Woodhead Publishing Series in Biomaterials
`
`Fundamental
`Biomaterials: Metals
`
`Edited by
`
`Preetha Balakrishnan
`
`Sreekala M S
`
`Sabu Thomas
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`

`Micro- and nanopatterning of
`biomaterial surfaces
`
`3
`
`Onur Sahin, Meiyazhagan Ashokkumar and Pulickel M. Ajayan
`Department of Materials Science & NanoEngineering, Rice University, Houston, TX,
`United States
`
`Abstract
`
`Patterning technologies have expanded drastically over the past two decades with
`continual development
`in current and future lithography methods. Specifically,
`micro- and nanopatterning techniques have been widely studied in a broad range of
`fields, from molecular biology research to communications technology. In general,
`micro- and nanofabrication techniques cover a variety of patterning methods;
`among them, photolithography is considered powerful since all integrated circuits
`(ICs) are fabricated using this method. Another common patterning technology is
`soft lithography, which includes microcontact printing (µCP), microtransfer mold-
`ing (µTM), replica molding (REM), micromolding in capillaries (MIMIC), injection
`molding, and many other processes. The purpose of this invited chapter is to pro-
`vide a brief overview of the different lithographic techniques specifically, photoli-
`thography, soft lithography, and electron-beam lithography, where we discuss the
`developments, issues, and major challenges associated with these technologies.
`Also in this chapter, we demonstrate a simple, inexpensive method for fabricating
`micrometer resolution protein patterns using a soft-lithographic technique. We
`believe that the contents discussed in this section, along with the demonstrated soft
`lithography procedure, will be helpful for a broad range of readers.
`
`Keywords: Patterning; biomaterials; nanoscale; PDMS; lithography; proteins
`
`3.1 Introduction
`
`Micro- or nanopatterning is the process of fabricating micro- or nanostructures,
`respectively, across the surface of the material, referred to as the substrate. While
`originally developed for the electronics industry, patterning technologies have
`found many uses in the studies of cellular biology, biomaterial engineering, and
`other related fields.
`In this chapter, we will focus on three common patterning technologies: (i) pho-
`tolithography; (ii) soft lithography; and (iii) electron-beam (e-beam) lithography.
`First, we describe photolithography, a process that shines light through a mask to
`ablate a photosensitive material, known as a photoresist, on the substrate according
`
`Fundamental Biomaterials: Metals. DOI: https://doi.org/10.1016/B978-0-08-102205-4.00003-9
`© 2018 Elsevier Ltd. All rights reserved.
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`68
`
`Fundamental Biomaterials: Metals
`
`Figure 3.1 Schematic showing different patterning techniques. (A) Soft lithography,
`microcontact printing, and microfluidics. (B) Photolithography (positive and negative).
`(C) e-beam lithography (positive and negative).
`
`to the pattern on the mask (Fig. 3.1B). There are two types of photolithography
`discussed: negative and positive photolithography. Second, we will discuss soft
`lithography, which refers to a group of patterning techniques that utilizes an
`elastomeric stamp, often polydimethylsiloxane (PDMS), to pattern a substance,
`known as the ink, on the substrate surface (Fig. 3.1A). In this, we will focus on two
`commonly reported soft lithography techniques: microcontact printing (µCP) and
`microfluidics. Finally, e-beam lithography is a maskless lithography process where
`a focused beam of accelerated electrons draws customized patterns on an e-beam
`resist coated on the substrate surface (Fig. 3.1C). As with photolithography, e-beam
`lithography also has a negative and positive modality.
`These patterning technologies are utilized by researchers to explore in vitro
`cellular behavior. Examples of fundamental cellular behavior include: (i) cellular
`differentiation [1], (ii) adhesion [2], and (iii) proliferation [3] as described below.
`Briefly, cellular differentiation is the process by which cells become more specialized
`by differentiating down a cellular lineage. For example, mesenchymal stem cells
`(MSCs) can differentiate into multiple cell types, including osteoblasts (bone cells),
`adipocytes (fat cells), chondrocytes (cartilage cells), or myocytes (muscle cells) [4].
`As the MSC differentiates down a cellular lineage, the cell morphology and function
`will change according to cues from the cell’s microenvironment. Thus, an MSC
`placed in an environment with osteogenic cues will differentiate down the osteoblast
`cellular lineage [5,6]. The mechanisms behind how these cues affect cellular differen-
`tiation are unknown and are an ongoing area of research. The in vivo cellular envi-
`ronment contains an intractably complex mix of mechanical and chemical cues [7].
