Evidence of a Mobile Precursor State in Nonspecific Protein

ARTICLE pubs.acs.org/Langmuir

Evidence of a Mobile Precursor State in Nonspecific Protein Adsorption Lei Shen and X.-Y. Zhu* Department of Chemistry & Biochemistry, University of Texas—Austin, Austin, Texas 78712, United States

bS Supporting Information ABSTRACT: We study the dynamics of nonspecific protein adsorption using nanometer
micrometer-scale patterns involving hydrophobic domains in hydrophilic matrices. We report the discovery of a critical requirement for the sizes of the hydrophobic/adhesive pads for protein adsorption: the area of each adhesive pad must be more than 2 orders of magnitude larger than the footprint of a protein molecule before irreversible adsorption occurs. We attribute this to the minimal surface area sampled by a mobile protein molecule in a precursor state before irreversible adsorption occurs. Kinetic analysis based on the precursor model quantitatively accounts for the experimental observation and reveals that the distance sampled by the mobile precursor state before irreversible adsorption increases with the size of the protein molecule.

’ INTRODUCTION The adsorption of protein molecules on solid surfaces is ubiquitous to a number of important processes and technologies, such as biosensing, microarrays, biomaterials, and nanomedicine. It also serves as a model for understanding biochemical processes occurring at cell surfaces. Although it is very desirably to exert molecular and nanoscopic control over protein adsorption, this process is notoriously nonspecific, particularly on hydrophobic surfaces. To adsorb irreversibly on a hydrophobic surface, a protein molecule is transformed from the native state in which it preferentially exposes hydrophilic domains in the aqueous solution to a disturbed or disrupted state on the hydrophobic surface. The 3D structure of a protein molecule must reorganize during the adsorption process to optimize interaction with the surface. It is believed that the contact area between a protein molecule and a surface is only 1 to 2 nm2 initially;1 it grows during the reorientation, reorganization, and unfolding process, which is known as the growing disk model for macromolecule adsorption.2
6 A similar model has been used by Seigel et al. in the kinetic analysis of nonspecific protein adsorption on hydrophobic surfaces of self-assembled monolayers on gold.7 Despite extensive research efforts on protein adsorption, the current understanding remains at a qualitative or phenomenological level (e.g., relating adsorption to surface chemical functionality,8,9 the chemical nature of protein surfaces and their footprints,10 and compositional heterogeneity on the scale of contact domains11
15). There is little molecular-level understanding of the dynamics of the protein adsorption and reorganization process. Here, we probe the dynamics of protein adsorption on hydrophobic surfaces using nanometer
micrometer patterns obtained from block copolymers. We report the surprising finding r 2011 American Chemical Society

that there is a critical requirement for the sizes of adhesive pads for protein adsorption: the area of each adhesive pad must be more than 2 orders of magnitude larger than the footprint of a protein molecule before irreversible adsorption occurs. We attribute this to the minimal surface area sampled by a mobile protein molecule (i. e., a precursor state) before irreversible adsorption occurs.

’ EXPERIMENTAL SECTION Polymer Thin Film Preparation. Polystyrene-block-poly(2-hydroxyethyl methacrylate) (PS200-b-PHEMA50, molecular weight MPS = 21 000, MPHEMA = 6300, and polydispersity index Mw/Mn = 1.10), PS60b-PHEMA150 (MPS = 6200, MPHEMA = 20 500, and Mw/Mn = 1.18) diblock copolymers, and polystyrene (PS760, MPS = 79 000, and Mw/Mn = 1.04) were obtained from Polymer Source, Inc. (Montreal, Canada) and used as received. All solvents used were reagent grade (Fisher). PS200-b-PHEMA50 (0.5 wt %) was dissolved in CHCl3. PS60-bPHEMA150 (0.5 wt %) was dissolved in a CHCl3/methanol mixed solvent (1:1 v/v) . A PS60-b-PHEMA150/PS760 polymer mixture was dissolved in THF (1.0 wt %). Each polymer thin film was spin coated onto a freshly prepared gold substrate (35 nm gold thin film thermally evaporated onto glass, with a 1 nm chromium adhesion layer) at 3000 rmp for 60 s. This was followed by solvent annealing in CHCl3/DMF (1:1 v/v) vapor for 3 h to allow microphase separation. We image the surface morphology of polymer thin films using an atomic force microscope (AFM, Agilent Technologies 5500) in ac mode. A silicon cantilever-mounted tip (10 nm radius of curvature, Received: February 15, 2011 Revised: April 18, 2011 Published: May 09, 2011 7059

