolytes with distinct molecular weights and different deposition time enabled a wider and more precise control on the porous structures in both nano- and micro-scales. Having gained a precise control over the pore size, layered multi-scale porous thin films were further built up with either micro-sized porous zone on top of nano-sized porous zone or vice versa. Then, we ` loaded novel hydrophobic anti-biofilm compounds (ABCs) into the porous multilayers. The release of ABCs, which is highly dependent on the porous structure, led to a good anti-biofilm performance on the substrate surface. We further applied the porous PEMs onto the atomic force microscopy (AFM) cantilever for the local delivery of hydrophilic proteins. We successfully demonstrated that the porous PEMs enabled much higher protein loadings than the conventional silanization with (3-aminopropyl) triethoxysilane (APTES). More importantly, the protein molecules could be locally delivered in a contact manner.  Lastly, we considered the development of the nano- and micro-scaled hierarchical surface structures from porous multilayers to achieve superwettability. We systematically investigated the effects of molecular weight of polyelectrolytes, deposition time during LbL assembly and pH for post treatment on the porous surface topography and wetting behavior. With the optimization of the processing conditions, we achieved a switch from superhydrophilicity to superhydrophobicity via a simple chemical vapor deposition (CVD) of fluorinated silane molecules onto the porous multilayers.           `                       Copyright by JING YU 2016                     ` v                      Dedicated to my parents and husband                        ` vi ACKNOWLEDGMENTS  To earn a PhD degree is never one persons work. I am so lucky that I have met a lot of people who offered tremendous help to make my PhD journey a lot easier. First and foremost, I am grateful to my advisor, Dr. Ilsoon Lee for always giving me the freedom of carrying out my own ideas, helping me dig out the potential of my work, and training me into an independent researcher. I am thankful to Dr. Lee for providing me with so many opportunities to become a professional. Without his insight, my research wouldn't be so well developed. Without his support, there is no way for me to graduate within four years. Without his mentoring, I don't think I would get the job that easily.  I also appreciate all the help and guidance from my committee members, Prof. Wei Liao, Prof. Lawrence Drzal, and Prof. K Jayaraman. I am grateful to Prof. Liao and Prof. Drzal for writing me recommendation letters when I was looking for jobs. I also had the opportunity to work in collaboration with Prof. Wei Liao, which provided me with a great opportunity to expand my research experience to membrane separation. I thank Prof. Jayaraman and Prof. Drzal for reminding me of the importance of polymer fundamentals. All the suggestions from my committee members make me realize my ignorance and encourage me to think about research from other perspective of views. I may disappoint them and myself for the limit knowledge I have gained during the past four years. But, this is a good lesson to keep in mind that there is no limit of learning. Our group is like a family to me. Here, I met one of my best friends, Dr. Oishi Sanyal. I admired her smartness, hard working and easy-going personality. She helped me a lot with my research and especially with scientific writing. She always encouraged me to lean in when I got ` vii frustrated with experiments. Her spirit was always with me during my last semester even though she left for postdoc last year. I deeply believe that one day, she is going to become a very successfully professor. I also had a great time working with Dr. Hong and Anna Song, and among the past members, I am very much thankful to Dr. Ankush Gokhale, Dr. Shaowen Ji and Dr. Wei Wang for all their help throughout my PhD. In addition, I appreciate the help from the undergraduate students, Brooke Meharg and Andrew Izbicki. I hope for the best for all the people that I mentioned above to achieve the success they truly deserve. While doing research, I also got a lot help from many facility centers at MSU. I appreciate Carol Flegler and Abigail Vanderberg from Center for Advanced Microscopy, Dr. Baokang Bi from W.M. Keck Microfabrication Facility Center, Prof. Dan Jones and Dr. Lijun Chen from MSU Mass Spectrometry Core, and Dr. Per Askeland from XPS from Composite Materials & Structures Center for offering all kinds of help. I also had great time working with Dr. Yongliang Yang. I appreciate him for sharing the protein delivery project with us. Besides research, I was also involved in the teaching assistance. I had a wonderful experience working with Dr. Fanelli during the last semester. I will always remember her warm personality, her kindness and her smile. She is one of the best instructors I have ever worked with. I especially thank all the students I assisted in CHE316 for being so independent and well-organized during the lab sessions, which saved me a lot of time to work on my dissertation. When I was reading all the good comments from the students, that was the first time I ever felt so satisfied and accomplished.  During my PhD, I am very lucky to become friends with a lot of nice people. My husband, Tianlong Song and I first met at MSU. He is the one who makes me feel that I may screw up a lot of things, but he is always around. I thank him for always seeing the good things in me. He ` viii may not be perfect, but he is the best that I could ever deserve. I am very thankful to Shengliu Wang for being my best friend for 11 years. She has never said no or let me down when I need help. I deeply appreciate my roommate Saisi Xue for standing up for me, dragging me out of many troubles and cooking delicious food for me. I didnt list the names of all the great people I met here, but I will always remember and cherish the time I spent with them. We will keep in touch. I wouldnt make this far without the guidance and encouragement from my family. I have the best parents in the world, who are also my best friends. They know me much better than I know myself. I especially thank my father for always seeing the potential in me. He is very smart and always knows how to instruct his students including me. He didn't give me up when I was one of the worst students in the class. He protected my self-confidence and taught me to challenge instead of simply following what the teacher said. He is also the one who provided me with the access of all kinds of scientific instruments and triggered my interest towards science and engineering. More importantly, if it werent him, I wouldnt have the courage to pursue this PhD. I also deeply thank my mother for always taking good care of me and the family. Her love and support always accompanied me when I was weak. The greatest thing I learnt from my parents is being a nice and kind hearted person. I deeply mourn the loss of my two grandfathers during my PhD, but their spirits of kindness and optimism always stay with me. I am very thankful to all my relatives who helped take care of my father while he was sick, offered help whenever needed, and shared happiness and joyful moments with me and my parents.  Thank you all!     ` ix TABLE OF CONTENTS  LIST OF TABLES ...................................................................................................................... xii  LIST OF FIGURES ................................................................................................................... xiii  KEY TO ABBREVIATIONS .................................................................................................. xvii  1. INTRODUCTION................................................................................................................. 1 1.1 Polyelectrolyte Multilayers (PEMs) .................................................................................. 1 1.1.1 Types of Polyelectrolytes ............................................................................................... 2 1.1.2 Growth Behavior of PEMs............................................................................................. 3 1.1.3 Surface Topography of PEMs ........................................................................................ 3 1.2 Porous PEMs ....................................................................................................................... 4 1.2.1 pH Induced Mechanism ................................................................................................. 4 1.2.2 Salt Induced Mechanism ................................................................................................ 5 1.2.3 Electrical Field Induced Mechanism ............................................................................. 6 1.2.4 Release Induced Mechanism.......................................................................................... 6 1.3 Applications of Porous PEMs ............................................................................................ 7 1.3.1 Drug Delivery ................................................................................................................ 7 1.3.2 Lithography and Contact Printing .................................................................................. 8 1.3.3 Superwettable Surfaces .................................................................................................. 9 1.3.4 Optical Devices ............................................................................................................ 10 1.3.5 Tissue Engineering....................................................................................................... 10 1.3.4 Other Potential Applications ........................................................................................ 11 1.4 Scope of the Thesis ............................................................................................................ 12 1.4.1 PEMs ............................................................................................................................ 12 1.4.2 Design of Functional Surfaces by Porous PEMs ......................................................... 13 REFERENCES ........................................................................................................................ 19  2. GROWTH BEHAVIOR AND UNIQUE SURFACE PATTERNS OF WEAK POLYELECTROLYTE MULTILAYERS: EFFECTS OF DEPOSITION TIME AND DISTINCT MOLECULAR WEIGHTS ................................................................................... 31 2.0 Abstract .............................................................................................................................. 31 2.1 Introduction ....................................................................................................................... 32 2.2 Experimental Section ........................................................................................................ 35 2.2.1 Materials ...................................................................................................................... 35 2.2.2 Layer-by-Layer Assembly ........................................................................................... 36 2.2.3 Film Characterization................................................................................................... 37 2.3 Results and Discussion ...................................................................................................... 37 2.3.1 Effect of Deposition Time on the Thickness of PAH/PAA Multilayers ..................... 37 2.3.2 Effect of Molecular Weight on the Film Growth of PAH/PAA Multilayers ............... 38 2.3.3 Effect of Deposition Time on the Surface Topography of PAH/PAA Multilayers ..... 43 2.3.4 Effect of Molecular Weight on the Surface Topography of PAH/PAA Multilayers ... 44 ` x 2.3.5 The Build-up of PAH/PAA Multilayers with Patterned Surface ................................. 48 2.4 Conclusions ........................................................................................................................ 51 REFERENCES ........................................................................................................................ 53  3. DEVELOPMENT OF MULTI-SCALE POROUS THIN FILMS FOR CONTROLLED RELEASE OF ANTI-BIOFILM COMPOUNDS ...................................... 57 3.0 Abstract .............................................................................................................................. 57 3.1 Introduction ....................................................................................................................... 58 3.2 Experimental Section ........................................................................................................ 63 3.2.1 Materials ...................................................................................................................... 63 3.2.2 Fabrication of Porous Thin Films ................................................................................ 64 3.2.3 The Loading and Release of ABC-1 ............................................................................ 65 3.2.4 Biofilm Test ................................................................................................................. 66 3.2.5 Preparation of Free Standing Porous PEMs................................................................. 67 3.2.6 Film Characterization................................................................................................... 67 3.3 Results and Discussion ...................................................................................................... 67 3.3.1 Effect of Molecular Weight of Polyelectrolytes and Deposition Time on the Formation of Porous PEMs .................................................................................................................... 67 3.3.2 Layered Multi-scale Porous PEMs .............................................................................. 76 3.3.3 Release of ABC-1 Molecules from Porous PEMs ....................................................... 78 3.3.4 Anti-biofilm Performance of ABC-1 Loaded Porous PEMs ....................................... 82 3.3.5 Free Standing Porous PEMs ........................................................................................ 84 3.4 Conclusions ........................................................................................................................ 85 REFERENCES ........................................................................................................................ 88  4. POROUS MULTILAYER-COATED AFM CANTILEVERS FOR PROTEIN DELIVERY ................................................................................................................................. 94 4.0 Abstract .............................................................................................................................. 94 4.1 Introduction ....................................................................................................................... 94 4.2 Experimental Section ........................................................................................................ 98 4.2.1 Materials ...................................................................................................................... 98 4.2.2 Fabrication of Porous PEMs on AFM cantilevers ....................................................... 98 4.2.3 Silanization of AFM Cantilevers ................................................................................. 99 4.2.4 The Loading of Protein Molecules .............................................................................. 99 4.2.5 Contact Release of Protein Molecules from Porous Multilayers ............................... 100 4.2.5 Solution Release of Weakly Bound Protein Molecules from Porous Multilayers..... 101 4.3 Results and Discussion .................................................................................................... 101 4.3.1 Porous Structures on the AFM Cantilever ................................................................. 101 4.3.2 Protein Loadings ........................................................................................................ 103 4.3.3 Contact Release of Protein Molecules from Porous Multilayers ............................... 105 4.3.4 Dissociation of Weakly Bound Protein Molecules .................................................... 107 4.4 Conclusions ...................................................................................................................... 108 REFERENCES ...................................................................................................................... 110  5. NANO- AND MICRO-SCALE HIERARCHICAL POROUS POLYELECTROLYTE MULTILAYERS FOR SUPERWETTABLE SURFACES .................................................. 114 ` xi 5.0 Abstract ............................................................................................................................ 114 5.1 Introduction ..................................................................................................................... 114 5.2 Experimental Section ...................................................................................................... 117 5.2.1 Materials .................................................................................................................... 117 5.2.2 Fabrication of Porous PAH/PAA Multilayers ........................................................... 118 5.2.3 Chemical Vapor Deposition (CVD) of Fluoroalkylsilane Molecules ........................ 119 5.2.4 Characterization ......................................................................................................... 119 5.3 Results and Discussion .................................................................................................... 120 5.3.1 Surface Topography and Wetting Behavior of Porous PAH/PAA Multilayers ........ 120 5.3.2 Effect of Molecular Weight of PAH on the Surface Topography and Wetting Behavior............................................................................................................................................. 123 5.3.3 Effect of Molecular Weight of PAA on the Surface Topography and Wetting Behavior............................................................................................................................................. 126 5.3.4 Effect of Deposition Time on the Surface Topography and Wetting Behavior ........ 126 5.3.5 Effect of pH for Post treatment on the Surface Topography and Wetting Behavior . 127 5.4 Conclusions ...................................................................................................................... 133 REFERENCES ...................................................................................................................... 135  6. THESIS SUMMARY AND FUTURE WORK .................................................................. 139 6.1 Thesis Summary .............................................................................................................. 139 6.2 Future Work .................................................................................................................... 141 REFERENCES ...................................................................................................................... 145            ` xii LIST OF TABLES  Table 2.1 Thickness of (PAH/PAA)20.5 multilayers fabricated under different pH conditions ... 43 Table 2.2 Surface roughness of (PAH8.5/PAA3.5)20.5 films ....................................................... 48  Table 5.1 The effect of pH for post treatment on the contact angle of porous (PAHL/PAAL)20.5 and (PAHH/PAAL)20.5 surfaces with the deposition time of 10s and1 min, respectively ............ 133                   ` xiii LIST OF FIGURES  Figure 1.1 Film deposition process via layer-by-layer assembly. Reproduced with permission from[2]. ........................................................................................................................................... 1  Figure 1.2 Schematics of the (a) 2.0/2.0, (b) 7.5/3.5, and (c) 6.5/6.5 PAH/PAA multilayer assemblies shown with PAA as the outermost layer. Reproduced with permission from [18]. ..... 2  Figure 1.3 Electric field induced morphological transitions in polyelectrolyte multilayers with the trigged release of Methylene Blue (MB). Reproduced with permission from [69]. ................. 6  Figure 1.4 Schematics for release induced mechanisms of PVP/PAA multilayers. Reproduced with permission from [71]. ............................................................................................................. 7  Figure 1.5 Schematic of porous multilayer films and implications on drug release for (a) microporous and (b) nanoporous films. Reproduced with permission from [4]............................. 8  Figure 1.6 Stable superhydrophobic coatings from polyelectrolyte multilayers: (A) SEM image of porous PAH/PAA multilayers with silica nanoparticles. (B) Water droplet on this superhydrophobic surface. Reproduced with permission from [55]. .............................................. 9  Figure 1.7 Chemical structure of ABC-1 ..................................................................................... 14  Figure 2.1 The effect of deposition time and molecular weight of polyelectrolytes on the thickness of (PAH8.5/PAA3.5)20.5 films. These thickness data were acquired with profiler. ...... 42  Figure 2.2 SEM images of surface morphology of (PAH8.5/PAA3.5)20.5 films. (The scale bar is  ..................................................................................................................... 45  Figure 2.3 AFM 3D images of the surface morphology of (PAHL8.5/PAAL3.5)20.5 films with different deposition time: (a) 10 s, (b) 1 min, (c) 5 min, (d) 10 min, and (e) 15 min ................... 46  Figure 2.4 AFM 3D images of the surface morphology of (PAHH8.5/PAAL3.5)20.5 films with different deposition time: (a) 10 s, (b) 1 min, (c) 5 min, (d) 10 min, and (e) 15 min ................... 47  Figure 2.5 AFM 3D images of the surface morphology of (a) (PAHL8.5/PAAL3.5)20.5, (b) (PAHH8.5/PAAL3.5)20.5, (c) (PAHL8.5/PAAH3.5)20.5 and (d) (PAHH8.5/PAAH3.5)20.5, respectively (The deposition time is 10s.) .................................................................................... 47  Figure 2.6 SEM images of PAHH8.5/PAAH3.5 films with number of bilayers as (a) 4.5, (b) 6.5, (c) 8.5, (d) 10.5, (e) 12.5, (f) 14.5, (g) 16.5, (h) 18.5 and (i) 20.5, respectively. (The deposition time is 10  .................................................................. 50  ` xiv Figure 2.7 The values of (a) thickness and (b) roughness of PAHH8.5/PAAH3.5 films as a function of number of bilayers (The deposition time is 10 min.) ................................................. 50  Figure 3.1 The effect of deposition time on (a) the thickness of PAHL/PAAL thin films before and after the post treatment and (b) the relative expansion of thickness and average surface pore size ................................................................................................................................................ 70  Figure 3.2 (a) and (b), (c) and (d), (e) and (f), (g) and (h), and (i) and (j) are the top-view and cross-sectional SEM images for porous (PAHL/PAAL)20.5 films with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. The arrow in each cross-sectional image indicates the interface between the glass substrate and the deposited film. ................................................. 71  Figure 3.3 (a) thickness of thin films before and after the post treatment and (b) the relative expansion of thickness and average surface pore size for (PAHL/PAAL)20.5, (PAHH/PAAL)20.5, (PAHL/PAAH)20.5, and (PAHH/PAAH)20.5, respectively. All the films were fabricated using deposition time of 10 s. ................................................................................................................. 74  Figure 3.4 (a) and (b), (c) and (d), (e) and (f), and (g) and (h) are the top-view and cross-sectional SEM images for porous (PAHL/PAAL)20.5, (PAHH/PAAL)20.5, (PAHL/PAAH)20.5, and (PAHH/PAAH)20.5 films, respectively. The arrow in each cross-sectional image indicates the interface between the glass substrate and the deposited film. All the films were fabricated using deposition time of 10 s. ................................................................................................................. 75  Figure 3.5 SEM cross-sectional ((a) and (b)) and top view (c) images of multi-scale porous thin films with nano-sized porous film as the bottom and micro-sized porous film as the top. The arrow in (a) indicates the interface between the glass substrate and the deposited film. (b) is an enlarged image of the red square area in (a). ................................................................................ 77  Figure 3.6 SEM cross-sectional ((a) and (b)) and top view (c) images of multi-scale porous thin films with micro-sized porous structure as the bottom and nano-sized porous structure as the top. The arrow in (a) indicates the interface between the glass substrate and the deposited film. (b) is an enlarged image of the red square area in (a). ........................................................................... 