DOI: 10.1021/la100363m 9695 Langmuir 2010, 26(12), 9695–9702 Published on Web 03/24/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society Self-Assembly of TMAO at Hydrophobic Interfaces and Its Effect on Protein Adsorption: Insights from Experiments and Simulations Gaurav Anand, Sumanth N. Jamadagni, Shekhar Garde, and Georges Belfort* The Howard P. Isermann Department of Chemical Biological Engineering, and The Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York Received January 25, 2010. Revised Manuscript Received March 8, 2010 We offer a novel process to render hydrophobic surfaces resistant to relatively small proteins during adsorption. This was accomplished by self-assembly of a well-known natural osmolyte, trimethylamine oxide (TMAO), a small amphiphilic molecule, on a hydrophobic alkanethiol surface. Measurments of lysozyme (LYS) adsorption on several homogeneous substrates formed from functionalized alkanethiol self-assembled monolayers (SAMs) in the presence and absence of TMAO, and direct interaction energy between the protein and functionalized surfaces, demonstrate the protein-resistant properties of a noncovalently adsorbed self-assembled TMAO layer. Molecular dynamics simulations clearly show that TMAO molecules concentrate near the CH 3 -SAM surface and are preferentially excluded from LYS. Interestingly, TMAO molecules adsorb strongly on a hydrophobic CH 3 -SAM surface, but a trade-off between hydrogen bonding with water, and hydrophobic interactions with the underlying substrate results in a nonintuitive orientation of TMAO molecules at the interface. Additionally, hydrophobic interactions, usually responsible for nonspecific adsorption of proteins, are weakly affected by TMAO. In addition to TMAO, other osmolytes (sucrose, taurine, and betaine) and a larger homologue of TMAO (N,N-dimethylheptylamine-N-oxide) were tested for protein resistance and only N,N-dimethylheptylamine-N-oxide exhibited resistance similar to TMAO. The principle of osmolyte exclusion from the protein backbone is responsible for the protein-resistant property of the surface. We speculate that this novel process of surface modification may have wide applications due to its simplicity, low cost, regenerability, and flexibility. Introduction There is considerable interest in understanding and controlling protein adsorption on solid substrates, with applications to surgical instruments, immunoassays, cell culture, contact lenses, drug delivery, biosensors, organ implants, membrane filtration, and chromatographic supports. 1-5 Despite significant effort, 3,6-8 a clear understanding of the mechanism of how substrate chem- istry influences protein behavior is lacking. Guidelines for design- ing protein-resistant surfaces have emerged from studies of protein interactions with self-assembled monolayers of alkane- thiolates on gold as model substrates. 4,6,9 Features of surfaces having low affinity for proteins include (i) hydrophilic character (i.e., high wettability), (ii) a large number of hydrogen bond acceptors, (iii) few or no hydrogen bond donors, and (iv) electrically neutral. 6 Poly(ethylene glycol) (PEG) 10-15 and zwitte- rionic surfaces 4,16-19 have received special attention, as they appear to repel proteins efficiently. The most successful methods for preparing protein-resistant surfaces involve complex covalent reaction schemes. 3,20 Here, we propose a novel and much simpler alternative for altering hydro- phobic substrates using a “formed in place” method. We show that the protecting osmolyte, 21 trimethylamine N-oxide (TMAO), self assembled on the hydrophobic undecanethiol SAM and consequently lysozyme adsorption reduced significantly over this TMAO painted surface. Specifically, we compare the adsorption characteristics of hen egg lysozyme (LYS) on hydrophobic CH 3 - SAMs, on our formed-in-place (TMAO)CH 3 -SAM surface, and on a (PEG)-SAM using a quartz crystal microbalance with dissipation (QCM-D). We also use atomic force microscopy (AFM) to measure the adhesion interaction between these different surfaces and glass slips covered with covalently tethered LYS. AFM measurements show that the protein-surface adhesion energy was significantly (1) Sluzky, V.; Tamada, J. A.; Klibanov, A. M.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 1991, 88(21), 9377–9381. (2) Shi, H. Q.; Tsai, W. B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398(6728), 593–597. (3) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17(20), 6336–6343. (4) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17(9), 2841–2850. (5) Hayden, O.; Lieberzeit, P. A.; Blaas, D.; Dickert, F. L. Adv. Funct. Mater. 2006, 16(10), 1269–1278. (6) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17(18), 5605–5620. (7) Sethuraman, A.; Han, M.; Kane, R. S.; Belfort, G. Langmuir 2004, 20(18), 7779–88. (8) Bearinger, J. P.; Terrettaz, S.; Michel, R.; Tirelli, N.; Vogel, H.; Textor, M.; Hubbell, J. A. Nat. Mater. 2003, 2(4), 259–264. (9) Anand, G.; Sharma, S.; Kumar, S. K.; Belfort, G. Langmuir 2009, 25(9), 4998–5005. (10) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142(1), 149–158. (11) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142(1), 159–166. (12) Lee, S.-W.; Laibinis, P. E. Biomaterials 1998, 19(18), 1669–1675. (13) Ma, H.; Hyun, J.; Stiller, P.; Chilkoti, A. Adv. Mater. 2004, 16(4), 338–341. (14) Scott, E. A.; Nichols, M. D.; Cordova, L. H.; George, B. J.; Jun, Y. S.; Elbert, D. L. Biomaterials 2008, 29(34), 4481–4493. (15) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Biomaterials 2005, 26(30), 5927–5933. (16) Azzaroni, O.; Brown, A. A.; Huck, W. T. S. Angew. Chem., Int. Ed. 2006, 45 (11), 1770–1774. (17) Sun, Q.; Su, Y. L.; Ma, X. L.; Wang, Y. Q.; Jiang, Z. Y. J. Membr. Sci. 2006, 285(1-2), 299–305. (18) Cheng, G.; Zhang, Z.; Chen, S. F.; Bryers, J. D.; Jiang, S. Y. Biomaterials 2007, 28(29), 4192–4199. (19) Li, G.; Cheng, G.; Xue, H.; Chen, S.; Zhang, F.; Jiang, S. Biomaterials 2008, 29(35), 4592–4597. (20) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19(6), 2388–2391. (21) Greenfield, N. J. Nat. Protoc. 2006, 1(6), 2733–2741.