Activity Measurements of Enzymes Conjugated to Superparamagnetic Iron Oxide Nanoparticles under an Alternating Magnetic Field

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Activity Measurements of Enzymes Conjugated to Superparamagnetic Iron Oxide Nanoparticles under an Alternating Magnetic Field
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english
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Bhattacharjee, Tapomoy
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University of Florida
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Master's ( M.S.)
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University of Florida
Degree Disciplines:
Chemical Engineering
Committee Chair:
RINALDI,CARLOS
Committee Co-Chair:
DOBSON,JON P

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Subjects / Keywords:
amf -- enzymes -- nanoparticle -- superparamagnetic
Chemical Engineering -- Dissertations, Academic -- UF
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Chemical Engineering thesis, M.S.
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theses   ( marcgt )
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Abstract:
Iron oxide magnetic nanoparticles have the capacity to dissipate the energy of an alternating magnetic field (AMF) in the form of heat. Heat is generated either due to Brownian relaxation, i.e., due to physical rotation of particles under AMF or due to Neel Relaxation, i.e., rotation of magnetic dipole only. This physical property can be used effectively for inducing local heating phenomena. Enzymes can be inactivated by inducing heat energy. Here the objective was to inactivate an enzyme using the benefit of local heating phenomena. Iron oxide nanoparticles synthesized by the co-precipitation of Ferric Chloride and Ferrous Chloride were used. Particle sizes were characterized using Dynamic Light Scattering (DLS). Relaxation properties of the nanoparticles were determined by AC susceptibility measurements. alpha-Amylase and beta-Galactosidase from Aspergillus Oryzae were used for heat inactivation study. Both alpha-Amylase and beta-Galactosidase were covalently conjugated to Iron Oxide nanoparticles. Conjugates were separated and washed. Remote inactivation studies were conducted under a magnetic field of 33.7 kA/m at a frequency of 255 kHz. Activity of alpha-Amylase was measured using starch iodine assay at 618 nm. Activity of beta-Galactosidase was measured using ONPG assay at 420 nm. Experiments done throughout this study confirms that both the enzymes are bound to the surface of Iron Oxide nanoparticles. Iron Oxide nanoparticles have also shown heat dissipation in presence of an AMF. Conjugated particles were washed properly and no presence of free enzyme was ensured. Activity of the conjugated particles, in case of both the enzymes, were measure at different temperatures with external heating. It was observed that enzyme bound to the IO nanoparticle can be inactivated by external heating. However, when placed under AMF, both the enzymes conjugated to IO nanoparticles, did not show any significant inactivation. Inference that can be drawn that local heating due to IO nanoparticles under AMF was not sufficient to inactivate the enzymes.
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by Tapomoy Bhattacharjee.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
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Adviser: RINALDI,CARLOS.
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Co-adviser: DOBSON,JON P.

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UFE0046842:00001


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ACTIVITY MEASUREMENTS OF ENZYMES CONJUGATED TO SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES UNDER AN ALTERNATING MAGNETIC FIELD By TAPOMOY BHATTACHARJEE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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2014 Tapomoy Bhattacharjee

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To my p arents

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4 ACKNOWLEDGMENTS Carlos Rinaldi, for granting me the freedom to pursue my academic and professional interests over the past one and a half years and Dobson of the Department of Biom edical Engineering and Material Science Engineering at University of Florida for engaging discussions and suggestions. I should also take this opportunity to thank my fellow colleagues Ana C. Bohorquez and Adam Monsalve for their continuous help and suppor t.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST O F ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ....... 14 1.1 Su perparamagnetic Iron Oxide Nanoparticle ................................ .................... 14 1.1.1 Nel Relaxation ................................ ................................ ....................... 15 1.1.2 Brownian Relaxation ................................ ................................ ................ 16 1.2 Local Heating Phenomena ................................ ................................ ................ 17 1.3 Objective ................................ ................................ ................................ ........... 18 1.4 Bio Conjugation Chemistry ................................ ................................ ............... 19 1.4.1 EDC/ NHS Chemistry ................................ ................................ .............. 19 1.4.2 Sulfo SMCC Chemistry ................................ ................................ ............ 20 1.5 Amylase ................................ ................................ ................................ ......... 21 1.6 Galactosidase ................................ ................................ ................................ 22 2 MATERIALS AND METHODS ................................ ................................ ................. 23 2.1 Materials ................................ ................................ ................................ ........... 23 2.2 Nanoparticle Characterization ................................ ................................ ........... 23 2.2.1 PEG550 Batch 1 ................................ ................................ ...................... 23 2.2.2 PEG550 Batch 2 ................................ ................................ ...................... 25 2.2.3 IO CMDX particles: ................................ ................................ .................. 28 2.3 Free Enzyme Activity ................................ ................................ ........................ 30 2.3.1 Amylase activity ................................ ................................ ................... 30 Galactosidase activity ................................ ................................ ........... 32 2.4 Conjugated Enzyme activity ................................ ................................ .............. 33 2.4.1 Amylase activity ................................ ................................ ................... 33 Galactosidase activity ................................ ................................ ........... 34 2.5 Bio Conjugatio n ................................ ................................ ................................ 34 2.5.1 For Amylase ................................ ................................ ......................... 34 2.5.2 For Galactosidase ................................ ................................ ............... 36 2.5.2. 1 Scheme 1 ................................ ................................ ....................... 36 2.5.2.2 Scheme 2 ................................ ................................ ....................... 37 2.6 Wash Procedure of Conjugated Enzyme ................................ .......................... 38 2.6.1 Amicon Ultra 15 Centrifugal Filter Units ................................ .................. 38

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6 2.6.2 Sephacryl S400 HR Columns ................................ ................................ .. 39 2.6.2.1 Amylase separation ................................ ................................ .... 39 Galactosidase separation ................................ ........................... 41 2.7 Inactivation Study ................................ ................................ .............................. 44 2.7.1 For amylase ................................ ................................ ................... 44 2.7.2 For galactosidase ................................ ................................ .......... 46 3 RESULTS ................................ ................................ ................................ ................ 48 3.1 Selectivity of Sulfo SMCC over EDC ................................ ................................ 48 3.2 Activity of free enzyme ................................ ................................ ...................... 50 3.2.1 For Amylase ................................ ................................ ......................... 50 3.2.2 For Galactosidase ................................ ................................ ................ 51 3.3 Activity of Enzyme Conjugated to Iron Oxide nanoparticle ............................... 52 3.3.1 For Amylase ................................ ................................ ......................... 52 3.3.2 For Galactosidase ................................ ................................ ................ 53 3.4 Activity under AMF ................................ ................................ ............................ 54 3.4.1 For Amylase: ................................ ................................ ........................ 54 3.4.1.1 Study 1: ................................ ................................ .......................... 54 3.4.1.2 Study 2: ................................ ................................ .......................... 55 3.4.1.3 Study 3: ................................ ................................ .......................... 57 3.4.2 galactosidase: ................................ ................................ ................ 59 3.4.2.1 IO CMDX conjugate Study 1: ................................ ......................... 5 9 3.4.2.2 IO CMDX conjugate Study 2: ................................ ......................... 60 3.4.2.3 PEG550 Conjugate Study 1: ................................ .......................... 61 3.4.2.4 PEG550 Conjugate Study 2: ................................ .......................... 62 3.4.2.5 PEG550 Con jugate Study 3: ................................ .......................... 62 3.4.2.6 PEG550 Conjugate Study 4: ................................ .......................... 63 4 CONCLUSION ................................ ................................ ................................ ........ 64 APPENDIX AMYLASE CONJUGATION TRIALS USING EDC/NHS CHEMISTRY ...................... 65 Scheme 1 ................................ ................................ ................................ ................ 65 Scheme 2 ................................ ................................ ................................ ................ 66 Scheme 3 ................................ ................................ ................................ ................ 67 Scheme 4 ................................ ................................ ................................ ................ 68 Scheme 5 ................................ ................................ ................................ ................ 69 Scheme 6 ................................ ................................ ................................ ................ 70 Scheme 7 ................................ ................................ ................................ ................ 71 LIST OF REFERENCES ................................ ................................ ............................... 72 BIOGRAPHIC AL SKETCH ................................ ................................ ............................ 76