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Table 1. Physiochemical Parameters of Fibrinogen (Fbn), Bovine Serum Albumin (BSA), and Horse Myoglobin (Myo) protein

Mw (kDa)

size (nm3)

na

specific volume (cm3/g)

Fbn

340

50  7  7

1.65

0.713

BSA Myo

67 17

874 2  3.5  4

1.57 1.40

0.734 0.743

42 N/m spring constant, 300 kHz resonance frequency) was used in all experiments. Proteins. We used three different proteins—fibrinogen from human plasma (Fbn), albumin from bovine serum (BSA), and myoglobin from equine skeletal muscle (Myo)—and all were purchased from SigmaAldrich (purity g98%). Their physiochemical parameters from the literature are listed in Table 1.16
20 Phosphate-buffered saline (PBS) was freshly prepared from sodium and potassium salts: NaCl (80 g), KCl (2 g), Na2HPO4 (2 g), and KH2PO4 (6.5 g) were dissolved in 1 L of DI water to give a solution of pH 7.4 at 25
C. Protein solutions in the concentration range of 5
50 μg/mL were prepared in PBS buffer immediately before use. Surface Plasmon Resonance (SPR). SPR measurements were carried out on a commercial instrument (SPRImager II, GWC Technologies Inc., Madison, WI). The surface plasmon was excited by collimated polychromatic p-polarized light directed at a gold film sample through a prism assembly at an angle of incidence of θ. The intensity change (ΔI) measured at a constant angle is directly proportional to the change in the bulk refractive index of the solution near the sensor surface. The effective thickness (d) of the adsorbed layer is given by d = (ld/2)[ΔI/s(na
ns)], where ld is the decay length of the evanescent field near the gold surface (typically 37% of the wavelength of light), s is a calibrated sensitivity parameter for the instrument, and ns = 1.334 is the refractive index of the solution.21 Parameter s was calibrated by a 1% ethanol/H2O mixture. On the basis of the effective thickness determined in the experiment, we can estimate the surface coverage (C) of adsorbed molecules as C(molecules/cm2) = d/V, where V is the specific volume (cm3/g) of the protein in the solution. All protein adsorption measurements were carried out at room temperature (25
C). The Au SPR sensor surface was in contact with a liquid flow cell. Initially, the sample was equilibrated with PBS buffer (pH 7.4) at a flow rate of 3 μL/s. Once a stable background was obtained, protein PBS buffer solution was injected into the cell at the same flow rate. The SPR signals from 16 areas (0.5  0.5 mm2 each) on the sample surface (18  18 mm2) were averaged and recorded as a function of time for a fixed time window. Then, a PBS buffer solution was injected into the cell to replace the protein solution and to wash away weakly adsorbed protein molecules. From the value change of the SPR baseline, the effective thickness and saturation coverage of adsorbed layers were calculated. In the range of flow rate investigated (∼0.5 to 5 μL/s), we observed no obvious differences in protein adsorption kinetics.

’ RESULTS AND DISCUSSIONS To generate well-controlled surface patterns on the nanometer
micrometer scale, we use thin films of diblock copolymer, polystyrene-block-poly(2-hydroxyethyl methacrylate), PS-bPHEMA, in the vertical cylinder phase.22 We use two block copolymers: PS60-b-PHEMA150 (MPS = 6200, MPHEMA = 20 500, and Mw/Mn = 1.18) and PS200-b-PHEMA50. These two polymers give hydrophobic domains in a hydrophilic matrix or vice versa, as shown by AFM images in Figure 1A,B, with diameters of the minority domain of approximately 15
20 nm. We choose PHEMA as the hydrophilic block because the ethylene glycol groups on PHEMA are known to be repulsive toward protein

Figure 1. AFM images taken in air for thin films of (A) PS60-bPHEMA150 and (B) PS200-b-PHEMA50 and PS60-b-PHEMA150/PS mixtures with different weight ratios (j) of PS homopolymer: (C) 0.1, (D) 0.2, (E) 0.4, and (F) 0.6 in PS60-b-PHEMA150/PS mixtures. The PS domains are higher than the surroundings by ∼2 nm in A and B and ∼60 nm in C
F. The scale bars are 100 nm in A and B and 1 mm in C
F.

