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Immobilization of Biomolecules on Poly(vinyldimethylazlactone)-Containing Surface Scaffolds Joshua E. Barringer,† Jamie M. Messman,‡ Abigail L. Banaszek,† Harry M. Meyer III,§ and S. Michael Kilbey II*,†,‡ Department of Chemical and Biomolecular Engineering, Clemson UniVersity, Clemson, South Carolina 29634, and Center for Nanophase Materials and Sciences and Materials Science and Technology DiVision, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, Tennessee 37831 ReceiVed September 5, 2008 We describe the successful development of a procedure for the step-by-step formation of a reactive, multilayer polymer scaffold incorporating polymers based on 2-vinyl-4,4-dimethylazlactone (VDMA) on a silicon wafer and the characterization of these materials. Also discussed is the development of a procedure for the nonsite specific attachment of a biomolecule to a modified silicon wafer, including scaffolds modified via drop-on-demand (DOD) inkjet printing. VDMA-based polymers were used because of their hydrolytic stability and ability of the pendant azlactone rings to form stable covalent bonds with primary amines without byproducts via nucleophilic addition. This reaction proceeds without a catalyst and at room temperature, yielding a stable amide linkage, which adds to the ease of construction expected when using VDMA-based polymers. DOD inkjet printing was explored as an interesting method for creating surfaces with one or more patterns of biomolecules because of the flexibility and ease of pattern design.
Introduction Polymeric materials are used in a variety of biomedical applications such as biosensors, restoratives for the repair of teeth and other bones, bioregulation, and drug delivery.1-4 Surfaces are frequently modified with biomaterial coatings to enhance therapeutic functionality, improve biocompatibility, or locally deliver treatment via implantation.3 Often, these surfacemodification applications require the immobilization of biomolecules on the solid support,4 which can be accomplished in a variety of ways. Among these, perhaps the most widely practiced method for nonsite specific immobilization of biomolecules is via amidation chemistry using N-hydroxy succinimide (NHS) esters.4 However, polymers containing NHS esters typically suffer from hydrolysis even at neutral conditions and undergo side reactions, including the formation of hydrolytically unstable ringopened conjugates and glutarimide-bound conjugates.5,6 With this in mind, we have been studying polymers based on 2-vinyl-4,4-dimethylazlactone (VDMA) as a platform for creating bioconjugates. Polymers containing VDMA are hydrolytically stable, and they form stable covalent bonds with amine and thiol groups commonly found in enzymes, proteins, and other biomolecules.7,8 Nucleophilic addition of a primary amine proceeds without a catalyst and at room temperature, yielding * Corresponding author. Current address: Center for Nanophase Materials Sciences, ORNL, and Department of Chemistry, University of Tennessee, Knoxville, Tennessee. † Clemson University. ‡ Center for Nanophase Materials Sciences, Oak Ridge National Laboratory. § Materials Science and Technology Division, Oak Ridge National Laboratory. (1) Grodzinski, J. J. React. Funct. Polym. 1999, 39, 99–138. (2) Heilmann, S. M.; Rasmussen, J. K.; Krepski, L. R.; Smith, H. K., II J. Polym Sci: Polym. Chem. 1984, 22, 3149–3160. (3) Khan, W.; Marew, T.; Kumar, N. Biomed. Mater. 2006, 1, 235–241. (4) Golova, J. B.; Chernov, B.; Kukhtin, A. U.S. Patent 20070238163A1, 2007. (5) Wong, S. Y.; Putnam, D. Bioconjugate Chem. 2007, 18, 970–982. (6) Devenish, S. R. A.; Hill, J. B.; Blunt, J. W.; Morris, J. C.; Munro, M. H. G. Tetrahedron. Lett. 2006, 47, 2875–2878. (7) Peterson, D. S.; Rohr, T.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2002, 74, 4081–4088.