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7 LIST OF TABLES Table page 2 1 beta gal conjugation scheme 1: Absorbance of sample and control after incubation ................................ ................................ ................................ ........... 36 2 2 beta gal conjugation scheme 2: Absorbance of sample and control after incubation ................................ ................................ ................................ ........... 37 2 3 Absorbance of the particles; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on, H 12 is the control ................................ ................................ ... 40 2 4 Absorbance of the starch iodine assay; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on, H 12 is the cont rol ................................ ............. 41 2 5 Difference of absorbance normalized by control; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on, H 12 is the control ................................ .. 41 2 6 Absorbance of the ONPG assay from column effluent; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on ................................ ........................... 43 3 1 for Starch Iodine assay of free enzyme after 45 min incubation at different temperature ................................ ................................ ..... 50 3 2 for ONPG assay of free b eta Galactosidase after 60 min incubation at different temperature ................................ .............................. 51 3 3 for Starch Iodine assay of conjugated enzyme after 45 min incubation at differen t temperature ................................ ......................... 52 3 4 for ONPG assay of conjugated beta Galactosidase after 60 min incubation at different temperature .......................... 53 3 5 Activity under AMF: Study 1: Absorbance after application of AMF .................... 54 3 6 Activity under AMF: Study 1: Absorbance of Control ................................ .......... 54 3 7 Activity under AMF: Study 2: Absorbance of after application of AMF ................ 55 3 8 Activity under AMF: Study 2: Absorbance of Control ................................ .......... 56 3 9 Activity under AMF: Study 3: Absorbance after application of AMF .................... 57 3 10 Activity under AMF: Study 3: Absorbance of Co ntrol ................................ .......... 57 3 11 IO CMDX Beta gal conjugate Study 1: Absorbance of sample and control ........ 59 3 12 IO CMDX Beta gal conjugate Study 2: Absorbance of sample and control ........ 60

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8 3 13 PEG550 (batch2) Beta gal conjugate Study 1: Absorbance of sample and control ................................ ................................ ................................ ................. 61 3 14 PEG550 (batch2) Beta gal conjugate Study 2: Absorbance of sample and control ................................ ................................ ................................ ................. 62 3 15 PEG550 (batch2) Beta gal conjugate Study 3: Absorbance of sample and control ................................ ................................ ................................ ................. 62 3 16 PEG550 (batch2) Beta gal conjugate Study 4: Absorbance of sample and control ................................ ................................ ................................ ................. 63 A 1 Scheme 2 reaction mixture preparation. ................................ ............................. 66

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9 LIST OF FIGURES Figure page 1 1 Energy of a single domain particle under applied field ................................ ....... 15 1 2 EDC Chemistry; R is IO nanoparticle and R' is the enzyme here ....................... 20 1 3 Sulfo SMCC Chemistry; R is the IO nanoparticle and R' is the enzyme ............ 21 2 1 DLS of PEG550 Batch 1 ................................ ................................ ..................... 23 2 2 Zeta potential of PEG550 Batch 1 particles ................................ ........................ 24 2 3 SAR measurement of PEG550 Batch 1 particles ................................ ............... 25 2 4 DLS of PEG550 Batch 2 ................................ ................................ ..................... 25 2 5 Zeta potential of PEG550 Batch 2 particles ................................ ........................ 26 2 6 M vs. H Plot for PEG550 batch 2 particles ................................ .......................... 27 2 7 SAR measurement of PEG550 Batch 2 particles ................................ ............... 28 2 8 DLS of IO CMDX particles ................................ ................................ .................. 28 2 9 Zeta potential of IO CMDX particles ................................ ................................ ... 29 2 10 M vs. H Plot for IO CMDX particles ................................ ................................ .... 29 2 11 SAR measurement of PEG550 Batch 2 particles ................................ ............... 30 2 12 Absorbane of starch iodine complex at different wavelength .............................. 31 2 13 beta Galactosidase assay ................................ ................................ .................. 32 2 14 Sample and contains washed particles after reaction; 'C2' refers to Negative Control and contains pure starch solution only; 'C1' refers to positive control and contains 2.5 ug of Amylase ................................ ................................ .......... 35 2 15 Wash using Amicon Ultra 15 Centrifugal Filter Units ................................ .......... 38 2 16 Starch Iodine assay of column effluent ................................ ............................. 40 2 17 ONPG assay of column effluent ................................ ................................ ........ 42 2 18 Particle effluent from the column ................................ ................................ ....... 44

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10 2 19 Coil for applying AMF ................................ ................................ ........................ 45 2 20 Inactivation study set up ................................ ................................ .................... 46 3 1 alpha Amylase (PDB ID: 6TAA) with Lysine residues marked in Yellow ............ 48 3 2 Terminal amine of alpha Amylase (PDB ID: 6TAA) ................................ ............ 49 3 3 Free alpha Amylase activity normalized by the maximum ................................ .. 50 3 4 Free beta Galactosidase activity normalized by the maximum ........................... 51 3 5 Normalized activity of alpha Amylase conjugated to Iron Oxide Nanoparticle .... 52 3 7 Activity un der AMF: Study 1: Temperature throughout the process ................... 55 3 8 Activity under AMF: Study 2: Temperature throughout the proce ss ................... 56 3 9 Activity under AMF: Study 3: Temperature throughout the process ................... 58 3 10 Activity of conjugated alpha Amylase under AMF ................................ .............. 59 3 11 IO CMDX Beta gal conjugate Study 1: Temperature throughout the process .... 60 3 12 IO CMDX Beta gal conjugate Study 2: Temperature throughout the process .... 61 3 13 Activity of conjugated beta Gal under AMF ................................ ........................ 63 A 1 Scheme 1: Starch Iodine assay with Washed particle s; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ................................ ................................ ...... 66 A 2 Scheme 2: Starch Iodine assay with Washed particles; '1' '6' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ................................ ............................... 67 A 3 Scheme 3: Starch Iodine assay with Washed particles; 'S' refers to Sample and cont ains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ................................ ................................ ...... 68 A 4 Scheme 5: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ................................ ................................ ...... 69 A 5 Scheme 6: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C1' refers to Negative Cont rol and contains pure starch solution only; 'C2' refers to positive control and contains 2.5 ug of Amylase with Starch solution ................................ ................. 70

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11 L IST OF ABBREVIATIONS AMF Alternating Magnetic Field DLS Dynamic Light Scattering IO Iron Oxide ILP Intrinsic Loss Power ONPG o nitrophenyl D galactoside SAR Spec ific Absorption Rate SQUID Superconducting Quantum Interference D evice

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degr ee of Master of Science ACTIVITY MEASUREMENTS OF ENZYMES CONJUGATED TO SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES UNDER AN ALTERNATING MAGNETIC FIELD By Tapomoy Bhattacharjee May 2014 Chair: Carlos Rinaldi Major: Chemical Engineering Iron oxide magnet ic nanoparticles have the capacity to dissipate the energy of an alternating magnetic field (AMF) in the form of heat. Heat is generated either due to Brownian relaxation, i.e., due to physical rotation of particles under AMF or due to Nel Relaxation, i.e ., rotation of magnetic dipole only. This physical property can be used effectively for inducing local heating phenomena Enzymes can be inactivated by inducing heat energy. Here the object ive wa s to inactivate an enzyme using the benefit of local heating phenomena. Iron oxide nanoparticles synthesized by the co precipitation of Ferric Chloride and Ferrous Chloride were used Particle size s were characterized using Dynamic Light Scattering (DLS). Relaxation properties of the nanoparticles were determined by AC susceptibility measurements. Amylase Galactosidase from Aspergillus Oryzae we re used for heat inactivation study. Both Amylase Galactosidase were covalently conjugated to Iron Oxide nanoparticles. Conjugates were separated and washed. Remote inactivation studies were conducted u nder a magnetic field of 33 .7 kA/m at a frequency of 255 kHz. Activity of Amylase wa s measured using starch iodine assay at 618 nm. Galactosidase was

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13 measured using ONPG assay at 420 nm. Experiments done throughout this study confirms that both the enzymes are bound to the surface of Iron Oxide nanoparticles. Iron Oxide nanoparticles have also shown heat dissipation in presence of an AMF. Conjugated particles were washed properly and no presence of free enzyme was ensured. Activity of the co njugated particles, in case of both the enzymes, were measure at different temperatures with external heating. It was observed that enzyme bound to the IO nanoparticle can be inactivated by external heating. However, when placed under AMF, both the enzymes conjugated to IO nanoparticles, did not show any significant inactivation. Inference that can be drawn that local heating due to IO nanoparticles under AMF was not sufficient to inactivate the enzymes

