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 CH3-SAM surface and are preferentially excluded from LYS. Interestingly, TMAO molecules adsorb strongly on a hydrophobic CH3-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 chemistry influences protein behavior is lacking. Guidelines for designing protein-resistant surfaces have emerged from studies of protein interactions with self-assembled monolayers of alkanethiolates 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) (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.
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electrically neutral.6 Poly(ethylene glycol) (PEG)10-15 and zwitterionic surfaces4,16-19 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 hydrophobic 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 CH3SAMs, on our formed-in-place (TMAO)CH3-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 (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.
Published on Web 03/24/2010
DOI: 10.1021/la100363m
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reduced for the TMAO-treated surface compared with the bare CH3 surface. This is consistent with the observed lower protein adsorption for the CH3-SAM þ TMAO surface and comparable to that for the PEG surface. The “formed TMAO layer” was stable over many hours and up to temperatures of 40 °C. To obtain molecular-level insights, we present results from molecular dynamics simulations of TMAO solutions at hydrophobic CH3-SAMs. Simulations show favorable binding of TMAO to CH3-SAM surfaces. Surprisingly, TMAO molecules bind to the surface in a “side-on” orientation and not with their polar oxygen atom pointing to the water phase. Our analysis highlights the role of hydration of surfaces and of TMAO molecules in determining both binding and orientational preferences. Additional simulations of TMAO-protein-water solutions strongly indicate exclusion of TMAO molecules from the protein vicinity. We hypothesize that binding of TMAO molecules at a CH3-SAM surface coupled with their preferential exclusion from the protein surface is responsible for making a TMAO-rich hydrophobic SAM interface protein-resistant. Our results provide a simple route toward making proteinresistant surfaces. In addition, they suggest possible extensions of this approach: other small molecules that bind to a given surface, but are excluded from the protein vicinity, would constitute excellent candidates to reduce nonspecific protein adsorption to surfaces. Surface modification by protecting osmolytes for protein resistance can potentially expand the existing repertoire of guidelines.
Experimental Section Materials. Lysozyme (LYS), ribonuclease A (RNase A), bovine serum albumin (BSA), immunoglobulin (IgG), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) hydrochloride carbodiimide (EDC), trimethylamine-N-oxide (TMAO), N,N-dimethylheptylamine-N-oxide (MHAO), betaine, taurine, sucrose, 1-undecanethiol, and 11-mercaptoundecanoic were purchased from Sigma-Aldrich Chemicals, St. Louis, MO. Triethylene glycol undecanethiol (SH-(CH2)11-(OCH2CH2)3-OH) was purchased from Prochimia, Sopot, Poland. An electron beam evaporator was used under a high vacuum (10-7 Torr) to coat glass coverslips (0.20 mm, Corning, New York) with a 15 nm layer of titanium (Ti, 99.999% International Advanced Materials, Spring Valley, NY) followed by a 50 nm layer of gold (99.999%, International Advanced Materials). All the solutions were prefiltered using 0.22 μm poly(vinylidine difluoride) (PVDF) membranes (Millipore corporation, Bedford, MA). Atomic force microscope (AFM) cantilevers modified with 10 μm borosilicate glass spheres followed by gold coating were purchased from Novascan, Ames, IA. Methods. QCM-D. A quartz crystal microbalance with dissipation (QCM-D) with a resolution of less than 1 ng/cm2 (D300 System, Q-Sense AB, G€ oteborg, Sweden) was used to measure protein adsorption. Gold-coated thin piezoelectric quartz discs oscillate (fundamental frequency ∼ 5 MHz) in shear mode at the resonant frequency by means of an oscillating electric field. The lateral amplitude of the vibrating crystal is 1-2 nm. Assuming no slip boundary condition, the change in frequency, Δf, induced by adsorption of mass Δm is related by22 Δf ð1Þ Δm ¼ -C n where C = 17.7 ng cmHz, n is the overtone number (n = 3,5,7), and f is the frequency of the overtone. Surface Functionalization. Seven different chemistries on AT cut gold QCM-D sensor crystals were tested for adsorption of 1 μM lysozyme solution in phosphate buffered saline (PBS): (1) (22) Sauerbrey, G. Z. Phys. A: Hadrons Nuclei 1959, 155(2), 206–222.
