Developing miniemulsion polymerization for use in the molecular imprinting of protein with nanoparticles

DEVELOPING MINIEMULSION POLYMERIZATION FOR USE IN THE MOLECULAR IMPRINTING OF PROTEIN WITH NANOPARTICLES TAN CHAU JIN NATIONAL UNIVERSITY OF SINGAPORE 2007 DEVELOPING MINIEMULSION POLYMERIZATION FOR USE IN THE MOLECULAR IMPRINTING OF PROTEIN WITH NANOPARTICLES TAN CHAU JIN (B. Eng. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE _____________________________________________________________Chapter Acknowledgements I would like to sincerely express my greatest gratitude to my supervisor, Dr. Tong Yen Wah, for his unreserved support and guidance throughout the course of this research project. His guidance, constructive criticisms and insightful comments have helped me in getting my thesis in the present form. He has shown enormous patience during the course of my PhD study and he constantly gives me encouragements to think positively. More importantly, his passion in scientific research will be a great motivation for my future career undertakings. In addition, I wish to express my heartfelt thanks to all my friends and colleagues in the research group, Mr. Zhu Xinhao, Mr. Khew Shih Tak, Mr. Chen Wen Hui, Mr. Shalom Wangrangsimakul, and Ms. Niranjani Sankarakumar and other staff members of the Department of Chemical and Biomolecular Engineering, especially Ms. Li Xiang, Ms. Li Fengmei, and Ms. Goh Mei Ling. Without their help, this project could not have been completed on time. Special acknowledgements are also given to the National University of Singapore for her financial support. Last, but not least, I would like to dedicate this thesis to my parents and younger brother, who have been standing by me all the time. Without their love, concern and understanding, I would not have completed my doctoral study. I _____________________________________________________________Chapter Table of contents Acknowledgements Table of contents Summary List of tables List of figures Nomenclature Chapter Introduction Chapter Literature review 2.1 Molecular imprinting 2.1.1 Traditional bulk imprinting 2.1.2 MIPs with controlled morphology 2.1.3 Molecular imprinting of protein macromolecules 2.1.4 Emulsion polymerization for molecular imprinting 2.2 Protein-surfactant interaction 2.2.1 Interfacial protein adsorption I II VI VIII IX XIII 6 10 11 13 20 25 26 Chapter The effect of protein structural conformation on nanoparticle molecular imprinting of ribonuclease A using miniemulsion polymerization 28 3.1 Introduction 28 3.2 Experimental section 29 3.2.1 Effect of ultraviolet (UV) radiation 30 3.2.2 Effect of high-shear homogenization 30 3.2.3 Effect of surfactants 30 3.3 Results and discussions 31 3.3.1 The effect of UV radiation 32 3.3.2 The effect of homogenization 35 3.3.3 The effect of surfactants 37 3.3.4 The effect of additive 40 3.3.4.1 The addition of electrolyte 42 3.3.4.2 The addition of a nonionic surfactant, PVA 45 3.4 Conclusions 49 Chapter Preparation of ribonuclease A surface-imprinted nanoparticles with miniemulsion polymerization for protein recognition in aqueous media 50 4.1 Introduction 50 4.2 Experimental section 52 4.2.1 Preparation of RNase A-imprinted and non-imprinted nanoparticles 52 4.2.2 Size measurement 53 4.2.3 Surface area measurement 54 II _____________________________________________________________Chapter 4.2.4 Determination of swelling ratio (SR) 4.2.5 Batch rebinding test 4.2.6 Competitive batch rebinding test 4.2.7 Kinetics study of MIP nanoparticles 4.2.8 Statistical analysis 4.3 Results and discussions 4.3.1 Size and morphology of the imprinted and non-imprinted particles 4.3.2 Batch and competitive rebinding tests 4.3.3 Rebinding kinetics study of MIP and NIP nanoparticles 4.3.4 Protein imprinting through miniemulsion polymerization 4.4 Conclusions 54 54 55 56 57 57 57 62 69 71 72 Chapter Defining the interactions between proteins and surfactants for nanoparticle surface imprinting through miniemulsion polymerization 74 5.1 Introduction 74 5.2 Experimental section 75 5.2.1 Preparation of surface-imprinted and non-imprinted nanoparticles 75 5.2.2 Morphological characterization 76 5.2.3 Batch rebinding test 76 5.2.4 Competitive batch rebinding test 77 5.2.5 Desorption study 77 5.2.6 Circular dichroism (CD) study 77 5.2.7 Statistical analysis 78 5.3 Results and discussions 78 5.3.1 Morphological features 78 5.3.2 Batch rebinding test 81 