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Novel polymer-metal nanocomposites for applications in detection and sensing

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Novel polymer-metal nanocomposites for applications in detection and sensing
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Chaparro, Dayling L
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University of South Florida
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PNIPAM
Microgels
Gold nanoparticles
Molecular imprinting
Interpenetrated networks
Dissertations, Academic -- Biomedical Engineering -- Masters -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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ABSTRACT: Detection of trace elements such as organic contaminants, explosive residues, and metal ions is an intellectually challenging task in science and engineering. It is also a topic of increasing importance due to its impact on society and the environment. Designing molecularly imprinted materials is one of the most promising approaches to explore sensing and detection applications. "Stimuli-sensitive" polymer materials are ideal candidates for these imprinted sensors as they are able to respond to changes in their environment and can be tailored by cross-linking the polymer chains. The responses can be amplified and transduced into measurable signals due to macromolecular properties provided by the use of a polymer. The purpose of the research in this project is to combine organic polymers with inorganic constituents to tailor the binding properties and the responses of the composite material for detection of metals ions in aqueous solutions.The research, here, is based on a thermally responsive polymer such as poly(N-isopropylacrylamide) (PNIPAM), which exhibits a well-known reversible volume phase transition in aqueous media around approximately 32 degrees C. Combining cross-linked microgels formed from PNIPAM and its copolymers with gold nanoparticles (GNP) imparts the composite material with optical properties such as intense visible absorption due to the unique surface plasmon absorption of these small nanoparticles. The use of copolymers allows incorporation of functional groups, such as carboxylic acid, that are potential sites for binding metal ions. Cross-linking of the metal ion binding polymer imprints the metal ion in the PNIPAM microgel network. In this research, design of the composite material was investigated using copolymers of NIPAM and acrylic acid (AA), copolymers of NIPAM and glycidyl methacrylate (GMA), and interpenetrating networks of PNIPAM and PAA.A broad spectrum of polymerization conditions were studied such as changes in cross-linking density as well as changes in the synthetic procedure. Techniques such as turbidometry, ultraviolet visible spectroscopy (UV-VIS), transmission electron microscopy (TEM), and dynamic light scattering (DLS) were employed to characterize the microgels as well as their composites with GNP. Preliminary investigation of imprinting the microgels with heavy metal ions such as copper was also performed. The novel polymer-metal nanocomposites explored here will serve as an important contribution for the current ongoing research efforts in designing materials in the nano-scale capable of sensing and detecting metal ions in solution with high selectivity.
Thesis:
Thesis (M.S.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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by Dayling L. Chaparro.
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Title from PDF of title page.
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Document formatted into pages; contains 69 pages.

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oclc - 181591978
usfldc doi - E14-SFE0002030
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ABSTRACT: Detection of trace elements such as organic contaminants, explosive residues, and metal ions is an intellectually challenging task in science and engineering. It is also a topic of increasing importance due to its impact on society and the environment. Designing molecularly imprinted materials is one of the most promising approaches to explore sensing and detection applications. "Stimuli-sensitive" polymer materials are ideal candidates for these imprinted sensors as they are able to respond to changes in their environment and can be tailored by cross-linking the polymer chains. The responses can be amplified and transduced into measurable signals due to macromolecular properties provided by the use of a polymer. The purpose of the research in this project is to combine organic polymers with inorganic constituents to tailor the binding properties and the responses of the composite material for detection of metals ions in aqueous solutions.The research, here, is based on a thermally responsive polymer such as poly(N-isopropylacrylamide) (PNIPAM), which exhibits a well-known reversible volume phase transition in aqueous media around approximately 32 degrees C. Combining cross-linked microgels formed from PNIPAM and its copolymers with gold nanoparticles (GNP) imparts the composite material with optical properties such as intense visible absorption due to the unique surface plasmon absorption of these small nanoparticles. The use of copolymers allows incorporation of functional groups, such as carboxylic acid, that are potential sites for binding metal ions. Cross-linking of the metal ion binding polymer imprints the metal ion in the PNIPAM microgel network. In this research, design of the composite material was investigated using copolymers of NIPAM and acrylic acid (AA), copolymers of NIPAM and glycidyl methacrylate (GMA), and interpenetrating networks of PNIPAM and PAA.A broad spectrum of polymerization conditions were studied such as changes in cross-linking density as well as changes in the synthetic procedure. Techniques such as turbidometry, ultraviolet visible spectroscopy (UV-VIS), transmission electron microscopy (TEM), and dynamic light scattering (DLS) were employed to characterize the microgels as well as their composites with GNP. Preliminary investigation of imprinting the microgels with heavy metal ions such as copper was also performed. The novel polymer-metal nanocomposites explored here will serve as an important contribution for the current ongoing research efforts in designing materials in the nano-scale capable of sensing and detecting metal ions in solution with high selectivity.
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Novel Polymer-Metal Nanocomposites for Applications in Detection and Sensing by Dayling L. Chaparro A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Engineering Department of Chemical Engineering College of Engineering University of South Florida Major Professor: Vinay K. Gupta, Ph.D. John T. Wolan, Ph.D. Norma Alcantar, Ph.D. Date of Approval: April 11, 2007 Keywords: pnipam, microgels, gold nanoparticles, molecular imprinting, inter penetrated networks Copyright 2007, Dayling L. Chaparro

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DEDICATION To my family

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ACKNOWLEDGEMENTS During my graduate education many people have been an influence to me helping to develop as a researcher and professional. In one way or another, my family and friends, professors, colleagues and co-workers have helped me to accomplish my goa ls. First and foremost, I would like to thank my advisor Dr. Vinay K. Gupta who has been an exceptional research mentor who provided me with an invaluable learning experienc e. I will always be appreciative of his interest in helping me augment my knowle dge, not only in this research but also in many other areas vital for the success of a gr aduate student. I would also like to thank my committee members, Dr. John Wolan and Dr. Norma Alcantar, which were an integral part of my graduate education. Special thanks to my lab partners: Mr. Adrian P. Defante, Mr. Cecil Coutinho, Mr. David Walker, Ms. Fedena Fanord, Ms. Justine Molas, and Dr. Jeung-Yeop Shim. They were an extraordinary group with whom I had many productive scientific discussions t ogether with lighter moments of relief. This was more than just a research group, but r ather a research family. Special recognition to Directors and Coordinators of the FGLS AMP Bridge to the Doctorate Program (NSF HRD # 0217675): Dr. Shekhar Bhansali, Dr. Ashanti Pyrtle, Mr. Bernard Batson, and Mrs. Eva Fernandez. This program provided m e with the financial support to pursue my graduate education and furthermore helped funding the dissemination of the results of my findings to the scientific communi ty.

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i TABLE OF CONTENTS LIST OF TABLES.............................................................................................................iii LIST OF FIGURES...........................................................................................................iv ABSTRACT.......................................................................................................................vi CHAPTER ONE: INTRODUCTION, MOTIVATION AND BACKGROUND...............1 1.1 Sensing and Detection Using Smart Polymers: Technical Background..................1 1.2 Composites of Polymeric Microgels and Metallic Gold Nanoparticles..................3 1.3 Molecularly Imprinted Materials.............................................................................5 1.4 Project Description...................................................................................................7 CHAPTER TWO: SYNTHESIS AND CHARACTERIZATION OF MICROGELS OF THERMALLY-RESPONSIVE POLYMERS...............................12 2.1 PNIPAM Microgels...............................................................................................12 2.1.1 Experimental Section....................................................................................12 2.1.2 Results and Discussions................................................................................15 2.2 Microgels Formed from Copolymers of P(NIPAM-AA)......................................17 2.2.1 Experimental Section....................................................................................17 2.2.2 Results and Discussions................................................................................18 2.3 Microgels Formed from Copolymers of P(NIPAM-GMA)...................................19 2.3.1 Experimental Section....................................................................................19 2.3.2 Results and Discussions................................................................................20 2.4 Microgel of PNIPAM with Interpenetrating Networks of PAA............................20 2.4.1 Experimental Section....................................................................................21 2.4.2 Results and Discussions................................................................................22 CHAPTER THREE: POLYMER-METAL NANOCOMPOSITES OF MICROGELS AND GOLD NANOPARTICLES.................................................29 3.1 Nanocomposites Based on PNIPAM Microgels and Gold Nanoparticles.............29 3.1.1 Experimental Section....................................................................................29 3.1.2 Results and Discussions................................................................................30 3.2 Nanocomposites Based on P(NIPAM-AA) Copolymer Microgels.......................31 3.2.1 Experimental Section....................................................................................32 3.2.2 Results and Discussions................................................................................35 3.3 Nanocomposites Based on P(NIPAM-GMA) Copolymer Microgels...................36 3.3.1 Experimental Section....................................................................................37 3.3.2 Results and Discussions................................................................................39 3.4 Nanocomposites Based on PNIPAM/PAA-IPN Microgels...................................42

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ii 3.4.1 Experimental Section....................................................................................42 3.4.2 Results and Discussions................................................................................43 CHAPTER FOUR: MOLECULARLY IMPRINTED POLYMERS (MIP).....................57 4.1 Molecularly Imprinted Polymers Based on PNIPAM/PAA IPN Microgel s.........57 4.1.1 Experimental Section....................................................................................57 4.1.2 Results and Discussions................................................................................59 CHAPTER FIVE: SUMMARY.........................................................................................63 REFERENCES..................................................................................................................65

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iii LIST OF TABLES Table 1: Formulation used for PNIPAM microgels..........................................................13 Table 2: Formulation used for P(NIPAM-AA) microgels................................................18 Table 3: Formulation used for P(NIPAM-GMA) microgels............................................20 Table 4: Formulation used for PNIPAM/PAA-IPN-I microgels......................................21 Table 5: Formulation used for PNIPAM/PAA-IPN-II microgels.....................................22 Table 6: Formulation used for P(NIPAM-AA) with CBA cross-linker...........................33 Table 7: Formulation used for molecularly imprinted PNIPAM/PAA microgels............ 59

