Biomaterials Surface Science E-Book
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- Herausgeber: Wiley-VCH
- Kategorie: Fachliteratur
- Sprache: Englisch
At the interface of biology, chemistry, and materials science, this book provides an overview of this vibrant research field, treating the seemingly distinct disciplines in a unified way by adopting the common viewpoint of surface science. The editors, themselves prolific researchers, have assembled here a team of top-notch international scientists who read like a "who's who" of biomaterials science and engineering. They cover topics ranging from micro- and nanostructuring for imparting functionality in a top-down manner to the bottom-up fabrication of gradient surfaces by self-assembly, from interfaces between biomaterials and living matter to smart, stimuli-responsive surfaces, and from cell and surface mechanics to the elucidation of cell-chip interactions in biomedical devices. As a result, the book explains the complex interplay of cell behavior and the physics and materials science of artificial devices. Of equal interest to young, ambitious scientists as well as to experienced researchers.
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Seitenzahl: 1157
Veröffentlichungsjahr: 2013
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Table of Contents
Related Titles
Title Page
Copyright
Preface
List of Contributors
Part I: Polymer Surfaces
Chapter 1: Part One: Polymer Surfaces
1.1 Introduction
1.2 Structuring and Modification of Interfaces by Self-Assembling Proteins
1.3 Structuring and Modification of Solid Surfaces via Printing of Biomolecules
1.4 Conclusion and Outlook
References
Chapter 2: Surface-Grafted Polymer Brushes
2.1 Introduction
2.2 Synthesis of Polymer Brushes
2.3 Stimuli-Responsive Polymer Brushes
2.4 Polyelectrolyte Brushes
2.5 Bio-Functionalized Polymer Brushes
Acknowledgment
References
Chapter 3: Inhibiting Nonspecific Protein Adsorption: Mechanisms, Methods, and Materials
3.1 Introduction
3.2 Underlying Forces Responsible for Nonspecific Protein Adsorption
3.3 Poly(Ethylene Glycol)
3.4 Surface Forces Apparatus (SFA)
3.5 Applications of Poly(Ethylene Glycol)
Summary
References
Chapter 4: Stimuli-Responsive Surfaces for Biomedical Applications
4.1 Introduction
4.2 Surface Modification Methodologies: How to Render Substrates with Stimuli Responsiveness
4.3 Exploitable Stimuli and Model Smart Biomaterials
4.4 Biomedical Applications of Smart Surfaces
4.5 Conclusions
Acknowledgments
References
Chapter 5: Surface Modification of Polymeric Biomaterials
5.1 Introduction
5.2 Effect of Material Surfaces on Interactions with Biological Entities
5.3 Surface Morphology of Polymeric Biomaterials
5.4 Surface Modifications to Improve Biocompatibility of Biomaterials
5.5 Surface Modifications to Improve Hemocompatibility of Biomaterials
5.6 Surface Modifications to Improve Antibacterial Properties of Biomaterials
5.7 Nanoparticles
References
Chapter 6: Polymer Vesicles on Surfaces
6.1 Introduction
6.2 Polymer Vesicles
6.3 Applications of Polymer Membranes and Vesicles as Smart and Active Surfaces
6.4 Current Limitations of Polymer Vesicles and Emerging Trends
6.5 Conclusions
Abbreviations and Symbols
References
Part II: Hydrogel Surfaces
Chapter 7: Protein-Engineered Hydrogels
7.1 Introduction to Protein Engineering for Materials Design
7.2 History and Development of Protein-Engineered Materials
7.3 Modular Design and Recombinant Synthesis Strategy
7.4 Processing Protein-Engineered Materials
7.5 Conclusion
References
Chapter 8: Bioactive and Smart Hydrogel Surfaces
8.1 Introduction
8.2 Mimicking the Extracellular Matrix
8.3 Hydrogels: Why Are They So Special?
8.4 Elastin-Like Recombinamers as Bioinspired Proteins
8.5 Perspectives
Acknowledgments
References
Chapter 9: Bioresponsive Surfaces and Stem Cell Niches
9.1 General Introduction
9.2 Stem Cell Niches
9.3 Surfaces as Stem Cell Niches
9.4 Conclusions
References
Part III: Hybrid & Inorganic Surfaces
Chapter 10: Micro- and Nanopatterning of Biomaterial Surfaces
10.1 Introduction
10.2 Photolithography
10.3 Electron Beam Lithography
10.4 Focused Ion Beam
10.5 Soft Lithography
10.6 Dip-Pen Nanolithography
10.7 Nanoimprint Lithography
10.8 Sandblasting and Acid Etching
10.9 Laser-Induced Surface Patterning
10.10 Colloidal Lithography
10.11 Conclusions and Perspectives
Acknowledgments
References
Chapter 11: Organic/Inorganic Hybrid Surfaces
11.1 Introduction
11.2 Calcium Carbonate Surfaces and Interfaces
11.3 Calcium Phosphate Surfaces and Interfaces
11.4 Silica Surfaces and Interfaces
11.5 Conclusion and Outlook
Acknowledgments
References
Chapter 12: Bioactive Ceramic and Metallic Surfaces for Bone Engineering
12.1 Introduction
12.2 Ceramics for Bone Replacement and Regeneration
12.3 Metallic Surfaces for Bone Replacement and Regeneration
12.4 Conclusions
References
Chapter 13: Plasma-Assisted Surface Treatments and Modifications for Biomedical Applications
13.1 Introduction
13.2 Surface Requisites for Biomedical Applications
13.3 Surface Functionalization of Inorganic Surfaces by Plasma Techniques
13.4 Applications of Plasma-Modified Surfaces in Biology and Biomedicine
13.5 Conclusions and Outlook
Acknowledgments
References
Chapter 14:Biological and Bioinspired Micro- and Nanostructured Adhesives
14.1 Introduction: Adhesion in Biological Systems
14.2 Fibrillar Contact Elements
14.3 Basic Physical Forces Contributing to Adhesion
14.4 Contact Mechanics
14.5 Larger Animals Rely on Finer Fibers
14.6 Peeling Theory
14.7 Artificial Adhesive Systems
14.8 Toward Smart Adhesives
Acknowledgment
References
Part IV: Cell–Surface Interactions
Chapter 15:Generic Methods of Surface Modification to Control Adhesion of Cells and Beyond
15.1 General Introduction
15.2 Survey on Generic Methods to Modify Material Surfaces
15.3 Results and Discussion
15.4 Summary and Conclusions
Acknowledgments
References
Chapter 16: Severe Deformations of Malignant Bone and Skin Cells, as well as Aged Cells, on Micropatterned Surfaces
16.1 Introduction
16.2 Experimental Methods
16.3 The Interaction of Bone Cells with Micropillars
16.4 The Deformation of Skin Cells as a Function of Their Malignancy
16.5 The Deformation of Fibroblasts of Different Cellular Ages
16.6 Discussion
16.7 Conclusions
Acknowledgments
References
Chapter 17: Thermoresponsive Cell Culture Surfaces Designed for Cell-Sheet-Based Tissue Engineering and Regenerative Medicine