`While far from a perfect imitation, patterning technologies allow researchers to
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`Micro- and nanopatterning of biomaterial surfaces
`
`69
`
`mimic and study these environmental cues in in vitro. For example, Kim et al. used
`µCP to control
`the differentiation of human-derived MSCs. When MSCs were
`cultured on linear graphene oxide patterns, they found that the elongated topography
`induced osteogenic differentiation. However, when a grid pattern of graphene oxide
`was used, the interconnected geometry induced neuronal differentiation [8]. In a
`similar experiment, Kilian et al. used µCP to argue that MSCs cultured on patterns
`with sharp edges, e.g., star shapes, tended towards an osteogenic lineage, whereas
`patterns with rounded edges, e.g., ovals, tended towards an adipogenic lineage. This
`difference was attributed to the sharp edges which increase cell myosin contractility
`and hence promoting osteogenic pathways [9].
`Cellular adhesion describes the process by which cells attach to their surround-
`ings. For a cell to attach, biomolecules on the surface of the cell must stick to
`another object, such as a substrate, another cell, or the extracellular matrix—a phe-
`nomenon known as adsorption [10]. There are two types of adsorption: chemisorp-
`tion and physisorption [11]. Chemisorption occurs when the cell attaches to the
`adsorbent surface through chemical bonds. For example, substrates can be coated
`with a cellular adhesion protein, such as an integrin, cadherin, selectin, or immuno-
`globulincell adhesion molecule [12], which promotes cellular attachment by cova-
`lently bonding the cell and substrate. Physisorption describes attachment through
`intermolecular interactions, such as hydrogen bonds, Van der Waals forces, and
`dipoledipole interactions. Physisorption usually does not require surface treatment
`of a substrate and is the mode of adsorption in common cell culture protocols using
`untreated glass or polystyrene Petri dishes. Self-assembled monolayers (SAMs),
`chains of molecules that bond with the substrate in an ordered structure, can be pat-
`terned on substrates and used to bind cells through both covalent (chemisorption)
`and noncovalent (physisorption) bonding [13]. The “head” of the chain binds to the
`substrate, and the “tail” binds to a biomolecule. SAMS can be functionalized to
`either promote or repel cellular adhesion. For example, alkanethiol chains can be
`patterned on metal substrates and then functionalized with fibronectin, an integrin
`adhesion protein [14]. Unpatterned regions are then coated with polyethylene glycol
`(PEG) terminated chains. The chains with fibronectin tails then promote cellular
`attachment, while the PEG-terminated tails repulse adhesion [15].
`As cells adhere to their surroundings, environmental cues continue to control
`pathways that determine cell division, a process also known as cellular prolifera-
`tion. Similar to cellular differentiation, cellular proliferation is controlled by a com-
`plex assortment of both mechanical and biochemical cues. Researchers can mimic
`these cues to understand the mechanisms controlling cellular proliferation [16]. For
`example, Thakar et al. patterned smooth muscle cells using µCP to alter cell shape
`in microgrooves and islands. It was found that cells constricted to a pattern had a
`more elongated cell shape and a decrease in proliferation, which was thought to be
`related to the change in nucleus shape impacting DNA synthesis [17].
`As discussed, patterning can aid scientists in understanding cellular behavior,
`such as differentiation, adhesion, and proliferation, by simulating in vivo environ-
`mental cues. There are other cellular functions such as cellular spreading [18] and
`migration [19] that are equally important and also studied through patterning
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`70
`
`Fundamental Biomaterials: Metals
`
`Table 3.1 Comparison of soft lithography, photolithography, and
`e-beam lithography
`
`Soft lithography
`
`Photolithography
`
`Resolution
`
`Bnm to µm in one
`dimension
`
`µm in two
`dimensions
`
`e-beam
`lithography
`
`nm in two
`dimensions
`
`Relative cost
`
`Cheapest
`
`Medium
`
`Most expensive
`
`Specialized
`equipment
`required
`
`Notes
`
`Access to
`photolithography
`equipment for one
`time use to make
`stamp
`
`UV light source and
`mask aligner,
`typically needing
`a clean room
`facility
`
`Electron microscope
`system with
`lithography
`accessories
`
`As opposed to other
`lithography methods,
`can be used for both
`planar and nonplanar
`substrates
`
`Necessary for soft
`lithography
`process in order
`to create stamp
`master
`
`Least common
`lithography
`method for
`biological
`patterning
`applications
`
`technologies. There are a large number of extensive review articles detailing exclu-
`sively about these studies [20,21] (Table 3.1).
