Adhesion Forces of the Blood Plasma Proteins on Self-Assembled

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Langmuir 1999, 15, 7639-7646

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Adhesion Forces of the Blood Plasma Proteins on Self-Assembled Monolayer Surfaces of Alkanethiolates with Different Functional Groups Measured by an Atomic Force Microscope Satoru Kidoaki and Takehisa Matsuda* Department of Bioengineering, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan Received March 26, 1999. In Final Form: June 18, 1999 To determine the adhesion force of representative blood plasma proteins (albumin (Alb), immunoglobulin G (IgG), and fibrinogen (Fib)) to foreign material surfaces, force-versus-distance curves were measured using an atomic force microscope (AFM) between the protein covalently immobilized AFM tips and the well-defined model surfaces of self-assembled monolayers (SAMs) of alkanethiolates terminated with different functional groups (CH3, NH2, OH, and COOH). From the f-d curves measured between the protein-immobilized tip and the SAM surface, the mechanical adhesion forces of the protein to the SAM surface (detachment force of protein from the surface) were determined. From the adhesion forces determined between the protein tip and the SAM surface and between like SAM-coated tips and surfaces (i.e., tip/ surface combinations: CH3/CH3, NH2/NH2, OH/OH, and COOH/COOH), the thermodynamic adhesion strength (work of adhesion and surface pressure of SAM on a protein surface) was also determined according to the Dupre´ equation and Johnson, Kendall, and Roberts adhesion theory. The relative strength of thermodynamic adhesion of the proteins to the SAM surfaces was found with statistical significance to be in the following orders: (a) For Alb and IgG, CH3- . (OH-, NH2-) > COOH-SAM surface; for Fib, CH3 . OH > NH2 > COOH. (b) On CH3-, NH2-, and OH-SAM surfaces, Fib exhibits higher adhesion than Alb and IgG.

Introduction Protein adsorption onto the surface of a foreign material is the initial event that occurs when a surface comes into contact with the living fluids such as blood, which is followed by a series of biological reactions such as platelet and leukocyte adhesion, blood coagulation, fibrinolysis, and thrombus formation. The biocompatibility of a material is known to be conditioned with the layer of protein initially adsorbed to its surface.1-3 Therefore, many studies on protein adsorption to various artificial surfaces have been carried out in order to understand the adsorption mechanisms involved, or to appropriately design biocompatible artificial surfaces (see reviews, refs 1-9). In the mechanisms of protein adsorption and desorption, the mass transfer property of the protein and the intrinsic properties of surface and protein are essential factors.1,4,5 * To whom correspondence should be addressed. Present address: Department of Biomedical Engineering, Kyushu University Graduate School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Telephone: (+81)92-642-6210. Fax: (+81)92642-6212. E-mail: [email protected]. (1) Horbett, T. A. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S. L., Peppas, N. A., Eds.; Advances in Chemistry Series 199; American Chemical Society: Washington, DC, 1982; pp 233-244. (2) Andrade, J. D., Ed.; In Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985; Vol. 2, pp 1-80. (3) Park, K.; Mosher, D. F.; Cooper, S. L. J. Biomed. Mater. Res. 1986, 20, 589-612. (4) Macritchie, F. Adv. Protein Chem. 1978, 32, 283-326. (5) Norde, W. Adv. Colloids Interface Sci. 1986, 267-340. (6) Ivarsson, B.; Lundstro¨m, I. CRC Crit. Rev. Biocompat. 1986, 2, 1-96. (7) Lundstrom, I.; Ivarsson, B.; Jonsson, U.; Elwing, H. In Polymer Surfaces and Interfaces I; Feast, W. J., Munro, H. S., Eds.; John Wiley & Sons, Inc.: New York, 1987; pp 201-230. (8) Tengvall, P.; Lundstrom, I.; Liedberg, B. Biomaterials 1998, 19, 407-422. (9) Elwing, H. Biomaterials 1998, 19, 397-406.

The former includes, for example, the concentrations of the protein and ionic species, the ionic strength, the diffusion coefficient of the protein, the flow rate of the solution, and the viscosity of the solution. The latter are determined by the molecular interaction between proteins and surfaces that involves van der Waals interactions, electrostatic interactions, hydrogen bonding, hydrophobic interactions, hydration force, steric interactions, charge transfer, or donor-acceptor interactions. Although the dependence of the amount of adsorbed protein or of the adsorption rate on these factors has been analyzed through adsorption isotherms and adsorption kinetic data, the strength of the molecular interaction between the protein and the surface is not well understood, mainly due to the following two difficulties: (a) the technical difficulty involved in direct measurement of the strength of the molecular interaction and (b) the complexity of the protein-surface interaction including the above-mentioned multicomponent interactions, which are imposed by the presence of various kinds of chemical functional groups on both the protein and material surfaces. Concerning these difficulties, recent developments in the technique of molecular force measurement using an atomic force microscope (AFM) and in the preparation of chemically well-defined surfaces are beginning to provide a useful strategy for investigating the protein-surface interaction. The AFM allows the measurement of the forceversus-distance curve (f-d curve) between the AFM tip and the sample surface, and has successfully been applied to force measurement of a system involving proteins: e.g., the measurement of single molecular forces between specific protein pairs such as avidin/biotin pairs10-13 or (10) Pierce, M.; Stuart, J.; Pungor, A.; Dryden, P.; Hlady, V. Langmuir 1994, 10, 3217-3221.