78  Figure 3.7 Accumulative release profiles of ABC-1 from different porous (PAH/PAA)20.5 thin films. ............................................................................................................................................. 79  Figure 3.8 The plots of release kinetics for (a) micro- and (b) nano-sized porous structures, respectively. .................................................................................................................................. 80  Figure 3.9 Bacterial colonies formed by the disaggregated V. cholera cells from the biofilm on the coupon surfaces ....................................................................................................................... 84  Figure 3.10 Photograph of a free standing porous (PAHL/PAAL)20.5 with 15min dipping and pH=2.0 for post-treatment in the exfoliation solution. The diameter of the Petri dish is 10cm. .. 85  ` xv Figure 4.1 SEM images of the AFM cantilever coated with porous PAH/PAA multilayers. Porous PAH/PAA multilayers were fabricated with deposition time of (a) - (c) 10 s, (d) - (f) 1 min, and (g) - (i) 5 min, respectively. ......................................................................................... 102  Figure 4.2 SEM images of the porous PAH/PAA multilayers on the Si3N4 wafer with deposition time of (a) 10 s, (b) 1 min, and (c) 5 min, respectively. ............................................................. 103  Figure 4.3 Fluorescence images of AFM cantilevers modified with (a) APETS and (b)-(c) porous PAH/PAA multilayers with deposition time of 10 s, 1 min and 5 min, respectively. .... 104  Figure 4.4 The fluorescence intensity of the AFM cantilevers modified with APETS and porous PAH/PAA multilayers with deposition time of 10 s, 1 min and 5 min, respectively. ................ 105  Figure 4.5 The fluorescence image of the dot pattern resulting from the contact release of fluorescence protein molecules from porous multilayer coated AFM cantilever (deposition time of 5 min): (a) directly after the protein incubation and (b) with three rinsing step with PBS solution after the protein incubation, and (c) immersed in PBS solution for two days after the  ............................................................................. 106  Figure 4.6 The dissociation of loosely bonded protein molecules from the porous PAH/PAA multilayers with deposition time of 10 s, 1 min and 5 min, respectively ................................... 108  Figure 5.1 (a)-(e) are the top-view SEM images of the porous (PAHL/PAAL)20.5 with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. (f)  (j) are the top-view SEM images of the porous (PAHH/PAAL)20.5 with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. (The post treatment was done at pH of 2.0.) (k)-(o) are the top-view SEM images of the porous (PAHH/PAAH)20.5 with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. ........................................................................................................................ 124  Figure 5.2 The values of (a) roughness and (b) contact angle for (PAHL/PAAL)20.5, (PAHL/PAAH)20.5, (PAHL/PAAH)20.5 and (PAHH/PAAH)20.5 with different deposition time, respectively. (The post treatment was done at pH of 2.0.) ......................................................... 125  Figure 5.3 XPS spectra of porous (PAHH/PAAL)20.5 film before (a) and after (b) the CVD process......................................................................................................................................... 127  Figure 5.4 SEM images of the porous (PAHL/PAAL)20.5 surfaces ((a)-(d) deposition time of 10 s; (e)-(h) deposition time of 1 min) with post treatment at pH of 1.8, 2.0, 2.2 and 2.4, respectively...................................................................................................................................................... 129  Figure 5.5 SEM images of the porous (PAHH/PAAL)20.5 surfaces ((a)-(d) deposition time of 10 s; (e)-(h) deposition time of 1 min) with post treatment at pH of 1.8, 2.0, 2.2 and 2.4, respectively...................................................................................................................................................... 130  ` xvi Figure 5.6 SEM images of the porous (PAHH/PAAH)20.5 surfaces ((a)-(d) deposition time of 10 s; (e)-(h) deposition time of 1 min) with post treatment at pH of 1.8, 2.0, 2.2 and 2.4, respectively...................................................................................................................................................... 131                ` xvii KEY TO ABBREVIATIONS  Chemicals  ABC-1                                    5-methoxy-2-[(4-methylbenzyl) sulfanyl]-1H-benzimidazole ABCs                                      Anti-biofilm compounds APTES                                   (3-aminopropyl) triethoxysilane BPEI    Branched poly (ethylene imine) BSA                                        Bovine serum albumin CHI                                         Chitosan DEN-COOH                           Carboxyl-terminated polyether dendrimer DI water          Deionized water HA                                          Hyaluronan HCL               Hydrochloric acid  KCl                 Potassium chloride KH2PO4                                                   Monobasic potassium phosphate LPEI                                        Linear poly (ethylene imine) MgCl2                                      Magnesium Chloride MMT             Montmorillonite NaCl                Sodium chloride Na2HPO4                                 Dibasic sodium phosphate NaOH              Sodium hydroxide OEGDA                                  Oligoethylene glycol dicarboxylic acid PAA    Poly acrylic acid ` xviii PAH    Poly allylamine hydrochloride PBS                                         Phosphate buffer saline PDAC               Poly (diallyl dimethyl ammonium chloride) PEG                Poly (ethylene glycol) PEI                  Poly (ethylene imine)    PES                Poly ether sulfone PMMA           Poly methyl methacrylate PVP                                         Poly (4-vinylpyridine) SPS    Sulfonated Polystyrene / Polystyrene sulfonate TPEDA                                   1N-[3-(trimethoxysilyl)propyl]ethylenediamine  Symbols A                                              Surface area of the drug-loaded porous samples Ci                                             Drug Concentration of sample i CA                                           Contact angle K                                              Release rate constant Mi                                            Total cumulative mass released per 1cm2 as of measurement i Mt                                            Total amount of drug released at time t M                                           Total amount of drug released as time goes to infinity n                                              Exponent characteristic of the release mechanism         nr                                             Refractive index t                                               Time Vi                                             Total volume of the release media prior to measurement  ` xix Terminologies                     Microcontact printing CVD                                        Chemical vapor deposition DPN                                        Dip-pen nanolithography LbL             Layer-by-layer MB                                          Methylene Blue  MF                Microfiltration NF                 Nanofiltration PE                 Polyelectrolyte PEM               Polyelectrolyte Multilayer  RO                Reverse Osmosis SLIPS                                       Slippery liquid infused porous surfaces UF                Ultrafiltration  Instrumentation AFM                Atomic force microscopy FRIT                                        Fourier transform infrared spectroscopy LC-MS             Liquid chromatography- Mass spectrometry SEM               Scanning electron microscopy XPS                           X-ray photoelectron spectroscopy ` 1 1. INTRODUCTION 1.1 Polyelectrolyte Multilayers (PEMs) Layer-by-layer (LbL) assembled PEMs have been considered as a versatile platform to design functional surfaces. J. J. Kirkland and R. K. Iler first applied the LbL technique using microparticles in 1966.[1] In 1990s, Gero Decher pioneered the LbL technique to build polyelectrolyte multilayers (PEMs) by the alternate deposition of polyanions and polycations onto the solid surfaces as shown in Figure 1.1.[2] The structure of the PEMs can be precisely controlled in nanometer scale. The LbL process is aqueous based, and there is no limit on the choice of substrate materials. Moreover, the polyelectrolytes can be replaced with functional components (i.e. proteins[3, 4], DNA[5], RNA[6], nanoparticles[7-9], copolymer micells[10, 11]) to functionalize the surface. Therefore, during the past two decades, these PEM films have been engineered for drug delivery systems[12-14], anti-bacterial surfaces[15, 16], substrates for cell adhesion and proliferation[17, 18], gas barrier[19, 20], gas separation[21], membrane filtration[22], anti-reflection coatings[23], electrical conductive devices[24], etc.      Figure 1.1 Film deposition process via layer-by-layer assembly. Reproduced with permission from[2]. ` 2 1.1.1 Types of Polyelectrolytes According to the degree of ionization, polyelectrolytes can be divided into strong and weak polyelectrolytes. Strong polyelectrolytes, such as poly(sodium 4-styrene sulfonate) (SPS) and poly(diallyldimethylammonium chloride) (PDAC) are fully charged in solution regardless of pH; while for weak polyelectrolytes, such as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA), the degree of ionization is highly dependent on the local pH.  The variation in charge density in weak polyelectrolytes leads to tunable multilayer architectures(Figure 1.4).[25-27] For PAA/PAH multilayers fabricated at pH 2.0/2.0, the carboxylic acid groups on PAA chain are partially ionized; while PAH is fully charged. To balance the charge on the outmost PAH layer, PAA chain segments need to form loopy shapes. For PAA/PAH multilayers fabricated at pH 3.5/7.5, the charge density of PAA is low, while PAH is highly charged. However, the adsorbed PAA on the surface will be fully ionized when dipped into PAH solution, leading to the absorption of a thick PAH layer. This thick layer of PAH will attract more PAA chains later on. For PAA/PAH multilayers fabricated at pH 6.5/6.5, both PAA and PAH in solutions are mthin bilayers.   Figure 1.2 Schematics of the (a) 2.0/2.0, (b) 7.5/3.5, and (c) 6.5/6.5 PAH/PAA multilayer assemblies shown with PAA as the outermost layer. Reproduced with permission from [18]. ` 3 1.1.2 Growth Behavior of PEMs Two types of multilayer growth behaviors were discovered. (1) Linear growth, which means the thickness or mass of the film increases linearly with the number of deposition steps. Linear growth always happens when the polyelectrolytes in the solution only interact with the oppositely charged polyelectrolytes on the film surface. (2) Exponential growth, which means the films grow exponentially with the number of deposition steps. The exponential growth behavior is based on the interlayer diffusion of polyelectrolytes within the multilayers during each deposition step. Diffusion of polymer chains within a swollen LbL films is dependent on  controlled by the parameters during LbL assembly, such as temperature[28], salt concentration[29-33], pH of polyelectrolyte solutions[26, 28, 34-39], molecular weight of polyelectrolytes[30, 33, 40-42], deposition time and number of bilayers [43-46].  1.1.3 Surface Topography of PEMs Besides film growth, surface topography of multilayers also changes with the number of bilayers, molecular weight of polyelectrolytes and the pH of polyelectrolyte solutions. For chitosan (CHI)/hyaluronan (HA) multilayers, the small islets showed up on the surface after the deposition of the first bilayer. The individual islets coalesced into larger vermiculate features with the increase of the number of bilayers.[40, 42] In addition, the transition from small islets to vermiculate patterns depends on the molecular weight of the polyelectrolytes. The low molecular weight polyelectrolytes could facilitate the generation of larger wormlike structures than the high ` 4 molecular weight polyelectrolytes.[42] Shen et al. [47] prepared poly(ethylene imine) PEI/PAA multilayers in the exponential growth regime, leading to the formation of micro/nano hierarchical structures. With a further step of fluorination, the surface of the multilayers became superhydrophobic.  1.2 Porous PEMs Porous polymeric films are in demand for a wide range of applications including foams[47], insulators[48], separator in electrochemical devices[49], membranes[50], catalytic supports[51], anti-reflection coatings[23, 52, 53], superhydrophobic coatings[54], cell scaffolds[55-57], and drug delivery systems[58]. Hierarchical (i.e., micro and nano sized) porous surfaces help achieve superhydrophobicity.[59] For controlled drug release, the release rate is highly dependent on the pore size.[13] A well-controlled porous structure (i.e., mesoporous shell and microporous core) enables a tunable drug release over time.[60] In addition, commercial nanofiltration/reverse osmosis membranes have an asymmetric structure with two distinct types of porous zones; the bottom one consists of micro-sized pores while the upper zone consists of nano-sized pores. LbL assembled PEMs followed by simple post treatments provides one of the most promising methods to generate porous polymeric frameworks. Based on different types of post treatments, the porous formation follows different mechanisms, which are summarized as follows. 1.2.1 pH Induced Mechanism  Rubner and coworkers first demonstrated the formation of porous networks using poly PAH/PAA multilayers.[23] The PEMs were fabricated with the PAH solution at a pH of 7.5 or ` 5 8.5 and the PAA solution at a pH of 3.5. The porous structure was formed after the post treatment, which includes acidic treatment within the pH range of 1.8-2.6, rinsing in DI water, drying and cross-linking[13, 23, 54, 61-64]. Immersion of PAH/PAA films in a low-pH aqueous solution causes rearrangement of the polymer chains[23, 62, 63]. This rearrangement is induced by the breakage of the ionic cross-links of PAA due to protonation of the carboxylate groups and charge repulsion among the free, positively charged amine groups of PAH. The rinsing step with DI water allows ion pairs to reform and form small water pocket by rejecting water from the film. By drying water out from the water pockets and cross-linking the polymer chains, stable porous films were obtained. Similar porous formation was also found in system of PEI/PAA multilayers.[65] The resultant porous films are stable for at least 18 months.[63] The pH induced porous PEMs are extensively studied due to the controllability on the porous structure. Both nano and micro sized porous films were able to be achieved by tuning the conditions of LbL assembly and the post treatment including pH of polyelectrolyte solutions[65], number of bilayers[13, 61, 64], pH of post treatment[13, 61, 63, 65, 66, 67], acid/base exposure time[65], and DI water rinsing time[63, 64].  1.2.2 Salt Induced Mechanism Caruso et al.[68] reported the formation of the porous PEMs by salt-induced structural changes in PAH/PAA multilayers. PAA/PAH multilayers were fabricated at pH of 5.0 for both polyelectrolytes with the presence of 0.2 M sodium chloride (NaCl). The porous structures formed by exposing the PEMs to pure water in less than 10 min when the salts diffused out, ` 6 leading to the rearrange of the polymer chains. With this approach, the pore size was limited to the nanometer scale.  1.2.3 Electrical Field Induced Mechanism Zacharia et al.[69] applied an electrical field on the linear poly(ethylene imine) (LPEI)/PAA multilayers. They found that under the electrical field, a porous structure formed due to the local changed in pH from the hydrolysis of water. The chain rearrangement first happened at the multilayer-electrode interface and propagates through the film. Therefore, micro-sized pores formed near the electrode, while nano-sized pores existed near the surface facing the electrolyte solution. The porous structure (i.e. pore size) is also dependent on the duration of the application of the field. In addition, small molecules were able to be released during the formation of porous structures under the electrical field, as shown in Figure 1.3.  Figure 1.3 Electric field induced morphological transitions in polyelectrolyte multilayers with the trigged release of Methylene Blue (MB). Reproduced with permission from [69]. 1.2.4 Release Induced Mechanism Zhang et al. [70, 71] developed another way to make porous LbL films is through the post treatment of hydrogen-bonded poly(4-vinylpyridine) (PVP)/PAA multilayers in base solution. ` 7 With the exposure to the sodium hydroxide solution at pH of 12.5 or 13, the hydrogen bonding broke, leading to the dissociation of PAA from the multilayer followed by the reconfirmation of PVP chains and the formation of porous structure, as shown on Figure 1.4. They further switched PAA with carboxyl-terminated polyether dendrimer (DEN-COOH) and confirmed that the dissociation of DEN-COOH is slower than that of PAA, leading to a smaller pore size.[72]   Figure 1.4 Schematics for release induced mechanisms of PVP/PAA multilayers. Reproduced with permission from [71]. 1.3 Applications of Porous PEMs 1.3.1 Drug Delivery It is well known that porous polymeric films/particles have been applied in many drug delivery systems.[58, 73, 74] Porous PEMs have shown great potential in drug delivery. Rubner et al.[13] first loaded ketoprofen and cytochalasin D into the porous PAA/PAH films and explored the release behavior from both nano- and micro- porous films (Figure 1.5). A zero-order release kinetic was achieved by the nanoporous films. And, the release of drugs could last over a period of many days. Moreover, they also designed a sandwich heterostructures with porous and nonporous regions stacking alternately in the films. It is possible to control the ` 8 loading optically with the sandwich structure acting as dielectric mirrors. It was also found that . The micro-sized porous structures provided a faster release of drugs with the release behavior followed the Fickian diffusion.   Figure 1.5 Schematic of porous multilayer films and implications on drug release for (a) microporous and (b) nanoporous films. Reproduced with permission from [4]. 1.3.2 Lithography and Contact Printing Besides small drug molecules, large molecules as proteins can also be delivered.[75, 76] Wu et al.[75] coated the AFM tips with nanoporous PAA/PVP multilayers. By controlling the post treatment time in KOH solution, the film thickness and pore sizes can be easily adjusted. They successfully incorporated a water-soluble fluorescent protein into the porous films on the reservoir for dip-pen nanolithography (DPN) to fabricate patterns with micrometer and submicrometer scales. Porous PAA/PVP multilayers were also coated on the PDMS stamps for [76]  ` 9 1.3.3 Superwettable Surfaces In order to achieve stable superhydrophobicity, Rubner et al.[55] applied porous PAA/ PAH multilayer films. The surface was then coated with silica nanoparticles and modified with (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane via chemical vapor deposition. The SEM image of the surface was shown in Figure 1.6 (a). The porous surface was able to provide superhydrophobic surfaces with a contact angle as high as 172o (Figure 1.6 (b)) and a sliding angle less than 2o.   Figure 1.6 Stable superhydrophobic coatings from polyelectrolyte multilayers: (A) SEM image of porous PAH/PAA multilayers with silica nanoparticles. (B) Water droplet on this superhydrophobic surface. Reproduced with permission from [55]. Zacharia et al.[67] applied a similar approach with porous branched poly(ethylene imine) (BPEI)/PAA multilayers. They further spin-coated the surface with fluorinated lubricant and created the slippery liquid infused porous surfaces (SLIPS). These surfaces were able to repel both water and decane with sliding angles as low as 3o within the temperature ranging from -10 to 95 oC. ` 10 1.3.4 Optical Devices Porous surfaces have been extensively studied for anti-reflection and anti-fogging applications. [23, 77-80] Rubner et al.[23] first developed the anti-reflection coatings from porous PAA/PAH multilayers. It was found that the index of refraction (nr) of the coatings must be between 1.2 and 1.3 in order to obtain efficient anti-reflection for glass and polymeric optical elements. The nanoporous structure induced at pH = 1.8 for 60 s was able to decrease the n to 1.25. With the addition of 0.1 M MgCl2 during the post treatment, the pH needs to be increased to 2.6 and the fabrication time was shortened to less than 10s to obtain an n value lower than 1.25. The designed nanoporous PEMs can provide good anti-reflection property at both visible and near-infrared wavelengths. They further expanded the application of porous PEMs to fabricate anti-fogging surface coatings[77]. A Bragg reflector was also developed by the porous PEMs.[81] A (PAH/SPS)50-[(PAH/PAA)4-(PAH/SPS)50]4 film was assembled and treated at pH = 2.3 while the PAH/PAA regions formed nanoporous structures and PAH/SPS regions remained unchanged. The sandwich structure could behave successfully like one-dimensional dielectric mirrors and vapor sensors by monitoring the change of refractive index.  1.3.5 Tissue Engineering Cells are sensitive towards the topography of material surfaces. Therefore, the cellular adhesion can be modulated by controlling nano- and micro-scale surface features. Cornel epithelial cells can adhere on the membranes with pore size ranging from 20 to 200 nm.[82, 83] Rajagopalan et al.[84] mimicked the pore size of the corneal membrane by the porous PAA/PAH ` 11 multilayers with pore diameter of 100 nm or 600 nm. Compared to micro-scale porous structures, the nanoporous structures enhanced the cellular response significantly, leading to higher cell proliferation and migration speeds.  1.3.4 Other Potential Applications  Solid-state polymer electrolytes are in great demand for applications in power sources, sensors and electrical devices due to their good mechanical properties and processability compared to liquid electrolytes.[85] Hammond et al. [86] loaded oligoethylene glycol dicarboxylic acid (OEGDA) as a plasticizing element into the porous PEI/PAA films via absorption from aqueous solution during the post treatment process. A relatively high ionic conductivity was obtained by this composite film, indicating a potential application in solar energy conversion and batteries. The development of free-standing films is extremely useful for the application in the fields of membrane separation, sensors, micromechanical devices, wound dressing, or even artificial organs.[87-90] The porous (PAH/PAA)20.5 thin films can be directly exfoliated from the substrates by an ion-triggered exfoliation[64, 91].  So far, porous PEMs have shown promising potential for the applications in a variety of research fields. However, one of the remaining issues for industrializing the LbL products is the fabrication efficiency. For the conventional dipping method, it takes 15  20 min to deposition one layer of polyelectrolyte. In order to LbL products more commercially feasible, two other methods, spray-assisted [92] and spin-assisted [93] coating, have been developed to shorten the deposition time to a few seconds. Different parameters are involved with different deposition ` 12 methods to control the film thickness, growth behavior, surface morphology, as well as the film properties. [93-96] Therefore, the optimization of the film properties is critical according to different fabrication methods. 1.4 Scope of the Thesis  1.4.1 PEMs The growth behavior of PAA/PAH multilayers has been studied by changing the pH conditions[27, 37], molecular weight of PAA[41], and number of bilayers[37] during LbL assembly. From previous studies, the molecular weight of PAA and PAH were mainly within the range of 5000 to 100,000 g/mol.[26, 35, 37, 41, 63, 97] In order to study the molecular weight effect thoroughly, we chose two distinct molecular weights for both PAA (15,000 g/mol (PAAL) and 225,000 g/mol (PAAH)) and PAH (15,000 g/mol (PAHL) and 900,000 g/mol (PAHH)). The application of polyelectrolytes with distinct molecular weights amplified the differences of chain mobility, and therefore enabled the observation of very detailed changes during the deposition steps. From previous studies[30, 33, 39, 41, 42], the molecular weight of polyelectrolytes affected the film growth randomly. However, the molecular weight of polyelectrolytes was always considered as an independent variable. Therefore, we built up the multilayers in both linear and exponential regimes by tuning the pH of polyelectrolyte solutions and then investigated the effect of molecular weight on the film growth by varying the deposition time from 10 s to 15 min. Besides growth behavior, we also investigated the effect of deposition time and molecular weight of polyelectrolytes on surface topography of multilayers.  ` 13 In Chapter 2 of this dissertation we have evaluated the effect of distinct molecular weight of polyelectrolytes and the deposition time on the film growth and surface topography of PAH/PAA multilayers. In addition, the LbL assembly is the preliminary step for developing the porous PEMs in the following chapters. The molecular weight of the polyelectrolytes and the deposition time influence the film thickness, composition and polyelectrolyte distribution of the multilayers, which may further affect the reorganization of polymer chains during the post treatment. 1.4.2 Design of Functional Surfaces by Porous PEMs (1) Anti-biofilm Surface Coatings. Recently, a series of novel benzimidazole molecules have been developed as anti-biofilm compounds (ABCs) with the ability to inhibit biofilm formation. It was found that biofilms of multiple bacteria pathogens, including Pseudomonas aeruginosa and a methicillin resistant Staphylococcus aureus can be inhibited efficiently by 5-methoxy-2-[(4-methylbenzyl) sulfanyl]-1H-benzimidazole (named ABC-1 with structure shown in Figure 1.7).[98] In order to generate a surface with good anti-biofilm properties, we want to take advantage of the novel anti-biofilm compounds, such as ABC-1. However, for small molecules like novel benzimidazole compounds, they don't have the ability to stay on the material surface by themselves and release as needed. Thus, a surface drug reservoir system is desired. LbL technique has already been applied for fabricating drug loaded PEMs by applying the drugs as one of the polyelectrolytes.[99] The release profile was linear for most cases, which means the release rate remains constant all the time. This is because the drug release is controlled by dissociation or degradation of polyelectrolytes. However, this approach requires the drug to ` 14 be hydrophilic, which can be alternatively deposited onto the surface with the other polyelectrolyte. However, the maximum concentration of ABC-This limits the amount of ABC-1 incorporated in the PEMs. Rubner et al. [13] first developed the porous PEM films and applied them for controlling drug release. With porous PAA/PAH films, hydrophobic drugs can be easily incorporated, and the release kinetics is highly dependent on the porous structure. Therefore, a precise control of pore size and size distribution is required.  Figure 1.7 Chemical structure of ABC-1 Instead of tuning the conditions of post treatment, we believe that the deposition conditions during LbL assembly could allow some new discoveries. For conventional LbL process, the deposition time is around 10 or 15 min per layer. Thus, slow processing becomes one of the major issues for industrializing PEM products. However, no research has been performed on the effect of deposition time on the porous structure, or how efficient the porous structure can be built up. Besides deposition time during LbL process, the molecular weight of polyelectrolytes not only affects the film growth behavior, polyelectrolyte distribution throughout the multilayers and surface topography, but also changes the chain mobility, which may significantly influence the reorganization of polymer chain during the post treatment. Therefore, the effects of deposition time and molecular weight of both PAA and PAH on the porous structures have been evaluated thoroughly in Chapter 3 of this dissertation. In addition, according to the previous ` 15 studies, the porous structure developed located in either nano- or micro-scale[13, 23, 65]. Even with micro- and nano- structure, both of the structures exist randomly on the surface [100]. Having gained a precise control over the pore size by tuning the deposition time and molecular weight of polyelectrolytes, we successfully built up the layered multi-scale porous thin films with either micro-sized porous zone on top of nano-sized porous zone or vice versa. We demonstrated that the release of ABC-1 could be highly controlled by varying the porous structure, and the anti-biofilm performance was also investigated. Moreover, we obtained the free-standing porous thin films through ion-trigged exfoliation.   (2) Protein Delivery. Cell membranes consist of a lot of functional protein receptors. The dysfunction of the protein receptors after bonded with pathogenic proteins (i.e. autoantibody) may cause tissue destruction or necrosis in the skin and originate many skin diseases. In order to study the disease model and mechanisms, the conventional injection of proteins or drugs into the cells wouldnt work. Meanwhile, the direct addition of proteins to the cell media prevents the study of cell-cell interaction. Therefore, a delivery in a contact manner to the membrane of a specific cell is required. Atomic force microscopy (AFM) has been developed as a nanorobot, which facilitates the applications of localized mechanical and chemical stimulations to a cell of interest as well as provides real-time cellular and molecular responses.[101] The advances of AFM based nanorobot allow the local delivery of drug/protein directly to the cell membrane, showing great application potential in the fields of drug delivery and cell biology.  ` 16 In Chapter 4, we deposited porous PAH/PAA multilayers onto the AFM cantilevers for protein delivery. We loaded anti-desmoglein (anti-Dsg) 3 autoantibodies and fluorescence labeled goat anti-mouse IgG as example proteins. The fluorescence labeled goat anti-mouse IgG can also behave as a secondary antibody for labeling anti-Dsg 3. Pemphigus vulgaris(PV) is an autoimmune skin disease caused by the dysfunction of desmosomal proteins, primarily desmoglein (Dsg) 3 on the cell membrane and the subsequent damage of the cellular adhesion within epithelial tissue.[102-104] The tissue destruction in the skin is associated with the anti-Dsg3 autoantibodies. However, the mechanism . [105] Fast LbL assembly was applied with the deposition time limited to 10 s, 1 min and 5 min to enhance the fabrication efficiency. We demonstrated that the porous multilayers enhanced the protein loadings significantly, compared to the traditional silanization of AFM cantilevers. We successfully proved that the protein molecules that were bound to the porous multilayers could be locally released in a contact manner. The porous PAH/PAA multilayers on the AFM cantilever provides a novel platform for localized protein or drug delivery and quantitative examination of the cell-protein interaction.  (3) Superwettable Surfaces. Surfaces with superwettability (i.e. superhydrophobicity and superhydrophilicity) have drawn extensive attention to address issues related to fouling[16, 106], corrosion[107], fogging[80], water collection[108], etc. In order to achieve superwettability, many techniques have been focusing on the modification of surface chemistry and topography [47, 55, 109-112], including etching[113], sol-gel[114], electrochemical deposition[115], phase ` 17 separation[116], electrospining[117], etc. However, the major concerns of these techniques are the involvement of hazardous chemicals, complicated processing procedures, high manufacturing cost, and poor controllability. Recently, LbL assembly has been considered as a method to fabricate superwettable coatings as the process is aqueous based, highly tunable, and there is no limit on the choice of substrate materials. Due to the nature of the polyelectrolytes, films created by LbL are naturally hydrophilic. Fluoroalkylsilane molecules can be grafted onto these hydrophilic surfaces by chemical vapor deposition (CVD), thus making them hydrophobic. However, without proper surface topography, it is hard to achieve superwettability. LbL assembly is good at generating surface with nano-scale roughness. In order to achieve stable superwettability, the LbL technique has been combined with the deposition techniques of nanoparticles. [55, 118] However, the combination of LbL assembly with other materials and techniques increased the complexity of fabrication and the processing time. The porous PEMs built out of PAA and PAH have been around for a while; however, no -sized particle deposition onto the micro-scale porous structures was always required to achieve superwettability.[55] Few attempts have been made to identify the transitional state when both nano- and micro-scaled structures could exist on the surface. Therefore, we aimed to obtain nano- and micro-scaled hierarchical structures from porous PAA/PAH multilayers, which in turn could lead to superwettability. In Chapter 4, we have mainly investigated the effects of molecular weight of polyelectrolytes, deposition time during LbL assembly and pH for post treatment on ` 18 the porous structure and surface wettability. We focused on optimizing the above parameters and demonstrated that the post treatment of PAA/PAH multilayers can directly help generate surfaces with superwettability. Summarizing, in this thesis, we first focused on investigating the effect of molecular weight of polyelectrolytes and deposition time on the film growth and surface topography of the PAA/PAH multilayers. Then the porous PAA/PAH multilayers were fabricated and successfully loaded with ABCs to improve the surface anti-biofilm properties. The effects of molecular weight of polyelectrolytes and deposition time on the porous structures and the release profiles were further studied. Layered multi-scale porous structures were also developed to achieve a more tunable release of ABCs. We further expanded the application of porous PAA/PAH multilayers to the fabrication of superwettable surfaces. The wettability of the porous surface was controlled by tuning the molecular weight of polyelectrolytes, the deposition time and the pH for post treatment. One of the major contributions of this thesis is to shorten the fabrication time of porous PEMs, making them more industrial feasible. With the novel porous structures developed in this thesis, we believe that we broadened the possible applications of the porous PEMs.      ` 19          REFERENCES           ` 20 REFERENCES     1. Iler, R., Multilayers of colloidal particles. Journal of colloid and interface science 1966, 21 (6), 569-594. 2. 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By varying the pH conditions, the PAH/PAA multilayers were fabricated via the layer-by-layer (LbL) assembly in both linear and exponential growth regimes. We found that the effect of Mw for both PAA and PAH on the film growth is highly dependent on the charge density and deposition time. In the linear growth regime, high Mw polyelectrolytes with low charge density could slow down the adsorption step, leading to the decrease of thickness compared to low Mw polyelectrolytes when the deposition time was limit. However, this trend could be reversed by increasing deposition time, for the adsorption of low Mw polyelectrolyte reached the equilibrium, while the adsorption of high Mw polyelectrolyte continued, and the larger coil size of high Mw polyelectrolyte enabled the surpassing of multilayer thickness compared with low Mw PAA. In the exponential growth regime, besides the slow adsorption, the application of high Mw polyelectrolytes further suppressed the interlayer diffusion, leading to the decrease of multilayer thickness regardless of deposition time. We further studied the effect of deposition time, Mw of polyelectrolytes, and the number of bilayers on the surface morphology in the exponential ` 32 growth regime. Surface roughness significantly increased with the application of high Mw PAA and long deposition time. Unique patterns including islet, ring and cantaloupe skin-like structures were formed in this work by tuning the above parameters. 2.1 Introduction Polyelectrolyte multilayers (PEMs) are formed by the alternate deposition of polyanions and polycations onto the solid surfaces via layer-by-layer (LbL) assembly.[1] These films have received extensive attention during the past two decades due to their potential applications in the fields of fabricating functional thin films and coatings.[1, 2] LbL assembled films have been engineered for drug delivery systems[3-5], anti-bacterial surfaces[6, 7], substrates for cell adhesion and proliferation[8, 9], gas barrier[10, 11], gas separation[12], membrane filtration[13, 14], anti-reflection coatings[15], electrical conductive devices[16], etc.  LbL assembly is considered as a versatile technique as it is highly tunable and can control the incorporation of functional materials, film thickness, composition and polymer distribution, according to different applications. There are two possible steps which may dominate the growth of the multilayers as shown in Schematic 1(a). One is adsorption, which is the step of polyelectrolyte molecules moving from the solution to the film surface. The other is the interlayer diffusion, indicating the movement of polyelectrolyte molecules from the surface into the film. So far, two types of multilayer growth behaviors have been reported based on the two steps. (1) Linear growth, where the thickness or mass of the film increases linearly with the number of bilayers. Linear growth is adsorption controlled with the polyelectrolytes in the ` 33 solution only interacting with the oppositely charged polyelectrolytes on the film surface. [17, 18] (2) Exponential growth, where the films grow exponentially with the number of bilayers. The exponential growth behavior is based on the interlayer diffusion within the PEMs, which always happens in weak polyelectrolyte systems if assembled with partially charged polyelectrolytes.[19-22] Recent study showed that the charge mismatch between poly(allylamine hydrochloride) PAH and poly(acrylic acid) PAA controlled by the pH conditions of the polyelectrolyte solutions was associated with interlayer diffusion, and in turn was able to control the growth behavior of the multilayers.[23]  Besides pH of polyelectrolyte solutions, the deposition time also affects the film growth significantly as the deposition process of polyelectrolyte is time-dependent. [24-27] Normally it would be expected that the increase of deposition time could lead to a thicker film. However, Barrett et al.[26] assembled the multilayers using PAH and a polyanion containing an azobenzene chromophore (P-Azo) and found a significant increase of the film thickness when the deposition time was less than 5 s. Similar results were obtained by from poly (ethylene imine) (PEI)/PAA/PEI/ montmorillonite (MMT) clay quadlayer system. [25] It has also been discovered that the PEI/PAA films grew linearly with deposition time of 1 s and exponentially with longer deposition time.[27] For short deposition time, due to the time limit for interlayer diffusion, the film growth is adsorption controlled, leading to a linear growth behavior.  Another important parameter which affects the growth behavior of the multilayers is the molecular weight of polyelectrolyte. For linear growth behavior, the film thickness increased ` 34 with the increase of molecular weight of polyelectrolytes because of the more coiled polyelectrolyte chains with high molecular weight.[18, 28] However, with the existence of interlayer diffusion, the film thickness increased with the decrease of the molecular weight of polyelectrolytes, indicating a fast diffusion of low molecular weight polyelectrolytes.[29, 30] For hyaluronan (HA)/chitosan (CHI) multilayers[31], in which CHI is the diffusing polyelectrolyte and HA is the nondiffusing species, the film thickness increased with the molecular weight increase of both polyelectrolytes. In the case of HA/ poly(L-lysine) (PLL) multilayers[32], the low molecular weight PLL can diffuse into the entire film during the deposition step; whereas the diffusion of the high molecular weight PLL was restricted to the upper part of the film. However, the film thickness still increased with the increase of molecular weight of PLL, while the increase of molecular weight of HA led to a decrease of the film thickness. In general, it is difficult to predict the effect of molecular weight of polyelectrolytes on the growth behavior of multilayers. The previous studies only considered the molecular weight of polyelectrolytes as an independent variable for influencing the film growth. [18, 28, 30-32] However, we believe the effect of molecular weight is intertwined with other parameters including the deposition time and the charge density of the polyelectrolytes.  PEMs show different growth behavior depending on the type of polyelectrolytes, deposition time as well as the molecular weight. In order to design LbL multilayers successively and extend their applications, it is necessary to understand the build-up of multilayers. PAH and PAA are among the most widely used polyelectrolytes used for a variety of applications. There are ` 35 studied in detail thus far. In the present work, the growth behavior of PAH/PAA multilayers was controlled in both linear and exponential regimes by fabricating the multilayers under different pH conditions. Two distinct molecular weights for PAA (15,000 and 225,000 g/mol) and PAH (15,000 and 900,000 g/mol) were chosen to study the molecular weight effect on the film growth in detail. The application of polyelectrolytes with distinct molecular weights amplified the differences of chain mobility, and therefore enabled the observation of very detailed changes during the deposition steps. In this work, we demonstrated that the effect of molecular weight of polyelectrolytes on the film growth is dependent on the deposition time and charge density. We also proved that high molecular weight polyelectrolytes could not only suppress the interlayer diffusion but also slow down the adsorption step significantly. In addition, by tuning the molecular weight of polyelectrolytes, deposition time and number of bilayers, we were able to obtain unique surface topographies of PAH/PAA multilayers ranging from nano- to micro-scale.  It is expected that the results reported herein will be of interest for a better understanding of the build-up of weak polyelectrolyte multilayers. 2.2 Experimental Section 2.2.1 Materials The sodium salt of Poly(acrylic acid) (PAAL, Mw=15,000 g/mol, 35% aqueous solution, and PAAH, Mw=225,000 g/mol, 20% aqueous solution) was purchase from Sigma Aldrich and Polyscience, respectively. Both poly(allylamine hydrochloride)(PAHL, Mw=15,000 g/mol and ` 36 PAHH, Mw=900,000 g/mol) were purchased from Sigma-Aldrich. All aqueous solutions were prepared using 18 MMillipore water, generating by a Barnstead Nanopure Diamond-UV  Glass slide, silicon wafer, polycarbonate, and polystyrene were used as substrates for LbL assembly and cleaned extensively prior to the deposition.  2.2.2 Layer-by-Layer Assembly All LbL films were assembled with a programmable Carl-Zeiss slide-stainer. Before depositing the multilayers, the substrate was cleaned by a plasma cleaner (Harrick Scientific Corporation, Broading Ossining, NY). The films were subjected to oxygen plasma for 20 min, producing hydrophilic moieties and negative charges on the surface. After the oxygen plasma treatment, polyelectrolyte solutions were prepared (10-2 M based on repeat unit of the polymer) with 18 MMillipore water and were pH-adjusted with 0.1M HCl or NaOH. The substrate was first dipped into PAH solution (without adjusting the pH) for 20 min to form a precursor layer, follow by three washing steps. Then, substrate was introduced in the aqueous solution of PAA adjusted to a desired pH for certain amount of deposition time, followed by three washing steps with pH adjusted DI water for sufficient amount of time. Subsequently, the substrate was immersed in the PAH aqueous solution adjusted to a desired pH with the same deposition time as PAA, and washed again three times with pH adjusted DI water. The deposition process was repeated until the desired number of bilayers was obtained. Different deposition time has been tried in this study including 10s, 1min, 5min, 10min, 15min and 30 min.  ` 37 2.2.3 Film Characterization Fourier transform infrared spectroscopy (FTIR) spectra were acquired using a Mattson FTIR spectrometer with a MCT detector. To determine the degree of ionization values for PAA and PAH, the aqueous solutions of PAA and PAH for fabricating multilayers were cast onto gold coated substrates, evaporated at room temperature and further dried at 85oC for 24 hours to remove the water completely. The resulting polymer films were used for FTIR characterization. The degree of ionization for PAA and PAH was characterized following the protocol listed in reference [20]. The thickness and surface roughness of the PEM films were measured in the dry state with a Dektak surface profiler. The surface topography were measured by an Asylum Barbara, CA) performed using tapping mode. A JEOL 6610LV Scanning Electron Microscopy (SEM) was used to observe morphology of the surface. All specimens were coated with gold before examination.  2.3 Results and Discussion 2.3.1 Effect of Deposition Time on the Thickness of PAH/PAA Multilayers The polyelectrolyte multilayers were constructed by the alternate deposition of PAH at pH = 8.5 and PAA at pH = 3.5. We applied FTIR to characterize the degree of ionization of polyelectrolytes with different molecular weight (data not shown).[20] It was found that when pH = 3.5, the degree of ionization of low molecular weight PAA was 20%; while the degree of ionization of high molecular weight PAA was 18%. Moreover, the degree of ionization of PAH ` 38 with low and high molecular weight at pH = 8.5 was 58 % and 61 %, respectively. Therefore, the molecular weight did not affect the degree of ionization of polyelectrolytes. According to the previous studies[21, 23, 30], the interlayer diffusion of both PAA and PAH led to an exponential growth in the multilayers. When weak polyelectrolytes are partially charged during LbL assembly, the mobility and swelling of the multilayers facilitate the interlayer diffusion.[22, 33] The thickness values of (PAH8.5/PAA3.5)20.5 films are presented in Figure 1 as a function of deposition time. As shown in Figure 1, the thickness of (PAH8.5/PAA3.5)20.5 films increased with the deposition time, which varied from 10s to 15min, for the adsorption of polyelectrolytes, interlayer diffusion and the reconstruction of the mulilayers always take time to reach the equilibrium.[22, 33] With shorter deposition time, the interlayer diffusion was highly limited, leading to a decrease of film thickness.  2.3.2 Effect of Molecular Weight on the Film Growth of PAH/PAA Multilayers  In order to study the molecular weight effect thoroughly, we fabricated PAH/PAA multilayers using PAA with molecular weight of 15,000 g/mol (PAAL) and 225,000 g/mol (PAAH)  and PAH with molecular weight of 15,000 g/mol (PAHL) and 900,000 g/mol (PAHH). Figure 1 also illustrates the effect of molecular weight of polyelectrolytes on the thickness of (PAH8.5/PAA3.5)20.5. In general, the increase of molecular weight of polyelectrolytes led to a decrease of film thickness. However, with a less increase in molecular weight of PAA, there was a more significant decrease in the film thickness, as compared to PAH. When the deposition time increased from 10 min to 15 min, for low molecular weight PAA systems, the increase of ` 39 thickness became very limited; whereas for multilayers with high molecular weight PAA, the thickness of the multilayers still increased significantly. This indicates that the increase of molecular weight of PAA slowed down the deposition process. Even though the increase of molecular weight of PAH led to a decrease of film thickness, it did influence the trend of thickness change with the deposition time as much as the molecular weight of PAA did. In addition, when the deposition time was shortened to 10s, the molecular weight of PAH almost did not affect the film thickness, while the increase of molecular weight of PAA led to a decrease of film thickness from around 466 nm to around 245 nm. The interlayer diffusion was highly suppressed when the deposition time was 10s, regardless of the molecular weight of polyelectrolytes. Thus, the suppression of film growth indicates that the low chain mobility of high molecular weight PAA with low the degree of ionization at pH = 3.5 may lead to a slower adsorption step; whereas the adsorption for PAH did not vary by the molecular weight probably due to its relatively high degree of ionization at pH = 8.5. In order to confirm this idea, we further assembled the multilayers at pH conditions of PAH10/PAA10 and PAH3.5/PAA3.5, respectively. The molecular weight of PAA was also varied. The thickness data are summarized in Table 1. The simplified scenarios for the deposition step of PAA under different LbL assembly conditions are illustrated in Schematic 1 (b) - (d). Based on the previous study[23], when assembled at pH conditions of PAH10/PAA10 and PAH3.5/PAA3.5, the multilayers followed the linear growth behavior with suppressed interlayer diffusion. According to the thickness data in Table 1, when the multilayers fabricated under ` 40 PAH10/PAA10, the increase of molecular weight of PAA led to an increase of the film thickness, which is consistent with previous studies[18, 28]. As presented in Schematic 1 (b), if the degree of ionization of PAA is very high, the adsorption is fast due to the strong electrostatic interaction between the multilayer surface and the molecular chain. With suppressed interlayer diffusion, the film thickness increased with the molecular weight of PAA because the polymer chains of high molecular weight PAA are more coiled than those of low molecular weight PAA. In the case of PAH3.5/PAA3.5, the interlayer diffusion is also suppressed. However, the film thickness decreased with the increase of molecular weight of PAA when the deposition time was 5 min. By increasing deposition time to 30 min, the thickness of the film fabricated with high molecular weight PAA further increased and surpassed the thickness of the film fabricated with low molecular weight PAA. When the degree of ionization of PAA is very low, the electrostatic interaction between the multilayer surface and polymer chains is weakened as shown in Schematic 1 (c). The increase of molecular weight of PAA further slowed down the adsorption step due to its poor chain mobility. Therefore, the multilayers with high molecular weight PAA were built up more slowly, leading to an initial decrease of thickness compared to low molecular weight PAA. With the increase of deposition time, the deposition of low molecular weight PAA reached the equilibrium while the deposition of high molecular weight PAA continued.  