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iv LIST OF FIGURES Figure 1: Schematic of the reversible volume phase transition that PNIPAM e xhibits due to thermal-stimuli........................................................................................9 Figure 2: Schematic of synthesis mechanism for the precipitation polymer ization of PNIPAM..............................................................................................................9 Figure 3: Schematic of the mechanism followed for the preparation of PNIP AMGNP nanocomposite.........................................................................................10 Figure 4: Schematic of the mechanism followed for the imprinting of metal ion in PNIPAM-GNP nanocomposite..........................................................................11 Figure 5: Turbidity data of PNIPAM-2%.........................................................................24 Figure 6: DLS of PNIPAM-4%-I......................................................................................24 Figure 7: Mean size of PNIPAM-4%-II............................................................................25 Figure 8: Changes in appearance of PNIPAM microgels upon heating the solution. ......25 Figure 9: TEM of PNIPAM-4%-I.....................................................................................26 Figure 10: Turbidity data of P(NIPAM-AA)-5%.............................................................26 Figure 11: Turbidity data of P(NIPAM-AA)-25%...........................................................27 Figure 12: DLS of P(NIPAM-GMA)................................................................................27 Figure 13: DLS of PNIPAM/PAA-IPN-II........................................................................28 Figure 14: DLS of PNIPAM/PAA-IPN-I.........................................................................28 Figure 15: Turbidity data of PNIPAM-2%-Citrate-GNP..................................................45 Figure 16: Changes in appearance of PNIPAM-2%-Citrate-GNP...................................45 Figure 17: TEM of (A) Citrate-GNP; (B) PNIPAM-2%-Citrate-GNP............................. 46 Figure 18: DLS of P(NIPAM-AA)...................................................................................46 Figure 19: UV-VIS of TOAB-GNP..................................................................................47

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v Figure 20: (A) DLS and (B) TEM of TOAB-GNP...........................................................47 Figure 21: DLS of P(NIPAM-AA)DMAP-GNP..............................................................48 Figure 22: UV-VIS of P(NIPAM-AA)DMAP-GNP........................................................48 Figure 23: TEM of (A) DMAP-GNP; (B) P(NIPAM-AA)DMAP-GNP..........................49 Figure 24: (A) UV-VIS and (B) DLS of Citrate-GNP......................................................49 Figure 25: Appearance of P(NIPAM-GMA)CYS............................................................50 Figure 26: DLS of P(NIPAM-GMA)CYS........................................................................50 Figure 27: (A) UV-VIS and (B) Appearance of P(NIPAM-GMA)CYS-GNP.................51 Figure 28: TEM of P(NIPAM-GMA)CYS-GNP..............................................................51 Figure 29: DLS of P(NIPAM-GMA)CYS-GNP-PNIPAM..............................................52 Figure 30: (A) UV-VIS and (B) Change in appearance of P(NIPAM-GMA)CYSGNP-PNIPAM................................................................................................52 Figure 31: TEM of P(NIPAM-GMA)CYS-GNP-PNIPAM.............................................53 Figure 32: (A) UV-VIS and (B) Change in appearance of P(NIPAM-GMA)CYSGNP-PNIPAM-AU.........................................................................................53 Figure 33: TEM of P(NIPAM-GMA)CYS-GNP-PNIPAM-AU......................................54 Figure 34: Thermal relationship between wavelength at maximum absorbance and mean size of P(NIPAM-GMA)CYS-GNP-PNIPAM-AU..............................54 Figure 35: DLS of Citrate-GNP and CTAB-GNP............................................................55 Figure 36: UV-VIS of Citrate-GNP and CTAB-GNP......................................................55 Figure 37: UV-VIS of PNIPAM/PAA-IPN-GNP.............................................................56 Figure 38: TEM of PNIPAM/PAA-IPN-GNP..................................................................56 Figure 39: DLS of PNIPAM/PAA-MIP-4%.....................................................................61 Figure 40: DLS of PNIPAM/PAA-MIP-7%.....................................................................61 Figure 41: DLS of PNIPAM/PAA-MIP-10%...................................................................62 Figure 42: DLS of PNIPAM/PAA-MIP-10% at different copper ion concentrations...... 62

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vi NOVEL POLYMER-METAL NANOCOMPOSITES FOR APPLICATIONS IN DETECTION AND SENSING Dayling L. Chaparro ABSTRACT Detection of trace elements such as organic contaminants, explosive residue s, and metal ions is an intellectually challenging task in science and enginee ring. It is also a topic of increasing importance due to its impact on society and the environment. Designing molecularly imprinted materials is one of the most promising approac hes to explore sensing and detection applications. “Stimuli-sensitive” polymer mate rials are ideal candidates for these imprinted sensors as they are able to respond to changes i n their environment and can be tailored by cross-linking the polymer chains. The responses can be amplified and transduced into measurable signals due to macromolecular propertie s provided by the use of a polymer. The purpose of the research in this project is to combine organic polymers with inorganic constituents to tailor the binding properties a nd the responses of the composite material for detection of metals ions in aqueous solut ions. The research, here, is based on a thermally responsive polymer such as poly(Nisopropylacrylamide) (PNIPAM), which exhibits a well-known reversible volum e phase transition in aqueous media around approximately 32C. Combining cross-linked microgels formed from PNIPAM and its copolymers with gold nanoparticles (GNP ) imparts the composite material with optical properties such as intense visibl e absorption

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vii due to the unique surface plasmon absorption of these small nanoparticles. The use of copolymers allows incorporation of functional groups, such as carboxylic acid, that ar e potential sites for binding metal ions. Cross-linking of the metal ion binding polymer imprints the metal ion in the PNIPAM microgel network. In this research, design of the composite material was investigated using copolymers of NIPAM and acrylic acid (AA), copolymers of NIPAM and glycid yl methacrylate (GMA), and interpenetrating networks of PNIPAM and PAA. A broad spectrum of polymerization conditions were studied such as changes in cross-linki ng density as well as changes in the synthetic procedure. Techniques such as turbidome try, ultraviolet visible spectroscopy (UV-VIS), transmission electron micro scopy (TEM), and dynamic light scattering (DLS) were employed to characterize the microgels as well as their composites with GNP. Preliminary investigation of imprinting the microgel s with heavy metal ions such as copper was also performed. The novel polymer-metal nanocomposites explored here will serve as an important contribution for the current ongoing research efforts in designing materials in the nano-scale capable of sensing and detecting metal ions in solution with high selectivity.

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1 CHAPTER ONE: INTRODUCTION, MOTIVATION AND BACKGROUND The broad aim of this thesis project is to develop a novel polymer-metal nanocomposite that can selectively bind heavy metal ions for applications in detecti on and sensing. The basis of the novel sensor is a “stimuli-sensitive” material because its intrinsic macromolecular properties allow for amplification of a signal In this research, we have used a thermally responsive polymer known as poly(N-isopropylacrylamide) or PNIPAM. To exploit the unique optical properties of gold nanoparticles (GNP) in the sensing, we have focused on the development of polymer-metal nanocomposites by combining GNP with cross-linked microgels of PNIPAM and its copolymers. For selective binding of the metal-ion, we have explored molecular imprinting in the PNIPAM microgels. The following sections provide an introduction to the materials, techniques, and concepts that are integral for the research in this thesis. 1.1 Sensing and Detection Using Smart Polymers: Technical Background Recent advances in materials science and engineering, and in particular, th e control of material properties, have been significant in the development of sensors 1, 2 The area of sensor technology is very broad and no simple classification is comple tely adequate. However, sensors can be categorized by either their chemical c omposition or their principle of operation 1-4 Effects such as thermal, electrical, mechanical, optical,

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2 and molecular interactions can be used for sensing 1-4 Sensors are widely used in all aspects of everyday life and their industrial applications include areas such as automotive, environment, and biomedicine 2, 3, 5, 6 The design and development of sensors requires many considerations such as the choice of the sensing element, issues of signal amplification, relay, and measur ement as well as suitable packaging of the sensor 1 The sensing element typically refers to a material that fundamentally interacts with the target analyte and can als o serve as a transducer by converting the interaction into a measurable output signal 1, 2 In this context, “smart materials”, which are able to respond to changes in their envi ronment, can be used in chemical sensors wherein a chemical change in the environment is transduced into a signal 2, 3, 5, 7, 8 One of the limitations in the use of “smart materials” is that the variations in environmental conditions and in analyte concentration need to be large to produce measurable responses 1, 3 “Stimuli-sensitive” polymers are a promising building block for sensing and detection because their intrinsic macromolecular properties allow for am plification of a signal that results from trace concentrations. The “stimuli-sensitive” m aterial used in this thesis research is a thermally responsive polymer known as poly(N-isopropylac rylamide) or PNIPAM. Since a cross-linked network of a polymer makes the network behave as one giant macromolecule, the research in this project focuses on colloidal, cross-li nked microspherical particles of PNIPAM or microgels. Synthesis of PNIPAM microgels was first reported by Pelton and Chibante in 1986 based on techniques performed by Philip Chibante in 1978 9, 10 Figure 1 illustrates a schematic of the reversible volume phase transition exhibited by PNIPAM nea r its

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3 lower consolute temperature (LCST) ~32C 10-13 At temperatures below LCST, PNIPAM microgels are very hydrophilic, swollen particles. However, upon incr easing the temperature to values above LCST the hydrophilic characteristic is los t. Here the microgels become hydrophobic and expel water from the cross-linked network as the chains collapse and tend to a globular shape. Because microgels of PNIPAM ca n reversibly respond in water to external stimuli such as temperature, these mat erials have been of great interest in applications such as chemical/biological sensors, d rug delivery, biomaterials, drug delivery and chemical/biological separations 5, 9, 13-16 PNIPAM microgels are nonionic but this property can be modified forming copolymer or interpenetrated networks with electrolyte materials such as m onomer or polymers of acrylic acid 10, 16-21 PNIPAM microgels, their copolymer and networks are typically synthesized by surfactant-free precipitation polymerizati on at ~70C using an initiator that such as potassium persulfate (KPS) 9-11 The cross-linking character of the microgels is achieved by adding a divinyl cross-linker such as methylene-bi s-acylamide (MBA). Figure 2 shows a schematic of procedures followed and ingredients used to synthesize PNIPAM microgels by precipitation polymerization. 1.2 Composites of Polymeric Microgels and Metallic Gold Nanoparticles Recently, interest in composite materials consisting of metal nanoparticle s and polymers in the form of microgels or networks has also increased. The addition of me tal nanoparticles with unique properties to a smart polymer leads to reversible m anipulation of these properties and greatly expands the range of material properties 2, 6, 22 The properties of the polymer-metal nanocomposites depend on the metal nanoparticle si ze,

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4 shape and concentration as well as the interaction with the polymer matrix 2, 6, 22 Nanometer sized, colloidal metal particles are of great interest since they can show sizedependent properties different from those exhibited by the bulk metal 2, 6, 22 The nanometer range at which these effects most significantly occur is bet ween 1-10nm in diameter 2, 6, 22 Electronic, optical, catalytic, and thermodynamic properties have been observed to be the most significant size-dependent properties of metal nanoparticl es 2, 6, 22 For gold nanoparticles, the surface plasmon absorption is one of the most attractive of these size-dependent properties because it leads to unique optical properties in t he visible spectrum of light. GNP solutions show conspicuous colors in solution because of their characteristic plasmon absorptions 2 This significant property makes GNP suitable for detection and sensing applications. There are many synthetic strategies to prepared colloidal gold and some of these can be traced back to the middle ages. However, all of the methods used before the 1990’s, produced highly unstable gold nanoparticles (GNP) with wide size distributions 6, 23-27 In 1994, Brust and Schiffrin demonstrated that uniform size of well dispersed GNP can be afforded by protecting their surfaces with alkanethiol molecules in orga nic media 23, 24 This method, which is very popular for producing 2-5nm GNP, involves the reduction of a gold salt by the use of sodium borohydride in the presence of a cappin g agent. Another common approach utilizes reduction of a gold salt and stabilization in presence of trisodium citrate to produce GNP that are greater than 10nm 28, 29 Several approaches have been developed to prepare polymer-metal nanocomposites in solution. These include combination of the nanoparticle with the polymer by simple diffusion mechanism, functionalization of the nanoparticle and/or