10.1021/la990357k CCC: $18.00 © 1999 American Chemical Society Published on Web 09/03/1999

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Figure 1. Scheme of the experimental setup.

antigen/antibody pairs14-18 and of nonspecific adhesion forces between proteins and material surfaces such as polystyrene19,20 or glass.20 For tailoring the surfaces of both AFM tip and the substrate, long-chain alkanethiolates [HS(CH2)nX, n > 10; X, functional groups] have been used, which adsorb to gold surfaces from solution and form well-packed, ordered, and highly oriented monolayers with the tail group X predominantly exposed to the monolayer-liquid interface, the so-called selfassembled monolayers (SAMs).21-24 In this study, to evaluate the adhesion strength between representative blood plasma proteins and well-defined model surfaces with close-packed functional groups at the outermost surface, we employed the AFM technique of force-versus-distance curve measurement and have measured the adhesion force between protein-immobilized AFM tips (albumin (Alb), immunoglobulin G (IgG), and fibrinogen (Fib) tips) and SAM surfaces with different functional groups (n ) 11, X ) CH3, NH2, OH, and COOH), as shown in Figure 1. The proteins were covalently immobilized on the AFM tip that was precoated with a (11) Lee, G. U.; Kidwell, D. A.; Colton, R. J. Langmuir 1994, 10, 354-357. (12) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415417. (13) Moy V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257259. (14) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368-1374. (15) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477-3481. (16) Allen, S.; Chen, X.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Sefton, J.; Tender, S. J. B.; Williams, P. M. Biochemistry 1997, 36, 7457-7463. (17) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H.-J.; Misevic, G. N. Science 1995, 267, 1173-1175. (18) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntherodt, H.-J. Biophys. J. 1996, 70, 2437-2441. (19) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Edwards, J. C. Langmuir 1997, 13, 4106-4111. (20) Sagvolden, G.; Giaever, I.; Feder, J. Langmuir 1998, 14, 59845987. (21) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (22) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Poter, M. D. Langmuir 1988, 4, 365-385. (23) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (24) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164.

SAM surface terminated with carboxyl groups through the condensation reaction between the amino groups in the protein and the carboxyl groups, according to a wellestablished method.25 First, the adhesion forces between like SAM tips and surfaces (i.e., tip/sample surface combinations; CH3/CH3, NH2/NH2, OH/OH, and COOH/ COOH) were measured in order to check the validity of our measurement. Second, the adhesion forces between the protein tip and the SAM surface were measured, and compared among different combinations of proteins and SAM surfaces. The surface functional group dependency on the adhesion strength of the proteins is discussed from both mechanical and thermodynamic viewpoints. Experimental Section Materials. Solvents and reagents, all of special reagent grade, were used without further purification. 1-Dodecanethiol was obtained from Wako Pure Chemicals (Osaka, Japan). 12Mercapto-1-dodecanoic acid, 11-mercapto-1-undecanol, and 11mercapto-1-undecylamine were synthesized as described in our previous studies.26,27 Bovine serum albumin (fraction V powder) (Alb; Sigma Chemicals, St. Louis, MO), sheep IgG (Organ Teknika, West Chester, PA), bovine fibrinogen (95% clottable) (Fib; Seikagaku Kogyo, Tokyo, Japan), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochrolide (EDPC; Sigma Chemicals), and N-hydroxylsuccinimide (NHS; Wako Pure Chemicals) were commercially available. Water was deionized with a Milli-Q reagent water system (Nippon Millipore, Ltd., Tokyo, Japan) to 18 MΩ cm resistivity (DI water). All measurements were made in phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 8.10 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4). Preparation of Au-Coated Surfaces and Au-Coated AFM Probe Tips. Gold films were prepared on glass microscope slide substrates and commercial Si3N4 tip-cantilever assemblies (Digital Instruments, Santa Barbara, CA) by thermal evaporation (VPC-260F, Shinku-kiko, Japan) with a 30 Å adhesion layer of Cr followed by 500 Å of Au. The Si3N4 cantilever used in this study was a 200 µm wide leg, flat triangle with a spring constant of 0.12 N/m. Prior to surface modification, the probes were cleaned by immersion in acetone and ethanol, and were subjected to nitrogen drying. High-purity Au wire (99.99%) was cleaned by (25) Carr, P. W.; Bowers, L. D. In Immobilized enzymes in analytical and clinical chemistry: Fundamentals and applications; John Wiley & Sons, Inc: New York, 1980; pp 171-173. (26) Tanahashi, M.; Matsuda, T. J. Biomed. Mater. Res. 1997, 34, 305-315. (27) Saito, N.; Matsuda, T. Mater. Sci. Eng. 1998, C6, 261-266.

