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A study the interaction forces between the bovine serum albumin protein and montmorillonite surface
Colloids and Surfaces A: Physicochem. Eng. Aspects 414 (2012) 104–114
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A study the interaction forces between the bovine serum albumin protein and montmorillonite surface Anh T.T. Tran ∗ , Bryony J. James Department of Chemical & Materials Engineering, The University of Auckland, Auckland 1142, New Zealand
h i g h l i g h t s
g r a p h i c a l
BSA coated tip was prepared and characterised. BSA–MMT adhesion forces were directly measured in liquid environment. BSA–MMT adhesion forces strongly depend on the solution pH. Addition of ethanol enhances the adhesion force. The BSA–MMT forces are due to electrostatic, hydrophobic, protein conformation.
The adhesion forces between the BSA protein and MMT surface were strongly dependent on solution pH and followed the behaviour of electrostatic force, hydrophobic force and the structure rearrangement of the protein.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 23 April 2012 Received in revised form 20 August 2012 Accepted 21 August 2012 Available online 10 September 2012 Keywords: Bovine Serum Albumin (BSA) protein Montmorillonite (MMT) Atomic Force Microscope (AFM) Adhesion force pH
a b s t r a c t
The interactions between Bovine Serum Albumin (BSA) protein and Montmorillonite (MMT) surfaces were investigated using an Atomic Force Microscope (AFM). The AFM tip was modified by coating with thin films of BSA on its surface while MMT surfaces were used as the substrates for analysis. The adhesion forces between them were measured at different pH values and ethanol concentrations. It was observed that protein–MMT surface adhesion forces strongly depended on the solution pH. Highest value of adhesion force was observed at the solution pH of 4.6 which is near the isoelectric point of protein (∼5.0). The adhesion forces then linearly reduced with both the increase and decrease of the solution pH. Variations of ethanol concentration also affected the measured adhesion forces, but in lesser extent than the pH effect. The maximum protein adsorption at the wine pH of 3.8 occurred with the ethanol concentration of 20%. It was found that the adsorption of BSA protein on the MMT surface followed the behaviour of electrostatic, hydrophobic interactions and the rearrangement of the protein structure. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction The adsorption of protein onto a solid surface has been attracting much attention in a wide range of disciplines, from materials science, environmental sciences, geophysics, biomaterials science and technology to biomedical processes. Thus, much progress has been made to better understand the protein adsorption mechanism. It has been suggested that the interaction between the proteins and
∗ Corresponding author. Tel.: +64 9 3737 599; fax: +64 9 3737 463. E-mail address: [email protected] (A.T.T. Tran).
the solid surface is a very complex process and depends on different factors including the nature of both protein and solid surface, and the surrounding environment [1,2]. During the last decades, several methods have been used to study the interactions between the protein and solid surface and measure their interaction forces [3–5]. Recently, direct microscopic observations with high resolution have been extensively used to investigate the interactions between two surfaces. Atomic Force Microscopy (AFM) is an ideal tool for force measurements at high spatial resolution, at a molecular scale and in a flexible operating environment. The interaction between AFM tips and a solid sample surface can be monitored and the information obtained can be associated with different
0927-7757/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2012.08.066
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Fig. 1. Micrograph of MMT particles. The insert image on the bottom right of the image shows the shape of a typical MMT particle.
physical–chemical processes including van der Waals force, hydrogen bonding, electrostatic force, and hydrophobic interaction [6–8]. Bentonite comprises predominantly montmorillonite (MMT, a member of the smectite group, My + nH2 O (Al2y Mgy )Si4 O10 (OH)2 . The base structure of bentonite is silicon which does not transfer to the wine. Thus, bentonite has been widely used in the beverage industry to remove the haze forming proteins in white wine [9,10]. The process, known as bentonite fining, refers to the addition of a bentonite slurry that is followed by the flocculation and settling of partially soluble components from wine. The adsorption of haze proteins in wine onto the bentonite is primarily driven by cation exchange where positively charged proteins are adsorbed onto the surface of negatively charged clay particles. Studies have shown that bentonite significantly swells and behaves like a series of small plates upon agitation in water. This results in a very large surface area which can adsorb several times as much in when bentonite is in its dry condition [11,12]. It has been reported that the adsorption of protein on the clay surface is very rapid. Studies in a model wine (ethanol with added bovine serum albumin) showed that the time to equilibrium adsorption was less than 30 s for a well dispersed sodium bentonite. The adsorption process of protein on the bentonite surfaces depends on various factors, including concentrations of bentonite and proteins, temperature, pH, and alcohol content of the wine [13–15]. However, while most studies have reported the kinetics of protein adsorption on the bentonite surface, there has been no study to examine the real physical interactions at the molecular scale [14,16–19]. In this work, we aim to investigate the physical interactions between the protein and bentonite surface in wine-like solution using Atomic Force Microscopy and to understand how these interactions relate to variation in pH and alcohol content. The technique used involved the attachment of protein onto the surface of the AFM tip. The adhesion forces between bentonite and protein were examined at various conditions of solution pH and ethanol concentrations. Studies of force measurements between the protein and the active MMT component of bentonite give a better insight into the fundamental knowledge of the important, and as yet little understood, surface interactions driving the adsorption of proteins using bentonite. This essential knowledge also helps us to develop methods for producing bentonite or bentonite-like surface for protein adsorption. 2. Material and methods 2.1. Materials Montmorillonite (MMT) particles supplied by Sigma–Aldrich, USA were approximately spherical and had an average size below
30 m (Fig. 1). Bovine serum albumin, BSA (Fraction V lgG free) was supplied in pellet form by Gibco, NY, USA. Other chemicals used for buffering included sodium phosphate, sodium chloride, and ethanol, all supplied by Sigma–Aldrich, US. All chemicals were used as received without further purification. 2.2. BSA coated tip and characterisation The interactions between bovine serum albumin protein and montmorillonite surfaces were systematically measured at different conditions of solution pH and ethanol concentrations using an Atomic Force Microscope (AFM). The AFM tip was modified by coating with thin films of BSA while MMT surfaces were used as the substrates for analysis. For the AFM tips, we started with clean, silicon nitride (Si3 N4 ) tips, pyramidally shaped, with a cantilever spring constant of 0.58 N/m (Fig. 2). These tips were then functionalised with BSA (Fraction V lgG free) protein thin films. After cleaning with acetone, the clean cantilevers were immersed in 50 mL BSA solution (0.02 g BSA in 5 mL of ethanol and 45 mL of distilled water, pH 7) and subsequently incubated for 48 h in a 37 ◦ C chamber. Before each use, the coated BSA cantilevers were rinsed in phosphate buffered saline (PBS: 0.1 M sodium phosphate, and 0.15 M sodium chloride, pH 7.2) to remove unbound BSA molecules [20,21]. The BSA coated cantilevers were subsequently left to dry at room temperature. Each new cantilever was freshly prepared before use. The BSA protein coating on the AFM tips was characterised using Scanning Electron Microscope (SEM, Philips FEGXL30, Eindhoven, The Netherlands). Prior to SEM imaging, the tips were coated with platinum using a sputter coater (Polaron SC 7640, UK) and examined at an accelerating voltage of 5 keV. To further examine the thin layer of BSA on the tip surface, the surface chemical compositions of the BSA powder, original silicon nitride tip and the BSA coated tip were analysed with XPS. The excitation source in use was Al K␣ (1486.6 eV). The pressure during analysis was between 1 × 10−9 and 1 × 10−8 Torr. To prepare the samples for the XPS examination, the cantilevers were mounted on a sample bar using double sided conductive carbon adhesive tape. Analysis of each sample started with a survey scan from binding energy 0 to 1100 eV. The pass energy was set at 160 eV with 3 sweeps × 1 eV steps × 180 ms dwell time. Once the peaks of individual elements were identified, high resolution scan of N 1s were collected to examine the presence of BSA protein on the cantilever surface. In the narrow scans, the pass energy was set at 20 eV with 3–5 sweeps, 0.1 eV steps size, and 60 ms dwell time. Binding energies were referenced to adventitious carbon at 284.5 eV.
