Recognition of Protein Adsorption onto Polymer Surfaces by Scanning

The University of Nottingham, Nottingham NG7 2RD, U.K.. J. Davies, A. C. Dawkes, and J. C. Edwards. Johnson & Johnson Clinical Diagnostics, Pollards W...
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Langmuir 1997, 13, 4106-4111

Recognition of Protein Adsorption onto Polymer Surfaces by Scanning Force Microscopy and Probe-Surface Adhesion Measurements with Protein-Coated Probes X. Chen,* M. C. Davies, C. J. Roberts, S. J. B. Tendler, and P. M. Williams Laboratory of Biophysics and Surface Analysis, Department of Pharmaceutical Sciences, The University of Nottingham, Nottingham NG7 2RD, U.K.

J. Davies, A. C. Dawkes, and J. C. Edwards Johnson & Johnson Clinical Diagnostics, Pollards Wood Laboratories, Nightingales Lane, Chalfont St Giles, Buckinghamshire HP8 4SP, U.K. Received February 18, 1997. In Final Form: May 22, 1997X In this paper, we demonstrate in situ recognition of protein adsorption onto a polymer surface with scanning force microscopy by probe-surface adhesion measurements and topography imaging with proteincoated probes. Albumin-coated probes have been employed in studies of albumin and fibrinogen adsorption to hydrophobic polystyrene surfaces. The adhesion between force microscope probes and sample surfaces were determined using profiles of retract force-distance curves. A large adhesion force profile resulted when the force-distance curves were measured on protein-free polystyrene surfaces. When the measurements were conducted on protein-exposed polystyrene surfaces, the force-distance curves showed negligible adhesion. The same coated probes were also used for in situ topographic imaging. Applications of this novel approach are described: first, the location of boundaries of preadsorbed protein films and, second, the dynamic detection of protein adsorption onto polystyrene surfaces. Two-dimensional adhesion energy maps were obtained by employing “layered imaging”. We also note that an increase in pressure exerted by the force microscope probe results in penetration of a protein film and contact of the probe with the underlying polystyrene.

I. Introduction Adsorption of protein molecules onto solid substrate surfaces has been attracting much attention, not only because of its interesting role in pure surface sciences but also because of its important role in surface technologies. For example, selective adsorption of protein onto polymer surfaces is a central issue in development of biosensors and immunoassays.1 Partially or totally adsorptionresistant or “nonstick” polymer surfaces are also interesting in many applications.2 In the surface adsorption study, direct microscopic observations with high resolution are often desired to understand the biomolecular structure and interactions involved. Scanning force microscopy (SFM, also called atomic force microscopy, or AFM) offers an ideal tool for protein adsorption study, in aspects of both high spatial resolution and convenient operation environment.3 A biomolecular SFM study generally involves immobilization of a sample species onto a solid substrate surface, by either chemical binding or physical adsorption. Physical adsorption, which has the advantage of simplicity, usually involves both electrostatic and hydrophobic interactions and is accompanied by desorption.4 In many systems, the adsorption-desorption process of biomolecules at solid-liquid surfaces is complex, often making it difficult to differentiate adsorbates from the substrate by using SFM, especially if they possess similar topographies. Here we present an in situ method * To whom correspondence should be addressed. Phone: 44(0)115 9515063. Fax: 44-(0)115 9515110. E-mail: pazxc@ vax.nott.ac.uk. X Abstract published in Advance ACS Abstracts, July 1, 1997. (1) Benmakroha, Y.; Zhang, S.; Rolfe, P. Med. Bio. Eng. Comput. 1995, 33, 811. (2) Schmidt, D. L.; Coburn, C. E.; Dekoven, B. M.; Potter, G. E.; Meyers, G. F.; Fischer, D. A. Nature 1994, 368, 39. (3) Hansma, H. G.; Hoh, J. H. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 115. (4) Brash, J. L.; Horbett, T. A. ACS Sym. Series 1995, 602, 1.