Blood Plasma Proteins on SAM Surfaces sonication in acetone and ethanol, and immediately evaporated at a pressure less than 2 × 10-5 Torr. To avoid excess heating of the Si3N4 probes during the evaporation process, which is believed to cause the cantilevers to bend, the following procedures were performed: (1) The Si3N4 probes were coated with 100 Å of gold at a rate of 0.5 Å/s (after coating with a 30 Å adhesion layer of Cr). (2) The evaporation was halted for 30 min in order to radiate heat. (3) Procedures 1 and 2 were continued until the thickness of the Au layer reached 500 Å. Here, the thickness and the deposition rate of Au films were monitored using a quartz crystal oscillator. Preparation of Self-Assembled Monolayers on AuCoated Surface. SAMs on the Au-coated surface of the AFM tips and of the glass substrates were prepared by immersion in 1 mM ethanolic solutions of ω-functionalized undecanethiolates, HS(CH2)11X (X ) CH3, COOH, OH, NH2), overnight at room temperature. Then, the substrates were rinsed in excess ethanol, dried under nitrogen, and immediately transferred into the custom-designed sample cell with PBS. The formation of SAMs has been confirmed in our previous studies, using X-ray photoelectron spectroscopy and wettability measurements.27 Protein Immobilization on the Au-Coated AFM Tips. Alb, IgG, and Fib were covalently immobilized on the Au-coated AFM probe tips by the coupling reaction between the primary amine groups of the proteins and the carboxyl groups on the HS(CH2)11COOH-SAM surface prepared on the AFM tips, which was carried out using the carbodiimide and hydroxylsuccinimide in the following procedure: (1) After carefully rinsing with ethanol and nitrogen drying, the carboxyl-functionalized tips were immersed in the 1:9 mixed solution of EDPC (25 mg/mL in water) and NHS (2.0 mg/mL in 1,4-dioxane) with gentle stirring for 15 min at room temperature. (2) The tips were thoroughly rinsed with DI water to remove any unreacted NHS, and then they were immersed in the protein solution (1.0 mg/mL in PBS) for 3 h at room temperature. Each protein solution was freshly prepared. (3) The tips were thoroughly rinsed with PBS in order to avoid multilayer adsorption of the protein. Protein immobilization on Au-coated glass substrate surfaces were also performed by the above procedure. Measurement of the Force-versus-Distance Curves. Force-versus-distance curves (f-d curves) between proteinimmobilized AFM probes and SAM surfaces were obtained with a Digital Instruments Nanoscope IIIa AFM (Dimension 3000). All f-d curve measurements were done in freshly prepared PBS in the sample cell, which was cleaned thoroughly prior to use. The protein-immobilized probes were equipped with a commercial fluid cantilever folder (Digital Instruments). The f-d curve was obtained by recording the AFM cantilever deflection caused by the vertical movement of the AFM tip through the following three processes: (1) the tip approaches the sample surface. Typically, the cantilever shows no deflection (approach trace; noncontact region). (2) The tip makes contact with the surface, and is pushed against the surface. The canilever is bent up with the applied force (contact region). (3) Z-piezo is retracted, and the tip is withdrawn from the surface. The cantilever is bent down until the tip reaches a distance at which the tip-sample contact becomes broken. Then, the cantilever deflection reverts to the original condition in the noncontact region (retract trace). The adhesive interaction between the proteins immobilized on the AFM tip and SAM surfaces was characterized from the hysteresis in the retract trace of the f-d curves, whose jump height from the bottom of the retrace line corresponds to the total adhesive force strength. The absolute scale of the measured adhesion forces was obtained through the following two steps. First, the ordinate in the f-d curve generated on Nanoscope IIIa software (change of the signal from the positionsensitive diode) was converted to the deflection of the cantilever in nanometers, using the gradient of the contact region in the retract line of the f-d curve. Next, the force applied to the cantilever was calculated in nanonewtons using Hook’s law and the spring constant of the cantilever. The spring constant (0.12 N/m) given by the probe manufacturer was used without further calibration in the present study. The reproducibility of the adhesion force measurements for a given protein-immobilized tip was examined by the following protocol. Typically, more than 30 f-d curves were obtained at

