Advanced Materials Interfaces

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Advanced Materials Interfaces

Edited by

Ashutosh Tiwari, Hirak K. Patra and Xuemei Wang

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v

Contents

Preface xiii

Part 1 Interfaces Design, Fabrication, and Properties

1 Mixed Protein/Polymer Nanostructures at Interfaces 3

Aristeidis Papagiannopoulos and Stergios Pispas1.1 Introduction 31.2 Neutral and Charged Macromolecules at Interfaces 41.3 Interfacial Experimental Methods 71.4 Interactions of Proteins with Polymer-Free Interfaces 91.5 Polymers and Proteins in Solution 111.6 Proteins at Polymer-Modified Interfaces 14

1.6.1 Steric Effects 151.6.2 Polyelectrolyte Multilayers: Electrostatic Nature

of Interactions 211.6.3 Counterion Release: Charge Anisotropy 23

1.7 Protein-Loaded Interfaces with Potential for Applications 261.8 Conclusions 30References 30

2 Exploitation of Self-Assembly Phenomena in Liquid-Crystalline Polymer Phases for Obtaining Multifunctional Materials 37

M. Giamberini and G. Malucelli2.1 Introduction 372.2 Amphiphilic Self-Assembled LCPs 412.3 Self-Assembled LCPs Through External Stimuli 442.4 Supramolecular Self-Assembled LCPs 482.5 Self-Assembled LCPs Through Surface Effects 542.6 Conclusions and Perspectives 57References 59

vi Contents

3 Scanning Probe Microscopy of Functional Materials Surfaces and Interfaces 63

Pankaj Sharma and Jan Seidel3.1 Introduction 643.2 Scanning Probe Microscopy Approach 65

3.2.1 Piezoresponse Force Microscopy 683.2.1.1 Advanced Modes of PFM 733.2.1.2 Enhancing Temporal Resolution 76

3.2.2 Conductive-Atomic Force Microscopy 793.2.3 Kelvin Probe Force Microscopy 81

3.3 Functional Material Surfaces and Interfaces 853.3.1 Ferroelectric Tunnel Junctions 863.3.2 Ferroic Domain Walls and Structural-Phase

Boundaries 933.3.3 Complex-Oxide Thin Films and

Heterostructures 953.3.4 Photovoltaics 104

3.4 Conclusion and Outlook 111References 114

4 AFM Approaches to the Study of PDMS-Au and Carbon-Based Surfaces and Interfaces 127

Giorgio Saverio Senesi, Alessandro Massaro,

Angelo Galiano and Leonardo Pellicani4.1 Introduction 1274.2 AFM Characterization of Micro–Nano Surfaces and

Interfaces of Carbon-Based Materials and PDMS-Au Nanocomposites 130

4.3 3D Image Processing: ImageJ Tools 1364.4 Scanning Capacitance Microscopy, Kelvin Probe

Microscopy, and Electromagnetic Characterization 1384.5 AFM Artifacts 1414.6 Conclusions (General Guidelines for Material

Characterization by AFM) 143Acknowledgments 146References 146

Contents vii

5 One-Dimensional Silica Nanostructures and Metal–Silica Nanocomposites: Fabrication, Characterization, and Applications 149

Francesco Ruffino5.1 Introduction: The Weird World of Silica Nanowires

and Metal–Silica Composite Nanowires 1505.2 Silica Nanowires: Fabrication Methodologies,

Properties, and Applications 1555.2.1 Metal-Catalyzed Growth 1585.2.2 Oxide-Assisted Growth 174

5.3 Metal NPs-Decorated Silica Nanowires: Fabrication Methodologies, Properties, and Applications 177

5.4 Metal NPs Embedded in Silica Nanowires: Fabrication Methodologies, Properties, and Applications 188

5.5 Conclusions: Open Points and Perspectives 197References 197

6 Understanding the Basic Mechanisms Acting on Interfaces: Concrete Elements, Materials and Techniques 205

Dimitra V. Achilllopoulou6.1 Summary 2056.2 Introduction 2076.3 Existing Knowledge on Force Transfer Mechanisms on

Reinforced Concrete Interfaces 2126.3.1 Concrete Interfaces 2126.3.2 Reinforcement Effect on Concrete Interfaces 2176.3.3 Interfaces of Strengthened RC Structural Elements 224

6.4 International Standards 2366.4.1 Fib Bulletin 2010 2376.4.2 ACI 318-08 2386.4.3 Greek Retrofit Code (Gre. Co.) Attuned

to EN-1998/part 3 2386.5 Conclusions 241References 242

7 Pressure-Sensitive Adhesives (PSA) Based on Silicone 249

Adrian Krzysztof Antosik and Zbigniew Czech7.1 Introduction 2497.2 Pressure-Sensitive Adhesives 250

