Characterization of Langmuir biofilms built by the biospecific interaction of arachidic acid with bovine serum albumin

Thin Solid Films 525 (2012) 121–131

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Characterization of Langmuir biofilms built by the biospecific interaction of arachidic acid with bovine serum albumin Patricia Pedraz a, Francisco J. Montes a,⁎, Ramón L. Cerro b, M. Elena Díaz a a b

Department of Chemical Engineering, University of Salamanca, Plaza de los Caídos 1-5, 37008 Salamanca, Spain Department of Chemical & Materials Engineering, The University of Alabama in Huntsville, 301 Sparkman Drive, 130 Engineering Building, Huntsville, Alabama 35899, USA

a r t i c l e

i n f o

Article history: Received 21 February 2012 Received in revised form 27 September 2012 Accepted 9 October 2012 Available online 30 October 2012 Keywords: Langmuir films Biofilms Bovine Serum Albumin Arachidic acid Biospecific interaction Brewster Angle Microscopy

a b s t r a c t Affinity between biomolecules and surface active materials induces the formation of a Langmuir-biofilm (L-biofilm), as the basis for the development of high resolution bioseparation processes. Experiments were performed to characterize the interaction between amphiphilic molecules and proteins, establishing the optimal conditions for bioseparation. In the model L-biofilm system, Bovine Serum Albumin (BSA) was the protein and arachidic acid was the amphiphilic molecule. The L-biofilm formation is promoted by interactions within the subphase (polar groups) and at the interface (non polar groups). The first stage of the process to create the L-biofilm is the migration of BSA from the water subphase towards the air–water interface. This step is followed by a two-step process that includes diffusion and rearrangement. This process has been modeled using a double exponential equation and it is dominated by diffusion, although rearrangement reveals as a faster process. Once the L-biofilm is formed, phase behavior isotherms first show compressible films with areas per molecule larger than the corresponding to pure arachidic acid, due to the penetration of BSA molecules into the acid monolayer. As compression progresses, BSA is squeezed out of the interface although remains attached to the acid in the subphase. The L-biofilm shows hysteresis, in contrast to the behavior of a pure acid L-film. A probable cause for this behavior is the folding of BSA in the L-biofilm upon compression and a slower unfolding during expansion with loss of surface active material in successive cycles of compression–expansion. Regarding the L-biofilm stability, experimental data show a phase of significant film reorganization, due to the presence of BSA, followed by migration of the L-biofilm towards the subphase. A key variable was subphase pH, because it induces changes in the conformational structure of BSA. Changes in structure affect diffusion, rearrangement and solubility and, therefore, L-biofilm formation, structure and composition. Brewster Angle Microscopy studies show the compactness of the L-biofilms, confirming a good level of L-biofilm formation at pH = 3.8 and 5.1 and a rather low formation at pH = 8.2. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The emergence of genomics and proteomics focused engineering research on the difficult and expensive processes of separation and purification of proteins. Even though separation of most proteins can be accomplished by several well-known standard methods, it is purification that constitutes the bottleneck, both in productivity and cost, of the global production process. Molecular recognition is one of the most selective biomolecule purification methods. Combining molecular recognition with the highly structured properties of Langmuir films (L-films) results in a very efficient biomolecule separation process. A controlled molecular recognition process can be achieved by a bioengineered L-film (L-biofilm)

Abbreviations: BSA, Bovine Serum Albumin. ⁎ Corresponding author. Tel.: +34 923294479; fax: +34 923294574. E-mail address: [email protected] (F.J. Montes). 0040-6090/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.tsf.2012.10.055

containing amphiphilic molecules [1]. The amphiphilic molecules can work as ligands having specific molecular affinities. A ligand is a molecule capable of binding and forms a complex with a biomolecule. The ligand-technique has been broadly and successfully used in affinity chromatography for years and now, the extension and refinement of its use in order to create unique L-films for purification purposes is hereby proposed. Since L-films are extraordinarily sensible to changes in its structure, a huge window opens for the development of a purification method with extreme separation capacity, because only biomolecules with very specific properties will be able to attach to the L-film. In short, our objective is to use a specific ligand to form an L-film and then use the ligand to “fish” a specific biomolecule (e.g. enzyme, protein) which is dissolved, alone or with others, in a water subphase. Once the L-biofilm is formed, the target protein is separated from the other solutes by transferring the L-biofilm onto a solid using the Langmuir–Blodgett (LB) deposition technique. In the experiments described here, an L-biofilm is formed by bonding a carboxylic acid (arachidic acid = ligand) and Bovine Serum Albumin (BSA). Since long chain carboxylic acids are not