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Figure 2. Representative force-versus-distance curves measured between like SAM tips and surfaces in pure water (a) and in PBS (b). The zero positions of tip-sample separation were chosen so as to indicate that the cantilever exhibits no deflection (i.e., zero applied force) at the position on the contact line. Dashed line: approaching trace. Solid line: retract trace. one location through repeated tip approach/retract cycles, and the measurements were also repeated at more than three locations on each sample. The frequency of the approach/retract cycle, 3.2 Hz, was chosen so as to minimize the noise fluctuation in a single f-d curve, and sequential f-d curves were collected by computer software at 1 s intervals. Some variation in f-d curves was seen in the measurement among locations on each sample and among samples. Thus, in order to discuss the adhesive force statistically, a series of experiments was performed with more than three newly prepared combinations of proteinimmobilized tips with SAM surfaces. Although, for the majority of functionalizxed tips, no significant change in adhesive force was observed for newly prepared combinations of the same sample, a few combinations failed to give reproducible force measurements; i.e., no adhesive jump could be observed at the rate of about one to ten combinations. This can be attributable to the immobilization failure of protein to the top of the AFM tip. Such measurement series were discarded.

Results Adhesion Force Measurement between Like SAM tip and Surface. Before measuring the adhesion force between a protein-immobilized AFM tip and self-assembled monolayer surface, it is necesary to assess the reliability of our force measurements. We carried out adhesion force measurements in pure water and in PBS solution between like SAM tips and surfaces (i.e., tip/ sample surface combination: CH3/CH3, NH2/NH2, OH/ OH, and COOH/COOH). Figure 2 shows representative f-d curves obtained for each combination between like SAM tips and surfaces in pure water and in PBS. Within f-d curves, the largest adhesive jumps in the retract trace were observed for the CH3/CH3 combinations both in pure water and in PBS. In f-d curves for all other combinations, with the exception of the COOH/COOH combination in PBS, small adhesive jumps were observed. In the f-d curve for the COOH/COOH combination in PBS, little detectable adhesive jump was observed probably due to the limit of the force resolution of the probe cantilever used in the present study. Here, to clarify the uncertainties

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Table 1. Summary of Adhesion Forces Measured between Like SAM Tips and Surfaces in Pure Water and in PBSa tip/surface combination CH3/CH3 NH2/NH2 OH/OH COOH/COOH

m 3 3 3 3

n 180 179 182 181

in pure water Fad (nN) (12.5)b

19.9 1.5 1.3 (0.3)b 0.6 (2.3)b

SD (nN)

m

n

Fad (nN)

(4.4)b

3 3 3 3

210 202 139 90

4.3 1.1 0.1 ∼0

11.6 1.0 1.3 (0.05)b 0.5 (1.1)b

in PBS SD (nN) γSB (mJ m-2) 1.3 0.3 0.1 ∼0

9.2 2.4 0.1 ∼0

SD (mJ m-2) 2.8 0.7 0.2 ∼0

a m, number of successfully prepared tip/surface combinations; n, total number of repetitive measurements; F , mean of adhesion force; ad γSB, mean of interfacial free energy between SAM surface and PBS (calculated using eq 3); SD, standard deviations. bFrom ref 29.

Figure 3. Representative force-versus-distance curves measured between the protein-immobilized tips ((a) Alb, (b) IgG, and (c) Fib tips) and SAM surfaces terminated with CH3, NH2, OH, and COOH groups. The zero positions were chosen to be similar to those in Figure 2. Dashed line: approaching trace. Solid line: retract trace.

in adhesion force measurement, we carried out measurements using three newly prepared tip-sample combinations for each sample condition. (The total number of measurements for each combination was between 100 and 200). Not only force variations but also some variations among the three measured distributions of adhesion force were observed in all tip-sample combinations tested. From repeated measurement of the height of adhesive jumps, mean values of adhesion force (Fad) for each tipsample combination were determined as shown in Table 1. It is noted that Fad in PBS solution was smaller than that in pure water for all tip-sample combinations tested. Adhesion Force between Protein-Immobilized Tips and SAM Surfaces. The f-d curves between the protein-immobilized AFM tips (Alb, IgG, and Fib tips) and the SAM surfaces with different functional groups (CH3-, NH2-, OH-, and COOH-terminated surfaces) were obtained in PBS (see the scheme in Figure 1). Figure 3 shows representative f-d curves for each combination of protein tip and SAM surface. Here, applied load was set to be between 2 and 5 nN. Below the loading force 2 nN, there were some observations that the hysteresis jump did not appear, indicating that the weak loading force cannot necessarily establish the adhesion between the protein and SAM surface. In the present study, in order to focus on the adhesion between them, we applied the loading force larger than 2 nN. The height of adhesive jump was not influenced so much by the change of loading force between 2 and 5 nN. In the f-d curves, irrespective of the kind of protein, exceptionally large adhesive hysteresis and multiple jumps in the retract trace were observed between the protein tips and CH3-SAM surface, compared with the SAM surfaces terminated with the other functional groups. Histograms of the frequency of the measured adhesion forces for the combination of proteins/CH3-SAM are plotted

