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Measurement of the Adsorption of Cytochrome c onto the External Surface of a Thin-Film Mesoporous Silicate by Ellipsometry J. Deere,†,‡ M. Serantoni,† K. J. Edler,§ B. K. Hodnett,†,‡ J. G. Wall,† and E. Magner*,†,‡ Materials and Surface Science Institute and Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland, and Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Received July 25, 2003. In Final Form: October 13, 2003
Introduction Since first described by Beck et al. in 1992,1 a large range of mesoporous silicate (MPS) materials has been synthesized. These materials are of interest as supports for catalysts,2 as adsorbents of metals and large organic molecules,3 and as a means of immobilizing proteins and enzymes.4-6 They exhibit highly ordered pore structures and very tight pore size distributions, which can be tuned by changing the preparation conditions. MPS can be prepared with pore diameters in the range 20-200 Å, a range sufficient to accommodate many proteins. The large surface areas of MPS, on the order of 1000 m2 g-1, enables high protein loadings (10 µmol g-1) to be readily achieved. As a result of their silicate inorganic framework, they are chemically and mechanically stable2 and are resistant to microbial attack. A number of authors have shown that the catalytic activities of a number of enzymes encapsulated within MPS are comparable to that of the aqueous enzyme. MPS have been used to immobilize a number of enzymes6-8 and may find applications in biosensors5 and biocatalytic8 and biomolecule separation7 systems. The interactions between proteins and MPS are governed by a number of factors, which, in addition to the pore size and surface area of the MPS, also include the charges on the protein and the silicate and the presence of hydrophobic/hydrophilic patches on the protein. For example, Stucky et al.7 have used MPS to sequester and release proteins of similar size but varying charge, while Liu et al.9 have prepared MPS modified with amino and * Author to whom correspondence should be addressed. E-mail:
[email protected]. Phone: (353) 61202629. Fax: (353) 61213529. † Materials and Surface Science Institute, University of Limerick. ‡ Department of Chemical and Environmental Sciences, University of Limerick. § University of Bath. (1) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.; Schmitt, K. D.; Chu, C. T.; Olson, D. H.; Sheppard, E. W.; McCullen, S. B.; Higgins, J. B.; Schlenker, J. L. J. Am. Chem. Soc. 1992, 114, 10834. (2) Johnson, B. F. G.; Raynor, S. A.; Shephard, D. S.; Mashmeyer, T.; Thomas, J. M.; Sankar, G.; Bromley, S.; Oldroyal, R.; Gladden, L.; Mantle, M. D. Chem. Commun. 1999, 1167-1168. (3) Thomas, J. M.; Raja, R.; Johnson, B. F. G.; O’Connell, T. J.; Sankar, G.; Khimyak, T. Chem. Commun. 2003, 10, 1126-1127. (4) Deere, J.; Magner, E.; Hodnett, B. K.; Wall, J. G. J. Phys. Chem. B 2002, 106, 7340-7347. (5) Washmon-Kriel, L.; Jimenez, V. L.; Balkus, K. J. J. Mol. Catal. B: Enzym. 2000, 10, 453-469. (6) He, J.; Li, X.; Evans, D. G.; Duan, X.; Li, C. J. Mol. Catal. B: Enzym. 2000, 11, 45-53. (7) Han, Y.-J.; Stucky, G. D.; Butler, A. J. Am. Chem. Soc. 1999, 121, 9897-9898. (8) Gills, I.; Ballesteros, A. J. Am. Chem. Soc. 1998, 120, 8587-8598. (9) Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124, 11242-11243.
carboxylate groups to substantially increase the binding capacity for horseradish peroxidase. While the main interest in using MPS to immobilize enzymes is to utilize their network of internal channels (and, consequently, their large surface areas), it is also possible for proteins to adsorb on to the external surfaces of MPS. This is particularly the case when the size of the proteins under investigation is larger than the diameters of the silicate pore sizes. To utilize techniques such as ellipsometry to ascertain the amount of externally adsorbed protein, it is necessary to use thin films of MPS instead of the normally used, powdered form. The synthesis of continuous films of mesoporous silica films was first reported by Ozin et al.10 Similar methods have been since used to deposit such films on a variety of different substrates.11,12 The growth and characterization of such films has been extensively reviewed.13 Currently, most preparations allow for pore networks that run parallel to the supporting substrate, but substantial effort is now being made to prepare films with pores running perpendicular to the substrate.14 There have been a number of reports on the use of ellipsometry to study protein adsorption at silicon and silica surfaces.15-18 Malmsten and Lassen17 have described the use of in situ ellipsometry to study the adsorption of human serum albumin, IgG, fibrinogen, and lysozyme at methylated silica surfaces. The amounts of protein adsorbed were similar to previous reports (by other methods), and the buildup of the adsorbed layers proceeded in a different manner for each protein. For instance, the adsorbed layer of lysozyme was more compact than