Langmuir 2004, 20, 8035-8041
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Surface Modification of Nanoporous Alumina Surfaces with Poly(ethylene glycol) Ketul C. Popat,† Gopal Mor,‡ Craig A. Grimes,‡ and Tejal A. Desai*,† Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, and Department of Electrical Engineering, Pennsylvania State University, State College, Pennsylvania 16802 Received April 12, 2004. In Final Form: June 30, 2004 Nanoporous alumina surfaces have a variety of applications in biosensors, biofiltration, and targeted drug delivery. However, the fabrication route to create these nanopores in alumina results in surface defects in the crystal lattice. This results in inherent charge on the porous surface causing biofouling, that is, nonspecific adsorption of biomolecules. Poly(ethylene glycol) (PEG) is known to form biocompatible nonfouling films on silicon surfaces. However, its application to alumina surfaces is very limited and has not been well investigated. In this study, we have covalently attached PEG to nanoporous alumina surfaces to improve their nonfouling properties. A PEG-silane coupling technique was used to modify the surface. Different concentrations of PEG for different immobilization times were used to form PEG films of various grafting densities. X-ray photoelectron spectroscopy (XPS) was used to verify the presence of PEG moieties on the alumina surface. High-resolution C1s spectra show that with an increase in concentration and immobilization time, the grafting density of PEG also increases. Further, a standard overlayer model was used to calculate the thickness of PEG films formed using the XPS intensities of the Al2p peaks. The films formed by this technique are less than 2.5 nm thick, suggesting that such films will not clog the pores which are in the range of 70-80 nm.
Introduction Nanoporous alumina surfaces have been widely used for sensor applications such as hydrogen sensing for industrial applications,1 ethylene sensing for environmental applications,2 wastewater sensing,3 humidity sensing for the food industry,4 ammonia sensing,5 glucose sensing for medical applications, and pH sensing.6 Refined anodization techniques have enabled the production of membranes with unique well-defined nanoarchitecture. This fabrication route allows for creation of nanopores with size scales from 5 nm to 10 µm with controlled topology depending on the voltage used for anodization. Hence, these nanoporous alumina films have potential applications in the field of biology due to close proximity in size scales to biological milieu. Our aim is to use these nanoporous alumina surfaces as membranes for targeted drug delivery applications. Of particular interest is the development and characterization of well-controlled, stable, and uniform surfaces capable of diffusion of biomolecules such as IgG and lysozyme and the blocking of antibodies and complement molecules from encapsulated cells. The majority of the membranes currently used for this applications are * Corresponding author. Mail: Department of Biomedical Engineering, 44 Cummington Street, Boston, MA 02215. Tel: 617358-2805. Fax: 617-358-2835. E-mail:
[email protected]. † Boston University. ‡ Pennsylvania State University. (1) Grimes, C. A.; Ong, K. G.; Varghese, O. K.; Yang, X.; Mor, G.; Paulose, M.; Ruan, C.; Dickey, E. C.; Pishko, M. V.; Kendig, J. W.; Mason, A. J. Sensors 2003, 3, 69. (2) Zhang, R.;Tejedor, M. I.; Anderson, M. A.; Paulose, M.; Grimes, C. A. Sensors 2002, 2, 331. (3) Yang, X.; Ong, K. G.; Dreschel, W. R.; Zeng, K.; Mungle, C. S.; Grimes, C. A. Sensors 2002, 2, 455. (4) Varghese, O. K.; Gong, D.; Paulose, M.; Ong, K. G.; Grimes, C. A.; Dickey, E. C. J. Mater. Res. 2002, 17, 1162. (5) Dickey, E. C.; Varghese, O. K.; Ong, K. G.; Poulose, M.; Grimes, C. A. Sensors 2002, 2, 91. (6) Cai, Q. Y.;Grimes, C. A. Sens. Actuators, B 2002, 79, 144.