`
`3.2 Photolithography
`
`In general, photolithography involves four components: (i) a substrate; (ii) a light-
`sensitive photoresist; (iii) a mask; and (iv) a UV light source. As shown schematically
`in Fig. 3.1B, photolithography begins by initially coating the substrate with a photore-
`sist. Then, UV light is shone through a mask with the desired pattern onto the photo-
`resist. For a positive photoresist, the exposed area becomes soluble, and can be
`washed away by developer solution in the final step. For a negative photoresist, the
`ablated area becomes insoluble, and the unexposed areas are washed away in the final
`step. The photomask can be made of a planar quartz disc with a micron layer thick
`chrome film deposited on top. As the UV light passes through the mask, light only
`transmits through the quartz; thus, the chrome layer design determines the pattern.
`An important consideration when using photolithography for biological studies is the
`biocompatibility of the photoresist. SU-8 is a popular photoresist for biological appli-
`cations due to its high level of safety in biological systems. Revzin et al. demonstrate
`the use of poly(ethylene glycol) diacrylate (PEG-DA) as a photoresist. In their
`experiment, a glass substrate is silanized and then coated with 1% w/v solution of
`0
`-dimethoxy-2-phenylacetophenone (DMPA).
`PEG-DA and the photo-initiator, 2,2
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`Micro- and nanopatterning of biomaterial surfaces
`
`71
`
`(A)
`
`Lift off
`
`(B)
`
`Etching
`
`mask
`
`1 - proteins immobilized
`on substrate
`
`photoresist
`
`substrate
`
`1 - positive
`photolithography
`
`2 - sucrose coating
`
`3 - positive photolithography
`
`2 - coat in
`adhesive proteins
`
`4 - etch with oxygen
`plasma treatment
`
`3 - remove
`photoresist
`
`5 - etch with acetone
`
`Figure 3.2 Schematic diagram demonstrating lift off (A) and etching process (B) to pattern
`cells using photolithography.
`Source: Modified from Sorribas H, Padeste C, Tiefenauer L. Photolithographic generation of
`protein micropatterns for neuron culture applications. Biomaterials 2002;23(3):893900.
`
`The PEG-DA/DMPA film is then exposed to UV light through a photomask. The
`exposed regions are then removed by rinsing with DI water (positive photolithogra-
`phy). Collagen was then coated onto the substrate, adhering to areas without PEG-
`DA. They then demonstrated the selective attachment of cells, such as hepatocytes or
`fibroblasts, to the patterned collagen [22].
`In another study, Sorribas et al. demonstrated two methods (as shown in
`Fig. 3.2) using photolithography to pattern neuronal cells. Adhesive proteins or pep-
`tides could be bound to the substrate by either cysteine groups or lysine residues. In
`the first method, named the lift off (3.2A), a positive photoresist, S-1813, was spun
`onto the substrate, exposed to UV light
`through a mask, and then removed
`(3.2A-1). Adhesive proteins were then coated on the exposed substrate and unre-
`moved photoresist (3.2A-2). Finally, the remaining photoresist was removed using
`acetone, leaving behind adhesive proteins (3.2A-3) that bound to neuronal cells.
`The second process was termed etching method (3.2B). In this protocol, proteins
`were first
`immobilized on the substrate,
`followed by a coating of sucrose
`(3.2B-1,2). Positive photolithography was then carried out on top of the sucrose
`(3.2B-3). The exposed sucrose was then treated with oxygen plasma, etching away
`the sucrose and proteins (3.2B-4). Finally, acetone was used to remove the photore-
`sist and sucrose (3.2B-5) [23]. Karp et al. reported another photolithography process
`using photo cross-linkable chitosan as the photoresist and demonstrated that numer-
`ous cell lines, including osteoblasts and myocytes, could be cultured to confluency
`on various pattern geometries [24].
`Photolithography is a powerful enabling technology for micropatterning; how-
`ever, its necessity for an expensive clean room facility presents a significant draw-
`back. Photolithography is commonly used to fabricate a master for soft lithography
`studies. Photolithographic patterns are also limited to micrometer scale resolution.
`
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`72
`
`Fundamental Biomaterials: Metals
`
`This is often sufficient since most biological studies only require micron level pre-
`cision. However, some experiments do require submicron resolution, which is often
`best accomplished through e-beam lithography.
