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Langmuir 2006, 22, 3844-3850
Dendritic Structures of Poly(Ethylene Glycol) on Silicon Nitride and Gold Surfaces Zhiyong Suo,† Fernando Tera´n Arce,† Recep Avci,*,† Kate Thieltges,† and Brenda Spangler‡ Image and Chemical Analysis Laboratory, Physics Department, Montana State UniVersity, Bozeman, Montana 59717, and Sensopath Technologies, Inc., Bozeman, Montana 59717 ReceiVed December 14, 2005. In Final Form: February 7, 2006 A hydrophilic silicon nitride surface was grafted with poly(ethylene glycol) monomethyl ether (average formula weight of 5000 Da) in a one-step protocol. The domains of stable dendritic structures of self-assembled monolayer islands on a silicon nitride surface were observed with atomic force microscopy. The moduli of elasticity of these dendritic structures in air and in KCl aqueous solution were compared. The value of the Young’s modulus of these structures is reduced by more than 3 orders of magnitude, from ∼12 GPa measured in air to ∼5 MPa in KCl solution. This dramatic reduction in elasticity was attributed to the swelling of the dendritic structures in aqueous solution, which was verified by the increased film thickness. These dendritic structures were not stable in the aqueous environment and could be removed by soaking in water for 22 h because of the hydrolysis of the silicate bonds. This fact was confirmed by the reduction of the C1s signal in the X-ray photoelectron spectroscopy experiments. These morphologies are not unique to silicon nitride substrate; similar features were also observed for thiolated poly(ethylene glycol) monomethyl ether molecules absorbed on a gold surface.
Introduction Oligo(ethylene glycol) (OEG) and poly(ethylene glycol) (PEG) have been widely used to prevent nonspecific protein and cell absorption on various surfaces.1-5 By modification with active functional groups (silane, thiol, etc.) or in conjugation with other biopolymers (poly-L-lysine, peptide, etc.), OEG/PEG can be grafted on various substrate surfaces including mica,6,7 silicon,8-11 silicon nitride,12,13 metals,5,14-17 and polymers.18 Successful * To whom correspondence should be addressed. E-mail: avci@ physics.montana.edu. Tel: 406-994-6164. Fax: 406-994-6040. † Montana State University. ‡ Sensopath Technologies, Inc. (1) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (2) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336-6343. (3) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605-5620. (4) Andrade, J. D.; Hlady, V.; Jeon, S. I. Hydrophilic Polym. 1996, 248, 5159. (5) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436. (6) Kiridena, W.; Jain, V.; Kuo, P. K.; Liu, G. Y. Surf. Interface Anal. 1997, 25, 383-389. (7) Raviv, U.; Frey, J.; Sak, R.; Laurat, P.; Tadmor, R.; Klein, J. Langmuir 2002, 18, 7482-7495. (8) Andruzzi, L.; Senaratne, W.; Hexemer, A.; Sheets, E. D.; Ilic, B.; Kramer, E. J.; Baird, B.; Ober, C. K. Langmuir 2005, 21, 2495-2504. (9) Sharma, S.; Johnson, R. W.; Desai, T. A. Biosens. Bioelectron. 2004, 20, 227-239. (10) Yam, C. M.; Lopez-Romero, J. M.; Gu, J. H.; Cai, C. Z. Chem. Commun. 2004, 2510-2511. (11) Zhu, X. Y.; Jun, Y.; Staarup, D. R.; Major, R. C.; Danielson, S.; Boiadjiev, V.; Gladfelter, W. L.; Bunker, B. C.; Guo, A. Langmuir 2001, 17, 7798-7803. (12) Colic, M.; Franks, G.; Fisher, M.; Lange, F. J. Am. Ceram. Soc. 1998, 81, 2157-2163. (13) Riener, C. K.; Stroh, C. M.; Ebner, A.; Klampfl, C.; Gall, A. A.; Romanin, C.; Lyubchenko, Y. L.; Hinterdorfer, P.; Gruber, H. J. Anal. Chim. Acta 2003, 479, 59-75. (14) Dalsin, J. L.; Lin, L. J.; Tosatti, S.; Voros, J.; Textor, M.; Messersmith, P. B. Langmuir 2005, 21, 640-646. (15) Tosatti, S.; De Paul, S. M.; Askendal, A.; VandeVondele, S.; Hubbell, J. A.; Tengvall, P.; Textor, M. Biomaterials 2003, 24, 4949-4958. (16) Huang, N. P.; Csucs, G.; Emoto, K.; Nagasaki, Y.; Kataoka, K.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 252-258. (17) Huang, N. P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17, 489-498. (18) Qiu, Y. X.; Klee, D.; Pluster, W.; Severich, B.; Hocker, H. J. Appl. Polym. Sci. 1996, 61, 2373-2382.
