Langmuir 2001, 17, 7645-7651
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Changes in Protein Adsorption on Self-Assembled Monolayers with Monolayer Order: Comparison of Human Serum Albumin and Human Gamma Globulin Stanislaw Petrash,† Tricia Cregger, Bin Zhao, Elena Pokidysheva,‡ Mark D. Foster,* William J. Brittain, Viktor Sevastianov,‡ and Charles F. Majkrzak§ Maurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio 44325-3909, Institute of Transplantology and Artificial Organs, Shukinskaya 1, Moscow 123182, Russia, and Radiation Reactor Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 Received July 30, 2001. In Final Form: September 12, 2001 The role that alkyl chain packing density in a self-assembled monolayer (SAM) plays in the adsorption of protein to the SAM surface was investigated using in situ neutron reflectivity (NR) and total internal reflection fluorescence (TIRF) measurements of the adsorption behavior of human serum albumin (HSA) and human immunoglobulin G (HGG). The proteins differ particularly in the fact that HSA has specific binding pockets for alkyl chains while HGG does not. NR results show that HSA adsorbs from a 1.0 mg/mL solution as a single layer on the SAMs, with the protein interpenetrating into a less densely packed SAM, but not into a more densely packed SAM. Likewise, the kinetics of the HSA adsorption to the SAMs varies markedly with the alkyl chain packing. In contrast, both the structure of the adsorbed layer and adsorption kinetics vary little with the alkyl chain packing density in the case of HGG adsorbing from solution. HGG also does not penetrate into loosely packed SAMs. NR results reveal that the HGG adsorbs as two layers, with the layer closest to the SAM being more tightly packed. When HSA and HGG compete for adsorption sites on a SAM, HGG effectively displaces HSA from a tightly packed SAM, but does not compete effectively with HSA adsorbed tenaciously onto a loosely packed SAM.
Introduction It has been suggested that engineering surfaces to adsorb specific proteins in a characteristic manner could be a pathway for developing biomaterials surfaces that evoke a desired response in a particular application.1 For example, adsorbing human serum albumin (HSA) onto polymer surfaces2-4 has been shown to provide some modest inhibition of thrombus formation by halting the usual cascade of protein adsorption events. However, this effect is diminished when the HSA molecules desorb from the surface. There have been attempts to increase the affinity of HSA for a surface by grafting alkyl chains to the surface of a polymer5,6 because albumin has several hydrophobic pockets for carrying fatty acid chains.7 Petrash and co-workers8 have also shown that when HSA is adsorbed to self-assembled monolayers of alkyl chains, * Corresponding author. Address: Department of Polymer Science, The University of Akron, Akron, OH 44325-3909. E-mail:
[email protected]. Phone: (330) 972-5323. Fax: (330) 972-5290. † Present Address: National Starch and Chemical Company, 10 Finderne Avenue, Bridgewater, NJ 08807. ‡ Institute of Transplantology and Artificial Organs. § Radiation Reactor Division. (1) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837. (2) Grasel, T. G.; Pierce, J. A.; Cooper, S. L. J. Biomed. Mater. Res. 1987, 21, 815. (3) Munro, M. S.; Eberhart, R. C.; Maki, N. J.; Brink, B. E.; Fry, W. J. J. Am. Soc. Artif. Intern. Organs 1983, 6, 65. (4) Lyman, D. J.; Knutson, K.; McNiel, B.; Shibatani, K. Trans. Am. Soc. Artif. Intern. Organs 1975, 21, 49. (5) Pitt, W. G.; Cooper, S. L. J. Biomed. Mater. Res. 1988, 22, 359. (6) Alvarez, C.; Bertorello, H.; Strumla, M.; Sanchez, E. I. Polymer 1996, 37, 3715. (7) Spector, A. A. J. Lipid Res. 1975, 16, 165. (8) Petrash, S.; Sheller, N. B.; Dando, W.; Foster, M. D. Langmuir 1997, 13, 1881.