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Fig. 2. SEM image of silicon nitride cantilever with a tip at the end.
2.3. MMT surface preparation and characterisation The MMT surface for AFM measurements was prepared by dusting MMT powder onto a stainless steel disc covered with a thin layer of epoxy resin (Araldite, Selleys). The samples were then left at ambient condition for 24 h to complete the curing and hardening process. In this study, the swelling of an MMT powder was investigated using Environmental Scanning Electron Microscopy (ESEM) (Quanta 200FEG, FEI, Eindhoven, The Netherlands). The samples were controlled for temperature using a peltier stage. Initially, the chamber pressure and sample temperature were set to 0.53 Torr and 20 ◦ C respectively, corresponding to a relative humidity of about 3%. The sample was left at this condition for 15 min. Then, the chamber pressure and temperature are set to the new values of 9.2 Torr and 10 ◦ C representing for the relative humidity of 100%. It took about 15 s for the sample temperature to reach the new value. Then images were taken every 30 s for 15 min to follow the swelling behaviour of the powder. To ascertain the affinity between the BSA protein and the MMT powder surface, 5 mL of MMT slurry (6 g MMT in 1 L of de-ionised water) were suspended in 25 mL of model wine solution. Model wine solution was made from 600 mg L−1 bovine serum albumin, 120 mL L−1 ethanol, 2 g L−1 potassium tartrate buffer, and deionised water with pH 3.8 which is based on the work of Blade and Boulton [14]. The mixture was shaken for 2 min at room temperature then centrifuged at the speed of 5000 rpm for 10 min. The buffer was carefully discarded and the residues were washed with distilled water several times and were dried at 100 ◦ C. The protein content in the clay material was then determined by X-ray Photoelectron Spectroscopy (XPS) (Kratos Ultra Axis DLD, Shimadzu, Manchester, UK). 2.4. Surface potential measurements In this study, zeta potential measurements were used to quantify the surface charge of pure BSA protein and MMT in solutions at different conditions of pH and ethanol content. The electrostatic potential was determined using a zeta potential analyzer (Zetasizer, Malvern Instruments). The instrument, using Smoluchowski’s equation, determines the electrophoretic mobility of the particles automatically and converts it to the zeta potential [22]. The zeta potential analysis was carried out at room temperature. A zero potential or isoelectric point (i.e.p.) indicates an uncharged surface. The sign of the surface is different for higher or lower pH in reference to the isoelectric point. The zeta potential of BSA–MMT mixture when they are close to contact at different values of pH and ethanol content was also
carried out. 5 mL of MMT slurry (6 g MMT in 1 L of de-ionised water) was suspended in 25 mL of model wine solution at particular value of pH. The mixture was shaken for 10 min at room temperature then centrifuged at 5000 rpm for 10 min. The buffer was carefully discarded. The residues were washed with distilled water several times to remove unbound BSA molecules and dried overnight at 70 ◦ C. Then, the powder was dissolved in distilled water with the absence/presence of ethanol. The final pH value was adjusted using potassium tartrate powder and NaOH solution. All the zeta potential results reported in this paper were an average of at least three measurements. 2.5. AFM measurements The adhesion forces between the BSA protein and MMT surfaces were carried out in contact mode, under liquid, at room temperature using the AFM (Nanoscope IIIa, with scanner serial 2318J, Digital Instruments, Santa Babara, CA 93103. The measurements in a liquid cell limited the formation of a thin liquid film on the substrate surfaces, and thus avoided the capillary force component to the measured forces by AFM. The composition of the liquid was controlled to explore the impact of pH and ethanol content. All images were recorded with the Nanoscope software (version 5.12), with integral and proportional gain of 2. The scanning speed was set at 1 Hz. To minimise the effect of surface roughness of the MMT on the force–distance curves, the force measurements were performed on scan areas of 1 m × 1 m or 2 m × 2 m in all cases. At the end of each measurement with a BSA coated tip, the tip was checked using SEM to ensure that no major degradation of BSA layer had occurred. Measurement time was limited to maximum 20 measurements of the same sample to avoid significant degradation of the BSA layer on the tip surface. For direct comparison between the uncoated and BSA coated tip, adhesion force measurements were conducted as follows. Firstly, we measured the interactions between the cleaned, uncoated silicon nitride tip as supplied by the manufacturer and the MMT surfaces at particular values of pH. Secondly, measurements were made with a BSA coated tip as a function of solution pH. Lastly, a set of measurements was made with a BSA coated tip as a function of ethanol content. In all measurements, the force–displacement curves were recorded under a model solution as a function of pH and ethanol concentrations. The model solution was made up of 2 g L−1 potassium D-tartrate (Sigma–Aldrich, US) monobasic [14]. The ethanol concentrations of the model solution were varied from 0% to 20%. The model solution pH ranging from 3.3 to 13.2 were adjusted using potassium tartrate and NaOH solution. In this study, the same silicon nitride tip was used in all measurements, and therefore it was possible to use the same value of spring constant for all calculations.
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Fig. 3. A typical deflection–distance curve between BSA coated tip and MMT surface in liquid at the pH 3.3.