5.3.3 Competitive batch rebinding test 84 5.3.4 Desorption study 86 5.3.5 Influence of the protein-surfactant interaction 87 5.4 Conclusions 93 Chapter Preparation of superparamagnetic ribonuclease A surface-imprinted submicrometer particles for protein recognition in aqueous media 94 6.1 Introduction 94 6.2 Experimental section 96 96 6.2.1 Preparation of Fe3O3 magnetite 6.2.2 Preparation of magnetic imprinted particles (mag-MIP) 96 6.2.3 Preparation of magnetic non-imprinted particles (mag-NIP) 98 6.2.4 Analysis and measurement 98 6.2.5 Determination of swelling ratio (SR) 98 6.2.6 Batch rebinding test 99 6.2.7 Competitive batch rebinding test 100 6.2.8 Adsorption kinetics study 100 6.2.9 Desorption kinetics study 100 6.2.10 Statistical analysis 101 6.3 Results and discussions 101 III _____________________________________________________________Chapter 6.3.1 Synthesis of mag-NIP and mag-MIP particles 6.3.2 Size determination using dynamic light scattering (DLS) 6.3.3 Morphological observation with FE-SEM and TEM 6.3.4 Specific surface areas and pore volumes 6.3.5 Thermogravimetric analysis (TGA) 6.3.6 Vibrating sample magnetometer (VSM) characterization 6.3.7 Determination of swelling ratio (SR) 6.3.8 Batch rebinding test 6.3.9 Competitive batch rebinding study 6.3.10 Rebinding kinetics study 6.3.11 Desorption kinetics study 6.4 Conclusions 101 101 103 107 107 109 111 112 115 118 119 121 Chapter Preparation of bovine serum albumin surface-imprinted submicron particles with magnetic susceptibility through core-shell miniemulsion polymerization 123 7.1 Introduction 123 7.2 Experimental section 126 126 7.2.1 Preparation of Fe3O4 magnetite 7.2.2 Preparation of superparamagnetic support particles 127 7.2.3 Aminolysis 127 7.2.4 Aldehyde functionalization 128 7.2.5 Immobilization of template BSA 128 7.2.6 Shell layer synthesis 128 7.2.7 Template removal 129 7.2.8 Preparation of non-imprinted particles from surface-modified support beads (iNIP) 129 7.2.9 Preparation of molecularly imprinted particles from unmodified core beads using free template (fMIP) 130 7.2.10 Preparation of non-imprinted particles from unmodified core beads (fNIP) 131 7.2.11 Analysis and measurement 131 7.2.12 Determination of estimated swelling ratio (SR) 132 7.2.13 Batch rebinding test 132 7.2.14 Competitive batch rebinding test 132 7.2.15 Adsorption kinetics study 133 7.2.16 Statistical analysis 133 7.3 Results and discussions 133 7.3.1 Preparation of the magnetically susceptible polymeric support beads 133 7.3.2 Surface immobilization of the template BSA molecules 134 7.3.3 Synthesis of the BSA surface-imprinted particles 139 7.3.4 Size measurements 142 7.3.5 Morphological observations 143 7.3.6 Swelling ratio (SR) measurements 148 7.3.7 Nitrogen sorption measurements 148 7.3.8 Thermogravimetric analysis (TGA) 149 IV _____________________________________________________________Chapter 7.3.9 Vibrating sample magnetometer (VSM) measurements 7.3.10 Batch rebinding test 7.3.11 Competitive batch rebinding test 7.3.12 Rebinding kinetics study 7.4 Conclusions 152 153 157 159 161 Chapter Conclusions 8.1 The importance of the template protein integrity 8.2 Successful fabrication of protein surface-imprinted nanoparticles 8.3 Template protein-surfactant interaction for effective imprinting 8.4 Incorporation of superparamagnetic property 8.5 Alternative approach of protein surface imprinting via a 2-stage core-shell miniemulsion polymerization 8.6 Suggestions for future work 8.6.1 The epitope approach 8.6.2 Packing the imprinted nanoparticles into columns 163 163 164 165 167 167 168 168 171 Reference 173 Appendix I List of Publications 179 V _____________________________________________________________Chapter Summary Molecular recognition can be briefly described as the capability of a host molecule to bind its specific ligand molecule through some forms of non-covalent interaction. Over years, extensive studies had been performed to investigate this recognition property and as a result, much understanding on the mechanism was derived. The biological importance of molecular recognition is well illustrated by its role as a main driving force for numerous biological processes that take place in living organisms. On the other hand, commercially, such property could be developed into valuable technologies for application in fields like analytical chemistry, bioseparation and catalysis. This seems especially