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Nov 23, 2004 - Structure of β-Casein Layers at the Air/Solution Interface: Atomic Force Microscopy Studies of Transferred Layers. Grigor B. Bantchev ...
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Structure of β-Casein Layers at the Air/Solution Interface: Atomic Force Microscopy Studies of Transferred Layers Grigor B. Bantchev and Daniel K. Schwartz* Department of Chemical Engineering, University of Colorado at Boulder, Boulder, Colorado 80309-0424 Received June 30, 2004. In Final Form: October 1, 2004 We report the nanoscale structural changes associated with the interfacial gelation of adsorbed β-casein layers as a function of aging time. Adsorbed layers were transferred to solid supports and imaged by atomic force microscopy. The aging of the layer was accompanied by the formation of distinct disk-shaped protein nanoparticles (∼20 nm in diameter). Under conditions where a gelled layer was expected (from previous interfacial rheology experiments), we observed ordering of the particles and the formation of elongated aggregates or linear rows. Brewster angle microscopy images were also obtained during the adsorption and gelation processes and during the degradation of the protein layer following addition of the surfactant sodium dodecyl sulfate (SDS). If SDS was added prior to interfacial protein gelation, the layer developed a foamlike morphology consistent with a fluid interfacial protein layer. However, if SDS was added after gelation, the protein layer was observed to fracture, consistent with the behavior of a solid phase.

Introduction In previous work,1 we reported measurements of the complex shear modulus (i.e., storage and loss moduli) of β-casein layers adsorbed at the air/water interface as a function of aging time. We found that initially the layer responded like a viscous 2D liquid, but that at higher coverage (higher concentration and longer aging times) it manifested predominantly elastic behavior. The frequency dependence of the shear moduli suggested that the observed transition was consistent with a 2D sol-gel transition described by percolation theory (see Winter,2 also Stauffer and Aharony3 for more details). Thus, solutions with concentrations >1.0 × 10-3 wt % formed an interfacial gel after 10-15 h of aging. Because β-casein is incapable of forming covalent cross-links (it contains no cysteine residues), the elasticity of the gelled layer presumably relies on physical interactions (e.g., van der Waals, H-bonding, hydrophobic interactions). Mackie et al. have studied the nanoscale structure of mixed proteinsurfactant layers.4-7 However, the nanoscale details of the structural changes associated with interfacial gelation remain poorly understood. In the present work, we present atomic force microscopy (AFM) images of β-casein layers transferred to a solid substrate by Langmuir-Schaefer deposition, as well as Brewster angle microscope (BAM) images of the layer, and correlate the structural features observed to the associated changes in the interfacial rheology under equivalent conditions. BAM observations indicated that the adsorbed protein layer was laterally homogeneous on * Corresponding author. Phone: 303-735-0240. Fax: 303-4924341. E-mail: [email protected]. (1) Bantchev, G. B.; Schwartz, D. K. Langmuir 2003, 19, 2673. (2) Winter, H. H.; Mours, M. Adv. Polym. Sci. 1997, 134, 165. (3) Stauffer, D.; Aharony, A. Introduction to Percolation Theory, 2nd ed.; Taylor & Francis, Inc.: Washington, DC, 1992. (4) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (5) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. (6) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 8176. (7) Mackie, A. R.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Patino, J. R. Biomacromolecules 2001, 2, 1001.