adsorption23 and the polymer thin film is stable in contact with aqueous solution (i.e., no swelling or reorganization, see Figures S1 and S2). Although the diblock copolymer film generates nearly uniform PS domains, it allows only very limited tuning of the domain size. To obtain larger domains, we adapt the approach of Russell and co-workers24 by mixing PS homopolymer into the PS-PHEMA diblock copolymer thin film. Microphase separation leads to domelike PS domains whose sizes increase with an increasing percentage of PS (Figure 1C,D). The height/diameter (h/d) ratio is ∼0.1 for images taken in air and doubles to ∼0.2 for images taken under the buffer solution (Figure S3). Note that despite the increase in PS domain sizes, their h/d ratios are approximately the same as that of the PS60-bPHEMA150 surface (Figure S4). Thus, the curvature of the PS domains remains about the same for 0 e j < 40% and is much smaller than those of the protein molecules. Note that when the PS homopolymer component is g40%, the domains merge into continuous networks (Figure 1E,F) and cross-sectional profiles of these large domains become plateaulike (Figure S4). Note that the low contrast regions in Figure 1C
F consist of PS60-bPHEMA150 vertical cylinder domains (Figure 1A). We use the SPR technique to quantify the adsorption of three protein molecules: human plasma fibrinogen (FBN), bovine serum albumin (BSA), and equine skeletal muscle myoglobin (MYO), with estimated sizes in the folded states of 50  7  7, 8  7  4, and 4  3.5  2 nm3, respectively. In each experiment, a gold SPR sensor coated with a particular polymer thin film is exposed to a liquid flow cell. We first equilibrate the sensor with PBS buffer solution and then inject a protein solution (50 μg/mL in PBS). The dimensions of the fluidic channel and the flow rate (3 μL/s) correspond to lamellar flow. After a fixed time of 180 s, we flow PBS to wash away weakly bound proteins and stop the adsorption process. SPR sensor response curves, summarized in Figure S5, allow us to quantify the number of protein molecules irreversibly adsorbed on each surface as a function of time. We use the short protein adsorption time (180 s) because we are interested in the initial adsorption kinetics; the amount of adsorbed protein increases nearly linearly with the time of exposure to the protein solution in this time window 7060

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Figure 3. Histograms of the PS domain sizes obtained from AFM images in Figure 1 for (A) j = 10% and (B) j = 20%. The average areas are (A) (2.4 ( 1.5)  104 nm2 and (B) (7.9 ( 4.7)  104 nm2.

Figure 2. Rate of initial protein adsorption (ra, b) as a function of the percentage (θPS) of PS homopolymer areas on the surfaces of the mixed polymer thin films for three protein molecules: (A) myoglobin (Myo), (B) bovine serum albumin (BSA), and (C) fibrinogen (FBN). The open circles are for PS200-b-PHEMA50. The dashed lines are linear relationships between ra and θPS. The solid lines are fits to the kinetic model based on a mobile precursor state, with the indicated fitting parameter of the root-mean-square displace (λ). See the text for details of the kinetic model.

(Figure S5). Thus, we approximate the initial rate of adsorption by the average adsorption rate within this time window. Figure 2 shows the rate of protein adsorption (b) as a function of the fraction of the surface area (θPS) that is PS homopolymer in the PS60-b-PHEMA150 matrix. We define θPS = 0 for the PS60b-PHEMA150 surface in the absence of additional PS homopolymer. The dashed lines are linear relationships, and the solid lines are fits to a kinetic model discussed later. The fractional surface area (θPS) of the PS homopolymer is quantitatively determined from AFM images. Here, the θPS value is close to the j value (weight ratio) defined above. We make two important observations. First, none of the three protein molecules adsorb onto the PS60-b-PHEMA150 surface (θPS = 0, i.e., without PS homopolymer added), despite the fact that this surface also consists of “sticky” PS domains (diameter ∼20 nm) with sizes larger than the projected areas on the surfaces of at least two of the protein molecules (MYO and BSA). The repulsive PS-PHEME block copolymer has also been implied in an earlier in situ study.25 This leads to the immediate conclusion that the irreversible adsorption of protein molecules requires that the sizes of the sticky pads to be larger than the projected footprint (not the initial contact area) of protein molecules. The question is, how much larger? The answer to the above question comes from the second observation (i.e., a comparison of the three protein molecules with different sizes). As we add PS homopolymer to the PS60-bPHEMA150 block copolymer, we form much larger hydrophobic

