Effect of Surface Wettability on the Adhesion of Proteins - Langmuir

Jul 30, 2004 - Besides significantly broadening the scope of available data on adhesion of proteins on solid substrates, we demonstrate for the first ...
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Langmuir 2004, 20, 7779-7788

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Effect of Surface Wettability on the Adhesion of Proteins Ananthakrishnan Sethuraman, Mina Han, Ravi S. Kane, and Georges Belfort* Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180-3590 Received March 2, 2004. In Final Form: June 4, 2004 Besides significantly broadening the scope of available data on adhesion of proteins on solid substrates, we demonstrate for the first time that all seven proteins (tested here) behave similarly with respect to adhesion exhibiting a step increase in adhesion as wettability of the solid substrate decreases. Also, quantitative measures of like-protein-protein and like-self-assembled-monolayer (SAM)-SAM adhesive energies are provided. New correlations, not previously reported, suggest that the helix and random content (as measures of secondary structure) normalized by the molecular weight of a protein are significant for predicting protein adhesion and are likely related to protein stability at interfaces. Atomic force microscopy (AFM) was used to directly measure the normalized adhesion or pull-off forces between a set of seven globular proteins and a series of eight well-defined model surfaces (SAMs), between like-SAM-immobilized surfaces and between like-protein-immobilized surfaces in phosphate buffer solution (pH 7.4). Normalized force-distance curves between SAMs (alkanethiolates deposited on gold terminated with functional uncharged groups -CH3, -OPh, -CF3, -CN, -OCH3, -OH, -CONH2, and -EG3OH) covalently attached to an AFM cantilever tip modified with a sphere and covalently immobilized proteins (ribonuclease A, lysozyme, bovine serum albumin, immunoglobulin, γ-globulins, pyruvate kinase, and fibrinogen) clearly illustrate the differences in adhesion between these surfaces and proteins. The adhesion of proteins with uncharged SAMs showed a general “step” dependence on the wettability of the surface as determined by the water contact angle under cyclooctane (θco). Thus, for SAMs with θco < ∼66°, (-OH, -CONH2, and -EG3OH), weak adhesion was observed (>-4 ( 1 mN/m), while for ∼66 < θco < ∼104°, (-CH3, -OPh, -CF3, -CN, -OCH3), strong adhesion was observed (e8 ( 3 mN/m) that increases (more negative) with the molecular weight of the protein. Large proteins (170-340 kDa), in contrast to small proteins (14 kDa), exhibit characteristic stepwise decompression curves extending to large separation distances (hundreds of nanometers). With respect to like-SAM surfaces, there exists a very strong adhesive (attractive) interaction between the apolar SAM surfaces and weak interactive energy between the polar SAM surfaces. Because the polar surfaces can form hydrogen bonds with water molecules and the apolar surfaces cannot, these measurements provide a quantitative measure of the so-called mean hydrophobic interaction (∼-206 ( 8 mN/m) in phosphate-buffered saline at 296 ( 1 K. Regarding protein-protein interactions, small globular proteins (lysozyme and ribonuclease A) have the least self-adhesion force, indicating robust conformation of the proteins on the surface. Intermediate to large proteins (BSA and pyruvate kinase-tetramer) show measurable adhesion and suggest unfolding (mechanical denaturation) during retraction of the proteincovered substrate from the protein-covered AFM tip. Fibrinogen shows the greatest adhesion of 20.4 ( 2 mN/m. Unexpectedly, immunoglobulin G (IgG) and γ-globulins exhibited very little adhesion for intermediate size proteins. However, using a new composite index, n (the product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib), to correlate the normalized adhesion force, IgG and γ-globulins do not behave abnormally as a result of their relatively low helix and random (or high sheet) content.

Introduction Although vast experimental literature exists on the adsorption of specific proteins to various polymeric surfaces in defined aqueous solutions, difficulties in determining the underlying reasons for the extent of adsorption have remained.1-4 Many researchers have addressed particular aspects of these difficulties through experiment, theory, and simulation. Heterogeneous substrates such as colloidal poly(tetrafluoroethylene),5-7 titanium oxide,8 polystyrene,5,9 and silica5,10-13 and flat * Corresponding author: G. Belfort. Phone: (518) 276-6948. Fax: (518) 276-4030. E-mail: [email protected]. (1) Norde, W. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; pp 21-43. (2) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 17, 1-63. (3) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340. (4) Norde, W.; Lyklema, J. J. Biomater. Sci. 1991, 2, 183-202. (5) Norde, W.; Zoungrana, T. Biotechnol. Appl. Biochem. 1998, 28, 133-143. (6) Vermeer, A. W. P.; Giacomelli, C. E.; Norde, W. Biochim. Biophys. Acta 2001, 1526, 61-69. (7) Giacomelli, C. E.; Norde, W. Biomacromolecules 2003, 4, 17191726.

