pubs.acs.org/Langmuir © 2010 American Chemical Society
Investigations of Chemical Modifications of Amino-Terminated Organic Films on Silicon Substrates and Controlled Protein Immobilization Joonyeong Kim,*,† Joungmo Cho,‡ Paul M. Seidler,† Nicholas E. Kurland,§ and Vamsi K. Yadavalli§ † Department of Chemistry, Buffalo State, State University of New York, 1300 Elmwood Avenue, Buffalo, New York 14222, ‡Department of Chemical Engineering, Goessmann Laboratory, 686 North Pleasant Street, University of Massachusetts, Amherst, Massachusetts 01003, and §Department of Chemical and Life Science Engineering, Virginia Commonwealth University, 601 West Main Street, Richmond, Virginia 23284-3028
Received March 24, 2009. Revised Manuscript Received November 30, 2009 Fourier transform infrared spectroscopy by grazing-angle attenuated total reflection (FTIR-GATR), ellipsometry, atomic force microscopy (AFM), UV-visible spectroscopy, and fluorescence microscopy were employed to investigate chemical modifications of amino-terminated organic thin films on silicon substrates, protein immobilization, and the biological activity and hydrolytic stability of immobilized proteins. Amino-terminated organic films were prepared on silicon wafers by self-assembling 3-aminopropyltriethoxysilane (APTES) in anhydrous toluene. Surface amino groups were derivatized into three different linkers: N-hydroxysuccinimide (NHS) ester, hydrazide, and maleimide ester groups. UV-visible absorption measurements and fluorescence microscopy revealed that more than 40% of surface amino groups were chemically modified. Protein immobilization was carried out on modified APTES films containing these linkers via coupling with primary amines (-NH2) in intact monoclonal rabbit immunoglobulin G (IgG), the aldehyde (-CHO) of an oxidized carbohydrate residue in IgG, or the sulfhydryl (-SH) of fragmented half-IgG, respectively. FTIR spectra contain vibrational signatures of these functional groups present in modified APTES films and immobilized IgGs. Changes in the APTES film thickness after chemical modifications and protein immobilization were also observed by ellipsometric measurements. The biological activity and long-term hydrolytic stability of immobilized IgGs on modified APTES films were estimated by fluorescence measurements of an adsorbed antigen, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (FITC-Ab). Our results indicate that the FITC-Ab binding capacity of half-IgG immobilized via maleimide groups is greater than that of the oxidized IgG and the intact IgG immobilized via hydrazide and NHS ester groups, respectively. In addition, IgGs immobilized using all coupling chemistries were hydrolytically stable in phosphate-buffered saline (PBS).
Introduction The controlled immobilization of proteins on solid substrates has been the subject of extensive research because of its application in the fabrication of immunoassay-based diagnostic devices.1-7 Although the details are different, the general requirements for protein immobilization include the physical and chemical stability of adsorbed proteins on a solid substrate. More importantly, retention of the biological activity of adsorbed proteins via siteselected immobilization is critical to the detection of target compounds with enhanced sensitivity and selectivity.5-10 Intensive efforts have been devoted to the development of various site-selected immobilization techniques. For example, *Author to whom correspondence should be addressed. Tel: 716-878-5114. Fax: 716-878-4028. E-mail:
[email protected]. (1) Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington, DC, 1995. (2) Proteins at Interfaces: Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; American Chemical Society: Washington, DC, 1987. (3) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760–1763. (4) Biosensor Principles and Applications; Blum, L. J., Coulet, P. R., Eds.; Marcel Dekker: New York, 1991; Vol. 14. (5) Brinkley, M. Bioconjugate Chem. 1992, 3, 2–13. (6) O’shannessy, D. J.; Brigham-Burke, M.; Peck, K. Anal. Biochem. 1992, 205, 132–136. (7) Karyakin, A. A.; Presnova, G. V.; Rubtsova, M. Y.; Egorov, A. M. Anal. Chem. 2000, 72, 3805–3811. (8) Turkova, J. J. Chromatogr., B 1999, 722, 11–31. (9) Johnson, D. L.; Martin, L. L. J. Am. Chem. Soc. 2005, 2018–2019. (10) Tsang, V. C. W.; Greene, R. M.; Pilcher, J. B. J. Immunoassay Immunochem. 1995, 16, 395–418.