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14 CHAPTER 1 BACKGROUND 1.1 Superparamagne tic Iron Oxide Nanoparticle Superparamagnetism is magnetic phenomena demonstrated by n anoscale ferromagnetic and ferri magnetic materials. In this case the nanoparticles demonstrate magnetic properties in presence of a magnetic field only. Superparamagneti sm occurs in case of Iron Oxide (IO) nanoparticles when the material is composed of very small crystallites (< 3 0 nm). Superparamagnetic Iron Oxide nanoparticles are generally made from ferrimagnetic iron oxides, such as magnetite (Fe3O4) and Fe2O3). 1 Superparamagnetic Iron O xide nanoparticles are widely used for numerous biomedical and bioengineering applicat ions such as MRI contrast enhancement 2 tissue repair, immunoassay, gene transfection 3, 4 tumor targeting, detoxification of biological fluids, hyperthermia 5 drug delivery and cell separation, etc. 6, 7 Superparamagnetic Iron Oxide nanoparticles are also useful as probes for measurement of physical properties such as viscosity of a fluid 8 In a recent study, Klyachko et. al. 9 have shown that low frequen cy alternating magnetic field c hanges the reaction kinetics of an enzyme when covalently bound to the surface of Sup erparamagnetic Iron Oxide nanoparticle. Iron oxide magnetic nanoparticles have the capacity to dissipate the energy of an alternating magnetic field (AMF) in the form of heat 10, 11 Heat is generated either due to Brownian relaxation, i.e., due to physical rotation of particles under AMF or due to Nel Relaxation, i.e., rotation of magnetic dipole only 12

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15 1.1.1 Nel Relaxation Single domain magnetic nanoparticles may respond to a time varying magnetic field by the rotation of its magnetic moment through realign ment of the magnetic spin without particle rotation. This phenomena is known as Nel Relaxation Relaxation time is defined as the amount of time the magnetization of the material remains in one stable direction after removal of an external field. R ealignm ent of the magnetic spin of these particles requires an activation energy E B [Fig 1 1 ]. IO nanoparticles usually relax by the Nel mechanism when kT is higher than this E B Figure 1 1 Energy of a single domain particle under ap plied field 13 For Superparamagnetic Iron Oxide nanoparticles relaxing by Nel mechanism Nel Relaxation time is given by: 14

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16 Where K the particle magnetocrystalline anisotropy constant, D m is the diameter of the magnetic core is a characteristi c time. 1.1.2 Brownian Relaxation Single domain magnetic nanoparticles may also respond to a time varying magnetic field by the rotation of the particle locked magnetic moment by physical particle reorientation through rotational Brownian motion This ph enomena is known as Brownian Relaxation In this case, r ealignment of the magnetic spin is not possible as E B is higher than kT at normal temperature. For Superparamagnetic Iron Oxide nanoparticles relaxing by Brownian mechanism in a Newtonian fluid, Bro wnian Relaxation time is given by: 14 Where Brownian Relaxation time, D h is the particl e hydrodynamic diameter, dimensional slip coefficient characterizing the amount of hydrodynamic slip at the nanoparticle surface. Under an A MF, these particles dissipate heat due to the viscous interaction of the particle surface with the surrounding fluid Relaxation occurs by the faster of these two mechanisms. An effective relaxation time is given by:

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17 1.2 Local Heating Phenomena Local energy delivery using magnetite particulate matter was first introduced by Gilchrist et al. in 1957 10 Local heating phenomena is widely used in case of magnetic fluid hyper thermia 15 In 2011, Creixell et. al. reported that epidermal growth factor conjugated iron oxide nanoparticles targeting the epidermal growth factor receptor (EGFR) result in a 99% reduction in cell viability and clonogenic survival when expo sed to an AMF, without a perceptible rise in medium temperature 5 In a more recent study, Polo Corral es et. al. h a ve demonstrated that the surface temperature of iron oxide nanoparticles in an alternating magnetic field (AMF) remains higher than the temperature of the surrounding medium through the temperature induced change in fluorescence of a thermores ponsive/fluorescent polymer consisting nanoparticle 16 Under the influence of an AMF single domain magnetic nanoparticles can dissipate heat by both relaxation mechanisms; both Nel and Brownian 1 This heat generation is dependent on the amplitude and frequency of the AMF applied. According 17 volumetric energy dissipation rate from a supe rparamagnetic nanoparticle (P) is given by: Where, is initial susceptibility of the nano particles, H is magnetic field amplitude,

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18 Here, 1.3 Objective Enzyme stability is related to enzyme structure and to factors in the microenvironment Temperature dependence of enzyme activity is related to the structure, thermodynamics and kinetics of the enzymatic reactio n 19 Different enzymes have previously been immobilized on magnetic nanoparticles fo r several application such as enzyme recovery 20, 21 bio sensing 22, 23 etc. The objective of this study was to use Superparamagnetic Iron Oxide nanoparticles with surface m odification, to control activity of an enzyme remotely. Previous attempts by Klyachko et. al. 9 incorporates a

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19 non heating magnetic field to change the reaction kinetics of an enzyme immobilized on the surface of IO nanoparticles predominantly by deformation of the enzyme by mechanical stress. As IO nanoparticles diss ipate heat under an AMF, enzymes covalently conjugated on to the surface of these nanoparticles also receive the energy. Here, the idea was to change the activity of an enzyme using this heat energy. In that way, by controlling amplitude, frequency and app lication time of AMF, activity of the enzyme can be controlled. 1.4 Bio Conjugation Chemistry 1.4.1 EDC/ NHS Chemistry EDC (or EDAC; 1 ethyl 3 (3 dimethylaminopropyl) carbodiimide hydrochloride) is used for conjugating biological substances containing carb oxylates and amines. Both EDC itself and the isourea f ormed as the by product of the crosslinking reaction are water soluble and may be removed easily by dialysis or gel fi ltration Here the Iron oxide nanoparticles were coated with dextran and thus contai ns carboxylic groups. Amylase enzyme contains amine groups. E DC reacts with carboxylic acids to create an active ester intermediate. In the presence of an amine nucleophile, an amide bond is formed with release of an isourea by product. EDC may be used t o form active ester f unctionalities with carboxylate groups using the water soluble compound NHS that couple rapidly with amines on target molecules

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20 Figure 1 2 EDC Chemistry; R is IO nanoparticle and R' is the enzyme here 24 1.4.2 Sulfo SMCC Chemistry Sulfo SMCC, sulfosuccinimidyl 4 ( N maleimidomethyl)cyclohexane 1 carboxylate, is a watersoluble analog of SMCC that possesses a negatively charge d sulfonate group on it NHS ring. Sulfo S MCC reacts with amine containing molecules to form stable amide bonds. Its maleimide end then reacts to a sulfhydryl containing compound to create a thioether linkage. Here the Iron oxide nanoparticles were coated w ith Poly Ethylene Glycol and contains amine groups. Amylase enzyme contains sulfhydryl groups.

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21 Figure 1 3 Sulfo SMCC Chemistry; R is the IO nanoparticle and R' is the enzyme 24 1.5 Amylase Amylase is a protein enzyme ( EC 3.2.1.1 ) that catalyzes the hydrolysis of internal 1, 4 glucan links in polysaccharides containing 3 or more 1, 4 linked D glucose units such as starch, yielding sma ller units like glucose and maltose 25 It was name d Amylase as the hydrolysis products are in the alpha configuration 26 Amylase has a molecular weight ranging from 51 kDa to 54 kDa depending on its source. Amylase form Aspergillus Oryzae (PDB ID: 6TAA) consists of 499 amino acids 27 The main substrate of Amylase is starch. Starch is a polymer of glucose linked to one another through the C1 oxygen, known as the glycosidic bond. Two types of glucose polymers are present in starch: amylose and amylopectin. Amylose is a linear

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22 polymer cons isting of up to 6000 glucose units with 1 4 glycosidic bonds 28 amylase is widely used in the starch industry to hydrolyze starch int o fructose and glucose syrups 29 amylase has its application in detergent 30, 31 textile 31 food, biofuel and paper industry as well 32 Activ ity of Amylase on a starch substrate can be monitored by a s pectrophotometric method. Amylose of starch forms a blue colored complex with Iodine which can be quantified using a spectrophotometer 33 This color of starch iodine complex is a result of iodine e ntrapped inside a helical chain of amylose 34 1.6 Galactosidase G alactosidase ( gal ; EC 3.2.1.23) is a hydrolase enzyme that catalyzes the hydrolysis of D galactosyl residues from polymers, oligosaccharides or secondary metabolites 35 It 4) galactosyl bonds in oligo and disaccharides and also catalyzes the reverse reaction of thehydrolysis, often called transglycosylation 36 Galactosidase from Aspergillus Oryzae (PDB ID: 4IUG) has 985 residues 36 This enzyme is mainly used for lactose removal from milk products and for the production of galactosylated products 37, 3 8 function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy s ources. The synthetic compound o nitrophenyl D galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o nitrophenol which has a yellow color Galactosidase activity can be measured by this method 39

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23 CHAPTER 2 MATERIALS AND METHODS 2.1 Materials Amylase, Potassium Iodide, Iodine and starch from Potato was bought from Sigma. EDC, NHS and Sulfo SMCC were bought from IO CMDx and PEG 550 Iron oxide nanoparticles were produced in the laboratory and provided by A C Bohorquez Sephacryl S400 HR gel filtration medium was bought from GE Healthcare Life Sciences Ambrell EasyHeat LI 8310 induction heater was used t o apply the alternating magnetic field. An AccuBlock Digital dry bath is used for heating. Spectrophotometric analysis was done using a Shimadzu UV 2600 spectrophotometer. 2.2 Nanoparticle Characterization 2.2.1 PEG550 Batch 1 Hydrodynamic diameter of PE G550 Batch 1 particles were measured by dynamic light scattering (DLS). Hydrodynamic diameter of these particles was 64 nm [Fig: 2 1 ] Figure 2 1 DLS of PEG550 Batch 1 Zeta potential of these nanoparticles were 15.3 +/ 0.72 mV [Fig: 2 2 ]

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24 Figure 2 2 Z eta potential of PEG550 Batch 1 particles SAR of PEG550 Batch 1 particles were measured at 37.5 kA/m field and 340 kHz frequency. To calculate the time dependent temperature rise 200 L of PEG550 suspension in water (0.36 mgFe/mL) was subjected to the AMF for 60 s and first 20 s of temperature data was used to calculate the slope [Fig: 2 3 ]. ILP was calculated from SAR.