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soluble in water, once they bind to BSA they hold the protein at the interface, allowing the subsequent LB deposition process. The affinity between BSA and long chain carboxylic acids is actually an important part of the biological role of BSA and of other proteins that act as carriers of hydrophobic molecules with low aqueous solubility. Hydrophobic molecules bind to hydrophobic pockets located on the protein structure. For instance, the central role of the carboxylic acid binding to albumin in the mammalian lipid transport system is to carry fatty acids that are otherwise insoluble in circulating plasma [2]. BSA contains six high-energy binding sites for long-chain fatty acids and several weak binding sites. Experimental evidence shows that from 6 to 13 molecules of long chain fatty acids can be bound per molecule of albumin. BSA presents a secondary structure predominantly helical [3] and three distinct homologous domains, two of them consisting on amino acid residues disulfide bonded. These properties, mainly the presence of several binding sites, make BSA a perfect target for a ligand like arachidic acid, ensuring the specificity of the affinity separation [4]. BSA is a globular protein of great conformational adaptability (“soft” protein) with the approximate shape of a prolate spheroid [5]. The blood plasma protein BSA represents 52–62% of the total plasma protein fraction [6]. Besides its carrying properties, the most important physiological function of BSA is to maintain the osmotic pressure and pH of blood. BSA has the ability to bind substances reversibly, especially negatively charged substances. That is the feature that defines BSA as a hydrophobic molecule carrier [7]. Isoelectric points (IEP) between 4.5–5.0 [8] and 4.7–4.8 [9] have been reported. As a consequence, the net charge of BSA is negative in a neutral solution. Depending on pH, the protein becomes a macrocation or a macroanion. In turn, the solubility of BSA, which depends on the interaction of water molecules and of ions present in the solution, is modified. Since the presence of charge groups helps to dissolve the protein in aqueous media, at the IEP, the solubility of BSA is greatly reduced [5]. Thus, a crucial experimental variable is the subphase pH because it modifies BSA stability and interface adsorption allowing control of the protein structure in favor of a higher adsorption at the air–water interface and, consequently, to a higher affinity separation. BSA conformational structure is also pH-dependent. At pH≈4.3–7, BSA is found in the most stable and compact form, the normal or N form, that resembles a heart-like or triangular shape [10]. At higher or lower pH values, BSA undergoes several structural transitions that are all reversible [10], [11]. In this way, at pH b 4.3, the N form transforms into the F form, characterized by a more elongated conformation, a significant loss in helical content and much lower solubility respect to the N form [11]. Below pH≈2.7, an even more expanded structure, the E form, is obtained. At alkaline conditions, pH>7, BSA undergoes a gradual conformational transition to the B form, characterized by a loosening of the molecule with loss of rigidity [5]. If solutions of BSA are maintained at pH 9 with low ionic strength and at 3 °C for 3 to 4 days, another isomerization occurs leading to the so called aged-form or A-form. The structure of B and A forms has not been determined to a sufficient extent and the only structural information available is that the transition from the N form to the B form involves a volume increase and a decrease in the helical content less significant than that corresponding to the N to F transition. In this article, a study of the interaction of BSA and arachidic acid at the air water interface is presented. The experimental determination of BSA adsorption kinetics at the air–water interface at different pH, the data analysis of surface pressure vs. area per molecule of the BSA-Arachidic Acid L-Biofilm and the study of the L-Biofilm at the air–water interface using Brewster Angle Microscopy (BAM) are the tools used is this study for the determination of the optimal conditions for a productive affinity separation. Our research group is currently performing broader studies that will determine the quality and amount of such separation.

2. Experimental section 2.1. Materials Crystallized and lyophilized bovine serum albumin (BSA ≥96%, CAS no. 9048-46-8; Mw=64,000 Da) was purchased from Sigma-Aldrich Chemical Co.; it was used as received without further purification. Arachidic acid (eicosanoic acid, 99%, CAS no. 506-30-9, Mw=312.5) was purchased from Acros Organic. Reagent grade chloroform, acetone and 2-propanol supplied by Sigma-Aldrich Chemical Co. were used for the cleaning of the trough. Ultrapure water (resistivity of 18.2 MΩ cm) was obtained from an Elgastat UHQ II system. The pH of the subphase was adjusted by addition of dilute solutions of hydrochloric acid (ACS reagent 37%, Sigma-Aldrich) and sodium hydroxide (ACS reagent 97%, Sigma-Aldrich) and measured by means of a pH-Meter BASIC 20+ (Crison Instruments, S.A.). Interfacial studies were performed using a 612D Nima Technology trough equipped with two polytetrafluoroethylene (PTFE) barriers, which move symmetrically. The trough has a maximum area of 600 cm 2 and it is also made of PTFE. A Wilhelmy plate attached to a force balance was used to measure the interfacial pressure. The plate consisted on a chromatography paper strip (Whatman's 3MM CHR). The subphase temperature was maintained constant by means of an external temperature controller unit Lauda RE 104. The subphase surface was cleaned with the help of a suction pump (Markos Mefar, model SP20). BAM images with dimensions 3.6 mm × 4 mm were recorded using a MicroBAM2 (Nima Technology), which allows the visualization of the resulting film structures at the center of the trough. The laser signal is captured by a VGA camera with a resolution of 6 μm per pixel at 30 fps. 2.2. Methods All experiments were carried out in the Langmuir–Blodgett trough described above at 21 °C [12]. Monolayers were prepared by spreading 75 μl of a 1 mg/ml arachidic acid in chloroform solution onto the subphase, consisting either on ultrapure water or a 10 −8 M BSA-ultrapure water solution. BSA subphase concentration was optimized in order to minimize the amount of protein used in each experimental run and, at the same time, to avoid any measurable surface pressure in the absence of arachidic acid during the experiments [13], in such a way that changes in the properties of the fatty acid monolayer could be attributed unequivocally to the interactions with BSA at the air–water interface. No salt was added to the subphase (ionic strength ≈ 0) in order to avoid any “salting-in” or “salting-out” effects. After the spreading, the solvent was allowed to evaporate for 15 min. The velocity of compression of the monolayer was set constant in all experiments at 40 cm 2/min. Following the experiments, the through was first cleaned with acetone, then with ultrapure water for an hour, latter with 2-propanol and finally with slightly acid ultrapure water overnight. 3. Results and discussion 3.1. Adsorption of BSA at the air–water interface Fig. 1 shows variation of surface pressure with time for a pure BSA solution. Since BSA is a surface active material, it adsorbs at the air– water interface by migration from the subphase. Three different pH values are shown: at pH = 5.1, the protein is in its N form and very close to the IEP. At pH = 3.8, the protein is in its F form and below the IEP, therefore, positively charged. Finally, at pH = 8.2, the protein is in its B form and above the IEP, i.e., negatively charged.

P. Pedraz et al. / Thin Solid Films 525 (2012) 121–131

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in Table 1 for the three pH values under study. The effect of pH is analyzed in Section 3.1.3. 3.1.2. BSA layer at the air–water interface As an increasing number of diffusion-transported BSA molecules populate the air–water interface, surface pressure increases. The mass transfer process leads to the formation of a BSA layer at the interface. In a subsequent step, the exposure of the molecules to the interface induces a structural rearrangement process (only observable at pH values of 3.8 and 5.1). At a given surface coverage, the available area cannot easily accommodate further protein molecules and the surface pressure increases slowly, showing an apparent tendency (not observed for the period of the experiments) to reach a plateau at large time values, at which adsorption equilibrium would be attained. It is evident from Fig. 1 (pH=5.1) that there are two different surface pressure increase rates: an initial faster increase (up to surface pressure values ≈10 mN/m) followed by a slower increase. Thus, Fig. 1 highlights the relative importance of the diffusion/rearrangement mechanisms, which can be analyzed comparing the kinetic values of a double exponential adsorption model [14,16] (Eq. (1)). Fig. 1. Adsorption curves of BSA at different pH values. BSA subphase concentration: 10−8 M. Circles: experimental data. Solid lines: fitting curves using the two-exponential model (Eq. (1)).