7.2.1 Goal of Cross-Linking 251

viii Contents

7.3 Significant Properties of Pressure-Sensitive Adhesives 2537.3.1 Tack (Initial Adhesion) 2537.3.2 Peel Adhesion (Adhesion) 2547.3.3 Shear Strength (Cohesion) 2557.3.4 Shrinkage 255

7.4 Silicone PSAs 2567.4.1 Properties 2567.4.2 Effect of Cross-LinkingAgent to the Basic

Properties Si–PSA 2607.4.3 Application 267

7.5 Conclusion 272References 273

Part 2 Functional Interfaces: Fundamentals and Frontiers

8 Interfacing Gelatin with (Hydr)oxides and Metal Nanoparticles: Design of Advanced Hybrid Materials for Biomedical Engineering Applications 277

Nathalie Steunou8.1 Introduction 2788.2 Physical Gelation of Gelatin 2798.3 Synthesis of Gelatin-Based Hybrid Nanoparticles and

Nanocomposites 2828.3.1 Preparation of Hybrid Composites by

Gelification and Complex Coacervation 2828.3.2 Processing of Gelatin-Based Hybrid Materials

into Monoliths, Films, Foams and Nanofibers 2888.3.3 Synthesis of Hybrid and Core–Shell

Nanoparticles and Nano-Objects 2908.4 Characterization of Gelatin-Based Hybrid

Nanoparticles and Nanocomposites 2948.5 Mechanical Properties of Gelatin-Based Hybrid

Nanoparticles and Nanocomposites 2968.6 Design of Gelatin-Based Hybrid Nanoparticles for

Drug Delivery 3028.7 Design of Nanostructured Gelatin-Based Hybrid

Scaffolds for Tissue Engineering and Regeneration Applications 310

8.8 Conclusions and Outlook 316References 318

Contents ix

9 Implantable Materials for Local Drug Delivery in Bone Regeneration 325

P. Díaz-Rodríguez and M. Landin9.1 Bone Morphology 3259.2 Bone Fracture Healing Process 3269.3 Current Materials for Bone Regeneration 327

9.3.1 Metals 3299.3.2 Ceramics 330

9.3.2.1 Biodegradable Ceramics 3309.3.2.2 Non-Absorbable Ceramics 332

9.3.3 Polymers 3329.3.3.1 Natural Polymers 3339.3.3.2 Synthetic Polymers 334

9.3.4 Composites 3359.4 Therapeutic Molecules with Interest in Bone Regeneration 336

9.4.1 Antibiotics 3379.4.2 Growth Factors 3399.4.3 Bisphosphonates 3409.4.4 Corticosteroids 3419.4.5 Hormones 3419.4.6 Antitumoral Drugs 3419.4.7 Others 342

9.5 Mechanism for Loading Drugs into Implant Materials and Release Kinetics 3439.5.1 Unspecific Adsorption 3449.5.2 Physical Interactions 3459.5.3 Physical Entrapment 3489.5.4 Chemical Immobilization 350

9.6 In Vitro Drug Release Studies 3509.6.1 Drug Release Kinetic Analysis 354

9.7 Translation to the Human Situation 3559.8 Conclusions (Future Perspectives) 356Acknowledgments 357References 357

10 Interaction of Cells with Different Micrometer and Submicrometer Topographies 379

M.V. Tuttolomondo, P.N. Catalano, M.G. Bellino

and M.F. Desimone10.1 Introduction 37910.2 Synthesis of Substrates with Controlled Topography 380

x Contents

10.3 Methods for Creating Micro- and Nanotopographical Features 381

10.4 Litography 381 10.4.1 Photolithography 381 10.4.2 Electron-Beam Lithography 382 10.4.3 Nanoimprint Lithography 383 10.4.4 Soft Lithography 384

10.5 Polymer Demixing 38410.6 Self-Assembly 38510.7 Cell Material Interactions 386

10.7.1 Lithography Method 386 10.7.2 Polymer Demixed 390 10.7.3 Cell Behaviour onto EISA obtained films 390 10.7.4 Biological Evidence 395

10.8 Conclusions 397Acknowledgements 399References 399

11 Nanomaterial—Live Cell Interface: Mechanism and Concern 405

Arka Mukhopadhyay and Hirak K. Patra11.1 Introduction 40511.2 Protein Destabilization 40711.3 Nanomaterials-Induced Oxidative Stress 408

11.3.1 Transitional Metal–Oxide Nanomaterials and ROS 409

11.3.2 Prooxidant Effects of Metal Oxide Nanoparticles 409

11.3.3 CNT-Induced ROS Formation 412 11.3.3.1 CNT-Induced Inflammation and

Genotoxicity and ROS 41511.4 Nucleic Acid Damage 41511.5 Damage to Membrane Integrity and Energy