17.1 Introduction
17.2 Characteristics of PIPAAm-Grafted Cell Culture Surfaces
17.3 Mechanisms of Cell Detachment from the Thermoresponsive Cell Culture Dish
17.4 Cell-Sheet-Based Tissue Engineering and Its Clinical Applications
17.5 Next-Generation Thermoresponsive Cell Culture Dishes
17.6 Conclusions
References
Chapter 18: Cell Mechanics on Surfaces
18.1 Introduction
18.2 What Is Elasticity and Stiffness?
18.3 Measuring and Quantifying Stiffness
18.4 Controlling Substrate Stiffness
18.5 Naturally Derived Scaffolds
18.6 Synthetic Scaffolds
18.7 Substrate Stiffness' Impact on Cell Behavior
18.8 When Stiffness In vivo Goes Awry: The Impact of Fibrosis on Function
18.9 Novel Surface Fabrication Techniques to Improve Biomimicry
18.10 Conclusion
Acknowledgment
Abbreviations
References
Chapter 19: Electrode–Neural Tissue Interactions: Immune Responses, Current Technologies, and Future Directions
19.1 Introduction
19.2 Immune Response to Neural Implants
19.3 Past and Current Neural Interfaces
19.4 Methods for Improvement of the Electrode–Tissue Interface
19.5 Conclusions and Future Directions
References
Index
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The Editors
Prof. Andreas Taubert
University of Potsdam
Institute of Chemistry
Karl-Liebknecht-Straße 24-25
14476 Potsdam-Golm
Germany
Prof. João F. Mano
University of Minho
3B's Research Group
Polymers Ave Park
S. Claudio do Barco
4806-909 Caldas das Taipas
Portugal
Prof. J. Carlos Rodrguez-Cabello
Universidad de Valladolid
Ctro. Investigacion Cientifica
Paseo de Belén, s/n
47011 Valladolid
Spain
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Preface
Biomaterials are nowadays well-established. It is generally accepted that synthetic and semi-synthetic materials are an asset for, among others, medical doctors trying to improve the quality of life of their patients. This can for example be achieved by replacing damaged organs or tissues with artificial hips, knees, heart valves, blood vessels, and so forth. To successfully do this, however, the clinicians do need a solid understanding of how the artificial materials interact with the body of the patient and which biological feedback loops may be triggered or altered by, for example, implantation. As scientists and engineers around the world learn more about the finer details of how advanced materials developed in their laboratories behave under certain circumstances, they specifically have to learn about how their materials interact with a living organism. Although the merging of biology, medicine, chemistry, physics, and materials science is not a new topic anymore, the advent of biomimetic materials chemistry has fundamentally changed, or more accurately, extended, the field of materials for the biological and biomedical sciences.
Biomimetic materials chemistry is essentially founded on the recognition that in many respects Nature is superior to human technology. It is much more “clever”, if you wish. Nature has developed strategies that go back millions of years to produce complex materials that still often outperform many materials made by man. As such, biomimetics is an important and inspiring field in its own right, but because the first contact between tissue and material is always at the surface, the surface of a material needs very special attention from scientists and engineers as well as from clinicians. Therefore, in the last decade a change in the paradigm of biomaterial design has taken place. While in the past, the predominant trend was the exploration of their value as biomaterial of many materials that were created for other applications, many times far away from medicine, nowadays a new generation of biomaterials, specifically designed for biomedical uses, is taking place. The one relevant fact of the success of such generation is the implication of biology in the foundations of the material design. Thas has allowed the creation of materials that not only provides of adequate mechanical compliance but that are able to directly interact with cells, creating by this way a totally new scenario in those materials mimic better than never the rich complexity and functionality of the natural extracellular matrix. As this new concepts are proven their efficiency in biomaterial design it is time to start summarizing all these efforts and compiling the ideas and work that are crafting that new trend.
Indeed, while there are numerous books on biomaterials as such, the editors of this book felt that there is a lack of a concise work summarizing the state of the art of biomimetic materials with a special focus on surface aspects. To a large extent, the book was inspired by the highly successful European Union-funded Research and Teaching Network “BioPolySurf,” which was initiated and coordinated by one of the editors (J.C.R.-C.) and brought together scientists from all over Europe and from chemistry, physics, polymer science, and biology and biomedicine. We therefore felt it timely to produce a concise yet complete book on how surfaces can be made and modified and how the different surfaces interact with biology in the broadest sense, from the basics of surface structuring to surfaces that can already nowadays be used for a specific application or even enable the communication with the macroscopic world, for example by integration into electronic circuitry. A number of the former BioPolySurf partners have agreed to contribute to the book, but we have also been lucky to find excellent contributors from almost every continent demonstrating that European projects do successfully act as nuclei for world-wide research networks.
The book starts out with a set of chapters on physical and chemical strategies for generating a specifically organized surface. Polymers for surface structuring (Alexander Böker, RWTH Aachen University), polymer brushes (Szczepan Zapotoczny, Jagiellonian University), PEGylated surfaces (Larry Unsworth, University of Alberta), stimuli-responsive surfaces (João Mano, University of Minho), biopolymer surfaces (Vasif Hasirci, METU Ankara), gradient surfaces (Muhammad N. Yousaf, York University), and polymer-based sensors (Wolfgang Meier, University of Basel) are the topics of what can be viewed as the first section of the book.