The results obtained are qualitatively equivalent for the three pH values. The surface pressure initially decreases, reaches a minimum and then starts increasing. The adsorption process can be divided into two phases. 1) An initial phase that is governed by diffusion of protein from the bulk to the interface followed by a rearrangement of the BSA at the air–water interface. The diffusion phase is characterized by a lag time [14,15] (tlag), which is the time that takes the surface pressure to increase from negative values to zero. 2) A final phase, governed by the incorporation of an increasing number of BSA molecules to the interface layer, produces an increase of surface pressure to positive values until a pressure plateau is reached. This phase is characterized by the kinetics of the adsorption process, described here by the double exponential model reported by Sharp et al. [16]. 3.1.1. Diffusion/rearrangement phase As the subphase protein concentration is 10 −8 M, the amount of protein initially present at the air–water interface is low, resulting in a 2-dimensional gas phase film. In this state, the protein molecules are far enough from each other so no interaction among them takes place and surface pressure does not reach measurable values. As time progresses, two simultaneous processes occur where BSA plays a major role. First, diffusion of BSA towards the interface, tends to increase surface pressure and second, rearrangement of new BSA molecules exposed to the interface which, according to the experimental results obtained in this work, results in a decrease of surface pressure. The emergence of those new molecules suddenly exposed to the interface is related to the experimental procedure of pouring a solution from a beaker to the trough. During this process, the area to which the solution is exposed is greatly increased and, consequently, an important number of molecules that were located at the bulk phase when the solution was in the beaker are suddenly exposed to the air– water interface after the solution is poured into the trough. The balance between the diffusion and rearrangement processes results in a minimum value of the surface pressure. Previous to the minimum surface pressure, the rearrangement dominates. Following the minimum surface pressure, the amount of BSA reaching the interface becomes large enough such that protein molecules interact with each other, causing an increase in surface pressure. As surface coverage proceeds, surface pressure reaches positive values. The time at which a minimum value of the surface pressure is obtained (tmin), as well as the lag time (tlag) calculated as the intersection between the time axis and the beginning of the pressure increase, are shown

h i h i ð−k tÞ ð−k tÞ πðtÞ ¼ πðt ¼ 0Þ þ A1 1−e 1 þ A2 1−e 2

ð1Þ

A1 and A2 are the relative contributions of diffusion and rearrangement to the increase in surface pressure and k1 and k2 are kinetic constants indicating the rates at which those two processes take place. The higher the values of k1 and k2, the faster the processes are. Physically speaking, A1 illustrates the effect over surface pressure of the molecules of BSA arriving by diffusion at the interface. Likewise, A2 illustrates the effect over surface pressure of the rearrangement of the BSA molecules already located at the interface. In addition, k1 indicates the speed at which BSA molecules arrive by diffusion at the interface and k2 indicates the speed of rearrangement. A1, A2, k1 and k2 in Eq. (1) are obtained by fitting experimental data of surface pressure vs. time. Raw data are rescaled such that the initial part of the curve belonging to the lag period is discarded, and the adsorption curves start from π (t= 0)= 0. To improve the curve fitting procedure one must take into account that A1 and A2 are linear fitting parameters while k1 and k2 are non-linear fitting parameters. Thus, the curve fitting procedure was split into a linear and a non-linear part because, under the assumption that a least-squares type of fitting should provide a global solution instead of a local solution. The experimental data and the curve fittings obtained are shown in Fig. 1. The fitting parameters and the regression coefficients are collected in Table 1. By comparing the values of the coefficients A1 and A2, it is clear that the effect over surface pressure of the molecules of BSA arriving by diffusion at the interface and represented by A1, is much stronger than the effect over surface pressure of the rearrangement of the BSA molecules already located at the interface, represented by A2. As a consequence, if we used a single exponent adsorption model in which only diffusion was taken into account (results now shown), the fitting to the experimental data would still be excellent. Experimental values of A2 are negative. This suggests that when a BSA molecule rearranges its structure at the interface, in order to

Table 1 Fitting parameters and regression coefficients corresponding to the use of Eq. (1) to model the adsorption kinetics of BSA at different pH values. pH

tmin, h

tlag, h

A1, mN/m

k1, h−1

A2, mN/m

k2, h−1

R2

3.8 5.1 8.2

0.36 0.69 2.02

0.52 0.99 7.56a

28.14 20.67 –

0.8 1.8 –

−1.67 −7.58 –

11.5 4.0 –

0.9998 0.9994 –

a

Obtained by extrapolation of the experimental data.