length scales >2 µm and grew gradually thicker with aging time. AFM images of appropriately transferred films were consistent with the BAM images at large length scales, but indicated the formation of distinct protein particles (∼20 nm in diameter) on the time scale of several hours. With additional aging, the particles organized into rows and appeared to aggregate in a “string of pearls” manner. Given the time-dependence of these structural changes, we hypothesize that interfacial gelation is associated with the formation of these aggregates. BAM observations provided further insight into the mechanical properties of aged β-casein layers. In particular, images of layers aged for different times (2 and 48 h) revealed different degradation mechanisms when exposed to the low-molecular weight surfactant sodium dodecyl sulfate (SDS). While a layer aged for only 2 h developed a foamlike morphology, described in the literature4-7 as consistent with a “liquid” layer, a layer aged for 48 h appeared to “fracture”, indicating solidlike mechanical properties. Materials and Methods Materials. β-Casein (Sigma C-6905, min. 90% β-casein by electrophoresis, lyophilized, essentially salt-free, Lot 108H7813) was used for the experiments. The actual lot analysis, according to the supplier, showed that β-casein was 95%; the contaminants were mainly γ-caseins (fragments of β-casein) ∼3%, R-casein ∼1%, and ∼1% of another variant of β-casein. No κ-casein was detected. Batch solutions were prepared by dissolving approximately 0.02-0.03 g of β-casein in 100 mL of phosphate buffer solution (water from Millipore UV+ system) with ionic strength 0.1 M (which is close to the ionic strength of bovine milk) and pH ) 7.4 (Fisher Sci, buffer B82). NaN3 (0.1 g/L) was added to the buffer solution to avoid bacterial contamination. The batch solution concentrations (about 0.03 wt %) were below the “CMC” of β-casein8 of 0.17 wt % and were kept in a refrigerator at ∼5 °C to avoid aggregation. A batch solution was not used longer than a week after its preparation. All experiments were performed at ambient temperature, 23.5 ( 1 °C. The bulk protein concentrations are reported in weight percent units. Small circular pieces of mica (9 mm diameter), used for most of the samples (Ted Pella high grade mica product No. 52-6), were cleaved immediately (8) Payens, T. A. J.; Brinkhuis, J. A.; van Markwijk, B. W. Biochim. Biophys. Acta Protein Struct. 1969, 175, 434.

10.1021/la048380f CCC: $27.50 © 2004 American Chemical Society Published on Web 11/23/2004

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Table 1. The Initial and Corrected Concentrations As Described in the Texta

C initial 2 × 10-5 wt % 1 × 10-4 wt % 1.1 × 10-3 wt % 5.5 × 10-3 wt % a

C after C after adsorption on adsorption on air/water interface all interfaces 1.2 × 10-5 wt % 9.1 × 10-5 wt % 1.1 × 10-3 wt % 5.5 × 10-3 wt %

C final

4.5 × 10-6 wt % 6 × 10-6 wt % 7.0 × 10-5 wt % 7.5 × 10-5 wt % 1.0 × 10-3 wt % 1.0 × 10-3 wt % 5.5 × 10-3 wt % 5.5 × 10-3 wt %

Values from the column “C final” were used further in the text.

prior to film deposition to expose fresh hydrophilic surface before the transfer. For experiments where the samples were transferred to a silicon substrate, silicon wafers were cut into small pieces (∼1 cm × 1 cm), rinsed with ethanol, and gently boiled in a Piranha solution (mixture of 70 vol % H2SO4 and 30 vol % of 30% H2O2) for 30 min. The clean silicon pieces were thoroughly rinsed with Millipore water and stored under water until use (within 24 h). In the cases where hydrophobic silicon was used, it was hydrophobized by placing the Piranha-cleaned pieces in ∼5% HF solution for 10 s, rinsed with Millipore water, kept dry, and used within 24 h. The HF etch results in a “passivated” hydrophobic silicon hydride surface. Glassware and Teflon parts were cleaned by boiling in Piranha solution for 30 min and were rinsed with Millipore water. Plastic tubing was cleaned with detergent (Alconox) and rinsed with copious amounts of Millipore water. Sample Preparation. Protein solutions were prepared by diluting the batch solution with buffer to the desired amount (110 mL) and concentration. The solution was introduced into crystallization dishes (75 mm diameter) and left to age. After the desired aging time, the layer was transferred to a solid support as described below and imaged using AFM. The layer transfer is a destructive procedure for the adsorbed layer, so solutions were discarded after a sample was transferred. Solutions in separate crystallization dishes were used for layers with different aging times. Interfacial adsorption of the protein resulted in a significant depletion of the bulk concentration. An estimate was made for the final protein concentration, based on the adsorption isotherm9 and interfacial areas within the container. Table 1 shows the initial protein concentrations and the approximate concentration after adsorption. The values were calculated using two possibilities, which we regard as extreme: (1) protein adsorbs only at the air/solution (22.7 cm2) and Teflon/solution (22.9 cm2) interfaces; and (2) the protein adsorbs on the glass surface (103.3 cm2) at the same density as at the air/solution interface. A third, more realistic, approximation (Cfinal) assumed that the adsorption on the glass surface was 75% less than at the air/solution interface. This approximation was reached by comparing the surface pressures of solutions contained within vessels with negligible surface area/volume ratios to the surface pressure of solutions contained within our standard deposition vessel.1 Our surface pressure measurements were cross-correlated with an isotherm taken from Hunter et al.9 Had we used the data of Graham and Phillips,10 a slightly weaker effect of adsorption would have been calculated. Several techniques were evaluated for sample transfer. In some cases, we exploited a Langmuir-Schaefer (LS) technique using mica, SiO2 (clean silicon wafer), or SiHx (hydrophobic etched silicon wafer). The layer was transferred simply by touching the substrate to the water surface; in all of the cases, the substrate was held parallel to the interface. While vertical LangmuirBlodgett (LB) deposition is easier to perform in a controlled manner, the layer is subject to flow and deformation during deposition. A well-executed LS transfer can result in a deposition without deformation of the layer. Following film transfer, the substrate after was held in a vertical position, and excess solution was removed by bringing the edge into contact with a Kimwipe or by gentle blowing with dried, filtered nitrogen. Other samples (9) Hunter, J. R.; Kilpatrick, P. K.; Carbonell, R. G. J. Colloid Interface Sci. 1991, 142, 429. (10) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 415.

