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In Situ Observation of the Surfactant-Induced Displacement of Protein from a Graphite Surface by Atomic Force Microscopy A. Patrick Gunning,* Alan R. Mackie, Peter J. Wilde, and Victor J. Morris Department of Food Quality and Materials Science, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. Received January 21, 1999. In Final Form: April 5, 1999 Atomic force microscopy has been used to visualize, in real time, the breakdown and removal of a β-lactoglobulin film from a graphite surface, by the addition of the nonionic surfactant Tween 20 to the liquid cell of the microscope. The initial stage of surfactant adsorption onto the graphite surface is seen to involve the nucleation of small surfactant domains within the protein network. These surfactant domains expand, compressing the protein network. The reduction in surface area covered by protein is compensated by an increase in the thickness of the protein film. Eventually, at sufficiently high surface concentration of surfactant, the protein network fractures allowing release of protein for displacement from the surface. The displacement mechanism observed at the graphite surface has been compared with the displacement by Tween 20 of a spread β-lactoglobulin protein layer from an air-water interface. In both cases a similar “orogenic” mechanism of displacement has been observed. The present studies provide a molecular model for the cleaning of protein films from surfaces.
Introduction Although both proteins and surfactants can act as stabilizers for foams or emulsions, it is known that addition of surfactant to a protein-stabilized interface results in the weakening and breakdown of the interfacial protein film.1 Until recently the exact mechanism for protein displacement was unclear despite extensive studies of the adsorption of proteins to various interfaces2-4 and the displacement of proteins from interfaces by competitive adsorption.5-10 Recent studies11 have shown that displacement of the milk proteins β-casein, β-lactoglobulin, and R-lactalbumin from an air-water interface, by the nonionic surfactant Tween 20, involves a novel “orogenic” mechanism. The surfactant adsorption at the interface involves the nucleation and growth of surfactant domains which compress the protein network, leading eventually to fracture of the protein network and the release of protein for displacement from the surface.11 In these studies the protein displacement was studied using a Langmuir trough and monitoring the surfactant adsorption through measurements of surface pressure. The structure of the mixed protein-surfactant films was investigated by (1) Wustneck, R.; Kra¨gel, J.; Miller, R.; Wilde, P. J.; Clark, D. C. Colloids Surf., A: 1996, 114, 255-265. (2) Al-Malah, K.; McGuire, J.; Sproull, R. J. Colloid Interface Sci. 1995, 170, 261-268. (3) Mackie, A. R.; Mingins, J.; North, A. N. J. Chem. Soc., Faraday Trans. 1991, 87, 3043-3049. (4) Cao, C.; Damodaran, S. J. Agric. Food Chem. 1995, 43, 3, 25672573. (5) Chen, J. S.; Dickinson, E.; Iveson, G. Food Struct. 1993, 12, 135146. (6) Clark, D. C.; Mackie, A. R.; Wilde, P. J.; Wilson, D. R. Faraday Discuss. 1994, 98, 253-262. (7) Dickinson, E.; Horne, D. S.; Richardson, R. M. Food Hydrocolloids 1993, 7, 497-505. (8) Horne, D. S.; Atkinson, P. J.; Dickinson, E.; Pinfield, V. J.; Richardson, R. M. Int. Dairy J. 1998, 8, 73-77. (9) Dalgleish, D. G.; Srinivasan, M.; Singh, H. J. Agric. Food Chem. 1995, 43, 2351-2355. (10) Euston, S. E.; Singh, H.; Munro, P. A.; Dalgleish, D. G. J. Food Sci. 1995, 60, 1124-1131. (11) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157-166.
imaging Langmuir-Blodgett films, pulled from the interface at defined surface pressures, using an atomic force microscope (AFM). Confirmation that the structures observed by AFM were representative of the structures of the mixed films at the air-water interface came from the demonstration that the changes in surface area occupied by surfactant, and measured from the AFM images, correlated with the changes in surface pressure measured at the interface on the Langmuir trough.11 A more direct approach to studying structural changes in mixed surfactant-protein films would be to observe the structural changes directly at the interface. Although such studies are feasible,12 they require specialized apparatus and the motion of the interface will reduce the ability to resolve structural detail. An alternative is to observe displacement of a protein layer from a solid hydrophobic surface in contact with a surfactant solution. In addition to acting as a model system for studying protein displacement from interfaces, the use of a solid surface provides a simple model for examining the “cleaning” of protein deposits from a surface. The adsorption of proteins to solid substrates has been used as a model system for studying protein adsorption at surfaces13,14 and displacement of protein from surfaces with surfactant.1,3,15 Thus the present study offers the possibility of visualizing the structural changes occurring at such solid surfaces. In the present study a layer of β-lactoglobulin molecules has been deposited onto a graphite surface from an air-water interface using Langmuir-Blodgett methods. The displacement of this protein layer by Tween 20 has been observed in real time by AFM. The displacement mech(12) Eng, L. M.; Seuret, Ch.; Looser, H.; Gunter, P. J. Vac. Sci. Technol., B 1996, 14, 1386-1389. (13) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 257265. (14) Dickinson, E.; Robson, E. W.; Stainsby, G. J. Chem. Soc., Faraday Trans. 1983, 79, 2937-2952. (15) Mackie, A. R.; Mingins, J.; Dann, R.; North, A. N. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; RSC Special Publication No. 82, RSC: Cambridge, 1991; pp 96-112.
