reading

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

Chapter 3: Scanning Probe Microscopy of Functional Materials Surfaces and Interfaces

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

4.2 AFM Characterization of Micro–Nano Surfaces and Interfaces of Carbon-Based Materials and PDMS-Au Nanocomposites

4.4 Scanning Capacitance Microscopy, Kelvin Probe Microscopy, and Electromagnetic Characterization

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

5.1 Introduction: The Weird World of Silica Nanowires and Metal–Silica Composite Nanowires

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

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

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

6.3 Existing Knowledge on Force Transfer Mechanisms on Reinforced Concrete Interfaces

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

8.5 Mechanical Properties of Gelatin-Based Hybrid Nanoparticles and Nanocomposites

8.7 Design of Nanostructured Gelatin-Based Hybrid Scaffolds for Tissue Engineering and Regeneration Applications

Chapter 9: Implantable Materials for Local Drug Delivery in Bone Regeneration

Chapter 10: Interaction of Cells with Different Micrometer and Submicrometer Topographies

Chapter 12: Bioresponsive Surfaces and Interfaces Fabricated by Innovative Laser Approaches

12.2 Pulsed Laser Methods Applied for the Grown of Inorganic and Organic Coatings

12.3 Combinatorial Laser Approaches: New Tool for the Fabrication of Compositional Libraries of Hybrid Coatings

The Advanced Materials Series provides recent advancements of the fascinating field of advanced materials science and technology, particularly in the area of structure, synthesis and processing, characterization, advanced-state properties, and applications. The volumes will cover theoretical and experimental approaches of molecular device materials, biomimetic materials, hybrid-type composite materials, functionalized polymers, supramolecular systems, information- and energy-transfer materials, biobased and biodegradable or environmental friendly materials. Each volume will be devoted to one broad subject and the multidisciplinary aspects will be drawn out in full.

Series Editor: Dr. Ashutosh Tiwari
Biosensors and Bioelectronics Centre
Linköping University
SE-581 83 Linköping
Sweden
E-mail: ashutosh.tiwari@liu.se

Publishers at Scrivener
Martin Scrivener (martin@scrivenerpublishing.com)
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Co-published by John Wiley & Sons, Inc. Hoboken, New Jersey, and Scrivener Publishing LLC, Salem, Massachusetts.
Published simultaneously in Canada.

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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 guaranteed 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 necessary 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 current 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 control 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 contents cover a wide range of the advanced materials interfaces with some academic and commercial purposes, with chapters focused on the fabrication 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 phenomena in liquid crystalline phases for obtaining multifunctional materials (Chapter 2). Jan Seidel discusses the scanning probe microscopy of functional materials surfaces and interfaces in Chapter 3. In an associated 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 applications 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 materials interfaces. Dimitra Achillopoulou looks at concrete elements, materials and techniques for understanding the basic mechanisms acting on interfaces (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, biosensing 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 biomedical engineering and sensing applications (Chapter 8). Implantable materials 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 elaborates 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 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, biosensors, engineering, and nanosystems. We hope that this book will provide a strong overview of advanced materials interfaces for scientists, researchers, 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 dedicated work to promote the birth of this book.

Editors
Ashutosh Tiwari, PhD, DSc
Hirak K. Patra, PhD
Xuemei Wang, PhD
May 13, 2016

Part 1

INTERFACES DESIGN, FABRICATION, AND PROPERTIES

Chapter 1

Mixed Protein/Polymer Nanostructures at Interfaces

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

Abstract

The modification of water/solid interfaces by adsorbed neutral or charged macromolecules is proved to provide an excellent environment for controlled protein 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]. 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 experimental studies of well-defined systems lay the ground for better understanding 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 polymer-modified interfaces are presented. The conformation of the surface-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 distribution of chains and proteins upon protein complexation are presented through important contributions from the literature, as the neutron reflectivity 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 presence 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 solutions. The formed layers can be very broadly categorized in (a) statistically adsorbed linear chains, (b) macromolecular brushes, and (c) polyelectrolyte multilayers (Figure 1.1).

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).