Transduction 41811.6 Conclusions 418References 419

Contents xi

12 Bioresponsive Surfaces and Interfaces Fabricated by Innovative Laser Approaches 427

F. Sima, E. Axente, C. Ristoscu, O. Gallet, K. Anselme and

I.N. Mihailescu12.1 Introduction 42812.2 Pulsed Laser Methods Applied for the Grown of

Inorganic and Organic Coatings 43012.3 Combinatorial Laser Approaches: New Tool for the

Fabrication of Compositional Libraries of Hybrid Coatings 434

12.4 Thin Bioresponsive Coatings Synthesized by Lasers 437 12.4.1 Bioactive Inorganic Coatings Obtained

by PLD 438 12.4.2 Bioactive Organic Coatings Obtained

by MAPLE 439 12.4.3 Bioactive Inorganic–Organic Coatings

Obtained by Pulsed Laser Techniques 440 12.4.4 Combinatorial Thin Coatings Libraries

Synthesized by C-MAPLE 442 12.4.4.1 Tailoring Cell Signaling Response by

Compositional Gradient Bioactive Coatings 442

12.4.4.2 Coatings for Protein Immobilization and Controlled Release 448

12.5 Conclusion and Perspectives 452Acknowledgments 453References 453

13 Polymeric and Non-Polymeric Platforms for Cell Sheet Detachment 463

Ana Civantos, Enrique Martinez-Campos, Maria E. Nash,

Alberto Gallardo, Viviana Ramos and Inmaculada Aranaz13.1 Introduction 46313.2 The Extracellular Matrix 46513.3 Platforms for Cell Detachment 466

13.3.1 Electroresponsive Platforms 466 13.3.1.1 Electroactive Self-Assembled

Monolayers 466 13.3.1.2 Polyelectrolyte-Modified Surfaces 469

13.3.2 Light-Induced Detachment 469 13.3.2.1 Photosensitive Inorganic-Based

Surfaces 469 13.3.2.2 Photosensitive Organic-Based

Surfaces 471 13.3.3 pH-Sensitive Surfaces 473

13.4 Degradable Platforms 475 13.4.1 Other Detaching Systems 476 13.4.2 Mechanical Platforms 476 13.4.3 Magnetic Platforms 479 13.4.4 Thermoresponsive Platforms 480 13.4.5 Clinical Translation 485

13.5 Conclusions 487References 487

Index 497

xii Contents

Preface

We all love Agent 007, but Bond wouldn’t be Bond without his instruments with smart interfaces. In each film we all expect to see Q demonstrating the assigned tools given to Bond for his next mission. It is almost guaran-teed that each and every piece of an instrument responds to Bond through well-integrated interfaces. For example, in the movie Skyfall, a Walther PPK/S 9mm short pistol is equipped with an advanced palm-print reader that activates the gun to fire only if it detects Bond’s palm. Following this logic, Bond’s gadgets are superior models of advanced materials interfaces. So it is up to us, the materials scientists, to design and develop the neces-sary material interfaces in the form of physical, chemical, and biological systems for the advancement of mankind. A profound understanding of different interfaces is thus a step forward into the future.

Interfaces are the key controller in nearly all advanced devices. In a wide spectrum of applications, from chemical catalysis to the Mars rover Opportunity, advancement is conducted by an elegance in our scientific understanding of manifold interfaces. Therefore, the implementation of cross-disciplinary systems is mostly interface driven. However, our cur-rent aspirations and confrontations in interface science involve more than a simple catalytic interface. For example, in medicine we want to direct, stimulate, and communicate with the diseased part to promote healing. This actually brings us to the “advanced material interface,” an interface that is fashioned by our accomplishments and that holds the keys to con-trol the material and/or device behavior in ways that consequently result in preferred outcomes. As the materials used for different purposes in our lives differ greatly, it has been difficult to develop a generalized concept regarding material interface, although many inspiring works have been conducted that have provided the interpretive foundation of advanced material interactions.