Eventually, the film thickness with high molecular weight PAA exceeded the film thickness with low molecular weight PAA due to the larger coil size of high molecular weight PAA.  ` 41    Schematic 2.1. Representations of (a) the definition of adsorption and interlayer diffusion during the deposition process and (b)  (d) the deposition process of PAA with different molecular weight under the condition of PAH10/PAA10, PAH3.5/PAA3.5, and PAH8.5/PAA3.5, respectively. (The charge density or zeta potential on the multilayer surface varied by changing the pH conditions. However, the deposition of the low and high molecular weight PAA were only compared under the same deposition conditions.) Schematic 1 (d) shows the case with interlayer diffusion taking place when the degree of ionization of PAA was relatively low. For low molecular weight PAA, the adsorption was fast, therefore the interlayer diffusion was the control step for film growth. Meanwhile, high molecular weight PAA suppressed the adsorption leading to the decrease of film thickness when the deposition time was 10 s. With the increase of deposition time, the interlayer diffusion was also suppressed by the high molecular weight PAA. Due to the time scale of the experiments, we did not try to figure out the deposition time required to reach the deposition equilibrium for high ` 42 molecular weight PAA. Moreover, similar results were observed for PAH under the condition of PAH10/PAA10, when PAH was weakly charged. As shown in Table 1, with the application of high molecular weight PAH, the multilayer thickness increased when the deposition time increased from 5 to 15 min, indicating a slow adsorption step. Therefore, it can be concluded that not only interlayer diffusion can affect the growth behavior of the PEMs, the adsorption step is also critical especially for high molecular weight polyelectrolytes with low degree of ionization.    Figure 2.1 The effect of deposition time and molecular weight of polyelectrolytes on the thickness of (PAH8.5/PAA3.5)20.5 films. These thickness data were acquired with profiler.   ` 43 Table 2.1 Thickness of (PAH/PAA)20.5 multilayers fabricated under different pH conditions   Samples Deposition time (min)  (PAHH 8.5/PAAL 3.5)20.5 5 1.523 ± 0.026 (PAHH 8.5/PAAH 3.5)20.5 5 0.931 ± 0.014 (PAHH 10/PAAL 10)20.5 5 0.017 ± 0.005 (PAHH 10/PAAL 10)20.5 30 0.060 ± 0.006 (PAHH 10/PAAH 10)20.5 5 0.065 ± 0.009 (PAHH 3.5/PAAL 3.5)20.5 5 0.118 ± 0.005 (PAHH 3.5/PAAL 3.5)20.5 30 0.123 ± 0.006 (PAHH 3.5/PAAH 3.5)20.5 5 0.108 ± 0.008 (PAHH 3.5/PAAH 3.5)20.5 30 0.129 ± 0.003  2.3.3 Effect of Deposition Time on the Surface Topography of PAH/PAA Multilayers The SEM images of the surface topography of (PAH8.5/PAA3.5)20.5 films are shown in Figure 2. For the multilayers fabricated with low molecular weight of PAA, the surfaces were relatively flat and featureless under the SEM. However, the detailed structure was not very evident from the SEM images. We further applied AFM to characterize the surface topography. Figure 3 and 4 consist of the AFM 3D images for (PAHL8.5/PAAL3.5)20.5 and (PAHH8.5/PAAL3.5)20.5, respectively. As shown in Figure 3(a) and 4(a), the surface contained a lot of small islets for short deposition time. With the increase of deposition time, the surface became smoother with larger peak and valley structures. For long deposition time, the polymer chains have enough time to relax and reconstruct on the surface as well as inside the multilayers, leading to a smoother surface; while for short deposition time, the polymer chains have limited ` 44 time to settle down on the surface, leading to the formation of the small islets. A progressive roughness increase is observed in Figure 2 with increasing deposition time for high molecular weight PAA systems. When the deposition time was 10 s, the surface was relatively flat. Thus, the AFM 3D images for four systems with the deposition time of 10 s are shown in Figure 5 for comparison. As shown in Figure 5 (c) and (d), the surface consisted of small sharp islets. This island like structure was significantly enhanced when the deposition time increased to 5 min in Figure 2. When the deposition time further increased to 10 or 15 min, a micro-scale network structure was formed on the surface. The surface RMS roughness data of all four multilayer systems are listed in Table 2. In general, the RMS roughness increased with the deposition time. 2.3.4 Effect of Molecular Weight on the Surface Topography of PAH/PAA Multilayers It is obvious from Figure 2 that the application of high molecular weight PAA enabled the formation of certain patterns on the multilayer surfaces and increased the surface roughness significantly, when the deposition time increased to more than 10 s. Even when the deposition time was 10 s, comparing to the surface of the multilayers built up with low molecular weight PAA (Figure 5 (a) and (b)), the height of the islets on the surface increased significantly with high molecular weight PAA (Figure 5 (c) and (d)), leading to the increase of RMS roughness. In all, the application of high molecular weight PAA led to the increase of surface roughness, which is also consistent with the RMS roughness listed in Table 2.    ` 45  (PAHL8.5/PAAL3.5)20.5 (PAHH8.5/PAAL3.5)20.5 (PAHL8.5/PAAH3.5)20.5 (PAHH8.5/PAAH3.5)20.5   10 s       1 min       5 min       10 min       15 min      Figure 2.2 SEM images of surface morphology of (PAH8.5/PAA3.5)20.5 films. (The scale bar is  The effect of molecular weight of PAH on the surface topography was not as significant as PAA. When deposited with low molecular weight PAA, the application of high molecular weight PAH slightly changed the surface topography by decreasing the size of the islets. For example, when the deposition time was 10s, compared to multilayers built up with the low molecular ` 46 weight PAH (Figure 3 (a)), the size of the islets for the multilayers with high molecular weight PAH (Figure 4(a)) . In addition, as shown in Table 2, the RMS roughness decreased with the increase of the molecular weight of PAH when the deposition time was longer than 10 s. When deposited with high molecular weight PAA, the application of high molecular weight PAH increased the number of islets on the surface as shown in Figure 5 (b) and (d). Meanwhile, the RMS roughness shown in Table 2 increased by depositing high molecular weight PAH. It is possible that the effect of the molecular weight of PAH on the surface RMS roughness was also influenced by the molecular weight of PAA.    Figure 2.3 AFM 3D images of the surface morphology of (PAHL8.5/PAAL3.5)20.5 films with different deposition time: (a) 10 s, (b) 1 min, (c) 5 min, (d) 10 min, and (e) 15 min   ` 47    Figure 2.4 AFM 3D images of the surface morphology of (PAHH8.5/PAAL3.5)20.5 films with different deposition time: (a) 10 s, (b) 1 min, (c) 5 min, (d) 10 min, and (e) 15 min     Figure 2.5 AFM 3D images of the surface morphology of (a) (PAHL8.5/PAAL3.5)20.5, (b) (PAHH8.5/PAAL3.5)20.5, (c) (PAHL8.5/PAAH3.5)20.5 and (d) (PAHH8.5/PAAH3.5)20.5, respectively (The deposition time is 10s.)  ` 48 Table 2.2 Surface roughness of (PAH8.5/PAA3.5)20.5 films  Samples Deposition Time Roughness (nm)    (PAHL8.5/PAAL3.5)20.5 10s 2.0  1min 3.8  5min 5.4  10min 9.8  15min 9.6   (PAHH8.5/PAAL3.5)20.5 10s 2.8  1min 3.5  5min 3.7  10min 5.5  15min 5.7    (PAHL8.5/PAAH3.5)20.5 10s 19.1 1min 99.3 5min 128.8 10min 140.2 15min 158.3   (PAHH8.5/PAAH3.5)20.5 10s 20.6 1min 119.6 5min 141.3 10min 187.8 15min 222.8  2.3.5 The Build-up of PAH/PAA Multilayers with Patterned Surface As shown in Figure 2, when the deposition time increased to 10 min, the deposition of both high molecular weight PAA and PAH formed a unique micro-scale network structure. It is interesting to study how this unique network structure was built up during the LbL assembly. Therefore, Figure 6 presents the surface morphology of PAHH8.5/PAAH3.5 system changing with the number of bilayers. The thickness and roughness values are summarized in Figure 7.  As ` 49 shown in Figure 6(a), we found that with the deposition of 4.5 bilayers, the surface consisted of some islet structures. When the number of bilayers increased to 6.5 (Figure 6(b)), some micro-sized circular ring structures were formed on the surface. With a further increase of number of bilayers to 10.5(Figure 6 (c)  (d)), the number as well as the size of the ring structures both increased. Later on, the ring structures started merging together and formed the network structures, as shown in Figure 6 (e)  (i). The thickness and surface roughness values of the multilayers shown in Figure 6 are summarized in Figure 7, accordingly. As shown in Figure 7(a), initially the thickness increased exponentially with the increase of number of bilayers. After the number of bilayers reached 16.5, the increment of thickness slightly decreased. Figure 7 (b) illustrates how the surface roughness changes over the number of bilayers. It is obvious that the RMS roughness always increased with the number of bilayers until the number of bilayers reached 16.5. The RMS roughness maintained almost the same when the number of bilayers increased from 18.5 to 20.5. The above results indicate that the polyelectrolytes tend to accumulate around rough area on the surface during the initial deposition steps. Therefore, the size of the ring structures and surface RMS roughness increased with the number of bilayer. Once the micro-scale network structure was formed, the polyelectrolytes started depositing more uniformly on the surface with the surface features maintained almost the same.   ` 50                Figure 2.6 SEM images of PAHH8.5/PAAH3.5 films with number of bilayers as (a) 4.5, (b) 6.5, (c) 8.5, (d) 10.5, (e) 12.5, (f) 14.5, (g) 16.5, (h) 18.5 and (i) 20.5, respectively. (The deposition    (a)                                                                           (b) Figure 2.7 The values of (a) thickness and (b) roughness of PAHH8.5/PAAH3.5 films as a function of number of bilayers (The deposition time is 10 min.) ` 51 2.4 Conclusions In this work, we investigated the effect of the deposition time and molecular weight of polyelectrolytes on the film growth and surface topography of PAH/PAA multilayers. The PAH/PAA multilayers were assembled in both exponential and linear growth regimes by adjusting the pH conditions. In exponential growth regime, the film thickness and surface roughness increased with the increase of deposition time. High molecular weight polyelectrolytes suppressed the interlayer diffusion, leading to the decrease of film thickness. When the deposition time was 10 s with the interlayer diffusion highly suppressed, the film thickness still decreased by the deposition of high molecular weight PAA, indicating that the adsorption step was slowed down. This was further confirmed by fabricating multilayers in the linear growth regime under the condition of PAH3.5/PAA3.5, where the deposition time may reverse the molecular weight effect on the film thickness. However, the slow adsorption of high molecular weight PAA was only observed when assembled at pH = 3.5 with low degree of ionization and poor chain mobility. When PAA was fully charged at pH = 10, the film thickness increased with the molecular weight of PAA due to the larger coil size of the high molecular weight PAA. The strong electrostatic force between the molecular chain and the charged multilayer surface facilitated the adsorption step of fully charged high molecular weight PAA. The slow adsorption of high molecular weight PAH was also observed when the multilayers were assembled under the condition of PAH10/PAA10. However, in exponential growth regime, due to the relatively high charge density of PAH at pH = 8.5, no obvious suppression of the adsorption step was ` 52 found. Therefore, the effect of molecular weight of polyelectrolytes on the film growth is dependent on the degree of ionization and the deposition time. We also found that the deposition of high molecular weight PAA enhanced the surface roughness significantly. Meanwhile, the effect of molecular weight of PAH on the surface topography was not as significant as PAA in the exponential growth regime. A micro-scale network structure was obtained by applying high molecular weight PAA with deposition time longer than 10 min. It was found that the build-up of the network structure started from the structure of small islets and then the formation of circular rings. The unique patterns formed in this work show great potential in the application for cell culture for the sensitivity of cells towards the surface topography.            ` 53          REFERENCES           ` 54 REFERENCES    1. Decher, G., Fuzzy nanoassemblies: toward layered polymeric multicomposites. science 1997, 277 (5330), 1232-1237. 2. Iler, R., Multilayers of colloidal particles. Journal of colloid and interface science 1966, 21 (6), 569-594. 3. Zhu, Y.; Shi, J.; Shen, W.; Dong, X.; Feng, J.; Ruan, M.; Li, Y., Stimuliresponsive controlled drug release from a hollow mesoporous silica sphere/polyelectrolyte multilayer coreshell structure. Angewandte Chemie 2005, 117 (32), 5213-5217. 4. Berg, M. C.; Zhai, L.; Cohen, R. E.; Rubner, M. F., Controlled drug release from porous polyelectrolyte multilayers. Biomacromolecules 2006, 7 (1), 357-364. 5. 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Langmuir 2007, 23 (4), 1898-1904. 33. Yoo, D.; Shiratori, S. S.; Rubner, M. F., Controlling bilayer composition and surface wettability of sequentially adsorbed multilayers of weak polyelectrolytes. Macromolecules 1998, 31 (13), 4309-4318.   ` 57 3. DEVELOPMENT OF MULTI-SCALE POROUS THIN FILMS FOR CONTROLLED RELEASE OF ANTI-BIOFILM COMPOUNDS 3.0 Abstract In this work, we focused on the design of porous polymeric films with nano and micro sized pores existing in distinct zones. The porous thin films were fabricated by the post treatment of layer-by-layer (LbL) assembled poly(allylamine hydrochloride) (PAH)/ poly(acrylic acid) (PAA) multilayers. In order to improve the processing efficiency, the deposition time was shortened to ~ 10 s. It was found that fine porous structures could be created even by significantly reducing the processing time. The effect of using polyelectrolytes with widely different molecular weights was also studied. The pore size was increased by using the high molecular weight PAH, while high molecular weight of PAA minimized the pore size to nanometer scale. Having gained a precise control over the pore size, layered multi-scale porous thin films were further built up with either micro-sized porous zone on top of nano-sized porous zone or vice versa. The release of a novel anti-biofilm compound, named ABC-1, from selected micro-sized, nano-sized and layered multi-scale porous structures was studied. We found that the amount of ABC-1 released could be tuned by the pore volume of the thin films, and the release rate was dependent on the surface pore size. With multi-scale porous structures, the duration of ABC-1 release was highly improved to more than 20 days. With evaporation method, a 99% of biofilm suppression of V. cholera was achieved by the ABC-1 loaded micro-scale porous PEMs. Moreover, through ion-` 58 trigged exfoliation, the porous thin films can be completely removed from the substrate to form free standing films.  3.1 Introduction Porous polymeric films are in demand for a wide range of applications including foams[1], insulators[2], membranes[3], catalytic supports[4], anti-reflection coatings[5], superhydrophobic coatings[6] and drug delivery systems[7]. For many applications, sophisticated porous structures with a precise control on the pore sizes ranging from nano to micro are desired. For example, hierarchical (i.e., micro and nano sized) porous surfaces also help achieve superhydrophobicity. [8] For controlled drug release, the release rate is highly dependent on the pore size.[9] A well-controlled porous structure (i.e., mesoporous shell and microporous core) enables a tunable drug release over time.[10] In addition, commercial nanofiltration/reverse osmosis membranes have an asymmetric structure with two distinct types of porous zones; the bottom one consists of micro-sized pores while the upper zone consists of nano-sized pores. Motivated by these prospects, the multi-scale porous thin films with well-defined micro and nano-sized porous regions were developed in this work. Layer-by-layer (LbL) assembly is considered as a highly versatile deposition technique for fabricating functional thin films and coatings.[11, 12] LbL assembled polyelectrolyte multilayers (PEMs) followed by simple post-treatment steps provides one of the most promising methods to generate porous polymeric frameworks. Rubner and coworkers first demonstrated the formation of porous networks using poly(allylamine hydrochloride) (PAH)/ poly (acrylic acid) (PAA) ` 59 multilayers. The PEMs were fabricated with the PAH solution at a pH of 7.5 or 8.5 and the PAA solution at a pH of 3.5. The porous structure was formed after the post treatment, which includes acidic immersion within the pH range of 1.8-2.6, rinsing in DI water, drying and cross-linking[5, 6, 9, 13-16]. Both nano and micro sized porous films were able to be achieved by tuning the post treatment conditions. In addition, free standing porous PAH/PAA films can be obtained through an ion-triggered exfoliation method[16]. Porous thin films can also be fabricated by salt-induced structural changes in PAH/PAA multilayers.[17] The porous structures were formed by exposing the PAA/PAH multilayers fabricated in the presence of salt to pure water. However, the pore size was limited to the nanometer scale. Another way to make porous thin films via LbL assembly is through the treatment of hydrogen-bonded poly(4-vinylpyridine) (PVP)/PAA multilayers in aqueous solution at pH of 12.5 when PAA was dissolved followed by the reconfirmation of PVP chains.[18, 19] Only micro-sized pores were obtained, and the stability of the hydrogen bonded LbL films over a broad range of pH is always an issue. Thus, in this work, we applied acidic treatment to induce the porous formation in PAH/PAA multilayer films. Immersion of PAH/PAA films in a low-pH aqueous solution causes rearrangement of the polymer chains.[5, 14, 15] This rearrangement is induced by the breakage of the ionic cross-links of PAA due to protonation of the carboxylate groups and charge repulsion among the free, positively charged amine groups of PAH. The rinsing step with DI water allows ion pairs to reform and form small water pocket by rejecting water from the film. By drying water out from the water pockets and cross-linking the polymer chains, stable porous films were obtained.  ` 60 In order to create distinct zones with different scales of the pore size, the pre-requisite was to have a very precise control on each of the zones independently and to understand the factors that affect the formation of those zones. Once those factors were identified, their combination could lead us to form multi-scale porous frameworks. Previous studies mainly investigated the effect of the number of layers[13, 16], pH[5, 9, 13, 15, 20, 21] and time[13, 16, 20] of the post treatment on the morphology of the porous PAH/PAA films. However, one major obstacle in commercializing any of these films is the long processing time that goes into fabricating the PAH/PAA films using LbL technique. Recently, several studies initiated using short deposition time to address this issue and apply for gas barrier films[22, 23]. The deposition time was shortened from conventional 15-20 min to less than 1 min. It has been found that different deposition time leads to varied film compositions and structures.[22] Since the formation of porous PAH/PAA films is mainly dependent on the interaction between PAA and PAH and the reorganization of polymer chains, the changes in film composition and polymer distribution may further alter the porous structure. However, no research has been focused on studying the effect of deposition time on the porous structure, or how efficiently the porous thin films can be built up. In addition, the mobility of the individual polymer chains also plays a crucial role. During the post treatment, the reorganization of polymer chains is highly influenced by the chain mobility and the interaction among functional groups. In this regard, the molecular weight of polyelectrolytes could be one of the critical and intrinsic parameters to tune the porous structure since it highly affects the chain mobility and the intramolecular and intermolcular interactions. It ` 61 has been reported that molecular weight of polyelectrolytes plays an important role during LbL assembly.[24-27] However, few studies focused on the molecular weight effect of polyelectrolytes on the porous structure.[21] In order to study the molecular weight effect thoroughly, we fabricated PAH/PAA multilayers using PAA with molecular weight of 15,000 g/mol (PAAL) and 225,000 g/mol (PAAH) and PAH with molecular weight of 15,000 g/mol (PAHL) and 900,000 g/mol (PAHH). In this study, we focused on the effect of deposition time and molecular weight of polyelectrolytes on the porous morphology in order to shorten the fabrication time and obtain a wider and more precise control on the porous structure at the same time. Having gained a precise control over the pore size, layered multi-scale porous thin films were further built up with either micro-sized porous zone on top of nano-sized porous zone or vice versa.  Biofilms can form on variable material surfaces where bacteria can attach, proliferate into multicellular communities and secrete protective polysaccharide layer outside.[28] Biofilms are notorious for their strong immune defense, high tolerance to antibiotic treatment and difficulty in removal.[29] It has been estimated by the US Centers for Disease Control and Prevention that more than 65% of hospital-acquired infections are related to biofilms. These infections lead to more than 500,000 deaths and $94 billion medical expense annually in the United States alone.[30] Biofilms are also responsible for enormous economic losses in the industrial field as well. The formation of biofilms causes contaminations, corrosion of industrial settings, and biofouling of ` 62 more than $200 billion to deal with these industrial problems.[31, 32] Thus developing strategies to inhibit the formation of biofilms is in urgent demand. It has been found that the existing antibiotics and disinfectants cannot prevent the biofilm formation effectively. Recently, a series of novel Benzimidazole molecules have been developed as anti-biofilm compounds (ABCs) with the ability to inhibit biofilm formation. It was shown that biofilms of multiple bacteria pathogens, including Pseudomonas aeruginosa and a methicillin resistant Staphylococcus aureus can be inhibited efficiently by 5-methoxy-2-[(4-methylbenzyl) sulfanyl]-1H-benzimidazole (named ABC-1) on various material surfaces, such as silicone catheters.[33] It is believed that the ABCs have the ability to stop bacteria switching to the attachment mode by making them lose the ability of sensing the existence of material surface. However, the inhibition mechanism of the ABCs is still under investigation. ABC-1 does not kill the bacteria and does not even inhibit the growth of Gram-negative bacteria significantly. It only affects the growth of Gram-positive bacteria at high concentration. This means ABC-1 minimizes selective pressure for resistance.[34] In order to generate a surface with good anti-biofilm properties, we want to take advantage of the novel anti-biofilm compounds, such as ABC-1. However, for small molecules like novel benzimidazole compounds, they don't have the ability to stay on the material surface by themselves and release as needed. Thus, a surface drug reservoir system is desired, and the porous PAH/PAA films is considered as an ideal candidate.  