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Self-Supporting Hydrogel Stamps for the Microcontact Printing of Proteins Naı¨s Coq, Ties van Bommel, Rifat A. Hikmet, Hendrik R. Stapert, and Wendy U. Dittmer* Philips Research, High Tech Campus, EindhoVen, The Netherlands ReceiVed January 5, 2007. In Final Form: February 16, 2007 In this work we explore a new hydrogel stamp material obtained from polymerizing 2-hydroxyethyl acrylate and poly(ethylene glycol) diacrylate in the presence of water for the microcontact printing of proteins directly on gold substrates and by covalent coupling to self-assembled monolayers of alkanethiols. At high cross-link density, the hydrogel is rigid, hydrophilic, and with a high buffer holding capacity to enable the unsupported printing of protein patterns homogeneously and reproducibly, with micrometer-range precision. The stamps were used to print antibodies to human parathyroid hormone, which were shown using immunoassay tests to retain their biological function with binding capacities comparable to those of solution-adsorbed antibodies.
Introduction The high-resolution deposition of biomolecules onto a substrate is desirable for applications that require miniaturization, spatial control, and multiplexing such as in bioanalytical detection using microarrays and biosensors.1,2 Microcontact printing (µCP) is a potentially biocompatible method for bringing biomolecules directly onto a large surface with a resolution of several micrometers. In this work we present a new hydrophilic stamp material containing water for the high-resolution printing of functional biological molecules. In µCP a relief-patterned stamp inked with the molecular solution is placed into contact with the substrate that is to be printed. After seconds to minutes of contact, depending on the diffusion time, the ink is deposited onto the surface, forming a negative of the stamp relief pattern. Printing of molecular inks has been investigated mainly using polydimethylsiloxane (PDMS) stamps3 because of the elasticity of the material and ease of stamp production. However, PDMS is highly hydrophobic, and the use of polar inks such as proteins and nucleic acids is problematic due to poor wetting and the inhomogeneous drying of inks on the surface of the stamp. For the printing of hydrophilic molecules the stamp surface can be modified, such as with an oxygen plasma treatment or by grafting hydrophilic molecules onto the stamp.4-6 Oxidative treatments need to be precisely controlled as the amount of oxidized PDMS species produced on the surface is dependent on the duration of treatment. Moreover, hydrophobic recovery due to the diffusion of bulk PDMS to the surface requires that care be taken that the stamps be used soon after oxidative treatment or within several days when stored in water and compromises the long-term stability of grafted * Corresponding author. E-mail:
[email protected]. (1) Sauer, S.; Lange, B. M. H.; Gobom, J.; Nyarsik, L.; Seitz, H.; Lehrach, H. Nat. ReV. Genet. 2005, 6, 465-476. (2) Quist, A. P.; Pavlovic, E.; Oscarsson, S. Anal. Bioanal. Chem. 2005, 381, 591-600. (3) Graber, D. J.; Zieziulewicz, T. J.; Lawrence, D. A.; Shain, W.; Turner, J. N. Langmuir 2003, 19, 5431-5434. (4) Delamarche, E.; Geissler, M.; Bernard, A.; Wolf, H.; Michel, B.; Hilborn, J.; Donzel, C. AdV. Mater. 2001, 13, 1164 ff. (5) Delamarche, E.; Donzel, C.; Kamounah, F. S.; Wolf, H.; Geissler, M.; Stutz, R.; Schmidt-Winkel, P.; Michel, B.; Mathieu, H. J.; Schaumburg, K. Langmuir 2003, 19, 8749-8758. (6) Azzaroni, O.; Moya, S. E.; Brown, A. A.; Zheng, Z.; Donath, E.; Huck, W. T. S. AdV. Funct. Mater. 2006, 16, 1037-1042.