Adsorption of macromolecular species from aqueous solutions in contact 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 separate 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 energetically 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 entropic 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 layer, is this of a macromolecular brush of chains in good solvent conditions (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 dispersions, especially polyelectrolyte brushes in aqueous media [14]. This way they can also prevent protein adsorption due to the high content of molecular 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 variety 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 create [15]. This effect is a powerful way to prevent colloidal aggregation and flocculation even at relatively high salt concentrations where the electrostatic 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 repulsion 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 polyelectrolyte 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].

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 components in the system. The power of these methods is in the fact that chemically equivalent isotopic nuclei can have significantly different scattering lengths, e.g. hydrogen versus deuterium. Using a hydrogenated polymer in a deuterated solvent (D₂O) 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 selective exchange of hydrogens with deuterons creates species of clearly different 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 matching). 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 I_i) hits the interface and the reflected intensity I_r (Figure 1.2) is measured as a function of the momentum transfer (q_z), i.e. the difference between the reflected and incident wave vectors. The measured quantity of interest is the reflectivity

. The x–y (interfacial plane) average of scattering lengh density profile ρ(z) defines R(q_z). Hence, a reflectivity experiment [21] provides the scattering length density profile perpendicularly to the plane of incidence defined by the planar interface (z-direction of Figure 1.2).

Figure 1.2 Schematic representation of NR for the characterization of a layer on the solid/liquid interface.

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 proteins with layers of polyelectrolytes can be visualized by the morphology 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 incidence 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 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.

Figure 1.3 SPR in the Kretschmann configuration.

Quartz crystal microbalance (QCM) is based on the piezoelectric properties 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 electrodes 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 protein 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, electrostatic, hydrogen bonding, and hydrophobicity are the ones that induce 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 –CH₃ (hydrophobic) and –OH (hydrophilic) modified gold surfaces showed the importance of shape and hydrophobicity of the proteins [33]. BSA is a globular protein, whereas Fg is an elongated one. BSA exhibits 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 parallel 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 myoglobin (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 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).

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.

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 complexation between polyelectrolytes and proteins is wide [37]. It includes small angle scattering techniques with neutrons or X-rays (SANS, SAXS), thermodynamics-related methods (ITC), microscopy (TEM, SEM, AFM), and rheological methods. SANS especially is very sensitive to the positions 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 contributions 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 proteins is not the sole parameter defining the complexation. The works of Dubin et al. [37] demonstrate the complexity of interactions between polyelectrolytes 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 attraction 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 D₂O/H₂O mixtures as solvents, it was possible to resolve the scattering contribution of the separate components within the complexes. It was found that for short polyelectrolyte 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 interaction 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 polyelectrolyte 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 could be dominated by sliding of the protein globules along the polyelectrolyte chains toward the center [42].

Hyaluronic acid is an intrinsically flexible polyelectrolyte. Mixed with lysozyme in aqueous solutions, it formed polyelectrolyte/protein complexes of rigid elongated morphology [43]. In more detail, the rod-like structures have the size of the hyaluronic acid chain contour length when the polymer is of low molecular weight. When the polymer’s molecular weight is high, the rod-like structures appear larger than the persistence length of hyaluronic acid. At low [–]/[+] charge ratios and salt contents, large clusters appear in solution.

As observed in Figure 1.5, when lysozyme is added to the solution the high-q (small length scale) regime is dominated by the form factor of the well-defined lysozyme globules. The low and intermediate q-range is gradually dominated by the characteristic fractal scattering of linear objects I ~ q⁻¹, which is the signature of rod-like formations [43]. Below [–]/[+]=2.2, clustering of complexes results to scattering from large objects with well-defined interfaces (I~q⁻⁴ upturn at low q).

Figure 1.5 Small angle neutron scattering data from lysozyme/hyaluronic acid mixtures at pH 4.7. From bottom to top lysozyme solution concentration increases, i.e. [–]/[+] ratio decreases. “2ph” indicates two-phase samples while the two curves on top are measurements from the dense phase. Straight lines indicate slope=–4 and –1.