were transferred in such a way as to leave exposed the original film/air interface (i.e., deposition from below). A region of the aged interface was separated (the layer on it was kept undisturbed), creating a fresh region of new interface, and the substrate was immediately immersed through this new interface into the solution. The substrate was then brought upward through the undisturbed aged interface, transferring the layer. For reasons stated below, the most reliable and valuable results were ultimately obtained using LS deposition on mica substrates. However, the qualitative similarity of film structure using other deposition methods and substrates serves as valuable corroborating evidence of transfer fidelity. To obtain transferred samples representative of the adsorbed layer, it was necessary to exchange the protein solution for buffer prior to film transfer. (Samples transferred without subphase exchange displayed large “droplets” visible by AFM; the droplets were presumably residue from bulk solution following evaporation.) Most samples were prepared in modified crystallization dishes, with an added spout at the bottom. The solutions with desired concentrations were prepared by introducing the buffer solution and the batch solution through the spout. A thin Teflon film with 19 mm diameter holes was placed at the solution/air interface, where it floated due to capillary forces. The floating film ensured that the contact lines moved along with the surface and thus prevented deformation of the layer during the subphase exchange. After the protein was allowed to adsorb at the interface for the desired time, the subphase was exchanged with a diluted phosphate buffer solution (pH ) 7.4, I ) 0.001 M). The dilution was done through the spout. It was carried out by removing part of the initial solution and carefully refilling with the diluted buffer. This procedure was carried out eight times, replacing ∼50 mL of solution each time. Each dilution (assuming ideal mixing) was by a factor of 2.5 or more, which brings the total dilution above 1000. In this way, the solution was rendered essentially protein free, which was confirmed by the significant decrease of the foam stability observed for the removed solution after several exchanges. The final solution taken from the beaker could not sustain bubbles for longer than 5 s. Because proteins are often found to adsorb irreversibly to hydrophobic interfaces,11,12 we assume that there was little significant desorption of the protein from the air interface during the subphase exchange. AFM Imaging. The transferred samples were studied using a Nanoscope III MM-AFM (Digital Instruments, a subsidiary of Veeco Metrology Group) under ambient conditions (i.e., samples were imaged dry). Two modes were utilized. The first was Tapping Mode, providing topographic and surface hardness information. Tapping mode is known to be less damaging to surfaces under investigation13 than contact mode because the transferred energy is less and because the tip does not exert a significant lateral force on the surface. Phase images are shown in this manuscript because they generally provided greater contrast than topographic images. The same features are apparent using both contrast mechanisms. The second method was contact mode, where the main information collected was the approximate thickness of the layer (as described below). While it provided similar topographic information for the layer, the images were of lower quality, because of shear deformation and wear by the AFM tip. To determine the approximate film thickness, we exploited the fact that contact mode can be used to intentionally damage the surface layer. In this experiment, an area was scanned in contact mode with a large load force to abrade the protein layer and open a hole reaching to the mica surface. Only mica was used as a substrate in this type of experiment, because it was much flatter, making it easier to determine when the protein layer was removed. The exact procedure was the following: First, an area 2 × 2 µm2 was scanned and an image was acquired. A subarea of 0.5 × 0.5 µm2 was then scanned with a applied normal (11) MacRitchie, F. Reversibility of Protein Adsorption. In Proteins at Liquid Interfaces; Miller, R., Ed.; Elsevier Science: New York, 1998; p 149. (12) Dickinson, E. J. Dairy Sci. 1997, 80, 2607. (13) Fritz, M.; Radmacher, M.; Cleveland, J. P.; Allersma, M. W.; Stewart, R. J.; Gieselmann, R.; Janmey, P.; Schmidt, C. F.; Hansma, P. K. Langmuir 1995, 11, 3529.