sheet synthetic polymeric membranes14-16 have been used for protein adsorption studies. Methods such as circular dichroism7,10 and attenuated total reflection Fourier transform infrared spectroscopy16-18 have been used to interrogate the conformational secondary structure of (8) Bentaleb, A.; Ball, V.; Haikel, Y.; Voegel, J. C.; Schaaf, P. Langmuir 1997, 13, 729-735. (9) Giacomelli, C. E.; Norde, W. J. Biotechnol. 2000, 79, 259-268. (10) Billsten, P.; Fresgard, P.-O.; Carlsson, U.; Jonsson, B.-H.; Elwig, H. FEBS Lett. 1997, 402, 67-72. (11) Wadu-Mesthrigr, K.; Amro, N. A.; Liu, G.-Y. Scanning 2000, 22, 380-388. (12) Giacomelli, C. E.; Norde, W. J. Colloid Interface Sci. 2001, 233, 234-240. (13) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Langmuir 2003, 19, 3848-3857. (14) Koehler, J. A.; Ulbricht, M.; Belfort, G. Langmuir 1997, 13, 41624171. (15) Koehler, J. A.; Ulbricht, M.; Belfort, G. Langmuir 2000, 16, 10419-10427. (16) Sethuraman, A.; Vedantham, G.; Imoto, T.; Przybycien, T.; Belfort, G. Proteins 2004, 56, 669-678. (17) Vedantham, G.; Sparks, H. G.; Sane, S. U.; Tzannis, S.; Przybycien, T. M. Anal. Biochem. 2000, 285, 33-49. (18) Vermeer, A. W. P.; Norde, W. J. Colloid Interface Sci. 2000, 225, 394-397.

10.1021/la049454q CCC: $27.50 © 2004 American Chemical Society Published on Web 07/30/2004

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adsorbed proteins on colloids and flat sheet surfaces, respectively. Several groups have attempted to simplify the investigation of protein adsorption processes through propitious choice of experimental conditions and theoretical assumptions. Thus, heterogeneous surfaces have been replaced by extremely well-defined surface chemistries at the molecular level using self-assembled monolayers (SAMs),19-21 low-temperature plasma,22-24 ultraviolet irradiation methods,25,26 and the grafting of polymers such as poly(ethylene glycol) and poly(vinyl pyrolidinone).27,28 Also, well-defined adsorbates (building blocks) such as amino acids and small peptides with known secondary structure have been used to simulate larger and more complex proteins.29,30 Others have compared the adsorption behavior and structural stability of wild-type proteins with those containing site-specific mutations to probe protein-surface and protein-protein interactions.31 Efforts to model protein adsorption have also shed light on several aspects of the adsorption process (an excellent summary has recently appeared in the literature).32 Lenhoff and co-workers have emphasized the effect of heterogeneous charge distributions on protein molecules and suggested preferred orientation during adsorption for ribonuclease A (RNase A, also see the experiments of Koehler et al. that confirm these predictions)14 and chymotrypsinogen A, because of their relatively large dipole moments.33-35 With regard to solution effects, the solvent composition (hydrogen or other ions) can influence protein adsorption through (i) electrostatics, by affecting the diffuse double layer, by changing the net charge of the substrate and the protein (depending on their isoelectric points, pI), and by inducing coadsorption of small ions,1,36,37 and (ii) disturbance of the water structure near hydrophobic surfaces.32,38 What are the criteria or rules that will help us choose surfaces with protein adhesive resistance? On the basis of mainly amount adsorbed using surface plasmon resonance (SPR), Whitesides’ group has suggested that protein adhesion resistant molecules need to be hydrophilic, to include H-bond acceptors, to not include H-bond donors, (19) Haeussling, L.; Ringsdorf, H.; Schmitt, F. J.; Knoll, W. Langmuir 1991, 7, 1837-1840. (20) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (21) Cheng, S.-S.; Chittur, K. K.; Sukenik, C. N.; Culp, L. A.; Lewandowska, K. J. Colloid Interface Sci. 1994, 162, 135-143. (22) Kiaei, D.; Hoffman, A. S.; Horbett, T. A.; Lew, K. R. J. Biomed. Mater. Res. 1995, 29, 729-739. (23) Lopez, G. P.; Ratner, B. D.; Rapoza, R. J.; Horbett, T. A. Macromolecules 1993, 26, 3247-3253. (24) Ulbricht, M.; Belfort, G. J. Membr. Sci. 1996, 111, 193-215. (25) Yamagishi, H.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 1995, 105, 249-259. (26) Pieracci, J.; Crivello, J. V.; Belfort, G. J. Membr. Sci. 1999, 156, 223-240. (27) Sheu, M. S.; Hoffman, A. S.; Ratner, B. D.; Feijen, J.; Harris, J. M. J. Adhes. Soc. Technol. 1993, 7, 1065-1076. (28) Chen, H.; Belfort, G. J. Appl. Polym. Sci. 1999, 72, 1699-1711. (29) Molnar, I.; Horvath, C. J. Chromatogr. 1977, 142, 623-640. (30) Basiuk, V. A.; In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; pp 45-70. (31) Malmsten, M.; Arnebrant, T.; Billsten, P. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; pp 95-113. (32) Roth, C. M. Lenhoff, A. M. In Biopolymers at Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; pp 71-94. (33) Yoon, B. J.; Lenhoff, A. M. J. Phys. Chem. 1992, 96, 3130-3134. (34) Roth, C. M.; Lenhoff, A. M. Langmuir 1993, 9, 962-972. (35) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11, 3500-3509. (36) Hannemaaijer, J. H.; Robbertsen, T.; Van den Boomgaard, Th.; Olieman, C.; Both, P.; Schmidt, D. G. Desalination 1998, 68, 93-108. (37) Palecek, S. P.; Mochizuki, S.; Zydney, A. L. Desalination 1993, 90, 147-159. (38) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464-3473.