Figure 4. Histograms of the frequency of the adhesion force measured in the repetitive force-distance curve measurements between the protein-immobilized tip ((a) Alb, (b) IgG, and (c) Fib tips) and CH3-SAM surface.

in Figure 4, in order to exemplify the typical variation of the adhesion measurement in the protein/SAM combination. The values of Fad and SD measured for each combination of protein tip and SAM surface are sum-

Blood Plasma Proteins on SAM Surfaces

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Table 2. Summary of Adhesion Forces Measured between Alb-, IgG-, and Fib-Immobilized Tips and SAM Surfaces Terminated with CH3, NH2, OH, and COOH groups in PBSa tip/surface combination

m

n

Fad (nN)

SD (nN)

Fib/CH3 Fib/NH2 Fib/OH Fib/COOH

3 3 3 3

212 239 229 248

4.6 1.1 0.8 0.1

1.3 0.4 0.6 0.1

Alb/CH3 Alb/NH2 Alb/OH Alb/COOH

3 3 3 3

185 208 206 207

4.5 0.8 0.3 ∼0

2.8 0.5 0.3 ∼0

IgG/CH3 IgG/NH2 IgG/OH IgG/COOH

3 3 3 3

217 171 208 183

3.7 0.9 0.1 ∼0

1.0 0.4 0.1 ∼0

a m, number of successfully prepared tip/surface combinations; n, total number of repetitive measurements; Fad, mean of adhesion force; SD, standard deviations.

Figure 5. Representative force-versus-distance curves measured between like protein-immobilized tips and surfaces. The zero positions were chosen to be similar to those in Figure 2. Dashed line: approaching trace. Solid line: retract trace.

marized in Table 2. Fad values of the Alb, IgG, and Fib on the CH3-SAM surface were around in 4 nN, which are considerably larger than those of protein/other-SAM pairs, irrespective of the protein. Fad was found to decrease in the order CH3 . NH2 > OH > COOH, irrespective of the kind of protein. In the combinations Alb/COOH and IgG/ COOH, detectable adhesive jumps in the f-d curve were not observed. Adhesion Force between Like Protein-Immobilized Tips and Surfaces. The f-d curves between like protein-immobilized tips and surfaces were also measured. In the f-d curves for three combinations (Alb/Alb, IgG/ IgG, and Fib/Fib), detectable adhesive jumps were not observed as shown in Figure 5, due to the limit of the force resolution of the probe cantilever. The adhesion force between like protein tip and surface was measured to be approximately zero irrespective of the kind of the protein (Table 3). Discussion Validity of Our Force Measurement. The adhesion force between like SAM tips and surfaces in an aqueous environment has been reported so far under different experimental conditions (e.g., thickness of Au layer deposited on the tip d 500-700 Å, solvent water, 1-pro-

Table 3. Summary of Adhesion Forces Measured between Like Protein-Immobilized Tips and Surface in PBSa tip/surface combination

m

n

Fad (nN)

SD (nN)

Alb/Alb IgG/IgG Fib/Fib

3 3 3

90 197 90

∼0 ∼0 ∼0

∼0 ∼0 ∼0

a m, number of successfully prepared tip/surface combinations; n, total number of repetitive measurements; Fad, mean of adhesion force; SD, standard deviations.