applications of PEG have also been reported in the fabrication of biocompatible materials to inhibit the biofouling process19-21 and to reduce undesired immune responses in the transplantation of organs22 and artificial devices23,24 in animals. Our motivation for this work largely stems from the need to prevent nonspecific interactions between bioactive molecules and the silicon nitride tips used in force microscopy measurements. To map the antibody-antigen interactions across a surface region of interest with atomic force microscopy (AFM), typically a tip modified with antibodies via a flexible linker is employed to probe antigen molecules present on cell surfaces or artificially immobilized on an abiotic substrate.13,25,26 During such measurements, if proper caution is not taken, the measured forces can be dominated by nonspecific tip-substrate interactions. It is desirable to inhibit such nonspecific interactions by modifying the tip surface with OEG/PEG molecules and allow the desired antibody-antigen interactions to take place during bioforce measurements when the tip is in contact with the measured surface. It has been reported that the hydroxyl groups of aliphatic alcohols can react with the silanol groups on silicon or silicon nitride surfaces and can survive for weeks in aqueous solution,27 which inspired us to avoid the time-consuming chemical modification and graft the commercially available PEG molecules directly onto silicon nitride surfaces. AFM has been used to measure the elastic properties of a monolayer or multilayer of organic molecules6,28,29, proteins,30,31 (19) Lee, Z. W.; Lee, K. B.; Hong, J. H.; Kim, J. H.; Cheong, C.; Choi, I. S. Bull. Korean Chem. Soc. 2005, 26, 1166-1167. (20) Sharma, S.; Desai, T. A. J. Nanosci. Nanotechnol. 2005, 5, 235-243. (21) Popat, K. C.; Mor, G.; Grimes, C. A.; Desai, T. A. Langmuir 2004, 20, 8035-8041. (22) Eugene, M. Cell. Mol. Biol. 2004, 50, 209-215. (23) Chang, S.; Lee, C.; Hsu, C.; Wang, Y. J. Biomed. Mater. Res. 2002, 59, 118-126. (24) Novikova, L.; Novikov, L.; Kellerth, J. Curr. Opin. Neurol. 2003, 16, 711-715. (25) Dupres, V.; Menozzi, F. D.; Locht, C.; Clare, B. H.; Abbott, N. L.; Cuenot, S.; Bompard, C.; Raze, D.; Dufrene, Y. F. Nat. Methodol. 2005, 2, 515-520. (26) Grandbois, M.; Dettmann, W.; Benoit, M.; Gaub, H. E. J. Histochem. Cytochem. 2000, 48, 719-724. (27) Trau, M.; Murray, B. S.; Grant, K.; Grieser, F. J. Colloid Interface Sci. 1992, 148, 182-189.
10.1021/la053389i CCC: $33.50 © 2006 American Chemical Society Published on Web 03/18/2006
Dendritic Structures on Silicon Nitride and Gold
or cells32 immobilized on a substrate surface. Compared to the intensive use of OEG/PEG to passivate surfaces against nonspecific protein absorption and to the study of the mechanical properties of alkane monolayers, very little has been reported on the measurement of the mechanical properties of OEG/PEG monolayers. We found that the self-assembly of PEG molecules on a silicon nitride surface could not form a complete continuous monolayer; instead it generated dendritic structures covering only part of the total surface area. In this paper, we present our results on the self-assembled dendritic structures of PEG molecules on silicon nitride and gold surfaces. The elasticity of these PEG dendritic structures was determined both in air and in KCl aqueous solution by means of nanoindentation and fitting to the Hertzian model. Experimental Section Materials. Hydroxyl-terminated poly(ethylene glycol) monomethyl ether (average formula weight of 5000 Da, PEG-5000-OMe) was purchased from Polysciences, Inc. (Warrington, PA) and used as received. Tetra(ethylene glycol) (OEG-4) was bought from Aldrich (Milwaukee, WI). Thiolated poly(ethylene glycol) monomethyl ether (HS-PEG-5000-OMe) was supplied by Sensopath Technologies, Inc. (Bozeman, MT). Silicon wafers with 2000 Å silicon nitride film deposited on both sides were purchased from University Wafer (South Boston, MA). Potassium chloride was bought from Sigma (St. Louis, MO). All the water used was purified by Milli-Q Ultrapure water purification systems (Billerica, MA). Preparation of Dendritic Structures on a Silicon Nitride Surface. A silicon nitride wafer was cut into 8 × 8 mm2 chips, which were then rinsed with CHCl3 and soaked in piranha solution (concentrated H2SO4/30% H2O2 ) 9:1, v/v) at room