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force of >34 nN for 30 min at a scan rate of 10 Hz (∼36 times) to remove a section of the adsorbed layer. Finally, a larger (imaging) scan was performed (1.5 × 1.5 µm2) using a lower force. For most samples, this process resulted in the exposure of a flat mica region. The difference in the height between the lower parts of the scanned region and the unscanned region was taken as the approximate layer thickness. Occasionally, however, the central area did not appear flat. In these cases, the images were not used to calculate film thickness. The average lateral diameter of particles in AFM images was measured by directly measuring the peak-to-peak distance between adjacent distinct features.14 To decrease the error of these measurements, we looked for sequences of approximately 10 features and measured the overall distance and the number of objects. The values obtained from different images were averaged, and the standard deviation was determined. For closepacked particles, the average peak-to-peak distance is an accurate representation of the lateral particle diameter, although the height difference from peak to valley provides little information because of the limited tip penetration due to the tip radius. This is in contrast with the situation for isolated particles, where the apparent height of a particle is a true representation of particle size, but the lateral size measurement is complicated by tip resolution effects. Our results do not indicate that these are spherical particles. In fact, they are disklike; the measurements described here determine the (larger) lateral diameter, while the thickness measurements determine the short diameter. Brewster Angle Microscopy. Brewster angle microscopy (BAM) relies on the fact that the p-polarized light incident at the Brewster angle does not reflect from an ideally sharp dielectric interface. When a layer is present on the interface, however, the Brewster condition is broken. The contrast in the reflectivity from a surface layer can be observed with a CCD camera through a microscope objective. A more detailed description of our BAM and Langmuir trough has been previously published.15 We have previously used this method to investigate the 2D phase behavior of and hydrodynamic phenomena within Langmuir monolayers.16-25 The solution (100 mL) was poured in a Langmuir trough (100 × 250 mm), with the barrier at the center of the trough. The interface was aspirated to remove surface contaminants, and BAM images were digitally recorded. During aging, the trough was covered to prevent contamination and to decrease evaporation of the solution. After an aging period, SDS solution (1 mL, 10-2 M) was injected on the side of the barrier that was not observed with the BAM. The SDS diffused through the subphase and came in contact with the protein layer within the BAM field of view. Sample Heterogeneity and Control Experiments. In contrast with interfacial films formed from synthetic and highly purified compounds that we have studied in the past,26-37 the samples we examined during the course of these studies had a greater degree of structural variation, from region to region within a given sample and between different samples. This included some features that could clearly be ascribed to impurities, as well as other observations that were more difficult to explain. We took several measures to be certain that the results reported here were representative of the true interfacial structure of (14) Simmons, B. A.; Taylor, C. E.; Landis, F. A.; John, V. T.; McPherson, G. L.; Schwartz, D. K.; Moore, R. J. Am. Chem. Soc. 2001, 123, 2414. (15) Kurnaz, M. L.; Schwartz, D. K. Phys. Rev. E 1997, 56, 3378. (16) Ignes-Mullol, J.; Schwartz, D. K. Phys. Rev. Lett. 2000, 85, 1476. (17) Ignes-Mullol, J.; Schwartz, D. K. Langmuir 2001, 17, 3017. (18) Ignes-Mullol, J.; Schwartz, D. K. Nature 2001, 410, 348. (19) Ivanova, A.; Kurnaz, M. L.; Schwartz, D. K. Langmuir 1999, 15, 4622. (20) Ivanova, A. T.; Ignes-Mullol, J.; Schwartz, D. K. Langmuir 2001, 17, 3406. (21) Ivanova, A. T.; Schwartz, D. K. Langmuir 2000, 16, 9433. (22) Kurnaz, M. L.; Schwartz, D. K. Phys. Rev. E 1997, 56, 3378. (23) Riviere, S.; Henon, S.; Meunier, J.; Schwartz, D. K.; Tsao, M. W.; Knobler, C. M. J. Chem. Phys. 1994, 101, 10045. (24) Schwartz, D. K.; Tsao, M. W.; Knobler, C. M. J. Chem. Phys. 1994, 101, 8258. (25) Ocko, B. M.; Kelley, M. S.; Nikova, A. T.; Schwartz, D. K. Langmuir 2002, 18, 9810.

