BIOMOLECULES IMMOBILIZATION ON ZIRCONIUM PHOSPHONATE SURFACES STUDIED BY SURFACE PLASMON RESONANCE ENHANCED ELLIPSOMETRY

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1 BIOMOLECULES IMMOBILIZATION ON ZIRCONIUM PHOSPHONATE SURFACES STUDIED BY SURFACE PLASMON RESONANCE ENHANCED ELLIPSOMETRY By ROXANE M. FABRE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

2 2010 Roxane M. Fabre 2

3 To my parents Marty and Michel To my sister Stéphanie 3

4 ACKNOWLEDGMENTS I would like to thank my advisor Prof. Daniel R. Talham and I am greatly thankful for his guidance throughout my graduate experience. He was always supportive of my work and I enjoyed our scientific discussions. I am grateful for the time he spent on reviewing my publications, oral presentations, posters and dissertation. I will always be thankful for his help and I respect him very much as a professor and person. I also enjoyed our rounds of golf. I also want to acknowledge the people I collaborated with, who helped enrich my research experience: Dr. Julien Monot, Dr. Marc Petit and Dr. Mathieu Cinier for the interesting work on protein microarrays and Dr. George Okeyo and Dr. Quentin Bricaud for the enjoyable collaboration on ellipsometry applications. My research experience in the Talham research group was a pleasure as I enjoyed the company of my fellow group members, and especially Dr. Monique Williams, Amandine Guiet and Matt Dumont. I also want to thank Prof. Gail E. Fanucci for her biochemistry advices and members of the Fanucci group, especially Natasha Pirman and Stacey Ann Benjamin. The chemistry department was a friendly place where people were always helpful and a special thanks to Maribel Lisk for her generous personality and Steve Miles for his analytical expertise on the ellipsometer instrument. Dr. Ben Smith, member of my committee, and Lori Clark have been always available and made my experience in Florida a successful experience. I appreciated the diverse and enjoyable friendships with Romain, Shreya, Soumya, Seni, Jen and Yongmo. I enjoyed the Florida weather and the weekends by playing golf and tennis so a special thanks to my Gainesville tennis teams and golf buddies. I can t thank everyone 4

5 but these people were always present on the court and outside: Emilie, Lan, Marissa, Yann and my coach Abdoulaye. My family has been present and supportive during my journey at the University of Florida and I cannot thank them enough. I would like to thank especially my parents, my sister, Cookie, my cat and all my family in the Netherlands. As well as my friends in France, Natacha, Ludivine, Elo, Petite Marie, Grande Marie, Anne-Laure and JP, who I missed but I knew that our friendships would remain through time and distance. 5

6 TABLE OF CONTENTS 6 page ACKNOWLEDGMENTS... 4 LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ABSTRACT CHAPTER 1 INTRODUCTION The Cell Membrane Model Systems to Solve Biological Mechanisms Methods of Investigation of Membrane Properties Biomolecules Immobilization Detection Methods for Biomolecules Immobilization Immobilization of Membrane Proteins on Surfaces Study Overview BIOPHYSICAL TECHNIQUES Surface Plasmon Resonance Enhanced Ellipsometry Ellipsometry Polarization of light Reflection at surfaces: Fresnel equations Nulling ellipsometry Modeling of the multilayer system Surface Plasmon Resonance Total internal reflection Surface plasmons Sensitivity of the technique Surface Plasmon Resonance Enhanced Ellipsometry Experimental set-up The SPREE experiment Kinetic models Langmuir model Two-step reaction model Imaging experiment Confocal Laser Scanning Microscopy Principles Fluorophores in Confocal Laser Scanning Microscopy... 55

7 Fluorescence Recovery after Photobleaching Instrumentation Experimental set-up Conclusions MISCELLANOUS EXPERIMENTS TO DEMONSTRATE THE POWER AND VERSATILITY OF ELLIPSOMETRY Introduction Multilayers System Analysis and 3D-Profile Imaging Experimental Section Materials Zirconium-phosphonate substrates Atomic force microscopy measurement Ellipsometric measurement Results and Discussion Multilayer system characterized by AFM Langmuir-Blodgett films on silicon characterized by ellipsometry Langmuir-Blodgett films on gold characterized by ellipsometry D imaging by ellipsometry Studies of Polyelectrolyte Heterostructures Containing Conjugated Polymers Introduction Experimental Section Substrate preparation and film deposition Ellipsometry measurement Results and Discussion Conclusions REVERSIBLE HIGH CAPTURE HISTAGGED PROTEINS TO BIPHOSPHONATE ADAPTORS STUDIED BY SPREE AND FLUORESCENCE TECHNIQUES Introduction Experimental Section Materials Protein Expression and Purification Microoarray Spotting and Incubation Conditions Microarray Analysis Surface Plasmon Resonance Enhanced Ellipsometry Results and Discussion Design of the Anchoring Linker Zr/phosphonate Surfaces and Linker Anchoring Protein Immobilization Microarray Experiments Comparison with Other Microarray Surfaces Conclusions

8 5 STABLE SUPPORTED LIPID BILAYERS ON ZIRCONIUM PHOSPHONATE SURFACES Introduction Experimental Section Materials Substrate Preparation Zirconium Phosphonate Modified Surfaces Lipid Vesicle Solutions and Formation of Supported Lipid Bilayers Ellipsometric and X-ray Photoelectron Spectroscopy (XPS) Measurements SPR Enhanced Ellipsometry (SPREE) Fluorescence Recovery after Photobleaching Results Symmetric Supported Lipid Bilayers Bilayer Stability Studied by SPREE Asymmetric Supported Lipid Bilayers Inner lipid monolayer Fusion of lipid vesicles on the inner monolayer studied by SPREE FRAP analysis of supported lipid bilayers Protein Binding to Supported Lipid Bilayers Discussion Conclusions SKELETONIZED SURFACES FOR THE STUDY OF TRANSMEMBRANE PROTEINS IN SUPPORTED LIPID BILAYERS Introduction Experimental Section Materials Zirconium Phosphonate-Modified Surfaces Supported Lipid Bilayers BK Ion Channel Expression Reconstitution of Integrin Instrumentation Results Characterization of the Skeletonized Zirconium Phosphonate Surfaces Incorporation of BK Ion Channel in Supported Lipid Bilayers Binding of Fibronectin to Integrin α 5 β Discussion Conclusions PHOSPHOPEPTIDES BINDING KINETIC STUDY ON ZIRCONIUM PHOSPHONATE SURFACE Introduction Experimental Section Materials and Solutions

9 SPREE Experimental Set-Up Results Zirconium Phosphonate Surface as an Enrichment Medium Maximum Binding Capacity on Zirconium Phosphonate Surfaces Kinetic Analysis of Phosphopeptide/Surface Interactions Study of Phosphorylated Peptide with ph Variation Phosphopeptides Study Conclusions and Future Work SUMMARY APPENDIX SUPPORTED LIPID BILAYERS ON ZIRCONIUM PHOSPHATE NANOPLATELETS LIST OF REFERENCES BIOGRAPHICAL SKETCH

10 LIST OF TABLES Table page 3-1 Description of the four distinct optic settings relative to the angles of the polarizer, compensator and analyzer Uncertainty of a single measurement of delta and psi with ellipsometer EP3. Refractive index and thickness of SiO 2 /Si (perfomed by Nanofilm) Results of thicknesses measurements, using the multilayer model on silicon substrates Optical parameters for the multi-layer model Thicknesses and adsorption times of symmetric lipid bilayers on zirconium phosphonate modified gold slides XPS analysis of the zirconium phosphonate and lipid monolayer films FRAP parameters of POPC doped with 2% NBD-PC Equilibrium and kinetic parameters for melittin adsorption to asymmetric bilayers using the two-state reaction model fit to the SPREE data Equilibrium and kinetic parameters for adsorption of BK ion channel to PCPS lipid membrane Optical parameters for the multilayer with the integrin-functionalized membrane Phosphorylated and non-phoshporylated peptides with their sequences Phosphorylated and non-phosphorylated peptides Association and dissociation rate constants of the interactions between phosphorylated peptides and zirconium phosphonate surfaces

11 LIST OF FIGURES Figure page 1-1 Schematic representation of a lipid bilayer Structure of phospholipids (a) and chemical formula of the different headgoups R (b) Models of cell membranes Main detection methods used in surface plasmon resonance: (a) direct detection; (b) sandwich detection method; (c) competitive detection methods Interaction of polarized light with a multilayer system Interaction of light with material Interaction of light with a three-layer system Schematic of the ellipsometry instrument with the different polarization states Total internal condition with the light passes from a denser medium (prism) to a less dense one (air) Surface plasmon resonance condition with the prism coated with the metallic layer Surface plasmon resonance simulation curve for Cr (2 nm)/au (28.5 nm) layer system Schematic of the surface plasmon resonance enhanced ellipsometry technique (SPREE) Typical SPREE sensorgram Typical SPREE sensorgram of phosphopeptide adsorbing to the zirconium phosphonate surface Schematic diagram of the optical pathway in laser scanning confocal microscopy Schematic representation of a typical FRAP experiment Flow chart of ellipsometry data analysis procedure Deposition steps for the formation of the zirconium phosphonate surfaces

12 3-3 Surface morphology of gold surface modified surface Schematic representation of the formation of the inner lipid monolayer by LB deposition on zirconium phosphonate surfaces Optical parameters of the multilayer system (SiO 2 /OTS/Zr-ODPA/DPPA) on silicon Fitting of the measured (dotted lines) and computed (solid lines) values of Ψ and Δ Experimental (dotted lines) and fit (solid lines) Ψ and Δ data for the multilayer (Si/SiO 2 /OTS/Zr-ODPA/DPPA) system Multilayer model for the determination of layer thicknesses on gold Ellipsometric measurement of a zirconium phosphonate modified surface (a) and the lipid monolayer (b) on a gold substrate D-profiles of zirconium phosphonate layer with different areas (100 μm 2 to μm 2 ) Schematic illustration of the gold modified surface with the hybrid lipid bilayer (left) and the lipid bilayer membrane (right) Ellipsometrically determined 2D height map of the mixed lipid layer surface D images of the mixed lipid layer surface Description of the LbL constructed polyelectrolyte films Thickness of heterostructure assemblies as a function of the number of buffer bilayer Chemical structures of bisphosphonate-mono-nta (a) and bisphosphonatebis-nta (b) Affitin immobilization on the zirconium phosphonate surface functionalized with mono- and bis-nta, probed in real time by SPREE Sensorgram for the interaction of affitin with a mono-nta functionalized surface Description of the different steps employed to test affitin activity on a microarray platform Detection of lysozyme captured by affitin spotted at 1 Μm on a Ni-NTAmodified zirconium phosphonate microarray

13 4-6 Fluorescence intensity versus concentration of AlexaFluor 647-labeled lysozyme in the incubation solution Fluorescence intensity versus concentration of spotted affitin on zirconium phosphonate slides Compared performances for lysozyme capture of H4 affitin immobilized on different substrates Schematic showing symmetric and asymmetric lipid bilayers on zirconium octadecylphosphonate (ODPA) modified surfaces Chemical structure of phospholipids POPC, POPG and DPPA Schematic of the SPREE experimental set-up showing the seven layers that correspond to the optical parameters in Table Monitoring fusion of POPC vesicles with different concentrations of DPPA to form symmetric supported lipid bilayers SPREE analysis following the effect of different solvent conditions on the stability of POPC/DPPA supported lipid bilayers Effect of dehydration on a) POPC supported lipid bilayers, b) POPG supported lipid bilayers, c) POPC/DPPA supported lipid bilayers Zoom on the SPREE experiments studying dehydration effect XPS multiplex spectra of the zirconium phosphonate layer before (top) and after (bottom) a DPPA monolayer is deposited XPS survey scan of zirconium phosphonate surface (a) and DPPA monolayer on zirconium phosphonate surface (b) SPREE analysis of the formation of the outer monolayer by vesicle fusion onto a POPC/DPPA monolayer Fluorescent images of vesicle fusion to form asymmetric bilayers with a DPPA inner layer Representative FRAP experiments performed on POPC lipid bilayers (a,b) or monolayer (c) doped with 2% NBD-PC Sensorgrams of the binding of melittin to asymmetric lipid bilayers with zwitterionic (POPC), anionic (POPG) and mixed lipid (POPC/POPG) Fluorescence intensity of the inner lipid layer containing 10:1 POPC:DPPA doped with 2% NBD-PC upon rinsing with SDS

14 6-1 Topology maps of the BK ion channel showing the position of the α- and β- subunits Schemes of the deposition technique of the skeletonized zirconium phosphonate surfaces for the incorporation of transmembrane proteins Images of the skeletonized surfaces on a glass wafer by AFM Images of the POPC lipid monolayer on the skeletonized surfaces on a glass wafer by AFM Incorporation of the BK ion channel into a lipid membrane supported on skeletonized surfaces Incorporation of the BK ion channel into a lipid membrane supported on nonskeletonized surfaces Binding of fibronectin to an α 5 β 1 -functionalized membranes supported on a skeletonized (a) and non-skeletonized surface (b) SPREE signal changes by phosphorylated (P-pp60 SRC) or nonphosphorylated (pp60 SRC) peptide adsorption Schematic showing the interactions at the surface The effect of flow rate on the adsorption of phosphorylated peptide P-pp60 SRC at a concentration of 80 μm, measured by SPREE Scheme illustrating the difference between the covalent linkage to the zirconium phosphate surface Sensorgrams of the binding of phosphorylated (a) and non-phosphorylated peptides (b) to zirconium phosphonate surfaces SPREE sensorgrams of the binding of phosphorylated and nonphosphorylated to zirconium phosphonate at different ph SPREE signal changes by one-phosphorylation site or three-phosphorylation site peptides on zirconium phosphonate surfaces SPREE signal changes by monophosphorylated peptide (1pY) on zirconium octadecylphosphonate surfaces Rate of Ψ signal obtained from adsorption of 1pY peptides studied by SPREE SPREE signal changes by trisphosphorylated peptide (3pY) on zirconium octadecylphosphonate surfaces

15 7-11 SPREE signal changes by monophosphorylated peptide composed of 13 amino acids (13AA) on zirconium octadecylphosphonate surfaces SPREE signal changes by monophosphorylated peptide composed of 6AA (6AA) on zirconium octadecylphosphonate surfaces A-1 Chemical formula of the phospholipids POPC, DPPA, POPG and DPPC A-2 Schematic of the zirconium phosphate nanoplatelets before (ZrP) and after (Zrp-Zr) zirconium treatment A-3 Schematic of the different steps involved in the formation of the nanoparticles coated with supported lipid bilayers A-4 SEM image of zirconium phosphate nanoplatelets A-5 SEM images of Zr-P nanoplatelets coated with POPC supported lipid bilayers A-6 Size distributions performed by DLS of lipids (a), nanoparticles (b) and the hybrid lipid/nanoparticle system (c) A-7 Mean zeta potential measurements of the zirconium phosphate before and after zirconium treatment A-8 Mean zeta potential measurements of different type of lipid bilayers A-9 Confocal images of zirconium phosphate nanoplatelets after zirconium treatment coated with POPC supported lipid bilayers (a) and naked (b) A-10 Fluorescence and transmitted light images of POPC (a), POPC/DPPA (b), DPPC (c) and POPG (d) supported lipid bilayers A-11 Zirconium phosphate nanoplatelets at different concentrations (a) 250 μg ml - 1 and (b) 500 μg ml -1 coated with POPC lipid bilayers A-12 Zirconium phosphate nanoplatelets after zirconium treatment coated with POPC (a) or POPC/DPPA (b) lipid bilayers

16 LIST OF ABBREVIATIONS AFM AOI BK BLM Brain-PS CLSM DLS DPPA dsdna FRAP GFP LB LbL LM LS MSE NTA ODM ODPA OTS POPC POPS ROI SDS Atomic force microscopy Angle of incidence Calcium-activated potassium channel Black lipid membrane Brain- phosphatidylserine Confocal laser scanning microscopy Dynamic light scattering Dipalmitoyl-sn-glycero-3-phosphate double-stranded deoxyribonucleic acid Fluorescence recovery after photobleaching Green fluorescent protein Langmuir-Blodgett Layer by layer Levenberg-Marquardt Langmuir-Schaefer Mean square error Nitrilotriacetic acid Octadecyl mercaptan Octadecylphosphonic acid Octadecyltrichlorosilane Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine Region of incidence Sodium dodecyl sulfate 16

17 SPR SPREE SUV TIR XPS Surface plasmon resonance Surface plasmon resonance enhanced ellipsometry Small unilamellar vesicle Total internal reflection X-ray photoelectron spectroscopy 17

18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOMOLECULES IMMOBILIZATION ON ZIRCONIUM PHOSPHONATE SURFACES STUDIED BY SURFACE PLASMON RESONANCE ENHANCED ELLIPSOMETRY Chair: Daniel R. Talham Major: Chemistry By Roxane M. Fabre August 2010 Membrane proteins are both structurally and functionally diverse. Present in biological membranes, their functions, mainly transport, signaling and surface recognition, depend on cell type and subcellular location. However, many questions on their interactions with cell membrane are still unanswered. For the thorough investigation of protein-lipid interactions, it is desirable to design simplified models of cell membranes. This work aims to find new approaches towards the development of membrane model systems to immobilize proteins. For the different models, zirconium phosphonate surfaces were used as a support for lipid bilayers. Two models, that provide stable, functional and reproducible membranes, have been developed to enable the immobilization of membrane proteins. In the first model, metal phosphonate chemistry was employed to increase the stability of lipid bilayers by means of covalent interactions. Phosphatidic acid lipid was used in a mixture of phospholipids to form covalent bonds with the latter surface. Additionally, the kinetic analyses of proteinbiosensor interaction were performed in order to verify the viability of these models as natural membrane. Membrane proteins were able to insert and maintained their 18

19 structure in the membrane as shown by binding experiments, performed by surface plasmon resonance enhanced ellipsometry (SPREE). However, the proximity of the inorganic support did not provide the space for the insertion of transmembrane proteins. The second approach, based on skeletonized surfaces as support for lipid bilayers, proved to be successful in the insertion of two proteins, integrin and BK channel. The immobilized proteins were shown to be functional and stable in lipid bilayers supported on skeletonized surfaces. Our findings provided then evidences that the model based on skeletonized surfaces behaved as natural membrane. Another model was developed to immobilize histagged proteins on zirconium phosphonate surfaces via synthetic linkers. The binding and activity of histagged proteins were studied and the results showed that proteins adsorbed in a functional conformation on the surface with high density. These linkers have thus the potential to be used in proteins microarrays. Also, zirconium phosphonate surface was used as an enrichment matrix for phosphorylated peptides. SPREE was employed to demonstrate the capability of the latter surface and to understand binding affinity of different peptides. The design, surface morphology, stability and characterization of the model systems were analyzed by complementary biophysical techniques. In this work, SPREE and ellipsometry provided insightful details on the kinetic binding, self-assembly monolayer formation and stability of the biosensors. Other techniques such as atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS) and confocal fluorescence microscopy were used to characterize the morphology of modified surface and the formation of supported lipid bilayers. 19

20 CHAPTER 1 INTRODUCTION Membrane biology is today a wide field that includes functional and structural aspects of the plasma membrane and complex interactions between lipids and proteins. Currently, the knowledge on cell membrane role and structure is being explored by a diversity of sciences including biotechnology, surface chemistry, biology, physics and engineering. Cell membranes, as one of the most important cell components involved in essential functions that define life, are central to understanding molecular biology, integrative cell biology and membrane dynamics. 1 Membrane functions range from cellular development, cell migration, signaling, ions transport, photosynthesis, respiration, fertilization and many more. 2 The plasma membrane is a complex environment for the folding and stability of proteins, progress has been made in solving protein-lipid interactions with the development of model systems. However, the high structural integrity of the lipid bilayer leads to complex mechanisms of protein insertion into the membrane and the understanding of these processes is still a challenge for researchers. The creation of functional model membranes is an important step to answer questions about membrane physicochemical properties. The Cell Membrane Natural cell membranes or lipid bilayer membranes act as selective barrier separating cell organelles and nucleus from the cytoplasm. They are central to life as they define cellular boundaries, organize reaction sequences and act in signal reception and energy transformation. However, their molecular composition was unknown until Research work on lipids started in 1917 by Irving Langmuir, when he discovered that lipid molecules at the air/water interface oriented in such a way that the 20

21 hydrophobic tails pointed to the air and the hydrophilic heads resided in the water. 3 In 1925, Gorter and Grendel used the Langmuir technique to determine that cell membranes are composed of two lipid layers. 4 This model, as shown in Figure 1-1, was accepted almost ten years later by the research community. Membranes are mainly composed of phospholipid bilayers that form a three dimensional lipid bilayer shell around the cell a few nanometers thick. However, in the 1970s, the development of electron microscopy provided techniques to study membranes in more detail. The cell appeared to be covered by a membrane, but also organelles were separated from the cytoplasm by a membrane. This other model was generally accepted until the development of technologies that allowed imaging of the membrane. Proteins were then discovered to be intrinsic components of the membrane that diffuse more or less freely. Stability is provided by hydrophobic interactions between lipid tails and hydrophobic residues of the proteins. Membranes contain two types of proteins: protein adjacent to the membrane, called peripheral proteins or embedded proteins, called integral proteins. Integral proteins play important roles in signaling and transport of molecules. Proteins in the inner cell surface acts as membrane receptors for filaments of the cytoskeleton (actin filaments, microtubules and intermediate filaments) and proteins in the outer cell membrane interact with the extracellular environment and with other cells. Interactions between proteins and cell membranes are an important key of the development of life at the molecular level. Eventually, further studies led to the modern view of the biological membrane that is composed of a diversity of lipids, sterols and membrane proteins that diffuse freely, called the fluid mosaic model. 21

22 Three different types of lipids are found in plasma membranes: phospholipids (most abundant), glycolipids and cholesterol. As seen in Figure 1-2a, phospholipid molecules have two hydrophobic fatty acyl tails and a hydrophilic group connected to a glycerol backbone. Phospholipids are esters of either glycerol or sphingosine, which is a long-chain, dihydric amino alcohol with one double bond. Phosphoglycerides, based on glycerol esters, are the most common phospholipids and have two ester bonds from glycerol to fatty acids plus one ester bond to phosphoric acid. Phosphoglycerides can be saturated or unsaturated, they can also differ in the number of carbons in the acyl chains and in the hydrophilic headgroup. In eukaryotic and prokaryotic membranes, zwitterionic lipids are mainly phosphatidylcholine (PC) and phosphatidylethanolamine (PE) while present negative lipids are mainly phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatydylkserine (PS) as described in Figure 1-2b. Figure 1-1. Schematic representation of a lipid bilayer. Cell membranes are mainly composed of phospholipids which are two fatty acid chains and a head group attached on a glycerol backbone. The hydrophobic tails are facing each other, while the hydrophilic head groups are exposed to the aqueous environment. 22

23 (a) (b) Figure 1-2. Structure of phospholipids (a) and chemical formula of the different headgoups R (b). Model Systems to Solve Biological Mechanisms The combination of membrane models and multiple scientific fields such as physics, chemistry, engineering and biology gave powerful information on the study of biological phenomena. This association created different approaches and techniques allowing the characterization of mechanism in the nanometers-scale. First model membranes were rather simple and became more complex in order to study membrane heterogeneity and lipid protein interactions. Such models should consist of a closed, spherical, single bilayer structure, in which membrane proteins can insert and maintain proper folding, structural functions, activity, and diffusion properties. The most popular membrane system used for biomolecular research is the lipid vesicle as seen in Figure 1-3a. Lipid vesicles provide a closed, stable and regular bilayer membrane. The diameter usually varies from nm for small unilamellar vesicles to nm for large vesicles. 5 Also, giant unilamellar vesicles, which size range from 10 to 100 μm, are popular in single-molecule optical microscopy. Lipid monolayers at the water/air interface, as represented in Figure 1-3b, were also used as model as they are easy to prepare and provide a regular and stable structure with control of the 23

24 composition. 6 However, this model is not a true representation of cell membranes as the outer leaflet is missing. In 1962, the first model of lipid bilayers was created by Mueller with black lipid membranes (BLMs), as seen in Figure 1-3c. 7 A septum with a μm diameter-hole separates two compartments. An organic solution lipid solution is then brushed over the hole and the compartment is filled with buffer. Dispersion of the solvent in the buffer eventually leaves a bilayer on the hole. However, this system is not highly reproducible and can lead to irregular BLMs. An alternative of those two systems is the formation of lipid bilayers on solid support separated by a thin aqueous layer as seen in Figure 1-3d. This model is a better representation of cell membrane as it is a fluid structure that reproduces the lateral mobility of all membranes components, delimits two aqueous compartments on both sides of the bilayer, and is defect-free to avoid channels for electrons, ions or small molecules. Figure 1-3. Models of cell membranes. (a) Lipid vesicle. (b) Lipid monolayer formed at the air/water interface. (c) Black lipid membrane. (d) Supported lipid bilayers. 24

25 Substrate-supported lipid bilayers, developed in 1985 by Tamm et al. on glass supports, offer a unique system to study membrane formation and biotechnological applications. Due to the high complexity of the membrane, systems for membrane models have been simplified to supported lipid bilayers. The purpose of those models is then to reduce the complexity of cell membrane to be able to study one biological phenomenon. The development of such model membranes gives the opportunity to study chemico-physical properties such as membrane dynamics and protein interactions. A large number of different types of supported membranes have been developed in the past two decades, including freely supported bilayers on hydrophilic surface where the hydrophobic phospholipid tails are shielded in the bilayer interior and the hydrophilic headgroups are situated at the surface and at the aqueous interface. This bilayer is separated from the support by a 10 Å water layer thus providing a degree of mobility for the lipids and a functional bilayer. Lipid bilayer membranes are created on the support using different methods as Langmuir-type approaches (Langmuir- Blodgett (LB) or Langmuir-Schafer (LS) depositions) and the spreading of vesicles on monolayer or hydrophilic surface. The Langmuir deposition involves a process of controlled dipping of a support through an organic amphiphatic monolayer. The vesicle approach consists of exposing a hydrophilic support by a lipid vesicle solution. Supported lipid bilayers have been formed mainly by direct fusion of lipid vesicles on glass or mica. 8 These techniques are widely used as it forms a defect free bilayer and is easy to prepare Other approaches involve the formation of monolayers or bilayers linked to chemically modified surface. Different modified surface have been used such as 25

