pubs.acs.org/Langmuir © 2010 American Chemical Society
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Designable Biointerfaces Using Vapor-Based Reactive Polymers Hsien-Yeh Chen† and Joerg Lahann*,†,‡,§, †
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Institute of Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany, ‡Department of Chemical Engineering, §Department of Materials Science and Engineering, and Department of Macromolecular Science and Engineering, University of Michigan, Ann Arbor, Michigan 48109 Received April 22, 2010. Revised Manuscript Received June 2, 2010 Functionalized poly(p-xylylenes) constitute a versatile class of reactive polymers that can be prepared in a solventless process via chemical vapor deposition (CVD) polymerization. The resulting ultrathin coatings are typically pinhole-free and can be conformally deposited onto a wide range of substrates and materials. More importantly, appropriately selected functional groups can serve as anchoring sites for tailoring biointerface properties via the immobilization of biomolecules. In this article, controlled surface chemistries are outlined that use functionalized poly(p-xylylenes) as reactive coatings, including alkyne-functionalized coatings for Huisgen 1,3-dipolar cycloaddition reactions or aldehydefunctionalized coatings. The reactive coatings technology provides flexible access to a range of different surface chemistries, enabling a broad range of potential applications in microfluidics, medical device coatings, and biotechnology. In this feature article, we will highlight recent progress in vapor-based reactive coatings and will discuss potential benefits and current limitations.
1. Introduction Alongside the genomic and proteomic revolution in modern biology came a growing need for novel technologies. The result has been an extraordinary push for biotechnology, which has sparked parallel efforts in advanced biomaterials including microstructured and nanostructured coatings. To date, myriad techniques have been developed for the spatially controlled distribution of proteins, polysaccharides, and nonfouling synthetic polymers such as polyethylene glycol (PEG).1,2 Existing immobilization approaches for biomolecules can be grossly divided into two categories: the physisorption of macromolecules onto substrates or covalent immobilization.3 Both physisorption and covalent immobilization have their advantages and disadvantages. Physisorption is simple and can typically be integrated into most biological processes without the alteration of existing formats. However, for applications that require longer performance times, the stability of physisorbed coatings may be limited. In some cases, physisorption also does not offer the same degree of molecular control as covalent immobilization. Thus, covalent immobilization strategies are typically considered to be superior for applications where defined biological structures have to be maintained over extended periods of time. On the basis of covalent immobilization strategies, microstructuring methods have been developed that involve soft-lithographic patterning, spatio-selective polymerization, photolithography, printing, and the masking or templating of surfaces. The interested reader should refer to recent review articles describing these trends.2,4 However, covalent coupling almost always requires the introduction of adequate functional groups on the substrate surface. Because the availability of suitable chemical surface groups is governed by the chemical makeup of the substrate material itself, *To whom correspondence should be addressed. E-mail: lahann@umich. edu. (1) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28–60. (2) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Biomaterials 2006, 27, 3044–3063. (3) Shin, H.; Jo, S.; Mikos, A. G. Biomaterials 2003, 24, 4353–4364. (4) Nie, Z. H.; Kumacheva, E. Nat. Mater. 2008, 7, 277–290.
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the choice of coupling chemistries is in most cases limited and covalent immobilization can involve rather complex reaction cascades. To address this dilemma, the ideal surface modification method will decouple the chemistry of the surface from the actual bulk composition.5 Following this approach, ultrathin polymer coatings, which essentially function as a “sticky” adhesion layer for the covalent immobilization of biomolecules, have been widely pursued.4 The ability to control surface properties independently from bulk material(s) not only expands the diversity of immobilization schemes but also ensures that a proven immobilization method can be easily transferred from one substrate to another without the need for continuous reconfiguration of the underpinning substrate chemistries. Among the different biomedical coating techniques, vaporbased processes combine a number of unique attributes. They are typically solventless (i.e., avoid solvents, plasticizers, initiators and other low-molecular-weight traces) and therefore often have superb biocompatibility. In addition, vapor-based coatings typically result in exquisite coating conformality of micrometer- or nanometer-sized features. This property can be attributed to the absence of dewetting effects that are typically encountered in solution-based coatings and can result in bridging and buckling of polymer coatings.6 Moreover, vapor-based coatings may follow cleaner reaction pathways because they may have a lower probability for side reactions, resulting in polymers with improved performance because of the enhanced linearity of the polymer chains and higher molecular weights. Several variations of CVD-based polymerization have been reported in the past.6-10 For example, Frank and co-workers11 have used a CVD technique to graft polypeptide chains onto (5) Castner, D. G. Nature 2003, 422, 129–30. (6) Tenhaeff, W. E.; Gleason, K. K. Adv. Funct. Mater. 2008, 18, 979–992. (7) Hanefeld, P.; Westedt, U.; Wombacher, R.; Kissel, T.; Schaper, A.; Wendorff, J. H.; Greiner, A. Biomacromolecules 2006, 7, 2086–2090. (8) Lahann, J. Polym. Int. 2006, 55, 1361–1370. (9) Senkevich, J. J.; Desu, S. B. Thin Solid Films 1998, 322, 148–157. (10) Cetinkaya, M.; Boduroglu, S.; Demirel, M. C. Polymer 2007, 48, 4130– 4134. (11) Chang, Y.-C.; Frank, C. W. Langmuir 1998, 14, 326–334.