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and are electrically neutral.39 They and others obtained extensive evidence to support these criteria using welldefined SAMs terminating in many functional groups. In most cases, single terminal groups were evaluated, while in others, mixed systems have been used.39 Luk et al.40 report a polyol-terminated SAM as a protein-resistant surface, while Kane et al.41 have designed protein-resistant surfaces based on osmolytes. In many of the protein adsorption studies mentioned, the propensity of a protein to adsorb onto a particular surface was measured by the amount of protein on the surface after some relatively short time period (hours). Many methods such as gravimetric, spectroscopic, inteferometric, and direct protein assays have been used to determine the quantity or thickness of the protein layer adsorbed on the chosen substrate.42 Our group has shown that this could result in erroneous conclusions when estimating the affinity of a surface to adsorb a protein. For example, during the adsorption of relatively rigid asymmetric molecules such as RNase A (hydrated dimensions 28 × 34 × 44 Å) onto mica from aqueous solution, a complete monolayer was attained in about 1 h in the flat-on orientation, but after 24 h of adsorption, the orientation changed to end-on.43 During this period, the amount of protein adsorbed onto the surface varied from 1.7 to 2.5 mg/m2, a 41% increase! Koehler et al.14,15 showed that adsorption of lysozyme (Lys) onto hydrophilic surfaces (hydroxyethyl methacrylate-modified polysulfone, PSf) was dominated by relatively small protein-polymer interactions. However, with a hydrophobic surface (unmodified PSf), protein-polymer and protein-protein interactions were both important and relatively large. Clearly, protein orientation and length of adsorption time can directly affect the amount adsorbed. Therefore, the effect of protein-protein interactions43 needs to be accounted for when trying to obtain a valid measure of the interaction between a protein and a solid substrate (i.e., protein-polymer interactions). For “soft” globular proteins (i.e., those with large adiabatic compressibilities, β) and especially those that have a high propensity to aggregate or change structure on adsorption, taking these considerations into account is especially important. Thus, directly measured adhesion or pull-off forces (rather than amount adsorbed) between a chosen protein and a particular solid substrate is likely to be a more fundamental and less ambiguous measure of protein-surface interactions. However, directly measured adhesion forces have their own peculiar complications, and these are addressed in some detail later. We present results of the direct measurement of the adhesion energy between different mostly globular proteins and surfaces created through SAMs with controlled molecular surface chemistry. We also measure the adhesive forces in aqueous solution between like-SAM layers and between like-protein layers. Protein-substrate interactions can then, in principle, be distinguished from protein-protein interactions. Experimental Section Materials. SAMs and Protein for Atomic Force Microscopy Adhesion Experiments. All materials and reagents were used as received. Alkanethiols, 1-undecanethiol, 11-mercapto-1-unde(39) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605-5620. (40) Luk, Y.-Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 96049608. (41) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 2388-2391. (42) Wang, Y.; Chang, Y.-C. Langmuir 2002, 18, 9859-9866. (43) Lee, C.-S.; Belfort, G. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 8392-8396.

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Table 1. Roughness-Corrected Sessile Contact Angles of Water on SAMs under Cyclooctane and Aira functional group

alkanethiol

θCOb (deg)

cos θCO

θairc (deg)

cos θair

-CH3 -OPh -CF3 -CN -OCH3 -OH -EG3OH -CONH2

HS(CH2)10CH3 HS(CH2)11OPh HS(CH2)2(CF2)9CF3 HS(CH2)11CN HS(CH2)11OMe HS(CH2)11OH HS(CH2)11(OCH2CH2)3OH HS(CH2)10CONH2

164 ( 2 156 ( 3 154 ( 2 144 ( 2 107 ( 2 62 ( 2 50 ( 3 20 ( 2

-0.96 -0.91 -0.90 -0.81 -0.29 0.47 0.64 0.94

107 ( 2 83 ( 2 115 ( 3 58 ( 2 78 ( 2 0.97

a Contact angle values are the mean of at least five measurements. b Contact angles of water under cyclooctane. c Contact angles of water under air.

Table 2. Protein Characteristics proteinc

MW (kDa)

pI

RNAse A Lys BSA IgG BGG Pyr Fib

14 14 69 155 170 237 340

9.5 11.1 4.8 6.1 ∼6.0 8.9 5.5

h × w × l (nm)

adiabatic compressibilitya 1012βs (cm2/dyn)

helix

3.8 × 2.8 × 2.2 4.5 × 3.0 × 3.0 14 × 4 × 4 Fab: 4 × 5 × 4 24 × 4.4 × 4.4 4.5 × 4.5 × 7.5 47 × 5 × 5

1.12 4.67 10.5 8.5 -

23 43 60 7 7 38 42

secondary structureb (%) sheet turns random 33 7 0 45 45 18 7

29 43 0 32 32 33 20

15 7 40 16 16 11 31

a The apparent adiabatic compressibility, β , of a solute is defined by β ) ∆V/∆P| , where P is the pressure applied at constant entropy, s s s S, and V is the volume.45 The flexibility of proteins is reflected in their adiabatic compressibility because it is directly related to the volume b fluctuation. Secondary structural components were obtained using the STRIDE algorithm.46 c All the molecules are monomer in solution except Pyr and Fib, which are tetramers.

