Thermosensitive nanomagnetic particles for bio separation

THERMOSENSITIVE NANOMAGNETIC PARTICLES FOR BIO-SEPARATION NABILA SHAMIM NATIONAL UNIVERSITY OF SINGAPORE 2007 THERMOSENSITIVE NANOMAGNETIC PARTICLES FOR BIO-SEPARATION NABILA SHAMIM BS.C CHEMICAL ENGINEERING BANGLADESH UNIVERSITY OF ENGINEERING AND TECHNOLOGY (BUET) DHAKA A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgement Acknowledgements I would like to express my sincere thanks and grateful acknowledgement to my supervisors; Associate Prof. M. S. Uddin, Associate Prof. K. Hidajat, and Associate Prof. L. Hong, for their valuable guidance, effective advice and encouragement through out the research program. I would also like to give my deepest appreciation to all the staff members in the Department of Chemical and Biomolecular Engineering and all my colleagues in the lab. Specially, I would like to express my gratitude to Dr. Peng Zanguo for his spontaneous and gracious suggestions that has led me to the successful completion of my research work. I would also like to extend my eternal gratitude to my parents, husband and my baby daughter Tajriya Ehsan for their love, support and dedication. Finally, my thanks to National University of Singapore for providing the Research Scholarship and to the department of Chemical and Biomolecular Engineering for providing the facilities to take this project through in its entirety. Nabila Shamim March 2007 i Table of contents Table of contents Acknowledgements i Table of contents . ii Executive summary vi Nomenclature . ix List of Figures xiii List of Tables . xvi Chapter Introduction . 1.1 General background on magnetic separation 1.1.1 Functionalized nano-magnetic particles 1.2 Research objective and significance . 1.3 Organization of the thesis . Chapter Literature Review 2.1 Magnetic Separation . 2.1.1 Classification of magnetic separation 2.1.2 Mechanism of magnetic separation . 2.1.3 Driving forces for protein adsorption at solid surfaces 2.1.4 Advantages of magnetic separation . 11 2.2 Magnetic particles . 12 2.2.1 Types of magnetic particles . 12 2.2.2 Preparation of magnetic particles . 13 2.2.3 Basic concepts of magnetic materials 15 ii Table of contents 2.2.4 Forces on magnetic particles 15 2.2.5 Properties of magnetic particles . 16 2.3 Surface modification of magnetic particles 17 2.4 Thermosensitive (PNIPAM) magnetic particles for separation 18 2.4.1 Preparation of thermosensitive nanomagnetic particles 20 2.4.1 Advantages and disadvantage of thermosensitive nanomagnetic particles . 23 2.5 Adsorption and Desorption . 24 2.5.1 Adsorption equilibrium 24 2.5.2 Desorption study 27 2.6 Evaluation of separated target . 28 2.7 Scope of the project 29 Chapter Materials and Methods 31 3.1 Materials . 31 3.2 Methods . 32 3.2.1 Preparation of surfactant coated magnetic nanoparticles . 32 3.2.2 Preparation of Thermosensitive Magnetic Nanoparticles 33 3.2.3 Introduction of Carboxyl Group on Thermosensitive Nanomagnetic Particles35 3.2.4 Adsorption experiments . 35 3.2.5 Desorption experiment . 37 3.3 Analytical methods . 38 3.3.1 Transmission Electron Microscopy (TEM) Measurement 38 3.3.2 X-ray Diffraction (XRD) . 38 3.3.3 Vibrating Sample Magnetometer (VSM) . 39 iii Table of contents 3.3.4 Thermalgravimetry Analysis (TGA) 39 3.3.5 Fourier Transform Infrared (FTIR) Spectroscopy . 39 3.3.6 Brunauer-Emmett-Teller (BET) Method . 40 3.3.7 Zeta Potential Analyzer 40 3.3.8 Dynamic Light Scattering 41 3.3.9 X-ray Photoelectron Spectroscopy (XPS) . 41 3.3.10 Circular Dichroism (CD) . 42 3.3.11 Fluorescence 42 3.3.12 Lysozyme Activity Measurement 43 Chapter Characterization of Nanomagnetic Particles . 45 4.1 Introduction . 45 4.2 Results and Discussion . 47 4.2.1 Characterization of PNIPAM Coated Nanomagnetic Particles . 47 4.3 Conclusions . 57 Chapter Adsorption/desorption of Bovine Serum Albumin (BSA) by Thermosensitive Nanomagnetic Particles 59 5.1 Introduction . 59 5.2 Results and Discussion . 60 5.2.1 Adsorption of BSA 60 5.2.2 Desorption of BSA . 71 5.2.3 Evaluation of conformational changes of BSA . 73 5.3 Conclusions . 76 iv Table of contents Chapter Adsorption/desorption of Lysozyme by Thermosensitive Nanomagnetic Particles 78 6.1 Introduction . 78 6.2 Results and Discussion . 