Published on Web 06/30/2010
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Figure 1. Schematic illustration of the system installation used for CVD (co)polymerization to prepare reactive coatings as well as polymer gradients. Scheme 1. CVD Polymerization of Substituted [2.2]Paracyclophanes to Prepare Functionalized Poly(p-xylylenes)
substrates, and Gleason and co-workers have shown that a wide range of polymer coatings can be prepared by plasma polymerization or initiated chemical vapor deposition (iCVD).6 In cases where hot filaments were used for initiation, radical polymerization can occur, which yielded chemically defined conformal coatings. In addition, patterning the surface with an initiator can result in microstructured polymers.12 An excellent review broadly covering the recent development of CVD polymers was published elsewhere, and the interested reader should refer to this article for detailed information.13 Wheras several vapor-based polymerization processes exist, the polymerization of [2.2]paracyclophanes (PCPs) to form poly(pxylylenes) (PPXs) following the Gorham process14 is among the best-established systems. During the Gorham process, thermal treatment of the starting material, [2.2]paracyclophanes, results in reactive species that are spontaneously deposited on the target surfaces that are typically maintained at or below room temperature. (12) Gu, H. W.; Xu, C. J.; Weng, L. T.; Xu, B. J. Am. Chem. Soc. 2003, 125, 9256–9257. (13) Alf, M. E.; Asatekin, A.; Barr, M. C.; Baxamusa, S. H.; Chelawat, H.; Ozaydin-Ince, G.; Petruczok, C. D.; Sreenivasan, R.; Tenhaeff, W. E.; Trujillo, N. J.; Vaddiraju, S.; Xu, J.; Gleason, K. K. Adv. Mater. 2010, 22, 1993–2027. (14) Gorham, W. F. J. Polym. Sci., Part A-1: Polym. Chem. 1966, 4, 3027–3039.
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Unsubstituted and mono- and dichloro-substituted poly(pxylylenes) have been marketed under the brand names parylene N, C, and D and are already widely used in packaging semiconductors and biomedical devices.15 For instance, the parylene C polymer is FDA-approved for coating the interior surfaces of blood bags or as base coatings for cardiovascular stents.16 For experimental details, a review of earlier work, and a discussion of physicochemical models of parylene deposition, we direct the reader to a comprehensive monograph.17 1.1. Functionalized Poly(p-xylylenes). Although conventional parylene coatings are typically used to provide inert packaging that suppresses potential adverse effects stemming from the underlying substrate, recent trends in biomedical engineering are targeting bioactive coatings that provide functional (15) Rodger, D. C.; Fong, A. J.; Wen, L.; Ameri, H.; Ahuja, A. K.; Gutierrez, C.; Lavrov, I.; Hui, Z.; Menon, P. R.; Meng, E.; Burdick, J. W.; Roy, R. R.; Edgerton, V. R.; Weiland, J. D.; Humayun, M. S.; Tai, Y. C. Sens. Actuators, B 2008, 132, 449–460. (16) Ragheb, A. O.; Bates, B. L.; Fearnot, N. E.; Kozma, T. G.; Voorhees, W. D., III; Gershlick, A. H. Coated Implantable Medical Device. U.S. Patent 6,774,278, Aug. 10, 2004. (17) Fortin, J. B.; Lu, T. M. Chemical Vapor Deposition Polymerization: The Growth and Properties of Parylene Thin Films; Kluwer Academic Publishers: Boston, 2004.
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Table 1. Comparison of CVD-based Polymer Coatings with Other Techniques system CVD polymer coating dip coating spray coating plasma polymerization Langmuir-Blodgett film self-assembled monolayer
stability
uniformity
thickness control
chemistry control
substrate dependency
process time
cost
scalability
vapor
good
good
good
good
low
medium
medium - high
medium
solution solution vapor
good good good
medium-poor medium good
medium medium good
medium medium poor
medium-low medium-low low
fast fast medium
low low medium-high
good good medium
solution
poor
medium-good
good
good
high
slow
medium
low
solution
poor
good
good
good
high
slow
high
low
guidance and intervention for the local cellular microenvironment through the presentation of immobilized biomolecules.2,18 Such an approach requires the availability of chemical groups that can be used as chemical anchors, to which biomolecules can be attached via covalent immobilization. For instance, the use of functionalized poly(p-xylylenes) has enabled the surface modification of metallic stents with a number of reactive coatings, including poly(p-xylylene-2,3-dicarboxylic acid anhydride), (18) Hersel, U.; Dahmen, C.; Kessler, H. Biomaterials 2003, 24, 4385–4415.
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which was instrumental in the covalent immobilization of r-hirudin.19 In a separate report, positive results have also demonstrated the high mechanical stability, good biocompatibility, and suppressed cytotoxicity of poly(hydroxymethyl-p-xylylene-co-p-xylylene)-coated stents in vitro and in vivo.20 (19) Lahann, J.; Klee, D.; Pluester, W.; Hoecker, H. Biomaterials 2001, 22, 817–826. (20) Schurmann, K.; Lahann, J.; Niggemann, P.; Klosterhalfen, B.; Meyer, J.; Kulisch, A.; Klee, D.; Gunther, R. W.; Vorwerk, D. Radiology 2004, 230, 151–162.
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Figure 2. Immobilization of an azide-containing ligand on the reactive polymer coating. The Cu(I) catalyst is microcontact printed onto a preadsorbed layer of biotin-based azide ligand on the alkyne-containing polymer coating.24 (Reproduced from ref 24 with permission. Copyright 2006 Wiley-VCH Verlag GmbH & Co. KGaA.)