canol, and 11-mercapto-1-undecanoic acid, were purchased from Sigma-Aldrich Chemicals (Milwaukee, WI). The remaining alkanethiols 11-phenoxy-1-mercaptoundecane, 1,1,2,2-tetrahydrofluoro-1-dodecanethiol, 11-cyano-1-undecanethiol, 11-methoxy-1-undecanethiol, 1-mercaptoundec-11-yl triethylene glycol, and 11-meraptoundecanamide were synthesized according to well-established protocols20,44 (Table 1). Proteins, RNAse A (bovine pancreas; R5125), Lys (chicken egg white; L6876), serum albumin (BSA, bovine; A7638), immunoglobulin G (IgG, bovine, I5506), γ-globulin (BGG, bovine; G5009), fibrinogen (Fib, human; F4883), and pyruvate kinase (Pyr, rabbit muscle; P9136), were purchased from Sigma-Aldrich Chemicals (Milwaukee, WI; Table 2). 1-Ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (Pierce) and N-hydroxysuccinimide (Fluka) were used for immobilizing the proteins. All buffers and protein solutions were filtered through 0.22-µm filters before use, and the force measurements were made in phosphate-buffered saline (PBS) pH 7.4. Methods. SAMs. Preparation of Gold-Coated Surfaces. Substrates were prepared by evaporation of 1.5 nm of titanium (99.999%, International Advanced Materials) and 50 nm of gold (99.999%, International Advanced Materials), onto glass coverslips (0.20 mm, no. 1-1/2, Corning), silicon substrates, and the atomic force microscope (AFM) probe tips. Evaporation of metals was conducted at a pressure of less than 1 × 10-6 Torr using an electron beam evaporator (Temescal BJD-1800, BOC Edwards, U.K.). The AFM probe tips were plasma-cleaned prior to deposition of gold. Preparation of SAMs on Gold-Coated Surfaces. The gold-coated AFM probe tips were immersed in solutions of the specified alkanethiols, and glass coverslips were immersed in a solution of HS(CH2)11COOH in ethanol (2 mM thiol) for 12 h, rinsed with ethanol, and dried under nitrogen. The SAMs were characterized using contact angle measurements and ellipsometry. The SAMs were immediately used after preparation for adhesion force measurements. The alkanethiols synthesized and used in this study are shown in Table 1. Protein Immobilization on the HS(CH2)11COOH SAM. Specific proteins were covalently immobilized on Au-coated silicon substrates by the coupling reaction between the exposed primary amine groups of the proteins and the carboxyl groups on the HS(CH2)11COOH SAM surfaces prepared on the Au-coated glass substrates. The procedure for the coupling reaction was carried (44) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569.

out as described in Lahiri et al.47 The immobilization involved two steps: generation of reactive N-hydroxysuccinimidyl esters from the carboxylic acid groups on the SAM surfaces and coupling of these groups with exposed amine groups on the proteins. The proteins were not immobilized with a preferred orientation because the covalent immobilization reaction occurred through many accessible lysine residues on the protein surface. AFM imaging of the protein-immobilized surface showed complete coverage of the surface with a root-mean-square roughness of 1.4 ( 0.1 nm. Adhesion Force Measurements. An AFM (AutoProbe CP, Veeco Instruments, Sunnyvale, CA) was used to measure the intermolecular forces. Briefly, the movement of the piezoelectric scanner was used to determine the separation distance, and the cantilever deflection was used to determine the force.48,49 The onset of the region of constant compliance (in which the slope of the cantilever deflection versus sample displacement curve equals the spring constant of the cantilever) was used to determine the zero distance, and the region in which force was unchanged was used to determine the zero force. All measurements were taken at an approach speed of 0.1 Hz. Triangular silicon nitride cantilevers were used (Type A, Veeco Instruments) with a nominal force constant supplied by the manufacturer of 0.05 N/m. Calibration of the cantilevers yielded a value of 0.048 ( 0.01 N/m using the frequency method.50 The cantilevers were then modified by attaching silica spheres with a nominal diameter of 2R ) 20.6 ( 1.3 µm (Duke Scientific, Palo Alto, CA) to the underside of the cantilevers using an inert epoxy resin (Epikote/ Epon 1002F, Shell Chemical Co., Houston, TX). The glue was previously shown to be inert during surface force measurements.51 The measured forces were normalized by the radius of the silica sphere, according to the Derjaguin approximation.52 The modified AFM cantilevers were plasma cleaned to remove any contaminating material from the surface prior to deposition of gold. Force measurements were obtained in an open liquid AFM cell (Veeco (45) Gekko, K.; Hasegawa, Y. Biochemistry 1986, 25, 6563-6571. (46) Frishman, D.; Argos, P. Proteins 1995, 23, 566-579. (47) Lahiri, J.; Isaacs, L.; Yien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790. (48) Ducker, W. A.; Senden, T. J.,; Pashley, R. M. Nature 1991, 353, 239-241. (49) Ducker, W. A.; Senden, T. J. Langmuir 1992, 8, 1831-1836. (50) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403-405. (51) Pincet, F.; Perez, E.; Belfort, G. Langmuir 1995, 11, 1229-1235. (52) Derjaguin, B. V. Kolloid-Z. 1934, 69, 155-164.