80 6.2.1 Adsorption of lysozyme on Thermosensitive Nanomagnetic Particles . 80 6.2.2 Desorption of Lysozyme from PNIPAM coated Nanomagnetic Particles 85 6.2.3 Evaluation of desorbed lysozyme 88 6.3 Conclusions . 93 Chapter Adsorption/desorption of BSA on MAA Coated Thermosensitive Nanomagnetic Particles 94 7.1 Introduction . 94 7.2 Results and Discussion . 96 7.2.1 Adsorption of BSA on Nanomagnetic Particles 96 7.2.2 Comparison between adsorption on PNIPAM and MAA-PNIPAM coated particles . 100 7.2.3 Desorption of BSA . 103 7.4. Conclusions 105 Chapter Conclusions and Recommendations . 107 8.1 Conclusions . 107 8.2 Recommendations for future work . 109 References 112 List of publications . 125 v Executive summary Executive summary Magnetic separation process involves magnetic particles, carrier liquids, complexes and target molecules. Magnetic separation is used for isolation and purification of chemicals (such as metal ions and organics) or biologically active compounds (such as proteins, enzymes, nucleic acids) out of a mixture. The principle of separating biological compound using the magnetic separation process is, at first the magnetic particles are combined with the intermediates to form a complex and then these complex particles are allowed to interact with the target molecules and finally the particles are separated from the mixture by using gradient magnetic field. However, bared magnetic particles can also be used for magnetic separation. The basic concept is to utilize the physical and specific chemical interactions between magnetic complex particles and target molecules. The interaction forces involved in magnetic separation process are electrostatic, hydrophobic and specific ligand interactions. In recent years, much interest has been focused on smart and intelligent stimuli responsive polymeric materials which can respond to small external stimuli such as pH, temperature and ionic strength. These stimuli responsive smart and intelligent polymeric materials can respond in shape or volume changes to small external stimuli such as temperature, pH, ionic strength etc. Among these, N-isopropylacrylamide (NIPAM) is the most widely studied thermosensitive polymer. It has a Lower Critical Solution Temperature (LCST) of 32oC in water. It collapses and shrinks above the LCST and swells and expands below the LCST of the polymer. Due to its well defined and reversible lower critical solution vi Executive summary temperature poly (N-isopropylacrylamide) (PNIPAM) has long been investigated as a versatile tools in biology. Thermosensitive polymer coated nanomagnetic adsorbents are synthesized by seed polymerization using surface modified nanomagnetic particles as the seeds. The Fe3O4 nanomagnetic particles are prepared by chemical precipitation of Fe2+ and Fe3+ salts in the ratio of 1:2 under alkaline and inert condition. The surface of these particles is modified by surfactants to achieve stability against agglomeration. These stable particles are then polymerized using N-isopropylacrylamide (NIPAM) as the main monomer, methylene-bis-acrylamide as the crosslinker and potassium per sulfate as the initiator. The thermosensitive adsorbents are characterized by using Transmission Electron Microscopy (TEM) and Vibrating Sample Magnetometer (VSM). TEM showed that the particle remained discrete with a mean diameter of 12 nm. Magnetic measurements revealed that the particles are superparamagnetic only with a small decrease of magnetism after binding with the polymer due to the increase in surface spin disorientation. Pure Fe3O4 spinel structure of these nanoparticles is indicated by the X-ray diffraction (XRD) patterns. The polymerization of NIPAM with the surface modified nanomagnetic particles is confirmed by Fourier Transform Spectroscopy (FTIR), Thermogravimetric Analysis (TGA) and X-ray Photoelectron Spectroscopy (XPS). Adsorption and desorption behavior of Bovine Serum Albumin (BSA) and lysozyme on surface modified magnetic nano-particles covered with thermosensitive (PNIPAM) polymer is investigated as a function of temperature, pH and ionic strength. The adsorption results exhibited both pH and temperature sensitivity. The result show that the temperature effect on adsorption/desorption behavior is mainly dependent on the vii Executive summary properties of the particles’ surface. The effect of pH is also investigated and observed the maximum amount of protein is adsorbed near the isoelectric point of BSA and lysozyme. Desorption results showed that more protein is desorbed when adsorption is done at lower temperature. Desorption efficiency is found to be greater than 80%. Conformational changes in BSA and lysozyme upon desorption from nanomagnetic particles are studied by circular dichroism and intrinsic fluorescence spectroscopy. Lysozyme desorbed by NaH2PO4 showed very little conformational changes while desorption by NaSCN showed significant conformational changes. 