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clean gold surface (Au), (2) TMAO adsorbed on gold from a 300 mM solution in PBS buffer for 24 h (Au þ TMAO), (3) -CH3 functional group terminated SAM (HS(CH2)10CH3) on gold (CH3-SAM), (4) TMAO adsorbed from a 300 mM solution in PBS buffer for 24 h on -CH3 functional group terminated SAM (CH3-SAM þ TMAO), (5) -CONH2 functional group terminated SAM on gold (HS(CH3)10CONH2) (CONH2-SAM), (6) TMAO adsorbed from a 300 mM solution in PBS buffer for 24 h on -CONH2 functional group terminated SAM (CONH2SAM þ TMAO), and (7) -EG3OH (HS(CH2)11(OCH2CH2)3OH) functional group terminated SAM on gold (PEG-SAM). SAMs were formed on the crystals by soaking in 2 mM solution of HS(CH2)10CH3 in ethanol for 12 followed by rinsing with ethanol and then drying under nitrogen. Gold-coated AT-cut quartz crystals, with a fundamental frequency of ∼5 MHz, were cleaned by immersion in a 1:1:5 mixture of H2O2 (30%), NH3 (25%), and distilled water at 60 °C for 20 min. The cleaned crystals were then washed with a distilled water/ethanol mixture (50:50) and then dried with nitrogen gas. They were then exposed to UV-ozone for 10 min and were thoroughly rinsed with ethanol and dried under nitrogen before use. Contact Angle. Sessile drop contact angles of clean deionized water drops on solid substrates in air were measured using an optical system (SIT camera, SIT66, Dage-MTI, Michigan, IN) connected to a video display. Each contact angle measurement was repeated 5 times, and an average value with error is reported. AFM. The “molecular puller” was a 1-D AFM (MFP-1 Asylum Research, Santa Barbara, CA). Borosilicate glass spheres of 10 μm diameters were attached to the AFM cantilevers tips. Gold-coated AFM cantilevers were functionalized with SAMs and TMAO in a similar way to gold-coated QCM crystals. Lysozyme was grafted onto a carboxy-SAM layer on coverslips by the N-hydroxysuccinimide chemistry.23 70-80 direct adhesion energy measurements in 10 mM PBS buffer at pH 7.4 and temperature 24 °C were obtained between the grafted lysozyme and the chemically modified cantilevers. The Derjaguin approximation24 was used to convert the measured adhesion force, Fa, into the adhesion energy of interaction per unit area, Ea, between two flat surfaces (large sphere of radius, R, and a flat substrate). The mean value for each surface was normalized by the maximum adhesion energy, Emax, measured between a particular surface and grafted LYS. For our study, Emax was between the CH3-SAM and LYS (0.328 ( 0.03 mN/m). Molecular Dynamics Simulations. The all-atom model of the TMAO molecule developed by Kast et al.25 was used. (See Supporting Information for force field parameters.) Although TMAO is electrically neutral, there is significant partial charge separation. The oxygen atom has a partial charge of -0.65e that is balanced largely by the buried nitrogen and partly by other atoms. The charge on oxygen is comparable to that on water oxygen (∼ -0.85e). Two different sets of detailed all-atom MD simulations were performed: (i) CH3- and CONH2-SAMs submerged in TMAO solution and (ii) lysozyme in TMAO solution, as described below. CH3- and CONH2-SAM Surfaces. We used self-assembled monolayer (SAM) surfaces, which have been well-characterized by our group recently.26-28 Briefly, the SAM comprises a 10 carbon atom long alkyl chain, attached to a sulfur atom on one end and to a CH3- or CONH2- headgroup on the other end. All headgroup atoms are represented explicitly, using the (23) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71(4), 777–790. (24) Derjaguin, B. V. Kolloid Z. 1934, 69, 155–164. (25) Kast, K. M.; Brickmann, J.; Kast, S. M.; Berry, R. S. J. Phys. Chem. A 2003, 107(27), 5342–5351. (26) Jamadagni, S. N.; Godawat, R.; Garde, S. Langmuir 2009, 25, 13092–13099. (27) Shenogina, N.; Godawat, R.; Keblinski, P.; Garde, S. Phys. Rev. Lett. 2009, 102(15), 156101. (28) Godawat, R.; Jamadagni, S. N.; Garde, S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15119–15124.
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AMBER-PARM 94 force-field parameters.29 A SAM slab spanning an area of 3.46 3.5 nm2 (56 headgroups) was used. The system was solvated with ∼1650 water molecules and 20 TMAO molecules, corresponding to a concentration of ∼0.5 M. Umbrella Sampling. To calculate the TMAO-SAM surface potential of mean force (PMF), we used umbrella sampling along the surface normal. A harmonic potential was applied on the TMAO center of mass in each of 13 sampling windows (0.1 nm spacing). The histograms from each window were combined using WHAM30 to generate the PMF, W(z). We also performed simulations of the protein lysozyme (PBD code 1LYZ) in 0.5 M TMAO solution. We used the all-atom AMBER PARM-94 force field29 to describe the protein. The temperature and pressure were maintained at 300 K and 1 atm, respectively, using the Berendsen coupling scheme.31,32 The allatom extended simple point charge (SPC/E) model32 was used for water. Electrostatic interactions were handled using the particle mesh Ewald method.33 All simulations were performed using GROMACS.34,35 Further details on MD simulations is in the Supporting Information.