The BSA coated probe–MMT surface adhesion forces were measured when the probe was brought into contact with the MMT surface. In principle, an AFM force–distance curve is a plot of tip–sample interaction forces vs. tip–sample distance. In order to obtain such a plot, the tip is ramped along the vertical axis and the cantilever deflection acquired. The measured adhesion force between the probe and the surface is determined from the deflection of the cantilever as a function of sample displacement using Hooke’s law. F =k·d
(1)
where k is the force constant of the cantilever; and d is the deflection of the cantilever. A typical force–distance curve between BSA coated tip and MMT surface in liquid at the pH 3.3 is shown in Fig. 3. The vertical axis of the graph represents the cantilever deflection whereas the horizontal axis is the distance between the sample and the tip. The physical aspects of AFM force measurements and the principle method for force calculation are well-described in the literature [23–27]. At each particular condition, adhesion forces were measured over at least 3 different areas of interest. In addition, we reduced the possibility of modification of the BSA coated tips by measuring pull-off force directly without scanning the samples relative to the tip. The tip–MMT surface adhesion forces reported in this paper are the average values of at least 100 measurements for each sample. 3. Results and discussion
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measurements. The presence of the debris on the BSA coated tip might reduce the accuracy of the force measurements between the BSA and MMT surface. In some cases, we speculated that the BSA layer might come off the tip apex. Thus, in order to minimise the effect of incomplete BSA covering the apex of the tip or the presence of the debris on the tip surface, we used the freshly prepared tip for a maximum of 20 force–distance curve measurements of the same sample. To further examine the composition of the original tip and the BSA coated tip, their surface chemical compositions were analysed with XPS. The surface composition of the bare tip, the BSA powder, and the BSA coated tip are given in Table 1. It is clear that the BSA coated sample comprises additional nitrogen functionality. This is further illustrated in Fig. 5 showing typical narrow scans of the N 1s peak for the tip, the BSA coated tip and the BSA powder sample. The nitride present in the silicone nitride tip gives a single N 1s peak at a binding energy 397.5, likewise the protein powder gives a single N 1s peak at a binding energy of 399.6 eV. The coated tip gives two N 1s peaks indicating the presence of a thin layer of protein on the tip’s surface. 3.2. Sodium montmorillonite (MMT) characterisation The swelling behaviour of MMT was examined using ESEM. Typical ESEM images of the swelling–shrinkage behaviour of the MMT particles, as humidity is increased from 3% to 100% and then reduced again, are shown in Fig. 6a–f. It was found that the particle area rapidly increased by about 15% within 30 s of the exposure to 100% relative humidity. Equilibrium swelling of 21% was reached within 2 min as shown in Fig. 6 g. Measurements were based on 3 aggregates of the different particle size at relative humidity of 100%. We found that the absolute error remained relatively low, and thus the errors were not included in the chart. It was not the intention of the current study to reproduce adsorption isotherms for MMT which have been well characterised elsewhere [14,28]. Swelling behaviour was assessed in this study to ensure that all AFM measurements were completed after maximum swelling. The atomic compositions of MMT powder and MMT powder after suspending in model wine solution are shown in Table 2. The presence of nitrogen on the surface of the MMT powder after suspending in model wine solution confirmed the adsorption of protein on the MMT surface. Thus, the combination of the data from the swelling test and XPS shows the fast adsorption of BSA protein on the MMT surface. This ensured that 2 min is long enough for the MMT particles swell to the maximum extent and the occurrence of interactions between BSA protein and MMT surface.
3.1. BSA coated tip characterisation 3.3. Zeta potential measurements Typical SEM images of the bare cleaned silicon nitride tip and BSA coated tip are displayed in Fig. 4a and b respectively. It may be seen from Fig. 4b that there was a layer distributed quite uniformly over the whole tip surface. The formation of the layer was subsequently identified as BSA protein by XPS analysis. A SEM image of a BSA coated tip after use for force measurements is shown in Fig. 4c. A thin layer of BSA remained relatively uniform, in particular at the apex of the tip. It was observed that there was some debris at the side of the pyramid. The debris would have probably originated from the MMT surface during the force
The zeta potentials of the pure MMT slurries, pure BSA solution, and MMT–BSA complex in the absence of ethanol at different pH values from 3.3 to 13.2 are shown in Fig. 7. It was observed that pure MMT slurries exhibited a low negative zeta potential and gradually decreased from −23 mV to −33 mV with increasing pH values. No charge reversal was observed at any stage throughout the range of the studied pH. It is known that the MMT clay has a 2:1 layered structure, in which a single layer of an octahedral aluminium magnesium sheet is sandwiched between two layers
Table 1 Surface composition (at%) of the samples. Tip
C
O
N
Si
Balance (Ca, S, Na)
N 1s binding energy (eV)
Silicon nitride tip BSA powder BSA coated tip
51.8 74.9 57.2
16.7 14.1 17.2
11.9 10.1 12.9
17.8
1.8 0.9 2.0
397.5 399.6 397.4, 399.6
10.7
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Fig. 4. SEM images of (a) a silicon nitride tip, (b) a BSA coated tip, and (c) a BSA functionalised tip used in force measurement.
Fig. 5. N1 s peak of (a) BSA powder, (b) original silicon nitride tip, and (c) BSA coated tip.
of tetrahedral silicon sheets. MMT has an isoelectric point in the range of 0.28–1.05 depending on slight variation in composition [29]. As the pH levels tested in this study ranged from 3.3 to 13.2, the observation that MMT surface was negatively charged, which
was probably related to the dissociation of a proton H+ to form the AlO− groups, are in consistent with other studies [29,30]. In contrast, the zeta potential measurements of BSA reflected the charged environment of BSA. BSA surface charge was positive at pH from 3.3 to 5.0. In this particular pH range, the maximum surface charge of 7.8 ± 0.5 mV was observed at the pH 4.6. Then the surface charge of the BSA solution shifted from positive values to negative values with increasing pH. A zero potential charge or i.e.p. of the BSA solution deduced from Fig. 7 was of approximately 5.0. This value is in agreement with those reported in literature which situated the i.e.p. of BSA between 4.8 and 5.8 [31,32]. It is known that BSA protein comprises long chains of amino acid. Part of these amino acids carries side chains of carboxyl or amino groups which can dissociate to COO− or NH3 + ions depending on pH [13]. At an acidic pH below 5.0, the amino groups tend to ionise to form NH3 + , resulting in the positive charge of the BSA solution. In contrast, the negative charge of the BSA solution relates to the formation of COO− groups from the ionisation of the carboxyl groups in the basic condition. When the pH equalled the isoelectric point of the protein, both NH3 + and COO− are presented, resulting in zero surface charge. The zeta potential of the MMT–BSA complex after adsorption was also examined. It was found that the surface charge of MMT–BSA complex was positive with the pH below 5.2 whereas it remained negative for pH values higher than 5.2. The zeta potential of MMT–BSA complex reached zero at pH of 5.2 which was close to the BSA’s zero charge point. At low values of pH, the zeta potential of MMT–BSA complex was significantly larger than that of the pure MMT particles but was comparable to that of BSA solution, suggesting that the BSA protein has effectively covered on the MMT surface. The larger zeta potential values of MMT–BSA confirmed that the
Table 2 Surface composition (at%) of the MMT powders. Tip
C
O
Si
Al
Mg
N
Balance (Ca, F, Na)
MMT powder MMT powder after suspending in model wine
11.0 16.9
52.3 49.9
24.5 21.7
8.3 7.2
1.8 1.5
1.5
2.1 1.3
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Fig. 6. Swelling–shrinkage behaviour of MMT particles (a and f) at relative humidity of 3%, (b–e) RH of 100%, (g) the area variation of MMT particle at relative humidity of 100%.
surface was covered by the adsorbed BSA molecules at high surface coverage. This further clarified that the positively charged BSA molecules effectively adsorbed onto the negatively charged MMT surface by electrostatic interaction which provided higher stability or stronger bonding of the colloid particles in aqueous media. When the pH increased, the MMT–BSA complex’s zeta potential became more negative, and was closer to that of MMT clay meaning that
pH increase induced stronger electrostatic repulsion between the negatively charged MMT surface and the BSA protein. The zeta potential of BSA and MMT–BSA complex with the presence of ethanol at pH 3.8 is illustrated in Fig. 7b. The zeta potential of pure MMT slurries remained unchanged with the addition of ethanol up to 20% of volume, and thus it was not included in Fig. 7b. However, ethanol addition caused an increase of the
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3.4.2. Effect of solution pH on measured adhesion force An approaching/retraction cycle of a typical deflection–distance curve between the BSA coated tip and MMT surface in liquid at the pH of 3.3 is shown in Fig. 3. The approaching curve described the interactions occurring when the tip and the sample surface were close to contact. The retraction curve described the adhesion created during the contact between the tip and the sample surface. In this study, both approaching curve and retraction curve of the interaction between BSA coated tip and the MMT surface were examined as a function of pH values. 3.5. Approaching curves
Fig. 7. Zeta potential measurements of (a) MMT, BSA and MMT–BSA complex as a function of pH, and (b) MMT–BSA mixture as a function of ethanol content at the pH 3.8.
positive surface charge of the BSA solution. The surface charge of the MMT–BSA complex was significantly higher than that of BSA solution, but showing a similar behaviour of zeta potential measurements. This confirmed the high coverage of BSA on the MMT surface as explained previously.