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minimize the exposure of its hydrophobic regions to the aqueous environment, the resulting effect is a decrease in surface pressure. This is also the effect that leads to the decrease of surface pressure observed during the lag phase. Considering that, at the concentration used in this research, the BSA molecule allocates itself at the interface as an oblate ellipsoid with dimensions of 40 Å × 40 Å × 140 Å [17], the projected area per molecule at the interface would be around 17,600 Å 2. According to the results obtained by Lu et al. [17], the projected area per molecule of BSA adsorbed at the air–water interface at pH = 5 and bulk concentration of 5.10 −4 g/L (in the order of the concentration used in this work), is 15,000 ± 2000 Å 2, a value slightly lower than the corresponding value before adsorption. This small reduction in the area per molecule of BSA could explain the decrease observed in surface pressure upon adsorption. The effect of pH on A1 and A2, indicates that when pH is increased from 3.8 to 5.1, diffusion of new molecules towards the interface has a smaller effect on surface pressure, and the rearrangement of those molecules has a larger effect on surface pressure, in the sense of a more powerful, still small, decrease of that pressure. The decrease of A1 when increasing from pH 3.8 to pH 5.1 could be related to the size of the protein. At pH = 3.8, the protein conformation is the F-structure, which is a more expanded structure than the N-structure adopted at pH = 5.1. With regard to A2, the effect of the rearrangement resulting from the exposure of the protein at the air–water interface is enhanced when pH changes from 3.8 to 5.1. This behavior must be related to the different capabilities of the F and N conformations to vary their structure at the air–water interface. Further details about BSA structures and its changes with pH are given in the next section. The kinetic constants k1 and k2 indicate the rate at which diffusion and rearrangement take place. From an analysis of the parameters listed in Table 1, we can conclude that diffusion is a slower process than rearrangement. It is also evident that diffusion becomes faster and rearrangement becomes slower when increasing pH. Again, considering the size of the protein at pH = 3.8 (F-form, expanded) and pH = 5.1 (N-form, compact), the smaller the molecule, the faster the diffusion, as predicted by the values of k1. From the values of k2 it appears that the rearrangement of the N-form is a more difficult process than the rearrangement of the F-form, probably due to the more compact structure of the N-form. 3.1.3. Effect of pH on BSA structure and solubility Fig. 1 shows that the formation of a BSA layer at the air–water interface is strongly influenced by pH. While at pH = 3.8, the protein diffuses and adsorbs in the time frame under study reaching surface pressure of around 20 mN/m, at pH = 8.2, the protein requires much longer times to diffuse towards the interface and to overcome the initial rearrangement period. At pH = 5.1, the behavior is intermediate. The kinetics of surface pressure at different pH values are directly related to two factors which depend on pH: the structural conformation of the protein and the net electrical charge. According to our results, the B structure shows a much greater solubility than that of the N and F forms. Additionally, comparing the experiments performed at pH = 4.5, 4.7, 5.1 and 6.5, all within the pH limits of the N form (see Fig. 2), the surface pressures obtained after 2.5 h at pH values close to the IEP (4.5, 4.7 and 5.1) were all similar, while at pH = 6.5, the surface pressure is much lower and comparable to the value obtained at pH = 8.2. Our results also show a loss of solubility of the F form with respect to that of the N form, a fact previously reported by Foster et al. [11]. The F conformer structure exposes most hydrophobic residues to the solvent [18] and hydrophobicity creates a dominant driving force for the BSA adsorption at the interface while inducing electrostatic repulsion of positively charged molecules at the interface. The B form is a more expanded structure than the N form. Thus, the B form could lead to higher surface pressures than the N form.

Fig. 2. Surface pressure values recorded after 2.5 h as function of pH.

However, the B-form does not adsorb at the air–water interface in an amount significantly enough to get beyond the gas phase. The B form of the protein exposes less hydrophobic residues to the water [18]. Since the negative charge prevails, electrostatic repulsions induce the formation of a depletion layer, hindering the protein adsorption at the interface. Finally, the N form, with its more compact structure, and therefore, smaller molecular projected area, adsorbs at the interface in a time frame larger than that corresponding to the F form. Although minimum solubility would be expected at the IEP, due to the absence of an electrostatic barrier for the adsorption, our results reveal that this is true for pH values where only the N form exists. Thus, solubility of BSA has a local minimum at pH values close to the IEP. At pH values around 4.7, maximum surface pressure values were recorded (Fig. 2). In addition, solubility highly increases as pH is changed away from pH 4.7, but still within the 4.3–8 range corresponding to the existence of the N form. However, in absolute terms, even when the molecule is positively charged, BSA surface pressures are higher when the F form prevails, indicating that the global minimum protein solubility is obtained when BSA stays in an F-conformation. The experimental results presented in this work differ considerably from previous reports [3,19], that show noticeably less differences in the adsorption kinetics for BSA at pH values at which the F, N and B forms are present. In previously published work [3,19] the N-form is reported as the one producing maximum surface pressures within the time of study, indicating that the experimental conditions in previously reported experiments [3,19] are significantly different from experimental conditions in our experiments, namely concentration of BSA and ionic strength. Indeed, changes in concentration and ionic strength can lead to very different adsorption kinetics.

3.2. Molecular recognition between BSA and arachidic acid To study the association of BSA molecules with the arachidic acid monolayers spread over the air–water interface, we measured the variation of surface pressure with time, while maintaining constant the area of the interface. The time of spreading was brought to 0 by eliminating the time ranging from the moment at which the subphase

P. Pedraz et al. / Thin Solid Films 525 (2012) 121–131

was added to the moment at which the interface was cleaned (circa 15 min). Fig. 3 shows an immediate increase of surface pressure, as the arachidic acid is spread over the gas–liquid interface. Surface pressure reaches a maximum that is a function of the pH of the subphase. The dynamics of surface pressure changes are also somewhat different for different subphase pH. At pH =3.8, surface pressure slightly decreases and then rises again quickly due to the prevailing influence of the diffusion process. By contrast, at pH=5.1 and pH=8.2, the rearrangement process is the time determining step and surface pressure decreases steadily. This secondary effect is more evident at pH=8.2 Since spreading of a pure layer of arachidic acid does not show substantial transport or arachidic acid towards the subphase, the initial increase can be justified by the interaction of the protein with the lipid monolayer. Thus, the protein initially present at the air water interface, rearranges to accommodate and interact with the newly added arachidic acid molecules. In the resulting protein-caboxylic acid monolayer at the air–water interface, the protein may penetrate into the lipid-hydrocarbon region interacting through short-range non-specific Van der Waals forces (Fig. 4(a)) or staying in separate 2D domains from the arachidic acid (Fig. 4(b)) [20]. Additionally, BSA molecules that are dissolved in the subphase will diffuse towards the interface to bind the polar domain of the carboxylic acid molecules. This process is promoted by the high affinity of BSA for

Fig. 3. Variation of surface pressure with time after spreading 75 μL of a 1 mg/mL arachidic acid solution over a 10−8 M BSA aqueous subphase at pH= 3.8, pH = 5.1 and pH = 8.2.