The overarching theme of what can be regarded as the second section is hydrogel surfaces. It contains contributions on synthetic protein-based hydrogels (Sarah Heilshorn, Stanford University), bioactive and smart hydrogel surfaces (Carlos Rodriguez-Cabello, University of Valladolid), and on bioresponsive surfaces and stem cell niches (Josep Planell, Catalonian Institute of Bioengineering). This section easily connects to the following part on organic/inorganic hybrid and inorganic surfaces with contributions on structure formation on small scales (Tobias Kraus, Leibniz Institute for New Materials), organic/inorganic hybrid surfaces (Andreas Taubert, University of Potsdam), inorganic surfaces (Maria Pau Ginebra, Barcelona Institute of Technology; Sanjay Mathur, University of Köln), and finally bionic surfaces and surface mechanics (Stanislav Gorb, University of Kiel).
The last section builds a bridge to biology and engineering as it comprises as set of chapters on cell-surface interactions and on the use of surfaces for cell engineering. Individual chapters discuss self-assembled monolayers and layer-by-layer surfaces as model substrates for cell adhesion (Thomas Groth, University of Halle), topography effects (Günter Reiter, University of Freiburg), surfaces for cell sheet production (Teruo Okano & Jun Kobayashi, Tokyo Women's Medical University), cell mechanics on surfaces (Adam Engler, University of California, San Diego), and electrode-tissue interactions (Mohammad Reza Abidian, Pennsylvania State University).
In summary, the editors believe that the selection of topics covers the key aspects of biomaterials surface science, from the chemistry and physics of fairly simple surfaces to highly structured, complex, and often multifunctional and multiresponsive surfaces that are well-adapted to a certain biological materials problem and beyond.
At this point it is important to acknowledge that this book would not have been possible without the help of all the authors mentioned above and their respective teams. These colleagues all agreed on a fairly tight schedule and delivered high quality overviews over their respective field of expertise within the general topic of biomaterials surface science. It is these people who advance the field not only by performing outstanding research, but also by letting others participate in their knowledge that make being a scientist a real pleasure.
Last but not least, the editors would also like to acknowledge the very efficient and friendly staff at Wiley-VCH, Dr. Martin Preuss, Dr. Bente Flier, and Ms. Bernadette Gmeiner, who provided much needed support over the course of the entire production process.
Andreas Taubert
João F. Mano
J. Carlos Rodríguez-Cabello
List of Contributors
Part I
Polymer Surfaces
Chapter 1
Part One: Polymer Surfaces Proteins for Surface Structuring
Alexander Schulz, Stephanie Hiltl, Patrick van Rijn, and Alexander Böker
1.1 Introduction
The use of proteins as an alternative for synthetic structures for the formation of new materials is a highly active topic in the research field [1, 2]. Properties and structures of proteins are generally well understood and this enables their use for other systems and in different settings/environments than the ones they are originally designed for [3]. Proteins themselves already display interesting properties with respect to catalytic activity, storage capabilities, and in being available in a wide variety of shapes and sizes. When introduced into systems not comprising a natural setting for these structures, the properties can be used, for example, to influence interfacial properties, for serving as a template for the deposition of inorganic materials, in modifications with synthetic moieties, or in combination with other biological structures.
Here we show different approaches and highlights of proteins at interfaces and the utilization in producing novel hybrid structures using their catalytic or coordinating properties for mineralization processes at liquid–liquid as well as liquid–solid interfaces. Additionally, at liquid–solid interfaces, a more localized degree of organization can be achieved via various deposition processes into a wide variety of patterns. The creation of patterns of biological species, including proteins, peptide fragments, antibodies, nucleotides, and so on, on solid surfaces allows for the development of biosensors and affinity essays.
1.2 Structuring and Modification of Interfaces by Self-Assembling Proteins
Nature offers a great diversity of proteins building complex superstructures that serve as a matrix for the growth of different materials. The process of biomineralization differs from organism to organism. In many cases, organisms build their biominerals by preorganizing a proteinous matrix that is subsequently mineralized. The mineralization can be guided by the insoluble matrix (by binding to crystals as well as by constraining the available space) and by soluble proteins and low-molecular-weight agents binding to the growing crystallites. We only discuss some very special systems in this chapter; a more general overview about biomineralization is given in the literature [4, 5]. Classical examples of proteins involved in biomineralization are collagen on the one hand, a protein assembling into fibrils and fibers [6, 7], and chitin on the other hand, assembling into different phases and also forming nematic phases [8, 9]. But there are also proteins that do not self-assemble in solution but at interfaces.
Self-organized protein structures on solid and liquid interfaces can be used for the tailored production of materials. The protein can serve as a starting point for nucleation, but it can also be a part of the forming material, yielding a composite material.
We discuss the assembly of long-chain polyamines occurring together with silaffins, the assembly of silicateins and hydrophobins, as well as some examples of possible modifications of the adsorbed proteins.
1.2.1 Formation and Modification of Protein Structures at Liquid Interfaces
In general, proteins are constructed from amino acids, some of them possessing apolar side chains, while others have polar or charged side chains. The best way to keep the apolar amino acids away from the surrounding aqueous phase is in most case the creation of a hydrophobic core. This core enables most of the apolar side chains to interact with each other via van der Waals forces, while the other amino acids can interact with surrounding water molecules. This construction is stable in solution – but it is not necessarily stable when the protein approaches an interface. The apolar phase might be a much more favorable surrounding to many apolar groups compared to the hydrophobic core. The resulting surface activity is different compared to classical surfactants as the protein does not necessarily present apolar groups to the surrounding phases in its native state. Thus, the protein often has to rearrange itself at the interface, so that the hydrophobic core turns inside out into the apolar phase, with the other groups remaining in contact with the aqueous phase. This leads to an energetically favored state of the protein that also reduces the interfacial tension. Obviously, this process often leads to dramatic changes in the secondary structure, making the adsorption irreversible or leading at least to a high activation energy for desorption. Adsorption can be analyzed with different models, which often distinguish between the diffusion to the interface and the process of rearrangement,sometimes including different conformations at the interface [10, 11].