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to identify protein adsorption onto polymer surfaces through the use of protein-coated SFM probes to measure probe-surface adhesion and to obtain complementary images. Interactions between SFM probes and sample surfaces depend on both probe and sample surface chemistry, geometry, and solution environment. Nevertheless, the problem essentially reduces to interactions between two surfaces, a geometry which has been studied using a variety of approaches.5-9 The advantage of using SFM in the study of such interactions results from its combination of high spatial and force resolution. For example, SFMs have been used to locate specific molecular interactions with a sensitivity to single molecular bonding forces, e.g. hydrogen bonding,10 avidin-biotin pairs,11-14 antibodyantigen pairs,15,16 and DNA strands.17 Indeed, SFM has been used for studies on a wide range of surface interactions by force-distance (f-d) measurements,18 e.g., local mechanical properties including friction,19,20 elasticity,20,21 (5) Israelachvili, J. N. Intermolecular and surface forces, 2nd ed.; Academic Press: London, 1991; Chapters 9-15. (6) Tabor, D.; Winterton, F. R. S.; Winterton, R. H. S. Proc. R. Soc. London, A 1969, 312, 435. (7) Derjaguin, B. V.; Rabinovish, Y. I.; Churaev, N. V. Nature 1978, 272, 313. (8) LeNeveu, D. M.; Rand, R. P.; Parsegian, V. A. Nature 1976, 259, 601. (9) Prieve, D. C.; Frej, N. A. Langmuir 1990, 6, 396. (10) Hoh, J. H.; Cleveland, J. P.; Prater, C. B.; Revel, J. P.; Hansma, P. K. J. Am. Chem. Soc. 1992, 114, 4917. (11) Pierce, M.; Stuart, J.; Pungor, A.; Dryden P.; Hlady V. Langmuir 1994, 10, 3217. (12) Lee, G. U.; Kidwell D. A.; Colton, R. J. Langmuir 1994, 10, 354. (13) Florin, E.-L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (14) Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257. (15) Stuart, J. K.; Hlady, V. Langmuir 1995, 11, 1368. (16) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (17) Lee, U. G.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771. (18) Butt, H.-J.; Jaschke, M.; Ducker, W. Bioelectrochem. Bioenerg. 1995, 38, 191.