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25 Figure 2 3 SAR measurement of PEG550 Batch 1 particles SAR of PEG550 Batch 1 was 1099 W/g of Fe in water and 947 W/g of Fe in 1.5% Agar ILP of PEG550 in water was 2.3 2.2.2 PEG550 Batch 2 Hydrodynamic diameter of PEG550 Batch 2 particles was 53 nm. Figure 2 4 DLS of PEG550 Batch 2

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26 Zeta potential of these nanoparticles were 11.62 +/ 1.89 mV [Fig 2 5 ] Figure 2 5 Zeta potential of PEG550 Batch 2 particles Saturation magnetization is measured with a SQUID and is found to be 26 kA/m.

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27 Figure 2 6 M vs. H Plot for PEG550 batch 2 particles SAR of PEG550 Batch 2 particles were measured at 37.5 kA/m field and 340 kHz frequency. To calculate the time dependent temperature rise 200 L of PEG550 suspension in water ( 2.8 mgFe/mL) was subjected to the AMF for 60 s and first 20 s of temperature data was used to calculate the slope [Fig: 2 7 ]. ILP was calculated from SAR. SAR of PEG550 Batch 2 in water was 540 W/g of Fe. ILP of PEG550 Batch 2 in water was 1.13 W/g of Fe.

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28 Figure 2 7 SAR measurement of PEG550 Batch 2 particles 2.2.3 IO CMDX particles: Hydrodynamic diameter of IO CMDX particles was 49 nm. Figure 2 8 DLS of IO CMDX particles Zeta potential of IO CMDX particles was 5.9 +/ 1.83 mV.

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29 Figure 2 9 Zeta potential of IO CMDX particles Saturation magnetization is measured with a SQUID and is found to be 200 kA/m. Figure 2 10 M vs. H Plot for IO CMDX particles SAR of IO CMDX particles were measured at 37.5 kA/m field a nd 340 kHz frequency. 200 L of IO CMDX suspension (10 mg particle/mL) in water was subjected

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30 to the AMF for 60 s and first 20 s of temperature data was used to calculate the slope [Fig: 2 11 ]. SAR of IO CMDX i n water was 598 W/g of Fe ILP of IO CMDX in w ater was 1.251 W/g of Fe. Figure 2 11 SAR measurement of PEG550 Batch 2 particles 2.3 Free Enzyme Activity 2.3.1 Amylase activity To know the absorption wavelength of the Sta rch Iodide complex formed, a 100 g/mL starch solution was prepared. Potassium Iodide and Iodine was added to water to a final concentration of 2.5mM to form the Iodine reagent. 20 L of 2.5mM Iodine reagent is added to the starch solution. A full range (3 00 700 nm) absorption scan was then made which gave the absorption wavelength as 618 nm.

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31 Figure 2 12 Absorbane of starch iodine complex at different wavelength Activity of Amylase was measured by a method described by Xiao et. al. 40 To quantify the activity of free enzyme (pH 7.4) at di fferent temperatures, 200 L of 2 mg/mL starch solution was incubated with 10 L of 24 g / mL free Amylase s olution for 45 min using a dry bath at 5 different temperatures (20C, 30C, 45C, 60C and 80C). After 45 min the reaction wa s quenched with 20 L of 1M HCl and 20 L of Iodine reagent wa s added to that. Absorbance of these solutions were measured at 618 nm. These are A sample Again, 200 L of 2 mg/mL starch solution was diluted with 10 uL PBS 1X and incubated for 45 min using a dry bath at 5 di fferent temperatures (20C, 30C, 45C, 60C and 80C). After 45 min the reaction wa s quenched with 20 uL of 1M HCl and 20 uL of Iodine reagent wa s added to that. Absorbance of these solutions were measured at 618 nm. These are A control. Activity was defi ned as U ( g of starch degraded per minute per g of enzyme used )

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32 Where, A ug is the absorbance for 1 g/mL solution. Normalized activity was used which was defined as: 2.3.2 Galactosidase activity Galactosidase activity was measure by the method described by Miller 39 Here galactosidase was assayed by measuring hydrolysis of the chromogenic substrate, o nitrophenyl Dgalactos ide (ONPG) as shown in Fig 2 13 The reaction was stopped by adding Na2CO3 which shifts the reaction mixture to pH 11. At this pH most of the o galactosidase is inactivated. The amount of o nitrophenol formed was measured by determining the absorbance at 420 nm. Figure 2 13 beta Galactosidase assay To measure Galactosidase activity a 5 U/mL solution of Galactosidase in PBS 1X was prepared. 250 L of 10 mg/ mL ONPG solution in PBS 1X was incubated with 20 L of Galactosidase solution for 60 min. 20 L of 0.5M Na 2 CO 3 solution was

PAGE 33

33 added to the reaction mixture to quench t he reaction Absorbance of the reaction mixture was measured at 420 nm. These are A sample Again, 250 L of 10 mg/ mL ONPG solution was incubated with 20 L of PBS 1X for 60 min and 20 L of 0.5M Na 2 CO 3 solution was added to it. Absorbance of this solution was measured at 420 nm. These are A control Activity was defined as U G ( g of o Nitrophenol produced per minute per U of enzyme used ). Where, A ug is the absorbance for 1 g/mL o Nitrophenol solution. Normalized activity was used which was defined as: 2.4 Conjugated Enzyme activity 2.4.1 Amylase activity To quantify the activity of conjugated enzyme (pH 7.4) at different temperatures, 200 L of 2 mg/mL starch solution was incubated wi th 10 uL of PEG550 conjugated Amylase solution for 45 min using a dry bath. After 45 min the reaction is quenched with 20 uL of 1M HCl and 20 uL of Iodine reagent is added to that. Absorbance of these solutions were measured at 618 nm. These are A sample here Activity normalized by maximum were measured at 5 different temperatures (20C, 30C, 45C, 60C and 80C).

PAGE 34

34 2.4.2 Galactosidase activity To measure conjugated Galactosidase activity, 20 0 L of 10 mg/ mL ONPG solution in PBS 1X was incubated with 20 L of conjugated Galactosidase solution for 60 min. 20 L of 0.5M Na 2 CO 3 solution was added to the reaction mixture to quench the reaction Absorbance of the reaction mixture was measured at 420 nm. These are A sample Again, 2 0 0 L of 10 mg/ mL ONPG solution was incubated with 20 L of PBS 1X for 60 min and 20 L of 0.5M Na 2 CO 3 solution was added to it. Absorbance of t his solution was measured at 420 nm. These are A control Now 20 L of conjugated Galactosidase was diluted using 2 0 0 L PBS. 20 L of 0.5M Na 2 CO 3 solution was added to it. These are A particle. Activity was defined as U G ( g of o Nitrophenol produced per minute per U of enzyme used ). Where, A ug is the absorbance for 1 g/mL o Nitrophenol solution. Normalized activity was used which was defined as: 2. 5 Bio Conjugation 2.5.1 For Amylase Different schemes were attempted to conjugate I ron Oxide nanoparticles to Amylase using EDC and NHS as cross linker Amylase coul d not be conjugated to IO

PAGE 35

35 CMDX nanoparticles using EDC and NHS as cross linker. Details of these procedures are described in Appendix A. Amylase was conjugated to PEG550 (batch 1) nanoparticles using Sulfo SMCC as cross linker. To do so, 5 mL of PEG550 p article suspension in water wa s taken (pH=5.81) The buffer wa s changed to PBS 1x using a 30 kDa membrane under centrifugation (pH=7.2); final volume wa s 6mL 2.2 mg of dry S ulfo SMCC was taken in two vials. 2.5 mL of PEG550 suspension wa s added to each vi al. Reaction wa s carried on for 1 hour under room temperature. These reaction mixtures we re washed with PBS 1x two times using 30kDa membrane using centrifuge. 2 mL of 45 mg/mL Amylase solution in PBS 1x was added to each vial. all vial is shakeke at anoth er is shaken at 25 C for 24 hours. After reaction these p articles were washed using a Sephacryl S 400 HR column Washed particles confirm presence of amylase on incubation with starch followed by the addition of iodine reagent. [Figure 2 14 ] For furth er conjugations 9 mg of Amylase was used and reaction was carried out at 25 C. Figure 2 14 Tapomoy Bhattacharjee. Scheme 8: Starch Iodine assay with Washed reaction; 'C2' r efers to Negative Control and contains pure starch solution only; 'C1' refers to positive control and contains 2.5 ug of Amylase. 10 th November, 2013.