Figure 4. AFM images of PS60-b-PHEMA150/PS surfaces with j = 0.2 (A
D) and 0.4 (E
H) obtained in PBS buffer solution (left) and FBN protein solution (right). (A, C, E, and G) Height images. (B, D, F, and H) Corresponding phase images. In images G and H, the locations of the FBN protein monolayers on PS islands are indicated. A cross-sectional profile of image G (green line) is shown in panel J; the height of the FBN monolayer is 1.6 ( 0.4 nm.

PS domains. Figure 3 shows histograms of domain areas for j = 10 and 20% (from Figure 1C,D). At j = 10%, the PS domain areas are distributed in the range of ∼5  103
8  104 nm2, with a mean value of (2.4 ( 1.5)  104 nm2; at j = 20%, the range broadens to ∼1  104
2  105 nm2, with a mean value of (7.9 ( 4.7)  104 nm2. The three protein molecules show different adsorption behaviors on these surfaces. For the smallest MYO, the amount of protein adsorption scales linearly with the total surface area of the PS homopolymer domains (Figure 2A). However, for the two larger protein molecules, the amounts of protein adsorption are clearly below those predicted by the linear relationship. For BSA (Figure 2B), appreciable protein adsorption is observed only for j > 10%, corresponding to hydrophobic domain sizes g(2.4 ( 1.5)  104 nm2, which is ∼430 times the projected size on the surface of the protein molecule in the native 7061

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Langmuir state. For the largest FBN (Figure 2C), the threshold is j ≈ 20%, corresponding to an average PS domain area of (7.9 ( 4.7)  104 nm2. However, the use of the average domain area underestimates the critical size requirement for protein adsorption. This is obvious when we compare the two histograms in Figure 3. Upon going from j = 10 to 20%, the most important difference is the appearance of larger domains in the range of ∼1  104
2  105 nm2. These larger islands are responsible for the adsorption of FBN molecules, and the lower size limit for protein adsorption is ∼1  104 nm2. This threshold size of the adhesive domain is ∼280 times larger than the projected area (∼350 nm2) of fibrinogen in the native state. A kinetic study based on the growing disk model concluded that the final footprint of an adsorbed FBN molecule on a hydrophobic self-assembled monolayer surface was ∼475 nm2.10 If we assume similar behavior on the hydrophobic PS surface, the threshold size of the adhesive domain is at least ∼210 times larger than the final footprint of the adsorbed fibrinogen molecule. The need for the critical adhesive domain size for the nonspecific adsorption of protein molecules is also confirmed by AFM imaging. Figure 4 shows AFM images obtained for the mixed polymer films (j = 20 and 40%) under a PBS buffer solution or an FBN protein solution (50 μg/mL in PBS). Note that the experimental timescale in this experiment is ∼2 h and the amount of protein adsorption is much higher than those obtained for the 180 s protein adsorption time in Figure 2. For j = 20%, the AFM images (A
D) show little difference for the surfaces immersed in the two solutions, indicating negligible protein adsorption, in agreement with the protein adsorption rate in Figure 2. When j is above the critical value (e.g., 40% shown in Figure 4E
H), AFM images taken for the surface immersed in FBN protein solution are clearly different from those of the control samples immersed in PBS buffer solution only. The presence of adsorbed protein molecules is evidenced by the observation of raised islands of 1.6 ( 0.4 nm in height on each PS domain (image G and cross-section J). These islands are made of much softer materials than those of either the PS domains or the PS60-b-PHEMA150 matrix, as shown by phase image H. The softer nature of these islands is consistent with adsorbed protein (FBN) molecules. The height of the adsorbed protein islands is a factor of ∼4 smaller than the small dimension (∼7 nm) of an FBN molecule in the native state, indicating substantial unfolding of the protein molecules in the adsorbed state. It is important to point out that the formation of continuous islands in the nonspecific adsorption of protein molecules, as seen in Figure 4G, does not contradict the requirement of critical domain size for the mobile precursor state. Specifically, we do not expect a large number of holes when the amount of nonspecifically adsorbed protein reaches saturation on a hydrophobic surface. A large adhesive area is needed for the mobile precursor state, not the final adsorbed state. In the case of monolayer protein adsorption, when it is near saturation, the last a few protein molecules can still adsorb through a precursor state on top of the occupied surface area, before it finally lands in the hole. This picture is well established in the field of thin film material growth. Note that for the ordered surface of the diblock PS200-bPHEMA50 thin film with PHEMA domains in the PS matrix (Figure 1B), the amounts of BSA and FBN adsorption (open circles in Figure 2) are the same as those on PS60-b-PHEMA150/ PS with the same percentage of surface PS. The drastic difference between these two surfaces in morphology and roughness does not seem to affect the amount of BSA or FBN adsorption. For the