26 alkylsilane monolayers on hydroxylated surfaces (silicon) or alkanethiolates on gold, silver and copper or carboxylic acids on metal oxides This area of synthesis and characterization of coatings of organic surfactants on metallic surfaces has developed in the last two decades. 18 However, supported membranes and hybrid bilayers cannot incorporate integral membrane proteins due to the proximity of the solid substrate. More complex models such as tethered bilayers or polymer-cushioned lipid bilayers have been created for the incorporation of transmembrane proteins. These models use weak nonspecific electrostatic interactions and strong specific interactions to link the membrane to the support. Developed methods are to deposit a lipid bilayer on a polymer/alkylthiol layer on gold or direct tethering of the membrane containing polymer molecules. 19,20 These polymer tethered lipid bilayers models were used to study mainly protein-membrane interactions. In a similar manner, Lang et al. and Bunjes et al. formed a stable bilayer tethered to the gold support via thiolipids or thiolpeptide molecules. Thiol peptides are composed of a small peptide with a thiol group at one end and a lipid at the other. 21,22 These tethered systems provide a fluid membrane and the aqueous layer between the membrane and the support required to study active transmembrane proteins. This works deals with the formation of cell membrane models on inorganic support that increases stability and allows incorporation of proteins. The preparation of these models makes use of the combined application of the LB technique and vesicle fusion on hydrophilic support. Our motivation for creating these models is to study membraneproteins interactions where proteins are in their functional state. 26

27 Methods of Investigation of Membrane Properties Over the years, several techniques have been employed to understand membrane physicochemical properties including thermodynamic approaches (differential scanning calorimetry and surface pressure-area isotherms), spectroscopic methods (fluorescence microscopy, infrared spectroscopy and nuclear magnetic resonance) and analytical measurements (quartz crystal microbalance, mass spectrometry). 2 Highly sensitive label-free analytical techniques such as AFM, SPR, and ellipsometry were used primarily in air but recently, new technologies allow surface studies in liquids. Recently, microscopy techniques such as electron microscopy, atomic force microscopy, fluorescence imaging have been used to obtain visual information of the membrane topology with great spatial resolution. A novel technique which is the combination of SPR and ellipsometry, called surface plasmon resonance enhanced ellipsometry (SPREE) has been used in this work to study membrane-protein interactions. This association of techniques provides information on binding kinetics without any labeling agents and is highly sensitive. Biomolecules Immobilization In this study, we only address biomolecules immobilization as adsorption of biological material on modified surfaces. The main approaches for biomolecules immobilization are based on physical absorption as well as hydrophobic and electrostatic interactions 23, covalent coupling and attachment of tag molecules 24 (biotinavidin, 25 histidine-chelated metal ion interaction, 26 DNA hybridization 27 ). In physical adsorption, ionic bonds and hydrophobic and polar interactions are the principal sources of binding but the main disadvantages are random orientation of proteins or DNA, for example, and weak attachment. Therefore, the other approaches have been widely 27

28 developed for protein immobilization. Covalent bindings are present through functional groups of exposed amino acids, leading to irreversible binding and high surface coverage. 28 Most common ones are amine chemistry used to bind lysine residue or thiol chemistry used to bind cysteine residues. Carboxyl groups can also be used to immobilize proteins with aspartic and glutamic residues and epoxy chemistry provide binding sites for serine and threonine. However, due to the presence of many functional groups in the protein, the protein can be randomly orientated and can lose its functions. 28 Another way is the use of the highly specific binding between two biomolecules such as the biotinylated protein-avidin interaction. Recently, DNAdirected immobilization has been developed, 27 this method is used in chip technology where the single-stranded DNA sequences are used for DNA immobilization with a complementary sequence. Another approach involves histidine-tagged protein and chelated metal ion. The binding is reversible and immobilized protein can be released through competition with another ligand (imidazole) or chelation agent (ethylenediaminetetraacetic acid, EDTA). Detection Methods for Biomolecules Immobilization SPREE technique can be applied to study immobilization of molecules of interest at the solid-liquid interface. An important feature of the surface modification is to prevent structural changes from biomolecules such as proteins, which can result in a loss of biological activity. 29,30 In biosensors, two biomolecules are interacting, one immobilized on the surface and the other in a buffer environment. The choice of molecules depends on the detection method (direct, sandwich and competitive as seen in Figure 1-4). Various molecules have been used to detect specific affinity in, for example, surface plasmon resonance (SPR). Antibodies remain the most frequently 28

29 used because of their high affinity and specificity against the target biomolecule. However, antibodies are usually expensive and laborious process is needed. Recently, other biomolecules have emerged such as single-chain antibody fragments, aptamers (single-stranded oligonucleotide sequences) and peptides. Peptides and aptamers, compare to antibodies, are inexpensive, more stable and easier to manipulate. In this work, the studied interactions are mainly direct such as the direct adsorption of proteins on lipid membrane. Figure 1-4. Main detection methods used in surface plasmon resonance: (a) direct detection; (b) sandwich detection method; (c) competitive detection methods. Immobilization of Membrane Proteins on Surfaces Protein immobilization is of great importance for proteomics studies as well as for drug discovery and environmental applications However, proteins are much more complex than DNA and different issues have been encountered with the development of 29

30 protein microarrays. Proteins are not stable in harsh conditions and are easily denatured. Therefore, it is important to preserve the functionality and the threedimensional structure of proteins. The immobilization of proteins on a support is desired in many biotechnological as well as scientific applications for detection or imaging biomolecules. An extensive number of protein microarrays has been designed for medical applications including antibody microarrays for prostate cancer detection, 34 antigen arrays to characterize sampled serum of human autoimmune diseases. 35 Development of protein arrays has focused on using different coating materials. Three different classes of bioactive material can be found: (1) ordered molecular organic layer (aldehyde, epoxy, or carboxylic esters), (2) gel or membrane-coated surface (agarose, nitrocellulose) and (3) supramolecular surfaces (dendrimers, avidin, PEG) are popular materials to coat glass support. 36,37 These modified surfaces enhanced biocompatibility, protect proteins from denaturing and increase binding ability. Other surfaces, including gold coated substrate, have been used with the increase of imaging techniques. For example, self assembled monolayers of thiol molecules is a common surface and provide good surface coverage. However, those surfaces are subjected to non specific interactions, yielding to background noise. A novel approach is then to study proteins in their natural environment. Membrane proteins are present in high abundance in cell membranes. It is well known that interactions between membrane proteins and lipid membrane play an essential role in the functioning of the cell membrane. Therefore, lipid messengers are key molecules in protein functions and cellular signals. 38,39 With their dual properties, proteins are membrane active species that can insert spontaneously in the lipid membrane. For 30

31 transmembrane proteins, the hydrophobic regions of proteins can interact with the acyl tails of the lipids as the hydrophilic residues can remain on the membrane periphery, interacting with the aqueous environment. For peripheral proteins, adsorption to the membrane occurs trough electrostatic, covalent and other interactions. The study of how proteins interact with membranes or become inserted into membranes is fundamental to understand signal transduction, protein sorting and endocytosis. Also, many diseases are caused by protein toxins able to reach the cytoplasm by passing through the membrane or by viruses that enter the cell by membrane fusion. By using cell membrane models to study protein insertion, some important aspects have to be taken into consideration such as keeping full retention of protein conformation, homogenous surface coverage, stability of the protein layer and minimizing non-specific adsorption. Membrane proteins usually keep their functional and active conformation in the presence of lipids, in fact, the physicochemical properties of the lipid environment and specific binding to lipid cofactors play a role in this conformation effect. Therefore, in this work, membrane models are being used to study proteins immobilization. Study Overview This research work aims to present several functional biosensors for the immobilization of biomolecules. While different biomolecules including proteins, lipids and peptides were studied, similar surface chemistry and analytical techniques were employed in this work. Zirconium phosphonate surfaces were used as binding matrix for biomolecules while surface plasmon resonance enhanced ellipsometry (SPREE) and fluorescence microscopy were used as characterization techniques. The first part, comprises of chapters 2, 3 and 4, is an introduction to analytical techniques and applications. The second chapter is a theoretical discussion of SPR, 31

32 ellipsometry, the combination of both techniques defined as SPREE and fluorescence recovery after photobleaching It introduces fundamental concepts and instrumentation requirements for the biophysical techniques. The next two chapters, 3 and 4, are devoted to the applications of ellipsometry and SPREE to biomaterials and polymers. While no chemical information can be obtained, these highly sensitive methods are powerful in characterizing biomolecule surface interactions. The second part, which consists of chapters 5 and 6, is a detailed discussion on the design of biosensors to study membrane and transmembrane protein adsorption in lipid bilayers. Chapter 5 focuses on the creation of a highly stable membrane model system that was characterized by AFM, ellipsometry, x-ray photoelectron spectroscopy (XPS). In order to verify the viability of the model, the binding of membrane proteins was studied by SPREE. In chapter 6, the latter model was improved for the immobilization of functional transmembrane proteins. For successful incorporation of proteins, the compromise has to be found between a natural environment that maintains their active form, and an environment that allows stability for characterization. In an approach to this problem, we have designed skeletonized surfaces to support lipid bilayers. The interactions of two different proteins, BK ion channel and integrin, with the membrane were then analyzed by SPREE. Finally, chapter 7 describes the use of zirconium phosphonate surfaces as a phosphopeptides enrichment matrix. Phosphorylation mechanisms are of growing interest and this inorganic matrix is being studied as well as binding affinity of different phosphopeptides to the surface. Kinetic analyses were performed on the peptidesurface interactions to better understand affinity binding relative to peptide 32

33 characteristics. This work is a combination of biophysical techniques and surface chemistry in the quest for the creation of biosensors to understand biomolecules immobilization. 33

34 CHAPTER 2 BIOPHYSICAL TECHNIQUES Surface Plasmon Resonance Enhanced Ellipsometry The increasing number of methods to synthesize or extract biomolecules requires more sensors to qualitatively or quantitatively detect interactions among those molecules such as antibodies, DNA, RNA, proteins, peptides and membrane lipids. Fluorescence techniques are mostly utilized but attaching fluorophores is often time and cost consuming. Another possible problem is photobleaching of the fluorescent labels. Layers of biomolecules are typically in the angstroms to nanometer range, well below optical wavelengths, thus conventional microscopy, which has a resolution limit in the micrometer range, is not applicable. However, some techniques like interferometry, ellipsometry and surface plasmon resonance are capable of analyzing very thin films. In the presented work, surface plasmon resonance enhanced ellipsometry (SPREE) has been used to study the biophysics and biochemistry of the binding of biomolecules such as lipid bilayers, proteins and peptides with a particular focus on protein-membrane interactions. In fact, biological interfaces are now important because fundamental phenomena occur on lipid membrane interfaces such as lipid-protein and protein-protein interactions. When used with appropriate metal coatings, SPREE is a powerful tool to study interactions in real time, such as binding events and structural changes, with a high level of sensitivity Ellipsometry is based on the Fresnel equations to determine layer thicknesses while excitation of surface plasmons in a metallic layer yields to sensitivity to refractive index changes of the material. The combination of both highly enhances the sensitivity of this technique. This method also 34

35 allows the study of multiple interface interactions such as chemical bond formation, charge transfer interactions, Van der Waals forces and acid-base chemistry. In this chapter the theory behind both techniques is described along with the instrumentation required to perform SPREE experiments, and some basics information on kinetic models. Ellipsometry Ellipsometry, widely used for surface and thin-film analysis of different materials such as metals, dielectric, semiconductors and polymers, is a very sensitive, nondestructive experimental technique. This technique is based on the change in the polarization of light on a reflecting system to determine optical constants as well as thicknesses of thin films and bulk materials. In a single measurement, film thickness with resolution on the order of angstroms and refractive index to three decimal places can be obtained. In this section, I introduced the theory of ellipsometry with fundamental equations and some background on the theory of light. Polarization of light Light is composed of an oscillating electromagnetic wave, however, ellipsometry uses only the electric components of the light as it interacts more with matter than the magnetic field does. The behavior with time of the vector field at a fixed point in space is called polarization. If the phase of the two components are the same then the polarization is linear; if the phase difference is ± 90 then the polarization is circular. In all other cases, the polarization is elliptical. In fact, the term ellipsometry comes from the word ellipse, which is the most general state of polarization of light. In principle, polarized light is reflected off a sample at an angle of incidence, φ, as shown on Figure 2-1. The change in polarization is related to the physical properties of the sample. 35

36 Figure 2-1. Interaction of polarized light with a multilayer system matrix R: The incident (E in ) and reflected (E out ) electric vector are connected through the E p,out = R pp E s,out R ps R sp E p,in R ss E s,in (2-1) Reflection at surfaces: Fresnel equations Light interacts with the surface as shown on Figure 2-2, an incident beam enters medium 1 and then at contact with the substrate, the incident beam can either reflect or refract. Figure 2-2. Interaction of light with material From this simple interaction, Fresnel reflection coefficients, for the perpendicular (s) and parallel (p) components of the electric field, can be determined as followed: r p,12 = N 2 cos φ 1 N 1 cos φ 2 N 2 cos φ 1 +N 1 cos φ 2 (2-2) 36

37 r s,12 = N 1 cos φ 1 N 2 cos φ 2 N 1 cos φ 1 +N 2 cos φ 2 (2-3) with N = n jk (2-4) where n is the index of refraction (ratio of the phase velocity in a material to the speed of light in vacuum) and k, the extinction coefficient. The reflectance ratios can directly be obtained from the Fresnel reflection coefficients:: R s = r s 2 (2-5) R p = r p 2 (2-6) The interaction of light with a three-layer system is drawn on Figure 2-3. Film thickness measurements utilize the interference between multiple light beams. The light traveling through the film rejoins the light reflected from the film surface. The various components of light will have different phases, depending on the additional optical distances they travel. The interference between multiple light beams will depend on the wavelength (due to different phase velocities) and angle of incidence (due to different path lengths). Film thickness is determined by analysis of the resulting interference oscillations. Figure 2-3. Interaction of light with a three-layer system. 37

38 Using the three-phase formalism, the total reflection coefficients and the film phase thickness, β, can be determined as followed: R p = R s = r p,12 + r p,23. e i2β (2-7) 1 + r p,12. r p,23. e i2β r s,12 + r s,23. e i2β 1 + r s,12. r s,23. e i2β (2-8) with β = 2π d N λ 2 cos φ (2-9) 2 where r 12 and r 23 are the Fresnel reflection coefficients for the interface between media 1 and 2, and 2 and 3, respectively. For a sample with multiple layers, the N- phase model is employed and the total reflection coefficients are calculated by the Airy formulas, the scattering matrix formalism and the 4*4 matrix formalism. Nulling ellipsometry The instrument used in this research is the Imaging EP 3 system (Nanofilm, Germany), which is a 532 nm-single wavelength ellipsometer capable of multilayer analysis by the variation of the angle of incidence. It is a powerful instrument with a lateral resolution of ± 1 µm and of ± 1 Å in the z-direction. The imaging ellipsometer operates on the principle of classical null imaging ellipsometry and real-time ellipsometric contrast imaging. The laser beam is elliptically polarized after it passes through a linear polarizer (P) and a quarter-wave plate (C) (Figure 2-4). The elliptically polarized light is then reflected off the sample (S) onto an analyzer (A) and imaged onto a CCD camera through a long working distance objective. In this PCSA configuration, the orientation of the angles of P and C is chosen in such a way that the elliptically polarized light is completely linear polarized after it is reflected off the sample. The ellipsometric null condition is obtained when the absolute minimum of light flux is 38

39 detected at the CCD camera. The angles of P, C, and A that obtained the null condition are related to the ellipsometric parameters delta and psi. Figure 2-4. Schematic of the ellipsometry instrument with the different polarization states. Modeling of the multilayer system In ellipsometry, incident monochromatic light is reflected or transmitted at a surface. For anisotropic materials, where the matrix R is diagonal (Rsp=Rps=0), the two complex reflection coefficients Rp (=Rpp) and Rs (=Rss) describes the two parameters and Ψ, measured by the ellipsometer: ρ = R p R s = R p R s e i(δ p δ s ) (2-10) Δ = δ p δ s (2-11) tan ψ = R p R s (2-12) The tangent of the angle psi gives the ratio of amplitude change for the polarization components, while delta denotes the relative phase shift of these 39

40 polarization components upon reflection. Reduction of those measured data with computerized optical modeling leads to a deduction of film thickness and the complex refractive indices. The optical model simulates those two parameters as a function of the optical properties of the sample. The model can also calculate optical properties such as index of refraction and thicknesses to simulate and Ψ in order to compare with the measured and Ψ. The fit results minimize the mean square error (MSE). 1 MSE = n m 1 y i y i n 1 σ i 2 (2-13) where y i is the measured data, <y i > the simulated data and m the number of fit parameters. Starting at a given value of thickness d, for example, the program calculates and Ψ and the MSE then increases or decreases the variable parameter d in the direction of the gradient of MSE with respect to the variable. The MSE decreases until a local minimum is found. There are many algorithms used to minimize the MSE of simulated and measured data and Ψ. The program uses Levenberg-Marquardt s algorithm, which is a combination of the inverse Hessian matrix and the gradient method. This method finds the minimum MSE with the smallest number of iterations of all methods, except when a strong correlation of fit-parameters exists. 43 A more detailed description of this algorithm can be found in chapter 3. Surface Plasmon Resonance Surface plasmon resonance (SPR) is an optical technique widely used in biological applications including biosensors, immunodiagnostics, kinetic analysis of antibody-antigen interaction as well as the study of binding of biomolecules to membranes. The advantage of this technique is its high sensitivity without the need for 40

41 fluorescent or other labeling agents. The SPR phenomenon was discovered in the early 20 th century and occurs on the condition of total internal reflection by thin layers of noble metals like Au, Ag and Cu. In principle, SPR sensors are thin-film refractometers that measure refractive index changes occurring at the surface of a metal film supporting a surface plasmon. The technique can monitor multistep adsorption and the measurements are made in real time providing dynamic kinetic information in a very sensitive and label-free biochemical experiment. Association and dissociation rates of binding processes are rapid and direct and only nm concentrations of protein are needed. Total internal reflection The first process to understand is total internal reflection (TIR). This condition occurs when a light beam propagating through a medium with a high refractive index reflects above a critical angle on a medium with lower refractive index (Figure 2-5). In SPR, typically, the incident light is passed through a dielectric medium like glass in a form of a prism, whose refractive index is greater than unity. As the angle of incidence increases, the transmitted beam gradually approaches tangency with the interface. At one minimum angle, the critical angle, the transmitted beam is parallel to the interface thus no energy can be transmitted across the interface. All the energy from the incident beam appears then in the reflected beam causing total internal reflection. However, the light beam leaks an electrical field intensity called an evanescent field wave into the low refractive index medium. If the total internal reflection interface is coated with a metallic layer of a specific thickness, the p-polarized component of the evanescent wave may penetrate the metal layer and excite electromagnetic surface plasmon waves propagating within the surface of the metallic layer (Figure 2-6). Only 41

42 the p-polarized light component can interact with the plasmons as the s-polarized component is not in the same plane of oscillation as the plasmons. For a non-magnetic layer, this surface plasmon wave will also be p-polarized and will create an enhanced evanescent wave. Figure 2-5. Total internal condition with the light passes from a denser medium (prism) to a less dense one (air). Figure 2-6. Surface plasmon resonance condition with the prism coated with the metallic layer. The angles θ r and θ 1 represent the angle of resonance and an angle inferior to the angle of resonance, respectively. Surface plasmons Surface plasmons describe the quantized nature of the energy of the surface electrons in the metal; the frequency of the surface plasmon depends on the bulk plasma frequency and on the dielectric constant of the medium in contact with the 42

43 metal. SPR occurs when a thin conducting film is placed at the interface between the two optical media (Figure 2-6). This particular configuration, called the Kretschmann configuration, is used to create an evanescent field on the gold surface where the polarized light is directed through a prism with high refractive index (n = 1.72) to the thin layer of gold in contact with the buffer solution with the low refractive index (n = 1.33). The effect of the high refractive index medium is to modify the wave vector of the light by decreasing the phase velocity of the photons. At a specific incident angle, greater than the TIR angle, the x-component of the k-vector of the evanescent field matches the wavevector of the plasmons oscillations at the metal/dielectric interface. If the reflectance R p of p-polarized light at a fixed wavelength is measured as a function of the incidence angle then a sharp minimum will be observed where this frequency matching condition is satisfied and SPR occurs (Figure 2-7). The minimum reflected light corresponds to the photon energy being coupled to the electrons in the film, a phenomenon called surface plasmon resonance. The observed minimum depends on different parameters of the reflecting system: refractive index (n), extinction coefficient (k) and thickness (d) of the different layers. Figure 2-7. Surface plasmon resonance simulation curve for Cr (2 nm)/au (28.5 nm) layer system 43

44 Sensitivity of the technique The surface selectivity of SPR comes from the excitation of surface plasmon polaritons at the metal-dielectric interface. Plasmons, although composed of many electrons, behave as if they were a single charged particle. Part of their energy is expressed as oscillation in the plane of the metal surface. Their movement, like the movement of any electrically charged particles, generates an electrical field. They propagate along the surface with amplitude decaying exponentially in the direction perpendicular to the interface, and can interact with molecules in the 100 nm range. Therefore, in our experiment, adsorption of biomolecules that changes the refractive index in the interfacial region can be monitored by SPREE. The SPR measurement highly depends on the optical properties of the metallic film layer. The quality of the SPR data depends on the film thickness, roughness but also on the adhesion layer. By varying the type of metal, the spectral resolution and sensitivity are different. In biological applications, often reproduced in a buffer environment, the film needs to durable and inert. The metal that fulfills those conditions the best was proven to be gold. In our experiment, a layer of 2 nm chromium and a 28.5 nm gold layer were sputtered on SF10 glass slides. Surface Plasmon Resonance Enhanced Ellipsometry Recently, the possibility of using the change in the phase of the reflected light through the SPR minimum to determine the refractive index of the medium has been investigated. The first work which suggested the use of ellipsometry for surface plasmon analysis appeared in 1976 by Abeles. 44 Under SPR conditions, ellipsometric parameters give a large enhancement of detection sensitivity compared to SPR techniques. SPREE, as seen on Figure 2-8a, is a technique that improves the precision 44

45 and sensitivity of the measurement When the laser light of the ellipsometer shines through the prism and the glass slide onto the gold film at the angle near the so-called surface plasmon resonance condition, the optical reflectivity of the gold changes very sensitively with the presence of biomolecules on the gold surface. This high sensitivity of the optical response comes from the efficiency of the collective excitation of conduction electrons near the gold surface. This configuration can come to a resolution of refractive index units for the SPREE technique compared to 10-5 for the ellipsometric measurements and in the order of 2*10-6 to 1*10-5 refractive index units for SPR only. 40 This method is also less sensitive to external light and intensity fluctuations of the incident light source than that of the SPR technique. The adsorption and desorption of biomolecules on the gold surface can be observed and quantified by monitoring the Ψ change as the increase in the resonance angle is proportional to the refractive index and thickness (surface coverage) of the adsorbed layer. Ψ as a function of the angle of incidence (AOI) spectra resemble typical SPR curves, which is quite obvious, since both dependencies represent the Fresnel R p amplitude. The minimum detectable signal change of the instrument used in this work was about 10 millidegrees in Ψ which results in a thickness precision of 0.1 nm. Experimental set-up The scheme of the experimental set-up is shown in Figure 2-8a. The gold-coated glass slide, with a chromium adhesive layer, is assembled with a 70μL sample cell, and a 60 SF10 prism (n=1.72) is mounted with the glass slide using diodomethane as an index matching fluid. The cell, as seen on Figure 2-8b, has inlet and outlet tubes 45

46 allowing injection of different solutions into the cell via a peristaltic pump with the possibility of varying the flow rate. A Peltier temperature control is linked to the cell and the temperature is set at 24 C for all experiments. The cell was sealed against the sample through a rubber O-ring. A laser beam (λ = 532 nm) is elliptically polarized after it passes through a linear polarizer and a quarter-wave plate. The elliptically polarized light is then reflected off the sample onto an analyzer and imaged onto a CCD camera trough a 10x working distance objective. The orientation of the angles of the polarizer and the analyzer is chosen in such a way that the elliptically polarized light is completely linear polarized after it reflects off the sample. The ellipsometric null condition is obtained when the minimum of light intensity is detected at the CCD camera. The angles of the different components are related to the optical properties of the sample. The software AnalysisR from the company Nanofilm in Germany is used to fit the experimental data to a particular model and we can then find the parameters of the system (thickness, refractive index and extinction coefficient of the layers). Real time measurements consist of recording the Ψ value at a fixed angle, a few degrees before the minimum angle to get the highest resolution, as a function of time. Figure 2-9a shows this specific angle (in orange) and Figure 2-9b represents the recorded kinetic data of psi at this specific angle. The experimental set-up was optimized to maximize sensitivity and to increase the signal to noise ratio. SPREE detection limits depend on the noise and baseline drift. Very long measurements degrade the detection limit since baseline drifts becomes increasingly problematic with increasing time. Potential sources of baseline drift are temperature fluctuations, which influence the refractive index which is related to the 46

47 signal. The cell was linked to a Peltier temperature control to minimize this effect. Changes in the flow rate and air bubbles are also sources of drift and can be minimized by constant flow rate but are a problem at elevated temperatures. In our experiment, measurements were performed using a peristaltic pump (Rainin, California) with digital control of the flow. For each experiment, sufficient time was allowed to obtain a stable signal thus getting the best signal to noise ratio. (a) (b) Figure 2-8. Schematic of the surface plasmon resonance enhanced ellipsometry technique (SPREE) (a). Image of the SPR cell composed of the SF10 prism in the Kretschmann configuration (b). The sample is situated under the prism and has a volume of 100μL. (a) (b) Figure 2-9. Typical SPREE sensorgram. The dip in the psi signal, seen in (a), is associated with the resonance phenomenon. The red curve shows from the shift in the angle of resonance after adsorption of molecules on the surface. By selecting an angle (in orange) before the angle of resonance, maximum signal change can be obtained. At this specific angle, psi can be recorded versus time (b) to obtain kinetic information. 47

48 The SPREE experiment The experimental procedure is uncomplicated. Figure 2-10 shows a typical adsorption sensorgram for a solution of phosphopeptide flowing over zirconium phosphonate surface. The psi signal is plotted as a function of time. The experiment starts with the buffer in contact with the surface (A). A biomolecule solution is then passed across the surface. At point B, phosphopeptides reach the surface and begin to adsorb or bind to the surface, yielding an increase in psi. At C, the adsorption reaches an equilibrium and at D, a buffer wash is performed to monitor any desorption. Kinetics information can be obtained by using an appropriate model. The psi signal in SPREE is not a simple report of association and dissociation at the surface. It is a combination of all chemical processes and transport processes of diffusion and flow. In our analysis, assumptions are made about the analyte, namely that the free analyte concentration remains uniform in space and constant in time. However, studies have been done on the limits of this assumption Several methods are being used to calculate the association and dissociation rate constants: linearization, analytical integration, 48 and numerical integration. 49 In this work, linearization was used to estimate the rate constants of peptides binding to zirconium phosphonate surfaces in a simple Langmuir interaction. As it is a simple one to one interaction, the association rate constant is calculated by linearization the association phase data at different peptide concentrations. During the dissociation, the formed complex is assumed to decay exponentially. However, more complex interactions, such as the one between membrane proteins and lipid bilayers, cannot be fitted by the simple bimolecular model. The alternative method is the non-linear analysis, where the data are fit to the sum of two-integrated rate equations resulting in two sets of association and dissociation 48