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5 polymer matrix to induce loading, in-situ reduction of metal nanoparticles in the polym er matrix, in-situ synthesis of polymer matrix in presence of preformed metal nanoparticles, as well as evaporation and sputtering, and the use of fluidized beds 6, 22 Synthesis of polymer-metal nanocomposites can be very tedious and difficult to achieve due to the great importance of emphasizing the best properties of both material for some applications. 1.3 Molecularly Imprinted Materials The fundamental basis of most chemical sensors is molecular interactions between a target analyte and a receptor. Improvements in sensor technology are, therefore, heavily dependent on optimizing this interaction and making it selective Molecular imprinting is one approach that allows this selectivity. Molecular imprinting is a new and attractive technique inspired by Fischer’s lock-and-key metaphor propose d in 1984 and is considered to be one of the keys for future science and technology in the development of molecular assemblies for molecular recognition applications 7 In recent years, extension of molecular imprinting to soft materials such as polymers has been actively investigated because of the flexibility in imprinting using chemica l cross-linking of the polymer 7, 8, 30 The concept of molecular imprinting in polymers is based on the complementary polymerization and immobilization (locking) of a template or substrate molecule normally in a porous polymer matrix 7 The template is first allowed to interact/bind with one of more functional monomers in the matrix in an orderly assembly. Immobilizat ion or locking of these interactions forms cavities that serve as recognition si tes

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6 complementary and selective for the template molecule. The rebinding process of the substrate relies on its diffusion to the recognition sites through the porous polymer matrix. In principle, many molecules, ranging from small ones such as drug s, hormones, metal ions to large ones such as proteins, could be molecularly imprinted 7, 8, 30 Several factors influence the preparation of a molecularly imprinted polymer, of whi ch the most important is the selection of the template molecule 7, 8, 30 Others include the type and availability of functional monomers to bind the template molecule, structure, siz e and solubility of template, and the application of the resultant imprinted material 7, 8, 30 For molecularly imprinted polymers (MIP), the imprinting method could be of two types: covalent or non-covalent 30 For the covalent MIP the polymerization should be carried out under conditions not altering the covalent linkage with the template. Upon rebinding the template in the imprinted polymer it is expected that the covalent li nkage is formed 30 Non-covalent (hydrogen bonding, electrostatic interaction, and coordinationbond formation) functional monomer-template connections, could be obtained in-situ by adding the components to the reaction mixture and removing the template after polymerization with a polar solvent 7, 8, 30 Synthesis of non-covalent imprinting is easier to achieve, the template is removed easily from the polymer and rebinding and releas e interactions are faster 7, 8, 30 On the other hand, covalent imprinting results in the imprinting of more accurate structure of guest-binding site and a wide vari ety of polymerization conditions can be used 7, 8, 30 Design of MIPs with improved recognition sites is still a challenge since s ynthetic techniques are relatively simple and limited when compared to biological syst ems 7, 8, 30

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7 MIPs have a many potential application including sensors devices where the MIP serves a recognition element. 1.4 Project Description This thesis project aims to develop novel polymer-metal nanocomposites with receptor sites that can selectively bind heavy metal ions for applications suc h as environmental sensing. The use of copolymers of NIPAM and acrylic acid or interpenetrated networks of poly(acrylic acid) with PNIPAM microgels was investigated to incorporate carboxylic acid groups for potential binding of metal ions. Use of the imprinting technique was explored to memorize the shape and binding of the metal ion within the polymeric matrix. Development of the polymer-metal nanocomposite was achieved by combining microgels based on PNIPAM with gold nanoparticles (GNP). Figure 3 shows a simplified schematic of how this procedure was performed. The mechanism followed for the imprinting technique and the expected responses of the composite upon molecular recognition of the target analyte are schematically illustrated in Figure 4. GNP in the composite microgel was used to enhance the optical properties of the composite and allow for colorimetric sensing and detection by transducing the molecular recognition into changes in surface plasmon absorption of the system. The following chapters provide detailed information on the research performed in this thesis project. Chapter 2 discusses the synthesis and characterization of PN IPAM microgels as well as microgels based on copolymers and interpenetrating netw orks. Chapter 3 describes several strategies for the preparation of nanocomposites based on microgels and GNP. Molecular imprinting procedures on interpenetrating type of

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8 microgels are summarized in Chapter 4. Finally, Chapter 5 provides a summary of the research and includes recommendations on modifications and improvements of the polymer-metal nanocomposites investigated here for applications in detection a nd sensing.

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9 Figure 1: Schematic of the reversible volume phase transition that PNIP AM exhibits due to thermal-stimuli. Monomer: NIPAM Crosslinker: MBA Initiator: KPS Figure 2: Schematic of synthesis mechanism for the precipitation pol ymerization of PNIPAM. 70C Precipitation of Microgels In Solution N 2 Purge Reaction Mixture Addition of Initiator Under N 2 PNIPAM O H H O H H O H H O H H O H H O H H O H H O H H O H H O H H O H H O H H Hydrophilic Increase in Temperature Hydrated Chains Expulsion of water collapse of chains Reversible Volume Transition MICROGELS Hydrophobic Oligoradic Precursor Particle Growing Particle PNIPAM Microgel N N O O HH N O OOS S O O O O O O K + K + Aqueous Media Changes in shape and phase separation

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10 Figure 3: Schematic of the mechanism followed for the preparation of PNI PAMGNP nanocomposite. GOLD NANOPARTICLES -COOH COOH -COOH -COOH -COOH -COOH -COOH -COOH -COOH -COOH -COOH -COOH -COOH -COOH PNIPAM MICROGEL WITH POLY(ACRYLIC ACID) FUNCTIONAL GROUPS NANOCOMPOSITE OF PNIPAM-GNP MICROGELS ~ 500 nm COOH COOH -COOH -COOH -COOH -COOH -COOH -COOH COOH -COOH

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11 Figure 4: Schematic of the mechanism followed for the imprinting of me tal ion in PNIPAM-GNP nanocomposite. Me+ COO Formation of carboxyl-metal ion complex upon incubation metal ion in composite Me+ Me+ Me+ Me+ Me+ Me+ Me+ COO COO COO Me+ COO COO COO Me+ COO COO Me+ COO -C O OO -O CMe Imprinting of metal ion in binding site provided by carboxyls Complementary shape of metal memorized in the polymeric matrix Removed C O O O O C Me+ C O OO -O CMe+ Me+ Me+ Expectation that this change will be most sensitive in the transition region Inter-particle distance of GNP changes and the plasmon absorption shifts Sudden recognition of analyte metal ion induces volume response COO

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12 CHAPTER TWO: SYNTHESIS AND CHARACTERIZATION OF MICROGELS OF THERMALLY-RESPONSIVE POLYMERS 2.1 PNIPAM Microgels The initial phase of the project focused on preparation of PNIPAM microgels to understand the impact of polymerization conditions. Towards this goal, effects of changes in amounts of monomer, solvent, cross-linker, and initiator were studied. Two microgel samples were prepared of 2 and 4% cross-linking. The cross-linking density was varied since it could be related to the pores size within the microgels and i s important in the preparation of composites composed of PNIPAM and small nanoparticles 9, 31-33 The cross-linking density also affects the response of the microgels low c ross-linking density makes the microgels more flexible when compared to higher cross-li nking 9, 31-35 2.1.1 Experimental Section Materials N-Isopropylacrylamide, 97% (NIPAM) was purchased from Aldrich Chemicals (WI) and purified by recrystallization from hexane. N,N’Methyline-Bis -Acrylamide (MBA) and Potassium Persulfate (KPS) were purchased from Aldrich Chemic als (WI) and used without further purification. The hexane used for purification of NIPAM was of reagent grade. Unless otherwise specified, the water used in all synthesis w as purified using an EasyPure TM UV system (Barnstead, IA).

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13 Synthesis of PNIPAM Microgels PNIPAM microgels were prepared by precipitation polymerization by usin g NIPAM monomer, cross-linker and a radical initiator in aqueous media. A fixed amount of recrystallized NIPAM monomer was dissolved in deionized water inside a thr ee neck round bottom flask. A corresponding amount of MBA (cross-linker) was added to the aqueous solution and stirred. A separate solution of KPS (initiator) was prepared by dissolving KPS in 10mL of deionized water. The amount of reactants and solvent for preparation of different samples is summarized in Table 1. Table 1: Formulation used for PNIPAM microgels. SAMPLE NIPAM (mg) MBA (mg) KPS (mg) H 2 O (ml) PNIPAM-2% 300 6 10 30 PNIPAM-4%-I 250 14 20 50 PNIPAM-4%-II 400 22 20 150 The stirred reaction mixture and the KPS solution were bubbled with nitrogen for 30-35 minutes. After bubbling with nitrogen, the reaction mixture was heated up to ~70C in an oil bath under a nitrogen atmosphere. The initiator solution was added to the reaction mixture in one shot once the reaction temperature was stable. The reac tion was allowed to proceed under nitrogen atmosphere and continuous stirring at ~70C for four hours. The resultant milky white microgel solution was cooled down to room temperature and purified. Typically, microgels were purified by centrifugation at 8500 rpm f or 30 minutes and redispersing in deionized water. The process was repeated at least four

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14 times. For dry samples, the PNIPAM microgels were dried overnight under vacuum usi ng a vacuum oven at 30-40C. Characterization In addition to turbidometry, two methods were commonly used to characterize PNIPAM microgels: Transmission electron microscopy (TEM) and Dynami c Light Scattering (DLS). DLS was useful to characterize the particle size a nd its distribution while TEM was used to probe the shape of the microgels. TEM characterization in this thesis was performed using a FEI Morgagni 268D instrument. For the analysis of PNIPAM-4% microgels, a drop of an aqueous solution containing the microgels was first placed on a Formvar-coated Cu grid. The ex cess of sample in the mesh grid was wiped off with a Kim-wipe after ~10 seconds. After allowing the sample to dry for ~1min, the grid was placed in the microscope. DLS, also know as photon correlation spectroscopy (PCS) and quasi-elastic light scattering (QELS) is a techniques to measure the size of colloidal parti cles 36 It measures the size by relating it to the Brownian motion of the particles. A laser light incident on the sample is scattered due to Brownian motion of the particles 36, 37 In DLS, the intensity of scattered light is measured as a function of time and can be related to the transl ational diffusion coefficient, which can be converted into a particle size using the Stokes-Einstein equation 36 The hydrodynamic diameter given by DLS corresponds to that of a sphere that has the same translational diffusion coefficient as the partic le 36 DLS measurements are sensitive to temperature, viscosity, density, and ionic conce ntration 36 DLS measurements for this thesis were performed using a Malvern Zetas izer Nano-S instrument. For characterization of PNIPAM-4% microgels, 1mL of diluted aqueous