Bantchev and Schwartz adsorbed protein layers. The characteristic images presented in the results section are representative of observations that satisfy the conditions described below. (1) Repetition. We prepared multiple replicate samples for every concentration and time point and acquired images at multiple locations on each sample. Samples were also prepared using several different deposition methods as discussed above. This approach permitted us to distinguish characteristic and repeatable observations from one-time artifacts due to impurities, etc. (2) Independent Control Measurements. As discussed below, the approximate film thickness was measured by AFM to compare this thickness with that expected from comparisons with previous reports. This allowed an independent check of at least one structural property of the transferred films. (3) Systematic Time-Dependence. We carefully analyzed the time-dependence of observed structures, because we believe that systematic trends in the evolution of structure as a function of aging time are consistent with the structure being native to and representative of the aged film. In contrast, structural artifacts that form during transfer from the liquid surface to a solid support are not likely to have a clear correlation with the duration of the pretransfer aging time.

Results and Discussion Layer Thickness. Although the destructive determination of approximate protein layer thickness by AFM is not particularly accurate as compared to a number of other methods, we regarded it as a critical internal control experiment to demonstrate that the transferred samples had properties consistent with that of the adsorbed layer at the liquid interface. Figure 1a shows the approximate adsorbed β-casein layer thickness as a function of aging time and solution concentration. There is significant scatter in the data. This is due, to a large extent, to the uneven thickness of the layer itself, but there were also some differences in the degree of tearing through the layer during the removal process, as well as uncertainties due to the roughness of the surface and damage to the layer with the AFM tip during imaging. Still, despite the scatter of the data, we observed a significant increase of the layer thickness with an increase of the bulk concentration and with aging time. For short aging times, the thicknesses of the layers increased quickly for high concentrations and slowly for low concentration solutions. At longer aging times, the thickness of the layer was increased at roughly the same rate for all solution concentrations. The absolute thickness was in reasonably good agreement with other methods used for the determination of protein adsorption (see Figure 1b).9,10,38-40 This is consistent with the idea (26) Kurnaz, M. L.; Schwartz, D. K. J. Phys. Chem. 1996, 100, 11113. (27) Schwartz, D. K. Surf. Sci. Rep. 1997, 27, 245. (28) Schwartz, D. K.; Garnaes, J.; Viswanathan, R.; Chiruvolu, S.; Zasadzinski, J. A. N. Phys. Rev. E 1993, 47, 452. (29) Schwartz, D. K.; Viswanathan, R.; Garnaes, J.; Zasadzinski, J. A. J. Am. Chem. Soc. 1993, 115, 7374. (30) Schwartz, D. K.; Viswanathan, R.; Zasadzinski, J. A. N. Langmuir 1993, 9, 1384. (31) Schwartz, D. K.; Viswanathan, R.; Zasadzinski, J. A. N. Phys. Rev. Lett. 1993, 70, 1267. (32) Sikes, H. D.; Schwartz, D. K. Science 1997, 278, 1604. (33) Takamoto, D. Y.; Aydil, E.; Zasadzinski, J. A.; Ivanova, A. T.; Schwartz, D. K.; Yang, T. L.; Cremer, P. S. Science 2001, 293, 1292. (34) Viswanathan, R.; Madsen, L. L.; Zasadzinski, J. A.; Schwartz, D. K. Science 1995, 269, 51. (35) Viswanathan, R.; Zasadzinski, J. A.; Schwartz, D. K. Science 1993, 261, 449. (36) Viswanathan, R.; Zasadzinski, J. A.; Schwartz, D. K. Nature 1994, 368, 440. (37) Zasadzinski, J. A.; Viswanathan, R.; Madsen, L.; Garnaes, J.; Schwartz, D. K. Science 1994, 263, 1726. (38) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847. (39) Russev, S. C.; Arguirov, T. V.; Gurkov, T. D. Colloids Surf., B: Biointerfaces 2000, 19, 89-100. (40) Sengupta, T.; Damodaran, S. Langmuir 1998, 14, 6457.