The collection of chapters in this book focuses on two key aspects, i.e., the design, fabrication and properties of advanced materials interfaces, and fundamentals and frontiers of relevant functional interfaces. The con-tents cover a wide range of the advanced materials interfaces with some

xiii

xiv Preface

academic and commercial purposes, with chapters focused on the fabrica-tion techniques, such as some recent development in the mixed protein/polymer nanostructures at interfaces by Stergios Pispas (Chapter 1). In view of the predominant functions of the self-assembly multifunctional materials, Giulio Malucelli reviews the exploitation of self-assembly phe-nomena in liquid crystalline phases for obtaining multifunctional mate-rials (Chapter 2). Jan Seidel discusses the scanning probe microscopy of functional materials surfaces and interfaces in Chapter 3. In an associ-ated chapter (Chapter 4), Giorgio Senesi emphasizes AFM approaches to the study of PDMS-Au and carbon based surfaces and interfaces, while Francesco Ruffino reviews the fabrication, characterization and appli-cations of one-dimensional gold-silica nanocomposites in Chapter 5. Meanwhile, some chapters of the book concentrate on the current research of the understanding of the basic mechanisms acting on advanced materi-als interfaces. Dimitra Achillopoulou looks at concrete elements, materials and techniques for understanding the basic mechanisms acting on inter-faces (Chapter 6). Adrian Antosik reviews pressure-sensitive adhesives (PSA) based on silicone in Chapter 7.

In the topic of fundamentals and frontiers of functional interfaces, bio-sensing techniques and biomedical applications are of upmost significance. Thus, Nathalie Steunou reviews the interfacing gelatin with hydroxides and metal nanoparticles for design of advanced hybrid materials for biomedi-cal engineering and sensing applications (Chapter 8). Implantable mate-rials take center stage in Chapter 9 with “Implantable materials for local drug delivery in bone regeneration”, authored by Patricia Diaz-Rodriguez. Martin Desimone is the author of Chapter 10 “Interaction of cells with different micrometer and submicrometer topographies”. Hirak K Patra reviews in Chapter 11 the mechanism and concern of the nanomaterial – live cell interface. In Chapter 12, Ion Mihailescu discusses recent advances in the study of bioresponsive surfaces and interfaces fabricated by innovative laser approaches. Inmaculada Aranaz reviews polymeric and non-polymeric platforms for cell sheet detachment in Chapter 13.

This book, Advanced Material Interfaces, consists of the highest level of understanding on interface know-how and applications. It not only elab-orates on the complex interfaces fashioned of solids, liquids, and gases, but also ensures a cross-disciplinary amalgam of physics, chemistry, materials science, engineering and life sciences. Advanced interfaces have a fundamental role in the operation of essentially all integrated devices. It is of utmost urgency to focus on how newly discovered fundamental constituents and interfacial progressions can be realized and used for precise purposes. Interfaces are associated with a wide multiplicity of the

Preface xv

application spectrum, from chemical catalysis to drug functions, and the advancement is funneled by fine-tuning our fundamental understanding of the interface effects.

The motivation for this book was to establish a starting point for elucidating and exploiting the different aspects of interfacial interactions with materials for science and technology. We have tried to cover many aspects of interfaces in different systems such as bioelectronics, bio sensors, engineering, and nanosystems. We hope that this book will provide a strong overview of advanced materials interfaces for scientists, research-ers, lecturers, undergraduate and graduate and PhD students in science.

We would like to thank all authors who are greatly appreciated for preparing the chapters with high quality and the production team for dedi-cated work to promote the birth of this book.

EditorsAshutosh Tiwari, PhD, DSc

Hirak K. Patra, PhDXuemei Wang, PhD

May 13, 2016

Part 1

INTERFACES DESIGN,

FABRICATION, AND PROPERTIES

3

Ashutosh Tiwari, Hirak K. Patra, and Xuemei Wang (eds.) Advanced Materials Interfaces, (3–36)

© 2016 Scrivener Publishing LLC

1

Mixed Protein/Polymer Nanostructures at Interfaces

Aristeidis Papagiannopoulos1 and Stergios Pispas2*

1Polymer and Colloid Science Group, Institute of Electronic Structure and Laser

(FORTH), Heraklion, Crete, Greece 2Theoretical and Physical Chemistry Institute, National Hellenic Research

Foundation, Athens, Greece

AbstractThe modification of water/solid interfaces by adsorbed neutral or charged mac-

romolecules is proved to provide an excellent environment for controlled pro-

tein loading and release. Advanced experimental methodologies that probe the

structural details of planar interfaces at nanometer length scales are presented.

The broad fields of polymers at interfaces and protein–polymer interactions in

solutions are introduced as a lay ground for the central subject of proteins at

polymer-modified interfaces. Important contributions to the literature are used

as paradigms to highlight the main findings and open subjects in the field, and at

the same time, the complementary use of experimental methods is illustrated. The

different kinds of interactions of proteins with macromolecular layers of various

conformations are broadly categorized, although the boundaries between cases

are by no means strict.

Keywords: Polymer interfaces, protein–polymer interactions, polyelectrolyte

multilayers, polyelectrolyte brushes, protein adsorption, counterion release,

protein charge anisotropy

1.1 Introduction

Controlling the properties of interfaces is a major research challenge because of the numerous practical applications in nano-bio technologies [1].