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15 solution in a cuvette was used. To obtain the changes in the size of PNIPAM microgel with temperature, the measurements were performed from 24-40C. 2.1.2 Results and Discussions Thermal behavior and volume phase changes of PNIPAM-2% were characterized by the use of a turbidometer. As shown in Figure 5, the turbidity of the solution increased during heating and decreased as it was cooled down. Changes in turbidity are an indication of the shrunken or swollen state of the microgels in solution. As seen in Figure 2.1, the solution became noticeably turbid when temperature reached 30C. A steep turbidity increase was observed up to 40C, where turbidity reached a constant value. The range from 30-34C was the volume phase transition (VPT) of the microgels where the microgels showed a significant change in volume by shrinking or swelling. The midpoint of this range ~32C can be used as a rough transition temperature. A small hysteresis in the transition phase was observed. However, the transition was rever sible and the solution returned to a transparent state after cooling down. DLS characterization of PNIPAM-4%-I, shown in Figure 6, illustrates how the mean size of the microgels decreases upon heating the solution from 24-38C. The mean size of the microgels changed from ~1 m to 500nm. The volume phase transition region was observed to be from 31-36C and the mid-temperature was 33.6C. DLS of PNIPAM-4%-II, in contrast to PNIPAM-4%-I, showed a smaller hydrodyna mic diameter (Figure 7). The size was calculated to be 550nm and 250nm, at a swollen and shrunken state, respectively. The smaller size of PNIPAM-4%-II was attribute d to polymerization with three times more solvent. PNIPAM-4%-II also showed a smoother size tre nd which

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16 is characteristic of a smaller polydispersity. Therefore, it is expect ed that these particles are more uniform in size and better dispersed in solution than PNIPAM-4%-I. The changes in appearance of PNIPAM microgels in solution due to thermal responses can be seen in Figure 8. The three pictures of a solution containing PNIPAM microgels illustrate the gradual change increase in turbidity as the soluti on is heated to its transition temperature. The solution is transparent at temperatures below LCS T and as the temperature reaches the LCST the solution starts becoming turbid. The PNIPAM solution changes from transparent to turbid or milky-white when the mean size of the mic rogels decreases and the refractive index contrast becomes large between the parti cles and the solvent. As seen in the TEM image shown in Figure 9, the size of the microgels was observed to be ~440nm at a shrunken state, which agrees with DLS characterization a t temperatures above LCST. In summary, precipitation polymerization is a useful synthetic technique for the preparation of PNIPAM microgels. The amount of cross-linker could be varied without changing the thermal behavior and VPT dramatically to trigger different st ates of densities in the microgels. This, in fact, could serve to indirectly control the pore s ize and flexibility of the polymer and potentially form composites by loading and re taining nanoparticles within the microgel network 33 The amount of solvent is an important material to synthesize monodisperse microgels of different sizes. It i s known that combination with surfactants such SDS (sodium dodecyl sulfate) in 1 mole%, can lead to the formation of microgels of even 100nm in diameter 11, 16, 33, 38

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17 2.2 Microgels Formed from Copolymers of P(NIPAM-AA) Copolymers of P(NIPAM-AA) microgels were prepared with the purpose of obtaining a material with functional groups capable of binding metal ions and metal nanoparticles while keeping the unique thermal-responsive properties of NIPAM. The functional groups provided by acrylic acid (AA) are carboxylic acid groups (COOH ). These not only have the potential to bind metal cations such as copper but also to modify physical properties of the microgels 16, 20, 38 When present in the microgels, COOH groups increase hydrophilic characteristics, which affect the transition t emperature of the microgel and leads to a higher LCST 16, 20, 38 Experiments described in this section were designed to understand the effects of acrylic acid monomer concentration on P(N IPAMAA) copolymer microgels and the changes in their VPT temperature. 2.2.1 Experimental Section Acrylic acid 99% and hydroxyl-ethyl-acrylate (HEA) 96%, were both purchased from Aldrich Chemicals (WI) and used without further purification. The syntheti c procedures and characterization techniques used for P(NIPAM-AA) microgels were similar to the ones discussed in section 2.1.1. Precipitation polymerization was carr ied in deionized water and initiated by KPS. Modifications to the procedure were mainly i n the addition of AA and HEA monomers to the reaction mixture along with NIPAM and MBA prior initiating the reaction according to the amounts shown in Table 2. Two copolymers of P(NIPAM-AA) cross-linked with MBA were prepared: P(NIPAM -AA)5% and P(NIPAM-AA)-25%. The percentage in the above naming designates the mole % of AA monomer used relative to NIPAM for the preparation of the copolymer.

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18 Table 2: Formulation used for P(NIPAM-AA) microgels. SAMPLE NIPAM (mg) AA (mg) HEA (mg) MBA (mg) KPS (mg) H 2 O (ml) P(NIPAM-AA)-5% 300 10 6 16 10 30 P(NIPAM-AA)-25% 300 46 14 17 11 30 2.2.2 Results and Discussions Turbidity measurements were used to characterize the thermal behavior and VPT of the P(NIPAM-AA) copolymer microgels. Figures 10 and 11, illustrate the turbi dity data of P(NIPAM-AA)-5% and P(NIPAM-AA)-25%, respectively. P(NIPAMAA)-5% exhibited a transition region from ~33-36C and a mid temperature of ~34C. As expected, copolymer microgels containing AA exhibited a slightly higher LCST in comparison to PNIPAM microgels due to the presence of COOH in their networks. P(NIPAM-AA)-25% showed a similar thermal-behavior and VPT to P(NIPAM-AA )-5% even though their AA molar percentage differed by 20%. This effect was attr ibuted to the increase of HEA composition in the preparation of the copolymer with 25 mole % AA. HEA increases size of porous voids and is favorable for making composites 16, 20, 39 However, it also enhances the hydrophobic forces in the microgels. Therefore, the thermal-behavior and changes in VPT induced by an increase in AA composition are inhibited to some extent by an increase in HEA composition in the synthesis of thes e copolymers. In summary, precipitation polymerization is also useful for synthesizing copolymers of PNIPAM microgels. The composition of AA can be varied to control COOH groups within the microgels as well as their hydrophilic forces and VPT.

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19 Hydrophobic monomers such as HEA counteract the interactions of AA and prevent the LCST from increasing to high values in copolymers of PNIPAM and AA. 2.3 Microgels Formed from Copolymers of P(NIPAM-GMA) In this section, thermo-responsive polymer particles were synthesized using a combination of Glycidyl methacrylate (GMA) and NIPAM monomers followi ng a procedure similar to those reported by Kawaguchi 40-42 GMA introduces epoxy groups in the microgel that can serve as reactive sites for immobilization of functional groups, such as amine (-NH) and thiol (-SH), that can further be used to hold gold nanoparticles in the microgel network using chemical interactions 40-42 2.3.1 Experimental Section Glycidyl methacrylate (GMA) stabilized 97%, was purchased from Acros Organics (NJ) and used after purification from inhibitors with neutral alumina by column chromatography. P(NIPAM-GMA) copolymer microgels were also prepared by precipitation polymerization using similar procedures explained in detail in se ction 2.1.1 and according to values in Table 3. However, since GMA is a hydrophobic monomer that gets consumed faster than NIPAM, some reaction conditions were adjusted to favor t he synthesis of a more uniform copolymer in terms of monomer compositions 40-42 To bring the reaction rate of these two monomers to an approximate value, the temperature at which the polymerization was carried out was increased to 80C and the reaction time extended to six hours. Purified GMA monomer was added to an aqueous solution containing NIPAM and MBA prior initiating the reaction. The reaction was ini tiated as

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20 before by adding KPS. The resultant copolymer was purified by centrifugati on as explained in section 2.1.1. Table 3: Formulation used for P(NIPAM-GMA) microgels. 2.3.2 Results and Discussions P(NIPAM-GMA) copolymer microgels were characterized by DLS. Fi gure 12 shows the hydrodynamic diameter of the microgels at a swollen and shrunken stat e was 450nm and 250nm, respectively. The transition region of the microgels ranged from 30-35C. As expected, the thermal behavior and VPT was similar to that observed on PNIPAM and P(NIPAM-AA) microgels. However, the presence of GMA made t he microgel solution looked opaque at ambient temperature. As a result, the jump in turbidity due to heating above LCST is not as significant as observed for PNIPA M microgels in Figure 7. 2.4 Microgel of PNIPAM with Interpenetrating Networks of PAA Interpenetrated networks (IPNs) of PNIPAM and AA were synthesized in thi s section by two methods-a two-step approach and a one-step method 15, 18, 19, 21 These were prepared with the purpose of obtaining a thermo-responsive material having as many carboxylic acid groups as possible within the microgel. According to literature, the hydrophilic PAA components in a IPN-type case are not chemically attach ed to the SAMPLE NIPAM (mg) GMA (mg) MBA (mg) KPS (mg) H 2 O (ml) P(NIPAM-GMA) 673 423 7 64 100

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21 PNIPAM network 19, 43-45 Therefore, they should not alter the LCST of the PNIPAM microgels and their behavior. The IPN-type material will be investigated fo r their potential application in the preparation of nanocomposites and molecularly imprinte d PNIPAM microgels. 2.4.1 Experimental Section A sodium salt of PAA (Typical Mw 15,000) and N,N,N 1 N 0 – tetramethylethylenediamine, 99% reagent (TEMED) were both purchased from Aldrich Chemicals (WI) and used without further purification. Synthesis of Microgels of PNIPAM with Interpenetrating Networks of PAA One-Step Approach: PAA was mixed together with NIPAM and MBA according to Table 4. Following precipitation polymerization procedures explained in section 2.1.1, the reaction was carried out under a nitrogen atmosphere and continuous stirring at ~70C for four an a half hours. The pH value of the reaction mixture was measured to be ~9.0 before addition of the initiator. The product, PNIPAM/PAA-IPN-I, was purified and characterized in a similar manner as in previous sections. Table 4: Formulation used for PNIPAM/PAA-IPN-I microgels. SAMPLE NIPAM (mg) PAA (ml) MBA (mg) KPS (mg) H 2 O (ml) PNIPAM/PAA-IPN-I 2000 3 80 40 400 Two-Step Approach : PNIPAM microgels were first prepared using procedures outlined in section 2.1.1. These microgels were mixed with AA in cold DI water. The round bottom