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carboxylic acids. The newly incorporated BSA molecules bind to the polar domain of the arachidic acid, forming a protein–acid double layer. In this case, BSA remains within the subphase joined to the acid that straddle the air–water interface due to its low solubility (Fig. 4(c)) [20]. Some of the BSA molecules may also reach the air– water interface and interact with the hydrophobic chain of the carboxylic acid (Fig. 4(a)) or form new protein domains (Fig. 4(b)). The formation of the protein-acid double layer might occur with (upper part of Fig. 4(c)) or without (lower part of Fig. 4(c)) significant conformational changes in the protein [20]. In all cases, the spreading of arachidic acid over the air–water interface allows it to combine with the BSA molecules present at the air–water interface and in the subphase. This combination results in a sufficiently populated layer (Fig. 4(a) and (b)) or compact/rigid (Fig. 4(c)) to produce a detectable increase in the surface pressure. Although the different configurations shown here differ considerably, the final effect of this structure is similar and corresponds to a film in a two dimensional liquid phase where its components interact. Dependence of the surface pressure on pH can be related to the different conformers existing at pH = 3.8, 5.1 and 8.2. As pointed out before in Section 3.1, the conformational solubility rank is B-form > N-form > F-form. In this way, the inclusion of BSA molecules at the arachidic acid monolayer as well as their binding to the polar carboxylic acid head groups, take place in much less extent as solubility increases. Nylander [20] noted that electrostatic effects do not seem to be definitive in this process. Although the pKa value of fatty acids in solution is about 5.7 [21], in monolayers, the pKa is in the order of 3–4 units larger. A case in point, the pKa reported for arachidic acid is 9 [22]. This large variation in dissociation constants indicates that simple electrostatic binding cannot explain the remarkable interaction between the protein and the arachidic acid at pH = 3.8 and 5.1, since no sensible dissociation of the carboxylic head group takes place at these pH values while the protein is positively charged and zwitterionic respectively. At pH= 8.2, the protein and a small fraction of the arachidic acid molecules are negatively charged. Changes in charge can contribute to the low degree of association of the B form of the protein to the lipid molecules. BAM images can provide information about the conformational characteristics of the BSA-arachidic acid layer. The dark background in Fig. 5 corresponds to the low reflectivity water surface, while the high reflectivity regions represent domains of surface active material. Independent of the subphase (Fig. 5(a) and (c)), the arachidic acid seems to organize in a heterogeneous film composed of floating, large two dimensional structures. However, as indicated by the recorded images, the presence of BSA in the subphase modifies the apparent structure of the film formed at the air–water interface. While the arachidic acid monolayer on a pure water subphase

Fig. 4. Schematic representation of proteins interacting with a long chain carboxylic acid such as arachidic acid [20]. (a) BSA penetrates into the lipid-hydrocarbon region interacting through short-range non-specific Van der Waals forces. (b) BSA stays in separate domains from the arachidic acid. (c) BSA binds to the arachidic acid, forming a protein–acid double layer, with (upper (c)) or without (lower (c)) BSA conformational change.

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displays the “sponge-like” structure typical of fatty acids [23] (Fig. 5(a)), the arachidic monolayer spread over a BSA 10 −8 M subphase presents a more compact aspect (Fig. 5(c)). Additionally, when the pH of the subphase is changed, apparent modifications of the macroscopic structure are detected. At pH= 3.8 (Fig. 5(b)) and pH = 5.1 (Fig. 5(c)), the BSA-arachidic acid layer compactness of the surface domains is noticeably larger than that of the domains formed at a pH= 8.2 (Fig. 5(d)). The sharper structure at pH 8.2 resembles the images obtained by monolayers of pure arachidic acid on an ultrapure water subphase. Finally, the reflectivity of the structures detected at pH = 3.8 and 5.1 is slightly different. The two-dimensional domains formed at pH =3.8 present higher reflectivity and, therefore, higher-density. These results agree with the reported poor adsorption/association characteristics of the BSA at pH=8.2 in contrast with the considerable protein-fatty acid interaction at acidic pH values. The protein–fatty acid interaction is precisely the cause of the more compact and smooth aspect of the floating film at pH=3.8 and 5.1. 3.3. Simultaneous study of surface pressure vs. area per molecule isotherms and BAM images After arachidic acid is added to the 10−8 M BSA subphase at different pH values and evaporation of the solvent (chloroform) takes places, the film was compressed and the evolution of surface pressure versus the available area was recorded and plotted. Fig. 6 shows surface pressure versus area up to the point previous to the collapse of the film. The pressure versus area isotherm of arachidic acid on pure water is shown as reference. The specific molecular area is defined as the ratio of the film area to the number of arachidic acid molecules. As it can be readily observed in Fig. 6, the shape of the resulting isotherms as well as the molecular area corresponding to a given surface pressure and the collapse pressure values are strongly influenced by

the concentration of protein in the subphase and by subphase pH. The arachidic acid isotherm is characteristic of the long chain fatty acids [21]. It is also evident that the presence of BSA in the subphase, definitely affects the compression pattern for the formed film at the air–water interface. Independent of subphase pH, there are, at least, two regions. A first region is characterized by an increase of the surface pressure as the interface is compressed and a second region is distinguished by a decreasing slope, indicating that surface pressure varies slowly upon compression. Within the first region, the BSA-arachidic acid mixed film is compressed with little or no loss of protein towards the subphase. The protein, that at low surface pressures unfolds at the interface, refolds upon compression [24] and gives, as a result, highly compressible films. To quantify the compressional elasticity of the films [25] as well as phase transitions [26] in the first region, an experimental value of compressibility (Cs) defined as s

C ¼−

  1 ∂A A ∂π T

ð2Þ

is calculated. A is the area per molecule of the film and π is the surface pressure. Compressibility can also be calculated theoretically, using the appropriate π − A equation of state [27]. The values computed for compressibility are shown in Fig. 7. The shape of the isotherms shown in Fig. 6 and the presence/absence of peaks in Fig. 7 reveal the phases and phase transitions at different surface pressures and pH conditions. From the isotherm shown in Fig. 6, it can be concluded that the arachidic acid film in pure water, initially in the gas state, enters the liquid expanded (LE) phase at an area per molecule of around 25 Å 2/molecule. At surface pressures of 21 mN/m and 43 mN/m, the arachidic acid undergoes two additional phase changes, from LE to liquid condensed (LC) and from liquid condensed to solid condensed state (S) respectively. At the solid condensed phase, the molecular

Fig. 5. BAM images before monolayer compression. (a) Arachidic acid in ultrapure water subphase at pH = 5.1. (b) Arachidic acid in a BSA 10−8 M aqueous subphase at pH = 3.8. (c) Arachidic acid in a BSA 10−8 M aqueous subphase at pH = 5.1. (d) Arachidic acid in a BSA 10−8 M aqueous subphase at pH = 8.2.