Langmuir 2010, 26(4), 2599–2608
antibodies are often immobilized on a surface in an oriented configuration using protein A (or protein G), which selectively binds to specific moieties in antibodies.11-13 However, low loading amounts and the long-term physical instability of adsorbed antibodies have limited the utility of protein A-based immobilization strategies. Instead, chemical conjugation using linkers (or coupling agents) has gained considerable attention as an alternative method.5-8 This conjugation technique provides direct and robust coupling of proteins to solid substrates via the formation of physically and chemically stable covalent bonds using various linkers on solid substrates. Despite many advantages of chemical conjugation, previous studies have shown that the orientation of immobilized proteins is significantly affected by the choice of linkers on the surface and the corresponding conjugation sites in adsorbed proteins.7,8 For example, immobilization via either glutaraldehyde or carbodiimide/N-hydroxysuccinimide (NHS) linkers results in the formation of layers of proteins with random orientations on the surface because of the ubiquity of conjugation sites, either primary amines or carboxyl groups, distributed throughout the protein. This is a significant issue in the immobilization of asymmetric proteins (e.g., immunoglobulin G) and often results in a reduction (11) (12) 1700. (13) (14)
Derek, A. P.; Martin, T. F.; James, N. M. Analyst 1994, 119, 2769–2776. Nakanishi, K.; Muguruma, H.; Karube, I. Anal. Chem. 1996, 68, 1695– Saha, K.; Bender, F.; Gizeli, E. Anal. Chem. 2003, 75, 835–842. Spitznagel, T. M.; Clark, D. S. Nat. Biotechnol. 1993, 11, 825–829.
Published on Web 01/22/2010
DOI: 10.1021/la904027p
2599
Article
of biological activity.14,15 Instead, well-defined, localized functional groups such as sulfhydryls or carbohydrates present in immobilizing proteins are preferred conjugation sites.7,16-18 The popularity of silicon wafers as solid substrates in many areas of science and technology mainly stems from the ability to incorporate a variety of functional groups by the formation of organosilane-based thin films.19-25 In particular, the formation of amino-terminated organic thin films on silicon substrates has been extensively studied because derivatizations of amino groups lead to the introduction of various chemical groups for many applications. This fact has greatly extended the utility of silicon wafers as solid substrates in the fabrication of biomedical devices by the controlled adsorption of biomolecules such as proteins and oligomeric nucleic acids.3,26-36 Recently, our research group reported the preparation of amino-terminated thin films on silicon substrates by self-assembling 3-aminopropyltriethoxysilane (APTES).24,25 The structure, stability, and reactivity of APTES thin films produced under various preparation conditions were characterized by Fourier transform infrared spectroscopy (FTIR), ellipsometry, and fluorescence microscopy. In particular, FTIR via a grazing-angle attenuated total reflection (GATR) method has proven to be a powerful technique for obtaining vibrational features of APTES thin films with a variety of thicknesses deposited on silicon substrates with enhanced sensitivity.37-40 Our results show that a larger number of reactive surface amino groups is available when the deposition of APTES is conducted for a longer time in organic solutions (e.g., toluene). In addition, these APTES films need to be cured at elevated temperature (e.g., 100 °C) for the production of physically durable films. Once the preparation conditions were optimized, we extended our research to explore