*Corresponding author: pispas@eie.gr

4 Advanced Materials Interfaces

Implant compatibility, protein separation and resistance, drug loading and release, tissue engineering, and antifouling are fields where the modern concepts can be exploited and generalized. Recent advances in experi-mental studies of well-defined systems lay the ground for better under-standing and potential theoretical description of the complex problem of bio- molecules or nano-drugs near polymer-functionalized interfaces. Polymers at interfaces offer great versatility due to their possibility for selective immobilization of components and stimuli responsiveness [2].

In this chapter, the central concepts of protein interaction with poly-mer-modified interfaces are presented. The conformation of the sur-face-attached macromolecular chains and the steric, electrostatic, and hydrophobic forces are key players in the binding of proteins on polymers and polyelectrolytes. Hence, the most commonly used polymeric layers, e.g. polyelectrolyte multilayers and neutral polymer or polyelectrolyte brushes are presented together with the main experimental methods used for their characterization in the first part of the chapter [3, 4]. Additionally, the interaction of proteins with solid surfaces in contact with water is introduced.

The main part of the chapter deals with the complexation of proteins with neutral polymer or polyelectrolyte layers in the water/solid interface. The key methods for studying the conformational changes and distri-bution of chains and proteins upon protein complexation are presented through important contributions from the literature, as the neutron reflec-tivity study of the distribution of deuterated proteins within PEG brushes [5]. The mechanisms of counterion release and the role of protein charge anisotropy are described, as they have been under investigation in the past decade and are still an open field of research. Finally, works with potential for applications are highlighted.

1.2 Neutral and Charged Macromolecules at Interfaces

The formation of polymeric interfaces is a field with long tradition in soft-matter research [6] because of its tremendous importance in industrial applications, food science, and biomedical research [2]. This discipline has evolved to the study of stimuli responsive interfaces created by the pres-ence of stimuli-responsive polymers. Since, in this chapter, the discussion will be focused on physical interactions between polymers and proteins, we will mainly discuss formation of polymeric interfaces from aqueous solu-tions. The formed layers can be very broadly categorized in (a) statistically

Mixed Protein/Polymer Nanostructures at Interfaces 5

adsorbed linear chains, (b) macromolecular brushes, and (c) polyelectro-lyte multilayers (Figure 1.1).

Adsorption of macromolecular species from aqueous solutions in con-tact with an interface depends on the interface/macromolecule interactions inside water. When the macromolecule contains hydrophobic groups, then their tendency to reduce their contacts with water forces them to sepa-rate from solution and become attached to the surface [7]. This effect can be reinforced by increasing the hydrophobicity of the interface, e.g. by polystyrene (PS) modification of a silicon surface [8]. Although energeti-cally it is favorable for all the hydrophobic groups of the chains to become attached on the surface, there are constraints [9] caused by the reduction of the chain conformational entropy (chain elasticity) and steric/electrostatic repulsions between chain segments near the interface. For homopolymer chains that contain segments with a moderate affinity to a surface entro-pic and steric restriction put a barrier to the amount of adsorbed polymer [10]. A random copolymer with hydrophilic and hydrophobic monomers is driven to the surface mostly due to its hydrophobic units [11]. In both cases, the segments bound to the surface are statistically distributed along the contour length (Figure 1.1a). The conformations are described by loops (free dangling chain parts between adsorbed segments), trains (continuous adsorbed chain parts), and tails free ends of adsorbed chains [6].

The conformation of adsorbed macromolecular chains is different than its conformation in solution. In a confined geometry the distances between monomers of different chains are fairly close, which increases the inter-chain interactions. Additionally, the interactions with the interface are very crucial since the last may create strong bonds for certain monomers, while others are free to move in solution. An example of great conformational change caused by confinement, due to interaction within a polymeric

Figure 1.1 Layers of macromolecular chains on surfaces: (a) statistically adsorbed linear

chains, (b) macromolecular brushes (grafting points at the chain ends are depicted as

black dots), and (c) polyelectrolyte multilayers (grey and black chain layers alternate).

(a)

(b) (c)

6 Advanced Materials Interfaces

layer, is this of a macromolecular brush of chains in good solvent condi-tions (Figure 1.1b). The chains do not feel any strong attraction from the surface except that their one end is bound to it (end-attached chains). If the distance (on the interface plane) between neighboring attaching points is much higher than the dilute-solution radius of gyration of a single chain, then the monomer concentration within the layer is high enough to overcome the entropic cost for stretching the chains outwards [12, 13]. Macromolecular brushes are very effective in stabilizing colloidal disper-sions, especially polyelectrolyte brushes in aqueous media [14]. This way they can also prevent protein adsorption due to the high content of molec-ular species that makes difficult for incoming ones (proteins) to penetrate and reach the surface.