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Fig. 6. Surface pressure vs. area per molecule of arachidic acid on 10−8 M BSA subphase at different pH values. The isotherm of arachidic acid on pure water is shown for comparative purposes.

area is about 20 Å 2/molecule, corresponding approximately to the footprint of the arachidic acid. Upon further compression, the film collapses for a surface pressure near π = 60 mN/m. The transitions from LE to LC and from LC to S are indicated by the two peaks in compressibility at approximately 20 mN/m and 40 mN/mn, shown in Fig. 7. An additional peak at a surface pressure of around 30 mN/m is detected but could not be identified with any phase transition in the corresponding isotherm. When proteins are present in the subphase, at pH 3.8 and pH 5.1, and after the spreading of the arachidic acid film, the initial recording

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shows that the complex film already exists in a LE state. Indeed, at these pH values, more protein molecules are thought to be incorporated into the film (Section 3.1). Upon compression, no clear phase changes can be detected at pH = 3.8 (which is characteristic of mixed films). At pH = 5.1, for a surface pressure of around 24 mN/m, a transition to a less compressible phase is detected from Figs. 6 and 7. However, in this state the new phase does not develop significantly as the film starts a different process, characteristic of the second region described next. At pH = 8.2, the film, initially in gas phase, evolves to a LE phase, although no phase transitions could be detected. In short, none of the BSA-arachidic acid films display a compressibility characteristic of a condensed state (the calculated values are mainly CS ≥ 0.01). Thus, only the pure arachidic acid, with compressibility coefficients descending abruptly to values under 0.005 m/mN shows the LC and S states. The more elastic BSA/arachidic acid films, instead of evolving to a condensed state due to the increase in surface pressure, undergo somewhat different changes. At surface pressures of ~ 27 mN/m (pH = 8.2), ~ 36 mN/m (pH = 5.1) and ~ 35 mN/m (pH = 3.8), we detect the presence of a second region in the surface pressure versus area isotherm, characteristic of Langmuir monolayers of proteins and enzymes (Fig. 6). In this region of the isotherm, the smaller variation of surface pressure with area is a result of the squeezing out of protein molecules from the air–water interface preceding the full collapse [28,29]. At pH = 3.8, the evolution from the first to the second region is smooth, while the changes in surface pressure are sharper for pH = 8.2 and pH = 5.1. These results indicate that at pH = 3.8 expulsion of proteins from the BSA populated interface proceeds at a slower pace. It is apparent from Fig. 6, regardless of surface pressure, that the presence of BSA in the subphase renders greater molecular areas. This effect is more pronounced as pH is decreased from 8.2 to 5.1 and from 5.1 to 3.8. The explanation is clear: BSA molecules penetrate the arachidic acid monolayer at the air–water interface, being the degree of penetration higher when the F conformer exists (pH = 3.8), lower with the B conformer (pH = 8.2) and intermediate with the N form (pH = 5.1). These results agree with the adsorption studies,

Fig. 7. Compressibility coefficient vs. surface pressure for arachidic acid in pure water and in a 10−8 M BSA aqueous solution at pH = 3.8, pH= 5.1 and pH = 8.2.

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solubility and structural characteristics of the different forms of the protein as a function of pH, as explained in Section 3.1. As a final point, it is interesting to notice that the collapse of the BSA containing films occurs at molecular areas close to the value corresponding to arachidic acid alone and at surface pressures remarkably lower than those at which a pure arachidic acid film collapses (60 mN/m). The first fact is directly related to the discharge of protein molecules from the arachidic acid monolayer as the surface pressure increases [20]. The first effect, ejection of proteins from the interface, is practically completed right before collapse. The second effect, surface pressures lower than arachidic acid at collapse, is related to the higher compressibility of the mixed films, as discussed above. Fig. 8 shows BAM images of BSA containing subphases at different pH values, recorded during interface compression. To facilitate comparison, we also included images obtained for a system of pure arachidic acid on pure water. As expected, initially the films are organized in large clusters floating at the air–water interface. As the available area is reduced, progressive disappearance of the dark regions recognized as the water surface, together with changes in the reflectivity of the domains are observed. At pH 3.8 and 5.1 and surface pressures around 25 mN/m, the interacting clusters, which give rise to surface pressures in the

LE region, form a homogeneous film. As surface pressure increases, we detect bright, parallel stripes with a recorded intensity level several orders of magnitude higher than that of the surrounding monolayer. Some of the stripes observed by the BAM may represent persistent folds of the monolayer, as this type of monolayers has a tendency to bend [30]. At 38 mN/m (a pressure close to the full collapse of the film), brighter areas representing the formation of out-of-plane or 3D structures are clearly detected. At pH 8.2, the film seems more similar to arachidic acid on a water subphase. The spongy interacting clusters show a slightly higher reflectivity than the one obtained at lower pH, which would indicate that the aliphatic chains are closer to be perpendicular to the interface. As the film is compressed, the free spaces between clusters disappear and a single film is formed. Again, less extensive bright areas are detected near collapse. 3.4. Compression/expansion cycles The results of three consecutive compression–expansion cycles of arachidic acid monolayers over 10 −8 M BSA subphases at variable pH values are shown in Fig. 9. The maximum surface pressure values at which the films were compressed were set to avoid entering the

Fig. 8. BAM images of arachidic acid in pure water and arachidic acid in 10−8 M BSA solution at different pH and surface pressure values.

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second region of the isotherms described in Section 3.3. These values of surface pressures are below values where significant formation of three dimensional structures preceding full collapse takes place for the three pH values. No waiting time between compression–expansion cycles was allowed. Hysteresis refers to the different path that compression and the decompression curves follow, due to conformational changes on the surfactant film caused by compression. It is shown in Fig. 9 that all arachidic acid-BSA films display some degree of hysteresis, where the expansion curve of the film takes place at lower specific areas than the compression part of the curve. This shift in behavior, that establishes the new path followed by the expansion sector, is a consequence of the new structural arrangements that molecules undergo after the first compression. Shifts in behavior are due to structural arrangements not returning to their original state shown before compression. Since pure fatty acids do not show hysteresis, the observed behavior must be assigned to the inclusion of BSA molecules in the film. The most likely explanation of the hysteresis phenomena occurring in the combined BSA-arachidic acid-film, for all pH values, is a combined process of protein folding at the air–water interface upon compression [24] and BSA loss from the interface towards the