important with the rapid growth of biopharmaceutical industry. However, in spite of their great versatilities, biomolecules are inherently fragile and they can be easily denatured under extreme conditions of temperature and pH. In addition to that, their high cost of production may cause their applications in some areas to be economically unfeasible. In recent decades, this has inspired chemists and engineers into developing mimicking synthetic materials that can overcome the inherent limitations of antibody molecules. Molecular imprinting is a state-of-the-art technique for preparing mimics of natural, biological receptors. It can be used to impart pre-determined molecular recognition property onto synthetic materials such as polymers. Much success has been achieved VI _____________________________________________________________Chapter with small molecules through the traditional method of bulk molecular imprinting. However, such approach, though simple, is not suitable for large molecules like proteins and oligosaccharides due to the inaccessibility of the imprinted binding sites to these bulky molecules. In addition, the crude post-treatment tends to produce imprinted polymer of inconsistent quality. Most of all, for its poor thermal dispersion, bulk polymerization is not suitable for industrial-scale application. In this project, we had developed an imprinting polymerization system that can overcome the limitations posed by the conventional imprinting methodology for protein imprinting. Miniemulsion polymerization had been chosen for this purpose while methyl methacrylate and ethylene glycol dimethacrylate were employed as the functional and cross-linking monomer respectively. On the earlier part, much effort was spent on understanding and optimizing the polymerization system for protein imprinting. Subsequently, protein surface-imprinted nanoparticles were successfully prepared through the modified, optimized miniemulsion polymerization system. The imprinted nanoparticles displayed significant molecular selectivity in an aqueous environment. One of the advantages of miniemulsion protein imprinting is that the system offers the option of incorporating desired property into the imprinted particles. Thus, in this contribution, we had imparted a superparamagnetic property into the protein-imprinted beads. This further widened the potential scope of application for the material in fields like magnetic bioseparation, bioimaging and cell labeling. VII _____________________________________________________________Chapter List of Tables Table 3.1 The surfactant system used for the RNase A structural CD study. 31 Table 4.1 The protocol for the preparation of imprinted and non-imprinted polymers under the conventional and optimized conditions of miniemulsion polymerization. 53 Table 4.2 Sizes of the polymeric particles prepared under the conventional (denaturing) and modified conditions. 59 Table 4.3 Calculated separation factors of the NIP and MIP nanoparticles based on the competitive rebinding test. 67 Table 4.4 Results of the rebinding tests illustrating the adsorption characteristics of the imprinted nanoparticles prepared. 69 Table 5.1 Results of the desorption study using different solvents. 87 Table 6.1 The miniemulsion polymerization reaction for RNase A imprinting. 97 Table 6.2 Results of the dynamic light scattering. 102 Table 6.3 Results from the nitrogen gas sorption measurements. 107 Table 6.4 The batch and competitive rebinding tests for mag-NIP and mag-MIP with different proteins. 115 Table 6.5 Selectivity parameters of the polymers. 117 Table 7.1 The surface atomic compositions of the support particles from the XPS widescan spectra. 136 Table 7.2 XPS analysis of the deconvoluted C1s peaks at each surface modification stage. 139 Table 7.3 Morphological features of the polymeric particles prepared. 143 Table 7.4 Results from the nitrogen gas sorption measurements. 149 Table 7.5 Results obtained from the batch rebinding tests. 