The protein charge anisotropy has been treated by Dubin et al. [44] in a systematic way. It was found that the non-trivial ionic strength dependence of the complexation with a polyelectrolyte is related to the non-uniformity of the electrostatic domains. The electric potential around a protein was visualized [44] by Delphi (Figure 1.6). Computer modeling with Delphi is based on a non-linear solution to the Poisson–Boltzmann equation in combination with the crystal structure of the protein and the charges of its amino acids. The outcome is the electrostatic potential around the protein as a function of pH and ionic strength.

Figure 1.6 Electric potential for BSA and lysozyme at pH 7 and I = 0.15 M. The surfaces correspond to 0.1 kT/e (grey: negative, dark grey: positive). Potential on protein surface is similarly colored.

The electric charge distribution is simplified by thinking of an electric dipole (not necessarily of equal positive and negative charge). The electrostatic interaction with another charge belonging to a polyion contains both an attractive and a repulsive term. The attractive term is expected to be larger in order for binding to take place, and this is supposedly achieved by the protein being oriented with the pole that it is oppositely charged to the macroion toward it. This is a proposed mechanism for complexation “on the wrong side” of the isoelectric point to take place. The net attractive force passes through a maximum as a function of ionic strength, which is reflected to the nonmonotonic ionic strength dependence of the amount of bound protein. It is predicted by the two-term interaction potential that its maximum corresponds to the Debye lengths on the order of the protein radius a result that is also found experimentally [44].

1.6 Proteins at Polymer-Modified Interfaces

The modification of surfaces with polymers aims at creating stimuli responsive interfaces for controlled loading and releasing procedures and biosensing applications. In practical applications, modification of surfaces with polymeric chains ranges from preventing proteins from adsorption to selectively separating them from solution. A polymeric layer at a solid/water interface offers an increased number of degrees of freedom for the interactions of the protein with the surface in contrast to an undecorated one. The presence of macromolecular chains provides an environment with tunable characteristic length scales (e.g. pore size), a degree of softness (i.e. chain elasticity), and often stimulus (pH, temperature, solvent, etc.) responsive nature.

1.6.1 Steric Effects

Although the interactions are much richer when electrostatic forces are included, the case of proteins near surfaces with neutral polymers is important both in conceptual and practical levels. Grafted flexible macromolecules impose kinetic and steric barriers [45] that control the adsorption of proteins or drug carriers. Their function can be tuned by the molecular weight, chemical structure, and surface coverage [46]. A protein that tries to reach a polymer grafted surface faces a kinetic barrier that is created by the polymers and the already adsorbed proteins. The incoming proteins force the grafted macromolecular chains to change conformation, and this in turn changes the potential barrier experienced by other proteins. Under a generalized diffusion approach, the increase of molecular weight of the tethered chains increases the potential barrier [46]. In more detail, there is an initial fast adsorption kinetics (i.e. the surface acts as an attractive sink) and a subsequent slower process where the incorporated proteins affect the layer configuration and hence the kinetics significantly. The second process becomes slower as the chain length increases. The initial fast process disappears above a certain value of polymer molecular weight. The desorption kinetics which are very slow become faster as the chain length increases, when the layer thickness is shorter than the protein size and slower in the opposite case. The polymer volume fraction near the surface is predicted to reduce (i.e. the chains stretch outward) in order for the protein to be accommodated.

An experimental realization of protein adsorption on a polymer-grafted surface is the case of Mb adsorbing a poly(ethylene glycol) (PEG) brush [5]. A silicon surface was modified by a 6 nm thick PS layer. The PEG brush was formed by transferring a monolayer of PS–PEG diblock copolymer chains from the air/water interface (Langmuir trough) onto the silicon surface (Langmuir–Schaefer technique). The PS–PEG chains are irreversibly attached to the PS layer by thermal treatment. The hydrophilic PEG-tethered chains act as the steric barrier for the incoming proteins and the PS-modified surface provides the short-ranged attractive potentials for the proteins (hydrophobic interaction). A NR experiment with contrast variation was performed to elucidate the volume fraction profiles of PEG and Mb. Mb was deuterated so that it has increased contrast to the normal hydrogen containing PEG. Experiments with four different kinds of solvent contrast were used (D₂O/H₂O mixtures from pure D₂O to pure H₂O), and the data from different contrasts were fit to the same model [5].