Typical approaching curves of the BSA coated tip–MMT interaction at different particular values of pH are shown in Fig. 11a. It was observed that the approaching curves of the tip–sample interactions varied with the pH values. Attraction forces were observed when the pH values were below the isoelectric point (5.0) of the BSA protein. There was no significant different in adhesion force at pH 3.3, 3.8 or 4.6. The adhesion force observed was 0.7 ± 0.3 nN approximately at the distance of 10 nm maximum. In contrast, repulsion forces were observed with increasing pH. As van der Waals forces are intermolecular forces [2,33] and unaffected by variations of the solution pH, the observed variations of the forces are not due to van der Waals. Instead, electrostatic force between the negatively charge MMT and positively charged BSA at the pH below 5.0 accounted for the observed attraction force. Increasing pH above the isoelectric point, the BSA protein was negatively charged as was the MMT surface, and thus a repulsion force was observed. The importance of electrostatic forces on the adhesion process in the retraction curves is discussed in more detail in the next section. 3.6. Retraction curves
3.4. Adhesion forces between BSA coated tip and MMT surfaces Samples of MMT were prepared on stainless steel discs, as described above, in order to probe the interaction of the BSA coated AFM tip with the MMT surface. The morphology of a thin layer of MMT powder on the stainless steel is shown in Fig. 8. The average surface roughness on a scan size 100 m × 100 m and 1 m × 1 m was 125 ± 35 nm and 3.3 ± 0.8 nm respectively. Throughout this study, the force measurements were carried out through the flat part of the MMT surface with a scan size of 1 m × 1 m or 2 m × 2 m which effectively eliminates the effect of surface roughness on the force measurements. 3.4.1. Force comparison between the bare silicon nitride and BSA coated tip with the MMT surface Prior to measuring the interaction force between BSA coated tip and the MMT surface, we conducted force measurements between the cleaned, uncoated silicon nitride tip and the MMT surface and then compared to BSA coated tip–MMT forces at particular values of pH. Typical force–distance curves measured at pH 3.8 of uncoated tip–MMT and BSA coated tip–MMT are shown in Fig. 9. It was observed that the distance at which the tip broke away from the MMT surface for the BSA coated tip was much larger than that for a bare tip. The corresponding adhesion forces at different values of pH with the absence of ethanol are shown in Fig. 10. At pH values of 3.3 and 7, the BSA–MMT adhesion forces were about two-fold that of the forces between the bare tip and MMT surface. However, the adhesion forces between the tip and the MMT surface were similar at the pH 10 whether the tip was bare or BSA coated. The similarity of the adhesion forces between the bare and BSA coated tip with MMT surfaces at pH 10 is discussed in more detail in the following section.
The interactions between the MMT surface and BSA coated tip in the retraction curves as a function of the solution pH were examined. A series of force–distance curves obtained at different pH values ranging from 3.3 to 13.2 between BSA coated tip and the MMT surface are shown in Fig. 11b. The corresponding forces are shown in Fig. 12. For these measurements, no ethanol was included in the pH buffer solution. It was observed that pH values had a strong effect on the adhesion force of the BSA protein onto the MMT surfaces. Large adhesion force between the BSA coated tip and MMT surface was observed with a maximum value of 10.2 ± 0.7 nN at pH 4.6. The adhesion forces then gradually decreased with an increasing pH. A minimum adhesion force of 1.3 ± 0.1 nN was found at pH 12 and then the interaction became completely repulsive at pH 13.2. There are a number of possible interaction forces acting between the two surfaces. These include surface tension, van der Waals, hydrogen bonding, electrostatic double-layer force, and hydrophobic interaction. The sum of interaction forces between the BSA coated tip and the MMT surface in air was found to be 24.5 ± 3.2 nN. This was nearly three times the maximum adhesion force in solution (at pH 4.6). The higher value in air was mainly due to the liquid–air interfacial tension arising from the formation of a thin layer of water adsorbed on the tip and the sample surface. The significant reduction of measured force in the liquid environment suggests that these surface tension forces are eliminated when measuring under fluid environment, which is in good agreement with the literature [34]. Thus, surface tension force is not a component part of the adhesion of protein to MMT. In the current study, the zeta potential measurements in the previous section showed the amphoteric nature of the BSA protein. The isoelectric point of the BSA protein was observed at the
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Fig. 8. 2D and 3D AFM images of MMT surfaces at a scan size of (a) 100 m and (b) 5 m.
pH 5.0 approximately. Below this point of zero-charged, BSA was positively charged whereas it was negatively charged with the pH above pH 5.0. However, the MMT surface was negatively charged throughout the range of studied pH from 3.3 to 13.2. Thus, the nature of the interaction force between MMT and BSA depended mainly on the surface charge of BSA protein. When the pH was below the isoelectric point of the BSA protein, a high attractive force was observed between negatively charged MMT surface and positively charged BSA protein. The attraction force observed at the pH of 3.3, 3.8 and 4.6 were not short range van der Waals force for such a large distance of 50 nm (Fig. 11b), but were undoubtedly long range electrostatic forces. The increase of the positively surface charge of the BSA protein with pH values from 3.3 to 4.6 (Fig. 7) corresponded to the increase of the attractive forces as observed in Fig. 12.
Fig. 9. Force–distance curve measured at pH 3.8 with (a) a clean, bare silicon nitride tip and (b) with a tip coated with BSA.
A maximum adhesion force was observed at the pH of 4.6 which which was close to the i.e.p. of BSA (5.0). The maximum forces observed close to protein i.e.p. in this study are consistent with other observations from the literature [2,35,36]. BSA protein comprises long chains of amino acid and has a low internal stability. Parts of these amino acids are hydrophobic and parts are hydrophilic. It has been reported that, near the isoelectric point, Coulomb attraction between oppositely charged residues in the protein promote the rearrangement of the molecules making the protein structure more compact [2,35–37]. This enhances the interactions between two hydrophobic molecules and thus reducing their undesirable interaction with water molecules. The soluble proteins then had a highly ordered protein structure with hydrophobic core and charged and polar side chains situated on the solvent-exposed surface where they interacted with surrounding molecules. Minimising the number of hydrophobic side
Fig. 10. Comparison of the adhesion force between the original tips (blue)/BSA coated tips (red) and MMT surfaces at specific pH values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Fig. 11. Force–distance curves obtained at different pH values between BSA coated tip and MMT surface with the absence of ethanol.
chains exposed to water resulted in a large entropy increase and a relatively small enthalpy effect [2]. Thus, Gibbs free energy Gads = Hads − TSads is more negative (where H, S, and T are the enthalpy, entropy and the absolute temperature respectively). In the current study this is demonstrated by a maximum adherence of BSA protein on the hydrophilic MMT surface at the pH near the isoelectric point. Thus, the maximum adhesion force observed at this particular pH was not electrostatic in nature but tied to the rearrangement of the protein structure. At pH above the isolectric point of approximately 5.0, both the MMT surface and BSA protein bear the same negative charge. The increase in surface negative charge of BSA with increasing pH values would cause an increase in repulsion of electronic double layers surrounding the BSA protein, and thus increase the potential energy barrier which hinders the approach to the MMT and then prevents attachment. Thus, a repulsion force between the MMT surface and BSA molecules was expected. However, the adhesion forces between the BSA and MMT surface at the pH range 5.6–10 took place as shown in Figs. 11b and 12. This suggested that the adhesion force between BSA and MMT surface was not solely due to electrostatic effects. BSA protein structure has a low internal stability. At a pH above the isoelectric point, the charge-charge interactions arose which easily affected the low internal stability structure of BSA protein.