© 1997 American Chemical Society

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and adhesion,19,22-26 various intermolecular forces including the DLVO forces and5,18,27-29 hydrophobic,30-33 structural,34 hydration,35 and steric36 forces. A number of groups have extended single point measurements to surface mapping of one or more of these local properties.20-26,37 While results are encouraging, there are still problems, including the influence of nonspecific forces, contact geometry, and system calibration. These factors can cause large deviations in experimental results. While nonspecific interactive forces interfere with the measurement of specific intermolecular forces, they may be exploited to probe other surface properties. The method presented here does not involve any specific ligandreceptor forces, instead, it exploits nonspecific hydrophobic interactions between a protein-coated probe and a sample. It has been shown that modified SFM probes not only can be used for studies of specific surface interactions but may also be employed for topographic scanning.38 The essential goal is to keep the same probe available for SFM imaging in situ, either subsequently or simultaneously. Experiments on both protein preabsorbed surfaces and protein dynamically adsorbing to surfaces have been performed. Discussions are focused on the complementary information obtained from in situ force-distance measurement, which provides a better understanding of the SFM images. II. Materials and Methods The SFM probe-sample configuration as employed here is schematically illustrated in Figure 1. The silicon nitride SFM probes are covalently coated with protein using a method similar to Vinckier et al.39 A slight modification is that the silanization of probe is realized by placing it into a room-temperature solution of 4% (3-aminopropyl)dimethylethoxysilane (Fluorochem Ltd., U.K.) in toluene. Each new probe was freshly coated before use. In this study, bovine serum albumin (BSA, Sigma Chemical Co.) was used for the probe coating, and both BSA and bovine fibrinogen (BFB, Sigma Chemical Co.) were used for the protein adsorption experiments. BSA and BFB were dissolved in 100 mM pH 7 potassium phosphate buffers prior to use, with concentrations from 0.001 to 1 mg/mL. (19) Koleske, D. D.; Lee, G. U.; Gans, B. I.; Lee, K. P.; DiLella, D. P.; Wahl, K. J.; Barger, W. R.; Whitman, L. J.; Colton, R. J. Rev. Sci. Instrum. 1995, 66, 4566. (20) Overney, R. M.; Meyer, E.; Frommer, J.; Gu¨ntherodt, H.-J.; Fujihira, M.; Takano, H.; Gotoh, Y. Langmuir 1994, 10, 1281. (21) Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hansma, P. K. Langmuir 1994, 10, 3809. (22) Torii, A.; Sasaki, M.; Hane, K.; Okuma, S. Sens. Actuators A 1994, 44, 153. (23) Mizes, H. A.; Loh, K. G.; Miller, R. J. D.; Ahuja, S. K.; Grabowski, E. F. Appl. Phys. Lett. 1991, 59, 2901. (24) Creuzet, F.; Ryschenkow, G.; Arribart, H. J. Adhes. 1992, 40, 15. (25) Radmacher, M.; Cleveland, J. P.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Biophys. J. 1994, 66, 2159. (26) van der Werf, K. O.; Putman, C. A. J.; de Grooth, B. G.; Greve, J. Appl. Phys. Lett. 1994, 65, 1195. (27) Senden, T. J.; Drummond, G. J.; Ke´kicheff, P. Langmuir 1994, 10, 358. (28) Drummond, C. J.; Senden, T. J. Colloids Surf. A 1994, 87, 217. (29) Senden, T. J.; Drummond, C. J. Colloids Surf. A 1995, 94, 29. (30) Rabinovich, Y. I.; Yoon, R. H. Langmuir 1994, 10, 1903. (31) Tsao, Y.-H.; Evans, D. F.; Wennerstro¨m, H. Langmuir 1993, 9, 779. (32) Tsao, Y.-H.; Evans, D. F.; Wennerstro¨m H. Science 1993, 262, 547. (33) Meagher, L.; Graig,V. S. J. Langmuir 1994, 10, 2736. (34) O’Shea, S. J.; Welland, M. E.; Rayment, T. Appl. Phys. Lett. 1992, 60, 2356. (35) Butt, H.-J. Biophys. J. 1991, 60, 1438. (36) Lea, A. S.; Andrade, J. D.; Hlady, V. ACS Sym. Series 1993, 532, 266. (37) Radmacher, M.; Tillmann, R. W.; Gaub, H. E. Biophys. J. 1993, 64, 735. (38) Xu, S.; Arnsdorf, M. F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10384. (39) Vinckier, A.; Heyvaert, I.; D’Hoore, A.; McKittrick, T.; Van Haesendonck, C.; Engelborghs, Y.; Hellemans, L. Ultramicroscopy 1995, 57, 337.