polymeric and, therefore, have relatively broad pore size distribution. Silicon membranes have been used for this application. They provide very well defined pore size distribution but have several problems associated with it such as fabrication cost, strength of material, and longterm stability. Nanoporous alumina membranes represent an alternative to silicon and polymeric membranes since they are easy to fabricate, chemically and thermally stable, and inert and can be produced with wide variety of pore size distribution. The surface of nanoporous alumina is inherently charged due to the equilibration of charged crystalline lattice defects within the surface.7 Depending on the net concentration of lattice defects, the surface may be positively or negatively charged with an attendant redistribution of oppositely charged lattice and electronic defects in the near surface region (known as the “space charge” region). This accumulation of charged defects at the surface and space charge region, which extends over several nanometers, leads to a net dipole moment. Thus, when biological material comes in contact with the surface, it may adhere, leading to pore clogging and subsequently membrane fouling. Hence, it is extremely important to modify the surface with a neutral material which will resist fouling and is inert and biocompatible before using the surface for further applications. Poly(ethylene glycol) (PEG) has been widely used to form nonfouling thin films on silicon surfaces in their solution form. Modification of silicon-based surfaces with PEG can be effected by either physical adsorption8-13 or (7) Frenkle, J. Kinetic Theory of Liquids; Oxford University Press: New York, 1946. (8) Amiji, M.; Park, K. Biomaterials 1992, 13, 682. (9) Amiji, M.; Park, H.; Park, K. J. Biomater. Sci., Polym. Ed. 1992, 3, 375. (10) Gingell, D.; Owens, N.; Hodge, P.; Nicholas, C. V.; O’Dell, R. J. Biomed. Mater. Res. 1994, 28, 505. (11) Grainger, D.; Nojiri, C.; Okano, T.; Kim, S. J. Biomed. Mater. Res. 1989, 23, 979.
10.1021/la049075x CCC: $27.50 © 2004 American Chemical Society Published on Web 08/10/2004
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covalent immobilization such as grafting and chemical coupling14-22 to create protein-resistant surfaces. It is relatively a simple molecule and has the following structure that is characterized by hydroxyl groups at either end of the molecule:
HO-(CH2CH2O)nCH2CH2-OH It is a linear or branched, neutral polyether available in a variety of molecular weights and is soluble in water and most organic solvents. Also, it behaves uniquely in an aqueous environment. The PEG chains are constantly in motion in an aqueous environment. It functions as a molecular windshield wiper, inhibiting any biomolecules that approach the surface, hence preventing biofouling.23-25 However, these films have not been applied to an alumina surface. Thus, in this work, we have covalently coupled PEG to the surface of nanoporous alumina to improve its nonfouling properties. The PEG-grafted alumina surfaces were characterized using X-ray photoelectron spectroscopy (XPS) to determine the chemical composition of the surface. Various concentrations of PEG and immobilization times were used. Further protein adsorption on unmodified and PEG-modified surfaces was also investigated. Finally, some theoretical calculations were made to determine the physical properties of PEG films on the surface. Experimental Section Reagents. Poly(ethylene glycol) (molecular weight ) 1000), fibrinogen (74%, 84% clottable), hydrogen peroxide, silicon tetrachloride, triethylamine, toluene, acetone, oxalic acid, chromic acid, phosphoric acid, hydrochloric acid, and copper chloride were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Aluminum sheets (99.9% purity, 0.5 mm thick) and 99.9% purity platinum foil were purchased from Alfa Aesar (Ward Hill, MA). Membrane Fabrication. Flat nanoporous alumina membranes were made from an aluminum sheet using a two-step anodization process. Aluminum sheeting was cut into pieces of size 1.5 cm × 1.0 cm. The pieces were subsequently cleaned by sonicating in acetone and deionized water and were dried using nitrogen. A thin layer of polymer was then spin coated on the cleaned aluminum pieces. The polymer used in this work was fingernail polish. The first anodization step was performed in 0.3 M oxalic acid for 6 h at 60 V using platinum as the cathode and polymercovered aluminum pieces as the anode. The pieces were then etched in a 4% (w/w) chromic acid and 8% (v/v) phosphoric acid mixture to remove the alumina layer that had formed on the unprotected side of the sheet. This anodization/removal step leaves a uniform concave nanoarray that