The focus of our group in this area has been on using the CVD polymerization of substituted [2.2]paracyclophanes to prepare a wide variety of novel poly(p-xylylenes) with functional groups appropriate for subsequent polymerization (Scheme 1). The actual CVD process used for the polymerization of substituted [2.2]paracyclophanes is an adaptation of the Gorham process developed for the deposition of conventional parylene coatings.14 In all cases, cyclic [2.2]paracyclophanes (dimers) are sublimated under reduced pressure (0.2-0.3 Torr) and subsequently introduced into a furnace at temperatures between 600 and 800 °C. At these temperatures, homolytic cleavage of the dimers can occur without thermal decomposition of the functional groups. After this initiation step, the quinodimethane molecules are transferred into the deposition chamber and will polymerize onto the substrate of choice, which can be maintained (21) Jiang, X.; Chen, H.-Y.; Galvan, G.; Yoshida, M.; Lahann, J. Adv. Funct. Mater. 2008, 18, 27–35. (22) Lahann, J.; Balcells, M.; Rodon, T.; Lee, J.; Choi, I. S.; Jensen, K. F.; Langer, R. Langmuir 2002, 18, 3632–3638. (23) Lahann, J.; Langer, R. Macromolecules 2002, 35, 4380–4386. (24) Nandivada, H.; Chen, H. Y.; Bondarenko, L.; Lahann, J. Angew. Chem., Int. Ed. 2006, 45, 3360–3363. (25) Nandivada, H.; Chen, H. Y.; Lahann, J. Macromol. Rapid Commun. 2005, 26, 1794–1799.
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at a controlled temperature between -40 and 60 °C. By synthesizing [2.2]paracyclophanes with a wide variety of functional groups,21-25 a range of polymer coatings with different reactive sites can be prepared. CVD polymerization of [2.2]paracyclophanes typically yields conformal coatings that exhibit mechanical integrity and low dielectric constants.17 A suitable installation for the CVD polymerization process is shown in Figure 1. A conventional one-source configuration yields polymers with a homogeneous functional group distribution, and binary or tertiary gradients can be synthesized from two-source or threesource configurations, respectively. Details on the preparations and applications of multifunctional and gradient CVD polymers will be further discussed in a later part of this article. It is now well-established that a diverse set of functionalized polymer coatings can be synthesized from substituted [2.2]paracyclophanes (Scheme 2). For example, carbonyl-functionalized [2.2]paracyclophanes have been utilized in various applications.26-28 Aldehyde-functionalized poly(p-xylylene) coatings maintain a high reactivity of functional groups after CVD polymerization, which has (26) Chen, H. Y.; Rouillard, J. M.; Gulari, E.; Lahann, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 11173–11178. (27) Chen, H.-Y.; Lahann, J. Anal. Chem. 2005, 77, 6909–6914. (28) Suh, K. Y.; Langer, R.; Lahann, J. Adv. Mater. 2004, 16, 1401–1405.
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Figure 3. Immobilization of oligosaccharides using a [(4-formyl-p-xylylene)-co-(p-xylylene)] coating and the corresponding fluorescence micrograph (bottom-right corner) after protein immobilization.25 (Reproduced from ref 25 with permission. Copyright 2005 Wiley-VCH Verlag GmbH & Co. KGaA.)
Figure 4. Example of immobilizing amino-functionalized biotin ligands onto pentafluorophenol ester-containing CVD polymers. A PDMS stamp was used for μCP to generate a patterned ligand on a polymer, and the fluorescence micrograph shows the self-assembly of fluoresceinconjugated streptavidin. Fluorescence micrograph of bovine aortic endothelial cells (BAECs) seeded for 24 h on a biotin- and antibodymodified polymer surface.22 (Reproduced from ref 22 with permission. Copyright 2002 American Chemical Society.)
been confirmed by the immobilization of disaccharides onto the resultant polymer coating.25 In principle, poly(p-xylylenes) have desirable properties for many biomedical surface modification applications. [2.2]Paracyclophanes, the starting material for CVD polymerization, are a well-understood class of molecules that can be synthesized or modified by a range of different chemical reactions.29 Broad diversification of the chemical anchor groups becomes a straightforward task enabling a highly modular coating platform. Thus, the CVD polymerization of functionalized [2.2]paracyclophanes can provide extraordinary versatility with (29) Hopf, H. Angew. Chem., Int. Ed. 2008, 47, 9808–9812.
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respect to the type of custom-tailored polymer coatings. Compared to currently employed biomedical coatings, vapor-based reactive polymers possess unusual features that can fulfill the increasing needs for sophisticated surface modification (Table 1). In addition, CVD polymerization follows well-defined activation and reaction pathways that typically yield very few side reactions. Well-defined linear polymer coatings are typically observed, an aspect in strict contrast to some of the other vapor-based methods, such as plasma polymerization. Finally, the introduction of more than one type of paracyclophane can result in CVD copolymerization,30,31 (30) Elkasabi, Y.; Chen, H. Y.; Lahann, J. Adv. Mater. 2006, 18, 1521–1526.
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Figure 5. Schematic illustrations of surface patterning techniques using CVD reactive coatings. Methods can be categorized into two groups: patterning post-CVD, in which μCP and photopatterning processes are applied after CVD coating, and patterning deposition during CVD, in which assisted molds or metal inhibitors are applied prior to the CVD process. Table 2. Patterning Techniques with Demonstrated Compatibility for CVD Polymerization post CVD or during the CVD process μCP photopatterning projection patterning stenciling vampir selective CVD
flexibility
continuous pattern formation
size limit
ease of preparation
substrate dimension
high-throughput production
post CVD post CVD post CVD
high high high
yes yes yes
submicrometer micrometer >10 μm
easy easy medium
2-D 2-D 2-D, 3-D
yes yes yes
during CVD during CVD during CVD
high high medium low (require metal inhibitor)
no yes yes
micrometer micrometer submicrometer
easy easy medium
2-D 2-D 2-D, 3-D
yes yes yes
which is a straightforward means to developing truly multifunctional groups for bio-orthogonal immobilization strategies.
2. Controlled Chemistries Using Diferent Functionalities 2.1. Click Chemistry. In recent years, efficient chemical reactions have been increasingly studied in materials science Langmuir 2011, 27(1), 34–48
and biotechnology for bioconjugation, drug discovery, materials science, and radiochemistry applications.32 Its prime representatives, the click reactions, offer a compelling set of features (31) Elkasabi, Y.; Yoshida, M.; Nandivada, H.; Chen, H. Y.; Lahann, J. Macromol. Rapid Commun. 2008, 29, 855–870. (32) Best, M. D. Biochemistry 2009, 48, 6571–84.