Table 1. Sessile Contact Angles of Water in Air on Different Surface Chemistries #
surfacea
sessile contact angleb θ°
wettability cos θ
1 2 3 4 5 6 7
Gold (Au) Au þ TMAO CH3-SAM CH3-SAM þTMAO CONH2-SAM CONH2-SAM þ TMAO (PEG)3-SAM
83 ( 2 83 ( 1 106 ( 2 78 ( 2 17 ( 1 22 ( 1 38 ( 1
0.11 0.12 -0.27 0.21 0.95 0.92 0.79
a
b
Self-assembled monolayers of alkanethiol on gold-coated mica. Mean of at least 5 measurements.
Results and Discussion Surface Characterization. Surfaces were characterized by measuring sessile water droplet contact angle, θ, in air (Table 1). Two important observations can be made: first, the contact angle on bare gold was similar to that of gold after TMAO was allowed to adsorb for 24 h indicating that TMAO does not bind strongly to a gold surface; second, there was a decrease in the contact angle from ∼106° for CH3-SAM to ∼78° for the TMAO-treated CH3-SAM. suggesting that TMAO binds to the hydrophobic CH3-SAM relatively strongly. This initial result provided the impetus to test the protein resistance of TMAO-treated CH3SAM surfaces, and also to test the stability and model the behavior of the adsorbed TMAO on such a surface. Lysozyme Adsorption. Adsorption isotherms of lysozyme (LYS, pI 11; MW 14 388 Da) on various surfaces are presented in Figure 1. The isotherms were measured at 24 °C in 10 mM PBS buffer at pH 7.4 for a 1 μM protein solution. The QCM-D sensor crystals were first equilibrated with the background PBS buffer (control), and a baseline was obtained before injection of protein solutions in the same buffer. In Figure 1, LYS solution was introduced at time t = 0. The proteins were then allowed to adsorb for 60 min. After 60 min, the LYS solution in the QCM-D chamber was flushed out and replaced by PBS buffer to remove nonspecifically and loosely adsorbed protein. The total mass of adsorbed protein dropped after this wash and remained steady thereafter for all cases. The extent of adsorption was largest on the bare gold surface (Au) (∼300 ng/cm2) followed by the gold surface coated with TMAO (Au þ TMAO) (∼200 ng/cm2). For the latter, the adsorption isotherm showed a positive slope with time, i.e., the amount adsorbed had not completely equilibrated, suggesting that the TMAO layer was unstable on gold and was being replaced by the protein. At long times, we expect it to reach the (29) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 2002, 117(19), 5179–5197. (30) Kumar, S.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A.; Rosenberg, J. M. J. Comput. Chem. 1992, 13(8), 1011–1021. (31) Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. J. Chem. Phys. 1984, 81(8), 3684–3690. (32) Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. J. Phys. Chem. 1987, 91 (24), 6269–6271. (33) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. J. Chem. Phys. 1995, 103(19), 8577–8593. (34) Berendsen, H. J. C.; Vanderspoel, D.; Vandrunen, R. Comput. Phys. Commun. 1995, 91(1-3), 43–56. (35) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7(8), 306–317.
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Figure 1. Adsorption isotherms of lysozyme on seven different surfaces obtained using the QCM-D at 24 °C. The mass of LYS adsorbed (ng/cm2) is shown with time. After 60 min, the nonadsorbed and loosely attached LYS molecules were removed by washing with PBS buffer for 10 min.
bare gold value of ∼300 ng/cm2, consistent with the wettability data in Table 1. Further, LYS adsorption on CONH2-SAM þ TMAO (∼80 ng/cm2) was similar to that on the bare CONH2-SAM alone (∼90 ng/cm2). However, it was for the hydrophobic CH3-SAM surface that a significant drop in the amount of LYS adsorbed was observed: from ∼135 ng/cm2 for the CH3SAM to about ∼30 ng/cm2 for the CH3-SAMþTMAO. The adsorption on the TMAO coated CH3-SAM, although higher, is comparable to that for the (PEG)-SAM (∼12 ng/cm2), a very protein-resistant surface.6 The drop in the amount of protein adsorbed was consistent with the increase in the wettability of the TMAO treated CH3-SAM. This result indicates that, under the conditions tested, TMAO bound relatively strongly to CH3-SAM, and LYS did not easily displace it. It also suggests the exciting possibility that, if the TMAO coating is stable, it could be used to coat similar hydrophobic surfaces and render them protein-resistant. Protein-Surface Interaction Forces. To characterize the interaction between lysozyme and the different surfaces, multimolecular force spectroscopy (MMFS) measurements were performed. Gold-covered AFM cantilevers were modified with 10 μm borosilicate spheres that were prepared with different chemistries (Table 1). The adhesion energy between lysozyme, which was grafted via its exposed lysines, onto gold-coated flat glass coverslips using succinimide chemistry23 and the functionalized cantilever tips was measured using AFM. The results, shown in Figure 2, are in agreement with the adsorption profiles monitored using QCM-D. The normalized adhesion energy, Ea/Emax, between the grafted lysozyme and both the bare gold (#1, 0.77 ( 0.15) and TMAO-treated gold surface (#2, 0.75 ( 0.12) was rather high, indicating that TMAO did not adsorb strongly on gold. In contrast, values for CONH2-SAM and CONH2-SAM DOI: 10.1021/la100363m
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Figure 2. Normalized adhesion energy, Ea/Emax, between seven different modified cantilever tips (with sphere of diameter 10 μm) and LYS grafted on a flat gold-coated glass coverslip. Each bar represents the mean obtained by a Gaussian fit over a set of 70-80 measurements, and the error is the standard deviation. The CH3SAM surface showed the maximum adhesion energy (Emax = 0.328 ( 0.03 mN/m): (1) Au, (2) Au þ TMAO, (3) CH3-SAM, (4) CH3-SAM þ TMAO, (5) CONH2-SAM, (6) CONH2SAM þ TMAO, (7) PEG-SAM. Insert: Diagram showing the CH3-SAM coated microsphere on the cantilever (top) interacting with tethered lysozyme (bottom) with energy Ea.