is crucial for achieving the narrow pore size distribution during the subsequent anodization step. With one side still protected by the polymer film, a second anodization was then conducted on the other side of the (12) Strasser, C. M.; Dejardin, P.; Galin, J. C.; Schmitt, A. J. Biomed. Mater. Res. 1989, 23, 1385. (13) Amiji, M.; Park, K. J. Colloid Interface Sci. 1993, 155, 251. (14) Emoto, K.; Harris, J. M.; VanAlstine, J. M. Anal. Chem. 1996, 68, 3751. (15) Kamath, K. R.; Danilich, M. J.; Marchant, R. E.; Park, K. J. Biomater. Sci., Polym. Ed. 1996, 7, 977. (16) Popat, K. C.; Sharma, S.; Desai, T. A. Langmuir 2002, 18, 8728. (17) Popat, K. C.; Johnson, R. W.; Desai, T. A. J. Vac. Sci. Technol., B 2003, 21, 645. (18) Sharma, S.; Johnson, R. W.; Desai, T. A. Appl. Surf. Sci. 2003, 206, 218. (19) Stark, M. B.; Holmberg, K. Biotechnol. Bioeng. 1989, 34, 942. (20) Tseng, Y. C.; Park, K. J. Biomed. Mater. Res. 1992, 26, 373. (21) Yin-Chao, T.; McPherson, T.; Yuan, C.; Park, K. Biomaterials 1995, 16, 963. (22) Lee, S.; Laibinis, P. E. Biomaterials 1998, 19, 1669. (23) Andrade, J. D.; Haldy, V. Adv. Polym. Sci. 1986, 73, 1. (24) Jeon, S. I.; Lee, L. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (25) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159.
Figure 1. Scanning electron micrograph of a nanoporous alumina membrane fabricated with 60 V anodization voltage resulting in pores sizes of 72 nm. sheet. This anodization was performed at the same voltage (60 V) as used in the first step. This results in a nanoporous layer of alumina which serves as the final membrane with a defined pore size architecture. The duration of the anodizing period controls the membrane thickness. Typically, the duration of the second anodization is 6 h. A window area of size 0.6 cm × 0.6 cm in the polymer film protecting one of the surfaces was then carefully removed using acetone and a cotton swab. The washed away polymer should not flow to the other side and block the alumina pores. To further clean the portion in the window where an unwanted thin layer of alumina formed underneath the polymer layer during the second anodization, a drop of 10% NaOH solution was applied for 15 min to completely remove it. The whole piece was then thoroughly rinsed in deionized water. Finally, an aluminum window with the edge still protected by polymer was achieved. Then the pieces were etched in a 10% (w/w) HCl and 0.1 M CuCl2 solution to remove the unprotected aluminum in the window, leaving a transparent alumina membrane in this area. The resulting alumina membranes had a pore size ranging from about 70 to 80 nm. Figure 1 shows a typical scanning electron microscope image of the membrane with a pore size of 72 nm, fabricated using 60 V anodization voltage. To remove the barrier layer existing on the aluminum etched window side, the pieces were further etched in 10% (v/v) phosphoric acid solution for 11/2 h at room temperature. Depressions formed in the alumina window help to hold this etchant solution. The protecting polymer layer on the edges was then removed using acetone. This procedure results in flat nanoporous alumina membranes supported in an aluminum frame. Figure 2 shows the detailed fabrication procedure for the membrane. Cleaning/Hydroxylating Nanoporous Alumina Surfaces. The surfaces were first boiled in 30% hydrogen peroxide for 15 min to introduce -OH groups on the surface for further modification. They were then boiled in deionized water for 15 min to clean the surface. They were then air-dried and stored in argon until further surface modification. Covalent Coupling of Poly(ethylene glycol) on Nanoporous Alumina Surfaces. PEG immobilization on the alumina surface was achieved by modifying a covalent coupling technique described by Sharma et al. for silicon surfaces.18 This technique forms more stable films compared to physical adsorption on the surface. In this technique, a PEG-silane couple is formed by reacting PEG with silicon tetrachloride in the presence of triethylamine as a catalyst. The reaction results in the formation of PEG-OSiCl3 which then reacts with the trace level -OH groups on the surface to form a network of Si-O-Si bonds resulting in the immobilization of PEG on the surface (Figure 3).26-28 The PEG-silane couple formation and its immobilization on the surface were performed in anhydrous conditions to prevent hydrolysis and undesired side reactions. In brief, PEG (molecular weight ) 1000) was dissolved in anhydrous toluene to form a PEG solution with various concentrations of 5, 10, 20, 40, and
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Figure 2. Schematic of the fabrication of a nanoporous alumina membrane by the two-step anodization process.