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22 flask containing the reaction mixture was placed in an ice bath to lower the solution’ s temperature to ~ -5C. This temperature was used to keep the microgels in a swol len state while polymerization of AA to PAA chains takes place within the PNIPAM networ ks. TEMED was used to accelerate the polymerization kinetics at -5 C. Table 5 provi des the amounts of the reagents used. The reaction proceeded under a nitrogen atmosphere and continuous stirring for six hours. The product, PNIPAM/PAA-IPN-II, was purified f our times by centrifugation, characterized and stored for further use. Table 5: Formulation used for PNIPAM/PAA-IPN-II microgels. 2.4.2 Results and Discussions PNIPAM/PAA-IPN-II was characterized by DLS as shown in Figure 13 The V PT of this IPN was similar to that observed for its seed, PNIPAM-4%-II. Howeve r, the hydrodynamic diameter in the swollen state was decreased by 50nm when compa red to the swollen diameter of PNIPAM-4%-II. During the preparation of PNIPAM/PAA -IPNII no cross-linker (MBA) was used to in the polymerization of AA. However, unreacte d vinyl groups within PNIPAM-4%-II can undergo cross-linking with some of the PA A chains and this cross-linking of PAA chains within the microgel can prevent the I PN microgel from swelling to the same extent as the pure PNIPAM microgel. The composition of AA was found to be 48% by mass (*measurements of total nitrogen content for estimation of AA content were done by Mr. Cecil Coutinho). SAMPLE PNIPAM-4%-II (mg) AA (mg) KPS (mg) TEMED (ml) H 2 O (ml) PNIPAM/PAA-IPN-II 100 300 6 0.06 100

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23 DLS characterization of PNIPAM/PAA-IPN-I is illustrated in Figu re 14. The size at the swollen state was ~300nm more than that of PNIPAM/PAA-IPN-II and the size ranged from ~800-450nm. The composition of AA was 52% by mass, which is closed to that obtained for PNIPAM/PAA-IPN-II. Thus, both one-step and two-step procedures proved to be useful in the preparation of PNIPAM microgels with enough AA content. However, the one-step approach was found to be simpler and more controllable than the one used to synthesize PNIPAM/PAA-IPN-II.

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24 Figure 5: Turbidity data of PNIPAM-2%. Figure 6: DLS of PNIPAM-4% -I. Lines are drawn as a guide. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 TURBIDITY (mV) 40 35 30 25 20 15 TEMPERATURE(C) Heating Cooling Transition: 30-34CTmid= 32C 1100 1000 900 800 700 600 500 MEAN SIZE (nm) 40 38 36 34 32 30 28 26 24 TEMPERATURE (C) Transition: 31–36CTmid = 33.5C

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25 Figure 7: Mean size of PNIPAM-4% -II. Lines are drawn as a guide. Figure 8: Changes in appearance of PNIPAM microgels upon heating the solut ion. 600 550 500 450 400 350 300 250 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 29-35CTmid= 32C Increasing Temperature

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26 Figure 9: TEM of PNIPAM-4% -I. Figure 10: Turbidity data of P(NIPAM-AA)-5%. 500nm 1.2 1.0 0.8 0.6 0.4 0.2 TURBIDITY (mV) 40 35 30 25 20 15 TEMPERATURE ( C) Heating Cooling Transition: 32-36CTmid: 34C

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27 Figure 11: Turbidity data of P(NIPAM-AA)-25%. Figure 12: DLS of P(NIPAM-GMA). Lines are drawn as a guide. 0.8 0.6 0.4 0.2 TURBIDITY (mV) 45 40 35 30 25 20 15 10 TEMPERATURE (C) Transition: 32-36CTmid= 32C 450 400 350 300 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 29-35CTmid= 32C

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28 Figure 13: DLS of PNIPAM/PAA-IPN-II. Lines are drawn as a guide. Figure 14: DLS of PNIPAM/PAA-IPN-I. Lines are drawn as a guide. 500 450 400 350 300 250 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 29-34CTmid= 31.5C 700 600 500 400 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 30-35CTmid= 32.5C

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29 CHAPTER THREE: POLYMER-METAL NANOCOMPOSITES OF MICROGELS AND GOLD NANOPARTICLES 3.1 Nanocomposites Based on PNIPAM Microgels and Gold Nanoparticles In this section the preparation of nanocomposites based on mixing PNIPAM microgels and GNP is explored. This method was selected over others because it is simple to perform. The approach was based on physical entrapment of GNP by diffusion through the pores of PNIPAM microgels. The diffusion process was achieved by me ans of the stimuli-responsive reversible behavior of PNIPAM. Citrate-stabiliz ed GNP of ~12nm in size and PNIPAM-2% ( cf Section 2.1) were the main components used for the preparation of the nanocomposite. 3.1.1 Experimental Section Citrate-stabilized GNP (JYS-I-1-1P) were synthesized by Dr. J-Y S him using procedures reported in literature 29, 46, 47 The nanocomposites based on PNIPAM-2% and citrate-stabilized GNP was prepared by a simple diffusion mechanism. The microgels were swollen and shrunken by altering the solution’s temperature below and above LCST to facilitate the diffusion of GNP through their pores. A 1mg/ml aqueous solution of PNIPAM-2% was mixed together with JYS-I-1-1P citrate GNP (~12nm) in a volume ratio of the microgel to GNP dispersion of 2:3. The solution was gently shaken for a few seconds, placed in an ice bath and stirred for 1 hour. The vial containing the resulting solution of PNIPAM-2%-Citrate-GNP was tr ansferred

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30 to an oil bath at 40C and stirred at same rate as before for an additional hour. The two steps of heating and cooling mentioned above constitute one cycle. The sample was subjected to three cycles over 6 hours. The nanocomposite sample was characteri zed and stored in the refrigerator for further use. In addition to techniques used in Chapter 2, UV-VIS spectroscopy was utilized to measure the light absorption of GNP and nanocomposites composed of microgels. UV-VIS spectroscopy characterization equipments used here were: Jasco Spectr ophotometer (Model V-530) and Ocean Optics USB-2000 Model. 3.1.2 Results and Discussions Turbidometry showed that the LCST for the nanocomposite was near 33C. In comparison to the PNIPAM-2% the VPT of the composite was narrower and significa ntly more discontinuous (Figure 15). The resultant nanocomposite solution was pink at temperatures below LCST (Figure 16). The pink appearance of the solution was due to the surface plasmon absorbance of the GNP. However, upon heating above LCST the turbidity of the polymer dominates the appearance. When the composite was purified by centrifugation to separate PNIPAM-2%Citrate-GNP from GNP that was outside the microgels, a dark colored preci pitate was obtained. This precipitate was re-dispersed in water. TEM images revealed tha t formation of the composite was not achieved using a simple heating and cooling cycle in our samples. Figure 17 (A) shows TEM of citrate-stabilized GNP and (B) shows the “composite” sample. An examination of the TEM reveals that the citrate-GN P was randomly dispersed throughout the sample and did not show any visible signs of

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31 entrapment within the spherical microgels (note: the lightly cross-linked mi crogels are not clearly observed in the image because they only weakly scatter the electr ons in TEM). Failure to entrap GNP can be attributed to possible separation of GNP from the microgel network upon purification due to large pore size and poor stability of GNP within the network. Minimization of the pore size of PNIPAM microgels is achievabl e by increasing cross-linking density and incorporation of functional groups in the mic rogel or on the surface of the GNP can help stabilize the GNP within the microgel. 3.2 Nanocomposites Based on P(NIPAM-AA) Copolymer Microgels Since results from section 3.1 suggest that functional groups that interact chemically with GNP can be useful to form nanocomposites, we used P(NIPMA-AA) copolymer microgels that incorporated thiol (SH) groups. Thiol groups have affi nity to both Au 3+ ions and colloidal gold 48, 49 By incorporating thiol groups our goal was to stabilize GNP within P(NIPAM-AA) copolymer microgels. The COOH groups from the acrylic acid could then be expected to bind metal ions and form a complex that could be used to prepare an imprinted polymer. The approach used to introduce thiols groups in P(NIPAM-AA) copolymer microgels was to combine two type of cross-linkers during the polymerization of NI PAM and AA ( cf section 2.2). The two chemical cross-linkers used to synthesize the copolymer microgels were MBA and N,N-cystamine-bis-acrylamide (CBA) 48, 49 Cross-links formed with MBA are chemically stable and inert, and do not have affinity towar ds gold 48, 49 CBA is a cross-linker containing reactive disulfide bridges that can form polymer

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32 chain transfer agents known as sulfhydryls 48, 49 In this manner, CBA can take part, not only in the branching of the polymer backbone through thiol-ethers bonds, but in the chemical stabilization of GNP within the copolymer microgel 48, 49 In this context, a second approach was also investigated to promote the interaction of GNP with the microgel. GNP was synthesized in an organic medium and transfer red to water using a positively-charged ligand known as 4-dimethylaminopyridine (DM AP) 50 As previously reported, DMAP ligand attaches to the surface of the colloidal gold nanoparticles and stabilizes the GNP in toluene as well as facilitates the phase transfer of GNP from toluene to water 50 By using DMAP and making the GNP surface positively charged, the expectation was that the deprotonated, negatively charged COOH groups of acrylic acid could influencing the loading of GNP via electrostatic att ractions. 3.2.1 Experimental Section Materials N,N-cystamine-bis-acrylamide (CBA) was purchased from Sigma-Aldric h Chemicals (WI) and used without further purification. Hydrogen tetrachloroa urate (HAuCl 4 ), tetraoctylammonium bromide (TOAB), sodium borohydride (NaBH 4 ) 4dimethylaminopyridine (DMAP) and mercapto-undecanoic acid (MUA) were al l purchased from Acros Organic (NJ). Synthesis of P(NIPAM-AA) Copolymer Microgels with CBA Cross-Linker P(NIPAM-AA) copolymer microgel with a composition of 26 mole% of AA wer e prepared by precipitation polymerization following similar procedures used to synt hesize copolymer microgels of PNIPAM and AA in Chapter 2. Since CBA is not soluble in