10.1021/la990069o CCC: $18.00 © 1999 American Chemical Society Published on Web 05/20/1999
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anisms observed directly at the graphite surface have been compared with those observed indirectly for the air-water interface. Experimental Section The milk protein used in the present study was β-lactoglobulin (L-0130, lot 91H7005) from Sigma Chemicals (Poole, U.K.), which has a molecular weight of 18.6 kDa. A solution was prepared at a concentration of 2 mg mL-1 in phosphate buffered saline (PBS, Sigma). The water used in the study was surface pure (γ0 ) 72.6 mN m-1 at 20 °C) cleaned using an Elga Elgastat UHQ water purification system. Tween 20 (molecular weight 1230) was purchased as a 10% solution (Surfact-Amps 20) from Pierce (Rockford, IL). Surface tension measurements were made using a wetted ground glass Wilhelmy plate and a Langmuir trough. The PTFE trough had dimensions 255 × 112 × 16 mm, a volume of 450 mL, with one fixed and one movable barrier. All experiments were performed at room temperature (20 °C) with the Elgastat purified water as the subphase. The β-lactoglobulin was spread at the air-water interface; the required volume was added carefully dropwise via a glass rod onto the surface and the rod then rinsed with buffer. Protein (64.8 µg) was added to the interface. For the real time imaging of protein displacement the protein film was transferred onto freshly cleaved highly oriented pyrolytic graphite (HOPG grade ZYH, Union Carbide, USA) using the Langmuir-Blodgett technique. The mounted piece of graphite was driven down through the interface and then back out again at a constant rate of 0.14 mm s-1. At this modest rate of dipping there is virtually no excess water left on the sample once it is recovered from the interface, because the sample dewets as it is removed from the interface. Nevertheless, the sample was allowed to air-dry for 10 min and then mounted in the liquid cell of the AFM under distilled water. The protein was transferred from a film spread initially at the air-water interface rather than adsorbed from a protein solution so that the final surface density on the graphite would be comparable to that used previously.11 The AFM used in the present study was manufactured by ECS Ltd. (Cambridge, U.K.). The probes used were Nanoprobe (Digital Instruments, Santa Barbara, CA) oxide-sharpened cantilevers with a quoted force constant of 0.38 N m-1. For the real time studies, images were acquired by “tapping-mode” in liquid at a frequency of 34.6 kHz. This was done by driving the cantilever near resonance by oscillating the whole liquid cell with an ac signal from the internal oscillator of a lock-in amplifier added to the Z piezo drive signal of the AFM. The lock-in amplifier was then used to detect both the amplitude and phase of the cantilever response. The amplitude signal from the cantilever is rectified by the lock-in and was used by the AFM feedback loop to control the force between sample and tip. The instrument used was an EG&G instruments dual channel lock-in amplifier, model 5302 (Wokingham U.K.). For imaging of samples transferred from the air-water interface onto mica (described later) the AFM was operated in constant force mode (dc mode). Images were acquired under redistilled 1-butanol and the imaging force was in the range 1-3 nN. For the dynamic studies the protein film on the graphite was first imaged under distilled water in order to confirm the presence of a uniform protein layer. Tween 20 stock solution was then added to the liquid cell in order to produce a final concentration of 4 mM surfactant. Addition of surfactant destabilized the imaging conditions, and the driving frequency had to be altered to regain control of the AFM and re-establish stable imaging conditions, a process which took only a few minutes. This effect probably arose due to adsorption of surfactant (or possibly protein) onto the probe, increasing the mass of the probe and lowering the resonant frequency of the probe-cantilever assembly. Adsorption onto the tip will also effect force-distance curves over the sample. Images were then acquired sequentially to follow adsorption of surfactant onto the graphite and the consequent displacement of protein from the surface. The collection time for individual images is approximately 2 min, and the entire sequence of images was collected over a period of about 26 min. Note that the gray scale which progresses linearly from black to bright
Langmuir, Vol. 15, No. 13, 1999 4637 white depicts a different height range in each frame. Average film thickness data presented in Figure 3 was determined from the separation of the peaks representing the substrate and protein layer in AFM image histograms. The investigation of the displacement of β-lactoglobulin from the air-water interface with Tween 20 has been described in detail elsewhere.11 Tween 20 was added to the subphase (final concentration 0.5 µM) and the adsorption to the interface monitored by measurements of surface tension. At defined surface pressures the interfacial structure was sampled by transferring Langmuir-Blodgett films onto freshly cleaved mica and then imaging the samples by AFM. The mica was dipped in and out at a constant rate of 0.14 mm s-1, air-dried for 10 min, and then imaged in the liquid cell of the AFM under butanol. It is worth mentioning that protein was only transferred onto the mica as it left the water phase.