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smaller MYO, the amount of protein adsorption on PS200-bPHEMA50 is 50% lower than the trend predicted for PS60-bPHEMA150/PS. This may be related to the inability of small protein molecules to bridge across the hydrophilic PHEMA domains on the former surface. A detailed study of how surface morphology and roughness affect protein adsorption is of definite interest and deserves future study. To further support the interpretation presented above, we carry out quantitative analysis of the adsorption kinetics presented in Figure 2. Although the actual adsorption process is very complex and involves multiple steps or protein unfolding, to a first approximation we can simplify the mobile precursor model as k a0

ka

M þ θPS h θ0 sf θ kd

ð1Þ

where M is a protein molecule in the solution; θPS is the relative coverage of the adhesive PS domain; θ0 is the coverage of the adsorbed protein molecule in the mobile precursor state; θ is the coverage of irreversibly adsorbed protein; and ka0 , ka, and kd are the rate constants. We assume that, after sufficient unfolding and dynamic sampling of the surface, the precursor state can desorb from the surface with probability p; the probability of irreversible adsorption is therefore (1
p). In the initial stage of protein adsorption (good for low protein coverage), the rate constants for protein desorption from the precursor state and adsorption into the irreversible absorbate state are related by kd = kop and ka = ko(1
p). Note that we are treating the initial stage of adsorption when the effect of occupied surface sites by irreversibly adsorbed protein molecules does not come into play. Under a steady-state approximation for θ0 , the initial rate (ra) of protein adsorption is given by ra ¼

dθ ¼ ka0 ½MθPS ð1
pÞ dt

ð2Þ

Now we turn to the dependence of the desorption probability (p) on the PS domain size. For each PS domain of radius R, what determines the probability of desorption is the dimension of each adhesive domain relative to the root-mean-square displacement (λ) of each protein molecule in the mobile precursor state ! Z ¥ rffiffiffi 21 x2 exp
2 dx ð3Þ p¼ πλ λ R which is a complementary error function; in the classic 2D random-walk problem, the mean square displacement is λ2 = 4Na2, where N is the total number of steps in the random walk and a is the step size.26 As shown in Figure 3, there is a broad distribution of PS islands sizes, with the mean size increasing with θPS. An analysis of images with θPS < 0.4 shows that the mean radius R increases nearly linearly with θPS: R ≈ 830(θPS) nm. If we apply this approximate relationship to all θPS, then we can then fit the experimentally measured adsorption rates as a function of θPS to eq 2 with λ and ka0 as the only fitting parameters. The fitting results are shown as solid curves in Figure 2. Despite the simplicity of the kinetic model, it satisfactorily describes the experimental results for all three proteins, with λ = 85 ( 40, 600 ( 180, and 2700 ( 1800 nm for MYO, BSA, and FBN, respectively. As expected, the root-mean-square displacement of each mobile precursor state increases with the size of the protein molecule. The need for adhesive domain sizes that are more than 2 orders of magnitude larger than the footprint of irreversibly 7062

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Figure 5. Schematic illustration of protein adsorption on hydrophobic domains (red) in a hydrophilic matrix (blue).