Figure 3. Reaction mechanism of PEG immobilization on alumina surfaces using the covalent coupling technique. 80 mM. Then, 0.4875, 0.975, 1.95, 3.9, and 7.8 mmol of triethylamine was added drop by drop to the PEG solutions of the above concentrations, respectively, at 25°C. The reaction mixture was gently shaken for 1 h. After this, 0.0875, 0.175, 0.35, 0.5, and 1.4 mmol of silicon tetrachloride was added, respectively, and the reaction mixture was further shaken for 15 min at room temperature. The reaction mixture was then filtered through a sintered glass funnel, and the filtrate was used directly to immobilize the surfaces without further purification since an excess of unreacted PEG is not expected to have harmful effects on the silanization process. To study the effect of PEG concentration (10, 20, 40, and 80 mM), immobilization of the PEGsilane couple with alumina surfaces was carried out for 1 h. Similarly, to study the effect of immobilization time (1, 2, 4, and 8 h), 5 mM concentration was used. After coupling, the surfaces were rinsed thoroughly with anhydrous toluene, acetone, and deionized water and were air-dried and stored in argon until further use. The modified surfaces were then characterized using XPS. Protein Adsorption. To investigate the nonfouling nature of PEG-modified alumina surfaces, interaction of proteins on them was investigated. Unmodified and PEG-modified alumina surfaces were transferred into wells of standard 6-well plates. A solution of 2 mL of fibrinogen (1 mg/mL) in phosphate-buffered saline (PBS) (pH ) 7.4) was added to each well. Adsorption was (26) Ulman, A. An introduction to ultrathin organic films; Academic Press: New York, 1991. (27) Wasserman, S. R.; Tao, Y.; Whitesides, G. M. Langmuir 1989, 5, 1074. (28) Vasant, E. F.; Voort, P. V. D.; Vraneken, K. C. Characterization and chemical modification of silica surfaces: Studies in Surface Science and Catalysis, Vol. 93; Elsevier: Amsterdam, 1995.
allowed to proceed in an incubator (0.5% CO2) for 1 h at 37 °C. Upon completion of adsorption, the surfaces were thoroughly washed with deionized water for removal of nonadsorbed proteins and salts from the buffer. The surfaces were then air-dried, and their composition was determined using XPS. X-ray Photoelectron Spectroscopy. To determine the surface composition of PEG-modified surfaces and protein adsorbed on unmodified and PEG-modified surfaces, XPS analysis was carried out. The surfaces were mounted on an XPS stage. Three spots per sample were analyzed. The analysis was conducted on a Kratos AXIS Ultra Imaging X-ray photoelectron spectrometer with a monochromatic Al KR X-ray small spot source (1486.6 eV) and a multichannel detector. The Kratos XPS samples had a surface area of about 400 µm by 700 µm. A concentric hemispherical analyzer (CHA) was operated in the constant analyzer transmission mode to measure the binding energies of emitted photoelectrons. The binding energy scale was calibrated by the Au4f7/2 peak at 83.9 eV, and the linearity was verified by the Cu3p1/2 and Cu2p3/2 peaks at 76.5 and 932.5 eV, respectively. Survey spectra were collected from 0 to 1100 eV with a pass energy of 160 eV, and high-resolution spectra were collected for C1s, N1s, and Al2p peaks detected with a pass energy of 10 eV. All spectra were referenced by setting the hydrocarbon C1s peak to 285.0 eV to compensate for residual charging effects. Data for percent elemental composition, elemental ratios, and peak fit analysis parameters were calculated using software supplied by Kratos with the XPS. Film Thickness from XPS Data. Film thickness can be determined from the attenuation of XPS signals from the alumina