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Figure 1. Schematic of experimental setup. Interaction between a surface-modified 20.6-µm diameter silica sphere attached to an AFM cantilever and a surface-modified glass coverslip. The two surfaces are brought together (compression), held, and pulled apart (decompression) in an AFM liquid test cell. The upper surface was covered with SAMs terminated with different functional groups (X) for the SAM-protein experiments (and with proteins for the protein-protein experiments). The lower surface was covered with protein covalently attached to SAM-COOH for the SAM-protein and the protein-protein experiments (and was covered with a SAM for SAM-SAM experiments). Instruments, Santa Barbara, CA). All parts of the cell were first cleaned by soaking in a phosphate-free detergent equivalent to a dichromate-sulfuric acid solution (RBS, Pierce, Rockford, IL) and then copiously flushing with deionized water followed by ethanol. The cell was then dried with ultrahigh purity nitrogen gas under pressure. All cell cleaning and assembly was performed in a laminar flow hood, and all measurements were taken at a temperature of 296 ( 1 K. Finally, the cell was installed into the AFM and the tip (with a surface-modified attached sphere) was brought into contact with the surface-modified glass coverslip (substrate) until force-distance measurements were obtained (Figure 1). The force of adhesion FAD is defined here as the maximum adhesive force observed on retraction (decompression) of the sample away from the tip. 〈FAD〉 represents the average of the maximum adhesive force from a set of 30-50 data points, using three sets of tip-sample systems. To compare the intermolecular forces between different tip geometries, the force was normalized by the radius, R, to give the energy of interaction (FAD/R). The loading force 5 nN and loading rate 0.1 Hz were kept constant throughout all the experiments. The area under the energy (FAD/R)-distance (D) curves is the work needed to separate the two surfaces and to pull apart a protein’s native structure (evident with a steplike decrease in the adhesive forces). Because the measurements involved an unknown number of adsorbed proteins, estimates using Jarzynski’s free energy inequality could not be obtained.53,54 Contact Angle. Contact angle measurements were used to estimate the interfacial energy and wettability of surfaces. The sessile contact angle method was used to measure the contact angles of a water drop on the solid substrates in air or in cyclooctane.38 For all systems, average values were obtained from multiple contact angle values (at least five) using an optical system (SIT camera, SIT66, Dage-MTI, Michigan, IN) connected to a video display. Corrections of the contact angles for roughness were obtained using the methods of Taniguchi et al.55,56 Following Sigal et al.,38 the wettability is defined as the cosine of the sessile contact angle (-0.97 < cos θ < 0.97). (53) Jarzynski, C. Phys. Rev. Lett. 1997, 78, 2690-2693. (54) Liphardt, J.; Dumont, S.; Smith, S. B.; Tinoco, I., Jr.; Bustamante, C. Science 2002, 296, 1832-1835. (55) Taniguchi, M.; Pieracci, J. P.; Belfort, G. Langmuir 2001, 17, 4312-4315. (56) Taniguchi, M.; Belfort, G. Langmuir 2002, 18, 6465-6467.

Results and Discussion Three sets of force measurements were performed: two sets of control experiments involving measurements between two SAM-covered surfaces and between two protein-covered surfaces and a set concerning the measurement of forces between protein and SAM-covered surfaces. Covalently immobilized protein was used in contrast to earlier work in which adhesion of adsorbed protein was studied.14,15,43 Both surface wettability and protein type affect the orientation43 and concentration of adsorbed protein.16,38 The same protein on different surfaces could not only adsorb with different orientations but also adsorb to different final concentrations. Both effects, orientation and surface concentration, were driven by the surface energy of the substrate, the exposed surface functionality of the protein, and the propensity of the protein to aggregate on the surface. Here, immobilization of the protein was random and depended on the sequence location of the surface-exposed lysines. This immobilization method resulted in tethered proteins with different orientations. The probe tip-substrate interactions, however, were due to noncovalent interactions such as hydrophobic, van der Waals, electrostatic, and hydrogenbond interactions. This research focused on the pull-off forces, the maximum of which we term the “adhesive force”. Pull-Off Forces between SAM-SAM Surfaces. A series of control experiments were conducted with different SAMs using interacting probe tips and glass coverslips covered with various alkanethiol SAMs in aqueous solution. In each case, the surfaces were covered with the same SAM such that self-interactions were measured. All the force curves were similar, and the maximum pull-offs occurred at zero separation. Large adhesive interactions were observed between SAM-SAM surfaces characterized with wettabilities 0.55. In Figure 2A, decompression curves (the two surfaces are pulled apart) for SAM-SAM interactions in pH 7.4 PBS buffer are presented. The data show very low adhesion between

Protein Adhesion to Self-Assembled Monolayers

Langmuir, Vol. 20, No. 18, 2004 7783 Table 3. Self-Interacting Pull-Off Forces between Covalently Attached Protein-Protein Covered Surfaces and n, the Protein Composite Index protein

〈F/R〉 (mN/m)

na

RNAse A Lys BSA IgG

0.5 ( 0.2 0.3 ( 0.2 1.8 ( 0.6 0.3 ( 0.1

1.52 2.05 20.00 10.48

protein 〈F/R〉 (mN/m) BGG Pyr Fib

0.2 ( 0.1 4.3 ( 1.2 20.4 ( 5.4

na 11.50 34.10 73.00

a The composite index, n, is obtained from the product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib.