1.2.1.1 Silaffins
We discuss silaffins as the first protein taking part in biomineralization processes. Silaffins consist of a phosphorylated backbone and polyamine side chains. These molecules occur in diatoms, in which they help to build various structures of silica being as beautiful as highly organized. These silaffins occur together as a mixture with other substances in nature, and the accompanying long-chain polyamines are especially important. These long-chain polyamines cannot be classified as proteins, but we discuss them in this chapter as they have functions similar to proteins that take part in the biomineralization of diatoms. While proteins such as collagen assemble into solid structures, the long-chain polyamine (most likely the crucial factor for typical structure formation of silica in diatoms [12]) phase separates into small droplets in aqueous solution. Silica precipitates on the surface of these droplets, embedding a fraction of the polyamines. When a critical amount of polyamine is co-precipitated within the silica, the droplet breaks down into smaller droplets because of the changes in phosphate concentration and pH value, and the precipitation proceeds afterwards at the freshly built surfaces. The silaffins do not give structure to the material, but accelerate the precipitation of silica. By this process, a hierarchical hexagonal material is built [12–14].
1.2.1.2 Hydrophobins
Hydrophobins are proteins capable of forming organized structures at interfaces via self-assembly. Filamentous fungi excrete these small, globular proteins that assemble into various structures. Many different hydrophobins exist, showing only a weak similarity in sequence, but exhibiting a typical pattern of eight cysteine residues building four disulfide bridges. These bridges also stabilize the secondary structures as some of the cysteines lie within helices or sheets [17]. Hydrophobins are commercially available in large amounts [18]. Despite their different sequences, fungi use different hydrophobins to lower the surface tension against water as well as to hydrophobize their spores and fruitbodies. This enables them to grow their fruitbodies out of the substrate into air or to infect new substrates coming from air [19, 20].
Hydrophobins are divided into two classes differing in terms of aggregate stability, as shown in Figure 1.1. Hydrophobins of class I build typical rod-shaped aggregates, termed rodlets, having a width of around 10 nm and a length of 100–250 nm [21]. These rodlets assemble into films at interfaces being extremely robust to detergents and fluctuations in pH; solubilization of the aggregates and their films is only possible via treatment with trifluoroacetic acid. In contrast, class II hydrophobins do not build rodlets, and their films are less stable and can easily be dissolved. These films also form characteristic patterns, although on a smaller lengthscale compared to the rodlets formed by class I hydrophobins [17, 20, 21].
Figure 1.1 (a) Atomic force microscopic (AFM) image of rodlets formed by the HGFI hydrophobin from Grifola frondosa. Rodlet formation is characteristic of class I hydrophobins. The rodlets were formed at the air–water interface in a Langmuir trough by multiple compression and lifted on a solid support for imaging, as described in [15]. (b) A surface membrane of HFBI imaged by AFM showing an organized structure. The film was formed at the air–water interface and lifted onto a mica support, as described in [16].
Source: Figure and description are taken from [17], reprinted with permission of Elsevier, Copyright 2009.
Furthermore, all hydrophobins possess a hydrophobic patch that is important for their surface activity. While most proteins have a hydrophobic core and a hydrophilic surface, the hydrophobic patch of hydrophobins is located on the surface. This leads to enhanced surface activity, as the protein is an amphiphile in its native state [17, 21]. Additionally, hydrophobins can rearrange at the interface like any other protein and they therefore adsorb irreversibly at the interface or need at least a much higher amount of energy to desorb in comparison to common surfactants [10, 11].
The quick formation of stable layers for different hydrophobins is followed by the decrease in interfacial tension and the increase in the dilatational modulus [18, 22]. The underlying processes can be understood by molecular dynamics (MD) simulations [23], and also by visualizing the structure via AFM and SEM, as can be seen in Figure 1.1 [17, 19]. The structures of HFBI show regular and nearly hexagonal features at liquid interfaces. The lattice parameters can be varied by the preparation technique of the interface or by protein engineering [24].
Films of the artificial hydrophobin H*Protein B on silica can serve as a template for the growth of layers of TiO2, consisting of polycrystalline anatase. The protein films are prepared by immersing a piranha-cleaned silicon wafer into a buffered hydrophobin solution at different temperatures for various periods. Afterwards, the coated wafer is transferred to an aqueous solution of titania at a controlled temperature to grow the titanium layer. The protein film does not only serve as a nucleation point, but as IR spectra show, it also gets incorporated into the layer of titanium dioxide. The roughness of the film can be controlled by the deposition time, and the mechanical strength in terms of hardness and Young's modulus was found to be much greater compared to layers prepared by chemical bath deposition [25]. This shows clearly that the use of proteins is not just another route to prepare materials, but rather a route to produce composite materials with superior properties.
Films of hydrophobin on liquid interfaces can serve as a matrix for subsequent mineralization (an example structure is shown in Figure 1.2). For example, an oil-in-water emulsion stabilized with the artificial hydrophobin H*Protein B can serve as a template for the creation of mineral microcapsules. In the first step, the protein adsorbs to the oil–water interface. Several oils are applicable for this process, and many of them work in the subsequent mineralization. The interfacial tension between oil and water is the important parameter that determines whether mineralization will take place or not. This knowledge enables to choose an oil that will match the desired properties of each process without having to test different oils in a screening. The protein is mineralized by a saturated solution of calcium phosphate with a suitable pH of 7.4 for the precipitation of hydroxyapatite, yielding oil-filled mineral capsules with a shell of hydroxyapatite. This process has several advantages. In most cases, the oil can be removed easily after the synthesis of the capsules, but it can also be used to solubilize compounds and keep them inside the capsules. Moreover, the process works under mild reaction conditions, and the resulting mineral phase is the same as in bones (nanocrystalline hydroxyapatite); consequently, the probability of getting a biocompatible material is high. The capsules can also withstand high temperatures up to 900 °C, and in addition to that, their morphology is tunable by thermal treatment. The morphology changes in two ways. First, the small crystallites begin to sinter together – this affects the mechanical properties as well as the porosity. Second, the mineral phase seems to change at high temperatures, accompanied by a drastic change in morphology (shown in Figure 1.2b). This enhances the scope of these capsules, as they could also be used as microreactors in processes that take place at elevated temperatures [22].