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substrates. The thickness can be obtained by using the standard uniform overlayer model, which is given by eq 1.29,30
( )
IAl ) I0Al exp -
t LAl
(1)
where I0Al is the intensity of Al peaks before surface modification, IAl is the intensity of Al peaks from the alumina surface after modification with PEG, t is the thickness of the film, and LAl is the electron attenuation length for the Al2p peak. To obtain film thickness from this equation, the electron attenuation length (LAl) for the Al2p peak needs to be calculated. The calculations are performed using NIST SRD-82 software31 based on the kinetic energy (KE) of the electrons, the photoionization asymmetry parameter (β), the inelastic mean free path (IMFP), and the transport mean free path (TMFP). The KE of the electrons is defined as the difference between the X-ray energy and the core electron binding energy of aluminum (BE). The X-ray energy for the Al KR source is 1486.6 eV.32 The values of BE were obtained from the NIST X-ray Photoelectron Spectroscopy Database SRD 20, version 3.1,33 and values of β were obtained from Band et al.34 The thickness of the layer of the adsorbed protein can be estimated in a similar way using the electron attenuation lengths and the standard uniform overlayer model as described earlier. In the case of protein adsorption, I0 is the intensity of the Al peak before protein adsorption (intensity of the unmodified/PEG modified surface) and I is the intensity of the Al peak after protein adsorption. The amount of absorbed protein in ng/cm2 was then estimated from thickness data using the method by Stenberg and Nygren.35
m ) dpF where m is the amount of protein adsorbed (ng/cm2), F is the density of adsorbed protein (g/cm3), and dp is the thickness of protein on the surface (Å). Calculation of PEG Surface Concentration and Grafting Density. The values of thickness obtained from the standard uniform overlayer model can be used to calculate the PEG grafting density. The PEG grafting density σ is given by eq 2.36
σ)
(La)
2
(2)
where a is the size of a monomer unit (∼3 Å)37,38 and L is the average distance between PEG chains grafted to the surface. (29) Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Langmuir 2004, 20, 429. (30) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059. (31) NIST Electron Effective Attenuation Length Database, version 1.0 (SRD-82); National Institute of Standards and Technology: Gaithersburg, MD, 2001. (32) Schweppe, J.; Deslattes, R. D.; Mooney, T.; Powell, C. J. J. Electon Spectrosc. Relat. Phenom. 1994, 67, 463. (33) NIST X-ray Photoelectron Spectroscopy Database, version 3.1; National Institute of Standards and Technology: Gaithersburg, MD, 2001. (34) Band, I. M.; Kharitonov, Y. I.; Trzhaskovskava, M. B. At. Data. Nucl. Data Tables 1979, 23, 443. (35) Stenberg, M.; Nygren, H. J. Phys. 1983, C-10, 83. (36) de Gennes, P. G. Macromolecules 1980, 13, 1069. (37) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (38) Szleifer, I. Biophys. J. 1997, 72, 595.
The average distance between PEG chains grafted to the surface L39 can be estimated by determining the surface concentration of PEG, Γ,40 which is given by eq 3.
L)
( ) M ΓNA
1/2
Γ ) Ft
(3)
where M is the molecular weight of PEG (1000 in this case), Γ is the surface concentration (gm/nm2), NA is Avogadro’s number (6.023 × 1023 mol-1), F is the density of the dry PEG layer (assumed to be constant, 10-21 gm/nm3),41 and t is the thickness of the PEG layer (nm). Therefore, by combination of eqs 2 and 3, a simplified relationship for σ can be obtained, which is given by eq 4.
σ)
( ) a2ΓNA M
(4)
Using eq 4, we can calculate the grafting density of PEG-modified surfaces.