Figure 2. Normalized force measurements for the SAM/SAM system. (A) Several typical normalized force-distance curves for decompression (pull-off) between surfaces covered with the same SAM chemistry on each substrate. (B) Summary of the maximum normalized pull-off (adhesive) forces between SAMcovered cantilever tips and SAM-covered glass coverslips versus wettability (cos θair, where θair is the sessile contact angle of a water drop placed onto the SAM in air) of the SAMs. In PBS buffer at pH 7.4. See Table 1 for list of symbols of SAM functional groups.

hydrophilic surfaces (>-5 ( 0.6 mN/m) and larger attractive forces with single jump-off points for the hydrophobic surfaces (-233 ( 9 mN/m). A summary of the pull-off forces versus wettability for all the SAM surfaces for SAM-SAM interactions (always the same SAM on both surfaces unless stated) is shown in Figure 2B. Very large adhesion was observed for the hydrophobic surfaces (-233 ( 9 mN/m, -CF3, -CH3, -OCH3, and -CN). The surface with the -OPh group showed the largest adhesion possibly due to π-π interactions of the phenoxy group. None of the other surfaces contained aromatic groups. The pull-off forces for the highly wettable surfaces were about 2 orders of magnitude lower (∼-5 ( 0.6 mN/m, -EG3OH, -OH, -CONH2). Adhesion was also measured for the mixed SAMs of undecanethiol and 11mercapto-1-undecanol with wettabilities of cos θ1 ) 0.57 (θ1 ) 55°) and cos θ2 ) 0.31 (θ2 ) 72°). All the force curves were generated in PBS at pH ) 7.4. Clearly, there exists a strong adhesive (attractive) interaction between the least wettable surfaces and minimal interactive energy between the hydrophilic surfaces. Because the polar surfaces can form hydrogen bonds with water molecules and the apolar surfaces cannot, these measurements provide a quantitative measure of the so-called mean hydrophobic interaction (∼-206 ( 8 mN/m, excluding -OPh) in PBS buffer. Because this value appears to be independent of a

particular substrate chemistry or hydrophobic surface, it suggests that the property of the solvent, and in this case the structure of water, is determining. Previous values of force measurements between hydrophobic surfaces have been reported to range from ∼20 mN/m57 to ∼1000 mN/ m58 with several reports between these values.59-61 The difference between these results and ours is likely due to the undefined radius of the pyramidal tip, different solution conditions, and the possibility of incomplete surface coverage. Pull-Off Forces between Like Immobilized Proteins. Interaction forces were measured between a series of proteins covalently immobilized on the tip and on the substrate (SAM-covered glass coverslip). Here, the same protein was covalently attached to the interacting probe tip and the SAM-covered glass coverslip. AFM images confirm complete protein coverage of the surfaces (data not shown). Table 3 lists the pull-off forces for the selfprotein measurements in PBS buffer at pH 7.4. Small globular proteins (RNAse A and Lys) have the least selfadhesion force, indicating robust conformation of the proteins on the surface. They also exhibit low values of adiabatic compressibility in solution (Table 2), indicating a more compact and “less flexible” structure. Increasing the loading force on the protein did not lead to any change in the normalized force-distance curve. RNase A has high sheet content (33%) while Lys contains only ∼7% sheet. IgG and BGG (45% sheet) exhibited very little adhesion. Larger proteins such as BSA (0% sheet) and Pyr (18% sheet) show measurable adhesion and also unfolding during retraction of the protein-covered substrate from the protein-covered AFM tip. The largest protein in the set, Fib (340 kDa; 7% sheet) shows the greatest adhesion of 20.4 mN/m. The normalized force-distance curves for these three proteins, BSA, Pyr, and Fib, were difficult to reproduce and showed steplike behavior during retraction. The “retraction” of the sample away from the tip likely leads to mechanical denaturation of the protein. Figure 3A shows multiple normalized decompressive force curves for separate runs as the Fib surfaces retract from one another. In some cases, contact between the Fib-covered probe tip and glass coverslip can be observed at separations as far as ∼1 µm, suggesting possible unfolding of the protein on the surface. Fib is known to denature on many surfaces.38,62,63 In an attempt to explain the effect of secondary structure and molecular weight of a protein on adhesion, we plot (57) Kokkoli, E.; Zukoski, C. F. Langmuir 1998, 14, 1189-1195. (58) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381-421. (59) Feldman, K.; Haehner, G.; Spenser, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134-10141. (60) Rabonovich, Ya. I.; Yoon, R.-H. Langmuir 1994, 10, 1903-1909. (61) Sinniah, S. K.; Steel, A. B.; Miller, C. J.; Reutt-Robey, J. E. J. Am. Chem. Soc. 1996, 118, 8925-8931. (62) Balasubramanian, V.; Grusin, N. K.; Bucher, R. W.; Turitto, V. T.; Slack, S. M. J. Biomed. Mater. Res. 1999, 44, 253-260. (63) Luck, M.; Paulke, B. R.; Schroder, W.; Blunk, T.; Muller, R. H. J. Biomed. Mater. Res. 1998, 39, 478-485.

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Figure 3. Normalized force measurements for the protein/ protein system. (A) Triplicate normalized force-distance curves for decompression (pull-off) between covalently immobilized Fib-Fib (same protein on both surfaces). In PBS buffer at pH 7.4. (B) Effect of secondary structure and molecular weight of a protein on the normalized self-adhesion force. Normalized adhesion force versus a composite index, n (product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib). In PBS buffer at pH 7.4. y ) 0.0043x2 - 0.0366x; R2 ) 0.9982. See Table 2 for list of symbols of the proteins and for their molecular weights and secondary structural components.

the normalized self-adhesion force versus a composite index, n (product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib), in Figure 3B. The choice of the components of n are based on the following: (i) Dobson et al.64-66 have suggested that the lowest energy state for secondary structure in a protein is likely to contain a high beta sheet content or a low helix and random content. (ii) Recent results from our group have demonstrated a helixto-sheet transition with time during adsorption of Lys on a hydrophobic surface.16 (iii) Experimental results from this work (Table 3) clearly demonstrate an increase in both self-interacting adhesion forces and jump-out distance with increasing molecular weight. The composite index, n, combines these two concepts into one parameter. A smooth increasing parabolic dependence with a correlation coefficient of R ) 0.9984 is observed in Figure 3B, indicating that n adequately tracks self-adhesion. This confirms that helix and random content (or by difference, (64) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329-332. (65) Chiti, F.; Webster, W.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi, G.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 96, 3590-3594. (66) Fandrich, M.; Fletcher, M. A.; Dobson, C. M. Nature 2001, 410, 165-166.