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subphase. During decompression, the folded protein does not unfold as fast as it was folded by compression, resulting in lower areas per molecule. This hypothesis can be confirmed by the significant lower degree of hysteresis taking place at pH = 8.2. At basic pH the protein, in its B form, will be present at the interface in much less extent than the F and N forms and, as a consequence, the folding/unfolding effect is less pronounced. For all pH values, the common feature is that successive hysteresis loops are reproducible and the shape of the loop after the first cycle is roughly the same after successive cycles. This reproducibility is a sign that after a complete expansion, protein molecules relax back to its original state. At all pH values, during the compression–expansion cycles, consecutive hysteresis cycles are displaced towards lower specific areas. The shift of the hysteresis cycles toward lower specific areas may be due to instability or dissolution of the biofilms. During the compression process, surface active molecules are expelled from the interface and move into the subphase. Therefore, as the film undergoes compression–expansion cycles, the initial amount of surface active material at the interface decreases. If the arachidic acid also becomes lost during this process, care should be taken when analyzing the displacement of consecutive cycles, since we can no longer perform an accurate calculation of the specific area. 3.5. Stability

Fig. 9. Compression–expansion cycles of arachidic acid on 10−8 M BSA subphase at different pH values.

Fig. 10 shows the increasing loss of the interfacial area occupied by films of pure arachidic acid and arachidic acid-BSA at pH values of 3.8, 5.1 and 8.2, when the films held at a constant surface pressure of 30 mN/m, 25 mN/m and 20 mN/m respectively. Stability is expressed in terms of the ratio A/A0, at constant surface pressure π. A is the interfacial film area at time t and A0 is the interfacial film area at zero time. A0 corresponds to the area measured when the target surface pressure is reached. The variation of A/A0 with time is monitored for 90 min in order to study the stability of the films. Possible mechanisms for the variation of A/A0 with time are reorganization, dissolution, or collapse [31]. The shape of the curves can give information on the particular cause of instability. If the curve is convex, the area losses are due to collapse in the interfacial films, while if it is concave, the area losses are due to dissolution of amphiphilic molecules to the subphase. The kinetics of the collapse and dissolution processes can be very complicated even though first-order processes have been proposed [32]. It is evident from inspection of Fig. 10 that the monolayer stability at fixed surface pressure is sensitive to subphase pH and to the presence of BSA. Firstly, the variation of A/A0 follows a concave curve for all cases under study except for arachidic acid on pure water at pH = 3.8. The concave curves are all equivalent in shape: an initial sharp decrease on the relative area followed by a slower but continuous decrease. This may be indicating the existence of at least two different mechanisms accounting for the decrease of area: reorganization, as the predominant cause of the decrease of A/A0 at the initial stage, followed by the dissolution of the monolayer into the subphase. The curve obtained for arachidic acid on pure water at pH = 3.8 is initially convex upwards, that is characteristic of film loss by collapse, giving rise to regions of multilayers on the surface [33]. This is expected since the monolayer is largely not ionized [33]. Secondly, despite the shape of the area loss versus time curves, the values obtained for the cases under study indicate that the presence of BSA, for pH values of 5.1 and 8.2, extends the reorganization region of the curve, in such a way that during the first 45 min of the experiment, 75–85% of the observed area loss takes place in the presence of protein. The percentage of area lost to compression is reduced to 65–75% without BSA, while at pH = 3.8 the protein seems to stabilize the film, impeding collapse. The former could be due to the importance of folding and rearrangement of the protein when the film is

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Fig. 10. Stability of pure arachidic acid and arachidic acid on 10−8 M BSA subphase at different pH values.

suffering from the continuous pressure and the later, to the cushion effect of the BSA molecules, in line with the greater compressibility observed for the arachidic acid-BSA films when compared to that of pure arachidic acid monolayers. Finally, the stability of the BSA-arachidic acid films is affected by pH in such a way that for increasing pH, the percentage of area losses is reduced (after 90 min, the area losses are 17%, 15% and 8% of the initial values for pH =3.8, 5.1 and 8.2 respectively). These phenomena can be related to the amount of protein incorporated into the film at the air– water interface and the consequent reorganization region of the area loss curve. At pH =3.8, the protein, in its F form, is less soluble than the N conformer (pH =5.1) and at pH =8.2, electrostatic repulsions hinder the adsorption of the protein at the interface. In short, increasing amounts of protein are obtained at the interface as pH decreases.

layer. The kinetics of the second phase are modeled using a double exponential equation. Kinetic constants show that diffusion has a larger effect over surface pressure than rearrangement, the latter being a faster process. Negative values of A2 obtained experimentally indicate that rearrangement of BSA leads to a small decrease of surface pressure based on a reduction of projected area per molecule, in line with data reported by Peters [5] and Lu et al. [17]. The effect of pH on the diffusion and rearrangement processes is related to the change in conformational structure of BSA, considering that at pH = 3.8, BSA is at its F-form and that at pH =5.1 BSA is at its N-form, which is a more compact configuration. BSA solubility is also pH related and, consequently, structurally related. Even though the F-form does not exist at the IEP, at this configuration the protein reaches a minimum in solubility, due to the higher number of hydrophobic residues exposed to the solvent. The steep increase in surface pressure observed after spreading arachidic acid is related to the formation of the L-biofilm at the interface. The formation of biofilms, which is also pH related, arises from BSA interacting with the polar group of the acid in the subphase, as well as from Van der Waals forces interacting between BSA and the non-polar hydrocarbon chain of the acid. BAM studies allow the description of the macroscopic structure of the L-biofilm at different pH. As reflectivity and, consequently, compactness of the biofilms formed at pH =3.8 and 5.1 are larger than those formed at pH =8.2, the poor adsorption/association characteristics of BSA at this pH are confirmed. Compression isotherms of the L-biofilm are affected by pH and two sections are observed in the isotherm, according to the changes in compressibility and the BAM images. In the first section, the L-biofilm is compressed with almost no loss of BSA towards the subphase, due to the refolding of the protein. The second section is the result of BSA being squeezed out of the L-biofilm. When comparing L-biofilms at different pH for a constant value of surface pressure, it is observed that films containing more BSA occupy more area per molecule, suggesting that in all cases some BSA molecules penetrate the acid monolayer. The ranking of penetration is F-form >N-form >B-form, that is, inverse to the solubility ranking. The L-biofilm collapses at a lower surface pressure but at approximately the same area per molecule than a pure acid L-film, suggesting that, at the collapsing moment, all BSA molecules have been ejected from the monolayer, but still many others are somehow attached to the acid in the subphase. When performing compression–expansion cycles over the L-biofilm, hysteresis cycles were found, in contrast to the behavior of a pure acid L-film. Hysteresis is probable due to the folding of BSA in the L-biofilm upon compression and a slower unfolding during expansion. BSA eventually relaxes to a more expanded conformation, following a displaced, although equivalent, compression path in a successive cycle. Cycle displacement is due to the loss of surface active material. Regarding the L-biofilm stability, which is affected by pH, data show that probably there are two mechanisms responsible for the loss of area: molecular reorganization, which is important initially and dissolution of the monolayer, which prevails latter. Acknowledgments Support for this project from CTQ2011-26905 (MICINN, Spain) is gratefully acknowledged.