49 rates Numerical integration has been used to fit very complex binding mechanism such as interactions between proteins and DNA, 49,53 but this approach was not used in this research work. Figure Typical SPREE sensorgram of phosphopeptide adsorbing to the zirconium phosphonate surface. Kinetic models SPREE has the ability to monitor adsorption of biomolecular reactions at a surface in real time without labeling agents. Adsorption depends on mass transport, the transport of analyte to the surface, and intrinsic adsorption kinetic rate, the binding of analytes with immobilized receptors at the surface. Since the slowest process will determine the rate measured by the instrument, the diffusion of analyte to the sensor surface is a major concern. To minimize the diffusion effect, a fast flow rate was used in the SPREE experiments. In this section, two models are described, one is the Langmuir model, describing one-to-one interaction and the second one described a twostep model used to analyze protein/membrane interactions. Langmuir model The analysis of the simple binding of an analyte A to a receptor B can be based on a simple model, called the law of mass action, represented by the following equation: 49

50 k on A + B AB (2-14) k off where A is the analyte and B the immobilized partner, k on is the association rate in s -1 M -1 and k off the dissociation rate in s -1. The binding is reversible and occurs when the analyte and receptor collide due to diffusion and flow, which bring the analyte to, and take it away from, the sensor surface. Once the binding has occurred, the formed complex AB remains for a random amount of time and then dissociation occurs. Equilibrium is reached when the rate of the complex formation equals the rate of the complex dissociation. The equilibrium dissociation constant, K D, is equal to the ratio of k off to k on. When the concentration of analyte equals the K D, half the receptors will be occupied at equilibrium. If the receptors have a high affinity for the analyte, the K D will be low, as it will take a low concentration of analyte to bind half the receptors. In this model, the reaction between the analyte and the immobilized partner follows first order kinetics, and during the association phase, the concentration of the ligand AB increases as a function of time according to: d cab (t) = k dt on. c A (t). c B (t) k off. c AB (t) (2.15) At time t, the concentrations of the immobilized partner and the analyte are: c B (t) = c B (0) c AB (t) and c A (t) = c A (0) (2-16) So, by substituting in equation (2-15): d cab (t) = (k dt on. c A (0) + k off ). c AB (t) k on. c A (0). c B (0) (2-17) At initial time, the concentration of the ligand is zero and the immobilized molecule has its highest concentration: 50

51 c AB (0) = 0 c AB (t) = k on. c A (0). c B (0) k on c A (0)+k off 1 e k onc A (0)+k off t (2-18) c B (0) = c AB (max) c AB (t) = k on. c A (0). c AB (max) k on c A (0)+k off 1 e k onc A (0)+k off t (2-19) The ellipsometric signal can be defined as: Δf(t) = f(t) f(0) = c AB (t) (2-20) f(t) = k on. c A (0). f(max) k on c A (0)+k off 1 e k onc A (0)+k off t + f(0) (2-21) Baseline equation: Association equation: Δf b (t) = t 0 + A A = f(0) (2-22) Δf ass (t) = f 1 1 e t τ + A (2-23) f 1 = k on. c A (0). f(max) k on c A (0)+k off and τ = 1 k on c A (0) + k off (2-24) During the dissociation phase, the concentration of the complex AB decreases as a function of time according to: d cab (t) = k dt off. c AB (t) (2-25) By integrating the equation (2-25): c AB (t) = c AB (0). e koff.t (2-26) The ellipsometric signal can then be defined as: Δf(t) = Δf(0). (e koff.t ) (2-27) Dissociation equation: Δf diss (t) = f 2 e koff.t + B f 2 = c AB (0) (2-28) 51

52 Two-step reaction model The two-step reaction model was used to analyze the binding of membrane or transmembrane proteins to the lipid membranes. The sensorgrams were analyzed by curve fitting using numerical integration analysis. 54 This model is used when the measured signal is the result of the sum of a fast and slow exponential decay and allows determination of the half time for both the fast and slow processes. This model applies well to the binding and insertion of proteins to the membrane. 55 The two-state reaction model can be represented as: P + L k a1 k a2 PL PL k d1 k d2 (2-29) where in the first step, the protein (P) binds to the lipids (L) to give the complex PL, and the complex PL is then changed to PL* in the second step, which cannot dissociate directly to P+L. The corresponding differential rate equations for the two-state reaction model are represented by: dψ 1 dt = k a1 [P] (Ψ max Ψ 1 Ψ 2 ) k d1 Ψ 1 k a2 Ψ 1 + k d2 Ψ 2 dψ 2 dt = k a2 Ψ 1 k d2 Ψ 2 (2-30) (2-31) where [P] is the protein concentration, Ψ 1 and Ψ 2 are the response units for the first and second steps, respectively and Ψ max is the equilibrium binding response. The association and dissociation rates for the first and second steps are k a1, k d1, k a2 and k d2. The association equilibrium or affinity constants for the first and second step are K 1 and K 2 and equal k a /k d. The total affinity constant K A (M -1 ) is the product K 1 K 2. 52

53 Imaging experiment The ellipsometer is composed of an objective and a CCD camera, two components that enable imaging of the sample. Modifying the optical properties of the sample changes the signal in the camera. Mapping of the complete image results in a two-dimensional map of the ellipsometric data that can be, via the model, calculated into thickness map of the sample. An imaging experiment is further described in the next chapter. The Imaging Ellipsometer EP3 from Nanofilm enables us to obtain 3D images of the lipid-protein binding. Using the Fresnel equations, for instance, the thickness of the protein can be calculated. In the film model, the adsorption of a protein onto a solid surface results in the change of Δ and Ψ. The surface concentration of protein Γ (ng/mm 2 ) is calculated using the following equation: Γ = K d p (2-32) where K=1.36g/mL is the density of the protein and d p (nm) is the optical thickness of the protein, a multilayer optical model is used to calculate the thickness of the protein layer by means of the software AnalysR designed by Nanofilm (Germany). Confocal Laser Scanning Microscopy Confocal laser scanning microscopy has been used over the last three decades in biological and medical fields. Both fluorescence and transmission are used in different biological applications. The basic concept in confocal fluorescence microscopy entails the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimen whose thickness exceed the immediate plane of focus. 53

54 Principles An epi-fluorescence laser scanning microscope is diagrammatically represented in Figure The laser system emits coherent light that passes through a pinhole aperture situated in the conjugated plane (confocal). The laser is then reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane. Secondary fluorescence emitted from points on the specimen pass back through the mirror and is focused as a confocal point at the detector pinhole aperture. Fluorescence emission occurring at points above or below the objective focal plane is not confocal with the detector pinhole and forms Airy disks in the aperture plane. Refocusing the objective in a different plane shifts the excitation and emission points on the specimen to a new confocal plane for the detector pinhole. Figure Schematic diagram of the optical pathway in laser scanning confocal microscopy 54

55 Fluorophores in Confocal Laser Scanning Microscopy Fluorescent probes play an important role in biological imaging of living cells and tissues. Many probes are designed with synthesized aromatic organic molecules that bind to biomolecules or to specific regions of the cell such as mitochondria, endoplasmic reticulum, nucleus and cytoskeleton. Fluorescent dyes have been employed for monitoring cellular integrity, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity. Fluorescent probe technology moved forward when the green fluorescent protein (GFP) was discovered from jellyfish More recently, the development of nanometer-sized quantum dots has provided a new area of confocal microscopy research. 59 Fluorophores are described according to their absorption and fluorescence properties (spectral profiles, absorbance and emission wavelengths and fluorescence intensity of emitted light). Fluorophores chosen for confocal applications must have a brightness level and signal persistence sufficient enough so images will not suffer from excessive photobleaching artifacts and low signal-to noise ratios. When the fluorophore is irradiated with a focused laser beam at high power, its emission is increased to dye saturation, depending on the excited state lifetime. In the excited state, fluorophores are unable to absorb another incident photon until they emit a lower-energy photon through the fluorescence process. When the rate of fluorophore excitation exceeds the rate of emission decay, the ground state population decreases and the molecules become saturated. Photobleaching occurs when a fluorophore loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. Upon transition from an excited singlet state to the excited triplet state, fluorophores may interact with another 55

56 molecule to produce irreversible covalent modifications. The triplet state is relatively long-lived with respect to the singlet state, thus allowing excited molecules a much longer timeframe to undergo chemical reactions with components in the environment. The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent on molecular structure and local environment. Some fluorophores bleach quickly after emitting only a few photons, while others that are more robust and can undergo thousands or even millions of cycles before bleaching. Fluorescence Recovery after Photobleaching Fluorescence recovery after photobleaching (FRAP) has become the most direct and elegant technique to characterize the lateral mobility of biomolecules such as lipids and proteins within biological membranes. FRAP is an optical technique that studies the translational dynamics of membrane components. 60 By inserting a fluorophore in the lipid bilayer, the diffusion coefficient of the biomolecule is measured by photobleaching the fluorescent molecules, typically in a small micron-sized region. The diffusion coefficient is then derived from the recovery of the fluorescence in the bleached area. 61 In 1976, Axelrod et al. developed a model to analyze and quantify the molecular mobility and interactions from FRAP data based on the lateral transport of a molecule undergoing Brownian motion. 62 This procedure can be applied to both Gaussian and uniform laser beam profiles but two conditions must be observed: the bleaching time should be <1/10 τ 1/2, where τ 1/2 is the time required to recover half of the fluorescence after bleaching and the ratio of the fluorescence immediately after photobleaching to the initial fluorescence should be <20%. 56

57 Instrumentation FRAP experiments were conducted with a confocal microscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and three lasers, a tunable argon ion laser (458, 488, 514 nm), a green HeNe laser (543 nm), and a red HeNe laser (633 nm) with three separate photomultiplier tubes (PMTs) for detection. The lipid fluorophore used is 1-palmitoyl-2- [6-[(7-nitro-2-1,3-benzooxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) with an excitation wavelength of 460 nm and an emission wavelength of 534 nm, therefore we used an Argon ion laser with a 457 nm beam with a Gaussian crosssectional intensity. The fluorescence intensity was recorded by a photomultiplier signal and by analysis of camera images for quantitative data. The cellular images were taken with a 20x objective. All analysis were conducted on the Fluoview 500 software, followed by processing of the data using Origin. Experimental set-up The FRAP technique measures diffusion rates of fluorescently tagged biomolecules such as proteins and lipids. In this method, a small spot of the biomolecule is continuously illuminated at low laser power (20%) and the emitted fluorescence is measured, F i. The laser power is then increased to 100% for a brief time to destroy the fluorescent molecules in the illuminated region of a few micrometers by rapid bleaching, causing irreversible photochemical bleaching of the fluorophores in that region, as shown in the first image of Figure The measured fluorescence is the fluorescence at time equal zero, F 0. After the light intensity is returned to its low level, the fluorescence is monitored to determine the rate at which unbleached molecules diffuse into the depleted region. Lipid molecules with a good lateral mobility 57

58 undergo Brownian motion and fluorophores situated outside of the bleaching region, diffuse throughout the sample and replace the non-fluorescent lipid in the bleached region. The fluorescence is measured until the recovery is complete, F,. Images are taken every 5s during the first minute after bleaching and every 10s thereafter. The kinetics of recovery can be empirically characterized by τ 1/2, which time required for the bleached spot to recover half of its initial intensity as seen on Figure Assuming a Gaussian profile for the bleaching beam, the mobile fraction and the diffusion constant D can be calculated from the following equations: Mobile fraction = F F 0 F i F 0 (2-33) D = w2 (2-34) 4τ 1/2 Figure Schematic representation of a typical FRAP experiment. Conclusions In the research work presented further on, characterization of biosensors is shown to be fundamental for the understanding of biological phenomena. SPREE and 58

59 fluorescence microscopy are two complementary biophysical techniques enabling rapid, sensitive, specific detection of chemical and biological analytes. In fact, SPREE allows the study of binding interactions between proteins and new developed biosurfaces, while FRAP provides information on the behavior of biological molecules in designed biosensors. Imaging techniques such as atomic force microscopy and quantitative technique such as X-ray photoelectron spectroscopy were also used to characterize the biosensors in more detail. 59

60 CHAPTER 3 MISCELLANOUS EXPERIMENTS TO DEMONSTRATE THE POWER AND VERSATILITY OF ELLIPSOMETRY Introduction This chapter aims to present practical understanding of ellipsometry to probe the adsorption of biomolecules and polymers on surfaces. While no chemical information about the surface is obtainable, ellipsometry is among the most sensitive analytical techniques available. Determination of thin films properties is important for many biological issues. For instance, surfaces modified with proteins or polymers are highly common methods to study behavior of living cells. Ellipsometry is a versatile optical technique for investigating thin films ranging from 0.1 to 300 nm and is used to determine optical properties and morphology of surfaces and adsorbing thin films. This technique is based upon measurement of changes that occur in the state of polarization of monochromatic light reflected of a surface. Ellipsometry is non-destructive, does not require labeling agents and measurements are usually fast. Measurements can be made in any transparent medium and may be performed on any reflecting or transparent surfaces. Ellipsometry also has advantages over other surface analytical methods. In fluorescence, a fluorescent probe attached to the biological material is required and the resolution is between 5 and100 μm, which is lower than for ellipsometry. However, the sensitivity of the imaging ellipsometer is nm or ng mm -2, comparable to fluorescence spectroscopy. Another important surface technique is atomic force microscopy (AFM), used to analyze the topography of a solid surface. AFM is the method with the highest spatial resolution (0.1 nm) but it takes several minutes to scan an area of 100 x 100 μm. Thus, if such resolution is not needed, imaging ellipsometry is 60

61 much faster and still provides medium spatial resolution (1.2 μm). Moreover, imaging ellipsometry can obtain profiles and maps of inner layers on a multi-layer stack and optical properties of the material (extinction coefficient, refractive index). Ellipsometry is able to determine optical constants of films by applying complex equations using appropriate software. The method consists of acquisition of data, processing of data and fitting of the data to a model. Data acquisition can be performed by different approaches but there is always one minimum where two real numbers, expressed as delta and psi (as explained in chapter 2) are determined by one single measurement. In ellipsometry, the optical parameters of the surface, such as film thickness and refractive indexes, are related to the measured angles trough complex equations that are neither linear nor invertible. Thus, data processing is a necessary step to solve surface parameters. Recently, ellipsometry is commonly used wherever thin films are found. Because of its capabilities, applications include research and development of optical and protective coatings, polymer and lithography materials and semiconductor integrated circuit manufacturing. 63,64 Ellipsometry can characterize many different material properties, as will be demonstrated in this chapter. Here, we present the use of ellipsometry as a sensitive technique for thin film studies including metallic, biomolecule and polymer layers. Multilayers System Analysis and 3D-Profile Imaging Surface modification is commonly used to specifically bind biomolecules such as DNA, proteins or lipids to the surface. Modification of surface properties by changing the surface functionality or by thin film deposition creates interesting physical and chemical properties for biological applications. Different properties can be influenced, 61

62 including hydrophilicity or hydrophobicity and the ability to form covalent bonds. Interactions between biomolecules and the surface will occur at the interface zone and the surface properties can greatly influence these interactions. Therefore it is necessary to combine surface modification to surface analysis to obtain information on the composition and structure of the top few atomic layers. Several analytical techniques have been used, including x-ray photoelectron spectroscopy, secondary ion mass spectrometry, Fourier transform infrared spectroscopy, scanning electron microscopy, contact angle measurement, ellipsometry and atomic force microscopy. This present work reports the characterization of thin films by atomic force microscopy and ellipsometry. Ellipsometry has been widely used for the measurements of thickness and optical properties of thin organic and inorganic films. This technique allows the determination of optical parameters and 3D-profiles. In the present study, imaging ellipsometry is used to study assembling of zirconium phosphonate surfaces and lipid monolayer on gold and silicon substrates. The classical Langmuir-Blodgett deposition technique, used for the stacking of homogenous monolayers, was used to form the thin films. The zirconium phosphonate inorganic surface has already been used for biomolecules immobilization including DNA and proteins AFM was also used to provide information of roughness and morphology of the surface. A combination of both techniques is often used as a means of better characterization of the system under study. Experimental Section Materials Reagents were obtained from commercial sources and used as received. The monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1-palmitoyl-2-62

63 oleoyl-sn-glycero-3-phosphocoline (POPC) were purchased from Avanti Polar Lipids (Alabaster, AL). The refractive index matching fluid diodomethane and zirconyl chloride, 98% were purchased from Aldrich. The buffer component trizma hydrochloride was from Sigma. SF10 glass slides were purchased from Schott glass (Duryea, PA) and gold (~28.5 nm) was evaporated on a 4 nm chromium adhesion layer on the clean glass slide by LGA Thin Films (Santa Clara, CA). All chemicals were used as received without further purification. Milli-Q water with a resistivity of 17.9 MΩ.cm was used for all experiments. The buffer, tris buffer, used throughout the experiments was a 10 mm trizma hydrochloride and 100 mm sodium chloride solution at ph 7.4. Zirconium-phosphonate substrates Zirconium phosphonate monolayers were prepared using a multiple-step Langmuir-Blodgett deposition technique. 71 Monolayers were transferred using a KSV 3000 Teflon-coated LB trough with hydrophobic barriers (KSV Instruments, Stratford, CT). The surface pressure was measured by a filter paper Wilhelmy plate. The aqueous subphase was 2.6 mm of CaCl 2 adjusted to ph 7.4 with a potassium hydroxide solution. Octadecylphosphonic acid was spread from a 0.30 mg/ml chloroform solution in the LB trough. The solvent was allowed to evaporate for 10 min and the monolayer was compressed at 10 mn min -1 to reach a surface pressure of 20 mn min -1. Hydrophobic substrates were then dipped down through the monolayer surface at 8 mm min -1 into a vial sitting in the subphase, transferring the layer. The vial containing the slide was removed from the trough and 3 mm of zirconyl chloride was added to bind a monolayer of Zr 4+ ions at the organic template. After four days, the slide was rinsed with water and kept in water before being used. 63

64 Atomic force microscopy measurement Atomic force microscopy imaging was performed using a Multimode AFM with a Nanoscope IIIa controller (Digital Instruments, Santa Barbara, CA) and commercially available silicon cantilever probes (Digital Instruments). Ellipsometric measurement Ellipsometric angles and spatially resolved contrast images were acquired using a commercial EP 3 -SW imaging system (Nanofilm surface analysis, Germany). This instrument has the capability of imaging ellipsometry, which combines the power of ellipsometry with microscopy, providing an x/y-resolution of approximately 1 μm as it is the smallest size of ROI with the 10x objective. The ellipsometer employed a frequency-doubled Nd:YAG laser (adjustable power up to 20 mw) at 532 nm. The laser beam was elliptically polarized after it passed through a polarizer and a quarter-wave plate. The elliptically polarized light was then refracted into a 60 SF10 prism and reflected from the gold film. After reflection, the light passed through a 10x objective and an analyzer, and imaged onto a CCD camera. Ellipsometry theory is based on the Fresnel reflection/transmission equations for polarized light at each boundary between different materials. This ellipsometric measurement is expressed in terms of amplitude ratio (Ψ) and phase shift (Δ): tan Ψ e iδ = Rp R s (3-1) where Rp and Rs are the complex Fresnel reflection coefficients for p- and s- polarized light. As seen in chapter 2, film thickness and refractive index can be derived from delta and psi, using complex equations. Therefore, a good knowledge of the system and a computer are essential for the determination of optical parameters. 64

65 While a single measurement can provide thickness data, the accuracy of single measurement can be increased by the selection of different types of measurement. In fact, ellipsometric nulling conditions can be fulfilled at four distinct optic settings, called zone, as described in Table 3-1. For instance, the 3-zone measurement finds minimum signal by rotating the polarizer and analyzer in the defined zone 3 and calculates delta and psi. It will also find the calculated angles for zones 1 and 2. The mean value at 2- or 4-zone measurements is free of systematic errors and increases the accuracy depending on the measurement condition, by an order of magnitude. The uncertainty for the measured angles and the optical parameters are shown in Table 3-2, as can be seen, the accuracy is increased for the 2- and 4-zone measurements. Table 3-1. Description of the four distinct optic settings relative to the angles of the polarizer, compensator and analyzer polarizer setting compensator setting analyzer setting Zone 1 Positive +45 positive Zone 2 Negative +45 negative Zone 3 Positive -45 positive Zone 4 Negative -45 negative Table 3-2. Uncertainty of a single measurement of delta and psi with ellipsometer EP3. Refractive index and thickness of SiO 2 /Si (perfomed by Nanofilm) 72 delta ( ) psi ( ) refractive index thickness (nm) Accuracy 1-zone ± 1 ± 0.5 ± 0.02 ± 1 (systematic error) Accuracy of mean of ± 0.1 ± 0.05 ± ± zones/4-zones Precision in thermal equilibrium ± ± ± ± For each layer, the angle of incidence varied from 60 to 77, as they provide the best sensitivity to material properties and ellipsometric angles values were recorded at every 0.5. Using Fresnel equations and an appropriate model, thicknesses of the layers can be determined. However, the equations resulting from the theoretical 65

66 description of the model are algebraically very complicated for even a single film on a bulk substrate. Consequently, the reverse computation can be problematic as the equations arisen from the models are generally transcendental and non-inverted. Expressions for optical constants or layer thickness cannot be directly obtained in terms of measured quantities (delta, psi and angle of incidence). The most common solution to this problem in ellipsometry used the Levenberg-Marquardt (LM) algorithm in regression analysis. 73 Mathematical models for many film structures and materials have been developed for ellipsometry. Computation of ellipsometric parameters, delta and psi, is not difficult when optical parameters of the studied system are known. Thus, numerical methods have been developed to solve this issue. The one used in the present study is the LM method. The strategy behind this technique is schematized on Figure 3-1. Figure 3-1. Flow chart of ellipsometry data analysis procedure. 66

67 In this method, a model is first created based on the known multilayer system. 74 Using estimated values for the materials properties, values of delta and psi are computed accordingly. The experimental data are then compared to the computed data in the least square sense by summing the differences between the experimental and computed values for each measured angle. The sum is the mean square error (MSE). If the initial estimate and measurements were perfectly correct then the MSE would be zero but this is rarely the case. The LM method then makes adjustments to the initial estimates to reduce the MSE as low as possible. Results and Discussion Multilayer system characterized by AFM Analytical techniques are being used more and more to probe structure and dynamics of biomolecules at surfaces. Preliminary characterization work has been performed on gold support by atomic force microscopy. Often, the adsorption of biomaterial requires homogenous surfaces for complete coverage. Therefore, it is important to characterize gold surface after modification with zirconium phosphonate film. The deposition of the zirconium phosphonate surfaces was followed by AFM as images were taken after each deposition steps. The formation of the inorganic surface involves two steps performed on a cleaned surface, in the first one, an ODM monolayer is deposited to render the surface hydrophobic then an ODPA monolayer is deposited followed by incubation in zirconium solution for four days (Figure 3-2). Figure 3-3a shows a typical AFM image of a plasma-cleaned gold surface. It can be seen from the image that the surface presents many scratches but remains flat. After deposition of the self-assembled ODM monolayer on the substrate by immersion in ethanol for 18 hours, the surface is again characterized by AFM, as seen on Figure 3-67

68 3b. Although cracks remain, coverage is uniform. Figure 3-3c shows the image acquired after the deposition of the zirconium phosphonate film. The surface is uniform and homogenous and we observe the disappearance of the scratches present on the gold substrate. Figure 3-2. Deposition steps for the formation of the zirconium phosphonate surfaces with the deposition of the ODM monolayer by immersion for 14 hours (1) followed by the LB deposition of ODPA and immersion for four days in zirconium solution (2). (a) (b) (c) Figure 3-3. Surface morphology of gold surface modified surface. (a) plain gold slide. (b) self-assembled ODM monolayer. (c) zirconium-phosphonate film. The z- scale is 50 nm for all images We found that the both ODM and zirconium phosphonate films formed uniform films after deposition on the gold surface and are then ready for the adsorption of biomaterials. 68

69 Langmuir-Blodgett films on silicon characterized by ellipsometry In this section, ellipsometry measurements of inorganic and lipid films on metallic surfaces has been used to obtain thicknesses of the multilayer system. To perform ellipsometric measurements, the surface must be flat, reflecting and have a size suitable for the experiments. Most metal surfaces are in agreement with those conditions, including gold and chromium. Gold surfaces bind thiol groups with high affinity. Therefore, simple surface modifications can be made with the use of small molecules containing thiols. The stability of those modified substrates is good and permits normal handling at room temperature. Silicon is also a highly suitable surface for ellipsometry experiments. Silicon wafers have a spontaneously grown, hydrophilic, silicon oxide layer that is very pure and chemically homogenous. 75 In this work, gold and silicon were used as the main substrates for ellipsometric measurements. As shown on Figure 3-4, the system consists of gold or silicon cleaned substrate coated with a self-assembled monolayer (ODM or OTS for silicon and gold, respectively). Then, a zirconium phosphonate film was transferred by LB technique. Finally, a lipid monolayer was deposited onto the inorganic film. Figure 3-4. Schematic representation of the formation of the inner lipid monolayer by LB deposition on zirconium phosphonate surfaces. 69

70 Layer thicknesses of the inorganic and lipid layers were determined after each deposition step by ellipsometry, as the hydrophobic surface has been well characterized. While the measurement of delta and psi is relatively simple, the correct interpretation of the data depends on the creation of a good model. What limits ellipsometry is not the measurement capability but accuracy of the model used. The model is created using the software AnalysisR from Nanofilm, Germany (Figure 3-5). It consists of four layers with known optical parameters and unknown thicknesses. Figure 3-5. Optical parameters of the multilayer system (SiO 2 /OTS/Zr-ODPA/DPPA) on silicon. Figure 3-6 shows the fit of a typical fitted data set for the delta and psi values. The system was composed of three layers of silicon dioxide, OTS and zirconium phosphonate film. Note the agreement between computed (solid lines) and measured (dotted lines) parameters. The model is therefore consistent with the real parameters composing the different layers. The calculated thickness of the zirconium phosphonate monolayer is 2.12 nm, consistent with previous works. 71,76 Moreover, the low value of 0.94 for the MSE indicates a value in agreement with the model. 70

71 Figure 3-6. Fitting of the measured (dotted lines) and computed (solid lines) values of Ψ and Δ for the measurement on the multilayer (Si/SiO 2 /OTS/Zr-ODPA) system. Measurements were taken each 0.5 from 60 to 77 A DPPA monolayer is then deposited on the surface by the LB method. The unknown value is now the thickness of the phospholipid monolayer, the model becomes more accurate when the number of unknown parameter is reduced. Therefore, the calculated thickness values for the OTS and inorganic layers are used in the new model to solve for the lipid thickness. The obtained results for the delta values (Figure 3-7) show a perfect superposition between the fitted and the experimental data. The value obtained for the thickness of the lipid monolayer is 2.39 nm and an MSE value of 0.72, under one, confirmed the good fit of the model. 71