With porous PAH/PAA films, hydrophobic ABC-1 was successfully incorporated. In this ` 63 deposition time and molecular weight of polyelectrolytes. More importantly, multi-scale porous structures have been successfully built up to achieve different release rate at different stage. The release profile was studied for nano-, micro, and multi-scale porous structures in detail. The bacterial test showed that a 99% of biofilm inhibition was achieved. The development of free standing porous PAH/PAA films enables the possible application in the field of wound healing and membrane filtration.  3.2 Experimental Section 3.2.1 Materials Poly (acrylic acid, sodium salt) solutions with different molecular weight (PAAL, Mw=15,000 g/mol, 35% aqueous solution, and PAAH, Mw=225,000 g/mol, 20% aqueous solution) were purchase from Sigma Aldrich and Polyscience, respectively. Both poly(allylamine hydrochloride) (PAHL, Mw=15,000 g/mol and PAHH, Mw=900,000 g/mol) were purchased from Sigma-Aldrich. All aqueous solutions were prepared using 18.2 MMillipore water at a concentration of 10 mM with respect to the repeat unit and adjusted to the required pH using 0.1M HCl or NaOH solutions. 5-methoxy-2-[(4-methylbenzyl) sulfanyl]-1H-benzimidazole (ABC-1) was kindly provided by Dr. Waters group in the department of Microbiology and Molecular Genetics at Michigan State University. Glass slides from Globe Scientific Inc. were cleaned by sonication for 20 min each in ethanol and DI water and then exposed to oxygen plasma generated by a Harrick plasma cleaner (Harrick Scientific Corporation, Broading Ossining, NY) for 20 min, producing hydrophilic moieties and negative charges on the surface. ` 64 Sodium chloride (NaCl), potassium chloride (KCl), dibasic sodium phosphate (Na2HPO4) and monobasic potassium phosphate (KH2PO4) are purchased from J.T. Baker. Phosphate buffer saline (PBS) was prepared with 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 and was adjusted to pH=7.4 with 1M hydrochloric acid. Ethanol was obtained from KOPTEC. 3.2.2 Fabrication of Porous Thin Films  All LbL films were assembled with a programmable Carl-Zeiss slide-stainer. After the oxygen plasma treatment, the glass substrates were immediately dipped into PAH solution (without adjusting the pH) for 20 min to form the precursor layer, followed by three washing steps. Then, the substrates were introduced in the aqueous solution of PAA (pH = 3.5) for required deposition time, followed by three washing steps with DI water (pH = 3.5) for sufficient time. Subsequently, the substrates were immersed in the PAH (pH = 8.5) aqueous solution with the same deposition time as PAA, and washed again three times with DI water (pH = 8.5). The deposition process was repeated 20 times. In total, 20.5 bilayers were deposited on the substrate, including the first PAH precursor layer. Deposition time of 10 s, 1 min, 5 min, 10 min and 15 min were applied in this work. The assembled polyelectrolyte multilayer films were immersed in the water solution with pH of 2.0 for 5 min followed by washing with DI water (pH =5.5) for 5 min. After the porosity induction, the films were dried and then heated at 180 oC for 2 hours to cross-link the films and prevent the porous structure from being distorted. This post treatment helped create porous films as described by other researchers.[5, 9, 15, 20] ` 65 For fabricating the layered multi-scale porous thin films, the bottom porous portion was first built up by repeating the previous steps and considered as the substrate for the following LbL assembly. The bottom porous portion was further introduced in the aqueous solution of PAA (pH = 3.5) for required deposition time and then PAH (pH = 8.5) aqueous solution with three washing steps in between. After 20 bilayers of PAA/PAH were built up, the entire thin film went through the post treatment again for making the top portion porous. Eventually, the entire thin film was further thermally cross-linked at 180 oC for 2 hours.  3.2.3 The Loading and Release of ABC-1 ABC-1 molecules were loaded into the porous PEM films through immersion in an ABC-1/ethanol solution (10mg/ml) for 6h. Then the samples were put in vacuum for one day to remove ethanol. After drying, samples were submerged in 250ml of PBS solution. The concentration of the ABC-1 was determined by 3200 QTRAP LC/MS/MS mass spectrometer using electrospray ionization. An Aecentis® Express C18 2.7  is used. Acetonitrile and 10mM ammonium acetate aqueous solution were used as the binary solvent system. A flow rate of 0.3 ml/min was used providing retention time of around 1.5 minutes for ABC-1.The oven temperature was set to 50 oC. The linear calibration range was selected from 5 to 500 nM with R2 = 0.993-0.997. Programming and quantification were carried out using ANALYST software. Corresponding to every time points, one milliliter solution was taken out from the release media. Usually, the samples were further diluted 10 times in order to fit in the calibration range.  ` 66 The total ABC-1 release from a single film was calculated according to the equation (1)                                                       (2) where Mi (g/cm2) is the total cumulative mass released from the 1cm2 area as of measurement i, A is the surface area of the drug-loaded porous samples, Ci (nM) is the concentration of sample i, Vi (ml) is the total volume of the release media prior to measurement i, and 285.2 is the molar mass of ABC-1.  An evaporation method was also applied for loading ABC-1. A coupon coated with the porous PEM films was first placed at the bottom of a glass scintillation vial and immersed with 3 ml ABC-1/ethanol solution (10mg/ml). Then, the scintillation vial was placed in a vacuum oven under 25 inch Hg vacuum over night. During the evaporation of ethanol, most of the ABC-1 compounds were wicked into the porous PEM films.  3.2.4 Biofilm Test The V. cholera was a derivative of El Tor C6706str2. V. cholerae cells were grown at 37°C with constant aeration in Luria-Bertani (LB) broth. The cell concentration of the bacteria media was 1×109 cells/ml, determined by standardizing the OD600 to 1.25. After the loading of ABC-1 into the porous films via the evaporation method, the coupons were immersed in the bacteria media and placed in a shaker at 220rpm at 35oC for 8 hours. Then, the coupons were taken out from the media and rinsed with PBS solution to remove the planktonic bacteria. The biofilms ` 67 formed on the coupon surface were disaggregated by vortexing for 30 s, sonicating for 3 min and vortexing for 30s. The solution was diluted serially and plated on the Trypticase soy agar (TSA) culture. The colonies were counted after the incubation for 24 hours to determine the ability of bacteria to attach to each sample. 3.2.5 Preparation of Free Standing Porous PEMs The porous (PAH/PAA)20.5 films are further immersed in DI water with pH adjusted to 12.0 for 30min to exfoliate the porous films from the substrate.  3.2.6 Film Characterization The thickness of the thin films before and after the post-treatment was measured in the dry state using a Dektak surface profiler. A JEOL 6610LV Scanning Electron Microscopy (SEM) was used to observe the surface and fractured cross-section morphology of the porous thin films. All specimens were coated with gold before examination under the SEM. 3.3 Results and Discussion 3.3.1 Effect of Molecular Weight of Polyelectrolytes and Deposition Time on the Formation of Porous PEMs In this work, the PAH/PAA multilayers were constructed by the alternate deposition of PAH at pH=8.5 and PAA at pH=3.5. According to the literatures, the degree of ionization of PAA in aqueous solution with pH=3.5 is less than 20%[35, 36], while the degree of ionization of PAH in aqueous solution with pH=8.5 is around 60%[35, 37]. Under this pH condition, a high ` 68 level of interlayer diffusion occurs in order for charge compensation to take place, leading to an exponential growth in the thickness of the multilayer films.[36, 38]  The variations in the thickness of (PAHL/PAAL)20.5 films as a function of the deposition time are shown in Figure 3.1(a). The deposition time varies from 10 s to 15 min. Before the post treatment, the multilayer thickness increases with the increase in deposition time as a result of time-dependent interlayer diffusion process. For short deposition time, the interlayer diffusion is suppressed, leading to thinner multilayers. This result is consistent with several previous studies[22, 38]. When the deposition time was increased from 10 to 15 min, the thickness almost remained the same. In fact, besides the change in thickness, the composition and distribution of PAA and PAH in the multilayers were also altered by deposition time.[22] All these factors affect the breakage of the ionic cross-links and the rearrangement of polymer chains, leading to different porous structures. The acid treatment was carried out under the condition of pH=2.0 for 5 min followed by 5 min of washing step with DI water. The porous structure was then thermally corss-linked at 180oC for 2 hours. The entire post treatment protocol was maintained the same throughout this study. Figure 3.2 shows the SEM images of the surface and cross-section of the porous structure with different deposition time. The values of average surface pore size are summarized in Figure 3.1(b). The average surface pore size increased sharply from approximately 108 to 259 nm when the deposition time changed from 10 s to 1 min. With deposition time further increased to 5, 10 and 15 min, the average surface pore size increased to approximately 305, 327 and 361 nm, respectively. In general, longer deposition time created ` 69 larger surface pore size. It is also obvious from Figure 3.2 that the inner pore size is different than the surface pore size. Micro-sized pores were successfully formed throughout the entire cross-section of the films. It is hard to measure the actual inner pore size, because the pores were highly interconnected. However, it is still obvious that the inner pore size increased as the dipping prolonged from 10s to 1 min. Figure 3.1(b) also shows the relative expansion of thickness as a function of deposition time. The value is not always proportional to the deposition time. This indicates that several intertwined factors like film composition, polyelectrolyte distribution, the nature of polyelectrolytes (i.e., chain mobility, hydrophilicity), mass lost during the post treatment[20], etc., influence the porous structure in a synergistic manner. The interlayer diffusion during LbL assembly definitely facilitated the formation of pores. Even though there are certain differences in thickness and pore size, the porous structures are very similar to each other when deposition time is longer than 5 min. Proper porous structure could be generated by the PAHL/PAAL multilayers assembled with deposition time of 10 s in a much faster way and with a smaller pore size.  ` 70          Figure 3.1 The effect of deposition time on (a) the thickness of PAHL/PAAL thin films before and after the post treatment and (b) the relative expansion of thickness and average surface pore size ` 71                                    Figure 3.2 (a) and (b), (c) and (d), (e) and (f), (g) and (h), and (i) and (j) are the top-view and cross-sectional SEM images for porous (PAHL/PAAL)20.5 films with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. The arrow in each cross-sectional image indicates the interface between the glass substrate and the deposited film. ` 72 Considering the efficiency of fabricating porous films, 10 s dipping was further applied to different molecular weight systems in order to study the molecular weight effect on the porous morphology. The acid treatment was still carried out by immersing PAH/PAA multilayers in pH=2.0 aqueous solution for 5 min followed by 5 min of washing with DI water. Figure 3.3(a) illustrates the effect of molecular weight on the thickness of the films before and after post treatment. Before porous treatment, the thickness for (PAHL/PAAL)20.5 is almost the same as (PAHH/PAAL)20.5. Similar results were also found for (PAHL/PAAH)20.5 and (PAHH/PAAH)20.5 films, which means the molecular weight of PAH does not affect the thickness of the films significantly. However, high molecular weight of PAA led to a decrease in thickness for multilayers fabricated using the same molecular weight of PAH. After the post treatment, the relative expansion of thickness for (PAHL/PAAL)20.5, (PAHH/PAAL)20.5, (PAHL/PAAH)20.5, and (PAHH/PAAH)20.5 are shown in Figure 3.3(b), respectively. With the same molecular weight of PAA, high molecular weight of PAH provided higher relative expansion of thickness; while with same molecular weight of PAH, high molecular weight of PAA limited the thickness expansion during the post treatment. SEM images for all four porous thin films are shown in Figure 3.4. It is obvious from the top-view images in Figure 3.4 that high molecular weight of PAH not only creates larger surface pore size but also leads to a less uniform pore size distribution. In addition, high molecular weight PAA lowers the pore size significantly, which is consistent with what has been reported previously[21]. The values of average surface pore size are presented in Figure 3.3(b). The surface pore size was able to be tuned from 25 to 133 nm with different molecular ` 73 weight combinations. Similar results can also be obtained for the inner pore size from the cross-sectional images in Figure 3.4, that high molecular weight of PAA led to a decrease in the inner pore size, while high molecular weight of PAH provided larger inner pores. According to the previous studies[14, 15], when PAH/PAA multilayers are immersed in pH = 2.0 aqueous solution, the carboxylate groups from PAA are protonated leading to the breakage of ionic cross-links, while the amine groups from PAH become fully charged. The intramolecular charge-charge repulsion for high molecular weight of PAH is much stronger than that of low molecular weight of PAH. This explains why the high molecular weight PAH caused larger pore size as well as higher relative expansion of thickness. Besides, the reorganization of polymer chain during post treatment is highly dependent on the chain mobility of the polyelectrolytes. PAA has very low charge density when immersed in pH=2.0 aqueous solution. The chain mobility is highly limited by using higher molecular weight of PAA, leading to a smaller pore size and consequently a lower relative expansion of thickness. It is apparent that the molecular weight of the polyelectrolytes plays a very important role in the formation of porous structures. However, the molecular weight effect doesnt only exist during the post treatment. During the LbL assembly, the molecular weight of polyelectrolytes also affects the adsorption and interlayer diffusion, leading to different film composition and distribution of polyelectrolytes.[25, 27, 39] It is hard to differentiate how these factors affect the porous structure independently, even though the deposition time of 10 s was applied when the interlayer diffusion is highly limited. But from ` 74 the above results, there is no doubt that changing molecular weight of polyelectrolytes enables a wider control of pore size and morphology.         Figure 3.3 (a) thickness of thin films before and after the post treatment and (b) the relative expansion of thickness and average surface pore size for (PAHL/PAAL)20.5, (PAHH/PAAL)20.5, (PAHL/PAAH)20.5, and (PAHH/PAAH)20.5, respectively. All the films were fabricated using deposition time of 10 s.  ` 75                                     Figure 3.4 (a) and (b), (c) and (d), (e) and (f), and (g) and (h) are the top-view and cross-sectional SEM images for porous (PAHL/PAAL)20.5, (PAHH/PAAL)20.5, (PAHL/PAAH)20.5, and (PAHH/PAAH)20.5 films, respectively. The arrow in each cross-sectional image indicates the interface between the glass substrate and the deposited film. All the films were fabricated using deposition time of 10 s.  ` 76 3.3.2 Layered Multi-scale Porous PEMs Based on the porous structures described above, multi-scale porous films were fabricated which constituted of a macro-sized porous zone on top of nano-sized porous zone (Figure 3.5) or the other way around (Figure 3.6). To fabricate these films, the bottom porous portion was made first using the usual protocol of LbL assembly followed by the post treatment. The PAA and PAH chains were completely reorganized during the acid immersion and DI water rinsing step, leaving both COO- groups and NH3+ groups on the surface. The cross-linking step causes the formation of amide bonds (-NHCO-) between the COO- groups of PAA and NH3+of PAH and preserves the porous structure from being altered by further immersion in aqueous solution[15, 40]. Some free carboxylate groups and ammonium groups remained in the films after the cross-linking[40]. The remaining free ammonium groups on the surface enabled the deposition of PAA at pH 3.5, when ammonium groups are completely charged and carboxylate groups become mostly protonated. The bottom porous thin film thereby acted as the substrate to further build up porous films on the top. Considering the quality of the porous structure and the fabrication efficiency, porous PAHL/PAAL thin film with 5 min dipping was selected as the micro-sized porous portion, while porous PAHL/PAAH thin film with deposition time of 10 s was chosen as the nano-sized porous portion.  As shown in Figure 5, porous (PAHL/PAAH)20.5 thin film with deposition time of 10 s was first built up as the bottom portion, followed by the porous (PAAL/PAHL)20 thin film with deposition time of 5 min. Two clearly defined zones with different pore sizes were fabricated by ` 77 this method, without any significant penetration of polyelectrolytes into the nano-sized bottom portion. In addition, the surface and cross sectional morphology for micro-sized top zone of the multi-scale porous films remained almost the same as the simple porous (PAHL/PAAL)20.5 films with deposition time of 5 min (Figure 3.2 (e) and (f)). Hence the substrate effect was minimal for the micro-sized porous top zone.  Figure 3.5 SEM cross-sectional ((a) and (b)) and top view (c) images of multi-scale porous thin films with nano-sized porous film as the bottom and micro-sized porous film as the top. The arrow in (a) indicates the interface between the glass substrate and the deposited film. (b) is an enlarged image of the red square area in (a). In the above mentioned scenario, the underlying porous portion had very small surface pore size, therefore the molecular weight of the polyelectrolytes used to build up the top porous portion, is not a matter of serious concern. However, if the bottom portion is made with relatively larger surface pore size, PAA with high molecular weight is required for the top porous portion. This is because the polymer chain size needs to be large enough for not diffusing into the porous bottom. In addition, after the bottom portion was thermally cross-linked, the surface became more hydrophobic, which helped trap air inside the porous structure and block the polyelectrolytes outside. As shown in Figure 3.6 (a) and (b), nano-sized porous structure of (PAAH/PAHL)20 with 10 s dipping has been successfully fabricated on top of the porous ` 78 (PAHL/PAAL)20.5 films with deposition time of 5 min. It is clear that no polyelectrolytes entered into the micro-sized porous bottom. Figure 3.6(c) shows the top view of this particular multi-scale porous thin film. Compared to Figure 3.4(e), it has been found that the number of pores decreases, while the pore size increases to 50 ± 19 nm. There was a slight change in the porous morphology for the nano-sized porous region of the multi-scale porous thin film from the simple porous (PAAH/PAHL)20 film with 10 s dipping. This is mainly because the micro-sized porous bottom has different charge density than the plasma treated glass slides, and the substrate effect is relatively more obvious when the film is very thin.  Figure 3.6 SEM cross-sectional ((a) and (b)) and top view (c) images of multi-scale porous thin films with micro-sized porous structure as the bottom and nano-sized porous structure as the top. The arrow in (a) indicates the interface between the glass substrate and the deposited film. (b) is an enlarged image of the red square area in (a). 3.3.3 Release of ABC-1 Molecules from Porous PEMs According to the porous structure developed above, four types of porous thin films were selected for investigating the release profile of ABC-1. Porous (PAHL/PAAL)20.5 with 5min dipping and (PAHL/PAAH)20.5 with 10s dipping are referred as the micro-sized and nano-sized porous structure in Figure 3.7, respectively. Multi-scale porous structures were also built up based on a combination of these two porous thin films. As shown in Figure 3.7, for micro-sized ` 79 porous structure, the release of ABC-1 is much faster than the others. The release of ABC-1 reached equilibrium at around 150 hours, and the total release amount is around g/cm2. For the first 48 hour, the average release rate of ABC-g/(cm2·h). However, it took nano-sized porous structure over 300 hours to reach a total release of g/cm2. There was an initial burst release due to remaining ABC-1 molecules weakly attached on the surface of the porous structures. After 48 hours, the release exhibited a zero-order release kinetics. The average release rate of ABC-1 from nano-sized porous structure is around 5.76×10-2 2·h). The amount of total release is different between nano-sized and micro-sized porous structure due to different pore volume in the porous films.   Figure 3.7 Accumulative release profiles of ABC-1 from different porous (PAH/PAA)20.5 thin films. In order to gain a better understanding of the release behaviors from micro- and nano-sized porous structures, a model developed by Peppas et al. was applied as follows:   ` 80  where Mt is the total amount of drug released at time t, M is the total amount of drug released as time goes to infinity, K is a release rate constant, and n is the exponent characteristic of the release mechanism. The value of n is equal to 1 for zero-order kinetics and is equal to 0.5 for Fickian diffusion. Equation (3) can be modified into Equation (4) as follows:  Therefore, we plotted vs. log t in Figure 3.8. As shown in Figure 3.8 (a), the n value for micro-sized porous structure is 0.503, indicating the release kinetics as Fickian diffusion. As shown in Figure 3.8 (b), the n value for nano-sized porous structure is 0.982, which is very close to zero-order kinetics. This is consistent with the previous studies[9] that the differences in the film morphology enabled the change of the release kinetics.   Figure 3.8 The plots of release kinetics for (a) micro- and (b) nano-sized porous structures, respectively.      ` 81 The release profile of multi-scale porous thin films with nano-sized porous structure on top of micro-sized porous structure is very similar to nano-sized porous structure for the first 300 hours. The reason can be attributed to the fact that the top nano-sized porous structure controls the release rate of ABC-1. The bottom micro-sized porous structure contains more pore volume, which allows more ABC-1 to be incorporated. Thus, the concentration of ABC-1 continuously increases after 300 hours. The average release rate of ABC-1 for multi-scale porous thin films with nano-sized porous structure on top of micro-sized porous structure is around 3.37×10-2 2·h) after the initial burst release. The n value of 1.01 was further calculated out with the subtraction of the initial burst release, indicating a zero-order release kinetic. The release rate of ABC-1 from nano-sized porous structure is slightly higher than that of multi-scale porous thin films with nano-sized porous structure on top of micro-sized porous structure. This is consistent with the SEM images shown in Figure 3.5 (a) and Figure 3.6 (a). Although the surface pore size of the multi-scale porous thin films with nano-sized porous structure on top of micro-sized porous structure is slightly larger, the number of pores is much less than that of nano-sized porous structure. For both nano-sized porous structure and the multi-scale porous thin films with nano-sized porous structure on top of micro-sized porous structure, there is a small initial burst release because some free ABC-1 molecules were left on the surface during the incorporation process.    