adsorbed protein molecules is unexpected. Although it is well accepted that the dynamics of protein adsorption on a hydrophobic surface consists of multiple states, from the weakly adsorbed initial state to the strongly adsorbed final state, our finding shows that the precursor to protein immobilization must be in a mobile state on the surface. This conclusion is consistent with previous studies on the lateral mobility of protein molecules at solid/liquid interfaces.27 For example, Tilton et al. showed the presence of both mobile and immobile fractions of a chemically modified BSA on two polymer surfaces, poly(methylmethacrylate) and poly(dimethylsiloxane);28 the mobile fraction was found to diffuse over micrometer distances, in qualitative agreement with the mean displacement distance of λ ≈ 0.6 μm obtained here from kinetic analysis for BSA on the PS surface. In the field of surface science, the concept of a mobile precursor state is well established for molecular chemisorption.29 In such a mobile precursor model for protein adsorption, the weakly adsorbed protein molecule samples the surface while reorganizing, unfolding, and exposing more hydrophobic domains. If the hydrophobic adhesive domain on the surface is below a critical size, then the protein molecule in the mobile precusor state does not have enough time/space to achieve sufficient reorganization for irreversible adsorption, before it encounters the repulsive hydrophilic surface and desorbs from the surface, as illustrated schematically in Figure 5. Thus, the dynamics of the mobile precursor state plays an essential role in determining the eventual outcome of irreversible protein adsorption. This finding is important to a number of applications. In biosensing, the presence of a mobile precursor state on the sensor surface should quantitatively change the way one carries out kinetic analysis. In biomaterial research, although it is known that protein adsorption is sensitive to nanometer patterns on the scale of protein adhesion domains,11
15 our discovery of a mobile precursor state extends the surface pattern sensitivity to sizes that are more than 2 orders of magnitude larger; this finding adds a new principle to biomaterial design. In protein separation and purification technology, one may also envision the control of surface adhesive pad size as an additional tool.

’ CONCLUSIONS We study the kinetics and dynamics of nonspecific protein adsorption using patterned surfaces on the nanometer
micrometer

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scale. Each pattern, formed from block copolymers, involves hydrophobic and protein adhesive domains in a hydrophilic and protein repulsive matrix. We show that the area of each adhesive pad must be more than 2 orders of magnitude larger than the footprint of a protein molecule before irreversible adsorption occurs. We attribute this to the minimal surface area sampled by a mobile protein molecule in a precursor state before irreversible adsorption occurs. This interpretation is supported by kinetic modeling based on a key concept in surface science: a mobile precursor state is most often a prerequisite to chemisorption. The critical size requirement discovered here may be utilized in the surface design of biomaterials and biosensors. As a future research direction, one may directly probe the dynamics of the mobile precursor state on the surface for protein adsorption using advanced microscopy and spectroscopy techniques. These challenging experiments, when combined with theories and simulations that are much more sophisticated than the simplistic model used here, will start to provide a quantitative understanding of the important protein adsorption process.

’ ASSOCIATED CONTENT

bS

Supporting Information. SPR responses from two block copolymer thin films in contact with aqueous PBS solution. AFM height images taken in air and under PBS buffer solution for thin films of PS60-b-PHEMA150/PS mixtures with different weight ratios (j) of PS homopolymer. Cross-sectional profiles from AFM height images for thin films of PS60-b- PHEMA150/PS mixtures with different weight ratios (j) of PS homopolymer. AFM height images taken in air for a thin film of PS60-bPHEMA150. Cross-sectional profile from the red line in the AFM image. SPR response for FBN, BSA, and Myo (50 μg/mL) adsorption on PS60-b-PHEMA150/PS mixture thin films with various surface ratio of PS homopolymer (%). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the U.S. Army Research Office (W911NF-08-1-0287) and the University of Texas at Austin. We thank Prof. T. P. Lodge and Dr. Athena Guo for valuable advices. ’ REFERENCES (1) Macritchie, F. Adv. Protein Chem. 1978, 32, 283. (2) Pefferkorn, E.; Elaissari, A. J. Colloid Interface Sci. 1990, 138, 187. (3) Van Eijk, M. C. P.; Cohen, S. M. A. Langmuir 1997, 13, 5447. (4) Van Tassel, P. R. J. Chem. Phys. 1994, 101, 7064. (5) Van Tassel, P. R. J. Colloid Interface Sci. 1998, 207, 317. (6) Brusatori, M. A.; Van Tassel, P. R. J. Colloid Interface Sci. 1999, 219, 333. (7) Seigel, R. R.; Harder, P.; Dahint, R.; Grunze, M.; Josse, F.; Mrksich, M.; Whitesides, G. M. Anal. Chem. 1997, 69, 3321–3328. (8) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biomol. Struct. 1996, 25, 55. (9) Ostuni, E.; Chapman, R. G.; Holmin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (10) Wertz, C. F.; Santore, M. M. Langmuir 2002, 18, 706. 7063

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