that of the other proteins. Other reports have focused on the use of in situ ellipsometry to measure the thickness of the protein layer. This study describes the use of ellipsometry to measure the extent of adsorption of the heme protein, cytochrome c (cyt c), onto the external surface of MPS. Experimental Section Materials. MPS thin films supported on 0.5-mm-thick silicon wafers were synthesized using a dip-coating alcohol vaporization method.13 The silicon wafers were cleaned with water, acetone, and chloroform just prior to film deposition; however, the native oxide layer was not removed. Film preparation followed the method of Grosso et al.19 An acidic solution of prehydrolyzed silica in ethanol was added to a second solution containing acidified cetyltrimethylammonium bromide (CTAB) in water/ ethanol. The molar ratios in the final solution were 1:20:4 × 10-3:5:0.1 TEOS/EtOH/HCl/H2O/CTAB. After aging for 1 h, the solution was spread uniformly onto a vertically suspended wafer (10) Yang, H.; Coombs, N.; Sokolov, I.; Ozin, G. A. Nature 1996, 381, 589-592. (11) Aksay, I. A.; Trau, M.; Manne, S.; Honma, I.; Yao, N.; Zhou, L.; Fenter, P.; Eisenberger, P. M.; Gruner, S. M. Science 1996, 273, 892898. (12) Zhao, D.; Yang, P.; Melosh, N.; Feng, J.; Chmelka, B. F.; Stucky, G. D. Adv. Mater. 1998, 10, 1380-1400. (13) Edler, K. J.; Roser, S. J. Int. Rev. Phys. Chem. 2001, 20, 387466. (14) Tolbert, S. H.; Scha¨ffer, T. E.; Feng, J.; Hansma, P. K.; Stucky, G. D. Chem. Mater. 1997, 9, 1962-1970. (15) Arwin, H.; Welin-Klintstro¨m, S.; Jansson, R. J. Colloid Interface Sci. 1993, 156, 377-382. (16) Malmsten, M. J. Colloid Interface Sci. 1994, 166, 333-342. (17) Malmsten, M.; Lassen, B. Proteins at Interfaces; American Chemical Society: Washington, D.C., 1995; Chapter 16. (18) Welin-Klintstro¨m, S.; Jansson, R.; Elwing, H. J. Colloid Interface Sci. 1993, 157, 498-503. (19) Grosso, D.; Babonneau, F.; Albouy, P. A.; Amenitsch, H.; Balkenende, A. R.; Brunet-Bruneau, A.; Rivory, J. Chem. Mater. 2002, 14, 931-939.
10.1021/la035358y CCC: $27.50 © 2004 American Chemical Society Published on Web 11/26/2003
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Langmuir, Vol. 20, No. 2, 2004 533
from a Pasteur pipet, and excess wicked from the bottom. The wafer was held vertical until the film had dried (5 min) and then heated in an oven at 70 °C for 1 h. Cyt c was obtained from Sigma and was purified using a published protocol.20 Water was purified (18.2 MΩ cm-1) using an Elgastat SPECTRUM system. Film Treatment. Three different batches of wafers were used. The first two batches were wafers of ∼2 × 2 cm, each coated separately. The third batch was a large single wafer, which was coated and then cut into ∼2 × 2 cm pieces using a diamond pen. The results reported here were obtained using this batch of samples. All the samples were processed as follows: (i) heated at 60 °C for 12 h, (ii) calcined at 450 °C for 6 h, and (iii) washed with water followed by adsorption of cyt c. Ellipsometry measurements were obtained at each step. With the first two batches, the protein was adsorbed from a 25 mM potassium phosphate buffer at pH 6.5, while with the third batch, the buffer solution was replaced with water. Protein adsorption was allowed to occur for 7 days at 4 °C. After adsorption, excess water/buffer was removed from the film surface by placing the film on a thermostatically controlled hotplate at 35 °C for 5 min. Ellipsometry. Measurements were performed on a variable angle spectroscopic ellipsometer (M-2000U J. A. Woollam Co., Inc., Lincoln, Nebraska, U.S.A.) in air at room temperature. The incident light beam was focused on to an area of about 1 × 2 mm2. Spectra were obtained at three different positions for each sample, using three angles of incidence (60, 65, and 70°) over the wavelength range 350-900 nm to develop appropriate models for the complex system under investigation. The data were fitted and modeled using the system software WVASE32 3.412d. The value of the mean-squared error (MSE), which represents the quality of the model used for the fitting process, is defined according to the Levenberg-Marquardt algorithm
MSE )
1 2N - M
N
∑ i)1
[(
) (
ψmod - ψiexp i exp σψ,i
2
+
)]
∆mod - ∆iexp i exp σ∆,i
1
2
2N - M
) χ2 (1)
where N is the number of (ψ, ∆) pairs, M is the number of variable parameters in the model, and σi is the standard deviation of the experimental data points.21,22 MSE values of less than 50 were considered acceptable. The film thickness values obtained were the arithmetic means of the results obtained for a series of samples, from the same preparation batch. The number of samples in each batch ranged from 18 (first step) to 4 (last step). Atomic Force Microscopy (AFM). The AFM images reported in this study were obtained using a TopoMetrix Explorer scanning probe microscope operating in noncontact mode. The triangular pyramidal silicon tips (Micoscopes SFM Probes model no. 1650-00 HFR) were of the following dimensions: 3-5-µm base, 10-15 µm in length with a tip radius