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Figure 1. Illustration of a protein-coated probe and a polymercovered sample surface with protein adsorption. The f-d measurement is achieved by varying the voltage applied on the piezoelectric crystal (z-piezo) which controls the SFM probe’s vertical displacement, while the lateral scanning is temporarily suspended. When the z-piezo extends, the probesample separation decreases, until the probe is brought into contact with the sample up to a preset point of maximum load. The z-piezo is then retracted and the probe is withdrawn from the sample surface. During a full z-piezo extension-retraction cycle, the recorded z-piezo voltage, which represents the relative probe-sample separation, and the deflection of the cantilever, which represents the probe-sample interactions, form an f-d curve. The SFM instrument employed was a TMX2000 Explorer (TopoMetrix Co., U.K.). Two types of scanners were used: a tripod-type scanner of 120 µm maximum lateral scan range with 10 µm vertical scan range and a tube-type of 3 µm maximum lateral range with 1 µm vertical range. All f-d measurements and SFM imaging were performed in liquid and the constantforce mode with contact forces as low as possible was employed in SFM imaging. To transfer the relative force scale (nA) recorded in the Explorer into the absolute force scale, two steps in calibration are performed. First, using the gradient of the contact region in the retract portion of the f-d curve, the signal shift (nA) is transferred into a corresponding probe vertical movement, or equivalently, the deflection of cantilever in nanometre (nm). The force applied on the cantilever in nanonewtons (nN) is then calculated from Hook’s law with the known spring constant of the cantilever. The spring constant (0.032 nN/nm) provided by the probe manufacturer is employed in the present study. Although this value possesses a large error, since the contrast in adhesion to be compared is much larger than the probe spring constant tolerance and, also, only the relative force difference is important in the in situ comparison, it is not necessary to exactly measure the spring constant of the individual cantilever as we routinely do in force measurement of specific molecular interactions.40 Silicon wafers (Agar Scientific Ltd., U.K.) were sequentially washed in chloroform, propanol, and methanol in an ultrasonic water bath, followed by rinsing with deionised water. Then 100 µL of a 0.5% polystyrene (Mw 45 000, Aldrich Chemical Co. Ltd.) in toluene solution was dropped onto the wafers spinning at a rate of 2000 rpm, resulting in a continuous and smooth coverage of polystyrene. The wafers were then glued with thermoplastic adhesive Tempfix (Agar Scientific Ltd., U.K.) onto the bottom of a home-made glass flow-through liquid cell of 20 mm diameter, allowing an easy refreshment or exchange of liquid in the cell. The liquid cell was fixed on the XY translation stage provided with the Explorer. Three types of experiments were performed, as follows. Method 1, Measurements on Protein Films Preadsorbed onto the Polymer Surface. After repeated control f-d measurements with the BSA-coated probes on several randomly chosen locations of a clean polystyrene surface in buffer, a 2 µL drop of BSA or BFB solution (0.1 mg/mL, pH 7) was placed on (40) Allen, S.; Chen, X.; Davies, J.; Davies, M. C.; Dawkes, A. C.; Edwards, J. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Biochemistry, in press.

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Figure 2. Force-distance curves and SFM images of a BSA film preadsorbed on a polystyrene surface with an BSA-coated probe. Polystyrene was dissolved in toluene with 0.5 mg/mL concentration and spin-cast on a silicon wafer. Half of one side of the sample surface was then covered with a drop of BSA solution of 0.1 mg/mL for 60 min. The sample was naturally dried in room temperature for 1 h before immersed in pH 7 phosphate buffer for both f-d measurements and SFM imaging. Typical f-d curves and SFM images obtained on the BSA-free region (a, b), the BSA-exposed region (c, d) and the boundary between the two regions (e, f) are presented, respectively. All SFM images were conducted in the contact mode with the 2 µm scanner. half of the polystyrene surface for 1 h. The surface was then rinsed with phosphate buffer from the protein-free side to the protein-exposed side. In this way, sample surfaces with two different domains, i.e., the bare polystyrene surface and the protein-exposed surface, were obtained. Force-distance measurements were conducted on both domains of the sample surface alternatively as the separation of the measuring points was steadily reduced. Such bracketing results in a final measurement location at the protein film boundary. Subsequent SFM imaging was performed in situ with the same probe. Tens of similar experiments with different protein solution concentrations were repeated to confirm consistence and variation. Method 2, a Dynamic in Situ Observation of Protein Adsorption onto Polymer Surfaces. After the polystyrenecoated wafers were mounted, control f-d measurements were carried out in buffer. The buffer was then replaced in a flowthrough fashion with BSA solutions. Time evolutions of f-d curves were recorded for solutions with different BSA concentrations (0.001, 0.01, and 0.1 mg/mL). When the f-d curves indicated that the surface was covered with protein molecules, SFM imaging was performed in situ with the same probe. Method 3, Adhesive Force Measurements on ProteinCovered Surfaces with Different Probe-Sample Contact Force Limits. The contact force limit is a preset threshold, which controls the maximum probe-sample contact force during the probe-sample approach. Immediately following the completion of method 2, f-d curves were measured and recorded while varying the contact force limit.