157 VIII _____________________________________________________________Chapter molecules in an imprinting system is similarly essential. Thus, in the early development of the miniemulsion polymerization as a protein imprinting system, great effort was put into studying the structures of the template protein molecules in a miniemulsion (Chapter 3). Three factors, namely the initiation method, the high-shear homogenization and the surfactant system, were singled out as the possible sources of protein denaturation. Modifications to the system were carried out accordingly based on the study. A redox initiation method was adopted to prevent any undesired distortion of the protein conformation by other means like UV and high temperature. The other important modification was made to the surfactant system used. Sodium dodecyl sulfate (SDS) is a common surfactant applied for miniemulsion polymerization but at the same time, it is also known to denature proteins. To effectively minimize such denaturing effect by SDS, a non-ionic, polymeric surfactant, poly (vinyl alcohol) (PVA) was added as a co-surfactant. This helped to preserve the integrity of the template protein molecules during the miniemulsion polymerization imprinting process while allowing certain degree of interactions between the template molecules and the surfactant micelles for successful protein surface imprinting. 8.2 Successful fabrication of protein surface-imprinted nanoparticles Based on the results of the protein conformations in a miniemulsion as studied, modifications to the system were made accordingly and protein surface-imprinted nanoparticles were successfully fabricated using the modified system (Chapter 4). The imprinted nanobeads sized about 40 nm and they displayed significant molecular 164 _____________________________________________________________Chapter selectivity towards the template RNase A nanoparticles in aqueous single- and binary protein adsorption systems, achieving an imprinting efficiency of 9.21 and a relative separation factor of 1.82. In addition to the preferential uptake of the template RNase A molecules, due to the high surface area of the nanobeads, the RNase A-imprinted nano-adsorbents also exhibited an exceptionally high loading of the target molecules (as high as 744.9 mg RNase A/g adsorbent), thus making it a promising separation medium material for large-scale bioseparation process. The RNase A adsorption kinetics of the imprinted nanobeads was also studied and was found to be favorable. Imprinted nanobeads (~ 80 nm) were prepared also prepared for comparison purposes based on the conventional miniemulsion polymerization protocol, which was found to be detrimental to the structural conformation of the template protein molecules. As expected, the imprinted particles failed to display molecular selectivity towards the template protein in the rebinding runs. This illustrated the importance and necessity for the preservation of template structural integrity in protein imprinting. 8.3 Template protein-surfactant interaction for effective imprinting In Chapter 4, a hypothesis was provided to explain the possible mechanism involved in the imprinting of template protein molecules during a miniemulsion polymerization. To have a better understanding of the mechanism, as presented in Chapter 5, proteins of different properties, namely Lys and BSA, were imprinted and the imprinted nanobeads were subsequently studied for their molecular recognition properties in adsorption experiments. The results were compared to that of the RNase A-imprinted 165 _____________________________________________________________Chapter nanoparticles. It was found that for Lys, the imprinted nanobeads displayed only limited imprinting effect while in the case of BSA-imprinted particles, the desired molecular selectivity was totally absent. CD analysis was then performed to study the template protein-surfactant interaction. The results showed that Lys molecules interacted significantly with the surfactant micellar system such that their structural configurations were totally changed. This provided a possible explanation for the limited imprinting effect observed for the Lys-imprinted nanoparticles. On the other hand, no configurational changes were observed for BSA molecules in the mixture. Nevertheless, this could signify a lack of template protein-surfactant interaction. Based on our hypothesis, the interaction between the template protein and the surfactant molecules is the major force that enables the protein molecules to be adsorbed to the surface of the surfactant micelles and partitioned across the oil-water phase boundary. This adsorption and partitioning of the protein molecules will thus allow the complementary binding sites for the template to be formed on the nanoparticle surfaces upon polymerization. Thus, the