PAGE 36

36 2.5.2 For Galactosidase Galactosidase to Iron Oxide nanoparticles. 2.5.2.1 Scheme 1 20 mg (2 mg core) IO CMDx particles were suspended in 2 mL DI water (pH= 5.4 ). 10 mg of dry EDC and 6 mg of Sulfo N HS was added to the particle suspension This was shaken for 15 min. 2 galactosidase was added to the reaction mixture. pH was adjusted to 7. 5 using 1M NaOH. This reaction is carried for 3 hours under room temperature. These particles were washed using two Sephacryl S 400 HR column collecting first 16 particle drops each time To see if there is any conjugated galactosidase bound to the particle, ONPG assay was used. 200 uL ONPG incubated with 30 uL particle solution for 3 hour. For control, 200 uL PBS incubated with 30 uL particle solution for 3 hour. The reaction was stopped by 30 L of 0.5M Na 2 CO 3 solution Absorbance was measured at 420 nm. Table 2 1 beta gal conjugation scheme 1: Absorbance of sample and control after incubati on Run Absorbance at 420 nm Sample control Run 1 0.477 0.336 Run 2 0.487 0.315 Run 3 0.509 0.332 Run 4 0.497 0.348 Average 0.493 0.333 SD 0.014 0.014 galactosidase on the surface of the particle.

PAGE 37

37 2.5.2.2 Scheme 2 1 mL of PEG550 particle suspension (2.8 mg Fe/mL) in water was taken. The buffer was changed to PBS 1x using a 30 kDa membrane u nder centrifugation. 2.2 mg of dry Sulfo SMCC was added to the particle suspension. Reaction was carried on for 1 hour under room temperature. This reaction mixtures were washed with PBS 1x two times using 30kDa membrane under centrifugation. 22.4 mg (~ 3 galactosidase was added to the vial. Reaction was carried on for 24 hour under 25 C temperature. After reaction these particles were washed using two Sephacryl S 400 HR column collecting first 16 particle drops each time. To see if there is an y galactosidase bound to the particle, ONPG assay was used. 200 uL ONPG incubated with 20 uL particle solution for 3 hour. For cont rol, 200 uL PBS incubated with 2 0 uL particle solution for 3 hou r. The reaction was stopped by 2 0 L of 0.5M Na 2 CO 3 solution. Absorbance was measured at 420 nm. Table 2 2 beta gal conjugation scheme 2: Absorbance of sample and control after incubation Run Absorbance at 420 nm sample control Run 1 0.826 0.397 Run 2 0.878 0.402 Run 3 1.048 0.439 Run 4 0.973 0.424 Average 0.931 0.416 SD 0.099 0.020 galactosidase on the surface of the particle.

PAGE 38

38 2.6 Wash Procedure of Conjugated Enzyme 2.6.1 Amicon Ultra 15 Centrifugal Filter Units At the initial stage Amicon Ultra 15 Centrifugal Filter Un its with 100 kDa molecular weight cutoff were used to separate conjugated particles from free enzyme. 2 mL of enzyme conjugated particles were washed 11 times for 10 min each at 2500 rpm. Each time the washed residue is re suspended in 2mL of PBS 1X. After each wash effluent were monitored using a UV spectrophotometer. This type of wash was discarded as traces of free enzyme were found even after 11 washes [Fig 2 15 ] Figure 2 15 Wash using Amicon Ultra 15 Centrifugal Filter Units

PAGE 39

39 2.6.2 Sephacryl S400 HR Columns To avoid any free enzyme a gel filtration step was introduced using Sephacryl S400 HR as separating medium. This separating medium has a MW cutoff of 9000kDa and nanoparticle cutoff of 31nm (Hydrodynamic diameter). To load these columns 12 mL of r aw media was diluted with 5 mL PBS 1X to form a slurry. Blank PD 10 columns were washed with 20% ethanol and filled with PBS 1X to remove any air bubble. The slurry was then added slowly and continuously while the solid media settles down along the column. The column was then washed thoroughly using PBS 1X. 2.6.2.1 Amylase separation To confirm the separation efficiency, 2mL of PEG550 (0.36 mg Fe/mL) particles were mixed with 9mg of Amylase and concentrated to 0.5 mL using a 30 kDa membrane under centr ifugation This mixture was then run through Sephacryl S400 HR columns. 95 droplets coming out of the column (after the first drop from the particle band) were collected in a 96 well plate. 100 uL of starch solution was added to each well. 96th well is a c ontrol and pure starch solution is added to it. Absorbance of each well was measured using a plate reader at 618 nm. This is absorbance of the particles [Table 2 3 ] This plate was shaken at 25C for 90 min. 20uL of 1M HCl wa s added to each well to quench t he reaction. 2 0 uL of Iodine reagent is added to each well. Absorbance wa s measured using a plate reader at 618 nm This is abso rbance of the assay [Table 2 4 ]. The difference of Table 2 3 and Table 2 4 is normalized by the difference of H12 of both table a nd plotted i n Table 2 5 It shows that the free enzyme starts to elute after 21 drops. A10 and A11, i.e., drop 10 and 11 has experimental error.

PAGE 40

40 Figure 2 16 Tapomoy Bhattacharjee. Starch Iodine assay of column effluent 6 th December, 2013. Table 2 3 Ab sorbance of the particles; A 1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on, H 12 is the control 1 2 3 4 5 6 7 8 9 10 11 12 A 0.058 0.068 0.093 0.161 0.139 0.118 0.083 0.14 0.202 0.336 0.209 0.138 B 0.213 0.218 0.213 0.192 0.164 0.134 0.0 99 0.105 0.163 0.242 0.198 0.154 C 0.141 0.125 0.11 0.103 0.096 0.09 0.086 0.083 0.081 0.077 0.076 0.072 D 0.086 0.073 0.073 0.069 0.069 0.066 0.066 0.062 0.062 0.061 0.062 0.058 E 0.057 0.06 0.058 0.067 0.058 0.056 0.056 0.056 0.057 0.055 0.054 0.051 F 0.054 0.052 0.056 0.053 0.054 0.053 0.051 0.051 0.054 0.051 0.054 0.048 G 0.045 0.049 0.049 0.052 0.054 0.052 0.049 0.049 0.05 0.049 0.048 0.048 H 0.05 0.048 0.048 0.049 0.048 0.048 0.049 0.06 0.047 0.049 0.049 0.048

PAGE 41

41 Table 2 4 Absorbance of th e starch iodine assay; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on, H 12 is the control 1 2 3 4 5 6 7 8 9 10 11 12 A 0.828 0.764 0.783 0.866 0.827 0.799 0.719 0.806 0.852 0.832 0.47 0.849 B 0.908 0.903 0.917 0.882 0.858 0.818 0.773 0.787 0.814 0.757 0.712 0.678 C 0.658 0.638 0.579 0.512 0.479 0.408 0.32 0.271 0.215 0.15 0.126 0.113 D 0.104 0.122 0.092 0.086 0.082 0.08 0.075 0.072 0.072 0.071 0.068 0.069 E 0.064 0.065 0.067 0.064 0.063 0.061 0.06 0.081 0.059 0.058 0.057 0.056 F 0. 058 0.057 0.056 0.056 0.055 0.053 0.054 0.054 0.055 0.053 0.052 0.051 G 0.055 0.052 0.051 0.052 0.052 0.052 0.051 0.058 0.05 0.05 0.05 0.057 H 0.051 0.05 0.05 0.049 0.05 0.05 0.049 0.049 0.049 0.051 0.05 0.812 Table 2 5 Difference of absorbance normal ized by control; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on, H 12 is the control 1 2 3 4 5 6 7 8 9 10 11 12 A 1.01 0.91 0.90 0.92 0.90 0.89 0.83 0.87 0.85 0.65 0.34 0.93 B 0.91 0.90 0.92 0.90 0.91 0.90 0.88 0.89 0.85 0.67 0.67 0.69 C 0.68 0.67 0.61 0.54 0.50 0.42 0.31 0.25 0.18 0.10 0.07 0.05 D 0.02 0.06 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 E 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.03 0.00 0.00 0.00 0.01 F 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 G 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.01 H 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 1.00 To o vercome this, enzyme conjugated Iron Oxide nanoparticles were separated from free enzyme usi ng two freshly prepared columns. O nly 16 drops were collected each time after the first particle drop. 2.6.2.2 Galactosidase separation To confirm the separation efficiency, 20 mg of beta gal was dissolved in 0.5 mL PBS 1X. This mixture was then run through Sephacryl S400 HR column. 192 droplets coming out of the column (after the introduction of protein solution) were collected in two 96 well plate. 200 uL of ONPG solution was added to each well. This plate was shaken at 25C for 120 min. 20uL of 0.5M Na2CO3 was added to each well to que nch the reaction [Fig 2 16 ]