III. Results and Discussion Method 1, Measurements on Protein Films Preadsorbed onto the Polymer Surface. All the f-d curves measured on bare polystyrene show a profile similar to that in Figure 2a. In the approach phase and before the probe contacts the sample surface, there is no significant interaction between the probe and the sample surface and, therefore, no change in the force signal, resulting in a horizontal line. The sloped line indicates a contact between the probe and the sample surface, causing a deflection of the cantilever and, therefore, a force signal change. The constant gradient of the sloped line indicates that a hard surface is in contact with the elastic SFM cantilever whose deflection follows Hooke’s law. When the probe is retracted from the surface, the second sloped line is parallel to the first one. However, it extends beyond the original probe-sample contact point by more than 100 nm, indicating that large adhesive forces exist between the probe and the sample surface. These forces cause the cantilever to stick to the sample surface until its spring force exceeds the adhesive forces, leading to a sudden jump of the cantilever back to the noncontact position. These large adhesive forces are due to strong interactions between the hydrophobic polystyrene surface and the protein molecules on the probe. The absolute adhesion strength may vary by about 2- or 3-fold in repeated

Recognition of Protein Adsorption

measurements, especially with different probes. This is a typical phenomenon for most adhesive force measurements and is possibly due to an inhomogeneous probe coating and variations in the probe-sample contact area. The SFM images of the bare polystyrene surface with the same BSA-coated probes measured in buffer show the expected smooth surface. A typical image is shown in Figure 2b. The curve at the left edge is due to a frictioninduced artifact. Such an artifact is observed when a large adhesive interaction exists between the probe and the sample surface. Note that the SFM data were acquired during the interval when the probe scanned from the left side of the scope area to the right side. When the data acquisition was taken during the reverse scanning, such a curve appeared at the right edge (images not shown). When the f-d measurements were conducted on the protein-exposed half-surfaces, only weak adhesion was observed in the retraction curves (Figure 2c). This observation results from the nonspecific interactions between protein molecules being much weaker than the interactions between the hydrophobic polystyrene surface and the protein-coated probe. This effect is particularly noticeable in a pH environment above the isoelectric points of both albumin and fibrinogen,41 e.g., at pH 7, where a strong repulsive force between protein molecules caused by their negative surface charge prevents any distinct adhesion between the protein-coated probe and the protein-covered sample surface. The SFM image obtained on the BSA-exposed halfsurface (Figure 2d) shows densely packed globular features, indicating a continuous albumin coverage. The typical diameter of these features is nearly 100 nm, which is about 10 times larger than the albumin molecule size. One possibility is that the coverage of albumin molecules may not be complete and the probe convolution effect may result in such enlarged features, if the probe apex radius is increased by the coating. However, if such a case happens, the f-d measurements should show typical adhesions when the BSA-coated probe touches the large bare polystyrene surface between the sparsely-distributed protein molecules. The consistent lack of adhesion observed in repeated f-d measurements excludes such a possibility. Therefore, the protein coverage must be continuous, and the 100 nm globular features are probably due to aggregation of albumin molecules. We also observed in our repeated experiments that such aggregation preferentially occurred for the higher protein concentrations above 0.1 mg/mL. Additional evidence for albumin aggregation comes from SFM images obtained with a normal noncoated probe, which showed similar features (images not shown). While adhesion profiles observed in the two different regions (the bare and the protein-coated) of the sample surface showed distinct differences and were repeatable, they displayed considerable variation in the intermediate boundary region. Figure 2e shows a typical f-d curve observed at the boundary which exhibits two typical features. First, the average adhesion strength is intermediate between the protein-exposed and the proteinfree regions. This can be seen from both the maximum adhesive force magnitude, indicated by the largest jump height, and the adhesion energy, indicated by the area above the retracting f-d curve and below the extending f-d curve. This result may be anticipated since, during the production of the protein-coated surfaces, the boundaries cannot be accurately controlled. Such a partial coverage of polystyrene surface divides the surface into small discrete subregions. This also causes the second (41) Smith, E. L.; Hill, R. L.; Lehman, I. R.; Lefkowitz, R. J.; Handler, P.; White, A. Principles of Biochemistry: General Aspects, 7th ed.; McGraw-Hill Book Company: New York, 1983; p 43.