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Figure 2. Representative phase contrast AFM images of layers adsorbed at the surface of 7.5 × 10-5 wt % β-casein solution after aging times of (a) 4 and (b) 48 h.

Figure 1. (a) Thickness measured from AFM images for layers adsorbed from β-casein solutions. The aging time is the elapsed time prior to subphase exchange. (b) Literature data for the adsorption of β-casein at the air-solution interface. The points at high concentrations are compiled from four sources. A density of 0.8 g/cm3 was assumed for the protein layer (i.e., 1 mg/m2 was counted as equivalent to 1.3 nm thickness). Error bars represent the standard deviations of multiple measurements. Lines are simply guides to the eye.

that the transferred samples were faithful replicas of the adsorbed layer and not, for example, artifacts of the transfer process itself. The fact that film thickness measured by AFM is slightly smaller than that measured by in situ methods may be due to the fact that films at the air/solution interface are swollen due to greater hydration. Also, in situ measurements may include an artificial additional thickness from proteins loosely associated with the adsorbed layer. Sample Transfer Methods. AFM images of samples transferred to mica, SiO2, or SiHx were qualitatively similar, in that the same types of features were observed as a function of solution aging time. However, the presence of random roughness on the silicon wafers made it difficult to distinguish surface texture due to the protein film from that of the underlying solid substrate. Mica substrates, on the other hand, are atomically flat and permitted unambiguous determination of protein layer structure. Protein layers transferred from “above” or “below” also resulted in qualitatively similar AFM images. However, the transfer of layers from below was much more elaborate and involved a greater risk of contamination. Indeed, AFM images of layers transferred from below sometimes displayed “holes” not otherwise observed, consistent with the presence of surface-active contaminants. Therefore, the images that will be reported here are for samples deposited from above onto mica substrates. This is somewhat counterintuitive from a Langmuir-Blodgett deposition perspective, because one would generally use a hydrophobic substrate for deposition from the hydrophobic side of an adsorbed layer. However, our empirical observations suggest that it is appropriate in this case. We speculate that the differences between the deposition of the protein layer as compared to what would be expected

Figure 3. Representative phase contrast AFM images of layers adsorbed at the surface of 1.0 × 10-3 wt % β-casein solution after aging times of (a) 0.5 and (b) 24 h.

for a simple molecular amphiphile can be ascribed to the strongly viscoelastic character of the adsorbed protein layer (which helps the layer maintain integrity) and the fact that there is not complete separation of hydrophilic and hydrophobic residues on the aqueous and vapor sides of the layer, respectively. Nevertheless, the qualitative similarity of film structure using a variety of substrates and deposition methods is an important finding. Combined with the consistency of the time-dependent thickness measurements with previous measurements of the native surface film, these observations suggest that the transferred films do indeed represent faithful replicas of the native protein film structure. Characteristic Nanoscale Appearance of Protein Layers. Low Concentration (5 × 10-3 wt %). Interfacial gelation was always observed at these concentrations in interfacial rheology experiments.1 The smooth images (Figure 5a) at early aging times evolved to rougher ones at intermediate and long aging times (Figure 5b). The increase of the roughness was associated with the appearance of more pronounced (both in height and in phase) features. At intermediate aging times, particles that were close to one another were hard to distinguish (Figure 5b), while at long aging time (Figure 5c), the individual features had deep dividing “valleys” between them, despite being quite close to each other. There was also a tendency toward clustering of the particles leading to the appearance of extended aggregates (see Figure 5c). In fact, the presence of linear clusters (sometimes well-organized as shown in Figure 4) was characteristic of conditions under which the layer is known to be gelled. Sizes of the Observed Features. The characteristic lateral size of the distinct features observed in the AFM images described above was of the order of 20 nm in all cases. Taking into account the typical layer thickness of 2-3 nm (Figure 1), this suggests the existence of protein aggregates in the shape of oblate ellipsoids. A more detailed analysis of feature sizes indicates a weak correlation with the layer aging and subphase concentration; values of these parameters are shown in Table 2. The