Figure 1.2Capsules synthesized from a hydrophobin-stabilized emulsion: (a) intact mineral capsule after 68 days of mineralization from a perfluorooctane/water emulsion; (b) capsules prepared from a perfluorooctane/water emulsion, sintered for 1 h at 900 °C. Structure of the mineral shell of capsules in dependence of oil: (c) perfluorooctane/water (50 days of mineralization); (d) silicone oil/water after partial washing with heptane, remains of silicone oil cover the surface at the right-hand side of the image (17 days of mineralization).
Source: Figures and descriptions are taken from [22], reprinted with permission of The Royal Society of Chemistry, Copyright 2011.
The biomimetic character of this approach of synthesis is not just the use of the protein as a simple matrix to start the mineralization. Proteins refold at interfaces to optimize the contact of hydrophobic groups with the apolar phase and the contact of the hydrophilic groups with the polar phase. It is feasible that the reorganization depends on the character of the apolar phase. The experiments show that the morphology of the mineral changes for different oils. This indicates that the protein does not just start the mineralization by heterogeneous nucleation, but also influences the mineral growth – a concept that is frequently used by matrix proteins in nature [5, 22].
1.2.2 Formation and Modification of Protein Structures at Solid Interfaces
Hydrophobins form stable films on hydrophobic solid as well as on liquid surfaces. The adsorption onto solid surfaces is often characterized by contact angle measurements. The contact angle of the hydrophobin EAS dissolved in water on Teflon (pure system: 108 ± 2°) changes to 48 ± 10° by adsorption of hydrophobin. The binding to the surface is strong, as the contact angle is still 62 ± 8° after washing with a hot solution of sodium dodecyl sulfate [19]. A commercially available class I hydrophobin can build remarkable stable layers on oxidized silica, changing the contact angle of water from 0° to 67°. This behavior emphasizes the amphiphilic properties of the molecule, as it is able to turn an apolar surface into a much more polar one and vice versa. The film withstands temperatures up to 90 °C without dissolving and shows a regular structure [25]. Films made of hydrophobin are also feasible to protect silicon against etching by alkaline solutions; hydrophobins can therefore serve as an alternative to classical lithography masks [26]. The microscopic structure of hydrophobin films was already explored by MD simulations. These simulations identified the important parts for the binding to hydrophobic surfaces for SC3 [23] and HFBII (a snapshot of the stable conformation at a silicone surface is shown in Figure 1.3) [27]. This knowledge enables molecular engineering to tailor the adsorption properties of these proteins to specific requirements.
Figure 1.3 (a,b) Representative tightly bound HFBII/Si(1 1 1) interface. The full hydrophobic patch is colored blue with the most adhesive residues colored red. Figure (b) gives a zoomed-in perspective view with the near-silicon methyl carbons shown as transparent van der Waals's spheres.
Source: Figure and description are taken with modifications from [27], reprinted with permission of Springer, Copyright 2011.
1.2.2.1 Silicateins
Silicateins are enzymes extracted from marine sponges, in which they hydrolyze different silica precursors under ambient conditions and physiological pH, without being very substrate-specific [28]. Special care has to be taken when immobilizing silicateins, as they become inactive if their secondary structure changes because of adsorption or constrictions in mobility of the active center. These constraints can be fulfilled using a spacing layer between matrix and silicatein layer. A quite general approach is to use a polymer layer together with a spacer. In the original system, nitrilotriacetic acid (NTA) binds to a gold surface via its thiol groups. The acidic groups, localized at the other end of the molecule, complex a nickel ion, which can be subsequently complexed by a His-tag attached to a silicatein (see Figure 1.4 for the adapted modification of WS2 rods) [29]. The silicatein keeps its catalytic activity, which is discussed in detail later. This system has also been adapted to use polymer layers [30], Fe2O3 [31], WS2 [32], or TiO2 [33] as a matrix. The variety of matrix materials shows that this system is well studied for many cases. There are also more straightforward ways that also preserve the catalytic activity of silicatein: gold surfaces can be modified with cystamine or cysteamine via their thiol groups. Afterwards, glutardialdehyde is added to link the silicatein covalently to the amine layer. Subsequently, the surface is mineralized by the addition of silica precursors [34]. All these processes share the need for a spacer in contrast to the systems at liquid/liquid interfaces described previously.
Figure 1.4 (a) Schematic representation of the fabrication of the biotitania/NT-WS2 nanocomposite. In the first step, the WS2 nanotube is functionalized with the multifunctional polymer ligand (gray) by complexation through Ni groups. The NTA tripod ligand is bound to the side groups of the polymer. In the next step, the silicatein-containing His-tag is attached to the NTA ligand by complexation of Ni ions through the His-tag. Finally, the water-stable precursor of titanium is hydrolyzed by the immobilized silicatein. (b) Scanning force microscopy (SFM) height image of surface-functionalized WS2 nanotubes. (c) Corresponding phase-contrast image shows the material contrast on the nanotubes. (d) Overview SEM image demonstrating the deposition of titania onto WS2 nanotubes. (e) Enlarged (HRSEM, high-resolution scanning electron microscopy) view of titania-coated WS2 nanotubes.
Source: Figures and description are taken with modifications from [32], reprinted with permission of WILEY-VCH, Copyright 2009.
We described several ways to bind silicatein to various surfaces without diminishing its activity. This remaining catalytic activity can be used to precipitate various materials from precursors, giving rise to several hybrid materials under mild reaction conditions: WS2 nanotubes coated with a layer of titania [32], gold nanocrystals grown on TiO2 nanorods (see also Figure 1.5) [33], magnetite particles covered with a layer of silica [31], and zirconium or titanium deposited onto a polymer substrate [30]. Furthermore, some of the materials are deposited in rather unusual shapes – such as triangular gold crystals on TiO2 nanorods (shown in Figure 1.5). The reason for this uncommon shape supposedly lies in the chiral surrounding of the reaction center of silicatein [33]. Furthermore, the layer thickness and roughness are determined by the specific reaction conditions. When these parameters are controlled carefully, the layers are smooth and can be adapted to the desired values, even when a simple system is used for the immobilization of the protein [34].