Results and Discussion PEG Surface Characterization. PEG-immobilized alumina surfaces were prepared using a covalent coupling technique as described earlier. Immobilization with PEG was carried out for four different PEG concentrations (10, 20, 40, and 80 mM) for a 1 h immobilization to study the effect of concentration. Similarly, to study the effect of immobilization time, 5 mM PEG concentration was used for four different immobilization times (1, 2, 4, and 8 h). PEG with molecular weight of 1000 was used since it is known to form thin films on inorganic substrates.15-17 XPS analysis was performed to ensure the presence of PEG moieties on the surface. Survey scans were taken to determine the elemental surface composition of various elements present, and high-resolution C1s scans were taken to study the carbon chemistries. The survey scan for the unmodified surface shows distinct peaks for Al2p (100 eV) and O1s (528 eV) as well as a small peak for C1s (285 eV) which is present due to impurities on the surface. After modification with PEG, there is an increase in C1s (285 eV) and Si2p (100 eV) peaks and a decrease in Al2p (72 eV). This trend is followed as the concentration of PEG and immobilization time increases. The presence of silicon is due to the silicon tetrachloride that is coupled with PEG. Table 1 highlights the elemental composition for both the effect of PEG concentration and the effect of immobilization time on the formation of PEG films obtained from the survey scans. For lower concentrations and smaller immobilization times, the difference in elemental composition for a modified and an unmodified surface is smaller, suggesting low PEG grafting density on the surface. However, with increase in concentration and immobilization times, the difference in elemental composition for modified and unmodified surfaces is significant, suggesting an increase in PEG grafting density. Since XPS is a depth-sensitive technique, these results confirm the presence of PEG on the surface. To further support the presence of PEG moieties on the surface, high-resolution C1s scans were taken (Figure 4a). There is an increase in the overall intensity of the C1s peaks after PEG modification. The high-resolution scan of a C1s peak for unmodified surfaces consists of only one (39) Harris, J. M. Poly(ethylene glycol): Biotechnical and biomedical applications; Plenum Press: New York, 1992. (40) Cuypers, P. A.; Corsel, J. W.; Jannssen, M. P.; Kop, J. M. M.; Hermens, W. T.; Hemker, H. J. J. Biol. Chem. 1983, 258, 2426. (41) Vogel, A.; Creswell, W.; Leicester, J. J. Phys. Chem. 1954, 58, 174.
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Table 1. Surface Elemental Composition for Unmodified and PEG-Modified Surfaces Obtained from XPS Survey Scans effect of PEG concentration (mM) %
unmodified
10
20
Al 32.24 ( 1.24 13.12 ( 1.22 C 13.33 ( 2.25 28.57 ( 1.58 O 54.43 ( 1.23 58.31 ( 1.54 Si 0 2.93 ( 1.54 C-O 0 70.8 ( 1.24
40
effect of immobilization time (h) 80
12.43 ( 1.33 6.21 ( 1.54 1.69 ( 1.24 35.87 ( 1.54 42.15 ( 1.57 53.62 ( 2.45 47.45 ( 1.59 48.01 ( 2.54 36.59 ( 2.58 4.25 ( 2.14 6.63 ( 1.54 8.10 ( 1.47 85.5 ( 2.41 92.1 ( 2.14 96.0 ( 1.57
1
2
4
24
29.46 ( 1.55 15.54 ( 1.24 53.75 ( 1.55 1.25 ( 1.64 35.8 ( 1.74
27.63 ( 1.64 17.87 ( 1.59 52.61 ( 1.21 1.89 ( 1.87 48.9 ( 2.34
18.82 ( 2.1 21.25 ( 1.66 56.02 ( 1.87 3.91 ( 1.57 59.8 ( 1.73
14.54 ( 1.47 27.54 ( 1.57 53.61 ( 1.55 4.31 ( 1.98 61.3 ( 1.87
Figure 4. High-resolution C1s scans for (a) unmodified and PEG-modified surfaces showing the C-O peak at negative 1.5 eV from the hydrocarbon peak. (b) Protein adsorption on unmodified and PEG-modified surfaces showing a decrease in protein adsorption with an increase in PEG concentration and immobilization time indicated by a decrease in C-N and NdC-O peaks. Table 2. Surface Elemental Composition for Protein-Adsorbed Unmodified and PEG-Modified Surfaces Obtained from XPS Survey Scans effect of PEG concentration (mM) %