panol, and hexane;28 d 500 Å, solvent water;29 d 1000 Å, solvent 0.01 M phosphate buffer30). Our measurements (d 500 Å, solvent water and PBS) were compared with those reported previously. The reported general feature that the largest adhesive jump appears on the f-d curve of the CH3/CH3 combination in the aqueous environment was reproduced in our observations (Figure 3). As for the value of the adhesion force, our results in pure water were compared with those in ref 29, where experimental conditions similar to ours were employed. Our measured mean values appear to be somewhat larger for the CH3/ CH3 and OH/OH combinations and to be somewhat smaller for the COOH/COOH combination than the reported mean values (in parentheses in Table 1). These differences may be attributable to minute differences in the experimental conditions whose complete standardization is quite difficult, such as tip geometry and surface roughness. In PBS, the largest force was observed for the CH3/CH3 combination (which is considerably larger than the other three combinations), followed by NH2/NH2. The smallest forces were observed for the OH/OH and COOH/COOH combinations. This strongly indicates that, also in PBS, the adhesion force due to hydrophobic interactions is much stronger than those due to hydrogen bonding or donoracceptor interactions. The reduced adhesion forces between like SAM tips and surfaces in PBS, irrespective of the type of terminal functional group, as compared with those measured in pure water, may be due to the salt effect involving the ionic strength. In particular, a marked decrease in the adhesion force for the CH3/CH3 combination in PBS may be derived from a change in the ordered structure of the hydrophobic hydration water layer, because the hydration force in water is modified by changes in the water structure on the addition of “structure makers” or “structure breakers” (such as salts) to the solution.31 A more detailed study of such ionic effects on the adhesion force for like CH3-SAM tip and surface is now under way and will be reported in the near future. Charactersistics of the f-d Curve between Protein Tips and SAM Surfaces. The observed profiles of the multiple adhesive jumps in the f-d curve between the protein tip and the SAM surface (Figure 3) are typical of f-d curves measured using protein-coated AFM tips [e.g., refs 12, 16-19, and 32], which have been interpreted in the literature to be due to the following effects: (1) nonlinear convolution of multiple unbinding processes of antigen/antibody pairs,12 (2) the breaking of different contact points between the antigen/antibody pairs,18 (3) (28) Han, T.; Williams, J. M.; Beebe, T. P., Jr. Anal. Chim. Acta 1995, 307, 365-376. (29) Sinniah, S. K.; Steel, A. B.; Miller, C. J.; Reutt-Robey, J. E. J. Am. Chem. Soc. 1996, 118, 8925-8931. (30) Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006-2015. (31) Drost-Hansen, M.; Clegg, J. In Cell-Associated Water; Academic Press: New York, 1979. (32) Oberleithner, H.; Schneider, S. W.; Henderson, R. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14144-14149.

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Figure 6. Schematic representation on the conformational behavior of protein immobilized on the AFM tip in the approach/retract cycle of f-d curve measurement.

the lateral movement of the sensor tip during the process of approach or withdrawal,18 and (4) the “spring” effect of partially unfolded protein which is loaded during the process of withdrawal of the tip and bridges between the tip and sample.18 In our measurement, it is noted that markedly longer range multiple jumps appeared in the f-d curves on the Fib/CH3 combination, compared with the Alb/CH3 and IgG/CH3 combinations (Figure 3). This may be due to the “spring effect” of large-sized proteins (interpretation 4). Fib is the largest protein (side dimension 450 Å33) within the three kinds of protein investigated here (side dimension Alb, 80 Å;34 IgG, 120 Å 35). Our interpretation on the conformational change of the protein immobilized on AFM tip in the approach/retract cycle of f-d curve measurement is schematically presented in Figure 6. In the enforced adhesion process of the measurement (approach trace), the protein on the AFM tip is pushed down against the surface with appropriate applied load, which induces deformation or denaturation of the protein to some extent (A-B). In the retract trace, the forcedly contacted and somewhat deformed protein may first be re-formed toward the original conformation to some extent before the detachment of the protein from the sample surface begins (B-C) and then be deformed with a stretching manner (C-D). After that, gradual detachment of the protein and the stretching deformation or denaturation proceed with a part of the protein surface adhered on the sample surface (D-E). In this process, the partial unfolding of protein may be induced. The degree of unfolding is influenced by the adhesion strength of protein to the sample surface, which is intrinsically determined by the sample surface character. Finally, the interface of protein/sample is broken and the protein refolds toward the native state (E-F). Though the native conformation may not necessarily re-form completely, such partial refolding may occur within the period of the approach/retract cycle of f-d curve measurements (approximately 300 ms in the present study). A single protein can refold within a few milliseconds when the refolding occurs through a fast-folding pathway.36 Adhesive hysteresis did not show a systematic time-dependent change through the repetitive process of approach/retract cycle of the tip, but showed random variation with the distri(33) Doolite, R. F. Annu. Rev. Biochem. 1984, 53, 195-229. (34) He, Xiao Min; Carter, D.C. Nature 1992, 358, 209-215. (35) Marquart, M.; Deisenhofer, J.; Huber, R. J. Mol. Biol. 1980, 141, 369-391. (36) Fischer, G.; Schmid, F. X. Biochemistry 1990, 29, 2205-2212.