þ TMAO were low; (#5, 0.06 ( 0.01) and (#6, 0.04 ( 0.01), respectively. Like the adsorption isotherms, values were very different for the CH3-SAM prior to and after treatment with TMAO; (#3, 1.0 ( 0.09) and (#4, 0.11 ( 0.01), respectively. This large drop in adhesion energy suggests that TMAO dramatically weakened the interaction between the CH3-SAM and lysozyme. As expected, the PEG surface showed the minimum adhesion energy when interacting with lysozyme (#7, 0.01). Thermal and Temporal Stability of the Adsorbed TMAO Layer. To determine the thermal stability of the adsorbed TMAO layer, we performed contact angle measurements as a function of temperature. TMAO was allowed to adsorb onto the CH3-SAM for 24 h. Sessile drop contact angles, θ, were then measured over the temperature range 23-55 °C. At each temperature, the sample was equilibrated for 30 min before the temperature was ramped up to the next value. Similar measurements were also carried out for the untreated CH3-SAM. The data in Figure 3a show that the wettability remained essentially constant at ∼-0.2 ( 0.02 (or ∼106.2°) for the CH3-SAM surface in the temperature range 23-55 °C, whereas for the CH3SAM þ TMAO surface, wettability remained fairly constant at ∼0.15 (or ∼78.2°) below 40 °C, and gradually approached that of the bare CH3-SAM surface at higher temperatures. This suggests that the TMAO layer was stable on the CH3-SAM up to ∼40 °C and that the TMAO molecules left the surface upon further heating. This also suggests the possibility that the nature of the surface can be toggled between protein-phobic and -philic by simply raising the temperature to about 40 °C;a very modest increase over ambient conditions. Practical application of using TMAO to reduce protein adsorption will require that the adsorbed osmolyte layer also be stable over time. To test this, we performed a lifetime stability experiment at 24 °C to measure the amount of TMAO adsorbed on a CH3-SAM substrate prior to and after rinsing with PBS buffer each for 600 min (Figure 3b). The amount of TMAO adsorbed reached a value of ∼240 ng/cm2after equilibration for 600 min in 0.3 M TMAO solution, which reduced to ∼100 ng/cm2 9698 DOI: 10.1021/la100363m
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Figure 3. (a) Wettability measurements as a function of temperature (30 min at each temperature) for a CH3-SAM surface (b) and CH3-SAM þ TMAO surface (after adsorbing TMAO for 24 h) ()). (b) Temporal stability of TMAO-coated onto CH3-SAM surface using QCM-D at 24 °C. TMAO was allowed to adsorb on the CH3-SAM surface for 600 min and then rinsed with PBS buffer for another 600 min to remove nonadsorbed and loosely attached TMAO.
Figure 4. Adsorbed mass (ng/cm2) of four different proteins (lysozyme (LYS), ribonuclease A (RNase A), bovine serum albumin (BSA), and immunoglobulin (IgG)) on CH3-SAM (black bars), and TMAO treated CH3-SAM (red bars). The inset shows the percentage reduction of adsorbed mass by the physisorbed TMAO on CH3-SAM as compared with CH3-SAM alone.