72 Figure 3-7. Experimental (dotted lines) and fit (solid lines) Ψ and Δ data for the multilayer (Si/SiO 2 /OTS/Zr-ODPA/DPPA) system Table 3-3. Results of thicknesses measurements, using the multilayer model on silicon substrates a. Data were then fitted to the multilayer model in order to calculate the thicknesses. Layer thickness (nm) MSE Zr-ODPA 2.12 ± DPPA 2.39 ± a Experimental data were obtained for a variation of the angle of incidence from 60 to 78 with a 0.5 step. Langmuir-Blodgett films on gold characterized by ellipsometry Gold substrates are used for the surface plasmon resonance experiments as described in chapter 5, so, we wanted to study the layered model on gold by ellipsometry, as well. Gold is known to have a poor adhesion when directly deposited on glass so a thin chromium adhesion layer was incorporated between gold and glass. The model with the estimated values is presented in Figure 3-8. This system is 72

73 composed of five layers, gold, chromium, ODM, zirconium phosphonate and the lipid monolayer. The thicknesses of gold and chromium have been measured by ellipsometry as well. Figure 3-9 shows the measured and fitted data for the ellipsometric angle delta, an almost perfect superposition is observed. The resulting thicknesses are 2.10 nm and 2.42 nm for the zirconium phosphonate and the lipid monolayer, respectively. The value of the MSE over the range of measurements remains low and acceptable. Therefore, the consistency of the solutions demonstrates that the global minima are correct for the model selected. Figure 3-8. Multilayer model for the determination of layer thicknesses on gold. Figure 3-9. Ellipsometric measurement of a zirconium phosphonate modified surface (a) and the lipid monolayer (b) on a gold substrate. Experimental data (dashed lines) were obtained for a variation of the angle of incidence from 60 to 78 with a 0.5 step. Data were then fitted to the multilayer model in order to calculate the thicknesses (solid lines). 73

74 3D imaging by ellipsometry The imaging ellipsometer has also the capability of imaging surfaces and quantifying thickness distribution. Thickness-maps can be obtained in less than a minute by imaging ellipsometry. For instance, Figure 3-10 presents four different areas of film thickness of the zirconium phosphonate modified surface. A delta map is obtained from recording a series of images with variable contrast (i.e. variable polarizer angle). Each of these images is well focused by using a 10x objective. The polarizer angles of minimum signal are interpolated for each pixel of the field of view. The map of minimum angles is converted into a delta map. The conversion of the delta map into the thickness map uses the same fitting algorithm used to calculate film thickness. This capability allows the study of the morphology of the surface. It gives similar information as AFM but each mapping takes only a few minutes. Figure D-profiles of zirconium phosphonate layer with different areas (100 μm 2 to μm 2 ). 74

75 Another system composed of two different lipid bilayer models was studied by imaging ellipsometry. In this system, an ODM layer was first immobilized on the gold slide by self-assembled deposition. Then, only half of the slide was covered with the zirconium phosphonate monolayer as shown in the schematic on Figure The gold substrate was set in the flow cell and POPC: DPPA (ratio 4:1) lipid vesicles were injected. As ODM creates a hydrophobic surface, adsorption and rupture of the vesicles created hybrid lipid bilayers. On the other half, lipid bilayer membranes were formed on the hydrophilic surface. A mapping of the μm area was performed, the refractive index of the mixed layer (ODM/ ODPA/ lipid layer) was averaged to 1.48 and the thickness of both layers were calculated using a five-layer model (SF10, chromium, gold, mixed layer and buffer). Figure Schematic illustration of the gold modified surface with the hybrid lipid bilayer (left) and the lipid bilayer membrane (right) Figures 3-12 and 3-13 show the results of a delta map. Around 100 contrast images were scanned incrementally over a degree change in polarization angle with the analyzer angle maintained at a constant value. These scans were then assembled to determine the null for each point. The 3D images demonstrated the presence of two layers with different thicknesses; the blue area represents the hybrid lipid bilayer and 75

76 the green area the lipid bilayer membrane. A thickness profile can also be obtained from the thickness map. The thickness profile (right image of Fig. 3-13) showed two averaged thicknesses in agreement with the expected values as shown in Figure (a) (b) Figure Ellipsometrically determined 2D height map of the mixed lipid layer surface (a). Green represents the lipid bilayer membrane on zirconium phosphonate surface and blue represents the hybrid lipid bilayer on ODM. Corresponding profile in (b) of the step between the hybrid lipid bilayer and the lipid bilayer membrane, represented by the red line on (a). Figure D images of the mixed lipid layer surface. The areas represented on the two maps are the same but the angle of capture is different. 76

77 Studies of Polyelectrolyte Heterostructures Containing Conjugated Polymers Introduction The buildup of polyelectrolyte multilayer coatings using the layer-by-layer (LbL) deposition technique was first introduced by Decher et al. in the early 1990s. 77 In this method, a polymer film is formed by exposing a charged surface to alternatively positively and negatively charged polymers. The relatively high concentration of polyelectrolyte in the adsorption solution results in an excess of ionic charge adsorbed and exposed to the solution, and thus the surface charge is reversed. This technique is interesting for different applications such as biomaterials. It holds a great advantage compared to other deposition techniques with high thickness and structure control on a nanometer scale. In fact, polyelectrolyte coatings can be controlled by the type of polymers, the number of deposited layers, the ionic strength at deposition and in some cases, the deposition ph. 81,82 In this study, we address the buildup behavior of LbL constructed polyelectrolytes films by measuring the thickness of the layer of two specific polymers with opposite charges, called the buffer layer, by ellipsometry. Experimental Section Substrate preparation and film deposition Prior to use, glass slides were cleaned by sonication in sodium dodecyl sulfate/water, water, acetone and then, isopropanol, for 15 min each and subsequently dried. Silicon Wafers were cleaned by immersion into a Piranha solution (concentrated sulfuric acid and 30% hydrogen peroxide solution 3:1) for 30 minutes and subsequently rinsed with Milli-Q water. (Caution: Piranha solution is extremely corrosive and must be handled with proper care). 77

78 Films were prepared using LbL self assembly technique using a programmable robot (Nonostrata StratoSequence IV). The polymers used for the buffer layer are poly(allylamine hydrochloride) (PAH) and polymethacrylic acid (PMA). The anionic polymer thyenyl modified poly(phenylene ethylene) type CPE (PPE-Th) and polyfluorene based CPE (PFl) and the cationic polymers PAH were used to construct the polyelectrolyte films. Substrates were alternatively dipped in solutions of cationic polyelectrolyte then anionic for 10 min each. Between each deposition step, substrates were rinsed three times by immersion in Milli-Q water for 3, 1 and 1 min respectively. After each rinsing cycle, the water was refreshed. All of the experiments were carried out at room temperature under ambient atmosphere. The typical film architecture is the following: substrate // (polycation/pfl) 1 // (polycation/polyanion) x // (polycation/ppe-th) 1 as described in Figure PPE-Th - PAH + Buffer PMA - Layer PAH + PFL - PAH + Figure Description of the LbL constructed polyelectrolyte films Ellipsometry measurement Ellipsometry was used to determine the thickness of the buffer layer (PAH/ PMA). Four-zone measurements were performed in air at a constant angle of incidence of 61.5 on Si/ SiO 2 substrate wafers. Samples were analyzed with the variation of the PAH/PMA buffer layer (x=0 to 7) so eight different samples were analyzed. Also, the LbL deposition process was performed three times so in total twenty-one different samples were prepared and studied by ellipsometry. For each sample, fifteen different 78

79 regions were considered to calculate the averaged thickness value. Different approaches have been used to convert ellipsometric data into physical properties. The common and simplest way is to assume a film complex refractive index value based on literature data to obtain film thickness. The thickness of the total polymer film was determined from a two-layer model (Si substrate/sio 2 /polymer/air) using a constant refractive index for the polymer layer of The measured angles Δ and Ψ were fitted by the optical model based on the Fresnel theory as a function of the optical parameters and the angle of incidence. Results and Discussion Figure 3-15 shows the averaged thicknesses for the heterostructure assembly. The LbL deposition technique was used to deposit the polyelectrolyte multilayer system. Eight different samples were studied with variation of the buffer layer. Firstly, the silicon dioxide thickness was determined by measuring the ellipsometric data on the silicon wafer minutes after the RCA cleaning procedure. The averaged value of the silicon oxide thickness, over 45 measurements, is 15 Å. This value is in the range of the oxide layer. 83 Then, the ellipsometric values were measured for the different samples and the optical total thicknesses of polymers were calculated using the two-layer model. We obtained a thickness of 21.5 ± 2.3 Å for the buffer layer-free system and ± 4.2 Å for the one system with seven buffer layers. A linear fit was performed on the results and a coefficient of determination close to 1 was obtained. The deposition is then homogenous and a uniform buffer layer is present in this system. The calculated buffer layer (PAH/PMA) thickness was 22.6 ± 1.8 Å (Figure 3-15b). The thickness increase after the addition of each buffer layer of Å is consistent with previous reports

80 Another system was studied where the buffer layer consisted of poly(diallyldimethylammonium chloride) (PDDA) as the polycation and poly(sodium 4- styrenesolfonate) (PSS) as the polyanion. However, no increase in thickness was observed after deposition of the sample with three buffer layers as seen in Figure 3-15a. Ellipsometry indicated that LbL deposition was not successful for this system. Figure Thickness of heterostructure assemblies as a function of the number of buffer bilayer (PDDA/PSS) (a) and (PAH/PMA) (b) separating pfl and PPE- Th (deposited from 1 mm solutions) measured by ellipsometry on silicon wafer with a 15Å silicon oxide substrate. The standard deviations for the ellipsometric thickness values are <4Å. Conclusions Ellipsometry is a now-well established methods for the determination of thin film properties such as thickness and refractive index. The use of angle variation to solve for thickness of thin films gave consistent results. Overall, MSE values were low and viable, demonstrating the good agreement between the measured and estimated data. Ellipsometry also provided optical parameters for different substrates, including silicon and gold. The models used throughout the ellipsometric study provided good fits (i.e. a low MSE) for essentially all of the measurements. 80

81 Moreover, imaging ellipsometry allows the study of other parameters, including 3D images of lipid layers, as described in this section. The imaging ellipsometer offered automatic recording of thin film thicknesses. This technique can measure delta and psi values in different regions of incidence simultaneously and mapping of the selected areas can be performed in less than a minute. Ellipsometry has been used in the determination of polymer thickness. The advantages of ellipsometry include its simple instrumentation, sensitivity to 0.1 nm range and speed of measurement. Its range of applications is large, however, the quality of the surface and adsorbing layer will affect the results therefore the surface should be uniform and highly reflecting. Moreover, the interpretation of the data depends highly on the model used and critical judgments should be made. 81

82 CHAPTER 4 REVERSIBLE HIGH CAPTURE HISTAGGED PROTEINS TO BIPHOSPHONATE ADAPTORS STUDIED BY SPREE AND FLUORESCENCE TECHNIQUES Introduction Proteins play a key role in cellular mechanisms, but most of their functions remain unknown. 85 Proteomics, which is the study of protein function, expression and localization on a cellular-wide scale, needs higher throughput analysis tools to achieve this goal. By analogy to work in genomics with DNA microarrays, 86,87 protein capture microarrays have been used successfully to identify differentially expressed biomarker proteins However, it is still an emerging technology, which needs selective methods for surface immobilization 85,93 and alternative strategies to antibodies for use as capture agents in protein microarrays. A significant challenge for protein microarrays is attachment on the chip surface. Immobilization of the protein by non-specific adsorption is often associated with problems such as a high background signal and significant loss of the protein probes during stringent washes. 28 Furthermore, random orientations of the immobilized proteins affect their activities, which decrease the sensitivity of detection. 94,95 Therefore, specific protein attachment through covalent coupling 85, or affinity interaction is considered to be a better strategy. 94 In the present work and in other studies, specific orientation of capture proteins is achieved by affinity interaction between a histag protein and a transition metal chelated to a nitrilotriacetic acid (NTA) moiety. Ni- NTA resins are routinely used for the purification of oligo-histagged proteins, which can be easily prepared by genetic fusion of an oligohistidine tag to the C- or N-terminus. Despite the moderate affinity (K D ~ 1 to 10 μm) and stability of individual Ni- NTA:oligohistidine complexes, this strategy has been successfully used in a microarray 82

83 format for the immobilization of about 6000 histag labeled yeast proteins, of which more than 80% were shown to retain their biological activity after immobilization. 117 However, most NTA surfaces used for the design of protein microarrays are obtained by spreading a functionalized polymer matrix on a glass surface, leading to inhomogeneous surface coatings without rigorous control of the NTA density. In previous work on DNA microarrays, our laboratories described a new ultraflat support with which the biological probes are bound to a monolayer-coated surface through an inorganic linkage, in contrast to existing systems based on the covalent attachment of the DNA probes via covalent organic bonds. We demonstrated the spontaneous linkage of oligonucleotide probes, phosphorylated at their 5 end, onto a zirconium-phosphonate modified surface. 70 More recently, we demonstrated that the phosphate/zirconium bond is strong enough for dsdna attachment and for studying DNA/protein interactions. 66 On the other hand, unmodified proteins are poorly adsorbed on such zirconium phosphonate surfaces and oligo-histagged proteins do not provide specific binding on these surfaces. In order to provide specific anchoring of oligo-histagged proteins to the zirconium phosphonate surface, our collaborators, Dr. Bruno Bujoli and the members of the Laboratoire de Synthèse Organique, in Nantes, France designed a bifunctional adaptor containing a multivalent phosphonic acid anchor at one extremity and a NTA group at the other. The phosphonate groups provide a stable bond to the zirconium interface by multipoint attachment. This affinity tag strategy provides a uniform orientation of proteins on the surface and high density coverage without the need to perform complicated chemistry on the protein targets or on the solid support. 83

84 Stable binding of the bifunctional adaptor is demonstrated, allowing reversible capture of histagged proteins. This technology is applied to a new class of small and stable capture proteins, the affitins, 118,119 which are shown to keep their binding properties when immobilized on the zirconium phosphonate surface and to exhibit a high signal to noise ratio relative to arrays prepared from other NTA functionalized supports. Experimental Section Materials Glass slides were purchased from Gold Seal Products (cat no. 3013, 3 x 1, thickness 0.93 to 1.05 mm). Reagents were of analytical grade and used as received from commercial sources, unless indicated otherwise. Hydrophobic glass slides were made using octadecyltrichlorosilane (OTS) following a method by Sagiv. 120 The zirconium octadecylphosphonate Langmuir-Blodgett monolayers were prepared on the hydrophobic slides as described previously. 121,122 Nexterion Slides E were obtained from Schott. FASTslide substrates were obtained from Whatman/Schleicher & Schuell. N, N -bis(carboxymethyl)-l-lysine hydrate and lyzozyme were purchased from Sigma Aldrich. Protein Expression and Purification. This work was performed by our collaborators at the Université de Nantes, France. H4 and B3 are recombinant proteins derived from Sac7d from Sulfolobus Acidocaldarius and evolved by ribosome display to bind lysozyme. 118,119 H4 was overexpressed in a recombinant E. coli BL21 strain carrying the corresponding plasmid. The culture was grown in Luria-Bertoni medium containing ampicillin (50 µg ml -1 ) at 37 C until an OD600 of Induction using isopropyl-1-r-d-thio-1-galactopyranoside 84

85 (0.5 mm) was continued overnight. The protein was purified on nickel-nitrilotriacetic acid (Ni-NTA) columns according to the manufacturer s recommendations (Qiagen, Courtaboeuf, France), eluting with a TBS 250 buffer (20 mm TrisHCl, ph 7.4, 150 mm NaCl, 250 mm imidazole). The protein concentration was measured using a Nanodrop 1000 spectrophotometer (Thermoscientific). The same procedure was applied to B3. Microoarray Spotting and Incubation Conditions To prepare the LB Ni-NTA substrate, the zirconium phosphonate modified slides were incubated overnight with a 1 mm NTA-adaptor aqueous solution. Slides were washed 3 times with ultrapure water and then incubated for 1 h with a 100 mmol L -1 NiCl 2 aqueous solution. The slides were washed again 3 times with ultrapure water and dried by centrifugation (1500 rpm, 1 min). The slides were then spotted with a microcaster TM 8-pin system (Whatman/Schleicher & Schuell). The spotted slides were placed overnight in an incubation chamber at 4 C and 75% humidity. To passivate the unspotted areas, slides were treated after spotting with a solution of 0.3% R-casein in a TBS (Tris-buffered saline) solution of TrisHCl (20 mm) and NaCl (150 mm) at ph 7.4. Incubation was performed by applying an Alexa 647-labeled lysozyme solution (1 μm in TBS-0.3% R-casein) to the substrate for 1 h at room temperature. Microarrays were washed 3 times with TBS-0.05% Tween 20 for 5 min, then once with TBS and ultrapure water. Finally, the slides were spun dry centrifuging at 1500 rpm for 1 min. All washes and incubations were performed in small staining jars at room temperature on an oscillating shaker. Microarray Analysis All microarrays were scanned on a Scanarray Gx apparatus (Perkin-Elmer) with a laser power and gain value of 60. Suitable excitation wavelength and emission filter 85

86 were used to detect Alexa 647: 650 nm (excitation), 665 nm (emission). The location of each analyte spot on the array was outlined using the mapping software Genepix (Axon laboratories, Palo Alto, CA). Surface Plasmon Resonance Enhanced Ellipsometry Surface Plasmon resonance enhanced ellipsometry measurements were performed on a commercial EP 3 -SW imaging system (Nanofilm Surface Analysis, Germany). The ellipsometer employed a frequency-doubled Nd:YAG laser (adjustable power up to 20 mw) at 532 nm. Linearly p-polarized light was directed through a 60 equilateral SF10 prism coupled to a gold coated SF10 slide via diiodomethane index matching oil in the Kretschmann configuration. The angle of incidence was kept at 64 for all kinetics experiments as this condition provided the highest sensitivity. Curve fitting of the kinetics data used the AnalysR software from Nanofilm, using a 1:1 Langmuir binding model. SPR slides were prepared by rendering the gold-coated slides hydrophobic with octadecylmercaptan before transferring the zirconium phosphonate layer. The slides were then immersed overnight in 1mM aqueous solutions of either the mono-nta linker or the bis-nta linker, followed by rinsing with ultrapure water and drying under a stream of nitrogen. The NTA-modified slides were placed in the SPR flow cell and treated with a 100 mm solution of nickel chloride. Protein binding onto the Ni-NTA surface was carried out by injecting 5 mm protein solutions in TBS. The surface was regenerated using 250 mm imidazole in TBS buffer. Results and Discussion Design of the Anchoring Linker In previous studies, we showed that oligonucleotides and dsdna can be immobilized on zirconium phosphonate surfaces and that phosphate terminated 86

87 oligonucleotides are selectively adsorbed over those without terminal phosphates. 66,70 It was also shown that dsdna phosphorylated at both ends binds better to the zirconium surface than monophosphorylated DNA. Based on these results, in order to make the zirconated phosphonate film suitable for oligohistidine-tagged protein immobilization, surface modification with either mono- NTA or bis-nta end groups was performed using two adaptors, respectively, which contained a similar scaffold bearing two phosphonic acid functions for binding to the zirconated surface (Figures 4-1). These two adaptors were synthesized in around 15 steps in good yield. 65 Similar adaptors with only one phosphonic acid moiety were found to bind poorly to the zirconated surface and were inadequate for the present application. (a) (b) Figure 4-1. Chemical structures of bisphosphonate-mono-nta (a) and bisphosphonatebis-nta (b) 87

88 Zr/phosphonate Surfaces and Linker Anchoring Zirconium phosphonate modified surfaces are generated by adsorbing Zr 4+ ions to surface phosphonate or phosphate groups. An active metal layer results when the phosphorylated groups are closely organized into a monolayer so that the Zr 4+ ions bind to the surface groups to form a layer while retaining some coordination sites that are free of the surface phosphate or phosphonate ligands. The starting phosphorylated surfaces can be prepared in different ways, including covalent attachment of the phosphorylated groups to silica or gold 128,130,131 and by Langmuir Blodgett deposition of an organophosphonic acid. 121,122, We have long utilized LB methods to prepare the zirconium phosphonate monolayers because the surface films formed in this way are of high quality, stable, and highly reproducible, and the surface chemistry can be applied to nearly any substrate material, permitting application of a wide range of surface analytical techniques on the same surface. 136 Modification of the zirconium phosphonate surface was thus investigated by incubating the substrate in a 1 mm aqueous solution of the desired NTA-adaptor, the binding of which was then monitored by X-ray photoelectron spectroscopy (XPS). XPS is a powerful surface analytical technique, which provides qualitative and quantitative information about the elements on a surface, and was recently shown to be an efficient, label free method for studying DNA surface coverage on zirconium phosphonate surfaces. The P/Zr ratio of 1.1 for the naked zirconated phosphonate monolayer indicates that it is almost fully reticulated by the zirconium ions. Upon reaction with the mono-nta adaptor, a significant increase of this ratio was observed (P/Zr = 1.4), which confirms binding of the bisphosphonate on the surface. Knowing the surface density of the zirconium ions within the monolayer, atoms cm -2, the mono-nta adaptor coverage can be estimated at

89 molecules cm -2, which corresponds to 1 NTA moiety every 1.58 nm 2. With the bis-nta adaptor, a P/Zr ratio of 1.18 was determined, which indicates half the surface coverage observed for the mono-nta molecule. However, as this adaptor presents two NTAs per molecule, the NTA density on the surface is roughly similar to that obtained with the mono-nta adaptor. Nickel complexation to the NTA end groups was then performed to make them capable of covalent binding to histidine-tagged proteins. Binding to the NTA-coated slides is evident as opposed to the bare zirconium phosphonate surface, with more Ni 2+ complexed to the bis-nta coated slide. Protein Immobilization The OPDA-Zr surfaces modified with mono-nta and bis-nta end groups were used to study the specificity and reversibility of their interaction with histidine-tagged proteins as well as the proteins activity when immobilized. For this purpose, monomeric proteins (affitins) with a single N-terminal oligohistidine tag were used. These affitins were derived from the Sac7d scaffold and selected by ribosome display to selectively recognize lysozyme. Binding of the H4 affitin on the zirconium phosphonate monolayer functionalized with nickel-loaded NTA groups, was first studied using SPR (Figure 4-2). As a control experiment, low and totally reversible binding of the protein was observed in the absence of nickel complexation to the NTA group (Figure 4-2a). In the case of both adaptors, the surface exhibits a high and specific binding of the histagged-affitin (Figure 4-2b and 4-2c). A higher phase shift amplitude was observed for the binding of the affitin to the mono-nta adaptor ( Ψ ~ 0.22 ) than for the bis-nta ( Ψ ~ 0.17 ), reflecting a higher amount of bound protein using the mono-nta adaptor. This result can be correlated to the higher surface coverage with this adaptor. 89

90 However, a significant dissociation of the affitin was observed with the mono-nta linker upon prolonged washing (~ 1 h) (Figure 4-2b), while the protein washing out was reduced in the case of the bis-nta linker (Figure 4-2c). This observation reflects the intrinsically low affinity of mono-nta towards a polyhistidine sequence (K D ~ 10-5 to 10-6 M), which can be improved when bis-nta functional groups are used. After washing, the amount of the proteins retained on the surface was similar for both adaptors. The specificity of the interaction is confirmed by the full reversibility of binding upon injection of high concentration of imidazole (Figure 4-3). The protein loading followed by imidazole washing cycle can be repeated without a decrease in binding amplitude, as shown in Figure 4-3 for 2 cycles. Figure 4-2. Affitin immobilization on the zirconium phosphonate surface functionalized with mono- and bis-nta, probed in real time by SPREE. Kinetic adsorption curves of affitin on (a) mono-nta without nickel, (b) mono-nta with nickel, (c) bis-nta with nickel. Experimental data (dotted line) were fitted (solid line) using a 1:1 Langmuir binding model for each step. Curves (b) and (c) were shifted by 0.05 and 0.2, respectively. 90

91 Figure 4-3. Sensorgram for the interaction of affitin with a mono-nta functionalized surface that shows binding of Ni 2+, adsorption and desorption of affitin and regeneration of the surface by 250 mm imidazole. This procedure was then repeated to show the reproducibility of the surface. Experimental data (dotted line) were fitted (solid line) using a 1:1 Langmuir binding model for each step. Microarray Experiments Having shown that both linkers can be efficiently immobilized on the zirconium phosphonate surface, making it suitable for histidine-tagged protein immobilization, the next step was to verify that the immobilized proteins are still functional and capable of binding lysozyme. Affitin immobilization on a microarray format was carried out according to Figure 4-4. All fluorescence experiments were performed by our collaborators in Nantes. The nickel-loaded NTA functionalized slides were spotted with affitin at fixed concentrations prior to a blocking step with R-casein, which due to its high phosphate content provides efficient saturation of the nonspotted areas, hindering nonspecific protein binding. The activity of the immobilized affitin was investigated upon incubation of the surface with AlexaFluor 647 labeled lysozyme overnight. To verify that immobilization of the affitin proceeds via the expected Ni-NTA/histidine-tag interaction, 91

92 the same experiment was performed on the naked zirconium octadecylphosphonate surface. To avoid interslide variation, each slide had only half of its surface functionalized with Ni-NTA groups, and incubation chambers with two compartments were used. Figure 4-4. Description of the different steps employed to test affitin activity on a microarray platform As an additional control, the affitins were spotted under two different conditions, one in a 25 mm imidazole-containing buffer to reduce nonspecific bonding to the Ni- NTA surface and the second in a 250 mm imidazole containing buffer to prevent histidine-tagged protein binding by competition (Figure 4-5). A negative control experiment was also performed with an affitin that does not bind lysozyme. At low imidazole concentration, only spots present on the surface functionalized with the Ni- NTA groups gave high signal intensities, which indicated that affitins were still functional after immobilization. As expected, a high concentration of imidazole (250 mm) prevented affitin binding on the Ni-NTA surface and subsequent lysozyme capture. Interestingly, a very low background value was observed in the absence of affitin, confirming the low nonspecific interaction of lysozyme with the surface when saturated with R-casein. However, nonspecific binding of the affitin on the naked zirconated 92

93 surface was not completely avoided by R-casein saturation since a low intensity signal was detected in that control. Different spotting concentrations of the affitins were explored, incubating at a fixed concentration of the lysozyme target. An increase in signal intensity was detected, with no saturation of the lysozyme capture, using affitin concentrations of μm (Figure 4-6), confirming the high density of Ni-NTA groups available on the surface of the slides. Both bisphosphonates adaptors led to similar fluorescence intensities at a given affitin concentration, in agreement with SPR data, which had shown that the amount of bound protein after washing is similar with the two linkers. However, the spot shape remains better defined on the bis-nta modified surface. On the mono-nta functionalized surface, the spot shape was irregular if affitin was spotted at high concentration, likely as a result of significant dissociation occurring during the washing steps. In the case of the bis-nta functionalized surface, stronger binding of the histidine tag by a bivalent interaction strongly reduces this effect (Figure 4-6). The sensitivity of the microarrays was explored by spotting with a constant affitin concentration and incubating with decreasing concentrations of AlexaFluor 647-labeled lysozyme, using the same slide divided into several independent incubation areas. For this experiment, two affitins differing in their affinity for lysozyme were chosen, H4, Kd ( M), and B3, Kd ( M). For the H4 affitin, the observed limit of detection was below 1 nm (Figure 4-7). As expected, the slide spotted with B3 affitin, which has a lower affinity for lysozyme, exhibited a detection limit around 0.1 μm, with low sensitivity even at high lysozyme concentration, likely because the weakly associating B3 lysozyme is lost during the washing process. 93