data show an increase in the lateral size of the features with the increase of the concentration. The effect seems to get weaker with the increase of the aging time, but still exists. A less pronounced effect was the change of the size with aging; there was a small decrease of particle size with increasing aging time. It is informative to estimate the number of molecules per protein aggregate. Assuming ellipsoidal particles, approximately 20 nm in diameter and 2.5 nm thick, one calculates a volume of ∼750 nm3. A rule of thumb for protein volume is (1.2 × molecular weight) Å3, which for the 25 kDa β-casein results in a volume of 30 nm3/molecule. This gives approximately 25 molecules/aggregate. BAM Observations. BAM images were recorded for protein layers that were expected to gel with aging time, adsorbed from 5.5 × 10-3 wt % β-casein solution. Initially, the protein adsorbed gradually, forming a layer that became brighter with age, indicating an increase in layer thickness. The layer was laterally homogenous (Figure 6a), and no domains were visible at the BAM’s resolution (several µm), which is consistent with the results reported in the literature.41 The apparent contrast in Figure 6a is due to illumination artifacts (interference and uneven illumination). The BAM results are also consistent with the fact that Martin et al.42 detected no change in the β-casein conformation for ∼8 h aging of the layer. Definite changes were observed in the behavior of the layer when SDS surfactant was injected in the subphase. The surfactant penetrated easily into the freshly formed layer (aged for 2 h) and caused the formation of dark “bubbles” in the BAM image. Presumably, the addition of surfactant to the interface initially compresses the remaining protein film; at later stages, protein is removed from the interface. Coarsening of the “bubble” structure eventually resulted in the formation of a 2D foam at the interface (Figure 6b). This foam ultimately broke and was transformed to small irregular-shaped islands (Figure 6c). These islands were eventually displaced from the surface after about an hour. On the other hand, the layer aged for 48 h showed greater rigidity and fractured upon penetra-

Figure 5. Representative phase contrast AFM images of layers adsorbed at the surface of 5.5 × 10-3 wt % β-casein solution after aging times of (a) 1.5, (b) 14, and (c) 24 h.

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6e) and sharp kinks. The islands were stable; while they rotated freely in the surfactant-dominated layer (darker region), they did not change their shape. After the initial rapid breakdown of the layer, there was some decrease in size of the remaining islands, but it was not accompanied by deformation. The islands were eventually displaced from the surface after about 1 day in contact with SDS. While the behavior of the layer aged for 2 h resembled the description of AFM and BAM experiments by Mackie et al.,4,7 the behavior of the layer aged for 48 h was quite different. The formation of round holes was attributed by Mackie et al. to the fluidity of the protein layer; the foamlike morphology is also consistent with this premise. However, our observation of cracks at long aging times demonstrates that the 48 h aged layer has solid behavior. Although some details of the experiments and results differ, recent results by Mackie et al.4,7 also suggest that holes formed in β-casein layers during displacement by surfactant (the nonionic Tween 20, in their case) are round for fresh layers but become irregular for aged protein layers. Conclusions We found that β-casein forms layers at the air/water interface that can be transferred with the LS technique to a solid substrate and investigated using AFM. The thickness of the transferred layers was qualitatively consistent with previous measurements using other methods 9,10,38-40 AFM images of transferred films demonstrated that the layer was initially smooth and essentially homogeneous with isolated particles that consisted of multiple β-casein molecules. The increase of concentration or aging time led to greater layer thickness accompanied by some overlapping of the particles. With the aging of the layer, the particles tended to form extended or linear aggregates and, especially for layers formed at higher concentrations, to become smaller and harder. This formation of aggregates correlates with the previously measured gelation of the layers. BAM images showed that the layer was homogeneous at large scales even after 48 h, suggesting that gelation is due to structural changes on smaller length scales. The degradation of aged layers upon introduction of SDS suggested an evolution from a fluid protein film at short aging times to a solid film after longer aging times, consistent with rheological measurements and the trend observed by other investigators.4,7 Figure 6. BAM images of layers adsorbed at the air interface of 5.5 × 10-3 wt % β-casein solution: (a) after 2 h aging, (b, c) layer aged for 2 h followed by the injection of SDS, and (d, e) layer aged for 48 h followed by the injection of SDS.

tion by SDS (Figure 6d). The islands formed were larger, and the island boundaries contained straight edges (Figure

Acknowledgment. This work was supported by the U.S. Department of Agriculture (Award No. 2002-3550312520). LA048380F (41) Rodrı´guez Patino, J. M.; Sa´nchez, C. C.; Rodrı´guez Nin˜o, M. R. Food Hydrocolloids 1999, 13, 401. (42) Martin, A. H.; Meinders, M. B. J.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. Langmuir 2003, 19, 2922.