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sheet and turn content) and size (molecular weight) are important. The measured self-interaction energy between unstable proteins (Table 3) is substantially lower than that between well-packed hydrophobic SAMs and is comparable to that between hydrophilic SAMs (Figure 2). Kidoaki & Matsuda67 have measured self-interaction forces for BSA, IgG, and Fib. They were unable to obtain a measurable force for these proteins using a pyramidal tip (Table 3).67 Pull-Off Forces between SAMs and Proteins. Intermolecular adhesion force measurements were generated between a series of SAM surfaces on the cantilever tip and proteins covalently immobilized to SAM-COOH on the glass coverslip. At pH 7.4, the carboxylic acid group was ionized and negatively charged (pKa 5.5). Adhesion between all the SAM surfaces and the SAM-COOH surface was negligible (data not shown). As previously for the self-SAM and self-protein analysis, raw data (normalized force-separation distance curves) for the proteinSAM interactions are shown first (Figure 4), followed by a summary of the adhesion behavior of most of the proteins on all the surfaces (Figures 5 and 6). Representative decompression measurements for RNAse A, Lys, and Pyr with five different apolar SAM surfaces are shown in Figure 4A-C, while a comparison of the decompression behavior of RNAse A, Lys, Pyr, Fib, Pyr, and BGG with one apolar surface, SAM-CH3, is shown in Figure 4D. One immediately notices that low molecular weight and relatively stable proteins such as RNAse A and Lys (∼14 kDa) return to the zero force line at relatively short separation distances, while large proteins such as BGG, Pyr, and Fib (170-340 kDa) return with a steplike profile at very large distances. The SAM-CN surface appears to stretch RNAse A to longer distances (∼30 nm) than the other surfaces (-CF3, 2 nm; -CH3, 15 nm; -OPh, 14 nm; -OCH3, 15 nm). With Lys, CF3- and OCH3-SAM surfaces exhibit very short pull-out distances (200 nm for -OPh, -CH3, and -CN, respectively. The remaining SAM surfaces of -CF3 and -OCH3 showed pull-out distances of ∼92°) to their “hydrophilic asymptotes” (right side, θco < ∼52°) within the same range of wettability (cos θco), suggesting that something other than the proteins and the SAM chemistry is involved, that is, possibly electrostatic effects or water structure. Both “asymptotes” increase with molecular weight of the protein (top to bottom, Figure 6). Also, it appears that the details of the surface chemistry are less important than whether a surface is within the hydrophobic group (-CH3, -OPh, -CF3, -CN, and -OCH3) or the hydrophilic (-OH, -CONH2, and -EG3OH) group. The normalized hydrophobic and hydrophilic asymptotic adhesive forces for large negative and positive values of cos θ (apolar and polar regions), respectively, are plotted against the composite index, n, for each protein in Figure 7. Adhesion on polar and apolar surfaces increases smoothly with the composite index, n, with good linear and parabolic fits for the polar and apolar surfaces, respectively. The difference between the values of the asymptotes as n f 0, that is, when the molecular weight approaches that of a very small stable protein (with no helical and random content), is ∼6.43 × 10-6 kJ/m2. A comparison of the adhesive energies for the three sets of measurements is instructive. The normalized force drops from -213 ( 5 mN/m for the SAM-CH3/SAM-CH3 system to -5.7 ( 2 mN/m for the SAM-CH3/RNAse A interaction. BGG and Pyr display a similar trend for the SAM-protein pull-off forces to the amount adsorbed previously observed by Sigal et al.38 using SPR. They measured adsorbed amount on similar SAM surfaces and, hence, accounted for protein-protein and protein-surface interactions. Work done previously to measure forces between alkanethiol-terminated SAMs (-CH3,67,68 -OH,67 and -NH2,67) and proteins (BSA,67 IgG,67 Fib,67 and HSA68) used a pyramidal tip, making comparisons with our results difficult (cannot use the Derjaguin approximation).52 However, our results (0-170 mN/m) are consistent with their reported values (range from ∼8 mN/m68 to ∼90 mN/ (68) Rixman, M. A.; Dean, D.; Macias, C. E.; Ortiz, C. Langmuir 2003, 19, 6202-6218.