unmodified
10
20
Al C O Si N (C-N + NdC-O)
24.71 ( 1.25 40.21 ( 1.54 23.19 ( 1.87 0 11.98 ( 1.11 63.9 ( 1.55
19.26 ( 1.45 35.54 ( 1.65 35.42 ( 1.54 1.24 ( 0.14 8.54 ( 0.44 58.8 ( 1.14
16.58 ( 1.47 30.14 ( 1.78 44.17 ( 1.87 1.99 ( 0.18 7.12 ( 0.47 52.3 ( 1.44
40
effect of immobilization time (h) 80
1
12.25 ( 1.45 9.58 ( 1.88 22.84 ( 1.55 24.54 ( 1.42 10.09 ( 1.45 37.57 ( 1.44 53.49 ( 1.33 71.79 ( 1.97 25.9 ( 1.66 4.25 ( 0.41 5.12 ( 0.47 0.75 ( 0.04 5.47 ( 0.45 3.42 ( 0.31 12.94 ( 1.51 48.7 ( 1.87 43.8 ( 1.82 56.7 ( 1.54
peak at 285 eV which is the C-C peak (carbon is present on the unmodified membranes due to impurities). The high-resolution C1s peak for PEG-modified surfaces consists of two well-defined peaks, a peak at 285 eV which is the C-C peak and a second peak at a shift of 1.5 eV from C-C, which is the C-O peak. Also, the intensity of the C-O in the C1s peak increases with increasing concentration and immobilization time, which suggests an increase in PEG grafting on the surface. Using the peak fit analysis software provided with the XPS instrument, the relative percentages of C-C and C-O in the C1s peak for PEG-modified surfaces were determined (Table 1). The relative percentage of C-O in the overall C1s peak is an indirect measure of PEG grafting on the surface. A convolution of Gaussian components was assumed for all peak shapes. Protein Adsorption. The interaction of PEG-modified surfaces with fibrinogen was investigated to better understand their behavior in physiological environments. Unmodified and PEG-modified surfaces were incubated with fibrinogen solution in PBS (1 mg/mL) for 1 h. The
2
4
24
18.54 ( 1.54 32.31 ( 1.74 39.22 ( 1.41 1.25 ( 0.14 8.68 ( 1.74 54.6 ( 1.78
16.52 ( 1.41 28.52 ( 1.11 44.9 ( 1.47 2.14 ( 0.16 7.92 ( 1.77 51.8 ( 1.59
13.70 ( 1.44 12.21 ( 1.24 66.09 ( 1.11 2.98 ( 0.44 5.02 ( 1.66 48.5 ( 1.47
surfaces were washed thoroughly and dried before the XPS analysis. Survey scans were taken to determine the elemental surface composition of various elements present. A higher amount of nitrogen is present on unmodified surfaces compared to PEG-modified surfaces. Since the nitrogen peak is the characteristic of protein adsorption on the surface, this suggests that PEG-modified surfaces adsorb less protein compared to unmodified surfaces. Further, with an increase in PEG concentration and immobilization time, the amount of nitrogen on the surface decreases. This indicates that as the amount of PEG on the surface increases, the protein adsorption decreases. Table 2 shows the elemental composition of proteinadsorbed surfaces. A more precise way to characterize protein on the surface is to determine the fraction of C-N and N-CdO peaks in the overall C1s peak. The C-N and N-CdO peaks are the characteristic of protein and are at a shift of 0.8 and 1.8 eV, respectively, from the C-C peak. Hence highresolution C1s scans were taken, and the peak fit analysis software provided with the XPS instrument was used to
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Figure 5. PEG grafting density and average distance between grafted chains for unmodified and PEG-modified surfaces: (a) effect of PEG concentration and (b) effect of immobilization time. Table 3. Theoretical Calculations of PEG Film Thickness and Protein Adsorption on Unmodified and PEG-Modified Surfaces (I0 ) 6218, LAl ) 2.728 nm) PEG concentration (mM)
thickness (nm)
protein adsorption (ng/cm2)
immobilization time (h)
thickness (nm)
protein adsorption (ng/cm2)
unmodified 10 20 40 80
0 1.66 ( 0.02 2.18 ( 0.05 2.45 ( 0.07 2.48 ( 0.06
4.521 ( 0.1 2.95 ( 0.08 2.45 ( 0.07 1.74 ( 0.04 1.25 ( 0.03
unmodified 1 2 4 24
0 1.14 ( 0.03 1.48 ( 0.04 1.72 ( 0.05 1.97 ( 0.07
4.521 ( 0.1 3.037 ( 0.09 2.479 ( 0.08 2.068 ( 0.08 1.472 ( 0.05