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Figure 6. Schematic drawing showing substrate-independent protocols of DNA nanopatterning via the combination of SuNS and CVD technologies. Tapping-mode AFM images (on the right) of DNA lines stamped on a variety of substrates including (a) silicon, (b) quartz, (c) polystyrene (PS), and (d) PMMA.46 (Reproduced from ref 46 with permission. Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA.)
Figure 7. Schematic illustration of using a photodefinable polymer coating for the confinement of hydrogel elements and for creating discontinuous surface patterns on 2-D (flat) and 3-D microchannels.47 (Reproduced from ref 47 with permission. Copyright 2005 American Chemical Society.)
including rapidity, high selectivity, specificity, and high yields. Several reviews and a monograph have been published.32-35 To widen the applicability of the click chemistry concept for biomolecule immobilization, alkyne-containing polymer coatings 40 DOI: 10.1021/la101623n
were synthesized via CVD polymerization.24 This approach decouples the actual click reaction from the need for continuously developing specialized modification protocols for different types of substrates. The same coating can be applied to a diverse set Langmuir 2011, 27(1), 34–48
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Figure 8. Schematic description of the 3-D microstructuring technique. The method comprises two process steps: deposition of the photodefinable CVD coating (step 1) and subsequent projection lithographic rendering (step 2). Fluorescence micrographs show that this technique can be applied to 3-D and complex surfaces including (a) microspheres,26 (b) fibers,48 (c) microchannels,49 and (d) stent devices.48 (The scheme and image a are reproduced from ref 26 with permission. Copyright 2007 National Academy of Sciences of the United States of America. Image c is reproduced from ref 49 with author’s permission.)
of substrates, such as plastic, metals, glass, or silicon. These CVD-coated substrates were then used as the basis for the spatially directed click reaction, namely, Huisgen 1,3-dipolar cycloaddition (Figure 2). Prior to CVD polymerization, ethynyl[2.2]paracyclophane was prepared from commercially available [2.2]paracyclophane. The resulting poly(4-ethynyl-p-xylylene-cop-xylylene) was then used for a click reaction with an azidecontaining biotin-based ligand in the presence of Cu(II) sulfate and sodium ascorbate. Sodium ascorbate generates Cu(I) ions in situ from CuSO4, which functions as the actual catalyst. To further ensure spatial control over the cycloaddition reaction, a microcontact printing (μCP) approach was developed on the basis of acetylene-functionalized reactive coatings. A patterned PDMS stamp was inked with a solution of CuSO4 and brought into contact with the substrate. Fluorescence microscopy was used to assess the spatially directed immobilization. This study clearly establishes that proteins can selectively bind to regions where the CuSO4 solution was microcontact printed. One of the key attributes of this approach is the flexibility of the bioconjugation (33) Lahann, J. Click Chemistry for Biotechnology and Materials Science; Wiley: Chichester, West Sussex, U.K., 2009. (34) Nwe, K.; Brechbiel, M. W. Cancer Biother. Radiopharm. 2009, 24, 289–302. (35) Nandivada, H.; Jiang, X. W.; Lahann, J. Adv. Mater. 2007, 19, 2197–2208.
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platform, which enables broad applicability to diagnostics, microarrays, biosensors, and biomedical device coatings. 2.2. Immobilization via Hydrazone Linkers. Hydrazone formation (i.e., the conversion of aldehydes with a hydrazine or hydrazide) is a widely used immobilization reaction with rapid reaction kinetics and high specificity. In a complementary effort, poly(4-formyl-p-xylylene-co-p-xylylene) polymer films were developed25 as biomedical coatings that enable the selective immobilization of biomolecules via formation of hydrazones. Here, the precursor was 4-formyl[2.2]paracyclophane, which is readily available from [2.2]paracyclophane. Again, 4-formyl[2.2]paracyclophane is first sublimated and then homolytically cleaved to yield poly[(4-formyl-p-xylylene)-co-(p-xylylene)] deposited on a cooled substrate. No signs of side reactions were observed. Moreover, this reactive coating exhibited good adhesion to a wide variety of substrates such as gold, silica, glass, and polydimethylsiloxane (PDMS) and showed excellent stability in aqueous and organic solvents. The selectivity of hydrazides and hydrazines toward aldehydes and ketones makes them suberb binding partners for the immobilization of biomolecules. For instance, dihydrazide linkers can covalently bind to the aldehyde groups of the reactive coating creating anchor sites for disaccharides (Figure 3).25 A similar DOI: 10.1021/la101623n
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Figure 9. Process of vapor-assisted micropatterning in replica structures (VAMPIR). Fluorescence micrographs showing the immobilization of QD and fluorescence-labeled biomolecules onto patterned surfaces.51 (Reproduced from ref 51 with permission. Copyright 2007 WileyVCH Verlag GmbH & Co. KGaA.)