the adsorbed protein layer is more resistant to elution by the common surfactant sodium dodecyl sulfate (SDS) when the alkyl chains in the SAM are loosely packed than when they are tightly packed. This suggests that the controlled deposition of hydrophobic SAMs may be one means to promote the passivation of a surface by the adsorption of HSA. SAMs are very robust in aqueous environments, give a well-defined surface, and are relatively easy to deposit.9,10 The packing density of the alkyl chains can be altered by varying the deposition time. Depositions allowed to progress to “completion” provide tightly packed, wellorganized layers of consistent monomolecular thickness, referred to here as “complete” SAMs. Shorter deposition times yield less densely packed and thinner SAMs, denoted here as “incomplete”. A simple explanation of the tenacity of HSA adsorption on loosely packed SAMs as resulting from the opportunity for interactions between the hydrophobic pockets and the individual chains in the SAM suggests itself. However, evidence of this sort of specific interaction is sought in the present work through further clarification of the adsorbed layer structure and adsorption kinetics as well as by comparison of the behavior of HSA with that of human gamma globulin (HGG), another important and widely studied blood protein.11 While HGG differs in several ways from HSA, of central interest here is the fact that it has no pockets for binding alkyl chains comparable to those in HSA. To observe if the HSA actually penetrates into a loosely packed SAM, neutron reflectivity (NR)12-16 has been used to sensitively probe in situ the structure of the adsorbed (9) Fragneto, G.; Thomas, R. K.; Rennie, A. R.; Penfold, J. Science 1995, 267, 657. (10) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (11) Kuby, J. Immunology; W. H. Freeman and Co.: New York, 1992; Chapter 2.
10.1021/la011192u CCC: $20.00 © 2001 American Chemical Society Published on Web 11/01/2001
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HSA and HGG layers on both complete and incomplete SAMs of deuterated hexadecyltrichlorosilane (d-HTS). Use of a deuterated SAM with NR provides contrast between the protein and SAM not available in the ex situ study of adsorbed layers done with X-ray reflectivity.8,17 Since a deuterated buffer solution does not penetrate a hydrophobic SAM appreciably, the neutron reflectivity data can be analyzed to yield a depth profile of protein concentration beginning within the SAM and extending out into the solution. Specific interaction of the protein with the SAM would also be expected to modify the kinetics of adsorption, and therefore, the kinetics of labeled HSA and labeled HGG adsorption to complete and incomplete SAMs were studied using total internal reflection fluorescence (TIRF).18-21 Measuring the kinetics of adsorption from solutions containing a single protein offered indications that indeed the behavior of HSA was quite sensitive to the SAM packing, while that of HGG was not. Specific interactions would also be expected to change the competitive adsorption of protein mixtures. It is known that, in general, when adsorption occurs from mixtures of proteins HSA adsorbs rapidly,6 but can then be displaced by other proteins in what has been labeled as the “Vroman effect”. If HSA could be tenaciously adsorbed to a surface this effect could be controlled. Measurements of competitive adsorption from a solution containing both HSA and HGG confirmed that HSA adhered more tenaciously to the loosely packed SAM and in that case was not easily displaced by the HGG. Experimental Section Reagents and Solutions. All reagents were obtained from Aldrich or Fisher, unless otherwise noted. Hexadecyltrichlorosilane-d33 (d-HTS) was synthesized by hydrosilation of 1-bromohexadecane-d33 (CDN Isotopes).22 Hydrogenous hexadecyltrichlorosilane (HTS) was purchased from Geleste, Inc., and was purified using a short-path distillation before use.22 A deuterated phosphate buffer solution (d-PBS) was prepared by dissolving 1.1833 g of potassium phosphate KH2PO4 and 4.3205 g of sodium phosphate NaHPO4 in 1 L of D2O to give a solution of pH of 7.2. A 1.0 mg/mL solution of delipidized HSA was made by dissolving 250 mg of HSA powder (Sigma, 99%, virtually globulin free, amino acid content < 0.005%) in 250 mL of d-PBS. A 1.0 mg/mL HGG solution was prepared in the same manner by dissolving 250 mg of HGG powder (Sigma, 99%, γ-Globulins prepared from Cohn Fraction II, III,