This resulted in the expanding of the protein structure. As such, the hydrophobic amino acids of BSA protein come into contact with the siloxane of MMT surface and adhere to the MMT surface by hydrophobic interactions [13], resulting in the adhesion between the BSA and MMT surface even though charges of both BSA and MMT were the same. Thus, the adherence of BSA onto the MMT surfaces was driven by different types of forces: electrostatic, hydrophobic and structural rearrangement in the BSA protein.
Fig. 12. Adhesion force between the BSA coated tip and MMT surface at different pH values with the absence of ethanol.
Fig. 13. Force–distance curves obtained at different ethanol content between BSA coated tip and MMT surface at the pH 3.8.
3.6.1. Effect of ethanol concentration on the adhesion force Measurements were also performed to examine the influence of ethanol concentration on the interaction between the BSA and the MMT surfaces. It has been reported that most grape wines have an ethanol concentration of about 12% (by volume) [9]. Therefore, in this study, ethanol concentration ranging from 0 to 20% was examined. Fig. 13 shows a series of force–distance curves between
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hydrophobic interactions. The dependence of measured forces as a function of pH and ethanol concentrations indicates that the MMT can be used as a cation exchange material.
References
Fig. 14. Adhesion force between the BSA coated tips and MMT surfaces as a function of ethanol content at the pH of 3.8.
BSA coated tip and MMT surface obtained at different ethanol content. In these measurements, the pH was maintained at a constant value of 3.8. Strong attraction forces were observed in all cases. A large distance from 50 nm to 100 nm approximate indicated that attraction force was not due to van der Waals force. The corresponding forces are illustrated in Fig. 14. It was observed that there were no significant changes in adhesion force with the increase of ethanol concentration from 0% to 12%. However, the adhesion force increased sharply when the ethanol concentration increased from 16% to 20%. Zeta potential measurement in Section 3.3 confirmed that the negative surface charge of MMT unchanged. However, positive surface charge of BSA increased with the ethanol content, thus increasing the electrostatic force between the negatively charged MMT surface and positively charged BSA. It was also observed that MMT particles have a strong affinity with water, as shown in Fig. 5. In water, MMT swells significantly enabling intercalation of protein molecules. Sun et al. [38] and Archarandio et al. [28] proposed that the presence of ethanol enhances the swelling of MMT and separates the silicate layers of the MMT particles. This results in the expansion and broadening of the MMT channel which enhances the approach of larger proteins to adsorb on the MMT surface and facilitate adherence. That, at high concentration of ethanol, the binding capacity of MMT is improved for larger protein that otherwise would not fit. An increase of adhesion force between MMT surface and BSA protein as a function of ethanol content in this is consistent with a study of Achaerandio et al. [28] who found that adsorption capacity of BSA on the bentonite surface tended to increase with increasing ethanol concentrations. 4. Conclusions The adsorption of bovine serum albumin protein on the MMT surfaces was systematically examined using an AFM. The tip was modified by incorporation of thin films of BSA. The force curves were measured at different conditions of solution pH and ethanol content. It was observed that interaction between the BSA protein and MMT surfaces were dependent on solution pH and ethanol content where pH had a stronger effect on the adhesion force. Highest adhesion force occurred at the pH of 4.6, which is near to the isoelectric point of proteins. This maximum adhesion force at this point related to the structure rearrangement in the protein to form a more compact structure. The adhesion forces decreased linearly with both an increase and a decrease of the solution pH. At pH values lower than the isoelectric point of BSA protein, the adsorption of BSA molecules onto the MMT surface followed the behaviour of electrostatic occurring between the positively charged BSA and negatively charged MMT. At pH values higher than the isoelectric point, the adhesion force was found to be dominated by
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[31] V. Vilker, C.K. Colton, K.A. Smith, The osmotic pressure of concentrated protein solutions: effect of concentration and pH in saline solutions of bovine serum albumin, J. Colloid Interface Sci. 79 (1981) 548–566. [32] D. Kuzmenko, E. Arkhangelsky, S. Belfer, V. Freger, V. Gitis, Chemical cleaning of UF membranes fouled by BSA, Desalination 179 (2005) 323–333. [33] F.O. Goodman, N. Garcia, Roles of the attractive and repulsive forces in atomicforce microscopy, Phys. Rev. B: Condens. Matter 43 (1991) 4728–4731. [34] F.L. Leite, P.S.P. Herrmann, Application of atomic force spectroscopy (AFS) to studies of adhesion phenomena: a review, J. Adhes. Sci. Technol. 19 (2005) 365–405.
[35] P. Bagchi, S.M. Birnbaum, Effect of pH on the adsorption of immunoglobulin G on anionic poly(vinyltoluene) model latex particles, J. Colloid Interface Sci. 83 (1981) 460–478. [36] P.G. Koutsoukos, C.A. Mumme-Young, W. Norde, J. Lyklema, Effect of the nature of the substrate on the adsorption of human plasma albumin, Colloids Surf. 5 (1982) 93–104. [37] K. Nakanishi, T. Sakiyama, K. Imamura, On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon, J. Biosci. Bioeng. 91 (2001) 233–244. [38] X. Sun, C. Li, Z. Wu, X. Xu, L. Ren, H. Zhao, Adsorption of protein from model wine solution by different bentonites, Chin. J. Anal. Chem. 15 (2007) 632–638.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
A study the interaction forces between the bovine serum albumin protein and montmorillonite surface Anh T.T. Tran ∗ , Bryony J. James Department of Chemical & Materials Engineering, The University of Auckland, Auckland 1142, New Zealand
h i g h l i g h t s
g r a p h i c a l
BSA coated tip was prepared and characterised. BSA–MMT adhesion forces were directly measured in liquid environment. BSA–MMT adhesion forces strongly depend on the solution pH. Addition of ethanol enhances the adhesion force. The BSA–MMT forces are due to electrostatic, hydrophobic, protein conformation.