temperature for 30 min, rinsed with water, and dried with nitrogen gas. Cleaned silicon nitride chips were heated by immersing them in pure PEG at 100 °C (no solvent was used: OEG-4 is liquid at room temperature and PEG-5000-OMe melts at 100 °C) under nitrogen atmosphere for 40 h. These samples were then extensively rinsed with deionized water until no visible marks were left on the surface; the samples were further cleaned by sonication for 5 min in a water bath. The surfaces were dried using dry nitrogen gas and were stored under ambient conditions. Preparation of Dendritic Structures on a Gold Surface. The silicon wafers covered with 100-nm gold film were rinsed with chloroform and cleaned in an O3 chamber for 30 min, rinsed with 100% ethanol and then with water, and dried with nitrogen gas. The cleaned gold surface was then incubated in a solution of HS-PEG5000-OMe in water (1 mM) for 17 h, rinsed extensively with water, dried with nitrogen gas, and stored in ambient conditions. X-ray Photoelectron Spectroscopy. The analysis was conducted on a Physical Electronics 5600ci XPS system equipped with monochromatized Al KR X-rays. The analysis area of the sample was ∼0.8 mm in diameter. Electron emissions were collected at 45° to the normal of the surface, and the spherical-sector-analyzer pass energy was selected as 11.75 eV for high-resolution scanning and as 46.95 eV for a survey to achieve optimum energy resolution and count rate. The data acquisition and data analysis were performed using RBD AugerScan 2 software. Atomic Force Microscopy. All measurements were carried out with a Nanoscope IIIa Extended Multimode atomic force microscope from Veeco (Santa Barbara, CA) with a 125 × 125 µm2 scanner (J-type). Imaging in air was performed in tapping mode to reduce tip-sample interactions by decreasing lateral forces with Olympus (28) Burns, A. R.; Houston, J. E.; Carpick, R. W.; Michalske, T. A. Langmuir 1999, 15, 2922-2930. (29) Liu, H.; Bhushan, B. Ultramicroscopy 2002, 91, 185-202. (30) Uricanu, V. I.; Duits, M. H. G.; Mellema, J. Langmuir 2004, 20, 50795090. (31) Suda, H.; Sasaki, Y. C.; Oishi, N.; Hiraoka, N.; Sutoh, K. Biochem. Biophys. Res. Commun. 1999, 261, 276-282. (32) Bowen, W. R.; Lovitt, R. W.; Wright, C. J. Biotechnol. Lett. 2000, 22, 893-903.
Langmuir, Vol. 22, No. 8, 2006 3845 tapping mode etched silicon probes (OTESPA7 type, purchased from Veeco). Low drive voltages were applied to the cantilever (3-15 mV), resulting in ∼5-30 nm free oscillation amplitudes (Ao). Setpoint values were chosen so that the working amplitude (Aset) to free amplitude ratios (Aset/Ao) were in the range of 0.7-0.9. The cantilevers of the silicon probes were specified to have a nominal spring constant of 42 N/m, a nominal tip radius of ∼10 nm and a typical resonance frequency of ∼300 kHz. Silicon nitride probes (MLCT-AUHW model, purchased from Veeco) were used for imaging and mechanical measurements in KCl aqueous solution. Each chip had five triangular probes and one rectangular probe, with a ∼60-nm-thick gold coating on the back of each probe to enhance reflectivity. Only cantilevers with nominal spring constants of 0.01 and 0.5 N/m were used. For each cantilever used to measure the elasticity of the dendritic structures, the spring constant was determined individually using the standard calibrated cantilevers bought from Vecco (CLFC-NOBO type, Santa Barbara, CA). The mechanical properties of the PEG monolayers were measured by acquiring either 256 or 1024 pairs of force vs displacement curves from a 10 × 10 or 5 × 5 µm2 area by subdividing the area into 16 × 16 or 32 × 32 equal-sized smaller areas (pixels) and acquiring a pair of force vs displacement curves from the center of each pixel. A pair of force vs displacement curves includes a loading and an unloading curve. The tip velocities for these measurements varied between 0.3 and 0.5 µm/s. Indentation curves were obtained from force vs displacement curves, as described elsewhere.33,34 Briefly, the deformation of the sample of interest (penetration depth) is obtained by subtracting the cantilever deflection from the displacement of the piezo. This value corresponds to the x axis of the indentation curve. Forces are calculated by multiplying the measured elastic constant of the cantilever by the cantilever deflection. A MatLab (MathWorks, Natick, MA) code was