PAGE 42

42 Figure 2 17 Tapomoy Bhattacharjee. ONPG assay of column effluent 25 th February, 2014 Absorbance was measured using a plate reader at 420 nm. This is absorbance of the assay. It shows that the free enzyme start s to elute afte r 92 drops (H8) [Table 2 6 ]

PAGE 43

43 Table 2 6 Absorbance of the ONPG assay from column effluent ; A1 12 refers to drop 1 12, B1 12 refers to drop 13 24 and so on 1 2 3 4 5 6 7 8 9 10 11 12 A 0.049 0.046 0.048 0.048 0.05 0.048 0.05 0.049 0.047 0.049 0.048 0.04 8 B 0.046 0.047 0.046 0.047 0.049 0.049 0.047 0.048 0.048 0.051 0.052 0.048 C 0.055 0.05 0.05 0.05 0.05 0.049 0.047 0.046 0.047 0.049 0.047 0.047 D 0.05 0.045 0.045 0.047 0.044 0.046 0.045 0.046 0.049 0.05 0.054 0.05 E 0.049 0.053 0.049 0.051 0.051 0.0 52 0.047 0.048 0.048 0.047 0.051 0.053 F 0.047 0.053 0.047 0.045 0.046 0.046 0.045 0.052 0.064 0.056 0.059 0.056 G 0.055 0.051 0.053 0.054 0.054 0.069 0.062 0.075 0.076 0.098 0.117 0.102 H 0.061 0.064 0.067 0.069 0.083 0.091 0.082 0.117 0.109 0.124 0.15 9 0.2 I 0.761 1.142 1.584 1.799 1.895 1.931 1.929 1.933 1.88 1.937 1.939 1.901 J 1.585 1.699 1.778 1.808 1.835 1.88 1.879 1.896 1.867 1.933 1.938 1.93 K 1.488 1.666 1.789 1.815 1.868 1.878 1.902 1.91 1.895 1.947 1.943 1.907 L 1.508 1.683 1.747 1.787 1. 843 1.867 1.872 2.175 1.882 1.903 1.924 1.891 M 1.49 1.678 1.803 1.816 1.893 1.893 1.896 1.908 1.901 1.914 1.915 1.871 N 1.546 1.722 1.806 1.841 1.875 1.892 1.896 1.9 1.876 1.887 1.868 1.815 O 1.581 1.707 1.825 1.778 1.815 1.816 1.826 1.795 1.743 1.755 1.717 1.651 P 1.498 1.602 1.63 1.645 1.663 1.644 1.652 1.647 1.592 1.569 1.524 1.484 Again, 20 mg of IO CMDX was dissolved in 0.5 mL PBS 1X. This suspension was then run through Sephacryl S400 HR column. 96 droplets coming out of the column (after the i ntroduction of particle suspension) were collected in a 96 well plate. It shows that the free particle starts to elute after 80 drops (G8) [Fig 2 17 ] If 16 drops collected from the first column, there will be some free enzyme in the effluent. But, a mount of this free enzyme is small and gets removed at the second column.

PAGE 44

44 Figure 2 18 Tapomoy Bhattacharjee. Particle effluent from the column 25 th February, 2014. 2.7 Inactivation Study 2.7.1 For amylase To remotely inactivate the enzyme bound to the surf ace of Superparamagnetic Iron Oxide Nanoparticles, an Alternating Magnetic Field (AMF) of 33.7 kA/m and 255 kHz is applied. In an 8 well strip 200 L of starch solution wa s taken in every well. The well strip wa s covered with a paraffin film and p laced ins ide the coil [Fig 2 18 ]. The coil was placed inside an incubator of constant temperature [Fig 2 19 ]. Starch stock solution was preheated inside the coil to a certain temperature. The we ll strip was then taken out and 10 L of conjugated particle solution w as added in every well. The well strip wa s again covered with a paraffin film and place inside the coil. AMF was applied for 90 min (33.7 kA/m, 255 kHz) The well strip was then taken out of the coil and 20 L of 1M HCl

PAGE 45

45 wa s added to each well to quench the reaction. 20 L of iodine reagent wa s added to each well Absorbance of each well was then measure d at 618 nm. For control the above mentioned procedure was followed without application of AMF. Figure 2 19 Tapomoy Bhattacharjee. Coil for applying AMF 20 th September, 2013.

PAGE 46

46 Figure 2 20 Tapomoy Bhattacharjee. Inactivation study set up 20 th September, 2013. 2.7.2 For galactosidase To remotely inactivate the enzyme bound to the surface of Superparamagnetic Iron Oxide Nanoparticles, an Alternating Mag netic Field (AMF) of 33.7 kA/m and 255 kHz is applied. In an 8 well strip 200 L of ONPG solution wa s taken in 3 well s in such a way that the samples goes at the center of the coil Other wells were filled with DI water. For PEG550 conjugates 10 L and for IO CMDX conjugates 3 0 L of conjugated particle solution was added in every well The well strip wa s sealed with a film and placed insi de the coil [Fig 2 18 ]. The coil was placed inside an incubator of constant temperature [Fig 2 19 ]. AMF was applied for 2hr for PEG550 conjugates and 3hr for IO CMDX conjugates The reaction was quenched after application of AMF using 0.5 M Na 2 CO 3 Absorbance of each well was then measure d at 420 nm.

PAGE 47

47 For control the above mentioned procedure was followed without application of AMF.

PAGE 48

48 CHAPTER 3 RESULTS 3.1 Selectivity of Sulfo SMCC over EDC E DC reacts with carboxylic acids to create an active ester intermediate. This ester intermediate is stabilized by NHS. In the presence of an amine nucleophile, an amide bond is formed with release of an isourea by product. In case of a protein, this amine nucleophile is generated by the terminal amine group and Lysine residue. To have this primary amine groups in a nucleophilic state pH of the reaction medium should be hi gher than the pKa of the individual residue. pKa of individual residues of Amylase (PDB ID: 6TAA) is obtained from The PROPKA Web Interface Amylase has 20 Lysine residue s and only 3 of them has no columbic interactio n with other residues [Fig 3 1 ]. Av erage pKa of these Lysine residue s is approximately 10. Amylase has only one terminal amine group with a pKa of 7.27. Figure 3 1 alpha Amylase (PDB ID: 6TAA) with Lysine residues marked in Yellow

PAGE 49

49 The terminal amine works as a nucleophile at pH of 7.4 (PBS). From The PROPKA Web Interface it is found that this terminal amine is 36% buried and is in columbic interaction with three Aspartic acid residues. So, it is inferred that this terminal amine nucleophile does not interact with the ester intermediate due to steric hindrance and columbic interaction with other residues. Figure 3 2 Terminal amine of alpha Amylase (PDB ID: 6TAA) In case of Lysine residues, pH of the reaction mixture has to be increased beyond 10 in order to produce amine nucleophile. Now, Half life of the intermediate ester is in between 4 5 hours at pH 7 and 0C. This half life decreases to 10 min at pH 8.6 and 4C. 24 So, it is inferred here that increasing the pH of the reaction medium beyond 10, force the intermediate ester to degrade immediately. Sulfo SMCC reacts with amine containing molecules to form stable amide bonds. Its maleimide end then reacts to a sulfhydryl cont aining compound to create a thioether linkage.