The adhesion forces between the BSA protein and MMT surface were strongly dependent on solution pH and followed the behaviour of electrostatic force, hydrophobic force and the structure rearrangement of the protein.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 23 April 2012 Received in revised form 20 August 2012 Accepted 21 August 2012 Available online 10 September 2012 Keywords: Bovine Serum Albumin (BSA) protein Montmorillonite (MMT) Atomic Force Microscope (AFM) Adhesion force pH
a b s t r a c t
The interactions between Bovine Serum Albumin (BSA) protein and Montmorillonite (MMT) surfaces were investigated using an Atomic Force Microscope (AFM). The AFM tip was modified by coating with thin films of BSA on its surface while MMT surfaces were used as the substrates for analysis. The adhesion forces between them were measured at different pH values and ethanol concentrations. It was observed that protein–MMT surface adhesion forces strongly depended on the solution pH. Highest value of adhesion force was observed at the solution pH of 4.6 which is near the isoelectric point of protein (∼5.0). The adhesion forces then linearly reduced with both the increase and decrease of the solution pH. Variations of ethanol concentration also affected the measured adhesion forces, but in lesser extent than the pH effect. The maximum protein adsorption at the wine pH of 3.8 occurred with the ethanol concentration of 20%. It was found that the adsorption of BSA protein on the MMT surface followed the behaviour of electrostatic, hydrophobic interactions and the rearrangement of the protein structure. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction The adsorption of protein onto a solid surface has been attracting much attention in a wide range of disciplines, from materials science, environmental sciences, geophysics, biomaterials science and technology to biomedical processes. Thus, much progress has been made to better understand the protein adsorption mechanism. It has been suggested that the interaction between the proteins and
∗ Corresponding author. Tel.: +64 9 3737 599; fax: +64 9 3737 463. E-mail address: [email protected] (A.T.T. Tran).
the solid surface is a very complex process and depends on different factors including the nature of both protein and solid surface, and the surrounding environment [1,2]. During the last decades, several methods have been used to study the interactions between the protein and solid surface and measure their interaction forces [3–5]. Recently, direct microscopic observations with high resolution have been extensively used to investigate the interactions between two surfaces. Atomic Force Microscopy (AFM) is an ideal tool for force measurements at high spatial resolution, at a molecular scale and in a flexible operating environment. The interaction between AFM tips and a solid sample surface can be monitored and the information obtained can be associated with different
0927-7757/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2012.08.066
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Fig. 1. Micrograph of MMT particles. The insert image on the bottom right of the image shows the shape of a typical MMT particle.
physical–chemical processes including van der Waals force, hydrogen bonding, electrostatic force, and hydrophobic interaction [6–8]. Bentonite comprises predominantly montmorillonite (MMT, a member of the smectite group, My + nH2 O (Al2y Mgy )Si4 O10 (OH)2 . The base structure of bentonite is silicon which does not transfer to the wine. Thus, bentonite has been widely used in the beverage industry to remove the haze forming proteins in white wine [9,10]. The process, known as bentonite fining, refers to the addition of a bentonite slurry that is followed by the flocculation and settling of partially soluble components from wine. The adsorption of haze proteins in wine onto the bentonite is primarily driven by cation exchange where positively charged proteins are adsorbed onto the surface of negatively charged clay particles. Studies have shown that bentonite significantly swells and behaves like a series of small plates upon agitation in water. This results in a very large surface area which can adsorb several times as much in when bentonite is in its dry condition [11,12]. It has been reported that the adsorption of protein on the clay surface is very rapid. Studies in a model wine (ethanol with added bovine serum albumin) showed that the time to equilibrium adsorption was less than 30 s for a well dispersed sodium bentonite. The adsorption process of protein on the bentonite surfaces depends on various factors, including concentrations of bentonite and proteins, temperature, pH, and alcohol content of the wine [13–15]. However, while most studies have reported the kinetics of protein adsorption on the bentonite surface, there has been no study to examine the real physical interactions at the molecular scale [14,16–19]. In this work, we aim to investigate the physical interactions between the protein and bentonite surface in wine-like solution using Atomic Force Microscopy and to understand how these interactions relate to variation in pH and alcohol content. The technique used involved the attachment of protein onto the surface of the AFM tip. The adhesion forces between bentonite and protein were examined at various conditions of solution pH and ethanol concentrations. Studies of force measurements between the protein and the active MMT component of bentonite give a better insight into the fundamental knowledge of the important, and as yet little understood, surface interactions driving the adsorption of proteins using bentonite. This essential knowledge also helps us to develop methods for producing bentonite or bentonite-like surface for protein adsorption. 2. Material and methods 2.1. Materials Montmorillonite (MMT) particles supplied by Sigma–Aldrich, USA were approximately spherical and had an average size below
30 m (Fig. 1). Bovine serum albumin, BSA (Fraction V lgG free) was supplied in pellet form by Gibco, NY, USA. Other chemicals used for buffering included sodium phosphate, sodium chloride, and ethanol, all supplied by Sigma–Aldrich, US. All chemicals were used as received without further purification. 2.2. BSA coated tip and characterisation The interactions between bovine serum albumin protein and montmorillonite surfaces were systematically measured at different conditions of solution pH and ethanol concentrations using an Atomic Force Microscope (AFM). The AFM tip was modified by coating with thin films of BSA while MMT surfaces were used as the substrates for analysis. For the AFM tips, we started with clean, silicon nitride (Si3 N4 ) tips, pyramidally shaped, with a cantilever spring constant of 0.58 N/m (Fig. 2). These tips were then functionalised with BSA (Fraction V lgG free) protein thin films. After cleaning with acetone, the clean cantilevers were immersed in 50 mL BSA solution (0.02 g BSA in 5 mL of ethanol and 45 mL of distilled water, pH 7) and subsequently incubated for 48 h in a 37 ◦ C chamber. Before each use, the coated BSA cantilevers were rinsed in phosphate buffered saline (PBS: 0.1 M sodium phosphate, and 0.15 M sodium chloride, pH 7.2) to remove unbound BSA molecules [20,21]. The BSA coated cantilevers were subsequently left to dry at room temperature. Each new cantilever was freshly prepared before use. The BSA protein coating on the AFM tips was characterised using Scanning Electron Microscope (SEM, Philips FEGXL30, Eindhoven, The Netherlands). Prior to SEM imaging, the tips were coated with platinum using a sputter coater (Polaron SC 7640, UK) and examined at an accelerating voltage of 5 keV. To further examine the thin layer of BSA on the tip surface, the surface chemical compositions of the BSA powder, original silicon nitride tip and the BSA coated tip were analysed with XPS. The excitation source in use was Al K␣ (1486.6 eV). The pressure during analysis was between 1 × 10−9 and 1 × 10−8 Torr. To prepare the samples for the XPS examination, the cantilevers were mounted on a sample bar using double sided conductive carbon adhesive tape. Analysis of each sample started with a survey scan from binding energy 0 to 1100 eV. The pass energy was set at 160 eV with 3 sweeps × 1 eV steps × 180 ms dwell time. Once the peaks of individual elements were identified, high resolution scan of N 1s were collected to examine the presence of BSA protein on the cantilever surface. In the narrow scans, the pass energy was set at 20 eV with 3–5 sweeps, 0.1 eV steps size, and 60 ms dwell time. Binding energies were referenced to adventitious carbon at 284.5 eV.
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Fig. 2. SEM image of silicon nitride cantilever with a tip at the end.