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33 water, it was dissolved in 250 L methanol before addition to the reaction mixture. Table 6 gives details on the amount of each reactant used to polymerize the thermo-responsi ve nanomaterial. The reaction proceeded for 4 hours. The product was purified by dialysis ( 2 weeks) and centrifugation (twice). The polymer, 260 mg, obtained upon drying it under vacuum was stored in a refrigerator for further use. P(NIPAM-AA) copolymer microgel were characterized by turbidometry, DLS and TEM. Table 6: Formulation used for P(NIPAM-AA) with CBA cross-linker. SAMPLE NIPAM (mg) AA (mg) HEA (mg) CBA (mg) MBA (mg) KPS (mg) H 2 O (ml) P(NIPAM-AA) 304 49 19 8 120 10 30 Synthesis of Gold Nanoparticles Stabilized by DMAP Gold nanoparticles (GNP) were synthesized in an organic medium by adapting the method reported by Brust et al 23, 24 A 38mM aqueous solution of hydrogen tetrachloroaurate (HAuCl 4 ) was added slowly to a 13mM solution of tetraoctylammonium bromide (TOAB) in toluene. The solution was stirred for 1.5 hr to allow the metal salt to be transferred to the organic phase. To reduce Au 3+ ions, a freshly prepared aqueous solution of 0.13M sodium borohydride (NaBH 4 ) was added dropwise to the mixture while stirring. The solution turned ruby red after the gold reduction wa s allowed to stir overnight. The organic layer was separated from the aqueous laye r and washed with water, 0.1M HCl, and 0.1M NaOH. The procedure was repeated three times and a final wash with DI water was performed. The organic layer containing the GNP stabilized by TOAB was allowed to dry with anhydrous sodium sulfate (NaSO 4 ) for 1

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34 hour. The TOAB-GNP recovered in 20mL of toluene was transferred to an amber bottle and stored in the refrigerator for characterization and further experiment s. TOAB-GNP was characterized by UV-VIS spectroscopy and TEM. For TEM measurement s, a drop of the toluene solution containing TOAB-GNP was placed in a Formvar-coated Cu grid. Particle sizing was also performed using DLS. DMAP-GNP was prepared by typically adding 20mg of solid DMAP to 250 L of the toluene solution containing TOAB-GNP. The displacement of TOAB by DMAP was allowed to proceed for 1 hour without shaking the solution. Displacement of TOAB by DMAP caused GNP to precipitate slowly out of solution. DMAP pellets appearance changed from white to black, suggesting GNP attachment. The supernatant, toluene was then removed carefully with a glass pipette to separate it from the black DMA P-GNP pellets. The samples were dried with a low flow rate of N 2 to help evaporate the remaining of toluene for 1 hour. The resulting DMAP-GNP were dispersed in wat er by adding 250 L of DI water to the tube containing the particles. The solution changed from transparent to turbid as dispersion of the particles in water phase was taken place DMAP-GNP solution (pH 11) was sonicated for 20 minutes to break down aggregates and then characterized by TEM. Nanocomposites Based on P(NIPAM-AA) Microgel with CBA Cross-linker To prepare the nanocomposites based on P(NIPAM-AA) copolymer microgel cross-linked with MBA and CBA, a mixing procedure was used. A volume of 0.45mL of positively charged DMAP-GNP solution was added to 0.5mL of 0.2mg/mL of P(NIPAM-AA). The sample was then sonicated, well-shaken, and stored in the refrigerator unt il further processing. Some sedimentation was observed the next day. After shaking the

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35 sample, 0.244mg of MUA (5mM in ethanol) was added to the sample to functionalize the GNP surface already loaded within the microgel. 3.2.2 Results and Discussions P(NIPAM-AA) microgels with CBA cross-linker showed a reversible volum e transition with temperature. As shown in Figure 18, the mean size of the microgels decreased from 1.2 m to 0.450 m upon heating. The solution changed from transparent to milky white as microgels collapsed. Figure 19 shows that the maximum absorbance peak of TOAB-GNP was at 515nm, which is indicative of well-dispersed GNP in solution. The TEM characterizati on (Figure 20(B)) shows that the particles ranged in size from 4 to 6 nm, which is consist ent with the shape of the spectrum in Figure 19. Figure 20(A) shows the size distribution of the particles using DLS. The mean size of TOAB-GNP was approximately 7 nm in diameter, which agrees well with results shown in Figures 19 and 20(B). The P(NIPAM-AA)DMAP-GNP composite showed a similar volume transition with temperature (Figure 21) but exhibited a mean size smaller than the microge l in Figure 18. This decrease in size can be attributed to electrostatic effect s from the DMPAGNP within the microgel and different additives such as HCl and DMAP. The visible absorption at 528nm of the composite materials in solution indicated that the GNP was not in a significant aggregated state within the microgels at ambient tem perature (Figure 22). The 15nm wavelength difference upon comparison of TOAB-GNP and P(NIPAM-AA)DMAP-GNP spectra (Figures 19 and 22, respectively) was attributed to t he phase transfer of GNP from organic to aqueous medium by DMAP ligand.

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36 TEM of P(NIPAM-AA)DMAP-GNP composites showed the presence of GNP inside microgels for the composite system (Figure 23). However, after dialy sis for one day it was observed that GNP were not retained within the copolymer microgel. Alternatively, purification via centrifugation led to the nanocomposite becoming adher ed to the walls of the centrifuge tube. Literature reports have indicated that a GNP complex stabilized with DMAP and/or MUA in water requires a basic environment 50, 51 However, redispersion of the nanocomposite in aqueous solution from the centrifuge tube walls was difficult even after raising the pH of the solution. Another drawback of this system was that microgels do not undergo phase transition at basic pH 38 3.3 Nanocomposites Based on P(NIPAM-GMA) Copolymer Microgels Since the approach described in section 3.2 using DMAP functionalized GNP did not provide a promising approach to preparing the nanocomposites, an alternative strategy based on P(NIPAM-GMA) copolymer microgels was explored to obtain a more stable composite that could exihibit unique and distinctive properties in response to changes in temperature. P(NIPAM-GMA) copolymer microgels have epoxy groups that can be used to introduce functional groups such as amine and thiol 40-42 Amine groups complex with gold ions and can facilitate metal nanoparticles nucleation and growth. However, the method adopted in our studies relied on interactions between negatively-charged GNP to the positive amine groups inside the copolymer microgels. This approach was preferre d over the in-situ synthesis of GNP because it is simpler and less intrusive. The in -situ synthesis method can lead to particle aggregation, polydispersity, and nucleation of

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37 particles outside the microgels, which can pose problems because the aggregation st ate of the colloidal gold influences their optical properties 2, 6 3.3.1 Experimental Section Materials Trisodium citrate (TSC) and cystamine2HCl was purchased from Sigma A ldrich (WI) and used without further purification. Synthesis of Gold Nanoparticle Stabilized by Trisodium Citrate Aqueous solutions of citrate-stabilized GNP (~6 nm) were prepared by reduc ing Au 3+ through the use of NaBH 4 A 9mL aqueous solution containing 0.250mL of 0.01M of HAuCl 4 and 0.500mL of 0.01M TSC was prepared in a scintillation vial. A volume of 0.250mL of ice cold 0.1M NaBH 4 freshly prepared was added to the solution in one shot while stirring. Immediately after adding the reducing agent, the solution turne d orange indicating the formation of GNP. The GNP solution was stirred for an additional two hours with open top to allow degassing of excess NaBH 4 from solution. Particles were characterized by DLS and UV-VIS spectroscopy. Amino-Functionalized P(NIPAM-GMA)CYS Copolymer Microgels Amino groups were introduced in P(NIPAM-GMA) copolymer microgels by allowing the epoxy groups in P(NIPAM-GMA) ( cf Chapter 2.3) to react with thiol groups of cystamine2HCl. A mixture of 30mg of P(NIPAM-GMA), 100mg of cystamine2HCl, and 50mL of water was transferred to a scintillation vial. The mixture was sti rred at room temperature at a pH value of 11 adjusted with 1M NaOH. The reaction continued for 24 hours. The amino-functionalized copolymer microgels (P(NIPAM-GMA)CYS) w ere

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38 purified by centrifugation (4 cycles). The yellow-brown precipitate was redispersed in water and purified by 3 days of dialysis. Nanocomposite Based on P(NIPAM-GMA)CYS Copolymer Microgels P(NIPAM-GMA)CYS (1mg/mL) was incubated with citrate-GNP in a volu me ratio of copolymer microgel to GNP of 1:5. Five incubations with addition of GNP followed by purification were performed for the formation of the nanocomposite. The solution appearance changed from the characteristic color of P(NIPAM-G MA) copolymer microgels (slightly opaque) to pink upon incubation with GNP. The nano-composite P(NIPAM-GMA)CYS-GNP was purified by centrifugation and char acterized by UV-VIS measurements. Second Polymerization with NIPAM : A mass of 2.5mg P(NIPAM-GMA)CYS-GNP was mixed with 100mg of NIPAM in 5mL of water to performed a second polymerization by precipitation polymerization. KPS (0.609mL of 0.015M) was added to initiate the reaction. The polymerization proceeded for 6 hours, at a stirring rate of 4rpm and a temperature of 70C. The resultant composite, P(NIPAM-GMA)CYS-GNP-PNIPA M was purified by centrifugation and characterized by DLS, TEM and UV-VIS. Seeded-Mediated Growth of GNP : The seed-mediated growth of gold was achieved by adding 2.5mg of P(NIPAM-GMA)CYS-GNP-PNIPAM and 0.383mL of HAuCl 4 to 20mL of DI water in a scintillation vial. The solution was placed in an ice bath and sti rred for 10 minutes. A volume of 0.625mL of hydroxylamine hydrochloride (reducing agent) was added to the solution while stirring. The composite solution was stirred for 40 minutes and purified by centrifugation (4 cycles). The composite P(NIPAM-GM A)CYSGNP-PNIPAM-Au was characterized by DLS, TEM and spectroscopy.

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39 3.3.2 Results and Discussions Figure 24(A) shows the maximum absorbance peak of citrate-GNP was at 521nm. The DLS characterization (Figure 24(B)) shows that the mean size of the par ticles was ~6 nm in diameter. Combining both characterization results it was concluded that the synthesized citrate-GNP were monodisperse and colloidally stable in soluti on. P(NIPAM-GMA)CYS copolymer microgels were opaque at room temperature. Figure 25 shows that turbidity increased slightly upon heating the solution above L CST. However, the opacity from the GMA core at room temperature reduces the magni tude of turbidity changes that is typically seen in microgels comprising of PNIP AM. DLS characterization of these amino-functionalized copolymer microgels (Fi gure 26) showed reversible volume transition and was similar to that observed in Figure 8 for P(NIPA MGMA). The results of P(NIPAM-GMA)CYS-GNP composite were promising in term s of loading and immobilization of GNP within the microgel network. Figure 27(A) shows t he UV spectrum for the composite. The maximum absorbance peak was of ~523nm at room temperature, which is the characteristic of GNP. The composite showed a pink appearance at room temperature (Figure 27(B)) but the increase in the solution’ s turbidity upon heating was barely detected (not shown). Absorbance peak at ~40C was ~527nm. The reversible change in absorbance peaks at temperatures below and above LC ST was indicative of immobilization and changes in interparticle distance of GNP w ithin the system. The shift to longer wavelengths of absorbance peaks in the UV spectr um and optical behavior of the composite suggested that proximity of GNP in the composite was a function of the swelling/shrinking degree of the system. However, since this cha nge