(15) Lu, B.; Smyth, M. R.; O’Kennedy, R. Analyst 1996, 121, 29R–32R. (16) Mallik, R.; Wa, C.; Hage, D. S. Anal. Chem. 2007, 79, 1411–1424. (17) Soltys, P. J.; Etzel, M. R. Biomaterials 2000, 21, 37–48. (18) Hirota, J.; Michikawa, T.; Natsume, T.; Furuichi, T.; Mikoshiba, K. FEBS Lett. 1999, 456, 322–326. (19) Haller, I. J. Am. Chem. Soc. 1978, 100, 8050–8055. (20) Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92–98. (21) Netzer, L.; Sagiv, J. J. Am. Chem. Soc. 1983, 105, 674–676. (22) Ulman, A. Chem. Rev. 1996, 96, 1533–1554. (23) Onclin, S.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2005, 44, 6289–6304. (24) Kim, J.; Seidler, P.; Wan, L.-S.; Fill, C. J. Colloid Interface Sci. 2009, 329, 114–119. (25) Kim, J.; Seidler, P.; Fill, C.; Wan, L.-S. Surf. Sci. 2008, 602, 3323–3330. (26) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287–12291. (27) Chrisey, L. A.; Lee, G. U.; O’Ferrall, E. Nucleic Acids Res. 1996, 24, 3031– 3039. (28) Saprigin, A. V.; Thomas, C. W.; Dulcey, C. S.; Patterson, C. H., Jr.; Spector, M. S. Surf. Interface Anal. 2004, 36, 24–32. (29) Hooper, A. E.; Werho, D.; Hopson, T.; Palmer, O. Surf. Interface Anal. 2001, 31, 809–814. (30) Charles, P. T.; Vora, G. J.; Andreadis, J. D.; Fortney, A. J.; Meador, C. E.; Dulcey, C. S.; Stenger, D. A. Langmuir 2003, 19, 1586–1591. (31) Flink, S.; Schonherr, H.; Vancso, G. J.; Geurts, F. A. J.; van Leerdam, K. G. C.; van Veggel, F.; Reinhoudt, D. N. J. Chem. Soc., Perkin Trans. 2 2000, 2141–2146. (32) Howarter, J. A.; Youngblood, J. P. Langmuir 2006, 22, 11142–11147. (33) Pereira, C.; Patrı´ cio, S.; Silva, R.; Magalh~aes, A. L.; Carvalho, P.; Pires, J.; Freire, C. J. Colloid Interface Sci. 2007, 316, 570–579. (34) Wei, H.; Zhou, L.; Li, J.; Liu, J.; Wang, E. J. Colloid Interface Sci. 2008, 321, 310–314. (35) Guo, W.; Ruckenstein, E. J. Membr. Sci. 2003, 215, 141–155. (36) Kovalchuk, T.; Sfihi, H.; Kostenko, L.; Zaitsev, V.; Fraissard, J. J. Colloid Interface Sci. 2006, 302, 214–229. (37) Olsen, J. E.; Shimura, F. Appl. Phys. Lett. 1988, 53, 1934–1936. (38) Rochat, N.; Chabli, A.; Bertin, F.; Olivier, M.; Vergnaud, C.; Mur, P. J. Appl. Phys. 2002, 91, 5029–5034. (39) Milosevic, M.; Berets, S. L.; Fadeev, A. Y. Appl. Spectrosc. 2003, 57, 724– 727. (40) Lummerstorfer, T.; Kattner, J.; Hoffmann, H. Anal. Bioanal. Chem. 2007, 388, 55–64.
2600 DOI: 10.1021/la904027p
Kim et al.
derivatizations of surface amino groups to incorporate chemical linkers for site-selected protein immobilization. In this article, we report the versatility of these APTES films by demonstrating chemical modifications of amino groups followed by the site-selected immobilization of proteins. Monoclonal immunoglobulin G (IgG) was chosen as a model protein because of its selective binding affinity toward the corresponding antigen, the availability of rich conjugation sites, and its asymmetric shape. In addition to primary amine groups, localized functional groups in IgG such as sulfhydryl (-SH) and aldehyde (-CHO) groups can be chemically created for site-selected conjugation by the reduction of disulfide bonds and the oxidation of carbohydrate moieties.5-10 Surface amino groups on APTES thin films were chemically converted to three different linkers: N-hydroxysuccinimide (NHS) ester, hydrazide, and maleimide ester groups. Modified APTES films containing these linkers were used as solid substrates for coupling monoclonal IgG via primary amine groups (-NH2),41-43 oxidized IgG via aldehyde groups (-CHO) in carbohydrate residues,17,18,44 and fragmented half-IgG via sulfhydryl groups (-SH) (Scheme 1).8,16,45,46 Fourier transform infrared spectroscopy with grazing-angle attenuated total reflection (FTIR-GATR), ellipsometry, atomic force microscopy (AFM), UV-visible spectroscopy, and fluorescence microscopy were employed to monitor the modifications of APTES films, protein coupling, biological activity (e.g., antigen binding capacity), and the hydrolytic stability of adsorbed proteins. On the basis of our experimental data, structures of modified APTES films and their effects on the biological activity of immobilized proteins are described in the article.