2.3. MMT surface preparation and characterisation The MMT surface for AFM measurements was prepared by dusting MMT powder onto a stainless steel disc covered with a thin layer of epoxy resin (Araldite, Selleys). The samples were then left at ambient condition for 24 h to complete the curing and hardening process. In this study, the swelling of an MMT powder was investigated using Environmental Scanning Electron Microscopy (ESEM) (Quanta 200FEG, FEI, Eindhoven, The Netherlands). The samples were controlled for temperature using a peltier stage. Initially, the chamber pressure and sample temperature were set to 0.53 Torr and 20 ◦ C respectively, corresponding to a relative humidity of about 3%. The sample was left at this condition for 15 min. Then, the chamber pressure and temperature are set to the new values of 9.2 Torr and 10 ◦ C representing for the relative humidity of 100%. It took about 15 s for the sample temperature to reach the new value. Then images were taken every 30 s for 15 min to follow the swelling behaviour of the powder. To ascertain the affinity between the BSA protein and the MMT powder surface, 5 mL of MMT slurry (6 g MMT in 1 L of de-ionised water) were suspended in 25 mL of model wine solution. Model wine solution was made from 600 mg L−1 bovine serum albumin, 120 mL L−1 ethanol, 2 g L−1 potassium tartrate buffer, and deionised water with pH 3.8 which is based on the work of Blade and Boulton [14]. The mixture was shaken for 2 min at room temperature then centrifuged at the speed of 5000 rpm for 10 min. The buffer was carefully discarded and the residues were washed with distilled water several times and were dried at 100 ◦ C. The protein content in the clay material was then determined by X-ray Photoelectron Spectroscopy (XPS) (Kratos Ultra Axis DLD, Shimadzu, Manchester, UK). 2.4. Surface potential measurements In this study, zeta potential measurements were used to quantify the surface charge of pure BSA protein and MMT in solutions at different conditions of pH and ethanol content. The electrostatic potential was determined using a zeta potential analyzer (Zetasizer, Malvern Instruments). The instrument, using Smoluchowski’s equation, determines the electrophoretic mobility of the particles automatically and converts it to the zeta potential [22]. The zeta potential analysis was carried out at room temperature. A zero potential or isoelectric point (i.e.p.) indicates an uncharged surface. The sign of the surface is different for higher or lower pH in reference to the isoelectric point. The zeta potential of BSA–MMT mixture when they are close to contact at different values of pH and ethanol content was also
carried out. 5 mL of MMT slurry (6 g MMT in 1 L of de-ionised water) was suspended in 25 mL of model wine solution at particular value of pH. The mixture was shaken for 10 min at room temperature then centrifuged at 5000 rpm for 10 min. The buffer was carefully discarded. The residues were washed with distilled water several times to remove unbound BSA molecules and dried overnight at 70 ◦ C. Then, the powder was dissolved in distilled water with the absence/presence of ethanol. The final pH value was adjusted using potassium tartrate powder and NaOH solution. All the zeta potential results reported in this paper were an average of at least three measurements. 2.5. AFM measurements The adhesion forces between the BSA protein and MMT surfaces were carried out in contact mode, under liquid, at room temperature using the AFM (Nanoscope IIIa, with scanner serial 2318J, Digital Instruments, Santa Babara, CA 93103. The measurements in a liquid cell limited the formation of a thin liquid film on the substrate surfaces, and thus avoided the capillary force component to the measured forces by AFM. The composition of the liquid was controlled to explore the impact of pH and ethanol content. All images were recorded with the Nanoscope software (version 5.12), with integral and proportional gain of 2. The scanning speed was set at 1 Hz. To minimise the effect of surface roughness of the MMT on the force–distance curves, the force measurements were performed on scan areas of 1 m × 1 m or 2 m × 2 m in all cases. At the end of each measurement with a BSA coated tip, the tip was checked using SEM to ensure that no major degradation of BSA layer had occurred. Measurement time was limited to maximum 20 measurements of the same sample to avoid significant degradation of the BSA layer on the tip surface. For direct comparison between the uncoated and BSA coated tip, adhesion force measurements were conducted as follows. Firstly, we measured the interactions between the cleaned, uncoated silicon nitride tip as supplied by the manufacturer and the MMT surfaces at particular values of pH. Secondly, measurements were made with a BSA coated tip as a function of solution pH. Lastly, a set of measurements was made with a BSA coated tip as a function of ethanol content. In all measurements, the force–displacement curves were recorded under a model solution as a function of pH and ethanol concentrations. The model solution was made up of 2 g L−1 potassium D-tartrate (Sigma–Aldrich, US) monobasic [14]. The ethanol concentrations of the model solution were varied from 0% to 20%. The model solution pH ranging from 3.3 to 13.2 were adjusted using potassium tartrate and NaOH solution. In this study, the same silicon nitride tip was used in all measurements, and therefore it was possible to use the same value of spring constant for all calculations.
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Fig. 3. A typical deflection–distance curve between BSA coated tip and MMT surface in liquid at the pH 3.3.
The BSA coated probe–MMT surface adhesion forces were measured when the probe was brought into contact with the MMT surface. In principle, an AFM force–distance curve is a plot of tip–sample interaction forces vs. tip–sample distance. In order to obtain such a plot, the tip is ramped along the vertical axis and the cantilever deflection acquired. The measured adhesion force between the probe and the surface is determined from the deflection of the cantilever as a function of sample displacement using Hooke’s law. F =k·d
(1)
where k is the force constant of the cantilever; and d is the deflection of the cantilever. A typical force–distance curve between BSA coated tip and MMT surface in liquid at the pH 3.3 is shown in Fig. 3. The vertical axis of the graph represents the cantilever deflection whereas the horizontal axis is the distance between the sample and the tip. The physical aspects of AFM force measurements and the principle method for force calculation are well-described in the literature [23–27]. At each particular condition, adhesion forces were measured over at least 3 different areas of interest. In addition, we reduced the possibility of modification of the BSA coated tips by measuring pull-off force directly without scanning the samples relative to the tip. The tip–MMT surface adhesion forces reported in this paper are the average values of at least 100 measurements for each sample. 3. Results and discussion
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measurements. The presence of the debris on the BSA coated tip might reduce the accuracy of the force measurements between the BSA and MMT surface. In some cases, we speculated that the BSA layer might come off the tip apex. Thus, in order to minimise the effect of incomplete BSA covering the apex of the tip or the presence of the debris on the tip surface, we used the freshly prepared tip for a maximum of 20 force–distance curve measurements of the same sample. To further examine the composition of the original tip and the BSA coated tip, their surface chemical compositions were analysed with XPS. The surface composition of the bare tip, the BSA powder, and the BSA coated tip are given in Table 1. It is clear that the BSA coated sample comprises additional nitrogen functionality. This is further illustrated in Fig. 5 showing typical narrow scans of the N 1s peak for the tip, the BSA coated tip and the BSA powder sample. The nitride present in the silicone nitride tip gives a single N 1s peak at a binding energy 397.5, likewise the protein powder gives a single N 1s peak at a binding energy of 399.6 eV. The coated tip gives two N 1s peaks indicating the presence of a thin layer of protein on the tip’s surface. 3.2. Sodium montmorillonite (MMT) characterisation The swelling behaviour of MMT was examined using ESEM. Typical ESEM images of the swelling–shrinkage behaviour of the MMT particles, as humidity is increased from 3% to 100% and then reduced again, are shown in Fig. 6a–f. It was found that the particle area rapidly increased by about 15% within 30 s of the exposure to 100% relative humidity. Equilibrium swelling of 21% was reached within 2 min as shown in Fig. 6 g. Measurements were based on 3 aggregates of the different particle size at relative humidity of 100%. We found that the absolute error remained relatively low, and thus the errors were not included in the chart. It was not the intention of the current study to reproduce adsorption isotherms for MMT which have been well characterised elsewhere [14,28]. Swelling behaviour was assessed in this study to ensure that all AFM measurements were completed after maximum swelling. The atomic compositions of MMT powder and MMT powder after suspending in model wine solution are shown in Table 2. The presence of nitrogen on the surface of the MMT powder after suspending in model wine solution confirmed the adsorption of protein on the MMT surface. Thus, the combination of the data from the swelling test and XPS shows the fast adsorption of BSA protein on the MMT surface. This ensured that 2 min is long enough for the MMT particles swell to the maximum extent and the occurrence of interactions between BSA protein and MMT surface.