For the multi-scale porous thin films with micro-sized porous structure on top of nano-sized porous structure, two slopes were found in the release profile. At the initial release stage, there is ` 82 a burst release of ABC-1 with an average release rate of 0.96 2·h). The n value was 0.51 for the initial release stage, indicating a Fickian diffusion. These proved that the top micro-sized porous structure controlled the initial release of ABC-1. However, the amount of the initial release is much less than that of micro-sized porous structure. In addition, after 100 hours, the average release rate of ABC-1 drops significantly to 2.56×10-2 2·h) and the n value became 0.70. It is probably because the ABC-1 molecules tend to settle down at the nano-sized bottom during the drying step after immersing porous structure in the ABC-1/ethanol solution. After the ABC-1 molecules were depleted from the top micro-sized porous structure, the ABC-1 molecules started releasing from the bottom nano-sized porous structure, while the top micro-sized porous structure became an obstacle for ABC-1 to release out. This explains the decrease of the release rate and the n value. The release for both multi-scale porous structures hasnt reached the equilibrium within 20 days. 3.3.4 Anti-biofilm Performance of ABC-1 Loaded Porous PEMs ABC-1 is known as an effective anti-biofilm compound towards V. cholera. The bacterial test was carried out by immersing the coupons into V. cholera media with a concentration of 1×109 cells/ml. The formed biofilm was dissociated from the surface and incubated on the TSA culture. The results for the plate counting of V. cholera colonies are shown in Figure 3.9. It is obvious that V. cholera can form biofilms easily on bare PS coupon. The decrease of the CFU values for coupons coated with only porous PEMs indicates the deposition of porous PEM films helped increase the surface anti-biofilm properties. This is because PAH contains amine groups ` 83 which provide the good anti-bacterial property.[41] At the same time, the porous PEMs swell in water, proving a moving surface which is hard for bacterial to attach.[42] The ABC-1 was loaded by both immersion method and evaporation method. After the incorporation of ABC-1, the anti-biofilm property on the surface was improved significantly as shown in Figure 3.9. We achieved a 99% suppression of biofilm formation with the ABC-1 loaded micro-scale porous PEMs via the evaporation method. The inhibition of biofilm formation for the ABC-1 loaded micro-scale porous PEMs via the immersion methods was not as efficient as the ABC-1 loaded micro-scale porous PEMs. This is mainly due to fact that more ABC-1 could be incorporated into the porous PEMS by the evaporation method, leading to a high ABC-1 concentration on the surface to suppress the biofilm formation. In addition, we also found that it is easier for the biofilm to form on the coupon coated micro-scale porous PEMs than on the coupon coated with nano-scale porous PEMs for the reason that micro-scale porous PEMs provided a rougher surface, which is easier for the bacteria attachment.[43] However, the release of ABC-1 from nano-scale porous structures was too slow to achieve a good biofilm inhibition. From our groustudies[33], the hydrogen bonded (PAA/Polyethylene glycol (PEG))5-(ABC-1/PEG)5.5, was fabricated. When immersed in the bacteria media, the hydrogen boned multilayers started dissociating from the surface due to the breakage of the hydrogen bonds after the pH change of the surrounding, thus the ABC-1 could be released. The dissociation of the polyelectrolytes from the coupon surface also provided a moving surface which is hard for the bacteria to attach.[41] In addition, PEG is well known as a type of anti-bacterial polymers which provide no binding side ` 84 for the bacteria attachment. [44, 45] However, the combination of ABC-1 release, the dissociation of the multilayers and the anti-bacterial property of PEG, the suppression of biofilm formation was only about 50 - 60 %. With the porous PEMs, we are able to incorporate more ABC-1 and achieve better anti-biofilm properties. Moreover, the fabrication of the hydrogen bonded multilayers also required longer processing time and the involvement of dimethyl sulfoxide (DMSO) to disperse ABC-1 in the aqueous solution.  Figure 3.9 Bacterial colonies formed by the disaggregated V. cholera cells from the biofilm on the coupon surfaces 3.3.5 Free Standing Porous PEMs The development of free-standing films is extremely useful for the application in the fields of membrane separation, sensors, micromechanical devices, wound dressing, or even artificial organs[46-49]. The porous (PAH/PAA)20.5 thin films can be directly exfoliated from the ` 85 substrates by an ion-triggered exfoliation[50, 51]. The exfoliation solution is simply an aqueous solution with pH adjusted to 12.0. After immersing the thermally cross-linked porous (PAH/PAA)20.5 films into exfoliation solution for 30min, a flexible free-standing porous (PAH/PAA)20.5 film was obtained (Figure 3.10). The exfoliation of the porous films was achieved by breaking the electrostatic interaction of the first PAH layer with the substrate, as the amine groups of PAH are highly protonated in the exfoliation solution. This method requires no sacrificial layer or any special precursor layer as described in previous studies[49-51]. In addition, after the exfoliation process, the surface of substrate is completely cleaned up and can be further used for another LbL assembly.  Figure 3.10 Photograph of a free standing porous (PAHL/PAAL)20.5 with 15min dipping and pH=2.0 for post-treatment in the exfoliation solution. The diameter of the Petri dish is 10cm. 3.4 Conclusions In this work, porous poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) films have been fabricated via Layer-by-Layer (LbL) assembly followed by a post-treatment under ` 86 acidic conditions. Multi-scale porous thin films have been developed for the first time with either micro-sized porous structure on top of nano-sized porous structure or vice versa. In order to build up the porous thin films more efficiently, the effect of deposition time on the morphology of porous films was investigated for the first time in this work. Compared to conventional 15 or 20 min dipping, we were able to shorten the deposition time to 10 s but still maintain fine porous structures. The molecular weight effect of both PAH and PAA were also studied. While an increase in the molecular weight of PAH led to an increase in the pore size due to the increase charge-charge repulsion during the post treatment. A decrease in the pore size was observed for high molecular weight of PAA. This is due to the decrease of chain mobility while PAA was almost not charged during the post treatment. In most cases, the pore size increased with the dipping time and the pH for the post treatment. By tuning the dipping time and molecular weight of polyelectrolytes, the pore size could be precisely controlled ranging from 20nm to over 10m. The layered multi-scale porous thin films were further fabricated by tuning the tipping time and molecular weight of polyelectrolytes.  For the first time, the multi-scale porous structures have been developed with either micro-sized porous structure on top of nano-sized porous structure or vice versa. (PAHL/PAAH)20.5 with 10s dipping and pH=2.0 for the post-treatment and (PAHL/PAAL)20.5 with 5min dipping and pH=2.0 for the post-treatment were selected as examples for nano-sized and micro-sized porous thin films, respectively. Multi-porous structures were further built up based on the two systems. The release profiles of ABC-1 from the selected nano-sized, micro-sized and multi-scale porous ` 87 structures were further studied. It has been proved that the release of ABC-1 can be controlled by tuning the porous structure to achieve initial burst release, sustained release, or a combination of both. For multi-scale porous structure, the release of ABC-1 could last for more than 20 days. V. cholera was picked for the bacterial test. ABC-1 was loaded via the evaporation method. We achieved a 99% suppression of biofilm formation with the ABC-1 loaded micro-scale porous PEMs. In addition, through an ion-trigged exfoliation, the porous thin films were completely removed from the substrate to form free standing films.              ` 88          REFERENCES           ` 89 REFERENCES    1. Goel, S. K.; Beckman, E. J., Generation of microcellular polymeric foams using supercritical carbon dioxide. I: Effect of pressure and temperature on nucleation. Polymer Engineering & Science 1994, 34 (14), 1137-1147. 2. Nguyen, C. V.; Carter, K. R.; Hawker, C. J.; Hedrick, J. L.; Jaffe, R. L.; Miller, R. D.; Remenar, J. F.; Rhee, H.-W.; Rice, P. M.; Toney, M. F., Low-dielectric, nanoporous organosilicate films prepared via inorganic/organic polymer hybrid templates. Chemistry of Materials 1999, 11 (11), 3080-3085. 3. Sanyal, O.; Sommerfeld, A. N.; Lee, I., Design of ultrathin nanostructured polyelectrolyte-based membranes with high perchlorate rejection and high permeability. 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ChemistryAn Asian Journal 2014.           ` 94 4. POROUS MULTILAYER-COATED AFM CANTILEVERS FOR PROTEIN DELIVERY  4.0 Abstract In this work, the porous poly (allylamine hydrochloride) (PAH)/ poly(acrylic acid) (PAA) multilayers were successfully deposited onto the atomic force microscopy (AFM) cantilever and behaved as the reservoir for protein delivery. Fast layer-by-layer (LbL) assembly was applied, which significantly reduced the fabrication time of porous multilayers. Compared to the traditional silanization of AFM cantilevers with (3-aminopropyl) triethoxysilane (APTES), the porous multilayers enabled much higher protein loadings. In addition, the protein loading increased with the thickness of the porous multilayers, which was controlled by the deposition time during LbL assembly. We further demonstrated that the protein molecules were bound to the porous multilayers via both strong electrostatic interaction and weak secondary interaction. The bound protein molecules enabled a local delivery in a contact manner.  4.1 Introduction Cell membranes consist of a lot of functional protein receptors.[1-3] The dysfunction of the protein receptors after bonded with pathogenic proteins (i.e. autoantibody) may cause tissue destruction or necrosis in the skin and originate many skin diseases, such as pemphigus vulgaris (PV), pemphigoid, and epidermolysis bullosa acquista.[4] In order to study the disease model and mechanisms, the conventional injection of proteins or drugs into the cells wouldnt work since the protein receptors only exist on the cell membranes. Meanwhile, the direct addition of ` 95 proteins to the cell media prevents the study of cell-cell interaction. Therefore, a delivery in a contact manner to the membrane of a specific cell is required.  Atomic force microscopy (AFM) has been developed as a nanorobot, which facilitates the applications of localized mechanical and chemical stimulations to a cell of interest as well as provides real-time cellular and molecular responses. [5] The advances of AFM based nanorobot allow the local delivery of drug/protein directly to the cell membrane, showing great application potential in the field of drug delivery and cell biology. In order to load proteins onto the AFM cantilever, the functionalization of the cantilever surface is in great demand. The most commonly used method is the aminofunctionalization with silane molecules such as (3-aminopropyl) triethoxysilane (APTES) and aminophenyl-trimethoxysilane (APhS).[6-8] This method enables the conjugation of a single layer of protein molecules through electrostatic interaction. Both mechanical and chemical signals can be applied to the cell.[7] However, the limit loading of proteins restricts the study of cell responses over a sufficient amount of time and impedes the establishment of the disease model.  Therefore, in this work, we coated the AFM cantilever with porous films to increase the loadings, maintain the functionalities, and achieve a controlled release of the protein molecules. Layer-by-layer assembly has been considered as a versatile technique for surface modification especially for biomedical applications as the process is aqueous based, highly tunable, and with no limit on the choice of substrate materials. LbL assembled multilayers followed by simple post-treatment steps with different pHs[9, 10], ionic strengths[11] and ` 96 electrical fields[12] provides one of the most promising methods to generate porous polymeric frameworks. Wu et al.[13] coated the AFM tips with hydrogen-boned poly(acrylic acid) (PAA)/ poly(4-vinylpyridine) (PVP) multilayers and induced nanoporous structures by dissolving PAA in the KOH solution (pH = 13). They successfully incorporated a water-soluble fluorescent biological activity. The porous structure behaved as an ink reservoir for dip-pen nanolithography (DPN) to fabricate patterns with micrometer and submicrometer scales. By controlling the post treatment time in KOH solution, the film thickness and pore sizes can be adjusted. However, the stability of the hydrogen bonded LbL films over a broad range of pH is always an issue. Long deposition time was required for assembling the hydrogen bonded multilayers due to the weak interaction between PAA and PVP. More importantly, this DPN approach could only enable a protein delivery in air but not in the cell media due to the large amount of free protein molecules in the porous films which could diffuse to the media and interact with other cells. Rubner et al. discovered the formation of porous networks by immersing poly(allylamine hydrochloride) (PAH)/PAA multilayers in an acidic aqueous solution with pH ranging from 1.6 to 2.6.[9, 14-19]. PAA and PAH are both weak polyelectrolytes with the degree of ionization dependent on the local pH. Therefore, the formation of porous structure is induced at acidic pHs by the breakage of the ionic cross-links in the multilayers due to the protonation of the carboxylate groups and charge repulsion among the free, positively charged amine groups of PAH. From the previous studies[9, 20-22], the porous structures can be well controlled in the nano and micrometer range ` 97 by manipulating the molecular weight of polyelectrolytes, the pH and the exposure time during the post treatment. The porous structure can be obtained even when the deposition time for PAA and PAH during LbL assembly was shortened from conventional 15-20 min to 10 s.[20] In addition, though crosslinked, free amine and carboxylate groups were remained in the porous multilayers, providing binding sites for protein molecules. The controllability on the porous structures, fast fabrication process and availability of protein binding sites make the porous PAH/PAA multilayers an ideal candidate to perform as reservoirs on the AFM cantilever for protein loading and delivery. In this work, we loaded anti-desmoglein (anti-Dsg) 3 autoantibodies and fluorescence labeled goat anti-mouse IgG as example proteins into the porous PAH/PAA multilayers on the AFM cantilever and studied the protein release. The fluorescence labeled goat anti-mouse IgG can also behave as a secondary antibody for labeling anti-Dsg 3. Anti-Dsg 3 autoantibodies is associated with PV, which is an autoimmune skin disease caused by the dysfunction of desmosomal proteins, primarily Dsg 3 on the cell membrane and the subsequent damage of the cellular adhesion within epithelial tissue.[23-26] In this work, we demonstrated a successful loading of proteins and release in a contact manner with the porous PAH/PAA multilayers coated AFM cantilever. Fast LbL assembly was applied with the deposition time shortened to 10 s, 1 min and 5 min. The porous PAH/PAA multilayers on the AFM cantilever provides a novel platform for localized protein or drug delivery and quantitative examination of the cell-protein interaction. ` 98 4.2 Experimental Section 4.2.1 Materials Poly (acrylic acid, sodium salt) (PAA, Mw=15,000 g/mol) 35wt% aqueous solution and poly(allylamine hydrochloride) (PAH, Mw=17,500 g/mol) were both purchase from Sigma  Millipore water at a concentration of 10 mM with respect to the repeat unit and adjusted to the required pH using 0.1M HCl or NaOH solutions. Silicon Nitride (Si3N4) Tips (DPN-10) were obtained from Bruker.   4.2.2 Fabrication of Porous PEMs on AFM cantilevers All LbL films were assembled with a programmable Carl-Zeiss slide-stainer. The AFM Tips were exposed to oxygen plasma generated by a Harrick plasma cleaner (Harrick Scientific Corporation, Broading Ossining, NY) for 20 min, producing hydrophilic moieties and negative charges on the surface. After the oxygen plasma treatment, the glass substrates were immediately dipped into PAH solution (without adjusting the pH) for 20 min to form the precursor layer, followed by three washing steps. Then, the substrates were introduced in the aqueous solution of PAA (pH = 3.5) for required deposition time, followed by three washing steps with DI water (pH = 3.5) for sufficient time. Subsequently, the substrates were immersed in the PAH (pH = 8.5) aqueous solution with the same deposition time as PAA, and washed again three times with DI water (pH = 8.5). The dipping process was repeated 20 times. In total, 20.5 bilayers were deposited on the substrate, including the first PAH precursor layer. Deposition time of 10 s, 1 min and 5 min were applied in this work. The assembled polyelectrolyte multilayer films were ` 99 immersed in the water solution with pH of 2.0 for 5 min followed by washing with DI water (pH =5.5) for 5 min. After the porosity induction, the films were dried and then heated at 180 oC for 2 hours to cross-link the films and prevent the porous structure from being distorted. This post treatment helped create porous films as described by other researchers.[9, 18, 20] A JEOL 6610LV Scanning Electron Microscopy (SEM) was used to observe the porous thin films on the AFM cantilevers. All specimens were coated with gold before examination under the SEM. 4.2.3 Silanization of AFM Cantilevers In order to compare with the porous PEM systems, the conventional chemical conjugation method was also carried out. The AFM tips were washed in chloroform for 1 hour and plasma cleaned in a Harrick plasma cleaner (Harrick Scientific Corporation, Broading Ossining, NY) for 20 min. The tips were  placed in a chamber with the presence (3-aminopropyl)triethoxysilane (triethylamine (TEA, Fisher) TEA at room temperature for 1 hour. Then the chamber was placed on a hot plate at 100 oC for 3 hours. 4.2.4 The Loading of Protein Molecules The pathogenic anti-Dsg3 antibody Px4-3 obtained from a patient with mucocutaneous PV by phage display was used and diluted in phosphate buffered saline (PBS) solution or cell culture medium at 1:50. Both porous PEM-coated and silanized tips were rinsed with PBS solution and then immersed in an anti-Dsg3 antibody Px4-, followed by rinsing with PBS solution for three times. After the loading of antibodies, the tips ` 100 were blocked with bovine serum albumin (BSA block) for 30 min and rinsed with PBS solution for three times. Alexa Fluor® 488 conjugated goat anti-mouse IgG (A11013, ThermoFisher Scientific) as the secondary antibody was diluted 300 times before use. The tips were then immersed in the secondary antibody solution for 1 hour, followed by rinsing with PBS solution for three times. The tips were always immersed in PBS solution prior to use. The protein loaded AFM cantilevers were imaged using a fluorescence microscope (Nikon Eclipse TE300) to determine the loadings of protein molecules.    4.2.5 Contact Release of Protein Molecules from Porous Multilayers 1N-[3-(trimethoxysilyl)propyl]ethylenediamine (TPEDA, Sigma-Aldrich) was mixed with ethanol with a volumetric ratio of 1 : 10. Plasma cleaned glass slides were immersed in the mixture for 30 min to form amino-terminated self-assembled monolayer. The modified glass slides were rinsed with ethanol and dried under N2. In the end, the modified substrates were placed in an oven at 60 oC for 1 hour to stabilize the monolayer. The amino-terminated substrates were used for contact release. The porous multilayer coated AFM cantilevers were first incubated in the florescent secondary antibody solution for 1 hour to stabilize the protein adsorption. The resulting cantilevers were either directly applied for the contact release test. After the protein incubation, the cantilevers were further rinsed with PBS solution for three times to remove the free protein molecules or immersed in PBS solution for two days and then applied for the contact release test. A micro-manipulator (Signatone S-926) was used to perform the contact release long with an optical microscope (Nikon TS 100) for positioning. The protein ` 101 loaded AFM tips were mounted onto the micro-manipulator, which allowed the contact of AFM tips with the amino-terminated substrates, forming a dot pattern of protein arrays. This pattern was recorded by the Nikon fluorescence microscope. The contact time was controlled to 15 s.  4.2.5 Solution Release of Weakly Bound Protein Molecules from Porous Multilayers After the removal of free protein molecules in the porous multilayers, the porous multilayer coated AFM cantilevers were further placed in the PBS solution. The change of fluorescent intensity on the AFM cantilever was monitored by the fluorescence microscope over time until the intensity maintained the same. 4.3 Results and Discussion 4.3.1 Porous Structures on the AFM Cantilever In this work, porous PAH/PAA multilayers deposited on the AFM cantilever were fabricated via LbL assembly of the multilayers followed by post treatment at pH = 2.0. Considering the fabrication efficiency, the deposition time during LbL assembly was shortened from conventional 15 min to 10 s, 1 min and 5 min, respectively. As shown in Figure 4.1, the AFM cantilevers were successfully coated with the porous PAH/PAA multilayers. With the increase of deposition time (Figure 4.1 (a), (d) and (g)), it is obvious that the surface of the AFM cantilever became rougher. When the deposition time was 10 s (Figure 4.1 (b)), the porous structure was not to bale to cover the AFM tip. With the increase of deposition time to 1 min (Figure 4.1 (e)), the height of the AFM tip was almost the same as the thickness of the porous multilayers. For the deposition time of 5 min (Figure 4.1 (g)), the tip was trapped inside the ` 102 porous multilayers. Therefore, the thickness of the porous multilayers increased with the increase of the deposition time. In addition, as presented in Figure 4.1 (c), (f) and (i), the surface pore size increased slightly with the increase of the deposition time. However, the porous structures on the AFM cantilever were different from the porous structures formed on the Si3N4 wafer as shown in Figure 4.2. It was found that the dimension and the shape of the substrates also affect the porous structures significantly. Due to the sharp edges and the small dimension of the AFM cantilevers, it is highly possible that certain amount of residual stress was trapped in the multilayers during the LbL assembly and relaxed during the post treatment, leading to different behavior of the chain rearrangement to form the porous structures.     Figure 4.1 SEM images of the AFM cantilever coated with porous PAH/PAA multilayers. Porous PAH/PAA multilayers were fabricated with deposition time of (a) - (c) 10 s, (d) - (f) 1 min, and (g) - (i) 5 min, respectively. ` 103  Figure 4.2 SEM images of the porous PAH/PAA multilayers on the Si3N4 wafer with deposition time of (a) 10 s, (b) 1 min, and (c) 5 min, respectively. 4.3.2 Protein Loadings In this work, we also applied the traditional silanization of AFM cantilevers with APTES, and compared the protein loadings with the method of depositing porous PAH/PAA multilayers. The loaded protein molecules were bound with the fluorescent secondary antibodies, which could show visible green light under the fluorescence microscope. The fluorescence intensity was proportional to the protein loadings. As shown in Figure 4.3(a)-(d), protein molecules were successfully loaded onto the AFM cantilever with both methods since green fluorescence showed up on all AFM cantilever. The fluorescence intensity is summarized in Figure 4.4. It is obvious that for the traditional silanization with APTEs, the green fluorescence was extremely weak and uniformly covered on the AFM cantilever due to the low protein concentration during the loading process. On the contrast, via the same loading process, bright green fluorescence exhibited uniformly on the AFM cantilevers with the porous multilayers. This indicates that the deposition of porous PAH/PAA multilayers enhanced the uniformity and loadings of protein molecules on the AFM cantilever significantly when compared to the traditional silanization with APTEs. For the porous PAH/PAA multilayers, even though they were thermally crosslinked, ` 104 certain amount of free amine and carboxylate groups were left inside the porous structure. Moreover, the porous structure offered a large surface area, which can provide more binding sites for the protein molecules. However, for the traditional silanization with APTEs, only a molecular layer of the amine groups was available for the adsorption of protein molecules.    Figure 4.3 Fluorescence images of AFM cantilevers modified with (a) APETS and (b)-(c) porous PAH/PAA multilayers with deposition time of 10 s, 1 min and 5 min, respectively. It was also found from Figure 4.3 (b)  (d) that the fluorescence intensity increased with the deposition time, which is consistent with the data listed in Figure 4.4. A longer deposition time enabled the generation of a thicker porous multilayer coating, which was able to provide more binding sites and therefore facilitate a higher loading of the protein molecules. This also indicates that the loading amount could be controlled by tuning the deposition time.  ` 105  Figure 4.4 The fluorescence intensity of the AFM cantilevers modified with APETS and porous PAH/PAA multilayers with deposition time of 10 s, 1 min and 5 min, respectively. 