a stable amide linkage.4,8 This property has been exploited to immobilize proteins on polymers of VDMA in solution8 and to construct multilayered films via a layer-by-layer approach, reacting poly(vinyl dimethyl azlactone) (pVDMA) with poly(ethylene imine) in an alternating fashion.9 Despite reports showing that polymers incorporating VDMA can be made by free radical10 and controlled (free) radical polymerizations11,12 and that proteins and other biomolecules can be immobilized onto pVDMA,4,7,8,10 thus far there have been no reports of using VDMA-based polymers as a surface-grafted platform to decorate interfaces with biomolecules. In this article, we demonstrate a straightforward method for creating surface-tethered layers containing pVDMA that can be subsequently functionalized by biomolecules containing primary amines. VDMA was copolymerized with vinylpyrollidone (VP), which is selected because of its low toxicity and biocompatibility,13 and the resultant copolymers were tethered to surfaces using a “primer” polymer layer approach.14 Poly(glycidyl methacrylate) (PGMA) was the base of the polymer support because it is readily grafted onto silicon wafers, providing a high surface density of epoxy groups available for subsequent modification,14 including reaction with ATRP initiators, carboxylic acids, and amines.14,15 Given previous successes in making patterned monolayers and polymer-modified surfaces (8) Drtina, G. J.; Heilmann, S. M.; Moren, D. M.; Rasmussen, J. K.; Krepski, L. R.; Smith, H. K., II; Pranis, R. A.; Turek, T. C. Macromolecules 1996, 29, 4486–4489. (9) Buck, M. E.; Zhang, J.; Lynn, D. M. AdV. Mater. 2007, 19, 3951–3955. (10) Stanek, L. G.; Heilmann, S. M.; Gleason, W. B. Polym. Bul 2005, 55, 393–402. (11) Schilli, C. M.; Mu¨ller, A. H. E.; Rizzardo, E.; Thang, S. H.; Chong, Y. K. AdVances in Controlled/LiVing Radical Polymerization; ACS Symposium Series 854; American Chemical Society: Washington, DC, 2003; Chapter 41, pp 603-618. (12) Fournier, D.; Pascual, S.; Fontaine, L. Macromolecules 2004, 37, 330– 335. (13) Nazarova, O. V.; Solovskii, M. V.; Panarin, Y.F.; Alekseyeva, S. V. Polym. Sci. U.S.S.R. 1987, 31, 428–433. (14) Liu, Y.; Klep, V.; Zdryko, B.; Luzinov, I. Langmuir 2004, 20, 6710– 6718. (15) Ko, S.; Jang, J. Biomacromolecules 2007, 8, 1400–1403.
10.1021/la802925g CCC: $40.75 2009 American Chemical Society Published on Web 12/10/2008
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Scheme 1. Schematic of the Multilayer Assembly Process, Including Principal Materials Useda
a Conditions: (1) 1 wt % PGMA in MEK and annealing (0.5 h, 110 °C); (2) 1 wt % 1,6-diaminohexane in ethanol and annealing (3 h, 80 °C); (3) 0.1 wt % p(VP-co-VDMA) in THF (18 h); and (4) 0.1 M dansylcadaverine in ethanol (24 h).
with drop-on-demand (DOD) inkjet printing,16-20 there is significant potential for advancements in biomaterial coatings created using this method. Thus, the attachment of a biomolecule to the multilayer polymer via DOD printing onto the scaffold was also explored. Dansylcadaverine was chosen as the model biomolecule because it has a single primary amine and a sulfonyl group, which facilitate attachment and characterization, and also because it is moderately soluble in ethanol, which is used in DOD printing. DOD inkjet printing uses printhead nozzles to release a single drop of ink precisely on a surface to form a printed pattern. A collection of these drops, which construct what is known as pixels, on the surface creates the desired patterned result. In this research, a gradient pattern was used. The volumes of the small drops that exit the inkjet printer are generally on the order of picoliters. The resolution of a printer denotes the distance between two adjacent ink drops, but the dissimilarity between printed pixels actually defines the pattern resolution. This feature gives the printer the ability to make pronounced visual graphics. Inkjet printing allows one to use small drop volumes of liquid solutions and experience a high printhead operating frequency, remarkable system reliability, and extremely regulated ink drop placement. For these reasons, DOD inkjet printing is finding widespread use as a patterning tool in thin film applications.