procedure has been used in the past to attach antibodies to the aldehyde-functionalized surface.36 Moreover, these reactive coatings can be applied to sealed PDMS microchannels, demonstrating the feasibility of surface modification within confined microgeometries.37 In this example, poly(4-trifluoroacetyl-pxylylene-co-p-xylylene) was prepared on the inner channel surface after assembly of the device and prior to conversion of the hydrazides, as described above for aldehyde-functionalized polymers. To assess the chemical activity of both reactive coatings, a biotin hydrazide ligand was used, followed by subsequent TRITC-streptavidin self-assembly. The results from fluorescence micrographs have provided evidence that a homogeneous distribution of functional groups can be created over the entire channel of the microdevice. 2.3. Anhydrides and Pentafluorophenol Esters. Common targets of biomolecule immobilization are amino groups, which are ubiquitous in proteins, peptides, and glycoproteins. To take advantage of these abundant chemical functionalities, reactive CVD coatings carrying anhydride as well as pentafluorophenol ester groups were developed.38 An example is shown in Figure 4 in which the resulting pentafluorophenol ester surfaces can be patterned readily using μCP with selected ink, and subsequent biological activities are demonstrated.22 The pentafluorophenol ester can be synthesized from [2.2]paracyclophane via a three-step synthesis.38 Alternatively, [2.2]paracyclophane-4,5,12,13-tetracarboxylic dianhydride can be synthesized from 4,5,12,13tetrakis(methyloxy carbonyl)-[2.2]paracyclophane by conversion with concentrated sulfuric acid. The tetra-ester is accessible through the Diels-Alder reaction of acetylenedicarboxylic acid methyl ester with hexatetraene.39 Once the corresponding (36) Gering, J. P.; Quaroni, L.; Chumanov, G. J. Colloid Interface Sci. 2002, 252, 50–56. (37) Chen, H. Y.; Elkasabi, Y.; Lahann, J. J. Am. Chem. Soc. 2006, 128, 374– 380. (38) Lahann, J.; Choi, I. S.; Lee, J.; Jenson, K. F.; Langer, R. Angew. Chem., Int. Ed. 2001, 40, 3166–3168. (39) Aly, A. A.; Ehrhardt, S.; Hopf, H.; Dix, I.; Jones, P. G. Eur. J. Org. Chem. 2006, 2, 335–350.
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paracyclophanes are synthesized, reactive coatings can be homogenously deposited on the substrates by means of CVD polymerization. In this specific case, the pyrolysis temperature was important for the quality of the reactive coatings, and a lower temperature (below 670 °C) can lead to better-defined coatings.23
3. Surface Micro/Nanostructuring Microstructures and local substrate chemistry can affect cellular responses and proliferation.2 For instance, cell responses to a synthetic extracellular matrix (ECM) depends on multiple substrate features such as the chemical composition, geometry and topological features, ligand organization, and substrate stiffness.40,41 Many engineering approaches aiming to control cell adhesion and spreading precisely through chemically and spatially designed surfaces have therefore been extensively researched.2,42 Examples include micromolding in capillaries, microcontact printing (μCP), replica molding, microtransfer molding, solvent-assisted micromolding, and capillary force lithography and are collectively called soft lithography.42 Similarly, surface patterns have also been fabricated using dip-pen lithography,43 imprinting lithographies,44 and a diverse range of photolithographical techniques.2 As part of our recent work, we have focused on the creation of microstructures and nanostructures by using reactive coatings prepared via CVD polymerization. The methods can be categorized into two groups (Figure 5). (i) Patterning post-CVD. In this category, homogeneously deposited reactive polymers are formed on the substrate during the first step and subsequent patterning steps, such as μCP or photopatterning, are then employed to form the desired patterns. (ii) Microstructured CVD deposition. In this (40) Lehnert, D.; Wehrle-Haller, B.; David, C.; Weiland, U.; Ballestrem, C.; Imhof, B. A.; Bastmeyer, M. J. Cell Sci. 2004, 117, 41–52. (41) Arnold, M.; Hirschfeld-Warneken, V. C.; Lohmuller, T.; Heil, P.; Blummel, J.; Cavalcanti-Adam, E. A.; Lopez-Garcia, M.; Walther, P.; Kessler, H.; Geiger, B.; Spatz, J. P. Nano Lett. 2008, 8, 2063–2069. (42) Xia, Y. N.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153–184. (43) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661–663. (44) Chou, S. Y.; Krauss, P. R.; Renstrom, P. J. Science 1996, 272, 85–87.
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Figure 10. Influence of structure dimensions on polymer depth during the VAMPIR process. (a) Imaging XPS Si 2s (150.0 eV) elemental map. (b) Imaging XPS F 1s (689.9 eV) elemental map. (c) Imaging ellipsometry thickness map. (d) Fluorescence micrograph showing biotin/ TRITC-streptavidin immobilization.51 (Reproduced from ref 51 with permission. Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA.)
category, surface patterns are formed via physical masking (assisted deposition) or are based on metal inhibitors (selective deposition), which have been previously applied to the substrates. After CVD polymerization, reactive polymers were found only over the expected surface areas. Table 2 summarizes the characteristic features and limitations of using these patterning techniques enabled by CVD-based reactive coatings. In the following section, we will address each approach in more detail and describe some specific examples. 3.1. Microcontact Printing (μCP). Microcontact printing (μCP) is a widely used approach to patterning surfaces on various types of substrates42 and can be simply applied to reactive coatings with a choice of inks on selected functional groups. In one example, μCP was used to pattern substrates by a PDMS stamp with a set of parallel lines of (þ)-biotinyl-3,6,9-trioxaundecanediamine.22 Microstructured substrates were stable for at least 7 days at room temperature. For the spatial control of cell attachment, biotin/streptavidin conjugation was used to immobilize a cellbinding antibody. As shown previously in Figure 4, the surface patterns were still recognized by cells.22 In an extension of this
initial work, we further showed that reactive polymer coatings could also be modified with DNA. By using supramolecular nanostamping (SuNS)45 technology on poly(4-formyl-p-xylyleneco-p-xylylene), controlled DNA attachment was resolved for feature sizes as small as 100 nm.46 The combination of SuNS with the CVD technology allowed for the extension of the nanopatterning protocols to a range of different substrates. In proof-of-concept experiments (Figure 6), nanopatterns were prepared on polystyrene, acrylic, and PDMS without an alteration of the immobilization protocol. Because of its simplicity, a broad spectrum of applications can be contemplated in the field of bio/ nanodevices. 3.2. Photopatterning. Another post-CVD micropatterning method involves the projection of ultraviolet light onto a photoreactive coating, such as the above-mentioned poly(4-benzoyl-pxylylene-co-p-xylylene). For example, photodefinable polymers have been used to immobilize an array of PEG hydrogels on the substrate surfaces28 or to prepare discontinuous surface patterns27 on 3-D microscale objects (Figure 7). Because of their solventless processing capabilities, CVD-based reactive polymer
(45) Yu, A. A.; Savas, T. A.; Taylor, G. S.; Guiseppe-Elie, A.; Smith, H. I.; Stellacci, F. Nano Lett. 2005, 5, 1061–1064.