In this study, we have significantly broadened the scope of available data on adhesion of proteins and solid substrates (seven proteins and eight SAM surfaces). In addition, we demonstrate for the first time that all seven proteins tested here behave similarly with respect to adhesion, exhibiting a step increase in adhesion as wettability of the solid substrate decreases. The step increase occurs within a narrow range of wettabilities of the solid substrates for all the proteins. Also, quantitative measures of protein-protein and SAM-SAM self-adhesive energies are provided. New correlations, not previously reported, suggest that the helix and random content (as measures of secondary structure) normalized by the molecular weight of a protein are significant for predicting protein self-adhesion and protein adhesion to solid substrates and are likely related to protein stability at interfaces. Three sets of measurements between like-SAMs, between like-proteins, and between proteins and SAMs are presented here. The first set provides an estimate of the attractive energy for the so-called mean hydrophobic interaction (excluding π-π interactions), while the second and third sets provide a direct method of assessing the conformational stability of adsorbed proteins. The third set also furnishes adhesive interactive energy between proteins and different surface functional groups and specifies the ranges of wettability in which hydrophobic, intermediate polarity, and hydrophilic interactions dominate. The main conclusions are given below for each set: SAM)SAM Interactions. (1) Large adhesive interactions were observed between uncharged SAM-SAM surfaces characterized with wettabilities 0.55. (2) The largest adhesion was observed for the hydrophobic surfaces (-233 ( 9 mN/m, -CF3, -CH3, -OPh, -OCH3, and -CN). (3) The surface with the -OPh group showed the largest adhesion possibly as a result of π-π interactions of the phenoxy group. (4) The pull-off forces for the highly wettable surfaces were about 1.5 orders of magnitude lower (∼-5 ( 0.6 mN/m, -EG3OH, -OH, -CONH2) than those for the apolar surfaces. (5) Clearly, there exists a strong adhesive (attractive) interaction between the least wettable surfaces and minimal interactive energy between the hydrophilic surfaces. Because the polar surfaces can form hydrogen bonds with water molecules and the apolar surfaces cannot, these measurements provide a quantitative measure of the so-called mean hydrophobic interaction (∼-206 ( 8 mN/m) in PBS buffer. Protein)Protein Interactions. (1) Small globular proteins (RNAse A with 33% sheet content and Lys with only ∼7% sheet content) have the least self-adhesion force, indicating robust conformation of the proteins on the surface. RNAse A and Lys have low values of the adiabatic compressibility (indicating a more compact structure). (2) Unexpectedly, IgG and BGG exhibited very little adhesion for intermediate size proteins. This can be explained by their low helix and random content (see the following). (3) Intermediate to large proteins BSA (60% helix, 0% sheet, is relatively unstable adsorbed to surfaces) and Pyr

Protein Adhesion to Self-Assembled Monolayers

(tetramer with 38% helix and 18% sheet) show measurable adhesion and also unfolding during retraction of the protein-covered substrate from the protein-covered AFM tip. Fib (42% helix, 7% sheet, tetramer) shows the greatest adhesion of 20.4 mN/m. The “retraction” of the sample away from the tip likely leads to mechanical denaturation of the protein. Fib is known to denature on many surfaces.38,62,63 (4) A smooth correlation estimating the propensity of a surface-bound protein to destabilize on interacting with other bound proteins of the same kind is presented. A normalized adhesion force was plotted against a composite index, n (the product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib; Figure 3B). This suggests that the helix and random content (or by difference, sheet and turn content) and size (molecular weight) are important. More data is needed to further test the efficacy of using the composite index, n, for predicting protein selfadhesion. Protein)SAM Interactions. (1) Small Proteins. Small and relatively stable proteins such as RNAse A and Lys (∼14 kDa) return to the zero force line at relatively short separation distances, while large proteins such as BGG, Pyr, and Fib (170-340 kDa) return with a steplike profile at very large distances, suggesting protein unfolding. The CN-SAM surface appears to stretch RNAse A to longer distances (∼30 nm) than the other surfaces (CF3-, 2 nm; CH3-, 15 nm; OPh-, 14 nm; OCH3-, 15 nm). With Lys, the CF3- and OCH3-SAM surfaces exhibit very short pull-out distances (200 nm for OPh-, CH3-, and CN-, respectively. The remaining CF3- and OCH3showed pull-out distances of 0.25. We, however, were able to detect measurable adhesion of these proteins at all wettabilities. For larger proteins, both the normalized adhesion force and the surface density of adsorbed protein films increased. Both studies were unable to distinguish between the effects of the different hydrophobic chemistries (-CH3, -OPh, -CF3). Sethuraman et al.16 have shown that the amount of Lys adsorbed onto a solid substrate strongly influences the propensity of Lys to lose helix in favor of sheet secondary structure. They and Sigal et al.38 also show that hydrophobic surfaces, such as SAM-CH3 or Teflon, adsorb more protein than (69) Oberhauser, A. F.; Hansma, P. K.; Carrion-Vazquez, M.; Fernandez, J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 468-472. (70) Gergely, C.; Voegel, J.-C.; Schaaf, P.; Senger, B.; Maaloum, M.; Horber, J. K. H.; Hemmerle, J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10802-10807.

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hydrophilic surfaces such as SAM-OH. Thus, proteins on a hydrophilic surface are less likely to interact with like proteins adsorbed onto the same surface. Also, the larger the protein, the more it adsorbed, especially onto apolar surfaces. Because all the solid substrates were relatively smooth (Rrms e 2 nm), roughness effects were ignored with respect to influence on protein adsorption and stability.71,72 Variability in the force measurements were possibly due (71) Denis, F. A.; Hanarp, P.; Sutherland, D. S.; Gold, J.; Mustin, C.; Rouxhet, P. G.; Dufrene, Y. F. Langmuir 2002, 18, 819-828. (72) Han, M.; Sethuraman, A.; Kane, R. S.; Belfort, G. Langmuir 2003, 19, 9868-9872.

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to defects on the SAM surfaces and the instability of the proteins in the adsorbed state. Acknowledgment. We thank G. C. Wang for allowing us to use their AFM during repair of our instrument and Brian Frank, Masahide Taniguchi, and John Pieracci for assistance. The funding support of the U.S. Department of Energy (Grant DE-FG02-90ER14114) and the National Science Foundation (Grant CTS-94-00610) is acknowledged. LA049454Q