poor imprinting efficiency of the BSA-imprinted particles can be attributed to the lack of protein-surfactant interaction. From our findings, it was concluded that the success of protein surface imprinting in a miniemulsion polymerization system is dependent on effective template protein-surfactant interaction. However, it would be of utmost importance that such interaction does not adversely affect the configurational characteristics of the protein molecules for successful protein imprinting. This understanding will allow us to better optimize the miniemulsion polymerization imprinting system, in terms of the surfactants used and the type of proteins to be imprinted, for its application. 166 _____________________________________________________________Chapter 8.4 Incorporation of superparamagnetic property In order to enhance the potential of the protein-imprinted particles as a bioseparation medium, a desired superparamagnetic property was incorporated. As shown in Chapter 6, iron oxide nanomagnetite was prepared and incorporated into the proteinimprinted particles during the miniemulsion polymerization. The final particles sized about 700-800 nm. The fabricated particles were regularly shaped and monodispersed. The successful encapsulation of the iron oxide magnetite was illustrated through direct TEM observation. The encapsulation efficiency was satisfactorily high and the magnetic property was also desired. This enabled the ease of material recovery and generation. Most of all, the magnetically susceptible imprinted particles exhibited significant molecular selectivity towards the template RNase A molecules in batch and competitive adsorption experiments. Equipped with the desired magnetic susceptibility, in addition to its imparted molecular affinity, the potential scope of application for the material is largely widened. 8.5 Alternative approach of protein surface imprinting via a 2-stage core-shell miniemulsion polymerization Up to this point, protein surface-imprinted beads had been successfully fabricated based on the inherent tendency of template protein molecules to interact with surfactant micelles and thus be adsorbed onto the micellar surface and partitioned across the oil-water phase boundary formed by the surfactant due to their amphiphilicity. Nevertheless, it is understood that different proteins behave differently and thus, some proteins like BSA and Lys as illustrated in Chapter 5, 167 _____________________________________________________________Chapter cannot be successfully imprinted through such direct application of miniemulsion polymerization. So an alternative approach based on a two-stage core-shell miniemulsion polymerization was employed to prepare surface-imprinted particles for these proteins (Chapter 7). In the investigation, BSA surface-imprinted particles with unique RBC-like morphology were successfully fabricated. The material displayed significant selectivity towards the template BSA molecules in both the single-protein and competitive adsorption experiments. In addition to that, Fe3O4 nanomagnetite was encapsulated in the imprinted core-shell particles, rendering them magnetically susceptible. For comparison purposes, core-shell imprinted particles based on nonimmobilized BSA molecules had also been prepared and poor imprinting efficiency was observed for them. This result coincided with our findings from Chapter that the direct application of miniemulsion polymerization for surface imprinting of BSA simply does not work and this can be probably due to the lack of protein-surfactant interaction which causes the BSA molecules to be unable to adsorb to the micellar surface. The difficulty was overcome by this alternative approach where the BSA template molecules were covalently immobilized onto the surface of the core particles. This illustrated the value of such immobilization strategy in the application of miniemulsion polymerization for proteins which are inherently incompatible. 