Results and Discussion Figure 1 shows a sequence of images obtained after addition of Tween 20 to the liquid cell. Each image is a three-dimensional map of the surface structure. The bright regions represent the rigid protein network, and the darker regions represent surfactant. The sequence demonstrates progressive adsorption of surfactant onto the graphite and the ultimate displacement of protein. The first image in Figure 1 shows the appearance of holes or domains in the protein film which are irregular in shape. It is constructive to compare the images in Figure 1 with those in Figure 2 acquired for protein-surfactant films pulled from the air-water interface onto mica at defined surface pressures. The starting point for the protein films was the same for both these studies, i.e., the same amount of β-lactoglobulin was spread on the airwater interface prior to the addition of Tween-20 and/or film transfer. The AFM images in Figure 2 are measured under butanol, which dissolves the surfactant, thus fixing the structure of the film. Consequently the black surfactant domains in Figure 2 are unambiguously identified, providing strong evidence that the dark areas seen in Figure 1 also correspond to surfactant domains. In Figure 1 the mobility of the surfactant molecules, their comparatively small size, at least in terms of cross section, and the large scan size make it impossible to resolve molecular detail within the surfactant domains. Subsequent images in the sequence in Figure 1 show that the irregular surfactant domains grow in size while the protein layer increases in thickness. The protein coverage shrinks resulting in protein areas linked by extended threads of protein which eventually break leaving a continuous surfactant phase containing islands of protein. During the early stages of surfactant domain growth, where the structure of the film is changing rapidly, the images are streaky indicating some instability in the imaging. In the later stages of the displacement process, where the changes in film structure are slower, the streakiness is reduced and the images are more stable. The similarity between the displacement patterns observed in Figures 1 and 2 suggest that the mechanisms are the same in both cases: an orogenic displacement mechanism.11 The orogenic mechanism involves the nucleation and growth of surfactant domains which reduce the area covered by protein at the surface or interface. In the previous detailed studies of protein displacement from the air-water interface11 it was noted that the reduction in surface area occupied by protein did not necessarily mean a reduction in the surface concentration of protein. The reduction in surface area was accompanied by an increase in film thickness, demonstrating that the proteins could reduce the interfacial contact area by reorientation or compression of the molecules, or buckling of the film,
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Figure 1. AFM “movie” sequence of the displacement of β-lactoglobulin from the graphite surface by the addition of surfactant Tween-20. The time interval between frames is typically 160 s. All frames in the sequence are of the same size and area on the substrate, although there is a small amount of drift. The scale bar in frame 1 is 1 µm. For values of the average film thickness (or height) see Figure 3.
without reducing the total surface concentration. Only in the final stages of collapse, after fracture of the protein
network, was protein actually lost from the surface. It should be noted that substantially higher levels of
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Langmuir, Vol. 15, No. 13, 1999 4639
Figure 2. Comparative AFM images of a spread β-lactoglobulin film which has been partially displaced by Tween-20 from an air/water interface. Image details, scan size, surface pressure (π) for each: (a) 1 × 1 µm, π ) 18.6 mN‚m-1; (b) 1.6 × 1.6 µm, π ) 20.2 mN‚m-1; (c) 3.2 × 3.2 µm, π ) 22.5 mN‚m-1; (d) 10 × 10 µm, π ) 27.1 mN‚m-1.