In aqueous environments, the use of macromolecules with ionizable groups, i.e. polyelectrolytes is very popular since it offers a great vari-ety of polymers (even otherwise intrinsically hydrophobic) to be used and also provides stimuli-responsive properties. In brushes made from end-attached strongly charged polyelectrolytes, the salt concentration of the solution acts as an external stimulus. Increasing the salt content, the electrostatic repulsions between monomers weaken, and the elasticity of the chains reduces the layer thickness. In particular, in a brush with high grafting density and high number concentration of counterions (osmotic brush), the salt content of the solution makes a difference to the brush characteristics only when it is higher than the counterion concentration within the brush. At low salt content, the counterions are localized within the brush and keep it fully extended by the high osmotic pressure they cre-ate [15]. This effect is a powerful way to prevent colloidal aggregation and flocculation even at relatively high salt concentrations where the electro-static repulsions are too weak to provide stability.

As already discussed, except from the brush conformation, where chains can be chemically grafted or physically adsorbed by a hydrophobic group or block at the end of the chain (amphiphilic block architecture), homopolymers, or random copolymers can become physically adsorbed on an interface. In that case, polymeric layers can be produced, but the range of thicknesses and adsorbed amounts that can be achieved is limited. Especially in the case of polyelectrolyte adsorption, the long range repul-sion between chains of a single species creates an energy barrier for new chains to reach contact with the surface that keeps the adsorbed amounts relatively low. A straight-forward way to create highly hydrated polyelec-trolyte layers [16] of desirable thickness, and adsorbed amount is the layer-by-layer deposition (Figure 1.1c) of alternating positively and negatively charged polyelectrolytes, i.e. polyelectrolyte multilayers [17].

Mixed Protein/Polymer Nanostructures at Interfaces 7

1.3 Interfacial Experimental Methods

One of the most powerful methods to investigate polymeric layers on solid/liquid [18, 19] and air/liquid interfaces [20] is neutron reflectivity (NR). As in all neutron scattering-related techniques, the scattering contrast is defined by the neutron scattering length density differences of the compo-nents in the system. The power of these methods is in the fact that chemi-cally equivalent isotopic nuclei can have significantly different scattering lengths, e.g. hydrogen versus deuterium. Using a hydrogenated polymer in a deuterated solvent (D

2O) creates adequate contrast for strong scattered

intensity. Additionally, when labeling one component between others is feasible, for example, one polymeric species within a mixture or a certain block of diblock copolymers, selective deuteration is used [19]. This selec-tive exchange of hydrogens with deuterons creates species of clearly dif-ferent neutron scattering length densities. Mixing the hydrogenated and deuterated version of the solvent (contrast variation) can produce a solvent of the same scattering length density as one of the species (contrast match-ing). Making one of the species effectively “invisible” from neutrons allows the conformation of the other species to be independently characterized.

In NR, a collimated neutron beam (with intensity Ii) hits the interface

and the reflected intensity Ir (Figure 1.2) is measured as a function of the

momentum transfer (qz), i.e. the difference between the reflected and inci-

dent wave vectors. The measured quantity of interest is the reflectivity

R qI q

Iz

r z

i

. The x–y (interfacial plane) average of scattering lengh

density profile ρ(z) defines R(qz). Hence, a reflectivity experiment [21]

Figure 1.2 Schematic representation of NR for the characterization of a layer on the

solid/liquid interface.

Solid/liquid interface

Thin layer

Solution

ki kr

qz

8 Advanced Materials Interfaces

provides the scattering length density profile perpendicularly to the plane of incidence defined by the planar interface (z-direction of Figure 1.2).

Atomic force microscopy (AFM) provides the roughness profile of a surface or in other words the height profile z(x,y) by measuring the force between a probe tip (cantilever) and the surface. In the tapping mode, the perturbation on soft samples is minimal in contrast to the contact mode. The oscillating cantilever helps to avoid lateral forces and displacement of weakly attached entities [22]. AFM provides 3-D images of the interfaces with vertical resolution (z-direction) in the order of 1 nm and lateral resolution several tens of nms. The interactions of pro-teins with layers of polyelectrolytes can be visualized by the morphol-ogy changes of the polyelectrolyte layers upon the complexation with the proteins [23, 24]. The roughness profile of a surface can be quantified by calculating its rms value or plotting z(λ), i.e. the height profile along a pre-defined contour on an AFM image. The self-similar structure and the characteristic length scales on a surface are provided by the power-spectral density (PSD) [25], which is related to the 2-D Fourier analysis of z(x,y).