8.6 Suggestions for future work 8.6.1 The epitope approach The epitope approach was first put forward by Rachkov et al. (Rachkov and Minoura, 2001) for protein imprinting. Instead of using the whole protein molecule as the 168 _____________________________________________________________Chapter template, a short peptide epitope that is sufficiently significant to represent the protein molecule is applied (Figure 8.1). With this approach, since only the peptide epitope, which is a small molecule, will be involved in the imprinting and the rebinding processes, the issue of limited diffusion often associated with the imprinting of bulky macromolecules can be avoided. In addition to that, such approach will be extremely valuable in cases where the target template proteins are difficult and expensive to prepare in the required quantities. Most of all, template-system compatibility has always been a major concern in protein imprinting due to the sensitive and flexible nature of protein molecules. With the epitope approach, such concern can be largely relieved. Figure 8.1 Schematic representation of the epitope approach of molecular imprinting (Bossi et al., 2007). To date, the epitope approach, though proved to be effective, has not been widely employed and related publications have been scarce (Nishino et al., 2006; Tai, et al., 2005; Brown and Puleo, 2007). Most work has focused on the preparation of epitopeimprinted polymer through thin-film fabrication and the traditional bulk imprinting 169 _____________________________________________________________Chapter system. So it would be of interest to apply such approach via the miniemulsion polymerization system that had been developed where monodispersed, regularly shaped epitope-imprinted nanoparticles could be directly synthesized. One of the possible strategies to be adopted is, first of all, a suitable sequence of peptide from a protein molecule has to be identified. Following that, the targeted epitope peptide sequence can be obtained through enzymatic or chemical proteolytic digestion. Preparative HPLC is then required as post-treatment to purify the peptide required. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) could be employed to verify the identity of the purified peptide epitope. Finally, epitope-imprinted nanoparticles for the targeted protein can be prepared via the miniemulsion polymerization. Figure 8.2 A schematic representation of obtaining peptide epitope through proteolytic digestion. 170 _____________________________________________________________Chapter 8.6.2 Packing the imprinted nanoparticles into columns Our previous investigative effort had focused on the understanding and development of an alternative imprinting system for the fabrication of protein surface-imprinted polymeric nanoparticles. From the practical application point of view, it would be of great interest to attempt to pack the imprinted nanoparticles into chromatographic columns and applied the columns in actual bioseparations so as to probe their properties as a separation medium. One of the biggest foreseeable challenges lies in the extremely high pressure associated with packing particles of sizes in nano-range. Special columns have to be designed to withstand such high pressure. In addition to that, special frits also need to be designed and custom-made for the purpose in order to hold the extremely small particulate packing. 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Tong, “Preparation of superparamagnetic Ribonuclease A surfaceimprinted sub-micrometer particles for protein recognition in aqueous media”, Analytical Chemistry, 79, 2007, 299-306. C. J. Tan; Y. W. Tong, “Molecularly imprinted beads by surface imprinting”, Analytical and Bioanalytical Chemistry, 389, 2007, 369-376. C. J. Tan, S. Wangrangsimakul, R. Bai, Y. W. Tong, “Defining the interactions between proteins and surfactants for nanoparticle surface imprinting through miniemulsion polymerization”, Chemistry of Materials, 2007 (accepted). C. J. Tan; K. H. Ker; H. G. Chua; Y. W. Tong, “Preparation of bovine serum albumin surface-imprinted submicron particles with magnetic susceptibility through core-shell miniemulsion polymerization”, Analytical Chemistry, 2007 (accepted). Conference publication C. J. Tan; Y. W. Yong, American Institute of Chemical Engineers Annual Meeting, San Francisco, CA, USA, November 2006. C. J. Tan; K. H. Ker; H. G. Chua; Y. W. Tong, International Conference on Materials for Advanced Technologies, Suntec City, Singapore, July 2007. Y. W. Tong; C. J. Tan, American Institute of Chemical Engineers Annual Meeting, Salt Lake City, Utah, USA, November 2007. 179 [...]