surfactant are needed to displace the β-lactoglobulin film from the graphite surface than those required for removal from the air-water interface. This is to be expected because the solid graphite presents a much more hydrophobic surface than air. Although it is difficult to be certain that at least some material was not progressively eroded from the interface, the increase in thickness with time suggests a mechanism similar to that observed in our previous work. In that case the conservation of film volume as the protein domains were compressed showed that during the majority of the compression phase very little adsorbed protein is solubilized. Only in the final stages of collapse was protein lost from the interface. The present images of protein displacement from graphite are very difficult to obtain, and the image quality is not as good as that obtained in the previous interfacial studies.11 Because of this it is not possible to carry out a detailed qualitative analysis of the images. However, measurements of average film thickness show that the contraction of the protein network is accompanied by an increase in film thickness (Figure 3), following the same trend as observed for protein displacement from the airwater interface.11 Average film thickness was measured
Figure 3. Protein film thickness data (average height of protein film derived from histogram analysis) versus time after addition of surfactant Tween-20.
rather than peak height which varies unpredictably as protein is lost to solution. The larger protein thicknesses observed in this study may indicate the presence of a bilayer. However, it should be noted here that the measurement of film thickness by tapping in liquid can be problematic when following dynamic events since tip contamination may alter the force-distance characteristics of the system.16 Because of the material adsorbed
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onto the tip, probe-sample interactions may alter and the displacements of the surface required to maintain the set amplitude deflection of the cantilever may not merely reflect relative thicknesses of the two domains. It has been shown previously that tapping mode experiments on biological samples where the scanner is oscillated at high frequency (as in this case) can result in false information in the vertical direction.17 Another explanation of the enhanced thickness observed by tapping under surfactant solution is that the protein layer is more hydrated and swollen than when imaging under butanol. However, irrespective of the absolute values, the changes in the average height of the protein layer with time are significant. Thus it can be seen that there is not a gradual loss of protein by erosion while the surfactant domains expand since this would involve no changes in the height of the protein layer. Rather the data suggest, in common with observations made on films transferred from the airwater interface,11 that the growth of the surfactant domains is opposed by elastic energy stored in the protein network. As a consequence the protein layer is forced to thicken in the competition for space at the interface and, as observed before, protein is only lost from the surface once the protein network has fractured.11 The orogenic displacement mechanism observed previously at airwater interfaces11 and apparently confirmed in the present studies can explain effects observed in recent scattering studies of competitive protein-surfactant adsorption at air-water interfaces,8 where an increase in thickness was observed prior to the displacement of protein from the interface. This effect was also observed in much earlier studies of foam films comprising β-lactoglobulin and Tween-20.6,18,19 In these previous studies, fluorescent labeling was used to show phase separation between the protein and surfactant. This labeling confirmed that it was the protein phase which increased the foam film thickness.18,19 The new mechanism presented here ex(16) Radmacher, M.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Science 1994, 265, 1577-1579. (17) Schabert, F. A.; Rabe, J. P. Science 1996, 70, 1514-1520. (18) Coke, M.; Wilde, P. J.; Russell, E. L.; Clark, D. C. J. Colloid Interface Sci. 1990, 138, 489-504. (19) Clark, D. C.; Coke, M.; Wilde, P. J.; Wilson D. R. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; RSC Special Publication No. 82; RSC: Cambridge, 1991; pp 272-276.
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plains the observed extensive adsorption of surfactant with little protein displacement from the surface. It also explains the increase in layer thickness before complete protein displacement from the surface. Given that the mechanisms of displacement are seen to be similar for air-water and solid surfaces, the heterogeneous nature of the displacement mechanism needs to be considered in interpreting scattering studies of competitive adsorption of proteins and surfactants to interfaces and solid surfaces. The present studies provide further evidence for a heterogeneous orogenic displacement mechanism. These studies also demonstrate the feasibility of using AFM to study adsorption processes, such as the biofouling of surfaces, or to investigate removal of biopolymers from surfaces, the “cleaning” of surfaces. Conclusions AFM has been used for in situ imaging of the displacement of the protein β-lactoglobulin from a liquid/graphite surface by the nonionic surfactant Tween 20. The displacement process has been shown to be similar to that previously observed for displacement of β-lactoglobulin from an air-water interface by Tween 20.11 In both cases the process has been shown to be orogenic and not simply a progressive erosion or exchange of individual proteins. The heterogeneous nature of the displacement mechanism needs to be considered in any interpretation of scattering studies used to investigate competitive adsorption of proteins and surfactants at interfaces and solid surfaces. Removal of protein layers from surfaces will be of importance in the “cleaning” of surfaces. In general it will be necessary to remove multilayers of proteins from the surface and the molecular mechanisms for the surfactantprotein interactions may vary with film thickness. The present studies illustrate a molecular mechanism for the removal of protein layers adjacent to the surface. Acknowledgment. This research was supported by the BBSRC core grant to IFR. Supporting Information Available: Video clip of film removal. This material is available free of charge via the Internet at http://pubs.acs.org. LA990069O