written to obtain the elastic modulus by fitting each of the force vs displacement curves in a force-volume file to the Hertzian model.35-37 Only the repulsive region of the loading curves was fitted. The origin of the indentation curves was determined from the intersection of the unloading curve with the horizontal line crossing the zero of the force axis. The Hertzian model is based on the premise that the contact is between a spherical tip and a flat surface; however, this premise is not always ideally met, in general because of imperfections of the tip geometry. For this reason, we experimentally determined the radius of the AFM tip for each set of experiments as described in the next paragraph. Furthermore, the sensitivity of the photodetector was calibrated using the linear part of the deflection of the cantilever, where the indentation of the surface is negligible. This can be achieved by using a hard surface. Fitting the indentation data to the Hertzian model in some cases does not give very good agreement, but the resultant Young’s modulus (E) still remains close to the values obtained by other techniques, to within ∼50% accuracy.38 The AFM tip radius was determined by imaging spherical gold colloidal particles with an average diameter of 30 nm (Ted Pella, Redding, CA) with the same tip after the elasticity measurement.39 The tip radius was estimated by modeling the AFM profiles of the particles as a sphere (AFM tip) in contact with another sphere (gold particle). The height of the profile gave the diameter of the gold particle. The tip radius was determined at the end of each forcevolume measurement, and this value was used to calculate the elastic modulus of the polymer monolayer. (33) Hues, S. M.; Draper, C. F. In Procedures in SPMs; Colton, E., Frommer, G., Guckenberger, H., Parkinson, R., Ed.; Wiley and Sons: Chichester, UK, 1998; Chapter 9.3. (34) Arce, P. F. M. T.; Riera, G. A.; Gorostiza, P.; Sanz, F. Appl. Phys. Lett. 2000, 77, 839-841. (35) Johnson, K. L. Contact mechanics; Cambridge University Press: Cambridge, U.K., 1985. (36) Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc. R. Soc. London, Ser. A 1971, 324, 301. (37) Pharr, G. M.; Oliver, W. C.; Brotzen, F. R. J. Mater. Res. 1992, 7, 613617. (38) Arce, F. T.; Avci, R.; Beech, I. B.; Cooksey, K. E.; Wigglesworth-Cooksey, B. J. Chem. Phys. 2003, 119, 1671-1682. (39) Vesenka, J.; Manne, S.; Giberson, R.; Marsh, T.; Henderson, E. Biophys. J. 1993, 65, 992-997.
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Figure 1. Grafting of hydroxyl-terminated PEG-5000-OMe on a hydrophilic silicon nitride surface.
Results and Discussion Formation of Dendritic Structures on Silicon Nitride. The hydrophilic silicon nitride surface has both amino and silanol groups, and the hydroxyl terminal groups of the PEG polymers react with the silanol groups to form a new silicate bond by releasing one water molecule (Figure 1).13,27,40 Because water is the byproduct, no solvent was used during the reaction to maintain a high concentration of the PEG polymers. Although PEG-5000OMe is solid at room temperature, it melts into a clear thick liquid at 100 °C, which in principle ensures an even and complete coverage of the chip surface. After the reaction, the silicon nitride chips were taken out, rinsed with water, and cleaned in a sonication bath containing water to remove the excess PEG-5000-OMe from the surface. XPS spectra clearly show the difference between the PEGgrafted sample and a control sample (clean silicon nitride chip without PEG). Both the C1s and the O1s signals show considerable increase for the sample with the grafted PEG-5000-OMe, while the N1s, Si2s, and Si2p signals are larger for the control sample (Figure 2a). Using the Si2p peak as the standard for assigning peak positions, the high-resolution scan of the C1s signal of the PEG grafted on the silicon nitride surface shows two components: a major peak centered at 286.5 eV, corresponding to the C-O signal of PEG molecules,41 and a minor peak at 285 eV, corresponding to the environmental hydrocarbon contamination of the sample in air (Figure 2b). For the control sample, only a peak at 285 eV was observed. These results suggest that the silicon nitride surface is covered by carbon- and oxygen-rich PEG molecules. However, because XPS can only probe the top layers of the sample (