Figure 1.5 HRSEM image demonstrating the hierarchical structure of the TiO2 nanowire/Au nanoparticle composite. Overview images of the TiO2/Au nanocomposites (top left) and a magnified view (right bottom) are given.
Source: Figure and description are taken from [33], reprinted with permission of WILEY-VCH, Copyright 2006.
In addition to the induction of mineralization processes by surface-bound proteins, the control of the exact protein position might be of interest. For this purpose, printing of proteins is a suitable technique that is discussed in the following section.
1.3 Structuring and Modification of Solid Surfaces via Printing of Biomolecules
The focus of this section is on different approaches used for structuring and modifying solid surfaces [35, 36] with biomolecules [37]. We address intaglio printing (IntP) [38] by means of wrinkling as a lithography-free method for the preparation of nanostructured substrates and its application in the assembly of bionanoparticles. Besides particle assembly on nanostructured corrugated substrates, the elastomeric templates serve as stamps in microcontact printing (μCP) and affinity contact printing (αCP) processes [39, 40] and also for fabrication of microfluidic devices [41, 42]. In general, IntP differs from μCP concerning the area of the stamp where from the ink is transferred. For IntP, particles are printed from wrinkle grooves, while for μCP, the ink is located at stamp protrusions. In contrast, in αCP, a target protein in solution complexes with a probe protein adsorbed on the stamp forming a protein complex, subsequently being transferred to a substrate.
1.3.1 Intaglio Printing Using Nanostructured Wrinkle Substrates
1.3.1.1 Wrinkling: Nanostructured Templates
Surface wrinkles form by release of strain in a bilayer system composed of a stiff thin layer resting on top of an elastic substrate. The long axis of the wrinkles develops perpendicular to the direction of the strain. Depending on the elastic properties of the system, wrinkle dimensions – wavelength and amplitude – range from nanometer to kilometer scale [43]. A large variety of materials is used to build up those bilayer systems with controlled solvent diffusion [44], deposition of metal films [45], and plasma oxidation of soft materials [46, 47] as prominent examples.
Here we address lithography-free preparation of wrinkles by plasma oxidation of polydimethylsiloxane (PDMS). The sample is uniaxially stretched in a custom-made apparatus according to Genzer et al. [48]. Subsequent plasmatreatment converts the top layer of the elastomer into glasslike SiO with variable thickness depending on the plasma treatment time. Strain release leads to surface wrinkling because of different Young's moduli of the components. Figure 1.6a shows an AFM height image of wrinkles and a cross-section revealing the sinusoidal shape of the structure, which is supported by the TEM cross-section (Figure 1.6b).
Figure 1.6 (a) AFM height image ( = 60 nm) of wrinkled PDMS substrate with wavelength nm and amplitude nm. The white line represents the position of the cross-section shown on the right side. (b) TEM image showing a cross-section of an epoxy replica of wrinkled PDMS ( nm and nm). (c) AFM height image ( nm) with corresponding height profile showing adsorption of virus (TMV, tobacco mosaic virus) in the wrinkle grooves ( nm and nm) [55].
Source: Reprinted with permission of The Royal Society of Chemistry, Copyright 2009.
In the case of PDMS, the thickness of the top layer is tunable by duration of the plasma treatment and directly influences the characteristic wrinkle dimensions (wavelength and amplitude ). is proportional to and the Young's modulus of the top layer as well as indirectly proportional to the Young's modulus of the substrate. The amplitude is proportional to and the compressive strain [49]. Besides systems composed of a variety of materials, a large number of wrinkle geometries is available, including linear, radial [50], and random [51] surface wrinkles as well as herringbone patterns [52] and wrinkle gradients [53, 54].
1.3.1.2 Assembly of Bionanoparticles on Wrinkles
In addition to the concept of wrinkling, we focus on the assembly of bionanoparticles, for example, the tobacco mosaic virus (TMV), on nanostructured wrinkled substrates as performed by Horn et al. [55]. TMV is a rod-like virus of 300 nm length and 18 nm diameter. The coat protein self-assembles into a helical structure with an inner channel of 4 nm. This work aims at optimized assembly conditions of TMV in wrinkle grooves and to avoid typical liquid crystal-like clustering of TMV during self-assembly to form uniform lines. Therefore, a set of parameters, namely, virus concentration and spin speed, were scanned to obtain statistic data on the assembly quality. As shown in Figure 1.6c, TMV aligns selectively in wrinkle grooves, presumably because of discontinuous dewetting during the spin coating process used for assembly. The easily tunable wavelength of the wrinkles predetermines the distance of the virus strings after the assembly.
Figure 1.7 combines SEM images from TMV assembly at different concentrations (a–c) and the virus occupancy and deviator parameters for the assembly at various concentrations and spin speeds (d). defines the number of particles adsorbed in the grooves, with 1 representing 100% occupation. The number of viruses adsorbed outside the grooves compared to the total virus number provides . With increasing concentration, the virus occupancy increases. In relation to the total number of viruses, the number of particles outside the grooves decreases, leading to a decrease of . With 90% of wrinkles filled with TMV and a minimum of viruses adsorbed outside the grooves, Horn et al. determined an optimal concentration of 0.9 mg ml for the TMV assembly. A screening of different spin speeds revealed maximum occupation () of wrinkles with TMV and a minimum of at 3000 rpm.
Figure 1.7 (a–c) SEM images of TMVs aligned on wrinkled PDMS substrates prepared from virus solutions of different concentrations. (d) Plot of virus occupancy (, squares) and virus deviator parameter (, circles) versus concentration and versus spin speed (dashed lines are guidelines for the eyes). Light and dark blue symbols belong to concentration axis, while orange and red symbols belong to the spin speed axis, as the black arrows also indicate [55].
Source: Reprinted with permission of The Royal Society of Chemistry, Copyright 2009.