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Figure 3. Images of BSA (a) and BFB (b) films preadsorbed on polystyrene surfaces with BSA-coated probes. Both were conducted in pH 7 phosphate buffer in the contact mode with the 120 µm scanner. Sample preparations were similar to that described in the legend of Figure 2.

typical feature, an intermittent contact of the probe to the polystyrene surface, so that the single large jump of the continuous polystyrene surface is replaced with a series of smaller jumps. The partial protein coverage also reduces the total contact area between the probe and the polystyrene surface, so that the adhesion energy is decreased. In our experiments, we found that it was not difficult to locate a 120 µm × 120 µm area in which a large adhesion was observed at one side while weak (or no) adhesion was observed at the other side both with a probability of higher than 95%. Such a difference was repeatable when measurements were moved from one side to another and vice versa, by changing the voltage applied on the piezoelectric crystals controlling the scanner’s lateral offsets. In such a way, the protein film boundaries were located before the commencement of imaging. The SFM image of the boundary region between the BSA-free and the BSA-covered surfaces (Figure 2f) looks similar to that of a protein fully-covered surface. However, by a careful comparison of repeated acquired images in these two regions, we found that the globular features in the boundary region always showed a slightly lower density and larger size. Together with the information provided by the f-d measurements, we know that the lower density results from the discontinuous coverage of albumin. However, because of the large size of the features, it is difficult to know whether they are separate albumin molecules or aggregates, even when the probe convolution effects are taken into account. Note that all SFM images shown in Figure 2 are obtained with the 2 µm scanner, which possesses a relatively higher resolution but a smaller scan range than the 120 µm scanner. Figure 3a is an image in the boundary region between the albumin-covered and albumin-free polystyrene surfaces obtained with the 120 µm scanner and reveals fractal-type structures formed during the drying process. Such structures were never observed in the dynamic measurements of either albumin or fibrinogen adsorption without dry process. The boundary region between the fibrinogen-covered and fibrinogen-free polystyrene surfaces (Figure 3b) shows different drying

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Figure 5. Dynamic observation of BSA adsorption onto a polystyrene surface. Retract f-d curves were measured on a polystyrene spin-cast surface with a BSA-coated probe after the surface was exposed to a BSA solution (0.01 mg/mL, pH 7, 100 mM phosphate buffer) for 0 min (I), 4 min (II), 5 min (III), 7 min (IV), 10 min (V), and 60 min (VI) without movement of the sampling location.

Figure 4. (a) Two-dimensional distribution of adhesion energy between a BSA-coated probe and a polystyrene surface preadsorbed with BSA, with (b) a simultaneous recorded surface topography (2 µm × 2 µm). The sample preparation was similar to that for Figure 2. The measurements were performed in pH 7 phosphate buffer at the boundary region between the BSAfree and BSA-covered polystyrene surfaces. The adhesion energies are extracted from f-d curves measured at each sampling point, with brighter color indicating higher adhesion energy. The topographic image is artificially shadowed for a better presentation.