94 Figure 4-5. Detection of lysozyme captured by affitin spotted at 1 Μm on a Ni-NTAmodified zirconium phosphonate microarray: white bar, buffer spotted only; light gray bar, affitin spotted with 250 mm imidazole; dark gray bar, affitin spotted with 25 mm imidazole. Fluorescence intensities were measured at 90% laser power and 50% photomultiplier gain. Figure 4-6. Fluorescence intensity versus concentration of AlexaFluor 647-labeled lysozyme in the incubation solution. Affitins H4 (black line) and B3 (gray line) were spotted (5 μm) on a mono-ni-ntafunctionalized ODPA/Zr surface. The gray dotted line corresponds to spotting of buffer. 94

95 Figure 4-7. Fluorescence intensity versus concentration of spotted affitin on zirconium phosphonate slides functionalized with mono-ni-nta (dark gray) and bis-ni- NTA (light gray) groups. Fluorescence data values correspond to the mean and the ecart type range for three replicates within one slide (inset) image obtained at 60% laser power and 60% photomultiplier gain Comparison with Other Microarray Surfaces The performance of our Ni-NTA-modified zirconium phosphonate surface was evaluated relative to other types of microarray substrates, including slides coated with a nitrocellulose-based matrix (FAST slides, Schleicher & Schuell), epoxide slides (Nexterion, Schott), and epoxide slides functionalized with Ni- NTA groups using NR,NR-bis(carboxymethyl)-L-lysine hydrate. All slides were spotted with the same affitin concentrations and incubated with a fixed concentration of the labeled lysozyme. The resulting fluorescence intensities are reported in Figure 4-8. Slides coated with nitrocellulose membrane gave the highest fluorescence signal, making it necessary to use a lower laser power in this case to avoid excessive background. However, even at lower laser power, the background signal is still high, resulting in a low signal-to-noise ratio (S/N ) 1.5. It is known that this polymeric material with defined microporosity binds 95

96 large amounts of protein in a noncovalent and nonspecific way. The poor signal-tonoise ratio could be related to the nature of the affitin, which is very small, and possibly easily washed out during the incubation process. Slides modified with a chemical reactive group, such as an epoxide, provide a covalent yet random immobilization of the proteins via reaction with amino groups present on the protein surface. At the highest concentration of spotted protein (10 μm), the fluorescence intensity using the epoxide E slide is about 50% lower than that obtained using the zirconium phosphonate slides functionalized with Ni-NTA. Figure 4-8. Compared performances for lysozyme capture of H4 affitin immobilized on different substrates: slide E, epoxide surface from Nexterion; slide E-NTA, Ni- NTA functionalized epoxide surface; mono-nta, mono Ni-NTA functionalized ODPA/Zr surface; FAST slide. The / indicates the fluorescence intensities were measured at lower laser power (30) and gain value (40) with the FAST slide. Somewhat surprisingly, when the epoxide E slides were functionalized with Ni- NTA groups using NR,NR-bis(carboxymethyl)-L-lysine hydrate, the observed fluorescence intensities were comparable to those obtained for the unmodified E slides. Although modifying the epoxide slides with Ni-NTA groups should provide an oriented immobilization of the affitins, similar to the Ni-NTA-functionalized zirconium 96

97 phosphonate surface, the observed fluorescence intensity was lower throughout the μm spotting concentration range. A possible reason might be that the zirconium phosphonate surface provides a higher density of NTA groups compared with the epoxide surface. Alternatively, the long spacer separating the NTA group from the phosphonic acid groups attached to the zirconium phosphonate surface may provide better accessibility to the proteins. Conclusions In this work, we demonstrate that a zirconium phosphonate modified surface can be efficiently functionalized with metal NTA groups in order to specifically bind histagged proteins in an oriented way. The NTA linkers bind the zirconated surface via a bis-phosphonic acid functional group with high avidity for the surface. These results further extend to the protein microarray field the applications of mixed organic/inorganic zirconium phosphonate surfaces, on which simple and irreversible modifications can be introduced without the need of chemical activation steps. Using these bifunctionnal adaptors, a high density of accessible NTA groups was obtained, allowing high efficiency of protein binding. Proteins immobilized in this way retain their ability to capture protein target with high sensitivity, and the modified zirconium phosphonate surfaces were shown to compete favorably with commercially available substrates designed for protein microarrays. Finally, we also show that the affitin scaffold can be used as capture agents on different types of microarray slides. The small size of this scaffold, which has been evaluated as an alternative to antibodies, allows high density of spotting and consequently high level of specific activity of the surfaces. 97

98 CHAPTER 5 STABLE SUPPORTED LIPID BILAYERS ON ZIRCONIUM PHOSPHONATE SURFACES Introduction Supported lipid bilayers have emerged as important biomimetic models for investigating many characteristics of cell membranes, ranging from fundamental studies of lipids to the mechanisms of membrane bound proteins. Several routes to these supported membrane models have now been described, the most commonly used of which are vesicle fusion onto a hydrophilic support to generate a bilayer and lipid monolayer transfer onto a hydrophobic support. 9,11, The latter approach can be used to form asymmetric bilayer structures. Many of these membrane mimics are viable models for some applications, but most fall short of being generally applicable. Bilayers supported on glass have good lipid mobility due to a thin layer of water between the bilayer and support, but these same systems are poor models for studies of transmembrane processes because the water layer is too thin. Approaches have been developed to add space between the bilayer and the support, including the use of polymer cushions or other spacers These strategies are effective, but often involve specialized reagents that are limited to a unique surface material. Another concern is bilayer stability, as they are typically sensitive to vibration or mechanical perturbation and are generally unstable to air. Recently, a number of strategies have been developed to generate air-stable supported bilayers, including the use of a nanoglassified gold surface, 144 lipopolymer membrane, 145 hydrogel mesh, 146 added cholesterol, 147 and modified lipids. 148 Each method fulfills its role of mimicking the cell membrane but many are also limited to specialized conditions. 98

99 The present work describes stable supported lipid bilayers that are formed on zirconium phosphonate modified surfaces. Zirconium phosphonate surfaces are known to efficiently bind phosphonates and phosphates, and we have previously used this surface to immobilize DNA and as a platform for oligonucleotide arrays. 66,70 We showed that the zirconium phosphonate surface selectively binds phosphorylated DNA over non-phosphorylated DNA, attributable to the specific binding of the terminal divalent phosphate in preference to weaker binding of the phosphodiester backbone. We consider several potential advantages to extending this surface to supported bilayers. Strong phosphate binding to the zirconium phosphonate surface could be used to lend stability to the supported bilayer structure. By including a percentage of phosphatidic acid groups in the bilayer, strong covalent linkages can be formed to the surface to anchor the assembly, increasing stability. Furthermore, our methods for preparing zirconium phosphonate modified surfaces can be extended to nearly any surface material including glass, gold or plastic, allowing the same chemistry for preparing the bilayers to be used for different supports. Often different analytical techniques require specific surfaces, such as gold for SPR, glass or quartz for fluorescence, or silicon for IR spectroscopy, and many procedures for preparing supported bilayers are specific for one surface or another. A technique that can be used for different supports can be helpful. Finally, it can be beneficial to identify supports that can be used to immobilize different biomolecular structures without extensive modification. Zirconium phosphonate has already been used to directly immobilize DNA, 67 phosphopeptides, 149 and phospholipids 150 and we show here that it can also be used for supported lipid bilayers. 99

100 Two methods for constructing supported lipid bilayers on zirconium phosphonate modified surfaces are illustrated in Figure 5-1. Different procedures are available for modifying surfaces with zirconium phosphonate layers, but we have long utilized LB methods in our laboratory and find this process to be efficient and highly reproducible. 71,76 Solid supports (gold or glass) are first rendered hydrophobic then coated with the zirconium phosphonate film. 76 Vesicle fusion to form symmetric bilayers is similar to procedures used on other surfaces. 139,158 Asymmetric lipid bilayers are formed by LB transfer of the inner lipid layer onto the zirconium phosphonate surface followed by fusion of vesicles of different lipid composition to add the distal layer. Figure 5-1. Schematic showing symmetric and asymmetric lipid bilayers on zirconium octadecylphosphonate (ODPA) modified surfaces. a) Vesicle fusion to form symmetric bilayers b) Langmuir-Blodgett transfer of the inner lipid layer followed by vesicle fusion to form asymmetric lipid bilayers Interactions between the zirconium phosphonate monolayer and lipid layers were studied by ellipsometry and X-ray photoelectron spectroscopy (XPS). Vesicle fusion 100

101 and the stability of the lipid assemblies under various conditions were investigated by surface plasmon resonance enhanced ellipsometry (SPREE). In addition, fluorescence recovery after photobleaching (FRAP) was used to study the fluidity of the supported membrane on the zirconium phosphonate film. To validate the model as a membrane mimic, interaction with the membrane protein melittin was studied as the composition of the lipid bilayers was changed, reproducing trends observed with other supported lipid bilayer systems. Experimental Section Materials Reagents were obtained from commercial sources and used as received. The monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocoline (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphorac-(1-glycerol)] (POPG) and 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4- yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased in chloroform from Avanti Polar lipids (Alabaster, AL). The refractive index matching fluid diodomethane and zirconyl chloride, 98%, were purchased from Sigma-Aldrich (St. Louis, MO). The buffer component trizma hydrochloride, sodium dodecyl sulfate (SDS) and ethanol were from Sigma. Slides used for SPREE experiments were made of 28.5 nm of gold evaporated on a 4 nm chromium adhesion layer on a clean SF10 glass slide (Schott Glass). Glass microscope slides for FRAP experiments were from Gold Seal, Portsmouth, N.H. Milli-Q water with a resistivity of 17.9 MΩ.cm was used for all experiments. The buffer used throughout the experiments was a 10 mm trizma hydrochloride and 100 mm sodium chloride solution, ph 7.4. The peptide melittin was obtained from Prof. G. E. Fanucci and used in the tris buffer. 101

102 Substrate Preparation Gold slides were cleaned using a solution of 15% ammonium hydroxide, 15% hydrogen peroxide and 70% water at C for 5 min then rinsed with milli-q water and dried with a flow of nitrogen. Gold slides were then plasma cleaned for 10 min, using a Harrick Plasma cleaner/sterilizer. Slides were immersed into 1 mm octadecylmercaptan (ODM) solution in ethanol for 16h to be rendered hydrophobic. Then, the substrates were rinsed with ethanol and dried under nitrogen. Glass slides were rendered hydrophobic with an octadecylltricholorosilane layer. Zirconium Phosphonate Modified Surfaces Zirconium phosphonate monolayers were prepared using a multiple-step Langmuir-Blodgett deposition technique. 71 Monolayers were transferred using KSV 3000 Teflon-coated LB trough with hydrophobic barriers (KSV Instruments, Stratford, CT). The surface pressure was measured by a filter paper Wilhelmy plate. The aqueous subphase was 2.6 mm of CaCl 2 adjusted to ph 7.4 with a potassium hydroxide solution. Octadecylphosphonic acid was spread from a 0.30 mg/ml chloroform solution in the LB trough. The solvent was allowed to evaporate for 10 min and the monolayer was compressed at 10 mn min -1 to reach a surface pressure of 20 mn min -1. Hydrophobic substrates were then dipped down through the monolayer surface at 8 mm min -1 into a vial sitting in the subphase, transferring the layer. The vial containing the slide was removed from the trough and 3 mm of zirconyl chloride was added to bind a monolayer of Zr 4+ ions at the organic template. After four days, the slide was rinsed with water and kept in water before being used. 102

103 Lipid Vesicle Solutions and Formation of Supported Lipid Bilayers Different compositions of DPPA, POPC and POPG lipid mixtures were used to form the small unilamellar vesicles. 9,159 The chloroform stock solution of the lipid mixture was dried via a nitrogen stream to form a uniform dry lipid film. The film was hydrated in tris buffer to obtain a concentration of 0.5 mg/ml with gentle vortex mixing leading to multilamellar vesicles. Five freeze/thaws were performed on the lipid solution to obtain large unilamellar vesicles. The lipid suspension was extruded 11 times through polycarbonate membranes with a pore diameter of 100 nm. Small unilamellar vesicles were also prepared by sonication for 3 10 min. During extrusion, the temperature was kept above the gel to liquid crystalline phase transition temperature. Vesicles were usually used within one day of preparation. Figure 5-2. Chemical structure of phospholipids POPC, POPG and DPPA. Symmetric supported lipid bilayers were formed by the adsorption and rupture of phospholipid vesicles directly on the zirconium phosphonate surface in a flow cell. Two steps were used to form asymmetric supported lipid bilayers. After the formation of the 103

104 zirconium phosphonate layer by LB deposition, the proximal monolayer was also formed using the LB technique. A 1 mm lipid solution in 3:1 chloroform/ methanol (v/v) was spread on pure water. After evaporation of the organic solvent, the monolayer was compressed at a speed of 10 mn min -1. Lipid monolayers were deposited on the hydrophilic surface at a constant surface pressure of 30 mn m -1 by pulling the substrate upward through the air-water interface at 8 mm min -1. The distal monolayer was formed by vesicle fusion in the flow cell. 160 Ellipsometric and X-ray Photoelectron Spectroscopy (XPS) Measurements Ellipsometry was used to characterize multilayer thin films and particularly the thickness of the inner layer of the supported lipid bilayer formed by the LB method. Ellipsometric angles and spatially resolved contrast images were acquired using a commercial EP 3 -SW imaging system (Nanofilm surface analysis, Germany). The ellipsometer employed a frequency-doubled Nd:YAG laser (adjustable power up to 20 mw) at 532 nm. Ellipsometry measures two parameters, Ψ and Δ that describe the change in light polarization. The values are related to the ratio of complex Fresnel reflection coefficients for parallel and perpendicular polarized light. For each layer, the angle of incidence varied from 54 to 71 and Δ values were recorded at every 0.2. Using Fresnel equations and an appropriate model, layer thicknesses can be determined. ρ = Rp R s = tan Ψ exp(jδ) (5-1) Δ = δ 1 δ 2 (5-2) tanψ = Rp R s (5-3) 104

105 XPS was performed using a UHV XPS/ESCA PHI 5100 system. Survey scans and multiplex scans (Au 4f, P 2p3, Zr 3d) were taken with a Al Kα X-ray source using a power setting of 300W and a takeoff angle of 45 with respect to the surface. Peak areas were determined using commercial XPS analysis software and Shirley background subtraction. SPR Enhanced Ellipsometry (SPREE) SPR provides the means to quantify the equilibrium constants and kinetic constants in very sensitive and label-free biochemical experiments. 161 Under SPR conditions, ellipsometric parameters give a larger enhancement of detection sensitivity compare to simple SPR techniques. 41,162 The minimum detectable signal change of the instrument used in this work was about 10 millidegrees in Ψ which results in a thickness precision of 0.1 nm. The angle of incidence that provides the highest sensitivity in Ψ measurements was chosen and was between 64 and 66 for the kinetics experiments. The ellipsometer, described in an earlier section, was coupled with an SPR cell to undergo SPR experiments in air or in liquid. Linearly p-polarized light was directed through a 60 equilateral prism coupled to a gold coated glass slide via diodomethane oil as an index matching fluid in the Kretschmann configuration. 163 After the vesicles were injected into the flow cell, the flow was stopped for 30 min to 1h to allow for vesicle fusion to occur on the surface. The formation of the lipid bilayer membrane was monitored by recording Ψ data versus time. Buffer was flowing for 30 min to remove free vesicles. The analysis programs AnalysR (Nanofilm, Germany) and BIAevaluation (Biacore) for the two-state model were used to further fit the experimental results. 105

106 Fluorescence Recovery after Photobleaching FRAP experiments were carried out on a confocal laser scanning microscopy system consisting of an Olympus IX-70 inverted microscope with an Olympus Fluoview 500 confocal scanning system. The NBD-POPC was excited with the 458-nm line of an argon ion laser, and emission was detected using a 505-nm long pass filter. A 20x objective lens was used for lipid bilayers and images were recorded using a CCD camera. The bleaching time was set to 25 s and background-corrected intensities of the bleaching spot were determined for each image taken until maximum recovery of fluorescence. A modified glass substrate was fixed at the bottom of the flow cell. Generally, 1 ml suspensions of 0.75 mg/ml lipid vesicles, fluorescently labeled with 2% NBD-PC, were applied. After 30 min of incubation with the vesicle solutions, the surface was washed extensively with tris buffer and the FRAP experiment was performed. Diffusion coefficients (D) and mobility (M) were obtained using the following equations: D = w2 (5-4) 4τ 1/2 M = F F 0 F i F (5-5) For which w is the width of the beam, τ 1/2 is the half-time of the fluorescence recovery, F 0 is the fluorescence before bleaching, F i is the fluorescence right after bleaching, and F is the fluorescence after recovery. 106

107 Results Symmetric Supported Lipid Bilayers The SPREE technique and a scheme of the different components of the supported bilayer assembly are shown in Figure 5-3. As shown on Table 5-1, a seven-layer model was used to fit the experimental data and calculate the thickness of the lipid bilayers assembled onto the zirconium phosphonate surfaces by vesicle fusion. The optical parameters for SF10, Cr, Au, ODM and buffer have been widely studied in the past. 164 The thickness of each of these layers was found by ellipsometry (Table 5-1), confirming the fabrication specifications of the Cr and Au layers and expectations from the literature for the ODM and zirconium octadecylphosphonate layers. 71,164 The experimental data were fit using AnalysR to the seven-layer model based on the Fresnel equations to obtain the bilayer thicknesses. Vesicles of POPC or POPG were used containing variable amounts of DPPA, chosen because the terminal divalent phosphate is expected to covalently bind to the zirconium phosphonate network. An objective was to determine appropriate percentages of DPPA that lend stability to the bilayers but are also low enough so that DPPA does not dominate the bilayer characteristics. By keeping the concentration of DPPA less than 50%, the fluidity of the vesicles allowed vesicle fusion. Lipid vesicles with a concentration of 0.5 mg ml -1 in tris buffer were introduced in the flow cell and Ψ values were recorded over time. SPREE sensorgrams for three different concentrations of DPPA mixed with POPC are shown on Figure 5-4. The kinetic curves are characterized by a baseline, absorption, and lipid desorption domains. There is an increase in Ψ corresponding to the binding and fusion of vesicles. After the signal stabilized, we assumed complete formation of the supported lipid 107

108 bilayers and running buffer was flowed through in the cell, causing a small decrease in Ψ corresponding to desorption of free vesicles in most experiments. Using the Fresnel equations, the seven-layer model was fit to the data to calculate the overall thickness of the lipid bilayers, reported in Table 5-2. The SPREE determined bilayer thicknesses range from 4.23 to 5.18 nm for all compositions studied and are within the range normally reported for supported lipid bilayers. 137 The time constant for vesicle adsorption increases as the concentration of DPPA increases. This result is explained by the higher gel to liquid crystalline transition temperature of saturated DPPA, which increases the rigidity of the lipid assembly, leading to slower kinetics. 165 Figure 5-3. Schematic of the SPREE experimental set-up showing the seven layers that correspond to the optical parameters in Table 1. Table 5-1. Optical parameters for the multi-layer model material refractive index extinction thickness (nm) coefficient Medium SF layer 1 Cr layer 2 Au layer 3 ODM layer 4 Zr-ODPA layer 5 lipid bilayer unknown Medium buffer

109 Figure 5-4. Monitoring fusion of POPC vesicles with different concentrations of DPPA to form symmetric supported lipid bilayers Table 5-2. Thicknesses and adsorption times of symmetric lipid bilayers on zirconium phosphonate modified gold slides lipid d (nm) adsorption time (min) POPC 5.18 ± POPC/DPPA (10%) 4.64 ± POPC/DPPA (50%) 4.23 ± Bilayer Stability Studied by SPREE The stability of supported lipid bilayers is an important concern. Confidence in the integrity of the model membranes following various stresses greatly increases their utility. In the present system, it was thought that occasional covalent linkages between DPPA and the zirconium phosphonate surface would greatly enhance the stability of the lipid bilayers. To test this idea, bilayers containing 10% DPPA were chosen, as this composition demonstrated good miscibility with other lipids at room temperature and was shown in Figure 5-4 to lead to fast formation of symmetric lipid bilayers. This ratio also complements the composition of mammalian cell plasma membranes, which often consist of 10-20% anionic lipids. 109

110 Following vesicle fusion and rinsing with buffer the effect of solvents and dehydration on bilayer stability were explored. Figure 5-5 shows the influence of ethanol and sodium dodecyl sulfate (SDS), an ionic detergent used for the permeabilization and solubilization of biological and model membranes. A low concentration of ethanol (5 %) has no effect on the POPC/DPPA supported lipid bilayer as no membrane is removed from the surface. The same results were obtained for POPC/POPG supported lipid bilayers (data not shown). On the other hand, SDS does remove some of the bilayers. A low concentration of SDS (0.5 %) removed 32% of the POPC/DPPA membrane and 2% of SDS removed 58% of the same membrane. POPC/POPG appeared to be less stable with 66% of membrane removed with 2% of SDS. Normally complete removal of the bilayers is expected. The fact that significant portions remain is evidence for a strong interaction with the zirconium phosphonate surface. The stability of the lipid bilayers to dehydration was also investigated within the SPREE flow cell. The 10% DPPA in POPC bilayer was compared to systems composed of only POPC or POPG lipids. Following vesicle fusion and rinsing with buffer, the bilayers were exposed to air for 200 minutes and rehydrated. The SPREE responses for each of the three systems are reported in Figure 5-6 in which dehydration is seen as a reduction in Ψ of around 7. Upon rehydration, the SPREE signal is recovered only for the DPPA-containing bilayer. According to Figure 5-7, 81% of the POPC membrane is removed from the surface and 96% of the POPG membrane is removed. In contrast, the bilayer containing 10% DPPA appears stable to dehydration, recovering greater than 95% of the membrane. 110

111 Figure 5-5. SPREE analysis following the effect of different solvent conditions on the stability of POPC/DPPA supported lipid bilayers. The SPREE measurement used an angle of incidence of Curves for the SDS solvent are shifted by 0.4 Figure 5-6. Effect of dehydration on a) POPC supported lipid bilayers, b) POPG supported lipid bilayers, c) POPC/DPPA supported lipid bilayers followed with SPREE. The angle of incidence was kept at

112 Figure 5-7. Zoom on the SPREE experiments studying dehydration effect on a) POPC supported lipid bilayers, b) POPG supported lipid bilayers, c) POPC/DPPA supported lipid bilayers followed with SPREE. The angle of incidence was kept at Asymmetric Supported Lipid Bilayers Inner lipid monolayer Monolayers of DPPA or a mixture of POPC/DPPA are easily transferred to the zirconium phosphonate surface using standard Langmuir-Blodgett deposition methods to form the inner layer of an asymmetric bilayer assembly. The complete process was characterized by XPS and ellipsometry for a DPPA monolayer. The gold XPS signal appears larger in the starting zirconium phosphonate layer and is attenuated after the lipid layer is added (Figures 5-8 and 5-9), showing clearly the presence of the lipid monolayer. The coverage of the lipid molecules can be estimated using the ratio of P to Zr, Table 5-3, based on the known Zr 4+ and ODPA coverage in the zirconium phosphonate modifying layer. 71 The zirconium phosphonate layer on gold shows a P to Zr ratio of 0.95 to 1 (Figure 5-8) which is consistent with the expected 1:1 stoichiometry 112

113 after considering the attenuation length of the photoelectrons and the depth of each species present in the film. 166 After transferring the lipid monolayer onto the zirconium phosphonate film, the P:Zr ratio is 1.6:1. This compares well with the expected stoichiometry of a DPPA monolayer on the zirconium phosphonate surface given the cross-sectional area per molecule of ODPA is 24 Å 2 76 and the cross-sectional area of DPPA is 40 Å 2 per molecule. The LB deposited lipid monolayer assembly was also characterized with ellipsometry in air. The thickness of the ODM, zirconium phosphonate, and DPPA layers were measured independently by ellipsometry, applying a seven layer model similar to that described in Table 5-1. The resulting thickness of the DPPA lipid monolayer, 2.42 nm, is consistent with the thickness of a lipid monolayer. 167 Figure 5-8. XPS multiplex spectra of the zirconium phosphonate layer before (top) and after (bottom) a DPPA monolayer is deposited Table 5-3. XPS analysis of the zirconium phosphonate and lipid monolayer films film type calculated relative intensities a surface coverage Zr 3d P 2p3 (mol cm -2 ) zirconium phosphonate 52 % 48 % DPPA monolayer on zirconium phosphonate 38 % 62 % a Relative intensities are determined from experimental peak areas normalized with atomic and instrument sensitivity factors. 113

114 (a) (b) Figure 5-9. XPS survey scan of zirconium phosphonate surface (a) and DPPA monolayer on zirconium phosphonate surface (b). 114

115 Fusion of lipid vesicles on the inner monolayer studied by SPREE Asymmetric lipid bilayers were formed by fusing lipid vesicles onto the hydrophobic surfaces generated after LB deposition of the inner DPPA or POPC/DPPA monolayers. Vesicle solutions formed from POPC and POPC/POPG (70/30) were introduced via the flow cell and the formation of the outer monolayer was followed by SPREE (Figure 5-10). The kinetics for the two lipid systems are similar. Fitting the data using the model in Table 5-2, the thickness of the outer POPC layer is 2.63 ± 0.01 nm and the outer mixed POPC/POPG layer is 2.66 ± 0.01 nm, consistent with the deposition of a monolayer via vesicle fusion. Fluorescence imaging also supports effective vesicle fusion. A DPPA monolayer on a zirconium phosphonate modified glass slide was placed in the fluorescence flow cell and the bilayer was completed by the fusion of POPC vesicles doped with 2% of the fluorescent lipid NBD-PC. Fluorescence images of free vesicles and of the outer lipid monolayer are shown in Figure The presence of non fused vesicles is clearly discerned from a uniform layer following vesicle fusion. 168 Figure SPREE analysis of the formation of the outer monolayer by vesicle fusion onto a POPC/DPPA monolayer. The angle of incidence was kept at The curve for POPC/POPG is shifted by