Experimental Section Materials and Preparations. Silicon wafers (1 cm × 1.2 cm) were purchased from Silicon Quest and cleaned immediately before use by immersion for 90 min in a piranha acid solution (3:1 v/v solution of sulfuric acid (EMD, 95-98%) and 30% hydrogen peroxide (VWR, 29-32%)), followed by rinsing with copious amounts of distilled, deionized water and drying with a stream of dry nitrogen. Caution! Piranha acid should be handled with extreme care because it reacts Violently with most organic materials. Methyl ethyl ketone (MEK) (Fisher, g99%), anhydrous ethanol (Fisher, 99.5+%), and tetrahydrofuran (Burdick and Jackson, g99%) were used as received. Benzene (ReagentPlus, thiophene free, g99%; Aldrich) was dried with calcium hydride, distilled under reduced pressure, and stored over nitrogen. 1-Vinyl-2-pyrrolidone (VP; g99%, 0.01% sodium hydroxide as inhibitor; Aldrich) was treated with calcium hydride overnight, distilled under high vacuum, and stored at reduced temperature over dry nitrogen. 2,2′-Azobis(2-methylpropionitrile) (16) Morgenthaler, S.; Zink, C.; Spencer, N. D. Soft Matter 2008, 4, 419–434. (17) Bietsch, A.; Zhang, J. Y.; Hegner, M.; Lang, H. P.; Gerber, C. Nanotechnology 2004, 15, 873–880. (18) Singh, B. K.; Hillier, A. C. Anal. Chem. 2007, 79, 5124–5132. (19) Sankhe, A. Y.; Booth, B. D.; Wiker, N. J.; Kilbey, S. M., II Langmuir 2005, 21, 5332–5336. (20) Pardo, L.; Wilson, W. C., Jr.; Boland, T. Langmuir 2003, 19, 1462–1466.
(AIBN; 98%, Aldrich) was recrystallized from anhydrous methanol at least three times, dried in vacuo, and stored under a blanket of dry nitrogen at 3 °C. 2-Vinyl-4,4-dimethylazlactone (VDMA; Isochem North America, LLC) was fractionally distilled under reduced pressure, and the middle fraction (∼70%) was used. Dansylcadaverine (Fluka, 99%) and 1,6-diaminohexane (Fluka, g99%) were used as received. Poly(glycidyl methacrylate) (PGMA) was prepared via free radical polymerization following the procedure of Luzinov et al.17 A number-average molecular weight (Mn) of 24 600 g/mol (relative to polystyrene standards) and a polydispersity of 1.61 were determined using size exclusion chromatography. Random copolymer p(VP-co-VDMA) was made from the AIBNinitiated polymerization of vinyl pyrrolidone (VP) and 2-vinyl-4,4′dimethylazlactone (VDMA) in benzene (Mw ) 184 300 g mol-1, PDI ) 2.25 with feed ratio of 3:1 VP/VDMA). In this reaction, AIBN (0.36 g, 2.2 mmol) was first added to a single-necked 100 mL Airfree round-bottom reaction flask equipped with a Teflon-coated magnetic stir bar and rubber septum. Dry benzene (42.0 mL, 0.471 mol) was then added, and subsequently, comonomers VP (13.5 mL, 0.126 mol) and VDMA (4.5 mL, 0.032 mol) were transferred via syringe while maintaining a dry, inert nitrogen environment. Three consecutive freeze-pump-thaw cycles were used to remove dissolved oxygen. Next, the reaction vessel was placed in an oil bath thermostatted at 65 °C and allowed to react for 65 min. The polymerization was quenched by immersing the flask and its contents in liquid nitrogen. The copolymer was precipitated by slowly adding (using a separatory funnel) the copolymer solution (∼5-10% in THF) to a large volume of a vigorously stirred hexanes/THF mixture (3:1 hexanes/THF) chilled to -30 °C. These conditions were chosen to preserve the azlactone functionality but still fractionally remove residual VP. Copolymer composition was determined using 1H spectroscopy. The mole fractions of VDMA and VP in the copolymer were determined from the integrated areas under the peaks centered at 1.37 and 3.23 ppm, respectively. The peak centered at 1.37 ppm represents the six methyl protons associated with the VDMA ring [C(CH3)2], and the peak centered at 3.23 ppm represents the two methylene protons adjacent to the nitrogen of the pendant VP ring (N-CH2). The resultant copolymer had Mn ) 81 900 g mol-1 and PDI ) 2.25 and was 30.7% VDMA as determined by NMR. Surface Modification Using p(VP-co-VDMA). Scheme 1 outlines the sequence of steps used to tether p(VP-co-VDMA) to silicon