(46) Thevenet, S.; Chen, H.-Y.; Lahann, J.; Stellacci, F. Adv. Mater. 2007, 19, 4333–4337.
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Figure 11. CVD polymerization approach to preparing a vapor-based initiator coating for poly[oligo(ethylene glycol) methyl ether methacrylate] modification via ATRP. A microstencil was used during CVD polymerization to direct initiator polymers onto defined surface areas. Using surface-initiated ATRP, a poly[oligo(ethylene glycol) methyl ether methacrylate] film was then selectively prepared in areas where the initiator coatings had been previously deposited. Fluorescence and phase-contrast micrographs show the biological activity of controlled protein and cell resistance on patterned surfaces.21 (Reproduced from ref 21 with permission. Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA.)
coatings could be ideally suited to creating reactive coatings on 3-D structures such as spheres. As shown in Figure 8, a projection patterning process that utilizes a digital micromirror is used to produce microstructures on 3-D substrates. In the first step, the poly(4-benzoyl-p-xylylene-co-p-xylylene) coating is applied to the substrate surface (e.g., microcolloids, microchannels, stent devices, etc.) via CVD polymerization. In the second step, in order to obtain spatially controlled surface patches, certain areas of the objects are illuminated with light at 365 nm using a high-throughput projection technique.26 Here, patterns were created using a programmable 1024 pixel 768 pixel digital micromirror. Although the entire substrate was coated with poly(4-benzoyl-p-xylylene-co-pxylylene) during CVD polymerization, only the illuminated areas underwent photochemical reaction. As a proof-of-concept experiment, biotin-streptavidin was used on the substrates tested for demonstration and the micrographs obtained by fluorescence microscopy have shown selectively immobilized streptavidin on microspheres (Figure 8a),26 fibers (Figure 8b),48 microchannels (Figure 8c),49 and stent devices (Figure 8d).48 The CVD polymerization process offers the advantage of conformity and 3-D coverage, and the projection patterning process circumvents the (47) Smith, J. P.; Hinson-Smith, V. Anal. Chem. 2005, 77 (21), 412 A. (48) Chen, H.-Y.; Lahann, J. Unpublished data. (49) Chen, H. Y.; Rouillard, J.-M.; Gulari, E.; Lahann, J. PMSE Prepr. 2006
44 DOI: 10.1021/la101623n
use of photomasks.50 Thus, the projection patterning technology based on CVD coatings offers a simple pathway for the surface modification of devices with 3-D and complex geometry.49 For example, PEO-amine-derived biotin ligands have been covalently attached to modified stents.48 3.3. Vapor-Assisted Patterning. We recently exploited the fact that reactive coatings can also be deposited within partially closed microgeometries37 for the simple fabrication of a range of different patterns. This technique results in chemical and topological surface microstructures that are accessible by simply masking certain areas of the substrate during CVD polymerization (Figure 9). We classified the resulting soft lithographic process as vapor-assisted micropatterning in replica structures (VAMPIR).51 Polymers studied with respect to the VAMPIR technique include poly(4-pentafluoropropionyl-p-xylylene-cop-xylylene) and poly(p-xylylene-4-methyl-2-bromoisobutyrate-co-p-xylylene). The lower limit of feature sizes accessible with VAMPIR was evaluated in PDMS replica structures, which were composed of varying distances between posts resulting in feature sizes of 150, 100, 50, and 25 μm. XPS imaging further confirmed that silicon
(50) Gao, X. L.; Zhou, X. C.; Gulari, E. Proteomics 2003, 3, 2135–2141. (51) Chen, H.-Y.; Lahann, J. Adv. Mater. 2007, 19, 3801–3808.
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Figure 12. Selectively deposited poly(4-vinyl-p-xylylene-co-p-xylylene). Polymer deposition occurs only in areas without the presence of titanium metal. The fluorescence micrograph shows fluorescently labeled O-methacrylate that was used via the olefin cross-metathesis reaction to confirm the reactivity of vinyl functional groups.52 (Reproduced from ref 52 with permission. Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA.)
signals could be detected only in areas that were masked during CVD polymerization. This was true for the entire range of feature sizes from 150 to 25 μm. Correspondingly, fluorine, a representative element of poly(4-pentafluoropropionyl-p-xylylene-co-p-xylylene), was detected only in areas not masked during CVD polymerization (Figure 10). The chemical reactivity of the VAMPIR microstructures was also verified by covalent binding of a biotin hydrazide followed by self-assembled TRITC-strepdavidin as shown in the fluorescence micrographs in Figure 10. In another example,21 a PDMS micromold was used during the deposition of poly(p-xylylene-4-methyl-2-bromoisobutyrate-cop-xylylene) to create an initiator coating on the substrate surface. Subsequently, atom-transfer radical polymerization (ATRP) was performed to form hydrogel films on the modified surface as shown in Figure 11. The stability of the polymer patterns was examined by immersing the coating in a range of different solvents, including methanol, ethanol, acetone, and chloroform, and also by a tape test in conjunction with a visual inspection. Microstructured poly[oligo(ethylene glycol) methyl ether methacrylate] hydrogel was evaluated by both protein adsorption and cell adhesion. Owing to the nonfouling property of poly[oligo(ethylene glycol) methyl ether methacrylate], fibrinogen adhered only to areas where no poly[oligo(ethylene glycol) methyl ether methacrylate] was present (i.e., areas without initiator (52) Chen, H. Y.; Lai, J. H.; Jiang, X. W.; Lahann, J. Adv. Mater. 2008, 20, 3474–3480.