In Figure 1.7, the volume fraction profiles of the components on the water/silicon interface are shown. The interfaces between components appear not sharp due to the nanoscale topological roughness of the solid surfaces (Si, SiOx: silicon oxides normally formed on Si) or the diffuse interfaces of soft components. In other words, polished hard surfaces always have some remaining modulation of their lateral profile and polymeric (or protein) layers have diffuse boundaries. The volume fraction profile of Mb consists of two layers. A dense inner layer is formed at the PS layer and a sparse layer on top of the initial one. The second layer is absent when the PEG chain length is high (one-layer adsorption). The total amount of adsorbed Mb drops as the grafting density of the brush increases. This experiment also provides the possibility of testing the desorption process. After adsorption of deuterated protein, the solution in contact with the surface was exchanged with a hydrogenated protein containing solution. If there was exchange between adsorbed and solution proteins, this should reflect on the NR profiles, but this was not the case. Evidently, any desorption process of Mb from the PS surface and the PEG brush is much slower than the experimental time or in other words the adsorption can be thought of as irreversible [5].

Figure 1.7 (a) Volume fraction profiles of components as defined by NR. (b) Schematic diagram of solid and diffuse layers.

In the previous work, the adsorption of Mb was referred to as “primary” adsorption, and it was mentioned that no “ternary” or “secondary” adsorption of Mb was observed. The three terms correspond to adsorption at the grafting surface, adsorption within the brush, and adsorption on the outer edge of the brush, respectively (Figure 1.8). Obviously, the two latter cases involve some kind of attractive interaction between the protein and the grafted chains. Apparently, there is no significant interaction between PEG and Mb to induce ternary or secondary adsorption. Indeed, these modes of adsorption can be predicted for proteins that have at least weak attractions with the chain monomers [47].

Figure 1.8 Sketch of adsorption modes of globular proteins from a macromolecular brush.

Interestingly in the adsorption of BSA on poly(ethylene oxide) (PEO) brushes, there is evidence of ternary adsorption and absence of primary adsorption [48]. The BSA-adsorbed amount in brushes of long chains shows an increase at low grafting densities and a decrease at high grafting densities. This can be explained by dominance of ternary adsorption at low grafting densities and excluded volume repulsion at higher grafting densities. A bimodal brush was also prepared in order to confirm the ternary adsorption scenario. This brush consisted of both long and short chains with a total grafting density where the primary adsorption is strongly suppressed. The results were similar to those of monomodal brushes with long grafted chains.

The interaction of a globular protein with a soft interface is influenced by the architecture of the macromolecular layer [49]. In a study of quaternized poly(4-vinylpyridine) (QPVP), two kinds of layer morphologies were tested. One where the QPVP homopolymer chains were statistically adsorbed on silicon oxide surface (Si substrate) and the other where the QPVP chains (of the same length) were end grafted on a PS-modified surface in order to avoid adsorption of chain segments (polymer brush). They tested the interaction of the oppositely charged human serum albumin (HSA). For similar surface QPVP coverage, the equilibrium amount of HSA in the layer was higher for the brush configuration. This indicated that the brush layer has more free charged segments available for binding in contrast to the statistically adsorbed chains that have a lot of contacts with the solid surface, and they may also form closed loops. The kinetics of adsorption was slower for the brush layer (Figure 1.9a). The brush configuration produces smaller and deeper “pores” for the protein globules, while the statistically adsorbed chains have wider and shallow ones.

Figure 1.9 (a) Adsorption kinetics of HSA on brush and statistically adsorbed QPVP chains and (b) adsorption isotherms normalized by the saturated adsorbed amount. Circles: QPVP brush 1.8 mg/m² (filled) and 2.3 mg/m² (open). Squares: Statistically adsorbed QPVP 1.2 mg/m² (filled) and 1.7 mg/m² (open).

This “striking” steric effect explained also the adsorption isotherms (Figure 1.9b) that were more abrupt for the statistically adsorbed layer [49]. The adsorbed proteins induce a barrier that needs an osmotic pressure increase to move them deeper into the layer so that new ones may be incorporated. The effect is more intense in the case of the brush configuration.