3.6. Conclusions References Affinity between BSA and long chain carboxylic acids induces the formation of an L-biofilm, which is the basis for the development of a high resolution bioseparation process. To establish the optimal conditions to undergo this type of bioseparation, a study of the behavior of the L-biofilm at the air water interface was conducted. Prior to the formation of the L-biofilm, BSA migrates from the water subphase towards the air– water interface following a two-step scheme: (1) a first phase governed by diffusion of protein from the bulk to the interface followed by a rearrangement of the BSA and, (2) a second phase governed by the incorporation of an increasing number of BSA molecules to the interface

[1] A. Brisson, W. Bergsma-Schutter, F. Oling, O. Lambert, I. Reviakine, J. Cryst. Growth 196 (2–4) (1999) 456. [2] C. Alvarez, H. Bertorello, M. Strumla, E.I. Sanchez, Polymer 37 (16) (1996) 3715. [3] B.A. Noskov, A.A. Mikhailovskaya, S.Y. Lin, G. Loglio, R. Miller, Langmuir 26 (22) (2010) 17225. [4] T. Peters, H. Taniuchi, C.B. Anfinsen, J. Biol. Chem. 248 (7) (1973) 2447. [5] T. Peters, All About Albumin: Biochemistry, Genetics,and Medical Applications San Diego, 1996. [6] N. Brandes, P.B. Welzel, C. Werner, L.W. Kroh, J. Colloid Interface Sci. 299 (1) (2006) 56. [7] T. Hu, Z. Su, J. Biotechnol. 100 (3) (2003) 267. [8] A. Kudelski, Vib. Spectrosc. 33 (1–2) (2003) 197.

P. Pedraz et al. / Thin Solid Films 525 (2012) 121–131 [9] T. Peters, Adv. Protein Chem. 37 (1985) 161. [10] D.C. Carter, J.X. Ho, Adv. Protein Chem. 45 (1994) 153. [11] J.F. Foster, V.M. Rosenoer, M. Oratz, M.A. Rothschild, Albumin Structure, Function and Uses, 1977. [12] S. Miao, H. Leeman, S. De Feyter, R. Schoonheydt, J. Mater. Chem. vol. 20 (2010) (Leuven, Belgium). [13] N.C. de Souza, W. Caetano, R. Itri, C.A. Rodrigues, O.N. Oliveira Jr., J.A. Giacometti, M. Ferreira, J. Colloid Interface Sci. 297 (2) (2006) 546. [14] P. Pal, T. Kamilya, M. Mahato, G.B. Talapatra, Colloids Surf. B: Biointerfaces 73 (1) (2009) 122. [15] C. Ybert, J.M. di Meglio, Langmuir 14 (2) (1998) 471. [16] J.S. Sharp, J.A. Forrest, R.A.L. Jones, Biochemistry 41 (52) (2002) 15810. [17] J.R. Lu, T.J. Su, R.K. Thomas, J. Colloid Interface Sci. 213 (2) (1999) 426. [18] P. Chen, R.M. Prokop, S.S. Susnar, A.W. Neumann, in: D. Möbius, R. Miller (Eds.), Proteins at Liquid Interfaces, Elsevier Science B.V., 1998, p. 303. [19] L.G.C. Pereira, O. Theodoly, H.W. Blanch, C.J. Radke, Langmuir 19 (6) (2003) 2349. [20] T. Nylander, in: D. Möbius, R. Miller (Eds.), Proteins at Liquid Interfaces, Elsevier Science B.V., 1998, p. 385. [21] E. Pezron, P.M. Claesson, J.M. Berg, D. Vollhardt, J. Colloid Interface Sci. 138 (1) (1990) 245.

131

[22] R. Johann, D. Vollhardt, H. Mohwald, Colloids Surf. A Physicochem. Eng. Asp. 182 (1-3) (2001) 311. [23] M. Haro, I. Gascon, R. Aroca, M.C. Lopez, F.M. Royo, J. Colloid Interface Sci. 319 (1) (2008) 277. [24] J. Sanchez-Gonzalez, J. Ruiz-Garcia, M.J. Galvez-Ruiz, J. Colloid Interface Sci. 267 (2) (2003) 286. [25] F. Behroozi, Langmuir 12 (9) (1996) 2289. [26] Z.W. Yu, J. Jin, Y. Cao, Langmuir 18 (11) (2002) 4530. [27] D. Vollhardt, V.B. Fainerman, Adv. Colloid Interf. Sci. 127 (2) (2006) 83. [28] P. Pal, M. Mahato, T. Kamilya, G.B. Talapatra, Phys. Chem. Chem. Phys. 13 (20) (2011) 9385. [29] T. Kamilya, P. Pal, G.B. Talapatra, Colloids Surf. B: Biointerfaces 58 (2) (2007) 137. [30] H.J. Zhang, G.C. Cui, J.B. Li, Colloids Surf. A Physicochem. Eng. Asp. 201 (1-3) (2002) 123. [31] G.L.J. Gaines, Insoluble Monolayers at the Gas–liquid Interfaces, John Wiley & Sons, New York, 1966. [32] B.P. Binks, Adv. Colloid Interf. Sci. 34 (1991) 343. [33] R. Aveyard, B.P. Binks, N. Carr, A.W. Cross, Thin Solid Films 188 (2) (1990) 361.