after washing and remained roughly constant over 600 min. Thus, the TMAO coating was stable in spite of extensive washing. Test for Larger Proteins and Additional Osmolytes on CH3-SAM Surface. We also tested the efficacy of TMAO on CH3-SAM surface to resist adsorption for other proteins and found that the layer is protein-resistant for proteins of similar size as LYS, but does not perform well for larger proteins. Figure 4 shows the adsorption of ribonuclease A (RNase A, MW: 14 kDa), lysozyme (LYS, 14 kDa), bovine serum albumin (BSA, 66 kDa), and immunoglobulin-γ (ΙgG, 150 kDa) on CH3-SAM surface and TMAO-treated CH3-SAM and it appears that the reduction in protein adsorption decreases exponentially with increasing molecular weight of the protein (insert in Figure 4). Larger proteins may require higher concentation of TMAO at the surface, but such a high concentration cannot be achieved under Langmuir 2010, 26(12), 9695–9702
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Figure 6. TMAO-SAM potential of mean force (PMF), W(z)/
Figure 5. Adsorbed mass of LYS (ng/cm2) on CH3-SAM (black
bars) and CH3-SAM treated with different osmolytes (TMAO, N, N-dimethylheptylamine-N-oxide (MHAO), betaine, sucrose, and taurine) (red bars).
adsorption-desorption equilibrium. The other reason for the lower effectiveness of TMAO with larger proteins might be that larger proteins displace noncovalently bound TMAO from the surface, a situation which could be remedied by a SAM presenting the N-oxide surface group that binds to the CH3-SAM more tightly. Such a SAM is, however, difficult to synthesize because the N-oxide of tertiary amines will oxidize the thiol group into the corresponding disulfide bond; moreover, further oxidation of disulfide functionality into thiosulfinates or sulfonates cannot be excluded. Hence, we also tested alternatives to TMAO by choosing different osmolyte molecules like betaine, taurine, and sucrose, but they were not found to be as effective as TMAO. However, N,N-dimethylheptylamine N-oxide hydrate, a higher alkyl homologue of TMAO and therefore structurally similar, was found to be as effective as TMAO in minimizing adsorption of small proteins like LYS but was again less effective for larger proteins like IgG (Figure 5). The effectiveness of TMAO at excluding proteins is consistent with it being the most protecting osmolyte,36 and as we show using molecular simulations, it can bind strongly to CH3-SAM surfaces. This significant binding is due to the relatively large fraction of hydrophobic surface area of the TMAO molecule. For comparison purposes, a plot of amount of adsorbed protein without water in molar units would be instructive. Since we did not know how much water adsorbed together with each protein, this plot was not undertaken. Subtracting mass of protein (surface plamon resonance) from mass of adsorbed water and protein (QCM-D) would give amounts of adsorbed water associated with the proteins.
Molecular Dynamics Simulations MD simulations focused on the following key questions: (i) Do TMAO molecules bind to CH3-SAM surfaces? And if so, what are the molecular origins of that binding? How do the bound TMAO molecules orient at the interface? (ii) How do TMAO molecules interact with a protein? And what is the origin of protein resistance of TMAO-treated CH3-SAM surfaces? Protecting osmolytes, such as TMAO, are thought to be preferentially excluded from a protein surface;37-39 we expect a similar mechanism (36) Street, T. O.; Bolen, D. W.; Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103(38), 13997–14002. (37) Lin, T.-Y.; Timasheff, S. N. Biochemistry 2002, 33(42), 12695–12701. (38) Liu, Y.; Bolen, D. W. Biochemistry 1995, 34(39), 12884–91. (39) Schellman, J. A. Biophys. J. 2003, 85(1), 108–25.
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kT, for CH3- (red) and CONH2-SAM (blue) surfaces from umbrella sampling simulations. Inset: TMAO density profile normalized by the density in the bulk water phase, predicted from the PMF, W(z), in the CH3- and CONH2-SAM systems.
at work if the surface concentration of TMAO molecules were enhanced. Also, TMAO molecules may bind to the surface via its three methyl groups with its polar oxygen pointing toward the bulk water phase, rendering the CH3-SAM-water surface relatively polar and reducing binding of other solutes. TMAO-SAM Potential of Mean Force (PMF). The TMAO-SAM PMF quantifies the free energy of interactions of TMAO molecules with the SAM surface. Figure 6 shows the TMAO-SAM PMFs for CH3- and CONH2-SAM systems. Both profiles show a minimum in free energy for TMAO near the surface followed by oscillations that reflect packing of the solvent at the interface. The difference is striking, however. At the CH3-SAM surface, binding is favored by a free-energy minimum of ∼2.7 kT (∼6.75 kJ/mol), whereas the minimum at the CONH2-SAM surface is shallower, only about ∼1 kT, roughly equal to the thermal energy of molecules. These PMFs can be used to predict the density profile of TMAO molecules using Fo exp(-W(z)/kT) (inset, Figure 6), where Fo is the density of TMAO in the bulk. Near the CH3 surface, the predicted TMAO density is about 15 times that in the bulk, suggesting stronger binding there. The binding at CONH2 surface is comparatively weak and would not significantly alter the nature of that surface as observed in Figures 1 and 2. Orientation of TMAO. One may expect the adsorbed TMAO molecules to orient with the three methyl groups in contact with the hydrophobic SAM surface, and the polar oxygen pointing toward the bulk water phase. Surprisingly, Figures 7 and 8a indicate that the TMAO molecules are oriented mostly “sideon”, i.e., with their N-O vector lying parallel to the surface. Specifically, the probability distribution of R, the angle made by the N-O vector with the surface normal (Figure 8a), shows a sharp peak at R ≈ 90°, confirming suggestions of the instantaneous snapshot in Figure 7. The preference for the “side-on” orientation is intriguing indeed. Clues into the factors governing the orientation come from examining density profiles of TMAO atoms and that of solvent water in the direction normal to the surface (Figure 8b). The atomic density profiles display typical stacking characteristic of liquids, and the first peaks of TMAO oxygen and nitrogen atoms are located at the same distance from the SAM surface, confirming the surface parallel orientation of the N-O vector. Importantly, the locations of the TMAO-O and water-oxygen peaks also coincide, indicating that the TMAO N-O vector aligns itself such that the oxygen fitted into the plane of high water-oxygen density. Figure 8b also shows the density profile for water hydrogens. That their first peak was also at about the same DOI: 10.1021/la100363m
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Figure 7. Snapshot from the simulation showing the CH3-SAM (blue) and TMAO molecules (oxygen, red; nitrogen, blue; carbon, cyan; and hydrogen, white). Water molecules are represented by small red dots.