4.3.3 Contact Release of Protein Molecules from Porous Multilayers The porous multilayer coated AFM cantilevers were incubated in the florescent secondary antibody solution for 1 hour to stabilize the protein adsorption. The resulting cantilevers were brought into contact with the amino-terminated substrates. As shown in Figure 4.5 (a), the dot pattern was successfully generated on the substrates due to the transfer of free protein molecules from the cantilever to the substrate. The result is consistent with the previous DPN studies.[13] However, the free protein molecules would diffuse out from the porous multilayers into the media, which lead to the possible protein interaction with all the cells and in turn highly limit the local delivery of the protein molecules to the cell membrane. Therefore, the AFM cantilevers were further rinsed with PBS solution for three times to remove the free protein molecules and then applied for the contact with the amino-terminated substrates. As shown in Figure 4.5 (b), the ` 106 dot pattern could be observed, indicating that the protein molecules that were bound with the porous multilayers can be transferred through a contact manner. Compared to the contact release of free protein molecules, the fluorescence intensity decreased because it is harder for bound protein molecules to be transferred to the substrates. The protein loaded porous multilayers on the AFM cantilever was further placed in the PBS solution for two days. As shown in Figure 4.5 (c), the dot pattern still could be observed, however with the intensity significant decreased, indicating that certain amount of bouned protein molecules may dissociate from the porous multilayers.          Figure 4.5 The fluorescence image of the dot pattern resulting from the contact release of fluorescence protein molecules from porous multilayer coated AFM cantilever (deposition time of 5 min): (a) directly after the protein incubation and (b) with three rinsing step with PBS solution after the protein incubation, and (c) immersed in PBS solution for two days after the protein incubation. (The scale bar is 10 .) ` 107 4.3.4 Dissociation of Weakly Bound Protein Molecules Figure 4.5(c) indicates that certain amount of protein molecules were able to dissociate from the porous multilayers if placed in the PBS solution. This was further confirmed by analyzing the change fluorescence intensity on the AFM cantilever during the immersion in PBS solution as shown in Figure 4.6. For porous multilayers with deposition time of 10 s, 1 min and 5 min, the percentages of the dissociation with respect to the protein loadings were 30.1, 27.7 and 31.2 %, respectively. Proteins contain the hydrophilic functional groups and hydrophobic backbones, which is similar to the porous PAH/PAA multilayers. The interaction between protein molecules and the porous multilayers is not limited to the strong electrostatic interactions. Some secondary interactions such as hydrogen bonding and hydrophobic-hydrophobic interactions may also exist. We believe that the weakly bound protein molecules could dissociate from the porous multilayers when immersed in the PBS solution. After 48 hours, the fluorescent intensity stopped decreasing for all the protein loaded porous multilayers, indicating that the remaining protein molecules were strongly bounded with the porous multilayers. The strongly bounded protein molecules were hard to be transferred from the porous multilayers to the amino-terminated glass substrates, leading to a significant decrease of fluorescence intensity shown in Figure 4.5(c). For the cell test, the contact time with a specific cell was set to only 15 min. Therefore, the amount of bound protein molecules dissociated to the media is very limit, which would not affect the surrounding cells. Instead, through the interaction with cell membrane, it would be easier for the ` 108 weakly bound protein molecules to dissociate from the porous multilayers, which would increase the availability of protein molecules and enable a sufficient delivery on the cell membrane.  Figure 4.6 The dissociation of loosely bonded protein molecules from the porous PAH/PAA multilayers with deposition time of 10 s, 1 min and 5 min, respectively 4.4 Conclusions We successfully deposited the porous PAH/PAA multilayers onto the AFM cantilever via fast LbL assembly followed by the post treatment at pH = 2.0. Anti-Dsg3 antibody Px4-3 and fluorescence labeled goat anti-mouse IgG as example proteins was successfully loaded into the porous multilayers. We demonstrated that the porous multilayers enhanced the protein loadings significantly, compared to the traditional silanization of AFM cantilevers with APTES. Three ` 109 porous multilayers with different thickness were fabricated by changing the deposition time during LbL assembly. We found that the protein loadings increased with the thickness increase of the porous multilayers. The contact release of protein molecules was carried out on amino-terminated glass substrates. We successfully proved that the protein molecules that were bound to the porous multilayers could be released in a contact manner. We further demonstrated that the bound protein molecules existed in the porous multilayers via strong electrostatic interaction and weak secondary interaction. The weakly bound protein molecules were able to dissociate from the porous multilayers over time when placed in the PBS solution. However, the dissociation would not affect the nearby cells due to the limit dissociation amount during the initial 15 min, which is the operation window for real cell test. In addition, we believe that when in contact with the cell membrane, under the applied force from the protein receptor, the weakly bound protein molecules could enabled a sufficient local delivery on the cell membrane.                ` 110          REFERENCES           ` 111 REFERENCES    1. Imokawa, G., Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Research 2004, 17 (2), 96-110. 2. Hocking, A. M.; Gibran, N. S., Mesenchymal stem cells: paracrine signaling and differentiation during cutaneous wound repair. Experimental cell research 2010, 316 (14), 2213-2219. 3. Gnecchi, M.; Zhang, Z.; Ni, A.; Dzau, V. J., Paracrine mechanisms in adult stem cell signaling and therapy. Circulation research 2008, 103 (11), 1204-1219. 4. Olivry, T., A review of autoimmune skin diseases in domestic animals: Isuperficial pemphigus. Veterinary dermatology 2006, 17 (5), 291-305. 5. Li, G.; Xi, N.; Yu, M.; Fung, W.-K., Development of augmented reality system for AFM-based nanomanipulation. Mechatronics, IEEE/ASME Transactions on 2004, 9 (2), 358-365. 6. Ebner, A.; Hinterdorfer, P.; Gruber, H. J., Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers. Ultramicroscopy 2007, 107 (10), 922-927. 7. Hu, K. K.; Bruce, M. A.; Butte, M. J., Spatiotemporally and mechanically controlled triggering of mast cells using atomic force microscopy. Immunologic research 2014, 58 (2-3), 211-217. 8. Barattin, R.; Voyer, N., Chemical modifications of AFM tips for the study of molecular recognition events. Chemical communications 2008,  (13), 1513-1532. 9. Mendelsohn, J.; Barrett, C. J.; Chan, V.; Pal, A.; Mayes, A.; Rubner, M., Fabrication of microporous thin films from polyelectrolyte multilayers. Langmuir 2000, 16 (11), 5017-5023. 10. Bai, S.; Wang, Z.; Zhang, X.; Wang, B., Hydrogen-bonding-directed layer-by-layer films: effect of electrostatic interaction on the microporous morphology variation. Langmuir 2004, 20 (26), 11828-11832. 11. Fery, A.; Schöler, B.; Cassagneau, T.; Caruso, F., Nanoporous thin films formed by salt-induced structural changes in multilayers of poly (acrylic acid) and poly (allylamine). Langmuir 2001, 17 (13), 3779-3783. ` 112 12. Cho, C.; Jeon, J.-W.; Lutkenhaus, J.; Zacharia, N. S., Electric field induced morphological transitions in polyelectrolyte multilayers. ACS applied materials & interfaces 2013, 5 (11), 4930-4936. 13. Wu, C.-C.; Xu, H.; Otto, C.; Reinhoudt, D. N.; Lammertink, R. G.; Huskens, J.; Subramaniam, V.; Velders, A. H., Porous multilayer-coated AFM tips for dip-pen nanolithography of proteins. Journal of the American Chemical Society 2009, 131 (22), 7526-7527. 14. Hiller, J. A.; Mendelsohn, J. D.; Rubner, M. F., Reversibly erasable nanoporous anti-reflection coatings from polyelectrolyte multilayers. Nature materials 2002, 1 (1), 59-63. 15. Sung, C.; Ye, Y.; Lutkenhaus, J. L., Reversibly pH-Responsive Nanoporous Layer-by-Layer Microtubes. ACS Macro Letters 2015, 4, 353-356. 16. Chia, K.-K.; Rubner, M. F.; Cohen, R. E., pH-Responsive Reversibly Swellable Langmuir 2009, 25 (24), 14044-14052. 17. Zhai, L.; Cebeci, F. C.; Cohen, R. E.; Rubner, M. F., Stable superhydrophobic coatings from polyelectrolyte multilayers. Nano letters 2004, 4 (7), 1349-1353. 18. Berg, M. C.; Zhai, L.; Cohen, R. E.; Rubner, M. F., Controlled drug release from porous polyelectrolyte multilayers. Biomacromolecules 2006, 7 (1), 357-364. 19. Sung, C.; Ye, Y.; Lutkenhaus, J. L., Reversibly pH-Responsive Nanoporous Layer-by-Layer Microtubes. ACS Macro Letters 2015, 4 (4), 353-356. 20. Yu, J.; Sanyal, O.; Izbicki, A. P.; Lee, I., Development of Layered Multiscale Porous Thin Films by Tuning Deposition Time and Molecular Weight of Polyelectrolytes. Macromolecular rapid communications 2015, 36 (18), 1669-1674. 21. Chen, X.; Sun, J., Fabrication of Macroporous Films with Closed HoneycombLike Pores from Exponentially Growing LayerbyLayer Assembled Polyelectrolyte Multilayers. ChemistryAn Asian Journal 2014, 9 (8), 2063-2067. 22. Cho, C.; Zacharia, N. S., Film stability during postassembly morphological changes in polyelectrolyte multilayers due to acid and base exposure. Langmuir 2011, 28 (1), 841-848. 23. Becker, B.; Gaspari, A., Pemphigus vulgaris and vegetans. Dermatologic clinics 1993, 11 (3), 429-452. ` 113 24. Ishii, K.; Harada, R.; Matsuo, I.; Shirakata, Y.; Hashimoto, K.; Amagai, M., In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris. Journal of Investigative Dermatology 2005, 124 (5), 939-946. 25. Anhalt, G. J.; Labib, R. S.; Voorhees, J. J.; Beals, T. F.; Diaz, L. A., Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease. New England Journal of Medicine 1982, 306 (20), 1189-1196. 26. Seiffert-Sinha, K.; Yang, R.; Fung, C. K.; Lai, K. W.; Patterson, K. C.; Payne, A. S.; Xi, N.; Sinha, A. A., Nanorobotic investigation identifies novel visual, structural and functional correlates of autoimmune pathology in a blistering skin disease model. PloS one 2014, 9 (9), e106895.            ` 114 5. NANO- AND MICRO-SCALE HIERARCHICAL POROUS POLYELECTROLYTE MULTILAYERS FOR SUPERWETTABLE SURFACES 5.0 Abstract We created both a superhydrophilic polymer surface and a superhydrophobic surface by using porous poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) multilayers with hierarchical surface structures. During post treatment at pH = 2.0, both nano- and micro-scale features were developed on the surface as a result of the combination of high molecular weight of PAH and low molecular weight PAA, along with a much shortened deposition time of 1 min. Though thermally cross-linked, the porous surface with hierarchical structure could achieve superhydrophilicity due to the remaining free amine and carboxylate groups on the porous structures. A switch from superhydrophilic to superhydrophobic surface was achieved via a simple chemical vapor deposition of Trichloro(1H, 1H,2H, 2H-perfluoro-octyl)silane. The effects of molecular weight of polyelectrolytes, deposition time during layer-by-layer (LbL) assembly and pH for post treatment on the porous surface topography and wetting behavior were investigated in detail. A variety of unique porous surface structures at different length scales were systematically studied by controlling the above parameters. 5.1 Introduction Surfaces with superwettability (i.e. superhydrophobic and superhydrophilic) play a key role in addressing problems related to fouling[1], corrosion[2], fogging[3], water collection[4], etc. ` 115 Many techniques including etching[5], sol-gel[6], electrochemical deposition[7], phase separation[8], and electrospining[9] have been applied to obtain the material surfaces with superwettability. The involvement of hazardous chemicals, complicated processing procedures, high manufacturing cost, and poor controllability are the major concerns for these techniques. In recent years, layer-by-layer (LbL) assembly has been considered as a method to fabricate superwettable coatings as the process is aqueous based, highly tunable, and with no limit on the choice of substrate materials. In order to achieve superwettability, surface chemistry and topography are the two key factors. Due to the nature of the polyelectrolytes, films created by LbL assembly are hydrophilic. Fluoroalkylsilane molecules can be grafted onto these hydrophilic surfaces by chemical vapor deposition (CVD), thus making them hydrophobic. The maximum contact angle (CA) on a flat surface is still around 118o, meanwhile a CA of 0o cannot be achieved without the increase of roughness.[10] Without proper surface topography, it is hard to achieve superwettability. LbL assembly is good at controlling surface roughness within nano-scale. However, special LbL conditions are required to enhance surface roughness. Previously, a superhydrophobic surface was obtained via fluorinating certain exponentially growing polyelectrolyte multilayers (PEMs), which facilitated the formation of micro/nano hierarchical structures.[11] The LbL assembly was carried out under a condition when the polyelectrolytes were weakly charged, leading to possible concerns about film stability and long processing time. In order to achieve stable superwettability, the LbL technique has been combined with the deposition of nanoparticles. ` 116 Mesoporous silica nanoparticles[12], titanium dioxide nanoparticles[13] and zinc oxide nanoparticles[14] have been applied during LbL assembly to fabricate superhydrophilic coatings. A stable superhydrophobic surface was achieved by electrodepositing gold clusters on polyelectrolyte multilayer followed by a further modification with n-dodecanethiol.[15] Porous poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) multilayer films were coated with silica nanoparticles and modified with semi-fluorinated silane to achieve the switch from superhydrophilicity to superhydrophobicity.[16] A similar approach was applied to porous branched poly(ethylene imine) (BPEI)/PAA multilayers and created slippery surfaces with low sliding angles and low contact hysteresis by the further addition of lubricant.[17] The deposition of nanoparticles could change the surface chemistry and enhance the surface roughness. However, the combination of LbL assembly with other techniques or materials increased the complexity of fabrication and the processing time. Therefore, in this work, only porous PAA/PAH multilayers were employed to simultaneously generate both nano- and micro-scale features on the porous surface which can show superwettability.  The PAH/PAA multilayers were fabricated by the alternate deposition of PAH at pH = 8.5 and PAA at pH = 3.5. The post treatment was carried out under an acidic pH, followed by rinsing with DI water, drying and cross-linking.[16, 18-23] To form the porous structure, the PAH/PAA multilayers undergo a rearrangement of the polymer chains at low pH[18, 20, 21], induced by the protonation of carboxylate groups on PAA chains and charge repulsion among the free positive amine groups on PAH chains. For porous PAA/PAH multilayers that have been generated so far, ` 117 the pore size locates in either nano- or micro-scale. No hierarchical surface structure has been discovered. Therefore, this work aims to obtain hierarchical structures with both nano- and micro-scale features existing on the surface of porous PAA/PAH multilayers, which in turn could lead to superwettability. It is expected that the hierarchical surface structures exist at the transition state between nano- and micro-scale surface topography. In order to gain a precise control on the porous structures and obtain hierarchical surfaces, the parameters including the deposition time during LbL assembly, molecular weight of polyelectrolytes, and pH for post treatment were investigated in detail. In this work, we optimized the above parameters and generated hierarchical structures with both nano- and micro-scale features existing at the same time. Superwettability was therefore achieved only with the porous PAH/PAA multilayers. The elimination of the involvement of other techniques, such as the deposition of nanoparticles on the microscale porous surface simplifies the fabrication process significantly. 5.2 Experimental Section 5.2.1 Materials Poly (acrylic acid, sodium salt) solution with Mw of 15,000 g/mol (PAAL, 35% aqueous solution) and two poly(allylamine hydrochloride) (PAHL, Mw=15,000 g/mol and PAHH, Mwdeionized (DI) water was used to prepare all aqueous solutions. Glass slides were purchased from Globe Scientific Inc. and used as the substrates for the deposition of polyelectrolyte multilayers. Before the LbL assembly process, substrates were cleaned by sonication for 20 min each in ethanol and ` 118 DI water, followed by an oxygen plasma treatment by a Harrick plasma cleaner (Harrick Scientific Corporation, Broading Ossining, NY) for 20 min, in order to clean the surface and make the surface negatively charged. All polyelectrolyte solutions were prepared at the concentration of 10 mM with respect to the repeat unit. 0.1M HCl or NaOH solutions were used to adjust the pH of solutions to desired values. 5.2.2 Fabrication of Porous PAH/PAA Multilayers A programmable Carl-Zeiss slide-stainer was used for the assembling of LbL films. After the oxygen plasma treatment, the glass substrates were immediately introduced to PAH solution (without adjusting the pH) for 20 min to form the precursor layer, followed by three washing steps with DI water. Then, the substrates were immersed in the aqueous solutions of PAA (pH = 3.5) and PAH (pH = 8.5) alternatively for the desired deposition time, with three washing steps in between. For the three washing steps, the pH of DI water was adjusted to the pH of the polyelectrolyte solution. The deposition process of PAA (pH = 3.5) and PAH (pH = 8.5) was repeated for 20 times. In total, we deposited 20.5 bilayers on the substrate, including the precursor layers. In this work, the deposition time was set to 10 s, 1 min, 5 min, 10 min and 15 min, respectively. For porous introduction, the assembled polyelectrolyte multilayer films were immersed in the water solution at a required pH for 5 min, followed by washing with DI water for 5 min. Then, the films were dried with nitrogen and heated at 180 oC for 2 hours to cross-link the porous structure. The pH for the post treatment varied from 1.8 to 2.4.  ` 119 5.2.3 Chemical Vapor Deposition (CVD) of Fluoroalkylsilane Molecules After the porous structures were crosslinked, trichloro(1H, 1H,2H, 2H-perfluoro-octyl)silane molecules were further deposited onto surface through a CVD process at 130oC for 2 hours, followed by heating at 180oC for 2 hours to remove free fluoroalkylsilane molecules. 5.2.4 Characterization  The surface chemistry of the porous films was determined by X-ray photoelectron spectroscopy (XPS) using a PHI 5400 ESCA spectrometer (Physical Electronics, Eden Prairie Msamples were irradiated with a non-monochromatic Mg X-ray source (1253.6 eV) operating at 300 W with a take- Scanning Electron Microscopy (SEM) was used to observe the surface morphology of the porous thin films. All specimens were coated with gold before examination under the SEM. The surface roughness was measured in the dry state using a Dektak surface profiler. The contact angle was measured by a VCA-2000 Video Contact Angle System (AST placed on the sample that was inclined slowly until the water droplet started to move. The sliding angle was then measured by a protractor.    ` 120 5.3 Results and Discussion 5.3.1 Surface Topography and Wetting Behavior of Porous PAH/PAA Multilayers The SEM images of the porous PEMs are presented in Figure 5.1 for (PAHL/PAAL)20.5, (PAHH/PAAL)20.5 and (PAHH/PAAH)20.5 with different deposition time, respectively. The post treatment was carried out at pH of 2.0. The RMS roughness and CA results are summarized in Figure 5.2 (a) and (b), respectively. The CA before the CVD process was lower than 60o, which means the surface was hydrophilic after the thermal cross-linking. During the thermal cross-linking, the COO- groups from PAA reacted with NH3+from PAH, forming the amide bonds (-NHCO-) to preserves the porous structure from being altered by further immersion in aqueous solution.[21, 24] The formation of the amide bonds should increase the hydrophobicity of the PEMs. However, some free carboxylate groups and ammonium groups remained in the films[24, 25], which maintained the hydrophilicity for the porous PEMs. The porous surface was turned from hydrophilic to hydrophobic by altering the surface chemistry through a CVD process of Trichloro(1H, 1H,2H, 2H-perfluoro-octyl)silane was applied. X-ray photoelectron spectroscopy (XPS) spectra of the porous multilayers were shown in Figure 5.3. One of the porous (PAHH/PAAL)20.5 films was randomly picked as an example. No detectable fluorine peaks was found before the CVD process (Figure 5.3(a)), whereas a strong fluorine peak at 688 eV showed up after the CVD process (in Figure 5.3(b)), indicating that the fluoroalkylsilane molecules were successfully grafted onto the porous surface by reacting with the free amine groups on PAH ` 121 chains. While after the CVD process, the CA increased to over 120o, which further confirms the successful grafting of fluoroalkylsilane molecules.[10], [26] (1) Porous (PAHL/PAAL)20.5. As shown in Figure 5.1 (a)-(e), the surfaces of the (PAHL/PAAL)20.5 films consisted of small pores, the size of which increased with the increase of deposition time. The surface RMS roughness also increased with the deposition time as shown in Figure 5.2 (a). Figure 5.2 (b) includes the CA values before and after the CVD process. When the deposition time was longer than 1 min, the CA before the CVD process decreased with the increase of RMS roughness, while the CA after the CVD process increased with the increase of RMS roughness. It is interesting that the porous (PAHL/PAAL)20.5 with deposition time of 10 s provided the lowest RMS roughness but the lowest CA before the CVD process and the highest CA after the CVD process.  (2) Porous (PAHH/PAAL)20.5. The surface of porous (PAHH/PAAL)20.5 with deposition time of 10 s contained both nano-sized pores and macro-sized bulge structures as shown in Figure 5.1(f). However, the surface RMS roughness is relatively low. When deposition time increased to 1 min, the surface exhibited a hierarchical structure with both nano-scale texture and micro-sized pores with sharp ridges (Figure 5.1(g)). The RMS roughness increased to around 224 nm. The CA before the CVD process was close to 0o, indicating a superhydrophilic surface. The mechanism behind is that the water could penetrate through the porous structure due to the rough hierarchical surface topography and the remaining hydrophilic functional groups in the porous structures. In addition, the hierarchical surface topography of porous (PAHH/PAAL)20.5 induced a ` 122 successful transition from superhydrophilicity (CA ~ 0o) to superhydrophobicity (CA = 155.6 ± 2.2o) after a simple CVD process of fluoroalkylsilane molecules as shown in Figure 5.2 (b). The sliding angle was also measured. It was found that the sliding angle for porous (PAHH/PAAL)20.5 with deposition time of 1 min is around 3o ( < 10o), indicating that the surface located in the Cassies state and owned a good self-cleaning property. When the deposition time reached 5 min,  surface started losing the nano-scale texture but contained the micro-sized pores with sharp ridges. The surface still exhibited superhydrophilicity and superhydrophobicity before and after the CVD process, respectively (Figure 5.2(b)). However, the sliding angle increased to around 10o due to the disappearance of nano-scale features. With the further increase of deposition time to 10 min and 15 min, the hydrophilicity and hydrophobicity of the porous surface both decreased. The sliding angle also increased to more than 20ostate. Based on the literatures[27-30], we believe that the size and the depth of the pores can strongly affect the transition. This also explains why it is hard to build a direct connection between the CA and surface RMS roughness.  (3) Porous (PAHH/PAAH)20.5. When the deposition time was as short as 10s, the surface of porous only (PAHH/PAAH)20.5 consisted of nano-sized pores, leading to a low RMS roughness. When the deposition time increased to 1 min, the surface became rougher and started showing micro-scale pores, leading to a decrease of CA before the CVD process and an increase of CA after the CVD process (shown in Figure 5.2(b)). With a further increase of deposition time to 5 ` 123 min (Figure 5.1(m)), both nano- and micro-sized pores existed on the surface, leading to a decrease of CA before the CVD process to 4.1o and an increase of CA after the CVD process to 149.3o. For porous (PAHH/PAAH)20.5 with deposition time of 10 or 15 min, the surface exhibited  superwettability as shown in Figure 5.2(b) due to the increase of surface roughness and the detailed topography. However, for porous (PAHH/PAAH)20.5, long deposition time was required to generate hierarchical structures and achieve superwettability. 