87% enzymatic activity is retained in the desorbed enzyme for desorption by NaH2PO4. Adsorption of BSA on carboxylated thermosensitive microspheres is also studied as a function of temperature, pH and ionic strength. These particles are prepared by adding methacrylic acid (MAA) in the polymerization process. Adsorption of BSA on the thermosensitive magnetic particles is mainly dependent on the properties of the particles’ surface. By increasing the temperature above the LCST of PNIPAM the particles shrank and are able to adsorb larger quantity of proteins, which is subsequently desorbed at lower temperature. It is believed that carboxylated thermosensitive particles adsorb proteins through hydrogen bonding. When the two extremes of hydrophobic interaction and hydrogen bonding interaction are compared, it is found that more protein are adsorbed using the later interaction. The adsorption equilibrium of BSA on thermosensitive nanomagnetic particles are fitted by Langmuir-Freundlich model. This study shows thermosensitive nanomagnetic particles are effective tools for the isolation and purification of biochemicals. viii Chapter: • Present study used single component system. Future study should focus on multi-component systems, starting with a binary system and then moving to a multi-component system. For multi-component, both simulated as well as actual protein source systems should be used. A selective and sequential adsorption approach may be tried. • Results from present study show that BSA undergoes conformational changes through adsorption/desorption on thermosensitive magnetic particles. Future research should focus on refolding of the denatured biomolecules. • In future, it will be interesting to compare the extent of conformation changes upon adsorption/ desorption of proteins on PNIPAM and MAAPNIPAM coated nano-magnetic particles • Surface-activated magnetic nano-particles have potential applications in cancer treatments though radiotherapy. 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Wang, Pressure dependence of the volume phasetransition of temperature-sensitive gels. Chemical Engineering Science, 51: pp.3235-3239, 1996. 124 List of Publications List of publications 1. Shamim, N., Z.G. Peng, L. Hong, K. Hidajat and M.S.Uddin, Synthesis and characterization of double surfactant coated magnetic particles, International Journal of Nanoscience, 4(2), pp 187-195, 2005. 2. Shamim, N., L. Hong, K. Hidajat and M.S.Uddin, Thermosensitive-polymercoated magnetic nanoparticles: adsorption and desorption of Bovine Serum Albumin, Journal of Colloid and Interface Science., 304 (1), pp 1-8, 2006. 3. Shamim, N., L. Hong, K. Hidajat and M.S.Uddin, Thermosensitive polymer coated nano-magnetic particles for separation of biomolecules, Separation and Purification Technology, 53 pp 164-170, 2007. 4. Shamim, N., L. Hong, K. Hidajat and M.S.Uddin, Thermosensitive polymer (Nisopropylacrylamide) coated nanomagnetic particles: Preparation and Characterization, Colloids and Surfaces B: Biointerfaces, 55, pp 51-58, 2007. 5. Shamim.N., L. Hong, K. Hidajat, and M.S. Uddin, Adsorption, desorption and conformational changes of desorbed lysozyme from thermosensitive nanomagnetic particles, Journal of colloid and interface science, accepted. 125 [...]