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coatings (Figure 11a,b). Similarly, murine fibroblast (NIH3T3 cells) grew only on areas that corresponded to nonmodified surfaces as shown in Figure 11c,d. 3.4. Selective Deposition. It has been reported by Vaeth and Jensen53,54 that members of the poly-p-xylylene family, such as parylene-N and parylene-C, can allow for substrate-dependent deposition. However these coatings are non-functionalized, because they lack anchoring groups to support further covalent immobilization.55 We have recently developed a reactive coating, poly(4-vinyl-p-xylylene-co-p-xylylene),52 whose formation can be inhibited by titanium. To identify this candidate, 4 nonreactive and 10 reactive poly(p-xylylenes) with a wide range of different functional groups, such as amines, alcohols, aldehydes, anhydrides, active esters, alkyne, and ketones, were compared. The CVD films were simultaneously deposited on a library of nine different metal surfaces: Cu, Ir, Ta, W, Pt, Ni, Ti, Ag, and Au. The presence of each polymer on the corresponding metal surface was confirmed by using infrared reflection absorption spectroscopy (IRRAS). To confirm the selective deposition of poly(4-vinyl-pxylylene-co-p-xylylene) on titanium further, we purposely prepared gold-patterned layers (that are non-inhibitory) on top of a titanium surface. Then we used fluorescein-labeled methacrylates via an olefin cross-metathesis reaction to verify the resulting (53) Vaeth, K. M.; Jensen, K. F. Adv. Mater. 1999, 11, 814–820. (54) Vaeth, K. M.; Jensen, K. F. Chem. Mater. 2000, 12, 1305–1313. (55) Herrera-Alonso, M.; McCarthy, T. J. Langmuir 2004, 20, 9184–9.
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Figure 13. (a) Preparation of a multifunctional copolymer by the CVD copolymerization of different substituted-[2.2]paracyclophanes. (b) Detection of the resulting exemplary bifunctional polymer surface; Atto 655 NHS ester (no. 2, red) and biotin hydrazide/TRITC-streptavidin (no. 1, green) were specifically used to target aminomethyl groups and keto groups, respectively. No cross reactivity was found in overlapped areas (no. 3, yellow).30 (Reproduced from ref 30 with permission. Copyright 2006 Wiley-VCH Verlag GmbH & Co. KGaA.)
polymer deposition behavior. The fluorescence data indicated that the deposition of poly(4-vinyl-p-xylylene-co-p-xylylene) occurred only in areas where no titanium was present (Figure 12). In addition, a microstructured poly[oligo(ethylene glycol) methyl ether methacrylate] hydrogel has also been created using this pathway, and the nonfouling property of such a surface was also demonstrated.52
4. CVD Copolymerization Biological interactions are hardly ever unitary and typically involve a complex cascade of immobilizations and the simultaneous manipulation of multiple types of biomolecules to a surface.56,57 However, almost all synthetic surface-modification concepts rely on a single type of surface cue. However, achieving a controlled simultaneous presentation of two or more biomolecules remains challenging.58 The precise control of multiple surface reactions, while avoiding cross reactivity between the different chemical groups, is key to overcoming challenges in this field. Further progress will be important for a number of applications, such as the regulation of cell shape, the development of advanced biological assays and scaffolds for regenerative medicine, and the (56) Keefer, L. K. Nat. Mater. 2003, 2, 357–358. (57) Nandivada, H.; Chen, H.-Y.; Elkasabi, Y.; Lahann, J. Reactive Polymer Coatings for Biological Applications. In Polymers for Biomedical Applications; Mahapatro, A., Kulshrestha, A. S., Eds.; American Chemical Society: Washington, DC, 2008; pp 283-298. (58) Kao, W. J. Biomaterials 1999, 20, 2213–2221.
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fabrication of increasingly complex micrototal analysis systems (μTAS)59 that all demand defined surface architectures and usually require precise immobilization of multiple biomolecules. To fulfill this need, our group has recently started to explore new avenues to developing bioorthogonal surface coatings using CVD copolymerization. Figure 13a illustrates the CVD copolymerization process for preparing multifunctional copolymers from different substituted [2.2]paracyclophanes. In a proof-of-concept experiment, we verified that both functional groups in copolymer poly[4-aminomethyl-p-xylylene-co-4-trifluoroacetyl-p-xylyleneco-p-xylylene] were available for further surface modification by reacting the copolymer with two fluorescent ligands that exhibited orthogonal reactivity. Atto 655 NHS ester was used for the specific detection of aminomethyl groups, and biotin hydrazide was used, followed by fluorescently labeled streptavidin, to target the keto groups on the copolymer surface. As anticipated, both ligands showed specific reactivity toward the targeting groups on the copolymer surface, and no cross-reaction was discovered (Figure 13b). An example31 of growing NIH3T3 fibroblasts on two CVD homopolymers, poly(4-aminomethyl-p-xylylene-co-pxylylene) and poly(4-trifluoroacetyl-p-xylylene-co-p-xylylene), and also a copolymer, poly[4-aminomethyl-p-xylylene-co-4-trifluoroacetyl-p-xylylene-co-p-xylylene], was demonstrated and compared (59) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637–2652.