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40 was very small, optical changes were not visible to the naked eye, presumably, due to the small size of GNP entrapped inside the microgel. TEM images (Figure 28) confirmed that the composite contained monodisperse and colloidally stable GNP. The images reveal ed that there was a high loading of GNP and that the particles were randomly dispers ed throughout the copolymer microgel. The samples wherein a second polymerization with NIPAM was performed exhibited an increase in mean diameter of approximately 250nm (Figure 29). This was probably caused by a high feed ratio of NIPAM to P(NIPAM-GMA)CYS-GNP composite during polymerization. The UV spectrum in Figure 30(A) showed that scattering from microgels dominated over the plasmon absorption of GNP at temperatures below and above LCST. The appearance of the solution, however, was not very different from that before the second polymerization (Figure 30(B)). TEM im ages in Figure 31 illustrate that GNP was retained in the composite after the se cond polymerization. The polymer matrix and complexion of GNP with amino groups were possibly stabilizing the GNP particles and prevented them from been affected by t he polymerization temperature. However, slight aggregation of the GNP in the outer shel l of the composites was observed. Unlike P(NIPAM-GMA)CYS-GNP, TEM images of P(NIPAM-GMA)CYS-GNP-PNIPAM also showed that the particle core-sh ell could not be distinguished anymore. It was concluded that a seeded growth of GNP by reduc tion of freshly added gold ions could overcome ambiguous color changes in the composite. The purpose of growing the GNP by seeded growth was to enhance the optical properties of the composite as the interparticle distance changes upon varying the temperature of the microgel. The UV spectrum of the P(NIPAM-GMA)CYSGNP-

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41 PNIPAM-Au composite showed a GNP absorbance peak at 550nm that shifted to the right by ~40nm to 590nm when heated well above LCST (Figure 32(A)). Photographs in Figure 32(B) illustrate that the composite optical response due to the volume phase transition can be observed with the naked eye. The solutions appearance changed dramatically from red to purple suggesting decrease in GNP interparticle distance as microgels collapsed upon heating. The color changes of the composite were reversi ble after cooling down the solution. TEM pictures confirmed the presence of larger GNP inside microgels (Fi gure 33). Figure 34 shows a plot of the wavelength of maximum absorption ( l max ) and the mean size of P(NIPAM-GMA)CYS-GNP-PNIPAM-Au composite as a function of temperature. An increase in l max was observed as temperature of the system increased. The value of l max increased continuously up to a temperature of 35C and then saturated. This trend was completely opposite to the trend in the mean size of the microgels a nd Figure 34 shows that the microgels collapsed with increasing temperature. The data suggests that beyond 35C, the interparticle distance of GNP cannot be reduced anymor e. The temperature range where the mean size and wavelength curves cross can be used as an optimal condition for binding of metal ions in sensing schemes. According to the graph this conditions occur at or near 32C, which coincides, not surprisingly, with the transition temperature of PNIPAM. From the experiment reported in this section it was concluded that optical properties and responses of thermally-responsive P(NIPAM-GMA)CYS-GN P composites can be enhanced by a combination of a higher amount of PNIPAM chains followed by seeded growth of the GNP inside the microgels. One of the drawbacks of this approach

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42 was the need to perform many multiple steps to obtain the final composite. This make s the preparation of the system to be complex and time consuming. Another limitation is the lack of functional groups in the final composite for binding metal ions and for exploiting any imprinting techniques. 3.4 Nanocomposites Based on PNIPAM/PAA-IPN Microgels To develop an enhanced composite system through easier and faster steps, we followed a strategy relying on interpenetrated networks of PNIPAM microg els and PAA in combination with positively charged GNP. IPNs of PNIPAM and PAA, as seen in Chapter 2, exhibited a thermal-behavior similar to PNIPAM microgels. PAA cont ains COO groups that can potentially bind metal cations. However, for this composite system, the COO groups can also be used to electrostatically stabilize GNP within the matri x of the IPN. To achieve this, a cationic surfactant, cetyl-trimethylamm onium bromide (CTAB), was used. The nanocomposites were prepared by mixing PNIPAM/PAAIPN-II with CTAB coated GNP. 3.4.1 Experimental Section Materials Cetyl-trimethylammonium bromide (CTAB), and ascorbic acid were both purchased from Sigma-Aldrich (WI). Synthesis of Gold Nanoparticles Stabilized by CTAB Citrate-GNP (~6nm) prepared and characterized as previously reported (se e section 3.3.2). A 0.1M solution of CTAB was prepared by heating it to 30C until

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43 dissolving the surfactant. To replace the citrate ion, 63 L of 0.01M HAuCl 4 500 L of 0.1M CTAB, and 12.5 L of a freshly prepared 0.1M ascorbic acid were added to the scintillation vial containing 3 ml of citrate-GNP solution. The mixture was gent ly stirred for 1 hour. The particles were characterized by DLS and UV-VIS. Nanocomposite Based on PNIPAM/PAA-IPN Microgels The thermo-responsive composites PNIPAM/PAA-GNP were prepared by mixing 250 L of 20mg/mL PNIPAM/PAA-IPN-II and 500 L of CTAB-GNP (1.017 x 10 13 particles) in a 1.5mL centrifuge tube. The solution was continuously stirred for si x hours using a vortex mixer. The product was purified by centrifugation and characteri zed by UV-VIS spectroscopy and TEM. 3.4.2 Results and Discussions DLS characterization of Citrate-GNP and CTAB-GNP particles is show n in Figure 35. The mean size determined was ~6nm and 8nm, respectively. The UV-VIS of Citrate-GNP, which is illustrated in Figure 36, shifted by 10nm to the higher wav elengths after substituting its surface with CTAB. The nanocomposite was easily prepared and redispersed in water after purifica tion by centrifugation. Figure 37 shows the UV spectrum for the composite at room temperature and at a temperature above LCST. Upon comparison of Figure 36 and Figure 37, the absorbance peak at room temperature was observed at same wavelength of CTAB-GNP. However, the scattering of PNIPAM/PAA IPN microgels domi nated that of CTAB-GNP in the composite at room temperature. When the composite was exposed to a temperature above LCST the original absorbance peak at 522nm shifted by 29nm to the

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44 higher wavelengths, which is indicative of a decrease in interparticle dis tance (Figure 37). However, a color change as in the case of P(NIPAM-GMA)CYS-GNP-PNIPAM -Au was not observed. Since the mean size thermal-behavior of the IPN composite is enough to induce such a dramatic shift in the UV spectrum of GNP, there is no need for a second polymerization with NIPAM. Growing of GNP is an option but the presence of COOH could lead to nucleation in through out the system other than in the CTAB-GNP seed. This could result in polydispersity, aggregation and changes in the characteristi c properties of GNP and the composite as a whole. Increase in the loading of GNP can be done easily by additional incubations of CTAB-GNP as performed for P(NIPAM-GMA)CYS-GNP. TEM of PNIPAM/PAA-IPN-GNP confirmed the presence of C TABGNP inside the matrix of the composite (Figure 38). The images show that the loading of GNP can be further optimized.

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45 Figure 15: Turbidity data of PNIPAM-2% -Citrate-GNP. Figure 16: Changes in appearance of PNIPAM-2% -Citrate-GNP. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 TURBIDITY ( mV) 40 35 30 25 20 15 TEMPERATURE (C) Heating Cooling Transition: 31-35CTmid= 33C Increasing Temperature

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46 Figure 17: TEM of (A) Citrate-GNP; (B) PNIPAM-2% -Citrate-GNP. Figure 18: DLS of P(NIPAM-AA). Lines are drawn as a guide. (A) (B) 1000nm 200nm 1400 1200 1000 800 600 400 MEAN SIZE (nm) 40 38 36 34 32 30 28 26 24 TEMPERATURE (C) Transition: 30-34CTmid= 32C

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47 Figure 19: UV-VIS of TOAB-GNP. Figure 20: (A) DLS and (B) TEM of TOAB-GNP. 0 5 10 15 20 0.1110100100010000Volume (%)Size (d.nm) Size Distribution by Volume 10 0nm (A) (B) 0.4 0.3 0.2 0.1 ABSORBANCE 1000 900 800 700 600 500 400 WAVELENGTH (nm) Max Absorbance= 515nm

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48 Figure 21: DLS of P(NIPAM-AA)DMAP-GNP. Lines are drawn as a guide. Figure 22: UV-VIS of P(NIPAM-AA)DMAP-GNP. 0.5 0.4 0.3 0.2 0.1 0.0ABSORBANCE 900 800 700 600 500 400 WAVELENGTH (nm) Max Absorbance= 528nm 900 800 700 600 500 400 300 MEAN SIZE (nm) 40 38 36 34 32 30 28 26 24 TEMPERATURE (C) Transition: 30-34CTmid= 32C

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49 Figure 23: TEM of (A) DMAP-GNP; (B) P(NIPAM-AA)DMAP-GNP. Figure 24: (A) UV-VIS and (B) DLS of Citrate-GNP. 0 5 10 15 20 0.1110100100010000Volume (%)Size (d.nm) Size Distribution by Volume 100nm 100nm (A) (B) 0.7 0.6 0.5 0.4 0.3 0.2 0.1ABSORBANCE 900 800 700 600 500 400WAVELENGTH (nm) Max Absorbance= 521nm (A) (B)

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50 Figure 25: Appearance of P(NIPAM-GMA)CYS. Figure 26: DLS of P(NIPAM-GMA)CYS. Lines are drawn as a guide. 520 500 480 460 440 420 400 380 MEAN SIZE (nm) 38 36 34 32 30 28 26 TEMPERATURE (C) Transition: 30-34CTmid= 32C Heating Cooling

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51 Figure 27: (A) UV-VIS and (B) Appearance of P(NIPAM-GMA)CYS-GNP Figure 28: TEM of P(NIPAM-GMA)CYS-GNP. 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1ABSORBANCE 900 800 700 600 500 400 WAVELENGTH (nm) Max Absorbance at T<32C: ~ 523nm Max Absorbance at T>32C : ~ 527nm (A) (B) 100nm

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52 Figure 29: DLS of P(NIPAM-GMA)CYS-GNP-PNIPAM. Lines are drawn as a guide. Figure 30: (A) UV-VIS and (B) Change in appearance of P(NIPAM-GMA)C YSGNP-PNIPAM. 800 600 400 200 MEAN SIZE (nm) 34 32 30 28 26 TEMPERATURE (C) Transition: 30-33CTmid= 31.5C (B) T > LCST T < LCST 0.40 0.35 0.30 0.25 0.20 0.15 0.10ABSORBANCE 900 800 700 600 500 400 WAVELENGTH (nm) (A)