`
`3.3 Soft lithography
`
`Soft lithography is a group of patterning methods that utilize an elastomeric stamp
`to deposit ink on a substrate. When compared to other patterning techniques, the
`benefits of soft lithography include not only a relatively lower cost, easier setup,
`and high throughput [25], but also a pattern resolution that can range from nanome-
`ter [26] to micrometer [27] precision. One drawback of soft lithography is the need
`to utilize another lithography method, such as photolithography or e-beam lithogra-
`phy, to fabricate the stamp master; however, this step needs only be done once, as
`once the master is fabricated, it can repeatedly be used to produce the stamps. The
`polymer PDMS is typically used to fabricate the mold due to its elasticity [28],
`optical transparency [29], hydrophobicity [30], biocompatibility [31], and gaseous
`permeability [32]. Based on the needs of the experiment, these properties can be
`tuned by various methods. Mechanical properties can be altered by changing the
`ratio of monomer solution and cross-linking agent [33]. Hydrophobicity and inert-
`ness can be tailored by engineering the surface chemistry of the stamp through UV
`ozone treatments [34] or wet-etching [35]. In this chapter, two modes of soft lithog-
`raphy will be discussed: (i) µCP; and (ii) microfluidics.
`
`3.4 Microcontact printing
`
`Microcontact printing refers to soft lithography methods where the ink is transferred
`by bringing the stamp in contact with the substrates. As schematically shown in
`Fig. 3.1A, a PDMS stamp is coated with ink and then placed onto a substrate. The
`ink is then selectively transferred based on the topography of the stamp. To com-
`plete the process, nonpatterned areas can be backfilled with an adhesion resistant
`layer. µCP can be used to pattern SAMs [36] as well as proteins [37], cells [38],
`and DNA molecules
`[39]. Common examples of patterned SAMs
`include
`(CH3(CH2)nSH) on metal
`substrates
`[40]
`and siloxanes
`alkane
`thiols
`(SiCl3(CH2)nCH3) on silicon or silicon dioxide substrates [41]. The thiol or
`
`the chain, bonding with the
`trichlorosilane groups function as the head of
`suitable substrate, while the methyl group acts as the tail. Protein adsorption can be
`controlled by patterning methyl-terminated SAMs to bind proteins, and backfilling
`with PEG-terminated SAMs to repel proteins [42]. As described by Thery et al.,
`µCP can also be used to pattern proteins without SAMs. In their protocol, fibronec-
`tin is coated directly onto the stamp while the substrate is given a UV ozone treat-
`ment
`to become more hydrophilic,
`thereby allowing the fibronectin to better
`adhere. Nonpatterned areas are then backfilled by poly-L-lysine-polyethylene
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`glycol, an adhesion resistant polymer [43]. Once fibronectin, or a similar adhesion
`protein, is patterned, cells can be introduced to grow on the adhesive patterns selec-
`tively. Dupont et al. utilized 3001000 µm2 fibronectin islands to investigate the
`role of mechanical cues in MSC differentiation. It was found that MSCs grown on
`hard substrates underwent osteogenic differentiation, whereas soft substrates
`induced adipogenic differentiation [44]. µCP can also be used to pattern DNA
`molecules, as shown by Lange et al. To accomplish this, both the PDMS stamp and
`the glass substrate were silanized. Finally, DNA diluted with deionized water was
`then coated on the stamp as the ink [45].
`
`3.5 Microfluidic patterning
`
`Microfluidic patterning (µFP) refers to utilizing an elastomeric stamp to create
`channels across a substrate where the ink flows through due to capillary forces. By
`placing the stamp on the substrate, a seal is set up in places where the stamp is in
`direct contact with the substrate, preventing the solution from flowing outside of
`the channels (Fig. 3.1A). Similar to µCP, µFP can be used to pattern proteins,
`ligands, and cells. Lee et al. demonstrates a µFP approach, also known as micro-
`molding in capillaries (MIMIC), to pattern protein repulsion and adhesion areas. In
`their approach, the surface of the substrate was initially coated with a protein adhe-
`sive material, polyelectrolyte (PEL). Following this, a diblock copolymer, poly(eth-
`ylene glycol)poly(D,L-lactide), was introduced into the channels at the stamp-PEL
`interface. After the PEGPLA had adsorbed onto the surface of the PEL, the stamp
`was removed, leaving behind a polymer barrier, which acted as a protein repulsor
`on top of the PEL. They could then load proteins, such as fluorescein isothiocyanate
`tagged bovine serum albumin, or cells, such as fibroblasts onto the substrate. Both
`proteins and cells selectively adhered to the PEL exposed areas [46].