Experimental Section Chemicals and Materials. Deionized water used in the preparation of chemical solutions and in the cleaning of the experimental apparatus was produced by a Millipore waterpurification system with a minimal resistivity of 18.0 MΩ cm. 3-Aminopropyltriethoxysilane (APTES, 99.0%, Acros), 1,1,1,3, 3,3-hexamethyldisilazane (HMDS, 98.0%, Acros), anhydrous toluene (99.8%, extra dray, water 0.1 mg/mL). From ellipsometric thickness measurements, the amount of immobilized half-IgG is estimated to be ca. 62 and 88% of the amount of IgG immobilized via NHS ester and hydrazide groups, respectively. Therefore, we could estimate that the binding capacity of half-IgG is about 3.2 and 1.3 times greater than that of IgGs adsorbed on the APTES surfaces via NHS ester and hydrazide groups, respectively. The antigen-binding capacity of IgGs on the surface appears to be well preserved for extended periods of time regardless of the conjugation sites. The amount of adsorbed FITC-Ab was reduced by ∼25% after silicon wafers with immobilized IgGs were kept in pH 7.2 PBS for 7 days (Figure 7). We assume that the reduced binding capacity of immobilized IgG on solid substrates after storage is caused by several factors including desorption via hydrolysis and structural rearrangements in conjugated IgGs. Our previous studies regarding the stability of APTES thin films in an aqueous solution via ellipsometry, FTIR, and fluorescence microscopy have shown that underlying APTES thin films are physically stable in water for a long period of time under the proper formation conditions.24,25 Our unpublished data indicates that the measured thickness of IgGs on silicon substrates DOI: 10.1021/la904027p
2607
Article
remained unchanged after a series of sonications, indicating that the IgG films are physically stable in an aqueous solution. Therefore, we assume that structural rearrangement in conjugated IgGs on the surface is mainly responsible for the reduction in the binding capacity. It is well known that the extent of structural rearrangement primarily depends on the surface coverage of the adsorbed protein.79-82 If there are fewer protein molecules on the surface, then adsorbed proteins are more likely to spread out until the process is prevented by neighboring proteins. Conversely, less structural rearrangement is allowed if more protein molecules are deposited on the surface. Considering rms roughness and surface features observed in AFM images in Figure 1, modified APTES surfaces are not completely covered by conjugated IgGs. Thus, a 25% reduction in biological activity within 48 h after immobilization probably results from the accompanying structural rearrangement. However, no further significant structural rearrangement is expected as indicated by the time-dependent binding capacity shown in Figure 7. Amide I modes in infrared spectra of surfaces with immobilized IgGs presented in Figures 2-4 are centered at ∼1655 cm-1. Because infrared spectra of IgG generally contain a vibrational feature of amide I close to 1630 cm-1 because of the presence of a β sheet, our infrared data indicates that the secondary structure of adsorbed IgGs was changed after immobilization on the surface.83 Previous studies have shown that the amount of β sheet decreases but the content of unordered structure in adsorbed IgG increases upon structural rearrangements on the surface.84 Although it is initially reduced by structural rearrangements on the surface, the biological activity of IgGs on silicon substrates appears to depend primarily on the (79) Kim, J.; Somorjai, G. A. J. Am. Chem. Soc. 2003, 125, 3150–3158. (80) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87–93. (81) Norde, W.; Giacomelli, C. E. Macromol. Symp. 1999, 145, 125–136. (82) Buijs, J.; van den Berg, P. T. W.; Lichtenbelt, J. W. T.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1996, 178, 594–605. (83) Infrared Analysis of Peptides and Proteins; Singh, B. R., Ed.; American Chemical Society: Washington, DC, 2000. (84) Buijs, J.; Norde, W.; Lichtenbelt, J. W. T. Langmuir 1996, 12, 1605–1613.
2608 DOI: 10.1021/la904027p
Kim et al.
choice of conjugation sites, which dictate the orientation of active sites on the surface.
Summary and Conclusions AFM, ellipsometry, FTIR-GATR, UV-visible spectroscopy, and fluorescence microscopy were used to monitor and characterize the grafting of three types of chemically reactive linkers on amino-terminated APTES films on silicon substrates. Intact and chemically modified monoclonal IgGs were immobilized on these functionalized surfaces, but their orientation was affected by the choice of chemical linkers as indicated by the binding capacity of FITC-labeled goat anti-rabbit IgG (FITC-Ab). This was attributed to the distribution and locality of the immobilization site(s) relative to the active site present in immobilized IgGs. Our results show that immobilized IgGs can be more biologically active on silicon surfaces when geometrically unique conjugation sites are used as in the case of carbohydrate moieties or sulfhydryl groups rather than primary amino groups. IgGs immobilized using these linkers were hydrolytically stable in an aqueous solution for extended periods of time, and their antigen-binding capacity was not significantly reduced. Acknowledgment. This work is supported by startup funds from the SUNY Research Foundation and Department of Chemistry, Buffalo State, SUNY. This research was also supported by the Buffalo State College Office of Undergraduate Research’s Small Grants Program and through the Early Undergraduate Research Program, a program partially funded through the College’s NSF-STEP grant (DUE-0431517). Several parts of a Nicolet Magna 550 infrared spectrometer were obtained from Department of Energy via the Used Energy-Related Laboratory Equipment (ERLE) Grant Program. Supporting Information Available: FTIR spectra in the range of 3800-2600 cm-1 and at 1300 and 700 cm-1 from intact and modified APTES films containing linkers and immobilized proteins. This material is available free of charge via the Internet at http://pubs.acs.org.
Langmuir 2010, 26(4), 2599–2608