116 Figure Fluorescent images of vesicle fusion to form asymmetric bilayers with a DPPA inner layer. a) Incomplete vesicle fusion and b) Homogeneous fluorescence following complete vesicle fusion and rinsing FRAP analysis of supported lipid bilayers Lateral diffusion FRAP analysis was performed to determine the fluidity of the model membranes. FRAP experiments were performed on zirconium phosphonate modified glass slides using a confocal microscope with lipid vesicles doped with a small amount of fluorescent lipid, and the diffusion coefficient and mobile fraction of the lipids were determined from the FRAP curves (Figure 5-12). The lateral fluidity of POPC lipid bilayers on zirconium phosphonate was compared to the same lipid system on a glass surface. For POPC doped with 2% NBD-PC, the average diffusion coefficient for bilayers on glass was determined to be 3.98 ± 0.11 μm 2 s -1, with a mobile fraction of 77.5% (Table 5-4). This value agrees well with previous results reported for lipid bilayers on glass. 169 On the other hand, for the same bilayer on the zirconium phosphonate film, the calculated diffusion coefficients were 5-fold lower (0.72 ± 0.04 μm 2 /s). Moreover, the mobile fraction was also very low, around 35%. These results indicate that the lipid bilayers have low fluidity on the zirconium phosphonate surface. The strong interaction with the zirconium phosphonate surface will be through the proximal layer, so to study the behavior of the distal layers, the fluidity of the asymmetric bilayers was investigated by including the fluorescent probes in only the outer layer, 116

117 assuming minimal exchange of lipids between layers. 141 The lateral diffusion of the outer layer is significantly higher, 2.86 ± 0.16 μm 2 /s, well within the range associated with fluid lipid bilayers. 8 The mobile fraction was also high, 76.0%, comparable to other of lipid bilayers with high fluidity. Figure Representative FRAP experiments performed on POPC lipid bilayers (a,b) or monolayer (c) doped with 2% NBD-PC. POPC vesicles were fused on a) glass, b) the zirconium phosphonate surface, and c) a DPPA monolayer previously transferred onto the zirconium phosphonate surface Table 5-4. FRAP parameters of POPC doped with 2% NBD-PC type of support fusion of POPC vesicles diffusion coefficient (μm 2 s -1 ) mobile fraction (%) glass bilayer 3.98 ± ± 0.8 Zr-ODPA film bilayer 0.72 ± ± 0.7 DPPA monolayer on Zr-ODPA film monolayer 2.86 ± ±

118 Protein Binding to Supported Lipid Bilayers Insertion and folding of proteins are among many cellular processes involving cell membranes. The ability to study such processes using accurate model systems is an important objective of artificial supported lipid bilayers. Therefore, the ability of the zirconium phosphonate supported lipid bilayers to bind a membrane protein was investigated. Melittin, a major component in honey bee venom, was selected as it is a well studied small protein and melittin-lipid membrane interactions have already been explored. 55, Depending on the composition of the lipid membrane, melittin acts in two different ways. It forms transmembrane pores in zwitterionic phospholipid membrane via the barrel-stave mechanism and acts in a detergent-like manner on negatively charged membranes. 171 The membrane lipid composition has a strong influence on the melittin-lipid interaction, so we varied the zwitterionic and anionic lipid composition in asymmetric supported bilayers and monitored the binding of melittin to the lipid membrane. To analyze the binding kinetics, the sensorgrams were analyzed by curve fitting using numerical integration analysis. 54 The binding mechanism of melittin to supported lipid membranes can be represented by a two-step reaction model. 55 The first step is the initial binding of the protein to the membrane surface, and the second is the insertion of melittin in the lipid membrane. The two-state reaction model can be represented as: P + L k a1 k a2 PL PL k d1 k d2 (5-6) where in the first step, the protein (P) binds to the lipids (L) to give PL, and the complex PL is then changed to PL* in the second step, which cannot dissociate directly 118

119 to P+L. The corresponding differential rate equations for the two-state reaction model are represented by: dψ 1 dt = k a1 [P] (Ψ max Ψ 1 Ψ 2 ) k d1 Ψ 1 k a2 Ψ 1 + k d2 Ψ 2 (5-7) dψ 2 dt = k a2 Ψ 1 k d2 Ψ 2 (5-8) where [P] is the protein concentration, Ψ 1 and Ψ 2 are the response units for the first and second steps, respectively and Ψ max is the equilibrium binding response. The association and dissociation rates for the first and second steps are k a1, k d1, k a2 and k d2. The association equilibrium or affinity constants for the first and second step are K 1 and K 2 and equal k a /k d. The total affinity constant K A (M -1 ) is the product K 1 K 2. Asymmetric bilayers were formed of POPC, POPG and POPC/POPG (70/30 w/w) outer layers with a POPC/10% DPPA LB inner layer. The asymmetric supported lipid bilayers were then exposed to 10 μm melittin and the peptide-lipid interaction was followed by SPREE in real time. Typical sensorgrams of the binding of melittin to the lipid membrane are shown on Figure The adsorption and desorption steps of the protein binding on the membrane are easily defined and using the two-step kinetic model the kinetic and equilibrium parameters were determined. The kinetic analysis was performed at least three times for each lipid composition and the averaged values for the rate constants and affinity constants are listed in Table 5-5. The affinity constant for melittin binding to POPG is almost 5-fold higher than the one measured for zwitterionic lipids, Also, the association rate constant for the first step is 4-fold higher for the anionic POPG and 2-fold higher for the mixed membrane than for zwitterionic POPC layer. This result indicates that electrostatic interactions control the 119

120 binding affinity of melittin to anionic lipids, consistent with what has been seen before, as melittin in a cationic form is attracted to anionic lipids via direct and fast electrostatic interactions. For the POPG layer, the association rates for the second step are slower, agreeing that the binding between melittin and anionic lipid membrane is controlled by the first step. The interaction between melittin and zwitterionic lipids is normally thought to be driven predominantly by hydrophobic interactions. However, for the zwitterionic (POPC) and mixed lipids (POPC/POPG) layers prepared here, the affinity constants are not very different, suggesting that the previously observed insertion into the membrane is incomplete. This result is similar to work previously observed for lipid monolayer models. 55 Figure Sensorgrams of the binding of melittin to asymmetric lipid bilayers with zwitterionic (POPC), anionic (POPG) and mixed lipid (POPC/POPG) outer layers. Peptide concentration = 10 μm. The data were fit using the two-state reaction model. The angle of incidence was kept at Curves for POPC/POPG and POPG are shifted by and 0.05, respectively 120

121 Table 5-5. Equilibrium and kinetic parameters for melittin adsorption to asymmetric bilayers using the two-state reaction model fit to the SPREE data a outer lipid layer b k a1 ( 10 4 M -1 s -1 ) k d1 (s -1 ) K 1 ( 10 4 M) k a2 (s -1 ) k d2 (s -1 ) K 2 K A ( 10 5 M -1 ) POPC POPC/POPG POPG a Data were averaged over three experiments b All lipid compositions are adsorbed onto a POP/DPPA (10%) lipid monolayer. Discussion The goal of the study was to demonstrate that zirconium phosphonate modified surfaces are practical substrates for supported lipid bilayers. Zirconium phosphonates have previously been shown to selectively bind phosphate terminated oligonucleotides, 67,70 phosphorylated peptides, 149 and phospholipids, 150 in applications that include bioarrays 67,70 and protein pull-down. 149,176 An important biomaterials objective is to develop surfaces and interfaces that can be used for a variety of applications without the need for function-specific pretreatment. We move closer to that objective by extending the utility of the zirconium phosphonate surface to supported lipid bilayers. Vesicle fusion is demonstrated to indeed produce supported bilayers on the zirconium phosphonate surface. As Figure 5-4 demonstrates, bilayer formation takes place on the same timescale as on other hydrophilic surfaces. 139,177,178 The rate of bilayer formation varies with lipid composition, but this observation is attributed to the different stiffness of the vesicles formed from different lipids. Increasing the percentage of the saturated DPPA slows down vesicle fusion on the surface. The zirconium phosphonate modified surface is terminated in oxide and hydroxide groups, and therefore provides a hydrophilic surface that interacts with the polar lipid 121

122 headgroups. At the same time, the Zr 4+ ions are known to form strong specific covalent bonds to dibasic phosphate and phosphonate groups, which can displace the oxide or hydroxide groups at the interface. However, less basic groups leave the zirconium ion coordination intact and are restricted to electrostatic and hydrogen binding interactions with the oxide/hydroxide groups. For example, we have previously shown that phosphate terminated oligonucleotides bind preferentially relative to modified DNA segments containing only phosphodiester groups. 66 We therefore decided to incorporate a phosphatidic acid into the lipid assemblies to determine if the stronger covalent interactions expected between the dibasic phosphate of the lipid headgroup and the zirconium ions could lead to enhanced stability of the lipid bilayers. As with other hydrophilic surfaces, supported bilayers of POPC or POPG on the zirconium phosphonate surface are unstable to dehydration (Figure 5-6). On the other hand, including just 10% of DPPA results in supported bilayers that are stable to dehydration (Figure 5-5). Evidence for the influence of DPPA is seen upon rinsing with the surfactant SDS, which normally removes most lipid assemblies. In the case of the DPPA-containing bilayers, lipid removal is incomplete (Figure 5-7), suggesting a strong interaction with the surface. However, after SDS rinse, the residual lipid is far more than the few percent DPPA used to form the bilayer, just over 50% of the bilayer is removed with 2% SDS. By fluorescently labeling the inner layer of an asymmetric bilayer, it is seen that SDS rinsing leaves the bottom layer attached to the surface (Figure 5-14). Even though only 10% DPPA is included in the layer, the whole layer remains, implying that covalent linkage of only a few molecules is enough to stabilize a larger cooperative lipid assembly on the surface. 122

123 The zirconium phosphonate strategy complements other recent examples of airstable bilayer systems. Jeon et al. formed a membrane with incorporated proteins that is completely enclosed in a hydrogel mesh. 146 Deng and coworkers created supported lipid bilayers supported by cholesterol groups. 147 A fluid and air stable lipopolymer membrane 145 was reported by Cremer and coworkers that mechanically strengthens membranes and increases its longevity three-times longer than regular lipid bilayers. Cheng et al. 144 studied the stability of various supported membranes (PC, DOPC, PE and more) on a nanoglassified substrate. A consequence of the stabilizing lipid-zirconium phosphonate interaction is reduced mobility of the lipids. The FRAP-determined lipid mobility of the bilayers is significantly smaller than reported for other supported bilayer models. 169 Even for the symmetric POPC bilayer, which does not include the covalent linkages to the support expected with phosphatidic acid, mobility is low. However, the reduced mobility is principally in the inner layer, and the outer leaflet is only minimally impacted by the surface-lipid interaction of the inner layer. By using Langmuir-Blodgett deposition of the inner layer followed by vesicle fusion to deposit just the outer layer, the stabilizing effect of the covalent interaction between DPPA and the zirconium surface is retained while permitting the composition of the outer layer to be varied. Indeed, lipid mobility and mobile fraction of the outer layer in the asymmetric bilayers is comparable to other viable model systems. 169,179 The viability of using the zirconium phosphonate supported bilayers as membrane mimics was demonstrated using the membrane binding peptide, melittin. Composed of 26 amino acids, residues 1-20 are predominantly hydrophobic and are 123

124 hydrophilic. 180 The latter region has four positive charges that bind to anionic lipids. The association of melittin with phospholipid bilayers has been shown to involve a two step binding mechanism. The initial binding step, K 1, is followed by insertion into the bilayer to form hydrophobic interactions. Electrostatic interactions dominate the binding of melittin to negatively charged membranes via K 1, whereas the protein forms subsequent hydrophobic interactions with zwitterionic membranes. 181 To perform the membrane binding analysis, asymmetric supported lipid bilayers were formed on the zirconium phosphonate surface with zwitterionic (POPC), anionic (POPG) and mixed (POPC/POPG) lipid outer layers. Previous studies have shown that melittin binding to supported bilayers is poorly described by the Langmuir binding model and that a two-state reaction model is more appropriate. As observed with previous bilayer membrane models, melittin associates faster with a significantly larger K 1 to the anionic POPG. Also in line with other model systems, the second (insertion) step is slower than the initial binding step for all membranes. At the same time, the insertion process, K 2, is more important for the zwitterionic POPC layer than for the POPGcontaining layers, in agreement with previous studies. The melittin binding studies yield similar results to those obtained with other supported bilayer model membrane systems. A feature of the zirconium phosphonate interface is that the chemistry is transferable to any commonly used analytical substrate. It is now routine to make common surfaces hydrophobic, and the zirconium octadecylphosphonate modifying layer can be deposited onto any hydrophobic surface. Therefore, the same biomolecule attachment chemistry can be used for analytical studies having different substrate requirements, including gold, silicon, glass, and plastic. This transferability lends 124

125 confidence when comparing results from different experiments that the system has not been perturbed by the different chemical transformations used to prepare different surfaces. A further convenience is that attachment to the zirconium phosphonate surface is via the phosphate group making it unnecessary to synthetically modify the biomolecules to be immobilized. Phosphopeptides, phospholipids, and oligonucleotides, as well as many proteins and DNA all naturally contain dibasic phosphate. Figure Fluorescence intensity of the inner lipid layer containing 10:1 POPC:DPPA doped with 2% NBD-PC upon rinsing with SDS. After the inner layer was deposited using LB methods, the outer POPC layer, without NBD-PC, was deposited by vesicle. The entire bilayer was exposed to SDS rinse Conclusions Zirconium phosphonate modified surfaces are shown to be a versatile platform for supported lipid bilayers. Inclusion of a small percentage of phosphatidic acid into the inner lipid layer to form covalent interactions with the zirconated surface significantly enhances the stability of the bilayer assemblies, retaining their structure following drying in air. A consequence of the strong surface-lipid interactions, the inner layer shows low fluidity; however the outer layer proves to be highly fluid, comparable to other supported 125

126 bilayer systems. The binding of melittin to bilayers containing anionic and zwitterionic lipids demonstrates that the supported lipid bilayers are a viable membrane mimic. Attractive features of the zirconium phosphonate modified surfaces include the ability to form symmetric or asymmetric bilayers on multiple substrates with composition control without requiring chemical modifications of the constituent lipids and the compatibility 126

127 CHAPTER 6 SKELETONIZED SURFACES FOR THE STUDY OF TRANSMEMBRANE PROTEINS IN SUPPORTED LIPID BILAYERS Introduction Cell membranes create a barrier between the cytoplasm compartment and the exterior, and communication is often driven by membrane proteins. Approximately 20-30% of the genome of organisms encodes for membrane-bound proteins 182,183 and account for two third of all protein drug targets However, membrane proteins are difficult to study in their natural environment because of the high complexity of cells. Also, due to the difficulties encountered to create a native like cell membrane, those proteins have been hard to characterize as models may prevent their lateral mobility and functionality. Supported lipid bilayers are interesting models to study cell membrane dynamics such as lipid diffusion, domain formation 187 and cell adhesion, 188 and interactions with membrane receptors. McConnell developed the formation of supported lipid bilayers on substrates by vesicle fusion. 9 This approach is now widely used to form other more complex systems used to study different processes such as signal transduction, ligand-receptor interactions and ion transport trough the membrane. 11,189 Many substrates have been used for the formation of supported lipid bilayers such as glass, mica, silicon, metal films and polymers. Interactions occur between the bilayers and the substrates trough van der Waals, electrostatic, hydration and steric forces and the membrane components properties can be perturbed. Membrane proteins, in particular, have a complex structure that contains large cytoplasmic and extracellular domains that protrude from the membrane and the close proximity of the substrate hinders the incorporation of transmembrane proteins. It can lead to denaturation of the proteins and decrease in mobility due to frictional coupling 127

128 with the support. Hence, it is important to develop models that reduce the substratebilayers interactions. Methods have been developed to increase the spacing between the membrane and the support such as cellulose films 19, polymer cushion 143,148,169,190,191, the use of nanoporous microbeads 192 or tethered molecules. 22,169, Interactions between the membrane and proteins have been investigated using lipid bilayers systems. For instance, a bacterial toxin forming channel was inserted in a solid supported membrane 200 and an ionic reservoir was created and showed to be functional in a tethered lipid membrane 201. Also, the transmembrane protein rhodopsin was incorporated into a laterally patterned membrane, with the preservation of its native functionality. 202 The present study was conducted using surface plasmon resonance enhanced ellipsometry (SPREE), this technique provides valuable kinetic information and surface coverage of protein binding to the lipid bilayers. In previous work, we developed a new approach to engineer stable supported lipid membranes. 203 This system uses a zirconium phosphonate modified substrate to tether the lipid membrane via a highly covalent bond between surface zirconium ions and divalent phosphate groups in lipids. As shown by the fluorescence recovery after photobleaching results, the inner layer of the supported lipid bilayer has low diffusion coefficient due to the interaction between lipid headgroups and the inorganic support. Moreover, the covalent interaction restricts the mobility and function of membrane components. In contrast to other approaches that elevate supported lipid bilayers from the support, our new model involves the creation of reservoirs beneath the membrane to incorporate transmembrane proteins. Skeletonized zirconium phosphonate surfaces have been designed using the Langmuir- 128

129 Blodgett technique. Skeletonized films yield holes in the zirconium phosphonate surface that the membrane can spam, increasing mobility and allowing incorporation of transmembrane proteins. To analyze the biological properties of this new model regarding transmembrane proteins, we performed binding studies and monitor the surface concentration of adsorbed ligand and binding affinities by SPREE. We have investigated the membrane binding process of two different proteins that play key roles in biological processes. The first one, BK ion channel, is expressed in a wide variety of fundamental cells including nerve cells by controlling firing patterns in neurons, muscle cells and endocrine cells. Ion channels are integral membrane proteins that create pores in the lipid membrane to transport ions through the membrane. The BK ion channel is a calcium activated potassium channel that possesses a seven transmembrane architecture. This ion channel is composed of a pore-forming tetrameric α dmain and a β domain comprising two α-helical transmembrane domains connected by a large glycosylated extracellular loop, with intracellular amino and carboxy termini, as can be seen on Figure 6-1. Figure 6-1. Topology maps of the BK ion channel showing the position of the α- and β- subunits (reprinted with authorization 204 ). 129

130 The second protein, integrin α 5 β 1, is part of the integrin family, cell adhesion molecules that interact with the extracellular matrix (ECM). Integrin-mediated adhesion leads to intracellular signaling processes that regulate cell survival, proliferation, and migration. Integrins can recognize a single ECM ligand, while others bind several different ECM proteins. In fact, the integrin α 5 β 1 binds to several ligands including fibronectin. Interactions mechanisms of these two proteins with the supported lipid bilayers on the skeletonized surface were analyzed by SPREE, and their binding to the membrane on the non-skeletonized surface was compared. The incorporation of the ion channel in the membrane was studied in real time and the functionality of the integrin was studied by the ligand binding assay. Experimental Section Materials 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoylsn-glycero-3-phospho-L-serine (POPS) were purchased from Avanti Polar Lipids (Alabaster, AL). Detergent-solubilized human integrin α 5 β 1 and bovine fibronectin were obtained from Chemicon (Temecula, CA). The refractive index matching fluid diodomethane and zirconyl chloride (98%) were purchased from Sigma-Aldrich (St. Louis, MO). Slides used for SPREE experiments were SF10 glass slide (Schott Glass) with 28.5 nm of gold evaporated onto 2 nm chromium adhesion layer. Glass microscope slides for the AFM experiments were from Gold Seal (Portsmouth, NH). Zirconium Phosphonate-Modified Surfaces Gold and glass slides were cleaned and then rendered hydrophobic by immersion in a 1 mm octadecylmercaptan solution in ethanol for 16 hours. Monolayers were then transferred using a KSV 3000 Teflon-coated LB trough with hydrophobic barriers (KSV 130

131 Instruments, Stratford, CT). A hydrophobic slide was dipped down through a Langmuir monolayer of either octadecylphosphonic acid to make a non-skeletonized surface or a mixture of octadecanol and octadecylphosphonic acid monolayer to form skeletonized films held at a constant pressure of 20 mn min -1 into a vial. Different mixtures were studied, from 0 to 50% in volume of octadecanol. The vial contains the slide immersed in the subphase was removed from the trough and zirconyl chloride was added to being a concentration of 3 mm to bind a monolayer of Zr 4+ ions at the organic template. The slide was rinsed with water and an additional ethanol rinse was performed on the mixed monolayers to remove the free alcohol molecules. Characterization of the skeletonized films was performed by atomic force microscopy. Supported Lipid Bilayers Unilamellar vesicles were formed following the same procedure as described previously. 203 Briefly, the chloroform lipid mixture was dried via nitrogen and the film was hydrated in tris buffer composed of 10 mm trizma hydrochloride and 100 mm sodium chloride at ph 7.4 to obtain a lipid concentration of 0.5 mg ml -1. After five freeze-thaw cycles, the lipid suspension was extruded 11 times trough polycarbonate membranes with a pore diameter of 100 nm. Supported lipid bilayers were formed by the adsorption and fusion of the vesicles on the hydrophilic modified surface. BK Ion Channel Expression Plasmid DNA encoding a histidine-tagged BK channel constructed with a C- terminal deletion from amino acid 347 was transcribed in vitro by the mmessage mmachine T7 ultra kit (Ambion, Austin, Texas). RNA was precipitated with LiCl, washed and centrifuged in ethanol (70%), dissolved in RNAse-free water and injected (46 nl per oocyte, ~ 50 ng RNA) into defolliculated stage V or VI oocytes maintained at C 19 in 131

132 ND 96 oocyte culture medium (96 mm NaCl, 2 mm KCl, 1 mm MgCl 2, 10 mm HEPES, 1.8 mm CaCl 2, ph 7.4) enriched with sodium pyruvate (2.5%), penicillin/streptomycin (1%) and horse serum (5%). Functional channel expression was verified by twoelectrode voltage clamping (TEVC). Oocytes expressing BK channel constructs were rinsed in high K buffer (400 mm KCl, 5 mm PIPES, ph. 6.8) supplemented with 100 µm phenylmethylsulfonylfluoride, 1 μm pepstatin, 1 µg ml -1 aprotinin, 1 µg ml -1 leupeptine, 1 µm p-aminobenzamidine and transferred to a 1 ml ground glass tissue grinder (Kontes Duall). Ground oocytes were solubilized using a buffer solution at a final concentration of 10 mm β-octyl glucoside containing 20 mm Tris buffer, 500 mm KCl and 20 mm imidazole, ph 7.5 with 5 µl/ml mammalian protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) added. The suspension was then agitated gently for 1 hour at 200 rpm on a platform shaker followed by centrifugation at 14,000 rpm at 4 C to separate the solubilized mixed micelles from cellular debris. 2 ml of the supernatant solutions were loaded onto 1 ml Histrap FF columns (GE Healthcare Life Sciences, PA, USA) for purification by immobilized metal ion affinity chromatography. Columns were equilibrated with binding buffer (10 mm β-octyl glucoside, 20 mm Tris buffer, 500 mm KCl and 20 mm imidazole, adjusted to ph 7.5) at a flow rate of 1 ml min 1 and washed with the same buffer. Unbound material was washed off using 5 column volumes of binding buffer, and then fractions of 1.5 ml eluted in a stepwise manner with an elution buffer containing 500 mm imidazole and all other components of the binding buffer at ph 7.4 and a flow rate of 1 ml min 1. Immunoblotting was used to determine the identity of purified protein samples. 132

133 Reconstitution of Integrin The proteoliposome preparation is based on a procedure described by Sinner et al. 195 A solution of 1 mg ml -1 POPC in chloroform was injected into a vial and dried via a nitrogen stream to a uniform dry lipid film. A volume of 20 μl at 100 μg ml -1 integrin α 5 β 1 was then added with 3mL of tris-mg buffer (10 mm trizma hydrochloride, 100 mm sodium chloride, 1 mm magnesium chloride, 0.2 mm calcium chloride, ph 7.4) and the mixture was vortexed for a few hours. The proteoliposomes solution was then extruded 11 times through polycarbonate membranes with a pore diameter of 100 nm. A volume of 2 ml of the proteoliposomes suspensions were used directly for vesicle fusion procedures done in the SPREE flow cell, the flow rate was 150 μl min -1. Functionality of the integrin was analyzed by the ligand binding assay to immobilized integrins. 195 Binding assays were conducted by adding fibronectin at a concentration of 15 μg ml -1 to the SPREE cell. After incubation for 30 min, the surface was rinsed with buffer until stabilization of the signal. Instrumentation Surface plasmon resonance enhanced ellipsometry (SPREE) measurements were performed on a commercial EP3-SW imaging system (Nanofilm Surface Analysis, Germany). The ellipsometer employed a frequency-doubled Nd:YAG laser (adjustable power up to 20 mw) at 532 nm. Linearly p-polarized light was directed through a 60 equilateral SF10 prism coupled to a gold-coated SF10 slide via diiodomethane index matching oil in the Kretschmann configuration. The angle of incidence was kept at 64 for all experiments because this condition provided the highest sensitivity. Curve fitting of the experimental data used the analysis programs AnalysR (Nanofilm) and BIAevaluation (Biacore) for the two-state model. 133

134 Atomic force microscopy imaging was performed in tapping mode using a Multimode AFM with a Nanoscope IIIa controller (Digital Instruments, Santa Barbara, CA) and silicon probes (Nanosensors, with dimensions: T= μm, W=26-27 μm, L=128 μm) were used. Results Characterization of the Skeletonized Zirconium Phosphonate Surfaces The Langmuir-Blodgett (LB) technique was used to form the skeletonized zirconium octadecylphosphonate modified surfaces (Figure 6-2). A mixed monolayer of ODPA and octadecanol was formed on the LB trough and deposited as a monolayer onto a hydrophobic support. Exposing the surface to Zr 4+ binds the metal ions to the surface, cross linking the phosphonate groups, leading to a stable monolayer. However, the alcohol molecules do not bind the Zr 4+ so that an ethanol rinse removes the free octadecanol leading to the skeletonized film. The skeletonized surfaces were then characterized by atomic force microscopy. Two different surfaces are shown on Figure 6-3, based on using 10% and 20% octadecanol which produced holes of approximately 100 nm and 500 nm, respectively. The holes in the surface can be spanned by lipid layers, as demonstrated upon deposition of an LB monolayer of POPC. Figure 6-4 shows AFM images of the lipid layer on the two surfaces derived from 10 % and 20% octadecanol. The lipid spans the holes, creating homogenous surfaces. If the holes are too large, such as those formed from a 50% octadecanol mixed layers, the lipid monolayer is unable to span the holes. 134

135 a) b) Figure 6-2. Schemes of the deposition technique of the skeletonized zirconium phosphonate surfaces for the incorporation of transmembrane proteins. The mixed monolayer is deposited by the LB technique on a hydrophobic surface. After incubation with zirconium ions, the surface is washed with ethanol to remove the non-covalently bound molecules (a). Deposition of the supported lipid bilayers on the skeletonized surface by vesicle fusion (b). a) b) Figure 6-3. Images of the skeletonized surfaces on a glass wafer by AFM. The support was rendered hydrophobic by an OTS layer and the skeletonized film was formed on the surface with different 1-octadecanol percent concentrations of 20% (a) and 10% (b). Image size (z-scale), 20 or 10μm (20 nm). 135