`In this chapter, we illustrate a simple technique to show the process of patterning
`proteins using µFP methods, as schematically depicted in Fig. 3.3. In our work, we
`patterned amyloid-beta, a protein involved in Alzheimer’s disease, using PDMS
`stamps with channel dimensions ranging between 2 and 5 µm and a height of
`B5 µm to form parallel linear patterns. First, the substrate (silicon or glass) and the
`PDMS pattern were plasma treated for about 510 min to generate hydroxyl func-
`tionalities on the surface, thus making the substrate and stamp more hydrophilic,
`which allows the protein solution to flow more quickly down the channels. Then,
`about B1 µg of protein dissolved in 1 mL, DMF was placed at one end of the
`plasma-treated substrate. The protein solution instantaneously flowed through the
`PDMS channels filling the capillaries uniformly. The solution was then allowed to
`dry for B16 h in ambient conditions before the PDMS stamps were gently peeled
`off from the substrate, which was then washed with excess water to remove any
`
`impurities. Finally, the formed protein patterns were annealed at (B50
`C) and then
`observed under an optical microscope. The formed patterns possessed the same
`parallel linear patterns as the negative stamp.
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`Fundamental Biomaterials: Metals
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`Figure 3.3 Schematic showing steps involved in the fabrication of protein pattern using
`microfluidic soft lithography technique.
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`3.6 Electron-beam lithography
`
`Electron-beam (e-beam) lithography is a maskless lithography method that utilizes
`an electron gun from a scanning electron microscope to pattern nanoscale features
`on a substrate surface. As opposed to photolithography, the resolution of e-beam
`lithography can reach precision levels down to 1 nm. Similar to photolithography,
`substrates for e-beam lithography are coated with Na resist that either cross-link
`when struck by electrons, rendering it less soluble in developer solution (negative
`e-beam lithography, Fig. 3.1D), or alters the resist to become more soluble (positive
`e-beam lithography, Fig. 3.1D). Examples of e-beam resist include PMMA for posi-
`tive e-beam and SU-8 for negative e-beam. Another popular resist for e-beam
`lithography is a PEG hydrogel. Myriad biomolecules and biological compounds
`have been patterned via e-beam methods. For example, nanopatterns have been
`demonstrated to immobilize calmodulin [47], bovine serum albumin [48], and biotin
`[49]. Christman et al. nanopatterned PEG to trap growth factors approximately
`100 nm apart within rectangular,
`triangular, and circular geometries
`[47].
`Nanopatterned PEG can also be functionalized to adhere to biomolecules, as dem-
`onstrated by Kolodziej et al. [50]. In their study, an aminooxy-terminated PEG
`hydrogel was patterned using e-beam lithography. RGD peptides were then intro-
`duced to the substrate, binding to the PEG locations. Finally, endothelial cells were
`brought onto the substrate, which selectively bound to the RGD proteins.
`
`3.7 Conclusion
`
`In summary, micro/nanopatterning is an enabling technology, allowing researchers
`to study the fundamental mechanisms of cellular behavior. The patterning methodo-
`logies discussed include soft
`lithography, photolithography, and e-beam litho-
`graphy. The needs of the experiment will determine which patterning modality is
`preferred. Soft lithography, a relatively simple, cost-effective patterning method,
`has been broadly adopted due to its ability to pattern within biological relevant
`length scales; however, for submicron resolution across two dimensions, other
`methods, such as photolithography or e-beam lithography, are often preferred.
`Photolithography is another conventional biological patterning process, not only to
`create stamp masters for soft lithography but also as a stand-alone method that can
`offer micron-resolution patterns across a large area of the substrate. Finally, e-beam
`lithography, while expensive and nonscalable, provides the highest resolution
`patterns with a significant amount of flexibility in pattern geometry due to the lack
`of a mask.
`The patterning methods discussed in this chapter focus on one- or two-dimensional
`patterns across a substrate; however, this model is often a poor predictor of biological
`functions that exist in three-dimensional environments. Thus, future research directions
`are incorporating multiple variables, including three spatial dimensions, time, electrical
`stimulation, temperature, shear forces, etc., to mimic the in vivo environmental cues
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`better. For example, Ho et al. demonstrated a lab-on-a-chip that mimicked liver lobule
`tissue by patterning hepatic and endothelial cells in a radial geometry using electric
`fields [51]. Though still nascent, these efforts hold potential in elucidating important
`biological mechanisms behind cellular function.
`
`Acknowledgments
`
`M.A.K. thank United States-India Education Foundation (USIEF) and Hartley L. Family
`foundation for financial support.
`
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