Figure 8. (a) Orientational distribution of the TMAO molecule at the interface and in bulk water. R is the angle made by the N-O vector of the TMAO molecule with the surface normal (schematic, panel d). The probabilities, P(R), were normalized by sin(R) due to the spherical geometry. The interface corresponds to the location where the TMAO-oxygen density profile (panel b) shows a peak. (b) Density profile of TMAO oxygen (red) and nitrogen (green) atoms, water oxygen (dark blue) and hydrogens (light blue), and CH3-SAM (gray) phase along the normal to the interface. (c) Average number of hydrogen bonds made by the TMAO oxygen with water molecules as a function of its orientation at the interface and in the bulk. (d) Schematic showing the orientation of the TMAO molecule for R = 0°, 90°, and 180°.
location is consistent with general understanding of water orientation at extended hydrophobic surfaces.40-44 Specifically, the plane of water molecules was primarily parallel to the hydrophobic SAM surface, with some tendency of the water hydrogens to point toward the surface, reflected in the tail of hydrogen density at shorter separations. By locating itself in the plane of (40) Chyuan-Yih, L.; McCammon, J. A.; Rossky, P. J. J. Chem. Phys. 1984, 80 (9), 4448–4455. (41) Lee, S. H.; Rossky, P. J. J. Chem. Phys. 1994, 100(4), 3334–3345. (42) Patel, H. A.; Nauman, E. B.; Garde, S. J. Chem. Phys. 2003, 119(17), 9199– 9206. (43) Richmond, G. L. Chem. Rev. 2002, 102(8), 2693–2724. (44) Pratt, L. R.; Pohorille, A. Chem. Rev. 2002, 102(8), 2671–92.
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Figure 9. Preferential interaction parameter, Γ, calculated for TMAO molecules, as a function of the distance, r from the surface of lysozyme and the CH3-SAM. Lysozyme, with the atoms colored according to the frequency with which a TMAO molecules is within 0.4 nm during a MD trajectory is also shown. Red and blue indicate high and low frequency, respectively.
maximum water oxygen and hydrogen densities, the TMAO oxygen was able to participate better in hydrogen bonding with water, while optimizing other interactions (see below). Thus, Figure 8b suggests that the structure and hydrogen bonding of water molecules near the CH3-SAM surface contributed to the orientation of TMAO molecules at the interface. We also calculated the number of hydrogen bonds the TMAO-oxygen atom makes as a function of its orientation (Figure 8c). In bulk water, the TMAO-O forms ∼3 hydrogen bonds on average. Figure 8c also shows the number of hydrogen bonds TMAO-O can form when it is located in the plane of maximum water-oxygen density and the rest of the TMAO molecule is rotated around it. For R = 0° (180°), the N-O vector would be pointing upward (downward). For R < 60°, there were significant steric interactions between methyl groups of TMAO and the surface, leading to a low probability of such orientations consistent with Figure 8. For R > 60°, the number of possible hydrogen bonds increased monotonically, reaching 3 for R > 150°. However, for R > 150°, methyl groups of TMAO would be fully detached from the surface leading to a significant loss of hydrophobic interactions between TMAO and the CH3-SAM surface. The collective effects of hydrogen bonding, steric repulsions, and hydrophobic interactions dictate the strong preference for “side-on” orientations for TMAO molecules at the CH3SAM interface. TMAO-Protein Interactions in Solution. A large body of work on protecting osmolytes suggests that these molecules are preferentially excluded from the protein vicinity.37-39,45,46 The exact mechanism of what leads to the exclusion is not clear. Seminal work from the Bolen group36,38,47 indicates that it is the unfavorable water-mediated interaction between the protein backbone and TMAO;the so-called osmophobic effect;that dominates TMAO effects on proteins.38 To explore whether we observe such an exclusion, and to understand specific protein-TMAO interactions in water, if any, we analyzed MD simulations of lysozyme in 0.5 M TMAO solution. We focused on the probability of observing a TMAO molecule within 4 A˚ of a given atom of a protein. The results in Figure 9 are striking indeed. Near a large fraction of the protein (45) Schellman, J. A. Biophys. Chem. 2002, 96(2-3), 91–101. (46) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. U.S.A. 2004, 101(17), 6433–8. (47) Bolen, D. W.; Rose, G. D. Annu. Rev. Biochem. 2008, 77(1), 339–362.