hydrophilic layers.7,8 In order to retain their functionality biological molecules such as proteins should be maintained in an aqueous environment under controlled pH and salt conditions. PDMS stamps do not provide this possibility, as polar molecules (e.g., water and salts) are not soluble in PDMS. In order to overcome these problems, stamp hydrogel materials have been developed. Standard polymer hydrogels such as agarose and polyacrylamide are commonly used in the electrophoresis and/ or purification of nucleic acids and proteins and can also be adapted to produce µCP stamps.9-12 Other hydrogels specifically developed for µCP include acrylic acids esterified to sugars.13,14 However, these standard and new hydrogels have the disadvantage that they are generally mechanically unstable and require a solid support or very thick stamps to function. The stamps produced from esterified acrylic acids are about 5% cross-linked, bound to a solid support, and enable printing of features of approximately 10 µm. Other hydrophilic stamp materials which have been investigated include poly(ethylene glycol)/urethane-related polymer composite and poly(ether ester).15,16 This report explores a rigid self-supporting hydrophilic hydrogel stamp material made by cross-linking at high density an acrylate monomer with poly(ethylene glycol) diacrylate in the presence of water or buffer that can be used to directly print proteins. We demonstrate in this work that our water-containing hydrogel stamps, in addition to being hydrophilic and enabling the high-precision printing of proteins, allow the inked proteins (7) Lawton, R. A.; Price, C. R.; Runge, A. F.; Doherty, W. J.; Saavedra, S. S. Colloids Surf., A 2005, 253, 213-215. (8) Efimenko, K.; Wallace, W. E.; Genzer, J. J. Colloid Interface Sci. 2002, 254, 306-315. (9) Mayer, M.; Yang, J.; Gitlin, I.; Gracias, D. H.; Whitesides, G. M. Proteomics 2004, 4, 2366-2376. (10) Stevens, M. M.; Mayer, M.; Anderson, D. G.; Weibel, D. B.; Whitesides, G. M.; Langer, R. Biomaterials 2005, 26, 7636-7641. (11) Weibel, D. B.; Lee, A.; Mayer, M.; Brady, S. F.; Bruzewicz, D.; Yang, J.; DiLuzio, W. R.; Clardy, J.; Whitesides, G. M. Langmuir 2005, 21, 64366442. (12) Burnham, M. R.; Turner, J. N.; Szarowski, D.; Martin, D. L. Biomaterials 2006, 27, 5883-5891. (13) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971-3975. (14) Martin, B. D.; Brandow, S. L.; Dressick, W. J.; Schull, T. L. Langmuir 2000, 16, 9944-9946. (15) Trimbach, D. C.; Al Hussein, M.; de Jeu, W. H.; Decre, M.; Broer, D. J.; Bastiaansen, C. W. M. Langmuir 2004, 20, 4738-4742. (16) Lee, N. Y.; Lim, J. R.; Lee, M. J.; Kim, J. B.; Jo, S. J.; Baik, H. K.; Kim, Y. S. Langmuir 2006, 22, 9018-9022.
10.1021/la0700321 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/22/2007
Hydrogel Stamps for Microcontact Protein Printing
to be kept in an aqueous environment, which permits them to preserve their biological activity after printing.