In surface plasmon resonance (SPR), electromagnetic surface waves along a metal/dielectric interface can be formed under certain conditions which are called surface plasmons. A laser beam is totally reflected on the dielectric (glass)/metal interface, and the angle of incidence is scanned in a broad range (Figure 1.3). The presence of the metallic layer creates surface waves that produce a reflectivity profile, which is a function of angle of inci-dence even in the total internal reflection regime. This profile contains the information of the laterally averaged refractive index of the formed layer (in principle similarly with NR), while practically the angular position of its minimum can provide the adsorbed amount on the metallic layer after

Figure 1.3 SPR in the Kretschmann configuration.

Laser

beam Detector

Prism

Silver layer

Adsorbed layer

Solution

Mixed Protein/Polymer Nanostructures at Interfaces 9

proper calibration. SPR is a highly sensitive method that is ideally suited for sensor applications [26] and has been successfully used for monitoring protein [27] and polyelectrolyte [28] adsorption.

Quartz crystal microbalance (QCM) is based on the piezoelectric prop-erties of quartz crystals [29]. For QCM, a quartz crystal wafer is cut in a form that allows stable oscillations in thickness shear mode. Oscillation at characteristic frequencies is induced by an electric circuit made of elec-trodes attached on the crystal. The difference of the characteristic frequency caused by an adsorbed substance (compared to the frequency of the pure crystal) depends on the adsorbed mass. In QCM, the bound mass oscillates at the same frequency and displacement as the underlying crystal. If the process is elastic, then no energy is dissipated. If the process is inelastic, it is accompanied by energy dissipation which provides information on the viscoelasticity of the adsorbed layer [30]. In studies on the water/solid interface, the hydrophilicity and roughness of the surface must be taken into account because both of them can cause the liquid from solution to follow the motion of the surface resulting to an increase in the apparent mass. Hydrophobic surfaces have the opposite effect because incomplete wetting may lead to entrapped air or vacuum.

Ellipsometry uses p-polarized visible laser light reflected on an interface to characterize nm range thin films. The ratio r

p/r

s of the amplitudes of

the p-polarized over the s-polarized reflected light is a complex number whose absolute value and phase angle contain information of the refractive index distribution normal to the interface [31]. It has been traditionally used to characterize the adsorbed amount and thickness of polymer and protein [32] layers. In general, the ellipsometry data are fitted to a model of the interface, i.e. slabs of certain thickness and uniform refractive index in order for the refractive index profile to be obtained.

1.4 Interactions of Proteins with Polymer-Free Interfaces

Proteins at the aqueous solution/solid interface play an important role in biocompatibility of implants and cell adhesion. The adsorption of a pro-tein on a surface is defined by the physico-chemical characteristics of the surface, the solution conditions, and the kind of the protein. These factors will affect the amount, the orientation, the conformation, and the mutual arrangement at the interface [33]. Forces such as van der Waals, electro-static, hydrogen bonding, and hydrophobicity are the ones that induce

10 Advanced Materials Interfaces

physical protein adsorption. Understanding these interactions is crucial for the description of polymer–protein interactions at interfaces.

The electrostatic part of the interaction between a protein and a charged surface is a problem of high complexity. It is known that the overall net charge of a protein is not enough to describe its electrostatic interaction with a charged surface due to the asymmetric distribution of charges on the protein [34]. A charged patch of the protein may be attracted by the surface charge, while the rest of the protein charge is repelled. This leads to adsorption with a preferred orientation. The distribution of ions in the region of the interface is significantly different from the one in the bulk. For example, the pH near an interface can be different from the solution pH. The charge of a protein that is near this interface will be in this way different from the one in bulk solution (charge regulation). Furthermore, adsorption of a charged particle will influence the electrostatic potential and charge distribution at the interface.

Surface chemistry is a factor that defines the adsorption of proteins [35]. A QCM study of adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) on –CH

3 (hydrophobic) and –OH (hydrophilic) modified gold sur-

faces showed the importance of shape and hydrophobicity of the proteins [33]. BSA is a globular protein, whereas Fg is an elongated one. BSA exhib-its a faster adsorption rate and final adsorbed amount on the hydrophobic surface in comparison to the hydrophilic one, while Fg shows similar rates and amounts for the two surfaces. In the case of BSA, there was a higher affinity for the hydrophobic surface (higher binding constant), whereas the adsorbed amount at saturation was higher on the hydrophilic surface. This was explained by a conformational change of BSA upon adsorption on the surface of higher affinity. The adsorption of Fg is a two-stage process. In the first stage, proteins adsorb rapidly and randomly with their long axis paral-lel to the surface. In the second stage, the proteins re-orient perpendicular to the surface in order to accommodate the increased number of incoming proteins and also decrease their unfavorable hydrophobic interaction with water. The helical structure of both proteins is denatured to a large degree by the interaction with the hydrophobic surface as it was found by grazing angle FTIR.