Dewetting of a continuous water film present on the substrate surface is supposed to be crucial for proper virus assembly. The thickness of the film influenced by the spin speed regulates the assembly quality. A thin water film starts dewetting on top of the wrinkles and locally raises the virus concentration in the wrinkle grooves and directs the virus arrangement. For a film thickness smaller than the wrinkle amplitude, a controlled dewetting is inhibited by hole formation in the film. Consequently, TMV sticks to the surface without preferential alignment.
Owing to swelling of PDMS in contact with organic solvents, the TMV lines need to be transferred to suitable plane substrates for further modification. The development of an appropriate printing process is summarized in the following section.
1.3.1.3 Intaglio Printing of Tobacco Mosaic Virus
As mentioned previously, the incision of an image into a surface-holding ink defines IntP. Horn et al. [38] transferred this definition to their system of TMV aligned in wrinkle grooves, with TMV acting as ink. For the printing process, an inked stamp (alignment of virus in wrinkle grooves by spin coating [55]) is pressed on a silicon wafer for 30 s. IntP results in regular virus stripes with line spacings from ≈300 nm to ≈1 μm characterized by SEM and AFM (Figure 1.8).
Figure 1.8 SEM images of TMV stripes with insets of AFM images ( nm and scale bar: 300 nm) are shown. The white lines in the AFM images represent cross-sections of the height profiles. The distances between the TMV stripes are (a) 405 nm, (b) 492 nm, and (c) 934 nm.
Source: Reprinted with permission of Wiley-VCH, Copyright 2010 [38].
Factors influencing pattern quality are prealignment of the virus (ink) on the stamp, hydrophilicity of the stamp and smoothness of the substrate to ensure quantitative particle transfer, and the stamp amplitude. With a stamp amplitude ranging from 20 to 65 nm, IntP is successful. Higher amplitudes result in a higher defect density of the pattern, or no pattern forms at all. During printing, a water film present after the spin coating presumably acts as transfer medium for the viruses. Water wets the channels build up of the Si wafer at the bottom and wrinkles on top inducing sufficient mobility of the viruses to move to the Si surface. Beyond an amplitude limit of 65 nm, incomplete wetting of the channels leads to incomplete or no particle transfer. Besides the amplitude limit, one has to hydrophilize the Si surface to obtain good wetting properties and successful printing. Treatment of the sample with oxygen plasma is a fast and convenient method for the hydrophilization.
Owing to addressable reactive groups on the virus surface, the TMV patterns can serve as templates for further mineralization and metallization reactions for the production of nanowires.
1.3.2 Microcontact Printing for Bioinspired Surface Modification
Besides IntP, microcontact printing [35, 39] (μCP) is a general approach to pattern self-assembled monolayers (SAMs) of functional molecules. During conformal contact between a topographically structured elastomeric stamp and a flat substrate, ink transfers to the flat substrate. Reactive inks bind covalently to the surface, while surface-active inks can also attach noncovalently. The topography of the stamp defines the resulting molecule pattern. Besides patterning of SAMs, μCP includes patterning of biomolecules, polymers, and colloids. Herein we focus on structuring of biomolecules on solid supports. The resolution of μCP is in the submicrometer range. Additionally, the process is carried out under ambient conditions and with only mild chemical treatments.
1.3.2.1 Microcontact Printing onto Self-Assembled Monolayers
Initially, μCP was shown for proteins on empirically chosen solid supports. Chen et al. [56] examined the influence of surface wettability of support and stamp on the quality of the resulting pattern. They printed proteins on two-component SAMs composed of polar and aliphatic alkanethiols. The fraction of polar groups incorporated into the SAM controls the wettability.
They found a minimum wettability necessary for successful μCP of protein onto the substrate (Figure 1.9a). Below a certain threshold, incomplete or no protein transfer occurs, while for ≥65% COOH SAM, the pattern transfers completely. The interaction between protein and substrate increases with increasing density of polar groups on the surface. To prove a relationship among wettability of the SAM, transferred protein amount, and chemical nature of functionality, they substituted polar COOH– groups with OH– (Figure 1.9b) and EG6OH groups (Figure 1.9c) (EGOH: hexa(ethylene glycol)-terminated alkanethiol). The resulting threshold value is slightly increased (70%) for OH SAMS, as the functional group provides only one site for polar interactions compared to COOH. EGOH with six interaction sites reduces the value to 4%. The threshold wettability, allowing complete protein transfer and performance of μCP under ambient conditions with proteins resistant to adsorption in aqueous conditions, suggest different mechanisms for adsorption and μCP. Additionally, the wettability of the stamp influences μCP results. Minimum SAM wettability for protein patterning increases for increasing stamp wettability. A model of competing attractive forces between stamp and substrate describes the results [56]. Polar groups on one of the surfaces increase the attractive forces, and increasing the hydrophobicity of the stamp allows patterning of proteins resistant to μCP from untreated stamps.
Figure 1.9 Printing of proteins on three different types of mixed SAMs. Micrographs of fluorescently labeled protein printed onto mixed SAMs of alkanethiol presenting –CH and (a) –COOH, (b) –OH, and (c) –EGOH functionalities. Each image is a representative image from experiments repeated a minimum of three times with similar results. Scale bar: 100 μm.
Source: Reprinted with permission of American Chemical Society, Copyright 2002 [56].
1.3.2.2 Microcontact Printing with Wrinkle Stamps
In the above-mentioned approach, PDMS stamps are replicas of a silicon master produced by photolithography with features on the microscale. Fery et al. [57] used PDMS wrinkles as stamps for μCP of fluorescently labeled macromolecules and bovine serum albumin (BSA) onto substrates coated with polyelectrolyte multilayers. Figure 1.10 shows an AFM image of printed BSA stripes with corresponding cross-section.
Figure 1.10 (a) AFM height image of printed BSA on a poly(ethylene imine) (PEI) activated glass surface. (b) Height profile of the printed BSA structures averaged perpendicular to the obtained pattern over all cross-sections is found to be 8 nm.
Source: Reprinted with permission of American Chemical Society, Copyright 2008 [57].