bution as is exemplified in Figure 4. This observation suggests that the irreversible denaturation of the protein did not proceed through the repetitive process, but a kind of periodic conformational change of the protein as mentioned above could occur. Mechanical Adhesion Strength and Thermodynamic Adhesion Strength between Protein and SAM Surfaces. During f-d curve measurement, a protein immobilized on the AFM tip is forcedly made in adhesion with a SAM surface by the descent of the tip in the first step, involving the exclusion of solvent molecules between the protein surface and the SAM surface. In the next step, the ascent of the tip, the interface between the protein and SAM (protein/SAM) is broken, creating two new interfaces (protein/PBS, SAM/PBS). In this situation, strictly speaking, Fad measured using an AFM is the force required to mechanically detach the forcedly adhered protein from the surface (which corresponds to a tensile test). As seen in the multiple adhesive jumps which appeared in the retract trace of the f-d curves for the protein/SAM combination, the tensile applied load reached the maximum value at the point of maximum deflection of the cantilever, and then remained over a relatively long distance of z-displacement of the tip. The profile means that a significant tip/sample interaction remains in the region (i.e., separation between protein and SAM surface is not yet completed). The complete separation (i.e., creating the two new interfaces protein/PBS and SAM/ PBS) is established just at the z-displacement of the tip where the cantilever deflection becomes zero. In this sense, more precisely, Fad should be defined as the “initial” vertical tensile detachment force of the protein tip from the SAM surface. In general, detachment forces depend on the kind of measurement method (vertical tensile, tensile shear, compression shear, peeling, etc.), and on the conditions of the interface failure (such as peeling speed, angle, adhesion area, etc.). Thus, it should be noted that Fad as the initial vertical tensile detachment force reflects only a part of the real adhesion strength between the protein and SAM surface, and does not necessarily reflect the force required for the real process of protein desorption (which is referred to here as the mechanical adhesion strength). On the other hand, the work of adhesion or the surface pressure as thermodynamic equilibrium quantities does not depend on the process of interface failure (thermodynamic adhesion strength). We appreciate the adhesion strength of the proteins to

Blood Plasma Proteins on SAM Surfaces

Langmuir, Vol. 15, No. 22, 1999 7645

SAM surfaces from both the mechanical and thermodynamic viewpoints. Fad can be related to the work of adhesion corresponding to the initial detachment process between the protein tip and SAM interface, Wps, by applying Johnson, Kendall, and Roberts (JKR) adhesion theory,37 the nature of which will be described later:

2Fad WPS ) 3πR

Table 4. Mean Values of Calculated Work of Adhesion between the Protein and SAM Surface WPS and Mean Values of Calculated Surface Pressure π (See Text), for Each Prepared Protein/SAM Combination tip/SAM surface combination

WPS

Fib/CH3 Fib/NH2 Fib/OH Fib/COOH

19.6 4.6 3.5 0.3

5.6 1.8 2.5 0.4

10.5 2.5 2.9 0.3

6.6 2.2 2.4 0.4

Alb/CH3 Alb/NH2 Alb/OH Alb/COOH

19.2 3.5 1.1 0.1

11.9 2.0 1.1 0.3

9.8 1.0 1.3 0.1

12.3 1.8 1.3 0.4

IgG/CH3 IgG/NH2 IgG/OH IgG/COOH

15.7 3.6 0.3 ∼0

4.5 1.5 0.4 ∼0

6.2 1.2 0.2 ∼0

5.7 1.8 0.5 ∼0

(1)

where the subscripts P and S denote protein and SAM surfaces, respectively. R is the tip radius. We apply the R value of 50 nm, measured from scanning electron microscope images of the present Au-coated and proteinimmobilized AFM tip (image not shown). WPS can be expressed by the following Dupre´ equation:

WPS ) γPB + γSB - γPS

(2) a

In this equation the subscript B denotes PBS. γPB, γSB, and γPS are the interfacial free energies of protein/PBS, SAM/PBS, and protein/SAM, respectively. In eq 2, γSB can be related to the work of adhesion between like SAMcoated tip and surface in PBS buffer WSS, which is calculated from Fad:

γSB )

1 W 2 SS

)-

Fad 3πR

(3)

Here, we supposed that γSS ) 0 for simplication, although γSS is not necessarily zero when the monolayers on the tip and sample are not bulk solid materials.29 JKR theory, which has frequently been used to relate the mechanical detachment force measured using an AFM and the thermodynamic work of adhesion,29,30,38,39 treats the adhesion between elastic solids with perfectly smooth surfaces under two main assumptions: i.e., the surfaces are “elastic” and “smooth”. The validity of the former assumption can be checked from the profile of the contact region in the f-d curve. It is known that, for an ideally elastic sample surface, the loading and unloading curves overlap while materials exhibiting plastic deformation show a hysteresis between the two curves.40,41 While hysteresis in the noncontact region of the f-d curves gives information on the interface character between tip and sample surface (such as adhesion), hysteresis in the contact region provides information on the hardness or elasticity of the sample surface.42 In Figure 3, contact lines are straight and little hysteresis is observed. Thus, the assumption of elasticity is valid for our case. On the other hand, the latter assumption that the surfaces are perfectly smooth may not be realized in a real experimental system. Irrespective of the uncertainty of the assumption, the experimentally measured adhesion force in the case between the like CH3-SAM tip and surface measured using an AFM in ethanol was in good agreement with the adhesion force calculated according to JKR theory, using (37) Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc. R. Soc. London 1971, 324, 301-313. (38) van der Vegte, E. W.; Hadziioannou, G. J. Phys. Chem. 1997, 101, 9563-9569. (39) Noy, A.; Frisbie, C. D.; Rozsnyai, L. F.; Wrighton, M. S.; Lieber, C. M. J. Am. Chem. Soc. 1995, 117, 7943-7951. (40) Doerner, M. F.; Nix, W. D. J. Mater. Res. 1986, 1, 601-609. (41) Weisenhorn, A. L.; Khorsandi, M.; Kasas, S.; Gotzos, V.; Butt, H.-J. Nanotechnology 1993, 4, 106-113. (42) Butt, H.-J.; Jaschke, M.; Ducker, W. Bioelectrochem. Bioenerg. 1995, 38, 191-201.