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Preparation of Stamps. A prepolymer solution comprising 2-hydroxyethyl acrylate (HEA) (Polysciences), poly(ethylene glycol) (400) diacrylate (PEGDA) (Kayarad) as the cross-linker, photoinitiator Darocure 1173 (Merck), and water was prepared. The optimized concentration is 72 wt % HEA, 18 wt % PEGDA, 10 wt % water, and 0.5 wt % Darocure. The prepolymer solution was spread by capillary action between a silicon master template and a glass slide (separated by glass spacers 0.7 mm thick) to the desired size and cured with UV exposure (1-5 min, 4 mW/cm2). Alternately, stamps were be made from a PDMS master, itself a replica of the original silicon master. Because of the O2 trapped in the PDMS, curing had to be completed in a N2 atmosphere and required longer exposure to UV light (∼10 min) to obtain a fully cured stamp. The resulting stamps were 704 µm thick (as the relief pattern from the wafer was 4 µm deep). They were stored in a high-humidity atmosphere and allowed to equilibrate for at least 24 h. Preparation of Substrates. The substrates used were 7 mm diameter silicon disks, sputtered with molybdenum (10 nm) and gold (80 nm) by physical vapor deposition. Before printing they were rinsed with 2-propanol and exposed to an oxygen plasma (3 min, 100 W, 0.15 mbar) to remove organic contamination. Mixed self-assembled monolayers (SAMs) of -COOH-functionalized alkanethiols (mole ratio, χ ) 0.01) on gold surfaces, treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS, 200 mM/50 mM, Sigma-Aldrich), were prepared according to a method previously described.17 Printing. Samples were printed with a solution of bovine serum albumin (BSA, Sigma-Aldrich) at a concentration of 0.1-10 mg/ mL or goat anti-human parathyroid hormone antibodies (anti-PTH, Future Diagnostics) at a concentration of 0.001-0.1 mg/mL in phosphate-buffered saline (PBS). The associated antigen is human parathyroid hormone (PTH). BSA was functionalized with fluorescein isothiocyanate (BSA-FITC, Sigma-Aldrich) for measurements with the fluorescence microscope. For printing, the polymer stamp, approximately 5 mm by 5 mm, was placed face up on a glass slide and a drop of protein solution (∼20 µL) was deposited on the stamp so as to cover the entire patterned surface for 2 min. The solution was removed, and the stamp was quickly dried under a N2 flow. To print, either the stamp was brought into contact with a clean gold substrate using slight pressure from a flat spatula to ensure contact, or for more precise printing, the substrate was brought down onto the stamp and a pressure was applied for 2 min by means of a 29.1 g weight. The weight, with the substrate sample attached via vacuum, was brought down onto the stamp with the relief pattern facing up from a height of several millimeters by means of an almost frictionless release mechanism. For better print homogeneity, especially over larger areas, it was found that the pressure-applying apparatus is more suitable. After printing, the sample was removed and the stamp was peeled off. Long contact times (>20 min) at the current weight used induce sagging and a deformation of the printed features as a consequence of diffusion. When stored in a water-saturated atmosphere, the stamp is stable for at least several months and can be used for repeated printing when care is taken to avoid mechanical stress on the thin stamps. Imaging of Printed Antibodies. FITC-functionalized rabbit anti-goat IgG antibodies (Sigma-Aldrich), which bind specifically to goat IgGs, were used to image the unlabeled printed anti-PTH antibodies under the microscope. The printed samples were first placed for 30 min in a blocking buffer containing 1% w/v BSA in PBS to prevent the anti-goat IgG from binding nonspecifically to the gold, which would otherwise create a high fluorescent background
in the nonprinted areas. The samples were then rinsed three times in PBS and incubated for 1 h, under slow agitation (150 rpm), with the labeled anti-goat IgG solution at a concentration of 0.83 ng/µL (in PBS, 0.1% w/v BSA and 0.05% w/v Tween 20). The samples were finally rinsed three times in a washing buffer containing 0.5% Tween 20 and PBS, to remove the remaining nonspecifically bound anti-goat IgG, and dried. Activity Tests and Immunoassays. Enzyme-linked immunosorbant assays (ELISAs) were conducted in order to determine the binding capacity of the printed antibody layer. All reagents and buffers unless otherwise stated were part of a kit supplied by Future Diagnostics. The printed anti-PTH samples were placed