By using PDMS wrinkles as stamps, the spacing between the protein lines decreases to nanometer scale. Additionally, the spacing is easily tunable by the wavelength of the wrinkled stamp, and expensive lithographic master production before the stamp casting is redundant. Fery et al. found limits below which stamping failed. The critical stamp dimensions are 40 nm in amplitude and 335 nm in wavelength, that is, below an aspect ratio of 0.11, μCP is not successful. Analysis of cross-sections from AFM images shows a mean height of 6–7 nm of the printed structure for stamp amplitudes exceeding 80 nm ( nm). Smaller stamp dimensions reduce the pattern height to zero, leading to disappearance of the features. With reduced stamp dimensions, the protrusions of the stamp approach each other accompanied by a loss in height/amplitude. Consequently, during printing, more protrusions contact the substrate and ink from the protrusions, and the wrinkle grooves are transferred.
As the hydrophobic PDMS surface is hydrophilized during plasma treatment, aqueous solutions are printable and the process is applicable to proteins or biological materials in general.
1.3.2.3 Microcontact Printing with Porous Stamps
Besides stamps produced by casting photolithographic masters and wrinkled stamps, porous stamps gained increasing attention for structuring dendrimers, nanoparticles [58], and proteins [58, 59]. Huskens et al. [58] produced porous stamps by one-step phase separation micromolding. Phase separation micromolding is a microfabrication technique to structure block copolymers, conductive, and biodegradable polymers under ambient conditions. The method takes advantage of the phase separation of polymer solutions occurring in contact with a structured mold. The microstructured polymer replica exhibits porosity. Figure 1.11 shows SEM images of porous stamps consisting of poly(etherimide) (PEI), poly(vinylpyrrolidone) (PVP), and poly(ethersulfone) (PES). N-methylpyrrolidone (NMP) serves as solvent for the polymers during stamp fabrication. Water-soluble PVP renders the stamp hydrophilic and provides a connected pore network without further modifications. Polymer composition and polymer concentration influence the degree of porosity and pore morphology.
Figure 1.11 (a, b) Scanning electron micrographs of porous stamps replicated from a silicon mold with 20 μm wide lines: Surface and cross-section images of PEI/PVP (in solution PEI:PVP:NMP = 18%:12%:70% w/w). (c–e) Fluorescence microscopy images (scale bars indicate 40 μm) and intensity profiles (insets) of HIgG–Fe (10–5 M, phosphate buffer saline (PBS) buffer) printed on N-[3-(trimethoxysilyl)propyl]ethylenediamine (TPEDA) functionalized glass slides in first to third prints using an oxidized PDMS stamp. Printing time was 5 min, and no reinking was used [58].
Source: Reprinted with permission of American Chemical Society, Copyright 2009.
The pores showing a maximum size of 2 μm act as ink reservoirs. Owing to their dimensions, proteins adhere only to the outer surface of common PDMS stamps. During printing, the larger part of the adsorbed protein remains on the substrate making reinking necessary after every printing step. A porous stamp overcomes this drawback. Huskens et al. [58] performed multiple printing steps with porous PES/PVP stamps and fluorescently labeled human immunoglobulin as ink. Proteins are trapped inside the pores of the stamp and subsequently transferred to the substrate upon conformal contact.Three consecutive printing cycles provide protein patterns with good quality.
Besides fabrication of porous polymeric stamps, modification of PDMS stamps with porous polyelectrolyte multilayers employing layer-by-layer (LbL) deposition was performed [59]. Post-treatment with base and cross-linking leads to porous stamps with pores acting as ink reservoirs. The polyelectrolyte multilayers consist of alternating layers of poly(4-vinylpyridine) (P4VP) and poly(acrylic acid) (PAA). The group studied multiple printing and the stability of the multilayer architecture during printing. The pore diameter ranges from several tens of nanometers to 200 nm. Contact angle measurements proved the stamp to be hydrophilic and therefore suitable for multiple printing steps of aqueous biological samples such as fluorescently labeled immunoglobulin. Cross-linking of the porous stamp structure, obtained by dissolution of PAA with base, is crucial as otherwise the film is partially or fully transferred onto the substrate upon μCP. BrCHBr acted as cross-linker in the gas phase and bound the multilayer covalently to the PDMS.
1.3.2.4 Enhanced Microcontact Printing
In this section, we focus on modified μCP processes utilizing porous surfaces, reactive μCP, and affinity microcontact printing (αCP). Additionally, we show examples for bioparticle assembly on printed surface patterns.
Zhang et al. [60] developed nanoporous silica surfaces for enhanced μCP of proteins. They prepared silica thin films on solid substrates via spin coating. Triblock copolymers act as directing agents and create porosity in the films. The porous layer is biocompatible resulting in minimal protein damage during printing. Conventional chemical surface modifications interact electrostatically/covalently with protein surface groups enhancing adsorption. Porous silica surfaces lead to more complete protein transfer compared to chemically modified surfaces. Especially, silica with a pore size similar or smaller than the dimensions of the protein yielded effective protein transfer concerning pattern completeness, protein layer thickness, and roughness. Protein layers on porous silica are thicker and more uniform compared to modified and untreated substrates. As protein function is retained after immobilization, porous silica serves as a basis for patterned immunoassays. Incubation with a secondary protein leads to deposition of this protein exclusively in areas of the primary protein [60].
With reactive μCP, chemical reactions are induced when ink is printed on a substrate, even when the reaction partners are unreactive under standard conditions [61]. On one hand, a feature of μCP is the short contact time necessary to form a dense monolayer of ink on the substrate, while on the other hand, it takes hours to prepare SAMs by adsorption from solutions. So the question was raised as to whether μCP is useful for acceleration of surface reactions. Generally, μCP reactions follow the rules of “click chemistry” showing high yields, mild conditions, and short reaction times. The scope of reactions ranges from condensations to cycloadditions, nucleophilic substitutions, and deprotections. The reactions benefit from several effects. Nanoscale confinement results in concentrated reagents in the contact area of stamp and substrate (concentration effect). The preorganization effect of one reacting group constrained and aligned on a surface accelerates the reaction. Reactions benefit from the pressure effect during conformal contact between stamp and substrate. The reactions are influenced by the micropolarity of the contact area (medium effect) [61]. An example for reactive μCP is the immobilization of