... Magnetic separation processes 6 Figure 2-2 Schematic diagram of the magnetic separation of non magnetic targets 8 Figure 2-3 Schematic diagram of interaction mechanisms with magnetic particles 9 Figure 2-4 Schematic diagram of adsorption desorption of target molecule on thermosensitive nanomagnetic particles 19 Figure 3-1 Preparation of thermosensitive (PNIPAM) coated nanomagnetic particles. .. nanoparticles 56 Table 5-1 Langmuir-Freundlich parameters for BSA adsorption on thermosensitive and bared nanomagnetic particles at different pH and temperature 71 Table 5-2 Desorption results of BSA from thermosensitive nanomagnetic particles 71 Table 5-3 The estimated percentage of α-helix contents from circular dichroism spectrum 75 Table 6-1 Langmuir parameters for. .. surfactant and thermosensitive polymer N-isopropylacrylamide (PNIPAM) coated inorganic materials and their use as a tool for bioseparation The adsorption equilibrium and effects of different parameters (such as pH, temperature and ionic strength) still need to be studied in details for thermosensitive nano-magnetic particles Moreover, conformational change of proteins during processes of extraction by thermosensitive. .. Evaluation of conformational changes of desorbed targets 6 Comparison of interaction forces for adsorption of protein on thermosensitive and carboxylated thermosensitive nano-magnetic particles 1.3 Organization of the thesis The present thesis is organized into eight chapters Chapter 1 give a brief introduction of magnetic separation, and thermosensitive polymer coated nano-magnetic particles, the objectives... preparing thermosensitive nanomagnetic particles and the characterizations of the nanomagnetic particles are described in Chapter 4 Chapter 5 present the adsorption desorption and conformational changes of desorbed BSA, which is known as soft protein Similar experimental studies for hard protein lysozyme have been detailed in Chapter 6 In Chapter 7 magnetic separation method is extended and applied for BSA... nano-magnetic particles also needs to be studied in details Desorption is another important parameter of separation process for the recovery of target molecule from the adsorbed surface Therefore, more focus should be given on desorption of protein from thermosensitive nano magnetic particles Finally, since most of the previous work focused only on one interaction forces, comparison of interaction forces... polymeric material coated nano-magnetic particles is that, separation could be achieved without significant change in the environmental factors (such as temperature, pH) Therefore, mild conditions of the biomaterials can be maintained throughout the process 1.2 Research objective and significance Many researchers focused on micro sized magnetic particles for separation of biomolecules However, no work so... NaH2PO4 (pH 4) and NaSCN (pH 6.0) from thermosensitive nanomagnetic particles8 9 xiv List of Figures Figure 6-6 Fluorescence emission spectra of native lysozyme (pH 4) and desorbed lysozyme by NaH2PO4 (pH 4) and NaSCN (pH 6) from thermosensitive nanomagnetic particles 92 Figure 7-1 Mechanism of protein adsorption on carboxylated thermosensitive nanoparticles 97 Figure 7-2... molecule on magnetic particles either individually or synergically Therefore, it is necessary to identify the critical steps in the adsorption, and thereafter to utilize a suitable interaction mechanism for the separation of desired products 2.1.4 Advantages of magnetic separation Compared to conventional separation processes, magnetic separation has the following advantages: • Magnetic separation can eliminate... time (Lee and Pope 1994) The conditions necessary for the formation of magnetic particles are essentially the same as for non-magnetic particles but some special precautions are necessary because of strong magnetic interactions among the particles The essential parameters are: 1 Separation of the nucleation process from the growing process 2 Protection of particles from aggregation 3 A controlled supply . iii 2.2.4 Forces on magnetic particles 15 2.2.5 Properties of magnetic particles 16 2.3 Surface modification of magnetic particles 17 2.4 Thermosensitive (PNIPAM) magnetic particles for separation. equilibrium of BSA on thermosensitive nanomagnetic particles are fitted by Langmuir-Freundlich model. This study shows thermosensitive nanomagnetic particles are effective tools for the isolation. parameters for BSA adsorption on thermosensitive and bared nanomagnetic particles at different pH and temperature. 71 Table 5-2 Desorption results of BSA from thermosensitive nanomagnetic particles