136 a) b) Figure 6-4. Images of the POPC lipid monolayer on the skeletonized surfaces on a glass wafer by AFM. The lipid monolayer was deposited by the LB technique on the skeletonized surfaces derived from 20% (a) and 10% (b) 1- octadecanol. Image size (z-scale), 10 μm (20 nm). The spanning of the holes is demonstrated by the smoothness of the surfaces. Incorporation of BK Ion Channel in Supported Lipid Bilayers To test the viability of the supported lipid bilayers, the BK ion channel protein was exposed to the bilayer and its incorporation was monitored using SPREE. Mixed POPC/POPS (80/20) lipid bilayers were formed on the skeletonized support by vesicle fusion. Figure 6-5 shows the formation of supported lipid bilayer followed by the binding studies of BK ion channel on skeletonized surfaces derived from 10% and 20% octadecanol. For the different supports, the formation of the supported lipid bilayer by vesicle fusion can be observed by the increase and stabilization of the ellipsometric signal. The increase of the signal corresponds to a thickness of around 5 nm which is consistent with the formation of lipid bilayers, using a value of 1.45 for the refractive index of the lipid membrane. After rinsing away the free vesicles with buffer, the BK channel was introduced above the supported lipid bilayer in tris buffer. An increase in the psi signal is observed, confirming the binding of the ion channel to the lipid bilayers. After the signal stabilized, desorption of non-incorporated proteins was performed by buffer rinsing. As shown in Figure 6-5, the desorption was greater for the skeletonized 136

137 surface derived from 10% octadecanol, with 65% of the ion channel desorbed, whereas only 38% of the protein was removed from the skeletonized surface derived from 20% octadecanol. For comparison, Figure 6-6 shows the control experiment at a supported lipid bilayer on a non-skeletonized zirconium octadecylphosphonate surfaces. After binding of the BK ion channel, the protein is completely removed from the membrane by rinsing with buffer. The full desorption of the transmembrane protein indicates that the protein could not incorporate into the membrane that is in close contact with the inorganic support. The kinetics of binding of BK ion channel on supported lipid bilayers was analyzed by curve fitting using numerical integration analysis. As a transmembrane protein, the binding of BK channel to the lipid membrane can be represented as a two-step interaction. 205 The first step describes the binding of the protein to the membrane surface and the second step is the incorporation of the BK channel into the membrane. The two-state reaction model can be represented as: k a1 k a2 P + L PL PL (6-1) k d1 k d2 where in the first step, the protein (P) binds to the lipid (L) to give PL, and the complex PL is then changed to PL* in the second step, which cannot dissociate directly to P and L. The association and dissociation rates for the first and second steps are k a1, k d1, k a2, and k d2. The affinity constants for the first and second step are K 1 and K 2, respectively, and equal k a /k d. The total affinity constant K A (M -1 ) is the product K 1 K 2. Kinetic data were calculated using the two-step interaction model for the bilayers on the skeletonized films and for the control (Table 6-1). The kinetic constants indicate that the 137

138 second step is far faster for the membrane that is supported on the skeletonized surfaces. In addition, the affinity constant is much higher for the protein inserted into the membranes supported on the skeletonized film. (a) (b) Figure 6-5. Incorporation of the BK ion channel into a lipid membrane supported on skeletonized surfaces. The kinetics study shows the formation of supported lipid bilayers followed by the insertion of transmembrane proteins as followed by SPREE. After the formation of the POPC/POPS lipid bilayers on the skeletonized films derived from 10% (a) and 20% (b) octadecanol, free vesicles were removed with buffer rinsing. The membrane was then incubated with the ion channel and binding interaction was monitored. The buffer used through experiments consisted of trizma hydrochloride and sodium chloride at ph 7.4. Figure 6-6. Incorporation of the BK ion channel into a lipid membrane supported on non-skeletonized surfaces. The kinetics study shows the formation of POPC/POPS supported lipid bilayers followed by the binding of transmembrane proteins as monitored by SPREE. 138

139 Table 6-1. Equilibrium and kinetic parameters for adsorption of BK ion channel to PCPS lipid membrane using the two-step binding model fit to the SPREE data a k a1 k d1 K 1 k a2 k d2 K 2 K A ( 10 3 M -1.s -1 ) ( 10-1 s -1 ) ( 10 4 M -1 ) ( 10-2 s -1 ) ( 10-5 s -1 ) ( 10 6 M -1 ) % skel. 10% skel. control a Data were averaged over three experiments. Binding of Fibronectin to Integrin α 5 β 1 The function of the integrin α 5 β 1 in the supported lipid bilayers was studied using a binding ligand assay. In this experiment, the binding capacity of the integrin receptor in the membrane to the ligand fibronectin is studied by SPREE. The skeletonized surface was immersed in a solution of integrin α 5 β 1 -POPC vesicles and formation of the proteincontaining bilayer was followed by SPREE (Figure 6-7). The experimental data were fit using AnalysR and the optical parameters from Table 6-2 to a seven-layer model based on the Fresnel equations. The optical thickness of the integrin-functionalized membrane over multiple experiments is 10.1 nm, which corresponds well to related measurements. 195 Upon exposure to fibronectin, the surface concentration of the bound ligand is calculated by a method previously reported 206 and used the following equation: Γ = 3d (n 2 n b 2 ) (n 2 + 2)(r(n b 2 2) υ(n b 2 1)) (6-2) where Γ is the surface concentration of the protein, d the layer thickness, n b and n, the refractive indexes of the buffer layer and the adsorbed layer, respectively, and r is the specific refractivity of fibronectin (0.243 ml g -1 ) and υ is the partial specific volume of fibronectin (0.729 ml g -1 ). 207,

140 After rinsing away free proteoliposomes, addition of fibronectin results in a binding signal of 0.69, corresponding to 51 ng cm -2 (Figure 6-7a). This surface concentration represents fibronectin partial coverage of 19%. Uptake of fibronectin indicates that transmembrane protein receptors are functional. On the other hand, insignificant binding signal of 0.17 was observed when the integrin -functionalized lipid bilayer was formed on the non-skeletonized surface (Figure 6-7b). Also, as a further control, a POPC lipid bilayer without the integrin on the skeletonized surface was incubated with fibronectin. As expected, the ligand does not bind the membrane surface without the integrin receptor (Figure 6-8). (a) (b) Figure 6-7. Binding of fibronectin to an α 5 β 1 -functionalized membranes supported on a skeletonized (a) and non-skeletonized surface (b). The kinetics study shows the formation of the functionalized membrane followed by the incubation of fibronectin with the surface. When the membrane is supported on the skeletonized surface, the increase in the signal is due to the strong binding of fibronectin to the integrin α 5 β 1. The buffer used for those experiments is composed of trizma hydrochloride, sodium chloride, magnesium chloride and calcium chloride at ph

141 Figure 6-8. Control experiment of the interaction of fibronectin with POPC lipid bilayers. Table 6-2. Optical parameters for the multilayer with the integrin-functionalized membrane material refractive index extinction coefficient thickness (nm) Medium SF layer 1 Cr layer 2 Au layer 3 ODM layer 4 Zr-ODPA layer 5 proteomembrane Unknown Medium buffer Discussion Models for supported lipid membranes have been increasing over the last decade but solving the problem of incorporating transmembrane proteins has not been reached. Several strategies have been developed to increase the spacing between the support and the membrane. Tethered polymer supported lipid bilayers have been popular 22,143,148,169,190 but other designs have arisen. Cremer et al. developed a system where lipid bilayers are supported on a double cushion, they obtain high fluidity of the annexin V transmembrane protein by FRAP. 143 A peptide-tethered artificial lipid membrane system was created to study the incorporation of integrins. 195 Nanoporous 141

142 silica microspheres were used as a support for lipid bilayers and membrane proteins were shown to be functional. 192 Skeletonized LB films were first reported by Blodgett taking advantage of the fact that fatty acids are much more soluble in organic solvents than are their corresponding fatty acid salts. 209,210 By varying the ph and metal in content of the subphase, metal carboxylate LB films can be deposited with different fractions of free acid. Upon rinsing the films with organic solvents, Blodgett reported skeletonized films, with tunable refractive index. Since then, the skeletonization process has seen other interesting applications such as vapor sorption at LB multilayers 211 and biochemical processes such as the binding of monoclonal antibody to a peptide at surfaces and in ultrathin films. 212 using zirconium phosphonate monolayer, metal in binding occurs in a separate step after monolayer transfer. Therefore, to achieve skeletonization, a mixed monolayer with octadecanol was used. After the zirconation step, the octadecanol can be washed away, leaving voids in the film. AFM analysis shows the creation of holes with diameters ranging from 50 to 200 nm across with depths of 1-2 nm. Two compositions using 10% and 20% octadecanol were selected for further study, as both provide viable supports for homogenous lipid layers as shown on Figure 6-4. For larger octadecanol concentration, the lipid bilayers would conform to the steps and holes on the surface instead of spanning them. To demonstrate that the model bilayers can support membrane-bound proteins, we studied the interactions of two different proteins with the supported bilayer. Generally, transmembrane proteins can be incorporated in the bilayer by either inserting directly into the preformed lipid bilayers 213 or by reconstitution into the lipid vesicles that are then fused on the substrates. 214 Both 142

143 mechanisms were investigated in this study. To observe the insertion of a protein into the bilayer, the BK channel protein was studied by SPREE. BK ion channels are calcium-activated potassium channels responsible for translocation of potassium across membranes. Structurally, BK channels comprise a pore-forming tetrameric α domain made of seven putative transmembrane segments and a β domain comprising two α- helical transmembrane domains connected by a large glycosylated extracellular loop, with intracellular amino and carboxy termini. As shown in Figure 6-5, the adsorption and fusion of lipid vesicles yield to the formation of supported lipid bilayers of around 5 nm. A slower kinetic is observed for the lipid bilayer formation on the skeletonized surfaces. The binding affinity of the BK channel is stronger when lipid bilayers are supported on a skeletonized surface. At the non-skeletonized support, the protein interacts with the POPC/POPS bilayer, but it is quickly removed upon buffer rinsing. On the other hand, protein binding is slower on the skeletonized support, but a significant amount of the mass increase is retained upon rinsing. The result indicates that the presence of reservoirs between the support and the membrane allows the insertion of the transmembrane protein into the membrane. Another difference between the skeletonized and non-skeletonized supports is that the binding kinetics of the BK channel appears to be slower at the skeletonized surfaces. Using a simple two-step model for the binding (equation 6-1) the association rate for the first step, k a1, decreases slightly at the skeletonized film, but the association rate for the second step, k a2, significantly increases. Intuitively, the first step, governed by electrostatic interactions with the bilayer interface, should be similar at the two supports. The second step, insertion into the membrane, is only significant on the skeletonized support. However, 143

144 significant restructuring of the protein is expected upon insertion, including reorganization of the solvent sphere. The insertion step is likely associated with a mass decrease as protein-lipid interaction replaces protein-solvent interactions. The simple kinetic analysis represented by equation 6-1 assumed no mass change associated with the insertion step, so to the extent that SPREE is sensitive to where solvolysis changes, the insertion step will appear to lower the overall association rate. The observed binding was in agreement with similar work performed by Dr. Okeyo on the BK ion channel. 204 Similar responses have been reported for other systems. Insertion of the instrinsic membrane protein CyaA into polymer tethered membranes also showed slower apparent kinetics than when associated with a non tethered control. 215 Upon washing with buffer, CyaA completely desorbed from the hybrid lipid bilayer but a similar binding behavior as BK channel with the skeletonized support is observed for CyaA on tethered lipid bilayers. Fusion of vesicles that have been reconstituted with a transmembrane protein represents an alternative route to protein-containing supported lipid bilayer. This approval was tested on the skeletonized supports with the integrin α 5 β 1 using the proteins ability to recognize its specific ligand, fibronectin, as an indicator of the viability of the supported lipid bilayer. The SPREE response is consistent with vesicle fusion on both the skeletonized and non-skeletonized supports. However, upon exposing the supported bilayer to fibronectin, ligand binding is only observed at the bilayer on the skeletonized film. The result indicates that the integrin inserts into the bilayer and retains its functionality when supported on the skeletonized surfaces. 144

145 Fibronectin binding can be compared to other immobilized integrin systems. Integrins α v β 3 and α 1 β 1 were incorporated into the tethered membrane via vesicle fusion. Functionality of the transmembrane receptors was confirmed by the binding of vitronectin and collagen type IV to the integrins α v β 3 and α 1 β 1, respectively, by surface plasmon spectroscopy. 195 Furthermore, the extent of ligand binding can be compared to other studies. Binding of the ligand vitronectin to immobilized integrin onto gold via ProLinker was also studied by SPR. 216 Integrin was immobilized as a single molecular monolayer and binding to vitronectin was observed. Integrins incorporated into the membrane, deposited onto the skeletonized surfaces, show similar behavior by binding to fibronectin. However, the coverage was only 19% in this system as functional proteins present in the membrane are in lower concentration. Conclusions In this study, the binding interactions of two different transmembrane proteins were analyzed by SPREE using an improved existing model. Skeletonized zirconium phosphonate surfaces were built on solid supports, and the optimized surface was characterized by AFM. Controlled skeletonized surfaces were used as a support for the study of transmembrane proteins incorporated into lipid bilayers. The main advantage of this system is that this surface can be deposited on any supports, allowing the study of the same interaction by different bioanalytical techniques. The formation of supported lipid bilayers by vesicle fusion is fast and reproducible and binding properties can be directly obtained by SPREE. This technique provided insightful information on the binding interactions and also on thicknesses and ligand coverage. The advantage of the biomimetic membrane was further demonstrated by the incorporation of two proteins with different adsorption approaches. Both proteins were able to incorporate in 145

146 the membrane. Moreover, kinetic binding properties and ligand binding assay show that transmembrane proteins retain their functionality and structural properties in the membrane. 146

147 CHAPTER 7 PHOSPHOPEPTIDES BINDING KINETIC STUDY ON ZIRCONIUM PHOSPHONATE SURFACE Introduction The reversible post-translational phosphorylation mechanism in proteins regulates almost all processes in cell life including cell growth, diffusion and differentiation, while abnormal phosphorylations can cause human diseases. It is estimated that one third of all intracellular proteins are phosphorylated. 217 Thus, identification and quantification of phosphorylation sites is of great importance to understand biological mechanisms that lead to diseases and cancers. Analytical techniques such as mass spectrometry, and MALDI specifically, have been widely used for the characterization and detection of phosphopeptides. However, the low relative abundance of phosphorylated peptides makes the analysis challenging. Therefore, different approaches have arisen for phosphopeptide pre-enrichment. 176, Immobilized metal affinity chromatography (IMAC), developed in 1975, is based on the interaction between metal-ligand complexes and specific functional groups such as phosphates. This technique is one of the most widely used to enrich phosphopeptides, using various metals such as Fe 3+, Al 3+, Zr 4+ and Ga 3+. However, IMAC surfaces allow nonspecific binding of nonphosphorylated acidic peptides which reduces sensitivity and specificity. Recently, metal oxide affinity chromatography (MOAC) technique was able to solve this issue. This technique has been more efficient in avoiding nonspecific enrichment. Indeed, the oxides of titanium, zirconium and aluminum are stable in a wide range of ph thus preventing nonspecific binding in acidic buffers. Zirconium alkyl-phosphonate surfaces have been recently used to enrich phosphopeptides. 149, The use of zirconium phosphonate increases the selectivity as compared with IMAC and MOAC techniques. Different types of 147

148 surfaces have been used such as nanoplatelets, polymer beads and nanoparticles. 149, A method developed by Xu et al. using crystalline α-zirconium phosphate nanoparticles was used to enrich phosphopeptide with higher sensitivity and selectivity. 225 Recently in the Talham group, Dr. Williams studied the phosphopeptides enrichment capacity of zirconium phosphate-based nanoparticles by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and high performance liquid chromatography with negative-mode electrospray ionization mass spectrometry (HPLC-(-)ESI-MS). 229 Despite the number of enrichment techniques, there has not been any global understanding of the kinetic binding of phosphopeptides to the surface, and the question of specificity of the different peptides has been so far elusive. Phosphopeptides can differ in factors such as length, number of phosphorylation sites and also site of phosphorylation. For instance, tri-phosphorylated peptides may have different binding affinities from short peptides. Kinetics studies could provide information on the relative enrichment of different type of peptides. In this study, SPREE has been chosen to study kinetic binding affinities of different phosphopeptides to zirconium phosphonate surfaces. SPR has become a major technique to monitor adsorption kinetics and real-time monitoring of molecular interactions at a gold surface Inamori et al. designed a phosphate capture molecule called biotinylated zinc (II) complex for phosphopeptides enrichement. 233 Detection and quantification of the phosphorylation of peptides was studied by SPR imaging. In their work, SPR allows the detection of phosphorylated peptide probe on the gold surface by using the chelate compound. The reaction rates and kinetics of phosphorylation by protein kinase were 148

149 also studied. In the following study, SPREE was used to study zirconium phosphonate surfaces as an enrichment medium for phosphopeptides. Kinetics analysis and ph studies were performed on different types of phosphopeptides to assess specific binding parameters. Three different groups were studied with each varied in one factor. First, phosphorylated and non-phosphorylated pp60c-src peptides were used to study zirconium phosphonate surface as an enrichement medium. The insulin receptor and the kinase domain of insulin receptor-2 phosphopeptides differed in the number of phosphorylation sites. In the third group, the EGF receptor substrate and the EGF phosphopeptides differed in length sequence. Experimental Section Materials and Solutions All peptides were acquired from Anaspec (California). The chosen peptides were pp60c-src and phosphorylated peptide pp60c-src, the insulin receptor and the kinase domain of insulin receptor-2 phosphopeptides, EGF receptor substrate and EGF phosphopeptides. The sequences of the different peptides are described in Table 7-1. All reagents were used as received. Table 7-1. Phosphorylated and non-phoshporylated peptides with their sequences Peptide name Sequence pp60c-src H-TSTEPQYQPGENL-OH pp60c-src, phosphorylated H-TSTEPQpYQPGENL-OH kinase domain of insulin receptor-2 H-TRDIpYETDYYRK-OH insulin receptor H-TRDIpYETDpYpYRK-OH EGFR phosphorylated, p583 H-DADEpYL-NH 2 EGF receptor substrate H-DADEpYLIPQQGFF-OH 149

150 SPREE Experimental Set-Up The SPREE experiments were performed with a Nanofilm imaging ellipsometer EP 3 (Germany) and a SPR flow cell in the Kretschmann configuration. The angle of incidence is set at 64 for all experiments, close to the resonance angle. Changes in the refraction index produced by the peptide-ligand interactions are detected as changes in the psi signal. The instrument can detect changes in refractive index of the order of The SF10 prism and the gold substrate are coupled together using an immersion oil. The flow cell is pressed against the gold surface where it is held by a clamp. The substrates were composed of 28.5 nm gold layer with 2 nm chromium adhesion layer. The running buffer used in all experiments consisted of 10 mm trizma hydrochloride and 100 mm sodium chloride at ph 7.4 if not specified. During the experiments, solutions were pumped through the flow cell using a peristaltic pump (Rainin, California) with digital control of the flow. The solutions were pumped at a rate of 25 μl min -1 if not specified. A baseline was first established by pumping buffer for 30 min and then the peptide was injected until saturation of the signal. Buffer was then flowed to monitor desorption of phosphopeptides. Results Zirconium Phosphonate Surface as an Enrichment Medium The specificity of the zirconium phosphonate surfaces for phosphorylated peptides was examined by SPREE by analyzing the binding affinity of H-TSTEPQpYQPGENL- OH, which is phosphorylated and H-TSTEPQYQPGENL-OH, which is nonphosphorylated. SPREE provides a reliable method for the estimation of kinetic parameters and binding is measured in real time without any labeling on the peptides. Figure 7-1 shows the increasing psi signal due to the interaction of the phosphorylated 150

151 peptide with zirconium immobilized on the surface. When the peptide solution was replaced by the buffer solution, a small decrease was observed due to desorption of non-specific binding after which the signal remains constant for at least an hour. The non-specific interaction between the non-phosphorylated peptide and the zirconium surface was also registered as a control (Figure 7-1). Low and non-specific binding between the non-phosphorylated peptide and the zirconium surface is observed. The surface was proven to be a successful enrichment medium for phosphopeptides. Figure 7-1. SPREE signal changes by phosphorylated (P-pp60 SRC) or nonphosphorylated (pp60 SRC) peptide adsorption on zirconium octadecylphosphonate surfaces. After stabilization of the signal, the peptide sample prepared in the running buffer at a concentration of 160 µm was injected into the cell. Maximum Binding Capacity on Zirconium Phosphonate Surfaces Assuming complete coverage of zirconium atoms on the surface of atoms cm -2, the coverage on the surface available in the flow cell, with an area of 35.3 mm 2, is atoms. The maximum concentration used in the peptide study is 160 μm, this value represents an amount of molecules per sample volume in the cell. With a peptide concentration of 1μM, the amount of zirconium atoms would be 151

152 equal to peptide molecules. Therefore, maximum surface coverage should be obtained for all concentrations used in this study. Kinetic Analysis of Phosphopeptide/Surface Interactions The transport of the peptide molecules to the surface is an important process in determining the binding kinetics. The rate of the adsorption is determined by mass transport and kinetics at the interface. The molecules can be brought to the surface by diffusion, a microscopic phenomena describing the random molecular motion driven by a concentration gradient, or by convection, a macroscopic phenomenon. The flux of molecules to the surface or out of the surface can be described by the mass transport rate k m. The role of the flow cell is to bring the analyte in contact with the surface and different processes are happening: convection, diffusion and reversible chemical reaction at the sensor surface (Figure 7-2). Figure 7-2. Schematic showing the interactions at the surface. The immobilized receptor B is attached to the sensor. A 0 is flowed through the cell, k m is the mass transport coefficient, used to describe the diffusion of analyte through the unstirred solvent layer. k a and k d are the association and dissociation rate, describing the formation of the complex AB

153 The overall rates of complex formation (k f and k r ) are then a function of mass transport rates and reaction rates: k a k m A 0 A + B AB k m k d (7-1) Therefore, the rate of association and dissociation may be limited by the kinetics or mass transport or both. The system is reaction-limited when the concentration at the surface is the same as the bulk concentration. In the other cases, those concentrations are different because the mass transport is too low to compensate for the difference in concentrations. For kinetic analysis, it is important that the system is limited by the reaction and not mass transport. Mass transport limitations occur when the kinetic rate is comparable or faster than the diffusion of analyte to the surface. The flow rate and the design of the flow cell are two parameters that can influence kinetic rates. If the adsorption is diffusion limited, then the adsorption rate is dependent on the flow rate; by increasing the flow rate, it is clear that the adsorption rate increases too. Also, an increase in the diameter of the tubing yields to a decrease in the adsorption rate. However, by increasing the flow rate, the process is then kinetically limited. In our study, the flow rate is 25 μl min -1 and the volume of the cell is 100 μl; for our kinetic analysis, we assume that the concentration of peptide at the surface is the same as the bulk concentration so we do not use a mass transport limited model. The assumption was made based on the following calculations. Mass transport can be described as: k m [A 0 ] k m [A] = k a [A][B] k d [AB] (7-2) The kinetic rate equation and the concentration of ligand B can be described by: 153

154 d[ab] = k dt a [A][B] k d [AB] [B] = [AB max ] [AB] (7-3) (7-4) Combining equations 7-2 and 7-4 results in: k m [A 0 ] + k d [AB] [A] = k m + k a ([AB max ] [AB]) (7-5) Combination of the kinetic rate equation gives: d[ab] = k dt f [A]([AB max ] [AB]) k r [AB] with k f = and k r = k a k m (k m + k a [B]) = k d k m (k m + k a [B]) = k a 1 + k a [B] k m k d 1 + k a [B] k m (7-6) (7-7) (7-8) A system is kinetically limited if the mass transport flux is much higher than the association rate, when k m >>k a [B]. In the other case, when the system is mass transport limited, k m can be described as 45 : k m = 0.98( D h )2/3 ( f bx )1/3 (7-9) The parameters are D for diffusion coefficient, f for the flow rate, h and b, the height and width of the flow cell, respectively. The value x is the distance from the flow cell entrance. To calculate k m, we insert the following parameters in the latter equation: D m 2 s -1 h = m f = 25 μl min -1 = m 3 s -1 b = m 154

155 x = m Inserting these values, we obtain: k m = m s -1. Assuming the ligand coverage calculated in the previous section, for kinetic limited reaction, k a << k m / [B] = m 3 mol -1 s -1. For fast interactions such as antigenantibody interactions, characteristics values are equal to or less than 10 2 m 3 mol -1 s -1. Therefore, our system is not limited by mass transport and the measurements are performed under binding reaction-controlled conditions. Binding experiments were performed at different flow rates as seen in Figure 7-3. Binding of phosphorylated peptides to the zirconium phosphonate surfaces was followed by SPREE and similar kinetics can be observed. These results demonstrated that the system is independent of the flow rate, therefore allows kinetically measurements. Figure 7-3. The effect of flow rate on the adsorption of phosphorylated peptide P-pp60 SRC at a concentration of 80 μm, measured by SPREE. Study of Phosphorylated Peptide with ph Variation To determine the effect of ph on the zirconium-phosphopeptide interactions, phosphorylated and the non-phosphorylated peptides at four different phs, 3, 4.5, 6 and 155

156 7.4 were studied. Zirconium phosphonate form covalent bonds to dibasic phosphates while carboxylates interact only electrostratically with the same surface, as seen in Figure 7-4. However, variation of ph might affect the type of interactions between zirconium phosphonate surface and dibasic phosphates present in biomolecules. The present study tries to understand the type of interactions involved as a function of ph. (a) (b) Figure 7-4. Scheme illustrating the difference between the covalent linkage to the zirconium phosphate surface formed by dibasic phosphate (a) and the nonspecific bonding experienced by weaker bases, such as carboxylate (b) or monovalent phosphodiester. For four different ph s ranging from 3 to 7.4, the binding of phosphorylated and non-phosphorylated peptides was studied by SPREE. Figure 7-5 shows the sensorgrams for both peptides at different phs. For all phs, phosphorylated peptides interact strongly with the zirconium phosphonate surfaces. However, the kinetics is faster for the binding of phosphopeptides at phs 4.5 and 6 and the dissociation is faster 156

157 at phs 3 and 7.4. Interactions between non-phosphorylated peptides and zirconium surfaces are weak and non-specific as dissociation removes most of the peptides. However, we observed higher non-specific binding at ph 3. (a) (b) Figure 7-5. Sensorgrams of the binding of phosphorylated (a) and non-phosphorylated peptides (b) to zirconium phosphonate surfaces at different ph ranging from 3 to 7.4. Concentration was 160μM for all peptides. Tris-buffer was used for all experiments. The flow was kept constant at 25 μl min -1. The reproducibility of the experiment was studied and each sensorgram in Figure 7-6 represent at least two different experiments for the phosphorylated and non- 157