PAGE 50

50 3.2 Activity of free enzyme 3.2.1 For Amylase Difference of a bsorbance of Control and Sample after follow ing the procedure described in C hapt er 2.3, is listed in Table 3 1 Normalized activity is plotted in Fig 3 3 Table 3 1 for Starch Iodine assay of free enzyme after 45 min incubation at different temperature 10 L of 24 g / mL free enzyme incubated with starch solution Temperature Run 1 Ru n 2 Run 3 Average SD 20 C 1.155 1.127 1.144 1.142 0.014 30 C 1.169 1.146 1.148 1.154 0.013 45 C 1.044 1.063 1.09 1.066 0.023 60 C 0.86 0.784 0.841 0.828 0.040 80 C 0.188 0.055 0.044 0.096 0.080 Figure 3 3 Free alpha Amylase activity normalized by the maximum

PAGE 51

51 3.2.2 For Galactosidase Difference of a bsorbance of Sample and Control after following the procedure described in chapt er 2.3, is listed in Table 3 2 Normalized activity is plotted in Fig 3 4 Table 3 2 for ONPG assay of free beta Galactosidase after 60 min incubation at different temperatu re 20 L of 5U / mL free enzyme incubated with 250 L of ONPG solution Temperature, C Run 1 Run 2 Run 3 Average SD 20 0.522 0.317 0.424 0. 421 0.103 30 0.589 0.601 0.636 0.609 0.024 45 0.885 0.886 0.882 0.884 0.002 60 0.721 0.750 0.749 0.740 0.016 70 0.169 0.173 0.161 0.168 0.006 80 0.010 0.012 0.017 0.013 0.004 Figure 3 4 Free beta Galactosidase activity normalized by the max imum

PAGE 52

52 3.3 Activity of Enzyme Conjugated to Iron Oxide nanoparticle 3.3.1 For Amylase Difference of absorbance of Control and Sample after following the procedure described in cha pter 2.4 is listed in Table 3 3 Normalize d activity is plotted in Fig 3 5 Table 3 3 for Starch Iodine assay of conjugated enzyme after 45 min incubation at different temperature 10 L of Conjugated particle incubated with starch solution Te mperature Run 1 Run 2 Run 3 Average SD 20 C 0.67 0.642 0.629 0.647 0.021 30 C 0.808 0.788 0.778 0.791 0.015 45 C 0.781 0.794 0.781 0.785 0.008 60 C 0.478 0.418 0.398 0.431 0.042 80 C 0.027 0.03 0.032 0.030 0.003 Figure 3 5 Normalized activity of alpha Amylase conjugated to Iron Oxide Nanoparticle

PAGE 53

53 3.3.2 For Galactosidase Difference of a bsorbance of Sample and Control after following the pr ocedure described in chapter 2.4 is listed in Table 3 4 Normalize d activity is plotted in Fig 3 7 Table 3 4 for ONPG assay of conjugated beta Galactosidase after 60 min incubation at different temperatu re 20 L of PEG550 conjugated beta galactosidase incubated with 200 L of ONPG solution Temperature, C Run 1 Run 2 Run 3 Average SD 20 0.168 0.18 0.156 0.168 0.012 30 0.261 0.233 0.243 0.246 0.014 45 0.397 0.431 0.411 0.413 0.017 5 0 0.406 0.45 0.447 0.434 0.025 60 0.389 0.412 0.439 0.413 0.025 7 0 0.003 0.025 0.023 0.017 0.012 Figure 3 6 Conjugated beta Galactosidase activity normalized by the maximum

PAGE 54

54 3.4 Activity under AMF Activity of enzyme co valently bound to the surface of t he superparama gnetic Iron Oxide nanoparticles was measured using the protocol mentioned in chapter 2.7 3.4.1 For Amylase: 3.4.1.1 Study 1: Incubator temperature: 45c Absorbance after Application of AMF: Table 3 5 Activity under AMF: Study 1: Absorbance after application of AMF Sample ID Absorbance at 618nm Absorbance Average SD 1 0.685 0.7 38 0.042 2 0.771 3 0.747 4 0.71 5 0.715 6 0.744 7 0.711 8 0.819 Absorbance of Control: T able 3 6 Activity under AMF: Study 1: Absorbance of Control Sample ID Absorbance at 618nm Absorbance Average SD 1 0.656 0.728 0.046 2 0.71 6 3 0.712 4 0.706 5 0.705 6 0.799 7 0.776 8 0.754

PAGE 55

55 Temperature profile throughout the process: Figure 3 7 Activity under AMF: Study 1: Temperature throughout the process 3.4.1.2 S tudy 2 : Incubator temperature: 45c Absorbance aft er Application of AMF: Table 3 7 Activity under AMF: Study 2: Absorbance of after application of AMF Sample ID Absorbance at 618nm Absorbance Average SD 1 0.591 0.593 0.043 2 0.582 3 0.563 4 0.558 5 0.576 6 0.553 7 0.646 8 0.672

PAGE 56

56 Absorbance of Control: Table 3 8 Activity under AMF: Study 2: Absorbance of Control Sample ID Absorbance at 618nm Absorbance Average SD 1 0.512 0.554 0.031 2 0.527 3 0.534 4 0.542 5 0.561 6 0.579 7 0.571 8 0.606 Temperature profile throughout the process: Figure 3 8 Activity under AMF: Study 2: Temperature throughout the process

PAGE 57

57 3.4.1.3 Study 3 : Incubator temperature: 50c Absorbance after Application of AMF: Table 3 9 Activity under AMF: Study 3: Absorbance after appli cation of AMF Sample ID Absorbance at 618nm Absorbance Average SD 1 0.942 0.899 0.027 2 0.939 3 0.891 4 0.869 5 0.885 6 0.893 7 0.896 8 0.875 Absorbance of Control: Table 3 10 Activity under AMF: Study 3 : Absorbance of Control S ample ID Absorbance at 618nm Absorbance Average SD 1 0.995 0.874 0.088 2 0.946 3 0.895 4 0.802 5 0.899 6 0.82 7 0.719 8 0.915

PAGE 58

58 Temperature profile throughout the process: Figure 3 9 Activity under AMF: Study 3 : Temperature thro ughout the process Now, from study 1, 2 and 3 it is observed that the absorbance of sample placed under AMF not significantly different from the absorbance of the control [Fig : 3 11 ]. As there is no statistical difference between these two absorbance, it can be concluded that the AMF does not have any significant effect on the activity.

PAGE 59

59 Figure 3 10 Activity of conjugated alpha Amylase under AMF 3.4.2 galactosidase: 3.4.2.1 IO CMDX conjugate Study 1: Absorbance of Sample and control: Table 3 11 IO CMDX Beta gal conjugate Study 1 : Absorbance of sample and control Sample ID Absorbance at 420 nm sample control 1 0.610 0.635 2 0.620 0.657 3 0. 629 0.674 Average 0.620 0.655 SD 0.010 0.020

PAGE 60

60 Temperature throughout the process: Figure 3 11 IO CMDX Beta gal conjugate Study 1: Temperature throughout the process 3.4.2.2 IO CMDX conjugate Study 2: Absorbance of Sample and control: Table 3 12 IO CMDX Beta gal conjugate Study 2 : Absorbance of sample and control Sample ID Absorbance at 420 nm sample control 1 0.531 0.555 2 0.653 0.678 3 0.689 0.688 Average 0.624 0.640 SD 0.083 0.074

PAGE 61

61 Temperature throughout the process: Figure 3 12 IO CMDX Beta gal conjugate Study 2: Temperature throughout the process 3.4.2.3 PEG550 Conjugate Study 1: Maintained temperature during the process: 50C Absorbance of Sample and control: Table 3 13 PEG550 (batch2) Beta gal conjugate Study 1: Abs orbance of sample and control Sample ID Absorbance at 420 nm sample control 1 0.685 0.799 2 0.718 0.717 3 0.766 0.658 Average 0.723 0.725 SD 0.041 0.071

PAGE 62

62 3.4.2.4 PEG550 Conjugate S tudy 2 : Maintained temperature during the process: 50C Absorbance of Sample and control: Table 3 13 PEG550 (batch2) Beta gal conjugate Study 2 : Absorbance of sample and control Sample ID Absorbance at 420 nm sample control 1 0.402 0.456 2 0.403 0.419 3 0.389 0.427 Average 0.398 0.434 SD 0.008 0.019 3.4.2.5 P EG550 Conjugate S tudy 3 : Maintained temperature during the process: 50C Absorbance of Sample and control: Table 3 15 PEG550 (batch2) Beta gal conjugate Study 3 : Absorbance of sample and control Sample ID Absorbance at 420 nm sample control 1 0.708 0.7 15 2 0.715 0.742 3 0.709 0.731 Average 0.711 0.729 SD 0.004 0.014

PAGE 63

63 3.4.2.6 PEG550 Conjugate Study 4: Maintained temperature during the process: 50C Absorbance of Sample and control: Table 3 16 PEG550 (batch2) Beta gal conjugate Study 4 : Absorb ance of sample and control Sample ID Absorbance at 420 nm sample control 1 0.638 0.616 2 0.629 0.601 3 0.618 0.618 Average 0.628 0.612 SD 0.010 0.009 Now, from the above studies it is observed that the absorbance of sample placed under AMF not significantly different from the absorbance of the control [Fig: 3 14 ]. As there is no statistical difference between these two absorbance, it can be concluded that the AMF does not have any signi ficant effect on the activity. Figure 3 13 Activity of conjugated beta Gal under AMF

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64 CHAPTER 4 CONCLUSION Experiments done throughout this study confirms that both the enzymes are bound to the surface of Iron Oxide nanoparticles. Iron Oxide nanoparticl es have also shown heat dissipation in presence of an AMF. Conjugated particles were washed properly and no presence of free enzyme was ensured. Activity of the conjugated particles, in case of both the enzymes, were measure at different temperatures with external heating. It was observed that enzyme bound to the IO nanoparticle can be inactivated by external heating. However, when placed under AMF, both the enzymes conjugated to IO nanoparticles, did not show any significant inactivation. Inference that ca n be drawn that local heating due to IO nanoparticles under AMF was not sufficient to inactivate the enzymes.