patterns. Near the edge of the fibrinogen film, there are radial line structures, due to the shrinking of the film during vaporization of water, similar to the albumin case. At the very edge of the film, there is a continuous “fence”, about 20 nm in width and 80 nm in height, much higher than the average roughness of the fibrinogen film. Such aggregation was never observed in the drying process of albumin. An extension of the method employed here to recognize protein adsorption onto polymer surfaces is a simultaneous acquisition of both topography and f-d curves in the boundary region between a BSA-covered surface and a bare polystyrene surface. Such data are shown in Figure 4. An adhesion energy distribution over the surface is extracted by using the Genesis Graphics System42 from (42) Williams, P. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B.; Wilkins, M. J. Nanotechnology 1991, 2, 172.

the f-d curves measured during a two-dimensional scan on the sample surface with a BSA-coated probe and shown in Figure 4a. For each surface point, the adhesion energy is calculated by E ) ∑f∆s, where f is the adhesive force, taken from the difference between the approach f-d curve and the retract f-d curve, and ∆s is the probe displacement, taken from the z scan step. Simultaneous topography data obtained with the same probe for each surface point are shown in Figure 4b. As expected, the lower adhesion energy regions, diagnostic of protein-protein interactions, correspond to regions of protein surface coverage in the topographic map, indicating a partial coverage of the polystyrene with BSA in this boundary area. Method 2, a Dynamic in Situ Observation of Protein Adsorption onto Polymer Surfaces. Time evolution of f-d curves with a BSA solution of 0.01 mg/ mL is shown in Figure 5. For clarity, only the retract f-d curves, which represent the probe-sample adhesions, are shown. Before the protein solution was introduced into the liquid cell, all f-d curves showed large adhesion forces due to strong probe-substrate interactions, similar to that in Figure 2a, as shown by curve I in Figure 5. About 4 min after the buffer was exchanged for the protein solution, changes in the f-d curves were observed. The most obvious is the decrease in adhesion force strength. In many cases the large and sharp jump associated with strong probe-substrate interactions disappeared, as shown by curves III and V in Figure 5. This indicates that the polystyrene surface was becoming covered with adsorbed protein molecules. In addition, a sharp jump related to a return of strong adhesion (curves II and IV in Figure 5) was also observed in the first 15 min, though the sampling point on the surface had not changed. The four curves (II-V in Figure 5) are randomly chosen examples, showing that the disappearance of the sharp jump in the complete f-d curve series was unpredictable. Two possibilities can be considered to account for such an irregularity. First, the partial coverage and random distribution of the adsorbed protein molecules, combined with thermal drift of the instrument, can lead to a touch of the probe to the bare polystyrene surface at one moment and to the protein-coated surface at the next. This is similar to the case when imaging at the boundary region between the bare polystyrene surface and the preadsorbed protein film, as described above in method 1. Second, this irregularity may also result from a dynamic

Recognition of Protein Adsorption

Figure 6. Dependence of adhesion on the probe-sample contact force limit. The adhesion is taken from the different between the free-level of the approach f-d curve and the lowest point of the corresponding retract f-d curve.

adsorption-desorption process of protein onto the polystyrene surface during the gradual formation of the protein monolayer. After 15 min, the sharp jumps in f-d curves appeared with decreasing frequency and were no longer observed after 1 h. This clearly indicates that the adsorption of protein molecules under these static conditions forms a continuous and stable film on the polystyrene surface after this time period. Subsequent in situ SFM imaging performed with the same probe confirmed this observation (images not shown). The experiments with BSA solutions of 0.001 and 0.1 mg/mL concentrations showed similar results, but, as anticipated, with different kinetics for protein monolayer formation. In the 0.001 mg/mL BSA concentration case, a strong adhesion was always observed during the 2-h period examined, indicating that complete protein surface coverage was not reached. At 0.1 mg/mL BSA concentration, only a few minutes were required for protein film formation and the complete loss of adhesion. Both findings were also confirmed by SFM imaging. Method 3, Adhesive Force Measurements on Protein-Covered Surfaces with Different ProbeSample Contact Force Limits. Following the loss of adhesion with a 0.01 mg/mL BSA solution in method 2, strong adhesion could be observed if the limit on the maximum probe-sample contact force was increased. We found that this force limit dependence of adhesion strength was reversible. This relationship is displayed in Figure 6. From the experiments described in method 1 and method 2, we know that strong adhesion indicates a contact of the protein-coated probe with the polystyrene surface. Therefore, when the surface is fully covered with protein, the observed strong adhesion at a high contact force limit indicates a deformation of the adsorbed protein film, suggesting that the probe penetrates through and touches the underlying polystyrene surface. The reversibility in adhesion strength indicates that the deformation of the protein film by the probe is recoverable, either by a movement of surrounding adsorbed protein molecules or by a rapid adsorption of protein molecules from solution. However, after another 40 min exposure to BSA solution, no large adhesion was observed till the contact force limit was set to the maximum value allowed by the measuring system. This means the probe is no longer able to touch the underlying polystyrene surface unless high forces are involved, indicating either a change in the packing of protein layer with time or an irreversible adsorption of protein molecules, which forms a firm monolayer or a multilayer protein film with increased thickness. To exclude the possibility that the