158 phosphorylated peptides. At ph 3, non-phosphorylated peptide does not completely desorbed from the surface after buffer rinsing and the binding difference is small between the two peptides. At both ph 4.5 and 6, the difference in binding for the phosphopeptides and non-phosphopeptides is larger and the kinetics is faster. At ph 7.4, the difference in both peptide binding is greater than for the other phs. We observed the highest binding for phosphorylated peptide at neutral ph. (a) (b) (c) (d) Figure 7-6. SPREE sensorgrams of the binding of phosphorylated and nonphosphorylated to zirconium phosphonate at different ph: 3 (a), 4.5 (b), 6 (c) and 7.4 (d). Black lines represent the phosphorylated peptides for different experiments. Grey lines represent the non-phosphorylated peptides for different experiments. The concentration was 160 μm and tris buffer was used. The flow was kept constant at x μl min

159 Phosphopeptides Study Using six different phosphorylated or non-phosphorylated peptides as described in Table 7-1, the relative binding was studied and compared by SPREE. Kinetic binding comparison can provide information on mechanism and conditions of enrichment as well as on the relative enrichment of different type of peptides. The first group, composed of pp60 and P-pp60, was studied in the previous section and demonstrated the efficiency of zirconium phosphonate surfaces as a phosphopeptide enrichment material. The second group, composed of 1pY and 3pY, is used to study the number of phosphorylation sites and the third group, composed of 6-AA and 13-AA, allows the study of the phosphopeptide length. Table 7-2. Phosphorylated and non-phosphorylated peptides peptide name Sequence Name pp60c-src H-TSTEPQYQPGENL-OH pp60 pp60c-spr, phosphorylated H-TSTEPQpYQPGENL-OH P-pp60 kinase domain of insulin receptor-2 H-TRDIpYETDYYRK-OH 1-pY insulin receptor H-TRDIpYETDpYpYRK-OH 3-pY EGFR phosphorylated, p583 H-DADEpYL-NH 2 6 AA EGF receptor substrate H-DADEpYLIPQQGFF-OH 13 AA Sensorgrams for the binding of four different types of peptides are shown in Figure 7-7. In Figure 7-7a, the peptides have the same sequence but differ in their lengths, thirteen and six amino acids. The kinetic is similar for both peptides. In Figure 7-7b, the peptides differ only in the number of phosphorylation sites, one is mono-phosphorylated and the other is tri-phosphorylated. The association step appears faster for the trisphosphorylated peptide while the amount of material on the surface is similar. Binding kinetic information is important to compare these different phosphopeptides. Therefore, a kinetic model was used to calculate the association and dissociation rate constants. 159

160 We used a Langmuir isotherm to deduce the kinetics of the adsorption of the peptides on the zirconium phosphonate surfaces. The Langmuir model is the most common model assuming that the reaction is reversible between the ligand (L) and the peptide (P) and that the number of receptors is fixed. The interaction can be described by: The corresponding kinetic equation is: k a P + L PL (7-1) k d dγ PL dt = k a C P Γ L k d C P (7-2) where Γ PL and Γ L stand for the surface concentration of the complex PL and the free ligand L respectively, and C P stands for the concentration of the running peptide. According to equation (7-1): Γ L = Γ PLtotal Γ PL (7-3) where Γ PLtotal stands for the total surface concentration of the complex PL. The increase in psi signal is proportional to the surface concentration of the complex PL: 235 Ψ = ργ PL (7-4) Ψ total = ργ PLtotal (7-5) Substituting equations (7-4) and (7-5) into (7-2) yields the following expression for the rate of the psi signal change: dψ dt = k aψ total C P (k a C P + k d )Ψ (7-6) Assuming constant concentration of the peptide in the flow cell, a plot of dψ/dt versus Ψ must give a straight line for first-order kinetics. A secondary plot of the slopes 160

161 of the first plot versus the concentration of the peptide yields a second straight line from which both rate constants can be evaluated. Determining the dissociation rate is straightforward by performing an experiment using a high concentration of peptides to maximize the coverage on the surface and then washing with buffer. Dissociation can be measured by the exponential decay and in this case, the concentration of the peptide is then zero so first-order kinetics can be described by: Ψ = Ψ 0 e k dt (7-7) By plotting ln(ψ 0 /Ψ) versus the time, the slope yields the dissociation rate constant. Dissociation rate constants were calculated from experiments in which maximum peptide binding was obtained in order to prevent rebinding on the surface. For all four peptides, four different concentrations ranging from 20 μm to 160 μm were studied. As an example, the binding of 1pY peptide at different concentrations to the zirconium phosphonate surface is shown in Figure 7-8. First, the derivative of Ψ was plotted against Ψ. The slope for the different concentrations was then plotted against concentrations. A straight line was obtained (Figure 7-9a) confirming that the interaction process fits well to pseudo-first order kinetics. The slope of the secondary plot yielded a second-order rate constant, k a, with value of (4.93 ± 0.11) 10 2 M -1 min -1. Dissociation rate constants obtained from equation 7-2 were often unreliable as the term k a C dominates the k d term. The dissociation rate constant was then calculated by equation 7-7. The slope of the plot ln(ψ 0 /Ψ) versus time yielded the dissociation rate constant, with value (8.02 ± 0.16) 10-3 min -1. The binding affinity was calculated to be M -1, consistent with high affinity binding. 161

162 Binding of the other peptides was studied by SPREE and the same calculations were performed (Figure 7-10, 7-11 and 7-12). The results for the kinetic rate constants and the equilibrium dissociation constants are shown in Table 7-2. Association rate constant is larger with an increase in the number of phosphorylation sites and slightly larger for smaller peptides. Dissociation is greater for longer peptides and monophosphorylated peptides. The equilibrium dissociation constants are in the micromolar range, in agreement with a modestly tight binding. The tris-phosphorylated peptide has a stronger binding affinity to the zirconium phosphonate surface than the monophosphorylated peptide. The shorter peptide has a slightly greater affinity for the surface than the longer peptide. Therefore, this kinetics binding comparison allows us to draw preliminary conclusions on the mechanism of phosphopeptide binding. (a) (b) Figure 7-7. SPREE signal changes by one-phosphorylation site or threephosphorylation site peptides on zirconium phosphonate surfaces. The sensor device was a gold slide was coated with zirconium phosphonate film, this chip was set in the SPREE apparatus. The running buffer was 10 mm triszma hydrochloride, 100 mm sodium chloride at ph 7.4(tris buffer). Concentration of 160 µm. 162

163 Figure 7-8. SPREE signal changes by monophosphorylated peptide (1pY) on zirconium octadecylphosphonate surfaces for concentrations ranging from 20 µm to 160 µm. Buffer is tris buffer at ph 7.4. (a) (b) Figure 7-9. Rate of Ψ signal obtained from adsorption of 1pY peptides studied by SPREE versus Ψ signal according to equation 7-6 for different peptide concentrations (μm): 20, 40, 80, 160 (a) and secondary plot of slopes versus peptide concentration (b). Figure SPREE signal changes by trisphosphorylated peptide (3pY) on zirconium octadecylphosphonate surfaces for concentrations ranging from 20 µm to 160 µm (a). Rate of psi signal increase versus psi signal according to equation 7-6 for different peptide concentrations (μm): 20, 40, 80, 160 and secondary plot of slopes versus peptide concentration (b). 163

164 Figure SPREE signal changes by monophosphorylated peptide composed of 13 amino acids (13AA) on zirconium octadecylphosphonate surfaces for concentrations ranging from 20 µm to 160 µm (a). Rate of psi signal increase versus psi signal and secondary plot of slopes versus peptide concentration (b). Figure SPREE signal changes by monophosphorylated peptide composed of 6AA (6AA) on zirconium octadecylphosphonate surfaces for concentrations ranging from 40 µm to 160 µm (a). Rate of psi signal increase versus psi signal and secondary plot of slopes versus peptide concentration (b). Table 7-3. Association and dissociation rate constants of the interactions between phosphorylated peptides and zirconium phosphonate surfaces calculated by the one-to-one interaction model peptide k a ( 10 3 M -1 min -1 ) k d ( 10-3 min -1 ) K D ( 10-6 M -1 ) P-pp ± ± ± pY ± ± ± pY ± ± ± AA ± ± ± AA ± ± ±

165 Conclusions and Future Work The binding affinity of different phosphopeptides with zirconium phosphonate surfaces was studied by SPREE. The data indicated that this surface can be used as an enrichment medium for phosphopeptides. Moreover, there is ph dependence on the binding affinity, low and neutral ph s achieved the highest and fastest binding. In addition, a binding comparison of the different type of peptides to the zirconium surface was made. It was seen that tris-phosphorylated peptide and smaller peptide have a stronger binding than mono-phosphorylated and longer peptide to the surface. Future work is ongoing to understand the ph dependence on the binding of phosphate biomolecules to the zirconium phosphonate surfaces. Infrared spectroscopy is currently used to study the binding of octadecylphosphonic acid to ATR germanium crystal at different ph s. Dynamic light scattering can also be used on zirconium phosphate nanoparticles to study zeta potential of the surface as a function of ph. In addition, a comparison between the zirconium phosphonate surface and zirconium phosphate nanoparticles, as well as the study of different metal such as lanthanides (terbium, europium and samarium) will be explored. 165

166 CHAPTER 8 SUMMARY The focus of the research presented in this dissertation was the development of biosensors for the immobilization of lipids, proteins and peptides. For the systems discussed in the different Chapters, zirconium phosphonate surface was used as a support for biomolecules immobilization. This surface has been a popular use for biomolecules immobilization such as DNA and proteins. In Chapter 3, proteins were directly immobilized on the surface via synthetic linkers. Also, it is becoming obvious that membrane-protein interaction plays an important role in the regulation of many cell processes. Ongoing research on these interactions and on the creation of new membrane models contribute to the understanding of these mechanisms. Therefore, in the other developed models, supported lipid bilayers were formed to support proteins. These systems allow the proteins to keep their natural environmental structure. As well as the design of biosensors, characterization by many techniques was an important aspect of this research work. As demonstrated in this work, by combining biophysical techniques, surface chemistry and biochemistry it was possible to gain insight in membrane-protein interactions and protein arrays. Biophysical techniques have been powerful in the study of biomaterial immobilization. As discussed in Chapter 2, SPR and ellipsometry were combined to form SPREE. This recent association allows direct kinetic measurement without any labeling and proved to be more sensitive than individual techniques. Applications in monolayer self assembly, biomolecules sensing and LbL polymer assembly were discussed and the obtained results demonstrated the utility of SPREE and ellipsometry. 166

167 Recently, proteins immobilization has become of high interest in the understanding of biological mechanisms. Chapter 3 described the design of biosensor to capture histagged-proteins. Proteins are immobilized to zirconium phosphonate surface by synthetic NTA-linkers. These linkers proved to be successful in the capture of affitins, small histagged proteins, and with high density capture. The different linkers were compared by studying binding affinities and kinetics by SPREE. The two linkers provided high density of available NTA groups, allowing high efficiency of protein binding. Proteins were also able to retain their ability to capture protein target with high sensitivity. Also, commercialized protein arrays were compared to the designed sensors and results validated them as competitive array technology. Inorganic surfaces have found a new application in biomolecules immobilization with DNA and with lipid bilayers and proteins in this work. The simple deposition of lipid bilayers on the zirconium phosphonate surface holds the promise to become simple but also useful strategy for the study of proteins binding. Two models, that provided stable, functional, reproducible membranes, have been developed that enable the immobilization of membrane proteins. In the first model, metal phosphonate chemistry was employed to increase the stability of lipid bilayers by means of covalent interactions (Chapter 4). Phosphatidic acid lipid was used in a mixture of phospholipids to form covalent bonds with the latter surface. Additionally, the kinetic analyses of proteinbiosensor interaction were performed in order to verify the viability of models as natural membrane. The principal objective of the models is to maintain protein function and structure. SPREE was employed to study insertion of proteins in the membrane. Membrane protein such as melittin was inserted and maintained its structure in 167

168 membrane as shown by binding experiments, performed by SPREE. However, the proximity of the inorganic support did not provide the space for the insertion of transmembrane proteins. The second model, based on skeletonized surfaces as support for lipid bilayers, proved to be successful in the insertion of two proteins, integrin and BK channel (Chapter 5). The immobilized proteins were shown to be functional and stable in the bilayers. Our findings provided then evidences that the model based on skeletonized surfaces behaved as natural membrane. Taken together, these new designs open doors toward the investigation of protein-lipid interactions. Also, the results that were obtained for different aspects of the models such as film design, surface morphology, stability and viability demonstrated the convincing benefits of using multiple biophysical techniques. In the context of this work we have also tested zirconium phosphonate surfaces as an enrichment matrix for phosphopeptides. The results illustrated the potential use of this surface for enrichment. Finding a procedure and kinetic data analyses of binding of phosphopeptides to zirconium phosphonate surfaces was another goal. Improvements of the methodology have been addressed in order to gain control of binding processes and to understand specific binding relative to different phosphopeptides. The SPREE technique was used to characterize binding affinity and kinetic model. Dependence in ph, number of phosphorylation sites and length of peptides were studied. Interesting results were obtained as ph 3 and ph 7.4 allowed high affinity binding. Further studies are ongoing by using IR technique to understand the role of ph on the binding to zirconium phosphonate surfaces. Phosphopeptides can differ in some aspects and high affinity is observed for longer and multiple phosphorylation sites peptides. 168

169 Several additional developments of these models can be already foreseen: (a) study of other histagged proteins with higher molecular weight; (b) development of methodology to study binding affinity of phosphopeptides to zirconium phosphonate surfaces; (c) comparison of affinity between zirconium phosphonate surfaces and zirconium phosphate nanoparticles; (d) use of different metals for the phosphonate chemistry; (e) complete ph study to understand the metal phosphonate chemistry by SPREE and IR. 169

170 APPENDIX SUPPORTED LIPID BILAYERS ON ZIRCONIUM PHOSPHATE NANOPLATELETS Introduction Lipid vesicles have been proven to be good models to mimic the curvature of cells but problems with size dispersity and chemical stability are often encountered. Hybrid lipid/nanoparticle systems present a growing interest in the biotechnology field for the design of biosensors, interface agents, and use with drug and gene delivery. An advantage is the biocompatibility of the lipid bilayers with cell membranes and their well characterized phase behavior and physiochemical properties. Also, membrane stability is enhanced because of the robust inorganic core coated with supported lipid bilayers. Different spherical supports have been used such as silica, polystyrene, and magnetic beads whose size range from tens of nanometers to several micrometers. 236 Models have been created in the last decade such as supported lipid bilayers on SiO 2 nanoparticles, the LipoParticles where polymer particles are coated with the lipid layers, 236,240 and supported lipid bilayers from different phospholipids on spherical silica beads using avidin-biotin interactions. 241 In this study, zirconium phosphate nanoplatelets were coated with supported lipid bilayers. Lipids with different head groups were used to study the stability of supported lipid bilayers on nanoplatelets. In particular, phosphatidic acid (PA) lipids were used to increase the stability as covalent interactions are expected between PA and zirconium ions. Characterization was mainly performed by confocal laser scanning microscopy (CLSM) as lipids were doped with fluorophores. 170

171 Experimental Section Materials Reagents were obtained from commercial sources and used as received. The monosodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) and 1- oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3- phosphocholine (NBD-PC) were purchased in chloroform from Avanti Polar lipids (Alabaster, AL). Zirconyl chloride (ZrOCl 2. 8H 2 O, 98%) and phosphoric acid, 85%, were purchased from Sigma-Aldrich (St. Louis, MO). The buffer component trizma hydrochloride was obtained from Sigma. Milli-Q water with a resistivity of 17.9 MΩ.cm was used for all experiments. Tris buffer used throughout the lipids experiments was a 10 mm trizma hydrochloride and 100 mm sodium chloride solution, ph 7.4. Vesicle Preparation DPPA, DPPC, POPC and POPG lipid mixtures were used to form the small unilamellar vesicles (SUVs). The chemical formula of the different lipids is given in Figure A-1. The chloroform stock solution of the lipid mixture was dried via a nitrogen stream to form a uniform dry lipid film. The film was hydrated in tris buffer to obtain a concentration of 0.5 mg ml -1 with gentle vortex mixing leading to multilamellar vesicles. Five freeze/thaws were performed on the lipid solution to obtain large unilamellar vesicles. The lipid suspension was extruded 11 times through polycarbonate membranes with a pore diameter of 100 nm. Vesicles were usually used within one day of preparation. 171

172 Figure A-1. Chemical formula of the phospholipids POPC, DPPA, POPG and DPPC. Preparation of Zirconium Phosphate Nanoplatelets This method was designed by Clearfield 242 and al. and described by the following procedure, the synthesis can be described by: ZrOCl 2 + 2H 3 PO 4 Zr(HPO 4 )2 H 2 O + 2HCl. A sample of 10 g ZrOCl 2 8H 2 O was refluxed with 100 ml of 3 M H 3 PO 4 in a Pyrex glass flask at 100 C for 24h. After the reaction, the products were washed and collected twice by centrifugation at 2900 g for 20 min. Then, the isolated nanoparticles were dried at 65 C for 24 h. The dried particles were ground with a mortar and a pestle into fine powders. The particles were then rinsed with water and centrifuged at 8,000 g for 5 min, and the process was repeated twice. After resuspension of the particles in acetonitrile: water (1:1), zirconium phosphate particles were collected by centrifugation and suspended in water. The resulting particles are called ZrP and were imaged by scanning electron microscopy (SEM). The resulting particles are not fully covered by zirconium on the surface, therefore the particles were incubated in 20 mg ml -1 ZrOCl 2 at room temperature overnight. Particles were collected by centrifugation, washed with water twice to remove excess zirconyl chloride, and then resuspended in water. These 172

173 particles are designated ZrP-Zr, as described in Figure A-2. To study the effect of ph on the particles, hydrochloric acid was added to the solution to create an acidic environment (ph 2). Particles treated this way are labeled ZrP-ZrH. Figure A-2. Schematic of the zirconium phosphate nanoplatelets before (ZrP) and after (Zrp-Zr) zirconium treatment. Deposition of Lipid Bilayers onto Zirconium Phosphate Nanoplatelets Nanoplatelets (500 µg ml -1 ) and SUVs (250 µg ml -1 ) of the desired lipid mixture were mixed for 1h at room temperature. The lipid bilayer surface is then much greater than the surface of the particles. The mixture was centrifuged at 10,000 for 20 min then rinsed with water and centrifuged (Figure A-3a). The latter process was repeated twice to obtain the hybrid system, as seen in Figure A-3b. (a) (b) Figure A-3. Schematic of the different steps involved in the formation of the nanoparticles coated with supported lipid bilayers (a). Schematic representation of the zirconium phosphate nanoparticles after zirconium treatment coated with supported lipid bilayers (b) 173

174 Confocal Laser Scanning Microscopy Fluorescence experiments were conducted with a confocal microscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and three lasers, a tunable argon ion laser (458, 488, 514 nm), a green HeNe laser (543 nm), and a red HeNe laser (633 nm) with three separate photomultiplier tubes (PMTs) for detection. The lipid fluorophore used, NBD-PC, has an excitation wavelength of 460 nm and an emission wavelength of 534 nm, therefore we used an Argon ion laser (457 nm) with a Gaussian intensity cross-sectional intensity. The fluorescence intensity was recorded by a photomultiplier signal and by analysis of camera images for quantitative data. The cellular images were taken with a 20x objective. The gain and photomultiplier values were kept the same for all experiments. All analyses were conducted on the Fluoview 500 software, followed by processing of the data using Origin. Quasi-Elastic Light Scattering Mean size distribution of nanoparticles, lipid vesicles and hybrid systems was determined at 25 C by a Precision Detectors PDDLS/ CoolBatch + 90T instrument. The data were analyzed with the Precision Deconvolve32 Program. The measurements were taken at 20 C and a 90 scattering angle, using a 683 nm laser source. Electrophoretic Mobility Measurements The electrophoretic mobility values were measured at 25 C using a Brookhaven ZetaPlus apparatus. Electrophoretic mobility (µe) was converted to zeta potential (ξ) according to Smoluchowski s equation: μ e = ε 0ε r η ξ (A-1) 174

175 where η, ε 0 and ε r are the medium viscosity, permittivity of vacuum, and relative permittivity, respectively. Results Characterization of Zirconium Phosphate Nanoplatelets A schematic of the zirconium phosphate nanoplatelets coated with lipid bilayer is shown in Figure A-3b. Lipid vesicles fuse on the highly hydrophilic surface to create supported lipid bilayers. The diameter of the zirconium phosphate nanoparticles are around 100 nm, as confirmed by the SEM images (Figure A-4). Different shapes can be observed but the dominant shape is hexagons. We also observe large aggregates due to interactions between nanoparticles. SEM images of lipid bilayers deposited onto the nanoparticles were also taken but the resolution was not sufficient to observe the membrane around the particles (Figure A-5). Moreover, the aggregation of the particles does not facilitate the observation of the ring of lipid bilayers around particles, as observed by Brisson et al by cryo-em. 238 Figure A-4. SEM image of zirconium phosphate nanoplatelets. The particles are about 100 nm in size. 175

176 Figure A-5. SEM images of Zr-P nanoplatelets coated with POPC supported lipid bilayers. Size Distribution of the Hybrid System Size distribution of the different systems was obtained by DLS. Measurements were performed at least five times for each system. However, results for the particles and hybrid system were not reproducible; the cause might be the large number of aggregates and also the non uniformity shape of the particles. In Figure A- 6, we can see an increase in the size between the nanoparticles alone and the lipid bilayers deposited onto the nanoparticles but the results are not consistent with a 10 nm lipid bilayers deposited onto the nanoparticles. 176

177 Figure A-6. Size distributions performed by DLS of lipids (a), nanoparticles (b) and the hybrid lipid/nanoparticle system (c). Zeta Potential Measurements of Hybrid Systems Zeta potential measurements indicate the functionalization of the nanoplatelets with a zirconium layer. Zirconium phosphate nanoparticles have a mean zeta potential of mv (Figure A-7). The addition of a layer of zirconium ions on the outside of the particles renders the particles more positively charged, as shown by the mean zeta potential value of mv. Supported lipid bilayers were then deposited onto the nanoparticles. Two types of lipids were used. The value of the zeta potential of the particles modified with a supported lipid bilayer of the zwitterionic POPC is similar to that of the nanoparticles alone (Figure A-8). Changes of only 0.8 mv and 3.04 mv for the particles before and after the incubation in ZrOCl 2, respectively, were obtained for the hybrid systems. However, when a bilayer of the anionic lipid (POPG) was deposited onto the nanoparticles, the value of zeta potential became more negative. Indeed, decreases of 8.14 mv and mv were obtained. This result shows the particles are modified and agrees with the deposition of neutral and anionic supported lipid bilayers. 177

178 Also, the results agree with the deposition of bilayers or multilayers. If a monolayer was formed, the particle surface would be hydrophobic and zeta potentials for the anionic lipids would then be closer to zero. Figure A-7. Mean zeta potential measurements of the zirconium phosphate before and after zirconium treatment. Figure A-8. Mean zeta potential measurements of different type of lipid bilayers (zwitterionic POPC and anionic POPG) deposited on the zirconium phosphate nanoparticles before and after zirconium treatment. Fluorescence Measurements of Nanoplatelets Coated with Supported Lipid Bilayers Confocal Fluorescence Microscopy experiments were performed on zirconium phosphate nanoplatelets coated with supported lipid bilayers, the latter are doped with 2% NBD-PC (fluorescent lipid). Different experimental conditions were studied such as lipid composition, nanoparticle concentration and ph. 178

179 POPC lipid fusion on the nanoplatelets was induced by mixing lipid vesicles doped with 2% NBD-PC and particles for 1h at room temperature. Mixtures were centrifuged (10,000g/20min) to separate the particles from vesicles. Nanoplatelets were then redispersed in pure water at specified ph. Figure A-9a shows fluorescent images of the hybrid lipid/ nanoplatelets system. Only lipids are doped with fluorophores and the centrifugation step removes all free vesicles. Therefore, presence of fluorescence is due to the presence of the hybrid system and not free vesicles. Higher fluorescence is observed when the extra zirconium layer was added onto the nanoplatelets. This result confirms that zirconium phosphate nanoplatelets coated with a zirconium layer is a better support for POPC supported lipid bilayers than without the zirconium layer. As a control, similar experiments were performed on naked nanoplatelets and as expected, no fluorescence was observed (Figure A-9b). Different lipid compositions were then studied. DPPC and POPG were chosen to study the effect of fatty acid hydrocarbon chain and headgroup (Figure A-10). Aggregates are obtained with the hybrid system DPPC/ZrP but more uniform fluorescence is obtained for DPPC/ZrP-Zr. The POPG lipid vesicles show similar results as POPC lipid vesicles, where supported lipid bilayers do no coat the ZrP nanoplatelets but deposit only onto ZrP-Zr nanoplatelets. The effect of concentration and ph were then studied, as seen on Figure A-11, a higher nanoplatelet concentration leads to a more concentrated and uniform hybrid system. Good results are obtained with an acidic environment but a basic environment leads to the formation of aggregates and non uniform hybrid lipid/nanoplatelets system. 179

180 The aim of the project was to increase the stability of supported lipid bilayers by using small amount of phosphatidic acid (PA) lipids in the bilayers. The presence of 10% of DPPA lipid should give higher stability to the supported lipid bilayers on zirconium phosphate nanoplatelets, as observed on zirconium phosphonate surfaces in chapter 3. High stabilization is expected due to the covalent interaction between PA and the outside zirconium layer. Therefore, stability experiments were performed by washing the lipid bilayer-nanoplatelets multiple times with water and different concentrations of SDS. For POPC modified nanoplatelets, the addition of 10% of DPPA yields to aggregation as seen in Figure A-12. Washing the hybrid systems with 0.5% SDS removes all POPC from the nanoplatelets. The same behavior is observed with addition of DPPA lipids (Figure A-12). Therefore, the stability of the hybrid system was not increased by the addition of PA lipids. (a) (b) Figure A-9. Confocal images of zirconium phosphate nanoplatelets after zirconium treatment coated with POPC supported lipid bilayers (a) and naked (b) (a) (b) 180

181 (c) (d) Figure A-10. Fluorescence and transmitted light images of POPC (a), POPC/DPPA (b), DPPC (c) and POPG (d) supported lipid bilayers deposited onto zirconium phosphate nanoplatelets before and after zirconium treatment. (a) (b) (c) Figure A-11. Zirconium phosphate nanoplatelets at different concentrations (a) 250 μg ml -1 and (b) 500 μg ml -1 coated with POPC lipid bilayers. Zirconium phosphate nanoplatelets coated with POPC lipid bilayers in acidic (b) and basic (c) environment for the same concentration of 500 μg ml

182 (a) (b) Figure A-12. Zirconium phosphate nanoplatelets after zirconium treatment coated with POPC (a) or POPC/DPPA (b) lipid bilayers. Successive water and SDS washes at different concentrations were performed to study the effect of PA on the lipid bilayer stability. Discussion In this project, we attempted to coat zirconium phosphate nanoplatelets with stable supported lipid bilayers. Although we managed to observe nanoplatelets by SEM, aggregation of the particles made it impossible to observe supported lipid bilayers on nanoplatelets. Brisson et al. studied the deposition of SUVs on silica nanoplatelets by cryo-em 6. Resulting images show the formation of supported lipid bilayers on the nanoplatelets by the presence of a continuous ring of electron-dense material separated 182