surfaces. The protocols implemented are derived from a considerable body of preliminary experiments that explored the effects of concentration and time to optimize the attachment of p(VP-coVDMA). As noted previously, silicon samples were freshly prepared prior to use, and ellipsometric and contact angle measurements were made between each step of the attachment procedure. We implemented the PGMA attachment procedure developed by Luzinov et al.14,21,22 Briefly, silicon wafers were dip-coated in a 1 wt % solution of PGMA in MEK and then immediately annealed for 30 min in an oven preheated to 110 °C. After the PGMA-modified
264 Langmuir, Vol. 25, No. 1, 2009 wafers cooled to room temperature, they were immersed in MEK and sonicated for 30 min to remove any unattached PGMA from the surface of the wafer and then dried with a stream of dry, filtered N2. The PGMA-modified silicon wafers were then immersed in a freshly made 1 wt % solution of 1,6-diaminohexane in anhydrous ethanol (EtOH). The wafer and solution were placed in a vacuum oven at 80 °C for 3 h, utilizing the vacuum (∼10 mmHg) to create an inert atmosphere. After they had cooled to room temperature, the wafers were sonicated for 30 min in EtOH and then dried with a filtered N2 stream. To attach the p(VP-co-VDMA) to the amine-functionalized layer, the wafers were immersed in a freshly made 0.1 wt % solution of p(VP-co-VDMA) in THF and allowed to react for 18 h at room temperature. After the reaction, the wafers were removed from the solution and sonicated for 30 min in THF before being dried with a stream of filtered, dry N2. Model biomolecule dansylcadaverine (DC) was attached by immersing the wafer in a freshly prepared 0.1 mM solution of DC in EtOH. The solution containing the surface was gently shaken for 24 h, and then the wafers were removed from the solution, rinsed with EtOH, and dried under a flow of filtered N2 . Characterization. Size Exclusion Chromatography. Size exclusion chromatography (SEC) was performed using a Waters Alliance 2695 separations module equipped with three Polymer Laboratories PLgel 5 µm mixed-C columns (300 × 7.5 mm2) in series, a Waters model 2414 refractive index detector, a Waters model 2996 photodiode array detector, a Wyatt Technology miniDAWN multiangle light scattering (MALS) detector, and a Wyatt Technology ViscoStar viscometer. Waters Empower 2 software was used to generate a conventional calibration curve based on low-PDI polystyrene standards {(162-6.04) × 106 g mol-1} and subsequently evaluate the molecular weight characteristics. Fourier Transform Infrared Spectroscopy (FTIR). The transmission spectra were obtained using a Bruker Optics Vertex 70 instrument with a KBr beamsplitter and DTGS detector. The IR beam was transmitted directly through the silicon wafer, which was polished on both sides. The background used was a spectrum obtained from a bare, cleaned silicon wafer. An aperture setting of 6 mm and a scanner velocity of 10 kHz were used. The Fourier transform parameters used are as follows: apodization function of BlackmanHarris 3-Term; phase resolution of 32 cm-1; phase correction mode of Mertz; and zero filling factor of 2. The acquisition parameters used were a mode of double-sided, forward-backward, and 254 scans at a resolution of 4 cm-1 with 512 background scans. Ellipsometry. Thickness measurements were made using a variable-angle Beaglehole Picometer Ellipsometer that utilizes a He-Ne laser light source (λ ) 632.8 nm). Measurements were conducted at integer values of the incident angle, ranging from 80 to 35° with a precision (0.1°. The thicknesses reported are averages of three ellipsometric measurements taken from selected areas near the center of a modified silicon wafer. The refractive indices, η, used for each layer are as follows: ηPGMA ) 1.525, ηdiamine) 1.5, ηp(VPco-VDMA) ) 1.5, and ηDC ) 1.5. After each step of the attachment procedure, the laterally averaged layer thickness was determined via ellipsometry. Spot-to-spot variations on any sample were found to be small (