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65 APPENDIX AMYLASE CONJUGATION TRIALS USING EDC/NHS CHEMISTRY Scheme 1 100mg of IO CMDx particle s were dis persed in 10 mL PBS 1x (1 mg core/mL) 6 mL of particle suspension was taken in 15 mL centrifuge tube and p H is taken down to 5.17 with 5 L 1M HCl 59 uL of EDC (10 mg/mL in PBS 1x) added to it followed by the addition of 35 uL of NHS (10 mg/mL in PBS 1x). This sample was m ixed in a shaker at 4 C at 600 rpm for 15 min The reaction mixture was w ashed with 30 kDa membrane under centrifugation (3200 rpm; 3 times, 5 min each) to remove excess EDC and NHS. Then 471 uL of 20mg/mL amylase solution in PBS 1x was added to it. This reacti on mixture was shaken for 4 hours at 4 C at 600 rpm. After the reaction was complete, the reaction mixture was w ashed with 10 0 kDa membrane under centrifuge. These washed particles were incubated with starch solution for 2 hours. On addition of iodine reag ent, dark blue color formation was seen which refers to the absence of amylase [Figure A 1] Conclusion: amylase was not conjugated to IO CMDx nanoparticles.

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66 Figure A 1 Tapomoy Bhattacharjee. Scheme 1: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only 1 st October, 2013. Scheme 2 420 mg IO CMDx particles were dispersed in 17.5 mL PBS 1x (pH: 7.4). 30 mg of amylase was dissolved in 5 mL PBS 1x. 360 mg EDC was dissolved in 3 mL PBS 1x Six set of reaction m ixture were prepared following Table A 1. Table A 1 Scheme 2 reaction mixture preparation. Sample amylase solution Particle suspension EDC solution 1 10uL 2.5mL 0.5mL 2 50 uL 2.5mL 0.5mL 3 0.1mL 2.5mL 0.5mL 4 0.2mL 2.5mL 0.5mL 5 0.5mL 2.5mL 0.5mL 6 1mL 2.5mL 0.5mL These reaction mixtures were shaken at room temperature at 600 rpm for 2 hours. All of these reaction mixtures were then washed with a 100 kDa membrane und er centrifuge to remove unreacted amylase and reaction by products. These washed particles were incubated with starch solution for 2 hours. On addition of iodine

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67 reagent, dark blue color formation was seen which refers to the absence of amylase. [Figur e A 2 ] Conclusion: amylase was not conjugated to IO CMDx nanoparticles. Figure A 2 Tapomoy Bhattacharjee. Scheme 2: Starch Iodine assay with Washed particles; '1' '6' refers to Sample and contains washed particles after reaction; 'C' refers to Contro l and contains pure starch solution only 25 th October, 2013. Scheme 3 24mg IO CMDx particles in PBS 1x were washed and suspended in phosphate buffer 10 mg EDC and 10 mg NHS eac h dissolved in 0.5 mL DI water wa s added to particle suspension. This mixture was s haken moderately at room temperature for 15min Excess EDC and NHS was washed times using 100 kDa membrane. Washed particles were suspended in phosphate buffer. Resultant pH was 7.7 Now 0.5 mL of 6 mg/mL amylase solution in PBS 1x was added to it. The reaction wa s conducted for 2 hours at room temperature. After the reaction was completed, the particle suspension was w ashed with 10 confirm any presence of amylase with starch iodine assay.

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68 Conclusion: amylase was not conjugated to IO CMDx nanoparticles. Figure A 3 Tapomoy Bhattacharjee. Scheme 3: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contai ns pure starch solution only 28 th October, 2013. Scheme 4 20mg of IO CMDx particle suspension (2 mg core) in PBS 1x was taken in 5mL vial and p H is taken down to 4.97 with 2 L 1M HCl. 118 L of EDC (0.1 mg/mL in PBS 1x) was added to it (EDC: COOH of part icle =1:1) Then, 35 L of NHS (0.2 mg/mL in PBS 1x) was added to it (NHS: COOH =1:1) This reaction mixture was m ixed in a shaker at room temperature at 300 rpm for 5 min. 523 L of 6mg/mL amylase solution in PBS 1x (Enzyme: COOH =1:1) was added to the reaction vial. The reaction is carried on for 2 hours at room temperature at 300 rpm. After the completion of the reaction, Particle suspension was w ashed with 100 kDa membrane under centrifuge to remove unreacted amylase m any presence of amylase with starch iodine assay.

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69 Conclusion: amylase was not conjugated to IO CMDx nanoparticles. Scheme 5 20 mg (2 mg core) IO CMDx particles were suspended in 2 mL MES buffer (pH=4.75) Particle s uspension was filtered using 0.2 s yringe filter. 1m L of 50 mg/mL EDC solution in MES was added to the particle suspension (10 times Stoichiometric amount) 1mL of 30 mg/mL of NHS solution in MES was added to the particle suspension (10 times Stoichiometric amount) This reaction mixture wa s shaken for 30 min. pH wa s adjusted to 7.1 using 1M NaOH. 1mL of 90 mg/mL alpha amylase solution in PBS (7.4 pH) added to this particle suspension. (10 times Stoichiometric amount) This reaction is carried for 2 hours under room temperature. After reacti on these particles were washed using a 100 kDa membrane W ashed particles were incubated with starch solution for 2 hours. On addition of iodine reagent, dark blue color formation was seen which refers to the absence of amylase. [Figur e A 4] Conclusion: amylase was not conjugated to IO CMDx nanoparticles. Figure A 4 Tapomoy Bhattacharjee. Scheme 5: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure star ch solution only 5 th November, 2013.

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70 Scheme 6 60 mg cIO CMDx particles were suspended in 6 mL MES buffer (pH=4.75) Particle solution wa s filt ered using 0.2u syringe filter. 1ml of 5 mg/mL EDC solution in MES was added to 2mL particle suspension (Stoichio metric amount) 1mL of 3 mg/mL of NHS solution in MES wa s added to the particle suspension (Stoichiometric amount) pH after addition of EDC and NHS is 4.9 This mixture is shaken for 30 min. 1mL of 90 mg/mL alpha amylase solution in PBS (7.4 pH) was then added to this particle suspension. (10X Stoichiometric amount) At this point, pH was adjusted to 9.9 using 0.1 M NaOH. This reaction wa s carried on for 12 hours under room temperature. After reaction these particle solution wa s washed using a 100 kDa memb rane under amylase with starch iodine assay. [Figure A 5] Conclusion: amylase was not conjugated to IO CMDx nanoparticles. Figure A 5 Tapomoy Bhattacharjee. Scheme 6: Starch Iodine ass ay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C1' refers to Negative Control and contains pure starch solution only; 'C2'

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71 refers to positive control and contains 2.5 ug of Amylase with Starch solution 7 th No vember, 2013. Scheme 7 60 mg of cIO CMDx particles were suspended in 6 mL MES buffer ( pH=4.78) Particle solution is filtered using 0.2u syringe filter. 1ml of 5 mg/mL EDC solution in MES was added to 2mL particle suspension (1X Stoichiometric amount) 1mL of 3 mg/mL of NHS solution in MES is added to the particle suspension (1X Stoichiometric amount) pH after addition of EDC and NHS was 4.9. Th e reaction mixture is shaken for 30 min.1mL of 90 mg/mL alpha amylase solution in PBS (7.4 pH) added to this part icle suspension. (10X Stoichiometric amount) pH at this point was 4.97. pH was adjusted to 7.04 using NaOH. This reaction is carried on for 1 3 hours under room temperature. amylase with starch iodine ass ay. [Figure A 6] Conclusion: amylase was not conjugated to IO CMDx nanoparticles. Figure A 6 Tapomoy Bhattacharjee. Scheme 7: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C2' refers to Negative Control and contains pure starch solution only; 'C1' refers to positive control and contains 2.5 ug of Amylase with Starch solution 8 th November, 2013.

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76 BIOGRAPHIC AL SKETCH Tapomoy Bhattacharjee had his schooling from Ramkrishna Vivekananda Mission Vidyabhaban, Barrackpore. In 2008, He received a National Merit Scholarship Award from Govt. of India for the period of 2008 to 2012. In 2012, he graduated with a Bachelo r of Chemical Engineering degree from Jadavpur University, India.