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Figure 14. Confocal micrographs of NIH3T3 fibroblasts grown on (a) a poly(L-lysine) (PLL)-coated surface (positive control), (b) a poly(vinyl chloride) (PVC) surface (negative control), (c) a poly(4-aminomethyl-p-xylylene-co-p-xylylene) (homopolymer), (d) a poly[4-aminomethyl-p-xylylene-co-4-trifluoroacetyl-p-xylylene-co-p-xylylene] (copolymer), and (e) a poly(4-trifluoroacetyl-p-xylylene-co-pxylylene) (homopolymer). Actin filaments (red dye) of NIH3T3s show spreading responses with respect to different surfaces.31 (Reproduced from ref 31 with permission. Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA.)
Figure 15. (a) Schematic of the custom-built two-source CVD system for creating polymer gradients; (b) fluorescence micrograph showing a gradient distribution by specifically targeting amino-and carbonyl gradients using an Atto 655 NHS ester and biotin hydrazide/TRITCstreptavidin, respectively, and (c) intensity profiles with respect to image b.60 (Reproduced from ref 60 with permission. Copyright 2009 WileyVCH Verlag GmbH & Co. KGaA.)
with a poly(L-lysine) (PLL)-coated surface (positive control) and a cytotoxic poly(vinyl chloride) (PVC) surface (negative control). The results indicated that actin filaments spread more on the Langmuir 2011, 27(1), 34–48
poly(4-aminomethyl-p-xylylene-co-p-xylylene) homopolymer, which is similar to those cultured on the PLL-coated glass substrate, whereas the actin cytoskeletal network was less pronounced DOI: 10.1021/la101623n
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on poly(4-trifluoroacetyl-p-xylylene-co-p-xylylene) and the negative control PVC surface. As anticipated, actin filament morphology showed a similarity between the above-mentioned two cases of homopolymers on the amino- and trifluoroacetylcontaining copolymer (Figure 14).
5. Gradients Gradient-based approaches are an emerging trend in many biomedical research areas including tissue engineering, diagnostic research, and cell/embryonic assays.61 Major efforts have recently been devoted to generating gradients with gradual changes in physical properties such as wettability, thickness, dielectric constant, temperature, morphology and also gradients with continuously varied chemical composition. Several excellent review articles addressing current technologies and challenges of using gradient surfaces in a variety of applications have since been reported.62,63 Although synthetic methods for producing these gradient surfaces such as bulk diffusion,64 microfluidic pathways,65 lithography,63 or a combination66 thereof showed preliminary promise, limitations remained for handling sophisticated biological signals and were often found to be lacking in the precise translation of chemical gradients into biological gradients,67 continuity in chemical resolution,68 and a general protocol for various substrates.69 Recently, we modified the CVD process by using a two-source CVD system (Figure 15a). As a proof-of-concept example, trifluoroacetyl-functionalized [2.2]paracyclophane and aminomethyl-functionalized [2.2]paracyclophane were used as the starting material for each source. These two monomers were then deposited countercurrently in the vapor phase to form (60) Elkasabi, Y.; Lahann, J. Macromol. Rapid Commun. 2009, 30, 57–63. (61) Genzer, J.; Bhat, R. R. Langmuir 2008, 24, 2294–2317. (62) Morgenthaler, S.; Zink, C.; Spencer, N. D. Soft Matter 2008, 4, 419–434. (63) Kim, M. S.; Khang, G.; Lee, H. B. Prog. Polym. Sci. 2008, 33, 138–164. (64) Zelzer, M.; Majani, R.; Bradley, J. W.; Rose, F. R. A. J.; Davies, M. C.; Alexander, M. R. Biomaterials 2008, 29, 172–184. (65) Jeon, N. L.; Dertinger, S. K. W.; Chiu, D. T.; Choi, I. S.; Stroock, A. D.; Whitesides, G. M. Langmuir 2000, 16, 8311–8316. (66) Luzinov, I.; Minko, S.; Tsukruk, V. V. Soft Matter 2008, 4, 714–725. (67) Chaudhury, M. K.; Whitesides, G. M. Science 1992, 256, 1539–1541. (68) Burdick, J. A.; Khademhosseini, A.; Langer, R. Langmuir 2004, 20, 5153– 5156. (69) Ballav, N.; Shaporenko, A.; Terfort, A.; Zharnikov, M. Adv. Mater. 2007, 19, 998–1000.
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linear gradients. We also were able to immobilize fluorescencelabeled ligands selectively onto the reactive polymer gradients (Figure 15b,c).60 Given the high demand for gradients in biotechnology and the wide-ranging applicability of CVD polymerization to a diverse set of substrates, polymer gradients based on CVD polymer technology have the potential to expedite biomaterial discovery for a range of biotechnological applications including tissue engineering, substrates for microbiological studies, and combinatorial research.
6. Future Outlook Reactive polymer coatings can improve the interfacial biocompatibility of biomaterials because they are compatible with complex biological features and represent designable interlayers. Many key features for biological applications have been added during the past few years. Recently, their uses in microfluidic devices, the fabrication of nonfouling surfaces, and the precise surface modification for complex 3-D devices have been demonstrated and were discussed in this article. These advanced coatings provide a technology platform that has the potential to improve active long-term control and the mimicry of biological systems. The development of these technologies will hold promise for enabling studies in biological and medical areas, such as the control of cell/cell, cell/protein, and protein/surface interactions with applications in biosensors, extracellular matrix substitutes, and microfluidics. Commercial systems for CVD polymerization in prepare poly(p-xylylenes) are available. However, the use of functionalized poly(p-xylylenes) in many cases still requires the custom synthesis of substituted [2.2]paracyclophanes. Other challenges include the development of coating technologies that enable the microstructuring of 3-D and complex substrates with feature sizes on the submicrometer scale. Finally, future advances may include high-precision modifications that will merge the reactive coatings technology with combinatorial approaches that can enable the high-throughput screening and development of novel biomaterials. Acknowledgment. We gratefully acknowledge support from the NSF in the form of a CAREER grant (DMR-0449462) and funding from the NSF under the MRI program (DMR 0420785).
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