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protein coating on the probe was damaged in such large contact force measurements, control f-d measurements with the same probe on another bare polystyrene surface were performed. These measurements still showed the typical strong adhesion. Resolution and Stability of the Protein-Coated Probe. To achieve a stable coating of the probe without a significant decrease in resolution, a uniform monolayer coating would be ideal. In practice, the steep geometry of the probe apex may introduce some difficulty in achieving such a complete and smooth coverage. Using probe geometry derivation routines discussed elsewhere,43,44 we estimate a typical effective probe radius of 20 nm for the protein-coated probes as compared to 5 nm for the clean probes. This increase in probe size is evidence that there exists aggregation of protein molecules on the probe. Such an increase in probe aspect would tend to decrease the effective resolution. However, it is wellknown that even without such a coating, the silicon nitride probes may also be readily contaminated and coated by some weakly-adsorbed protein from solution, causing a similar reduction in resolution. We found no relation between the probe quality and different “curing” processes discussed in the literature, though the curing process has been proved to be important in obtaining a good monolayer coating.45 In the event that the protein layer on a probe becomes damaged, we have found that the coating integrity may be reestablished with a slightly reduced stability by simply dipping the probe into protein solution. Such a recovery may be repeated a number of times. In contrast, simply dipping a clean probe into a BSA solution without the previous silanization treatment does not result in a stable f-d measurement. IV. Conclusions In this study, we have shown that, with a protein-coated probe, f-d measurements are capable of recognizing protein absorption onto polystyrene surfaces, either statically or dynamically. In general, such methods can be applied to other situations, as long as the f-d curves between the probe and the sample show some character distinguishable from those between the probe and the substrate. We have proved that a protein-coated probe can be used for f-d measurements and in situ SFM imaging, both separately and simultaneously, without a prohibitive reduction in image quality. In a preliminary study, we have also shown that the adhesion measurement between the probe and the protein-covered substrate is dependent on the probe-surface contact force limit, possibly due to deformation of protein films by the probe. Acknowledgment. The authors acknowledge the support and funding of the postdoctoral fellowship to X.C. from the BBSRC and Johnson and Johnson Clinical Diagnostics. S.J.B.T. is a Nuffield Foundation Science Research Fellow. LA970172I (43) Williams, P. M.; Shakesheff, K. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B. Langmuir 1996, 12, 3468. (44) Williams, P. M.; Shakesheff, K. M.; Davies, M. C.; Jackson, D. E.; Roberts, C. J.; Tendler, S. J. B. J. Vac. Sci. Technol., B 1996, 14, 1557. (45) Vrancken, K. C.; Van Der Voort, P.; Possemiers, K.; Grobet, P.; Vansant, E. F. In Chemically Modified Surfaces, Proc. Symp. on Chemically Modified Surfaces; Pesek, J. J., Leigh I. E., Eds.; Roy. Soc. Chem.: Cambridge, U.K., 1994; p 46.