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53 Figure 31: TEM of P(NIPAM-GMA)CYS-GNP-PNIPAM. Figure 32: (A) UV-VIS and (B) Change in appearance of P(NIPAM-GMA)C YSGNP-PNIPAM-AU. 50 0nm 100 0nm T > LCST T < LCST (B) 0.5 0.4 0.3 0.2 0.1ABSORBANCE 1000 900 800 700 600 500 400 WAVELENGTH (nm) Max Absorbance at T<32C: ~ 545nm Max Absorbance at T>32C : ~ 564nm (A)

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54 Figure 33: TEM of P(NIPAM-GMA)CYS-GNP-PNIPAM-AU. Figure 34: Thermal relationship between wavelength at maximum absorbance and mean size of P(NIPAM-GMA)CYS-GNP-PNIPAM-AU. Lines are drawn as a g uide. 500nm 800 700 600 500 400 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) 558 556 554 552 550 548 546 544 WAVELENGTH (nm) MEAN SIZE WAVELENGTH AT MAX ABSORBANCE

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55 Figure 35: DLS of Citrate-GNP and CTAB-GNP. Figure 36: UV-VIS of Citrate-GNP and CTAB-GNP. 1.2 1.0 0.8 0.6 0.4 0.2 ABSORBANCE 1000 900 800 700 600 500 400 WAVELENGTH (nm) Max Absorbance of CTAB-GNP= 522nm Max Absorbance of Citrate-GNP= 512nm 0 5 10 15 20 25 0.1110100100010000Volume (%)Size (d.nm) Size Distribution by Volume Citrate-GNP CTAB-GNP

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56 Figure 37: UV-VIS of PNIPAM/PAA-IPN-GNP. Figure 38: TEM of PNIPAM/PAA-IPN-GNP. 500 nm 1.2 1.0 0.8 0.6 0.4 0.2 ABSORBANCE 1000 900 800 700 600 500 400 WAVELENGTH (nm) Max Absorbance at T> 32C= 552nm Max Absorbance at T< 32C= 522nm

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57 CHAPTER FOUR: MOLECULARLY IMPRINTED POLYMERS (MIP) 4.1 Molecularly Imprinted Polymers Based on PNIPAM/PAA IPN Microgels One of the tasks in the thesis project was to study imprinted microgels for bindi ng heavy metal ions such as copper. The thermo-responsive polymer selected was PNIPAM/PAA-IPN microgels because their polyelectrolyte units provide a large amount of binding sites for the metal ion. The preparation of the MIP was explored by performing a second polymerization on the microgels in presence of the target anal yte at various cross-linking densities. The goal was to memorize the shape of the analy te, copper (Cu 2+ ) in this case, to make an MIP based on PNIPAM/PAA-IPN microgels that could selectively bind the target analyte. Since PNIPAM/PAA can respond re versibly to thermal stimuli, the recognition ability of the imprinted microgel could be c ontrolled by temperature. 4.1.1 Experimental Section Materials 2-methylsulfoxide (DMSO) was purchased from Sigma Aldrich (WI) and 4,4azobis (4-cyanovaleric acid) was a sample from Wako Chemicals. Copper acetat e (II) was purchased from Acros Chemicals (NJ).

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58 Synthesis of MIPs Based on PNIPAM/PAA-IPN Microgels The carboxylic acid sites in PNIPAM/PAA-IPN-II microgels were neutralized with 1M aqueous NaOH. A three-fold excess amount of 1.5M copper acetate (II) was added to the microgel solution relative to amount of PAA in the IPN. Mixing was continued for 24 hours to exchange the Na + with Cu 2+ Purification of PNIPAM/PAA-IPN-Cu 2+ to remove the unbound Cu 2+ consisted of dialysis for 1 day and 4 cycles of centrifugation at 8500 rpm for 30 minutes. The polymer was vacuum dried overnight at 30C. For the second polymerization on PNIPAM/PAA-IPN-Cu 2+ to imprint the target ion the solvent was changed to DMSO 8, 45, 52, 53 Microgels can be redispersed in DMSO but they do not undergo volume phase changes in this solvent. After dispersing dried PNIPAM/PAA-IPN-Cu 2+ in 1mL of DMSO and adding NIPAM and MBA according to values in Table 7, the reaction mixture was stirred and bubbled with N 2 for 30 minutes. The initiator solution in DMSO, 1mol% of ACVA, was also bubbled with N 2 for 30 minutes. The reaction mixture was heated to 60C, initiated by ACVA, and the reacte d was allowed to proceed for 4 hours. The product was mixed with water and centrifug ed 4 times to separate the microgels from DMSO. After removing the bound Cu 2+ by adding several aliquots of 0.1M HCl, the product was rinsed several times with DI water and purified by centrifugation. Redispersion of the resultant precipitate in wate r was achieved by prolonged sonication and mixing with a vortex mixer. The imprinted polymer was tested using DLS.

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59 Table 7: Formulation used for molecularly imprinted PNIPAM/PAA micr ogels. SAMPLE IPN (mg) Cu 2+ [1.5M] (ml) NIPAM (mg) MBA (mg) DMSO (ml) PNIPAM/PAA-MIP-4% 10 0.116 100 5 1 PNIPAM/PAA-MIP-7% 10 0.116 100 10 1 PNIPAM/PAA-MIP-10% 10 0.116 100 15 1 4.1.2 Results and Discussions DLS characterization of PNIPAM/PAA-MIP cross-linked at 4, 7 and 10% is shown in Figures 39, 40, and 41, respectively. The mean size and volume phase transition of PNIPAM/PAA-MIP-4% were very similar to that observed for PNIPAM/ PAA-IPN microgels. However, the mean size of PNIPAM/PAA-MIP-7% and PNIPAM/PA A-MIP10% microgels decreased at swollen state by ~70 and ~100nm, respectively. The resultant mean size discrepancy for the MIPs cross-linked at 7 and 10% was attri buted to the increase in cross-linking density inside the microgels and reduction in swe lling. Since our results showed that cross-linking took place within the PNIPAM/PAA-IPN, the next step was to determine if recognition sites for Cu 2+ were formed. To accomplish this, the volume phase changes of 1mg of PNIPAM/PAA-MIP-10% in aqueous solution were investigated from 20-35C with different concentrations of Cu 2+ It was expected that the swelling degree of the MIP at various metal ion concentr ations would reflect the occurrence of the templated sites. As the microgels shrin k in response to temperature and the –COOH groups come in close proximity, we expected that the MIP would show a recognition for the Cu 2+ ions and that this would be manifested in the size. Figure 42 shows a plot of PNIPAM/PAA-MIP-10% solution of 70 moles of COOH groups and 0 moles, 10 moles, 15 moles, and 20 moles of Cu 2+ At concentrations of

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60 10 moles and 15 moles the mean size of the MIP decreased and at 20 moles of Cu 2+ the mean size was that of the MIP containing no copper. The imprinting of PNIPAM/PAA-IPN microgels by polymerization in DMSO decreased colloidal stability in water. This possibly influenced the prel iminary data shown in Figure 42, which does not clearly show effects from the interaction with Cu 2+

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61 Figure 39: DLS of PNIPAM/PAA-MIP-4%. Lines are drawn as a guide. Figure 40: DLS of PNIPAM/PAA-MIP-7%. Lines are drawn as a guide. 500 450 400 350 300 250 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 29-34CTmid= 31.5C PNIPAM/PAA-IPN PNIPAM/PAA-MIP-7% 500 450 400 350 300 250 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 29-34CTmid= 31.5C PNIPAM/PAA-IPN PNIPAM/PAA-MIP-4%

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62 Figure 41: DLS of PNIPAM/PAA-MIP-10%. Lines are drawn as a guide. Figure 42: DLS of PNIPAM/PAA-MIP-10% at different copper ion concentr ations. Lines are drawn as a guide. 500 450 400 350 300 250 MEAN SIZE (nm) 40 35 30 25 20 TEMPERATURE (C) Transition: 29-34CTmid= 31.5C PNIPAM/PAA-IPN PNIPAM/PAA-MIP-10% 450 400 350 300 250 200 MEAN SIZE (nm) 34 32 30 28 26 24 22 20 TEMPERATURE (C) 0moles Cu 2+ 10moles Cu 2+ 15moles Cu 2+ 20moles Cu 2+

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63 CHAPTER FIVE: SUMMARY In summary, the research described in this thesis has studied four distinct thermally-responsive materials: PNIPAM microgels, copolymeric mic rogels of P(NIPAM-AA), copolymeric microgels of P(NIPAM-GMA), and interpenet rated networks of PNIPAM/PAA. Both P(NIPAM-AA) and PNIPAM/PAA-IPN microgels provide polyelectrolyte units with carboxylic acid groups that can be used for the preparation of composites with GNP and also for the potential binding of metal cations. However, experiments described in Chapter 2 show that a higher concentration of carboxylic acid sites within the microgel network can be achieved by PNIPAM /PAAIPN. Among the two methods to synthesize these IPN microgels, the one-step approach was found to be simpler. Copolymer microgels of P(NIPAM-GMA) were found to give functional groups that could be further used for the effective immobilization of GNP i n the matrix of the microgels. Among the strategies investigated for preparation of nanocomposites of thermally-responsive microgels and GNP, the most promising approach was found to be based on the PNIPAM/PAA-IPN microgels with carboxylic acid groups that we re useful for the interaction with positively functionalized GNP coated with a cationic surfactant CTAB. In this system, the COOH groups can also serve as the useful sites for t he binding of metal cations. An additional benefit of this approach as compared to composites described in Chapter 3, namely PNIPAM-2%-CitrateGNP, P(NIPAM-AA)DMA P-GNP,

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64 and P(NIPAM-GMA)CYS-GNP-PNIPAM-AU, was that the system of PNIPA M/PAAIPN-GNP was simple and quick to synthesize. The experiments detailed in this thesis show that a lightly cross-linked sam ple, PNIPAM-2%-CitrateGNP, did not retain the GNP. This was possibly because of the lack of functional groups within the microgel network that can serve as driving force for the loading of GNP and as their stabilizer inside the matrix. A system that did poss ess functional groups from acrylic acid, P(NIPAM-AA)DMAP-GNP, failed to embed the GNP in the microgels because the GNP with a positively charged ligand such as DM AP had poor transferability of GNP from organic to aqueous medium and was difficult to reproduce. P(NIPAM-GMA)-GNP samples showed high loading of GNP within the microgel as well as promising thermal response of the composite and plasmon absorpti on shifts for the embedded GNP. However, the presence of GMA in the system limited t he optical properties of the nanocomposite. A preliminary study of imprinted microgels for binding heavy metal ions such as copper was performed using PNIPAM/PAA-IPN microgels. The imprinting of PNIPAM/PAA-IPN microgels required additional steps such as polymerizat ion in an organic (DMSO) solvent, which caused subsequent problems of colloidal stability in water. We believe that this influenced the experiments that were done to test t he selectivity of the MIP for Cu 2+ Therefore, results obtained from PNIPAM/PAA-IPNMIP were not definitive. Future work on addressing these limitations is necessar y.

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