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surface, shown in blue, there is very small probability of observing a TMAO molecule, indicating exclusion of TMAO molecules from the protein vicinity. The small fraction of the protein surface that displays direct interactions with TMAO molecules is shown in red in Figure 9. The polar oxygen of TMAO is expected to form hydrogen bonds with basic residues such as arginine, aspargine, and lysine.46 These are indeed the residues with which TMAO interacts most often (see Supporting Infomation). Even in those cases, the local context (e.g., chemistry, topology) had significant effects, and not all arginines or aspargines displayed sizable interactions with TMAO. The exclusion of TMAO from the protein can be quantified more globally by calculating the so-called preferential interaction coefficient, Γ,48-50 defined as NW =NTMAO ð2Þ Γ ¼ NTMAO 1 nW =nTMAO where NW and NTMAO are the number of TMAO and water molecules within a cutoff distance, r, of the protein surface and nW and nTMAO are the number of respective molecules in the bulk. A negative value of Γ indicates depletion, and a positive value indicates local enhancement of molecules in the vicinity. Figure 9 shows Γ values for TMAO molecules calculated as a function of cutoff distance from lysozyme. A similar calculation for TMAO molecules near CH3-SAM is also shown. The exclusion of TMAO from the vicinity of protein is indicated by negative values of Γ. The broader exclusion zone is about 1.0 nm thick, indicating that the effect of the protein on TMAO propagates up to about three hydration layers. In contrast, the enhancement of TMAO near the CH3-SAM is apparent in the positive value of Γ in the vicinity. The two sets of simulations: (i) TMAO solution at a CH3SAM surface, which shows significant enhancement of TMAO in the vicinity of the interface, and (ii) lysozyme in TMAO solutions, which showed that TMAO was largely excluded from the protein surface, provide indirect evidence for a mechanism by which the TMAO treated CH3-SAM resists protein adsorption. Because TMAO concentrated near the CH3-SAM interface and was inherently excluded from proteins, it seems reasonable to observe protein resistance near a TMAO-covered CH3-SAM surface. It also suggests that other osmolytes and small molecules, which are excluded from the protein surface, can similarly assist in reducing protein adsorption, if such molecules also bind relatively strongly to the surface in question. The above mechanism relies on the preferential exclusion of TMAO near protein playing a major role in protein resistance. Alternatively, it may be that protein resistance is only a result of TMAO adsorption on CH3-SAM making it somewhat hydrophilic. To test this, we performed simulations of binding of a hydrophobic polymer (of freely jointed 25 hydrophobic monomers26) in the presence of CH3 and TMAOþCH3-SAM systems. Jamadagni et al.26 have shown that binding of this polymer to the surface was rather sensitive to the hydrophobicity of the interface, with the polymer binding strongly to the CH3-SAM and being repelled by hydrophilic -OH or -CONH2 SAMs. If TMAO treatment makes the CH3 surface hydrophilic, then it will likewise reduce the strength of polymer binding to the surface significantly. Interestingly, our calculations (see Supporting Information) indicate that TMAO had a negligible effect on the (48) Parsegian, V. A.; Rand, R. P.; Rau, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97(8), 3987–92. (49) Ghosh, T.; Kalra, A.; Garde, S. J. Phys. Chem. B 2005, 109(1), 642–51. (50) Athawale, M. V.; Dordick, J. S.; Garde, S. Biophys. J. 2005, 89(2), 858–866.
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binding of the hydrophobic polymer to the CH3-SAM surface. This observation is consistent with results of Athawale et al.,50 who showed that TMAO did not significantly affect the strength of hydrophobic interactions at pair or many-body levels. In principle, one would similarly like to calculate the binding freeenergy of a protein onto the CH3-SAM in the presence and absence of TMAO. However, such a calculation is computationally prohibitive due to the length and time scales involved, as well as due to conformational space (e.g., protein orientations) that needs to be sampled in such simulations. The results on gold surfaces are still puzzling. Contact angle, QCM-D, and AFM measurements indicate that TMAO does not bind strongly to gold surfaces, and therefore, TMAO treatment of gold did not affect binding of lysozyme. Why TMAO does not bind to a hydrophobic gold surface is not clear. Because of the lack of reliable and efficient classical force fields to describe interactions with gold, simulations to understand TMAO-gold interactions were not performed. We speculate that there could a few reasons why TMAO does not bind to gold: (i) Contact angle measurements indicated that the gold surface was less hydrophobic than CH3-SAMs (Table 1). (ii) The gold surface is smoother than CH3-SAM surfaces, and surface roughness might play a role in limiting binding of TMAO. Specifically, any imperfections in the experimental CH3-SAM (which are not accounted for in simulations) will likely enhance TMAO binding there and correspondingly enhance protein resistance. (iii) TMAO is a small molecule that can fit into the interstitial gap (owing to it being of the same length scale as the SAM headgroups) between flexible CH3-SAM chains, and such a mechanism is not possible at a bare gold surface.