... development of a new strategy and technique for the molecular imprinting of protein macromolecules The main considerations include (1) the compatibility of the imprinting system with the template protein molecules; (2) the imprinted polymer should address the issue of limited diffusion that is often associated with the imprinting of macromolecules and (3) the viability of the imprinting system for industrial... Molecular imprinting technology has gained considerable momentum in its development since 1990, as witnessed by the increase in the number of publications on the related field Protein imprinting has been a focus of research for many chemists and engineers working in the field of molecular imprinting This is because the successful creation of synthetic polymers that can recognize proteins, though being very... their investigation, instead of imprinting the entire protein molecule, they imprinted just a small peptide fraction of the protein This short peptide is the epitope of the 16 _Chapter 2 protein It was proposed that the epitope represented the protein molecule and the imprinted polymer would be able to bind the protein specifically by recognizing the epitope representing... bits where their applications in certain areas will be restrained Secondly, with the creation of imprinted cavities within the polymer bulk, limited diffusion is often encountered for the removal and rebinding of template molecules This is especially important for the imprinting of macromolecules like proteins and oligosaccharides Thirdly, most of the bulk imprinting polymerizations and rebinding studies... This thesis focused on the investigative work that had been conducted to develop miniemulsion polymerization as a viable protein -imprinting system In the early part, much research effort was spent on studying and optimizing the various parameters of the miniemulsion polymerization system to ensure its compatibility with the inherently fragile template protein molecules Based on the study, the polymerization. .. issues of solubility with the mainstream molecular imprinting methodology where organic solvents are mainly used as porogens for the imprinting polymerization systems Secondly, proteins are molecules with very flexible structures that can be changed easily by environmental conditions (Bossi et al., 2007) In addition, through the classical monolithic approach of imprinting, the removal and rebinding of the. .. illustrate the applicability of miniemulsion polymerization as an effective protein surface imprinting system - To incorporate superparamagnetism into the final imprinted polymeric products 4 _Chapter 1 - To adopt an alternative approach of surface imprinting for proteins which cannot be imprinted directly via the direct application of miniemulsion polymerization due to their inherent... probably be of interest Hypothesis From the rationale above, it is therefore hypothesized that the tendency of protein molecules to adsorb and be bound to the oil-water interface can be used as a means for protein surface imprinting via miniemulsion polymerization Besides that, the high surface area to volume ratio of nano-sized imprinted particles will provide a sufficiently high template protein loading... however, for its sufficiently satisfactory imprinting efficiency, convenience and ease of template removal, non-covalent approach remains as the popular, mainstream methodology for molecular imprinting Subsequently, the pre -polymerization imprinting mixture will be polymerized in the presence of a cross-linker monomer, which serves to strengthen and fix the positions of the functional moieties for the creation... _Chapter 1 List of Figures Figure 2.1 Schematic illustration of the principle of molecular imprinting 8 Figure 2.2 Micro-contact patterning approach for protein imprinting (Shi et al., 1999) 16 Figure 2.3 Metal-ion mediated protein imprinting on methacrylate-derivatized silica particle surface (Kempe et al., 1995) 18 Figure 2.4 Molecular imprinting of nucleotides at the oil-water interface (Tsunemori . on the development of a new strategy and technique for the molecular imprinting of protein macromolecules. The main considerations include (1) the compatibility of the imprinting system with. NATIONAL UNIVERSITY OF SINGAPORE 2007 DEVELOPING MINIEMULSION POLYMERIZATION FOR USE IN THE MOLECULAR IMPRINTING OF PROTEIN WITH NANOPARTICLES TAN CHAU JIN (B. Eng. (Hons.),. selectivity in an aqueous environment. One of the advantages of miniemulsion protein imprinting is that the system offers the option of incorporating desired property into the imprinted particles.