determine the percentages of C-N and N-CdO in the overall C1s peak (Table 2). A convolution of Gaussian components was assumed for all the peaks. There is an increase in the intensity for the C-C peak; however, the intensities of C-N and N-CdO peaks and their percentages in the C1s peak are decreased in the PEG-modified membranes. This suggests lower protein adsorption on PEG-modified membranes. Theoretical Analysis. Thickness of Film from the Standard Overlayer Model. The film thickness can be calculated using the standard overlayer model which utilizes the intensity of the Al2p peak before and after modification. Since XPS is a depth-sensitive characterization technique, any modification of the surface chemistry will result in changes in the intensity of the peak for the base material of the surface (which is aluminum here). Hence, this model gives an accurate measurement of the thickness of the film formed on the surface utilizing the variation of intensities. Table 3 summarizes the thickness of PEG films formed for various concentrations and
immobilization times with the covalent coupling technique described here. As can be seen, the thickness of the PEG film is less than 2.5 nm for all the variations in concentrations and immobilization times, suggesting formation of very thin films which will not clog the pores. Further, the amount of protein adsorbed on the surface was calculated using the thickness from the standard overlayer model (Table 3). There is a 70% reduction in protein adsorption after PEG modification. Calculation of Grafting Density. The values of thickness obtained from the standard uniform overlayer model can be used to calculate the PEG grafting density. Grafting density is an important parameter since it is the measure of formation of PEG chains on the surface for various conditions. Figure 5 shows the grafting density and average distance between PEG chains grafted. As the concentration and the immobilization time of PEG increase, the average distance between grafted PEG chains decreases. This might be because more PEG molecules are available for covalent attachment which results in
Modification of Nanoporous Alumina Surfaces
higher grafting density. However, the grafting density seems to saturate at higher PEG concentrations and higher immobilization times. This might be because the surface is completely saturated and no more covalent attachment of PEG is possible. Conclusions The application of nonfouling thin films such as PEG to alumina membranes is useful in drug delivery applications to prevent membrane fouling in long-term usage. PEG was covalently grafted to alumina surfaces, and the films were characterized using XPS. XPS results confirm the presence of PEG moieties on the surface. The highresolution C1s spectrum shows the presence of a C-O peak at negative 1.5 eV from the hydrocarbon peak, which suggests the presence of PEG moieties on the surfaces. With an increase in concentration and immobilization time, the intensity of the C-O peak also increases.
Langmuir, Vol. 20, No. 19, 2004 8041
Theoretical calculations show that the optimal value of grafting density is achieved for high PEG concentration and immobilization times when uniform coverage on the surface is achieved. Also, the standard overlayer model was used to estimate film thickness. The thickness of the films formed by this technique for various PEG concentrations and immobilization times was less than 2.5 nm. Thus the formation of films should not cause pore blockage. Further, about 70% reduction in protein adsorption can be achieved with PEG films formed by the covalent coupling technique. Acknowledgment. The authors acknowledge Elisabeth Shaw of the Center of Materials Science and Technology of the Massachusetts Institute of Technology, Cambridge, for training on XPS and funding by the National Institutes of Health (EB00570-01). LA049075X