Conclusions Design of protein-resistant surfaces is important for a variety of applications. Current methods of creating such surfaces involve complex chemical reactions focused on making the surface more polar and hydrophilic.51 Such methods cannot be employed easily to render surfaces of synthetic membranes or chromatographic resins in modules and columns protein-resistant. We proposed a simple method to achieve this goal. Using QCM-D, we showed that the osmolyte trimethlyamine-N-oxide (TMAO) adsorbed onto hydrophobic CH3-SAM surfaces and significantly reduced the adsorption of lysozyme. Using QCM-D, AFM, and sessile contact angle measurements, we demonstrated that this formedin-place TMAO coat is stable up to 40 °C and cannot easily be removed by rinsing with PBS for 10 h. To further understand the molecular basis of the experimental results, we performed detailed MD simulations, mimicking the experimental systems. Surface-TMAO potentials of mean force profiles showed that binding of TMAO to a CH3-SAM surface was favored by a significant free energy minimum, whereas that minimum was shallow for TMAO near a CONH2-SAM. With respect to orientational preference of the adsorbed TMAO molecules, we showed that they do not adsorb with the polar oxygen pointing toward the bulk water phase (end-on), but instead adopted a mostly side-on orientation. In such orientations, the polar oxygen atom can form more favorable hydrogen bonds with water, and also maintain some hydrophobic contacts between the methyl groups of TMAO and the CH3-SAM surface. Simulations of lysozyme in TMAO solutions showed that TMAO interacted weakly with most of the protein, except for some (basic) residues with which hydrogen bonding was possible. (51) Taniguchi, M.; Belfort, G. J. Membr. Sci. 2004, 231(1-2), 147–157.
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Calculation of the preferential interaction parameter, Γ, indicated that TMAO was preferentially excluded from the protein surface and, in separate simulations, accumulated near a CH3-SAM surface. Additional simulations of the binding of a hydrophobic polymer to the CH3-SAM in the presence and absence of TMAO showed that the strength of binding of the polymer was largely unaffected by the addition of TMAO. The simulations thus provided a molecular basis to explain the experimental results: TMAO molecules bound strongly to the CH3-SAM surfaces and, as a protecting osmolyte, was largely excluded from the lysozyme surface. Thus, binding of lysozyme molecules to the TMAO-rich SAM-water interface became unfavorable, leading to a significant drop in protein adsorption. These results suggest that other small molecules, including natural protecting osmolytes, which bind strongly to a surface, but are excluded from a protein surface, could also reduce the binding affinity of proteins for those surfaces. To test the generality of the approach, we have also used the QCM-D to measure the adsorption of several other proteins (RNase A, BSA, and IgG) with CH3-SAM and TMAO-coated CH3-SAM surfaces and found out that TMAO is most effective with smaller proteins (∼14 kDa). Additionally, we compared the adsorption behavior of LYS with several different osmolytes (betaine, sucrose, and taurine) and a homologue of TMAO (N,N-dimethylheptylamine N-oxide hydrate) and found that only the TMAO-like molecule,
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N,N-dimethylheptylamine-N-oxide (MHAO), was effective in minimizing protein adsorption of small proteins and worked almost as efficiently as TMAO. The process of osmolyte selfassembly is simple, self-forming, and self-healing in aqueous solution and can be used after device assembly with complex geometries and narrow channels that are normally inaccessible to other covalent surface modification methods. It could also be useful for medical applications, since TMAO is naturally found in living cells. Acknowledgment. We acknowledge the partial support of the U.S. Department of Energy, DOE (DE-FG02-90ER14114 and DE-FG02-07ER46429) and the National Science Foundation (Grant No. CTS-94-00610) for this work. S.G. thanks partial financial support of NSF-NSEC (DMR-0642573) grant. We thank Ravi Kane and Lasana Power at Rensselaer Polytechnic Institute for insightful discussions on the properties of osmolytes and for suggesting alternative osmolytes, respectively. Supporting Information Available: Details of the force field used to model TMAO, and results on the interaction of TMAO with different amino acids of lysozyme and how TMAO affects the binding of hydrophobic polymers to hydrophobic CH3 SAM surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.
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