SD

π

SD (mJ m-2)

SD, standard deviations.

the value of interfacial free energy between CH3-SAM and ethanol obtained from the contact angle measurement.43 Therefore, JKR theory may also be worthwhile for a rough comparison of the thermodynamic adhesion strength between proteins on the AFM tip and sample surfaces. The calculated values of γSB and WPS and their standard deviations are shown in Tables 1 and 4, respectively. The surface pressure, π, at the interface between the protein surface and the SAM surface in PBS (which is a measure of interfacial free energy change on the protein surface) can be determined from eq 2 and is tabulated in Table 4.

π ) γPB - γPS ) WPS - γSB

(4)

Here, it should be noted that the present values of π were evaluated for the initial detachable region of the protein/ SAM interface. The mean values of π and their standard deviations were not calculated from Fad and SD in Tables 1 and 2, but recalculated from the total data which were generated by randomly combining all the individual values of measured adhesion force for the SAM/SAM and protein/ SAM combinations. On the basis of Fad and π values, we can compare the adhesion strength among each combination of protein and SAM surface from both the mechanical and thermodynamic viewpoints. From the statistical comparison of the Fad values of a protein among the different combinations with CH3-, NH2-, OH-, and COOH-terminated SAM surfaces, which was carried out by employing the Mann-Whitney U-test, the mechanical adhesion strength is found to decrease in the order CH3 . NH2 > OH > COOH, for Alb, IgG, and Fib (for all comparisons, with a statistical significance level of P < 0.01). On the other hand, the thermodynamic adhesion strength evaluated by π is found to decrease in the order CH3 . (NH2, OH) > COOH for AlB and IgG and CH3 . OH > NH2 > COOH for Fib (for all comparisons, P < 0.01, except for Fib/NH2 versus Fib/OH, P < 0.05). Markedly large Fad, WPS, and π for the CH3-SAM combination are noted, irrespective of the kind of protein, as was pointed out under Results. These results are in good agreeement with the well-known feature that the amount of adsorbed protein and adsorption rate are larger for hydrophobic surfaces than for hydrophilic surfaces (e.g., (43) Frisbie, C. D.; Rozsnai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071-2074.

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refs 5, 44, and 45), and confirm the general feature that the predominant force operating in the protein adsorption process in an aqeous system is the hydrophobic effect. The hydrophobic interaction observed between hydrophobic amino acids residues of protein and the CH3-SAM surface is found to be significantly stronger than other interaction components that include van der Waals interactions, electrostatic interactions, and hydrogen bonding, etc. From the comparison of the values of Fad and π of a SAM surface among the combinations with Alb, IgG, and Fib, Fib is found to exhibit particularly the highest adhesion strength (on both the mechanical and thermodynamic adhesion) for CH3-, NH2-, and OH-SAM surfaces, irrespective of whether the surface is hydrophobic or (44) Brash, J. L.; Uniyal, S.; Chan, B. M. C. Artif. Organs (Suppl.) 1981, 5, 475-477. (45) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988, 124, 28-43.

Kidoaki and Matsuda

hydrophilic (for all comparisons, P , 0.05). Studies of the adsorption of these three proteins (Alb, IgG, Fib) to various polymer surfaces showed that the adsorption constant, which is determined from the Langmuir isotherm equation, is highest for Fib, followed by Alb, and is lowest for IgG.45 This is in good agreement with our results. For the COOH-SAM combination, statistical significance was not detected. Detailed study for the adhesion force of the proteins on various artificial surfaces including polymer brush will strongly be needed with the study of protein adsorption behavior on surfaces. These studies are ongoing in our laboratory. Acknowledgment. We thank Prof. Kazue Kurihara of Tohoku University for her technical advice on AFM force curve measurement. LA990357K