5.3.2 Effect of Molecular Weight of PAH on the Surface Topography and Wetting Behavior As shown in Figure 5.1, the surface topography of porous (PAHH/PAAL)20.5 varies a lot from porous (PAHL/PAAL)20.5. In general, the surface pore size of (PAHH/PAAL)20.5 was larger than that of (PAHL/PAAL)20.5, and the pore size distribution was wider. According to our previous studies[25], high molecular weight PAH could provide stronger intramolecular charge repulsion during the post treatment, leading to more drastic chain rearrangement and. Therefore, the application of the high molecular weight PAH could increase the surface pore size as well as the surface roughness. This is consistent with the values of surface RMS roughness shown in Figure 5.2 (a) that the surface of (PAHH/PAAL)20.5 was rougher than that of (PAHL/PAAL)20.5. This further led to a lower CA before the CVD process and a higher CA after the CVD Process (Figure 5.2(b)).    ` 124                          Figure 5.1 (a)-(e) are the top-view SEM images of the porous (PAHL/PAAL)20.5 with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. (f)  (j) are the top-view SEM images of the porous (PAHH/PAAL)20.5 with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. (The post treatment was done at pH of 2.0.) (k)-(o) are the top-view SEM images of the porous (PAHH/PAAH)20.5 with deposition time of 10 s, 1 min, 5 min, 10 min and 15 min, respectively. ` 125   (a)    (b)  Figure 5.2 The values of (a) roughness and (b) contact angle for (PAHL/PAAL)20.5, (PAHL/PAAH)20.5, (PAHL/PAAH)20.5 and (PAHH/PAAH)20.5 with different deposition time, respectively. (The post treatment was done at pH of 2.0.) ` 126 5.3.3 Effect of Molecular Weight of PAA on the Surface Topography and Wetting Behavior As shown in Figure 5.1, the surface topography of porous (PAHH/PAAH)20.5 further differs from  (PAHL/PAAL)20.5 and (PAHH/PAAL)20.5. Comparing to (PAHH/PAAL)20.5, high molecular weight of PAA limited the surface pore size. This is consistent with our previous studies[25] that high molecular weight PAH was weekly charged during the post treatment, therefore could hinge the rearrangement of polymer chain. This also explains that surface RMS roughness of (PAHH/PAAH)20.5 was lower than that of (PAHH/PAAL)20.5. However, due to the existence of high molecular weight PAH, the surface contained pores with very different sizes when the deposition time increased to more than 1 min, leading to different surface wettability. 5.3.4 Effect of Deposition Time on the Surface Topography and Wetting Behavior For all three porous films, it is obvious that the deposition time affects the surface topography significantly. During the LbL assembly, the film thickness, composition and polyelectrolyte distribution were all changed by different deposition time. These changes further affect the polymer chain rearrangement during the post treatment, leading to different porous structure, surface topography and wettability. In the case of (PAHH/PAAL)20.5 films, the amount of high molecular weight PAH deposited in the film increased with the increase of deposition time. Therefore, the surface roughness increased due to the increased charge-charge repulsion among PAH chains. A hierarchical structure was generated when the deposition time was 1 min. With the further increase of deposition time, the surface lost the nano-scale features. When the deposition time decreased to 10 s, the surface was not rough enough to achieve superwettability. ` 127 With the application of high molecular weight PAA instead of the low molecular weight PAA, the surface became hierarchical when the deposition time increased to more than 5 min. The - and micro-scale surface topography is due to the suppression of the porous structure formation by high molecular weight PAA. In all, the molecular weight of polyelectrolytes and the deposition time influence the porous surface topography in a synergistic manner.   (a)                                                                    (b) Figure 5.3 XPS spectra of porous (PAHH/PAAL)20.5 film before (a) and after (b) the CVD process. 5.3.5 Effect of pH for Post treatment on the Surface Topography and Wetting Behavior         Several previous studies have shown the effect the pH for post treatment on the pore size.[18, 21, 22, 31] Thus, the pH for post treatment is critical for controlling the structure topography. According to the data in Figure 5.2(b), we were able to achieve superwettability when the deposition time was as short as 1 min. Thus, considering the fabrication efficiency, we only investigated the pH effect on the surface wettability with deposition time of 10 s and 1 min. ` 128 The post treatment was carried out at pH = 1.8, 2.0, 2.2 and 2.4, respectively. The surface SEM images of the porous (PAHL/PAAL)20.5, (PAHH/PAAL)20.5 and (PAHH/PAAH)20.5 are presented in Figure 5.4 - 5.6, respectively. In general, the surface pore size increased with the pH of post treatment, which is consistent with the previous studies [18, 21, 22]. Some exceptions were found when the post treatment was carried out at pH = 1.8, especially for the PEMs fabricated with short deposition. For example, no porous structure was observed from Figure 5.4(a). Also, for porous (PAHL/PAAL)20.5 with deposition time of 1 min, the pore size of post treatment at pH = 1.8 (Figure 5.4(e)) was larger than that of post treatment at pH = 2.0 (Figure 5.4(f)). Similar result was also found for porous (PAHH/PAAL)20.5 that pore size in Figure 5.5(a) was larger than that of Figure 5.5(b) as well as Figure 5.5(e). These are due to the drastic breakage of ionic cross-links when the PEMs were exposed to a low pH, leading to a significant dissociation of polyelectrolytes from the surface.[31] In addition, the dissociation of polyelectrolytes was more obvious for short deposition time, since fewer amounts of materials were deposited. It was also found from Figure 5.6 that the film stability increased with the deposition of high molecular weight PAA due to its low chain mobility during the post treatment.            ` 129                                      Figure 5.4 SEM images of the porous (PAHL/PAAL)20.5 surfaces ((a)-(d) deposition time of 10 s; (e)-(h) deposition time of 1 min) with post treatment at pH of 1.8, 2.0, 2.2 and 2.4, respectively. ` 130                                      Figure 5.5 SEM images of the porous (PAHH/PAAL)20.5 surfaces ((a)-(d) deposition time of 10 s; (e)-(h) deposition time of 1 min) with post treatment at pH of 1.8, 2.0, 2.2 and 2.4, respectively.     ` 131                                      Figure 5.6 SEM images of the porous (PAHH/PAAH)20.5 surfaces ((a)-(d) deposition time of 10 s; (e)-(h) deposition time of 1 min) with post treatment at pH of 1.8, 2.0, 2.2 and 2.4, respectively.  Table 5.1 lists the CA values before and after the CVD process according to the porous surfaces shown in Figure 5.4 - 5.6. As shown in Figure 5.4(c)-(h), the surface of porous ` 132 (PAHL/PAAL)20.5 only contained pores with diameter over 100 nm. Therefore, the CA before and after the CVD process were mainly around 56o and 125o, respectively. The only exception is the porous surface with deposition time of 10 s and pH of 2.0 for post treatment due to the existence of both nano- and micro-scale features on the surface as introduced before. Similar results were found for porous (PAHH/PAAH)20.5. The surfaces only consisted of holes with different sizes when the deposition time was 10 s (Figure 5.6 (a)-(d)). Even when the deposition time increased to 1 min (Figure 5.6 (e)-(h)), the surface was not able to achieve superwettability. For porous (PAHH/PAAL)20.5, it is obvious from Figure 5.5 that lower pH for post treatment facilitated the formation of nanoscale features on the surface. When post treatment was carried out at pH = 1.8, the surface mainly contains nano-scale porous features as shown in Figure 5.5 (a) and (e). However, only nano-scale roughness is not enough for achieving superwettability. When the pH increased to 2.0, both nano- and micro-scale features existed on the surface (Figure 5.5 (b) and (f)), leading to a decrease of the CA before the CVD process and an increase of the CA after the CVD process. If the pH for post treatment was further increased to 2.2 (Figure 5.5 (c) and (g)), the nanoscale features started vanishing, resulting in an increase of CA before the CVD process and a decrease of CA after the CVD process. It is interesting that the surface pore size exhibited a bimodal distribution for the porous (PAHH/PAAL)20.5 films induced at pH = 2.4. In sum, the pH for post treatment is critical to the surface topography of porous PEMs. A Low pH for post treatment would facilitate the formation of nano-scale features as well as jeopardize the film ` 133 stability, whereas a relatively high pH for post treatment would help form micro-scale features. An intermediate pH was required to obtain a possible hierarchical structure.  Table 5.1 The effect of pH for post treatment on the contact angle of porous (PAHL/PAAL)20.5 and (PAHH/PAAL)20.5 surfaces with the deposition time of 10s and1 min, respectively  Sample Deposition Time pH of post treatment Contact Angle before CVD (o) Contact Angle after CVD (o) (PAHL/PAAL)20.5 10 s 1.8 56.1 ± 1.0 116.2 ± 5.4 2.0 30.6 ± 3.4 143.5 ± 2.7 2.2 55.3 ± 1.8 128.3 ± 1.4 2.4 57.2 ± 2.4 124.8 ± 2.3 1 min 1.8 12.5 ± 3.9 126.0 ± 1.3 2.0 56.4 ± 5.3 125.2 ± 0.9 2.2 54.7± 1.8 126.2 ± 1.9 2.4 57.6 ± 1.3 121.2 ± 0.6 (PAHH/PAAL)20.5 10 s 1.8 27.6 ± 2.7 125.4 ± 3.1 2.0 13.8 ± 3.2 141.3 ± 1.4 2.2 29.6 ± 3.0 128.9 ± 1.3 2.4 56.3 ± 3.7 129.0 ± 1.6 1 min 1.8 33.9 ± 2.2 124.8 ± 3.4 2.0 < 5 155.6 ± 2.2 2.2 50.0 ± 1.3 142.6 ± 0.5 2.4 45.6 ± 1.4 138.3 ± 1.6 (PAHH/PAAH)20.5 10 s 1.8 34.9 ± 2.5 125.2 ± 2.0 2.0 24.4 ± 0.6 127.5 ± 2.9 2.2 40.0 ± 2.8 123.6 ± 2.4 2.4 44.7 ± 1.7 124.1 ± 2.7 1 min 1.8 29.0 ± 2.5 129.8 ± 0.8 2.0 17.9 ± 7.2 132.9 ± 1.9 2.2 26.2 ± 3.4 125.7 ± 2.2 2.4 41.6 ± 0.7 126.0 ± 2.9 5.4 Conclusions         In this work, we found that the deposition time, molecular weight of polyelectrolytes and pH for post treatment affect the surface topography of porous PEMs in a synergistic manner. For (PAHL/PAAL)20.5, no superwettable surfaces could be generated regardless of the deposition time ` 134 and the pH for post treatment.  For porous (PAHH/PAAL)20.5, a hierarchical surface with both nano- and micro-scale features was achieved with deposition time of 1 min and porous treatment at pH = 2.0. With the further increase of deposition time or pH for post treatment, the surface lost the nano-scale features; whereas the surface lost the micro-scale features if the deposition time or pH decreased. For porous (PAHH/PAAH)20.5, the existence of high molecular weight PAA hinged of micro-scale features on the porous surface. Therefore, the surface became superwettable when the deposition time increased to over 5 min. When the deposition time was less than 5 min, no hierarchical surface structure could be formed regardless of the pH for the post treatment. In general, short deposition time as well as low pH for post treatment could facilitate the formation of nano-scale structure on the porous surface. High molecular weight PAH could help generate rough surfaces; whereas high molecular weight PAA could limit the surface roughness. With the optimization of above parameters, we successfully fabricated a surface with hierarchical structure by depositing porous (PAHH/PAAL)20.5 with deposition time of 1 min and porous treatment at pH = 2.0 and achieved superwettability. The decrease of deposition time from conventional 15 or 20 min to only 1 min significantly improves efficiency of fabricating porous multilayer films.    ` 135          REFERENCES           ` 136 REFERENCES    1. Genzer, J.; Efimenko, K., Recent developments in superhydrophobic surfaces and their relevance to marine fouling: a review. Biofouling 2006, 22 (5), 339-360. 2. Chen, Y.; Chen, S.; Yu, F.; Sun, W.; Zhu, H.; Yin, Y., Fabrication and anticorrosion property of superhydrophobic hybrid film on copper surface and its formation mechanism. Surface and Interface Analysis 2009, 41 (11), 872-877. 3. Lai, Y.; Tang, Y.; Gong, J.; Gong, D.; Chi, L.; Lin, C.; Chen, Z., Transparent superhydrophobic/superhydrophilic TiO 2-based coatings for self-cleaning and anti-fogging. Journal of Materials Chemistry 2012, 22 (15), 7420-7426. 4. Zheng, Y.; Bai, H.; Huang, Z.; Tian, X.; Nie, F.-Q.; Zhao, Y.; Zhai, J.; Jiang, L., Directional water collection on wetted spider silk. Nature 2010, 463 (7281), 640-643. 5. Kwon, Y.; Patankar, N.; Choi, J.; Lee, J., Design of surface hierarchy for extreme hydrophobicity. Langmuir 2009, 25 (11), 6129-6136. 6. Shirtcliffe, N.; McHale, G.; Newton, M.; Perry, C., Intrinsically superhydrophobic organosilica sol-gel foams. Langmuir 2003, 19 (14), 5626-5631. 7. Shirtcliffe, N. J.; McHale, G.; Newton, M. I.; Chabrol, G.; Perry, C. C., DualScale Roughness Produces Unusually WaterRepellent Surfaces. Advanced Materials 2004, 16 (21), 1929-1932. 8. superhydrophobic surface. science 2003, 299 (5611), 1377-1380. 9. Han, D.; Steckl, A. J., Superhydrophobic and oleophobic fibers by coaxial electrospinning. Langmuir 2009, 25 (16), 9454-9462. 10. Wang, S.; Liu, K.; Yao, X.; Jiang, L., Bioinspired surfaces with superwettability: new insight on theory, design, and applications. Chemical Reviews 2015, 115 (16), 8230-8293. 11. , A.; Rosenhahn, A.; Shen, J.; Grunze, M., pH-amplified exponential growth multilayers: A facile method to develop hierarchical micro-and nanostructured surfaces. Langmuir 2008, 25 (2), 672-675. ` 137 12. Zhang, L.; Qiao, Z.-A.; Zheng, M.; Huo, Q.; Sun, J., Rapid and substrate-independent layer-by-layer fabrication of antireflection-and antifogging-integrated coatings. Journal of Materials Chemistry 2010, 20 (29), 6125-6130. 13. Kommireddy, D. S.; Patel, A. A.; Shutava, T. G.; Mills, D. K.; Lvov, Y. 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ChemistryAn Asian Journal 2014, 9 (8), 2063-2067. ` 138 24. Harris, J. J.; DeRose, P. M.; Bruening, M. L., Synthesis of passivating, nylon-like coatings through cross-linking of ultrathin polyelectrolyte films. Journal of the American Chemical Society 1999, 121 (9), 1978-1979. 25. Yu, J.; Sanyal, O.; Izbicki, A. P.; Lee, I., Development of Layered Multiscale Porous Thin Films by Tuning Deposition Time and Molecular Weight of Polyelectrolytes. Macromolecular rapid communications 2015, 36 (18), 1669-1674. 26. Guo, C.; Wang, S.; Liu, H.; Feng, L.; Song, Y.; Jiang, L., Wettability alteration of polymer surfaces produced by scraping. Journal of Adhesion Science and Technology 2008, 22 (3-4), 395-402. 27. Lafuma, A.; Quéré, D., Superhydrophobic states. Nature materials 2003, 2 (7), 457-460. 28. Barbieri, L.; Wagner, E.; Hoffmann, P., Water wetting transition parameters of perfluorinated substrates with periodically distributed flat-top microscale obstacles. 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We fabricated the porous PAH/PAA multilayers via LbL assembly followed by the post treatment at acidic pHs. Therefore, the conditions of both LbL assembly and the post treatment would affect the porous structures in a synergetic manner. Therefore, in Chapter 2, we first studied the effect of deposition time, molecular weight of polyelectrolytes, and number of bilayers on the growth behavior and surface topography of the multilayers during the LbL assembly. The following chapters (Chapter 3 - 5) of this thesis emphasized on the effect of deposition time, molecular weight of polyelectrolytes, and the pH for the post treatment on the structure of the porous multilayers. We found that fine porous structures could be created even by significantly reducing the deposition time to 10 s. The pore size was increased by using the high molecular weight PAH, while high molecular weight of PAA minimized the pore size to nanometer scale. In addition, the pore size increased with the increase of the pH for the post treatment. By tuning the above  Having gained a precise control over the porous structures, the porous multilayers were designed for different applications. In Chapter 3, in order to generate a surface with anti-biofilm ` 140 properties, the porous multilayers were deposited onto the substrates and successfully loaded with a novel anti-biofilm compound, named ABC-1. In addition, the layered multi-scale porous thin films were further built up for the first time with either micro-sized porous zone on top of nano-sized porous zone or vice versa to achieve sustained release and/or two-stage release with different release rates. We found that the amount of ABC-1 released could be tuned by the pore volume of the thin films, and the release rate was dependent on the surface pore size. With the layered multi-scale porous structures, the duration of ABC-1 release was highly improved to more than 20 days. A 99% of biofilm suppression of V. cholera was achieved by micro-scale porous PEMs loaded with ABC-1 via the evaporation method. In addition, a free standing porous film was obtained through ion-trigged exfoliation, which showed great potential in the applications of membrane filtration and wound dressing. While in Chapter 3 porous PEMs enabled the control release of hydrophobic molecules, we further proved in Chapter 4 that porous PEMs can also be applied for the delivery of hydrophilic proteins. We successfully deposited the porous PAH/PAA multilayers on to the AFM cantilever for local protein delivery. Anti-Dsg3 antibody Px4-3 and fluorescence labeled goat anti-mouse IgG as example proteins was successfully loaded into the porous multilayers. We demonstrated that the porous multilayers enhanced the protein loadings significantly and enabled the release of protein molecules that were bound to the porous multilayers in a contact manner. We further demonstrated that the bound protein molecules existed in the porous multilayers via strong electrostatic interaction and weak secondary interaction. The weakly bound protein molecules ` 141 were able to dissociate from the porous multilayers when immersed in the PBS solution. However, the dissociation would not affect the nearby cells due to the limit amount in 15 min of cell test. In addition, we believe that when in contact with the cell membrane, under the applied force from the protein receptor, the weakly bound protein molecules could enabled a sufficient local delivery on the cell membrane. In Chapter 5, we further expanded the application of porous multilayers to generate superwettable surfaces. By tuning the deposition time, molecular weight of polyelectrolytes, and the pH for post treatment, we fabricated the porous multilayers with nano- and micro-scaled hierarchical structures successfully. A switch from superhydrophilic to superhydrophobic surface was achieved via a simple chemical vapor deposition (CVD) of Trichloro(1H, 1H,2H, 2H-perfluoro-octyl)silane. More importantly, the deposition time was shortened from conventional 15 min to 1 min.  6.2 Future Work Based on the previous studies[1-3], PEMs have been widely applied for surface modification of membranes in the field of water treatment.  Commercial Ultrafiltration (UF) and Nanofiltration (NF) membranes have been modified by PEMs to yield higher rejection as well as fluxes than commercial RO membranes.[1, 2] In addition, the PEMs also improve the fouling resistance of the membranes due to the nature of polyelectrolytes.[3] The commercial NF membranes include a non-woven poly(ethylene terephthalate) (PET) fabrics as the bottom support layer, a micro-sized porous polysulfone as the middle layer prepared by solvent casting ` 142 method, and nano-sized porous polyamide as the skin layer synthesized by interfacial polymerization.[4] The structure of NF membranes is very similar to the layered multi-scale porous PMEs shown in Chapter 3. More importantly, we adopted a simple bottom-up approach to fabricate the layered multi-scale porous PEMs. It is expected that we can mimic the structure of commercial NF membranes by tuning the molecular weight of polyelectrolytes, operation conditions for LbL assembly and post treatments. For mimicking the structure of RO membranes, we can further deposit a few bilayers of non-porous PEMs on the porous layers as an additional barrier for unwanted ions. This would allow the rejection of even small monovalent ions. In Chapter 5, we also proved that porous PEMs are hydrophilic, which is ideal for developing high perm-selective membranes. This should potentially eliminate the demands for operation under high pressure and in turn decrease the energy consumption during the membrane filtration. In addition, the porous films made from polyelectrolytes can provide good anti-fouling and anti-bacterial properties due to the fact that polyelectrolytes contain charged functional groups. So far, in industry, different processing techniques are required to fabricate different components of the membrane. Our bottom-up approach will highly simplify the processing techniques. In addition, the implementation of fast LbL assembly will further improve the efficiency of membrane fabrication. Overall, we believe the fabrication of layered multi-scale porous PEMs will provide a new direction for fabricating membranes with better performance. The application of porous PEMs as membranes can be further expanded to Oil/Water separation. With the rapid growth of industry, large production of oily wastewater leads to an ` 143 inevitable challenge on the environmental protection as well as the desire of clean water for daily life. One of the novel and effective approaches for oil/water separation is the modification of membrane surface with superwettability.[5] Membranes designed with superhydrophobic-superoleophilic or superhydrohpilic-superoleophobic surfaces have shown great potential on oil/water separation based on the different surface tensions between water-membrane and oil- membrane.[6, 7] Surface chemistry and topography is decisive towards the wettability of membrane.[8-10] We believe that the fabrication of porous PEMs offers an ideal approach for oil/water separation. In Chapter 5, we demonstrated the formation of nano- and micro- hierarchical structure on the surface of porous PAH/PAA multilayers, which in turn achieved superwettability. We also proved that the porous structure was highly tunable by changing the parameters during LbL assembly as well as post treatment. Another issue with oil/water separation lies in the membrane fouling.[5] Though PEMs have already shown good antifouling property[11], components with good anti-fouling properties such as clay[3] and PEG[12] could be further added during the fabrication of porous PEMs. Therefore, with proper design of the fabrication conditions and possible combination with traditional filtration membranes, even emulsified oil/water separation could be achieved with high flux, excellent selectivity and low energy consumption.         The concern with the application of porous PEMs in membrane filtration locates in their mechanical properties. The development of mechanically robust coatings on complex surfaces is always in great demand. The coating should be able to stand for certain mechanical and chemical ` 144 stresses. However, it has been noticed that the mechanical properties of porous PEMs is relatively weak, although the free standing films have been successfully exfoliated from the substrate in Chapter 3. Poor mechanical properties may highly limit the real life applications to stand for certain pressure and shear stress. In order to enhance the mechanical property of the porous thin films, we suggest the incorporation of nanofillers such as graphene oxide[13], cellulose nano-whisker[14] and clay[15] during LbL assembly to fabricate porous composite films. It has been proved by our group that clay PEMs can provide better anti-fouling performance of the NF membranes.[3] Therefore, porous clay-PEM composites can further enhance the mechanical as well as fouling resistance if served as membranes. However, it is hard to predict how the nanofillers will affect the porous structure formation. The design of the multilayers and the optimization of fabrication conditions are required to apply the porous composite films for real applications.          ` 145          REFERENCES           ` 146 REFERENCES    1. Sanyal, O.; Sommerfeld, A. N.; Lee, I., Design of ultrathin nanostructured polyelectrolyte-based membranes with high perchlorate rejection and high permeability. Separation and Purification Technology 2015. 2. Sanyal, O.; Liu, Z.; Meharg, B. M.; Liao, W.; Lee, I., Development of polyelectrolyte multilayer membranes to reduce the COD level of electrocoagulation treated high-strength wastewater. Journal of Membrane Science 2015, 496, 259-266. 3. Sanyal, O.; Liu, Z.; Yu, J.; Meharg, B. M.; Hong, J. S.; Liao, W.; Lee, I., Designing fouling-resistant clay-embedded polyelectrolyte multilayer membranes for wastewater effluent treatment. Journal of Membrane Science 2016, 512, 21-28. 4. Tang, Z.; Qiu, C.; McCutcheon, J. R.; Yoon, K.; Ma, H.; Fang, D.; Lee, E.; Kopp, C.; Hsiao, B. 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