In a NR study, the conformational changes and possible denaturation of proteins upon adsorption can be implicitly defined by the capability of accurate layer thickness measurements. For example, when myoglo-bin (Mb) adsorbs on octadecyltrichlorosilane (OTS)-modified surfaces from low concentrations, a dense protein layer is formed with a thickness (1.3 nm) significantly lower than the diameter of the protein (4.0 nm) in its

Mixed Protein/Polymer Nanostructures at Interfaces 11

native conformation [36]. On PS (a less hydrophobic substrate) although the protein adsorbed amount is similar to the previous case, the protein diameter is affected to a smaller extent (thickness ~2.1 nm), i.e. the protein is less flattened. At higher solution concentration, another less dense layer is formed on top of the denatured one. This layer has a thickness similar to one protein diameter meaning that the protein keeps its bulk solution conformation (Figure 1.4).

1.5 Polymers and Proteins in Solution

The development of drug carriers, protein separation, food industry, and biosensors applications has driven a lot of research work toward the investigation of optimal systems and fundamental understanding of the interactions involved. The range of experimental methods for com-plexation between polyelectrolytes and proteins is wide [37]. It includes small angle scattering techniques with neutrons or X-rays (SANS, SAXS),

Figure 1.4 Neutron scattering length density profiles obtained from NR for protein

adsorption on OTS (a) and PS (b) modified silicon substrates from high (1) and low

(2) protein solution concentrations. The dashed lines represent the profiles without

included roughness. Reprinted from [36], Copyright 2012, with permission from Elsevier.

6

4

2

0

–2

6

4

2

0

–2

–1

0

1

2

3

0 50 100

OTS

OTS

Layer 2

Layer 2

Layer 1 1a 1b

2a 2bLayer 1

Layer 1

Layer 1

z (Å)

SL

D (

10

–6Å

–2)

SL

D (

10

–6Å

–2)

SL

D (

10

–6Å

–2)

–1

0

1

2

3

SL

D (

10

–6Å

–2)

150

0 50 100z (Å)

150

0 50 100z (Å)

150

0 50 100z (Å)

150

H2O

H2O

H2O

H2O

SiO2

SiO2

Si

SiO2Si

Si

SiO2

Si

PS

PS

(a) (b)

12 Advanced Materials Interfaces

thermodynamics-related methods (ITC), microscopy (TEM, SEM, AFM), and rheological methods. SANS especially is very sensitive to the posi-tions of the atoms nuclei and gives clear information on the morphology at length scales between 1 and 1000 nm (combined with USANS). Contrast variation in SANS provides flexibility in separating the scattering contri-butions of the different species in solution and makes this method ideal for complexation experiments where the scattering length densities of the interacting components are adequately different [38].

In polyelectrolyte–protein complexation, electrostatics is an important interaction between the two charged components. The net charge of pro-teins is not the sole parameter defining the complexation. The works of Dubin et al. [37] demonstrate the complexity of interactions between poly-electrolytes and proteins. The distribution of positive and negative charges on a protein (charge patches) is of equal importance and seemingly leads to the attraction between objects of same charge. Charge regulation is also potentially an effect that causes proteins to bind to polyelectrolytes of same charge. The works of Ballauff et al. have dealt with this problem by small angle scattering techniques and pointed on the electrostatic nature of attrac-tion at the “wrong side” of the isoelectric point [39, 40]. The origin of the preferable state of complexed proteins and spherical polyelectrolyte brushes of same charge was explained by the gain in entropy via counterion release.

The complexation of lysozyme with the oppositely charged PSSNa in semi-dilute conditions for PSSNa is an example of utilization of contrast matching in SANS [38]. Using deuterated PSSNa and D

2O/H

2O mixtures

as solvents, it was possible to resolve the scattering contribution of the sep-arate components within the complexes. It was found that for short poly-electrolyte chains and excess of protein the initial polyelectrolyte network structure was changed to aggregated state. In excess of polyelectrolyte, the form factor of lysozyme is altered to one closer to an excluded volume chain one. This was a direct evidence of protein unfolding due to interac-tion with the polyelectrolyte. The effect of destruction of lysozyme’s α-helix was confirmed by FTIR.

Ballauff et al. used SAXS to resolve radial distribution of electron density in model spherical core–shell polyelectrolyte brushes [41] and polyelec-trolyte microgel nanoparticles [42]. The radial distribution of BSA on the nanoparticles showed that the protein binds mostly on the polyelectrolyte and not on the hydrophobic core, which was highlighted as signature of domination of the electrostatic interactions. Additionally, they have found with time-resolved SAXS experiments that the loading of nanoparticles