in microtiter plates, washed in PBS, and blocked for 1 h in blocking buffer. They were incubated for 2 h in PTH solution and biotinylated anti-PTH (secondary antibody), at a concentration of 1µg/mL under slow agitation (200 rpm). The samples were rinsed three times in washing buffer and left to incubate for 1 h in a solution of 1µg/mL HRPstreptavidin in 50% v/v Stabilzyme buffer (SurModics, Inc.). The streptavidin binds strongly to the biotin attached to the secondary antibodies. The remaining free HRP enzyme was removed by washing three times in washing buffer, and thereafter a solution of commercial chemiluminescence substrate for HRP was added (SuperSignal Pico ELISA Chemiluminescence Substrate, Pierce Biotechnology). The measurements were taken after 5 min of reaction with a FLUOstar OPTIMA plate reader (BMG Labotechnologies). Within one study, the same stamp, reinked in between printings, was used to print samples for all the data points. Error bars represent samples made in duplicate. The variation in relative light units (RLUs) between studies is a consequence of the differing printed areas for different stamps. From-solution immobilized antibody molecules were adsorbed from solution onto Au overnight or onto SAMs (activated for 7 min with EDC/NHS and then rinsed in water) for 30 min. After incubation, the samples were washed three times in wash buffer. For assays comparing the printed with solution-adsorbed antibodies, the RLU signals were normalized with the area printed. Fluorescence Microscopy, AFM, and XPS. Fluorescence images were obtained with a DMLM Leica fluorescence microscope with a Photometric Coolsnap HQ CCD camera, ultrahigh-pressure mercury lamp, and Leica filter cube L5. Atomic force microscopy (AFM) was completed with a Veeco Dimension 3100 in tapping mode at a scan rate of 0.3 Hz with a silicon nitride tip, NSC16/50 (MikroMasch). X-ray photoelectron spectroscopy (XPS) measurements were carried out in a Quantera from Phi (Q1) using monochromatic Al KR radiation with a measuring spot of 100 µm. During the measurements the angle between the axis of the analyzer and the sample surface was 90°. The spot was rastered across an area of 1200 µm × 500 µm in order to reduce the effects of radiation damage. For the same reason, the measurements have been carried out with all neutralizing beams (electron flood gun and slow ions) switched off. The thickness of the biological layer above the Au substrate was calculated using the layer model described in the cited reference.18 Contact Angle Measurements, Rheometry, and Water Holding Capacity. Static water contact angle measurements were performed on an OCA30 model from DataPhysics. Each measurement is based on an average of three points taken at different positions on the sample. The Young’s moduli of the stamps were determined with a Physica MCR 300 rheometer at 25 °C under a water-saturated atmosphere. The normal force applied was ramped from 0-5 N at a rate of 0.1 N/5 s, using an 8 mm plate. To determine the actual amount of water contained in the stamps after polymerization, the material was dried overnight in a desiccator under vacuum and the percentage weight change was calculated. The water/buffer holding capacity was calculated from the ratio ([Mw - Md]/Md) of the weights of the stamps before (Md) and after soaking for 1 h in solution (Mw).
(17) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790.
(18) van der Marel, C.; Yidirim, M.; Stapert, H. R. J. Vac. Sci. Technol., A 2005, 23, 1456-1470.
Experimental Methods
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Figure 1. (a) Left: Young’s modulus (E) of hydrogel stamps with HEA and 18 wt % PEGDA for various concentrations of water in the prepolymer solution. Right: Young’s modulus of hydrogel stamps with HEA and 10 wt % water for various concentrations of PEGDA. (b) The actual amount of water present in the stamp after curing as a function of the prepolymer water concentration for hydrogel stamps containing 18 wt % PEGDA and HEA. (c) The weight of water or PBS that can be adsorbed by the cured stamp as a function of the prepolymer water concentration for hydrogel stamps containing 18 wt % PEGDA and HEA.
Results and Discussion Hydrogel Stamp Properties. In order to achieve a mechanically robust hydrogel that is self-supporting, the PEGDA concentration was increased above 10 wt %, which corresponds to 10% cross-link density. By further varying the PEGDA and water content of the prepolymer solution, stamps of different stiffness were obtained (Figure 1a). Stamps with high PEGDA and moderate water (