3.1. BSA coated tip characterisation 3.3. Zeta potential measurements Typical SEM images of the bare cleaned silicon nitride tip and BSA coated tip are displayed in Fig. 4a and b respectively. It may be seen from Fig. 4b that there was a layer distributed quite uniformly over the whole tip surface. The formation of the layer was subsequently identified as BSA protein by XPS analysis. A SEM image of a BSA coated tip after use for force measurements is shown in Fig. 4c. A thin layer of BSA remained relatively uniform, in particular at the apex of the tip. It was observed that there was some debris at the side of the pyramid. The debris would have probably originated from the MMT surface during the force
The zeta potentials of the pure MMT slurries, pure BSA solution, and MMT–BSA complex in the absence of ethanol at different pH values from 3.3 to 13.2 are shown in Fig. 7. It was observed that pure MMT slurries exhibited a low negative zeta potential and gradually decreased from −23 mV to −33 mV with increasing pH values. No charge reversal was observed at any stage throughout the range of the studied pH. It is known that the MMT clay has a 2:1 layered structure, in which a single layer of an octahedral aluminium magnesium sheet is sandwiched between two layers
Table 1 Surface composition (at%) of the samples. Tip
C
O
N
Si
Balance (Ca, S, Na)
N 1s binding energy (eV)
Silicon nitride tip BSA powder BSA coated tip
51.8 74.9 57.2
16.7 14.1 17.2
11.9 10.1 12.9
17.8
1.8 0.9 2.0
397.5 399.6 397.4, 399.6
10.7
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Fig. 4. SEM images of (a) a silicon nitride tip, (b) a BSA coated tip, and (c) a BSA functionalised tip used in force measurement.
Fig. 5. N1 s peak of (a) BSA powder, (b) original silicon nitride tip, and (c) BSA coated tip.
of tetrahedral silicon sheets. MMT has an isoelectric point in the range of 0.28–1.05 depending on slight variation in composition [29]. As the pH levels tested in this study ranged from 3.3 to 13.2, the observation that MMT surface was negatively charged, which
was probably related to the dissociation of a proton H+ to form the AlO− groups, are in consistent with other studies [29,30]. In contrast, the zeta potential measurements of BSA reflected the charged environment of BSA. BSA surface charge was positive at pH from 3.3 to 5.0. In this particular pH range, the maximum surface charge of 7.8 ± 0.5 mV was observed at the pH 4.6. Then the surface charge of the BSA solution shifted from positive values to negative values with increasing pH. A zero potential charge or i.e.p. of the BSA solution deduced from Fig. 7 was of approximately 5.0. This value is in agreement with those reported in literature which situated the i.e.p. of BSA between 4.8 and 5.8 [31,32]. It is known that BSA protein comprises long chains of amino acid. Part of these amino acids carries side chains of carboxyl or amino groups which can dissociate to COO− or NH3 + ions depending on pH [13]. At an acidic pH below 5.0, the amino groups tend to ionise to form NH3 + , resulting in the positive charge of the BSA solution. In contrast, the negative charge of the BSA solution relates to the formation of COO− groups from the ionisation of the carboxyl groups in the basic condition. When the pH equalled the isoelectric point of the protein, both NH3 + and COO− are presented, resulting in zero surface charge. The zeta potential of the MMT–BSA complex after adsorption was also examined. It was found that the surface charge of MMT–BSA complex was positive with the pH below 5.2 whereas it remained negative for pH values higher than 5.2. The zeta potential of MMT–BSA complex reached zero at pH of 5.2 which was close to the BSA’s zero charge point. At low values of pH, the zeta potential of MMT–BSA complex was significantly larger than that of the pure MMT particles but was comparable to that of BSA solution, suggesting that the BSA protein has effectively covered on the MMT surface. The larger zeta potential values of MMT–BSA confirmed that the
Table 2 Surface composition (at%) of the MMT powders. Tip
C
O
Si
Al
Mg
N
Balance (Ca, F, Na)
MMT powder MMT powder after suspending in model wine
11.0 16.9
52.3 49.9
24.5 21.7
8.3 7.2
1.8 1.5
1.5
2.1 1.3
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Fig. 6. Swelling–shrinkage behaviour of MMT particles (a and f) at relative humidity of 3%, (b–e) RH of 100%, (g) the area variation of MMT particle at relative humidity of 100%.
surface was covered by the adsorbed BSA molecules at high surface coverage. This further clarified that the positively charged BSA molecules effectively adsorbed onto the negatively charged MMT surface by electrostatic interaction which provided higher stability or stronger bonding of the colloid particles in aqueous media. When the pH increased, the MMT–BSA complex’s zeta potential became more negative, and was closer to that of MMT clay meaning that
pH increase induced stronger electrostatic repulsion between the negatively charged MMT surface and the BSA protein. The zeta potential of BSA and MMT–BSA complex with the presence of ethanol at pH 3.8 is illustrated in Fig. 7b. The zeta potential of pure MMT slurries remained unchanged with the addition of ethanol up to 20% of volume, and thus it was not included in Fig. 7b. However, ethanol addition caused an increase of the
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3.4.2. Effect of solution pH on measured adhesion force An approaching/retraction cycle of a typical deflection–distance curve between the BSA coated tip and MMT surface in liquid at the pH of 3.3 is shown in Fig. 3. The approaching curve described the interactions occurring when the tip and the sample surface were close to contact. The retraction curve described the adhesion created during the contact between the tip and the sample surface. In this study, both approaching curve and retraction curve of the interaction between BSA coated tip and the MMT surface were examined as a function of pH values. 3.5. Approaching curves
Fig. 7. Zeta potential measurements of (a) MMT, BSA and MMT–BSA complex as a function of pH, and (b) MMT–BSA mixture as a function of ethanol content at the pH 3.8.
positive surface charge of the BSA solution. The surface charge of the MMT–BSA complex was significantly higher than that of BSA solution, but showing a similar behaviour of zeta potential measurements. This confirmed the high coverage of BSA on the MMT surface as explained previously.
Typical approaching curves of the BSA coated tip–MMT interaction at different particular values of pH are shown in Fig. 11a. It was observed that the approaching curves of the tip–sample interactions varied with the pH values. Attraction forces were observed when the pH values were below the isoelectric point (5.0) of the BSA protein. There was no significant different in adhesion force at pH 3.3, 3.8 or 4.6. The adhesion force observed was 0.7 ± 0.3 nN approximately at the distance of 10 nm maximum. In contrast, repulsion forces were observed with increasing pH. As van der Waals forces are intermolecular forces [2,33] and unaffected by variations of the solution pH, the observed variations of the forces are not due to van der Waals. Instead, electrostatic force between the negatively charge MMT and positively charged BSA at the pH below 5.0 accounted for the observed attraction force. Increasing pH above the isoelectric point, the BSA protein was negatively charged as was the MMT surface, and thus a repulsion force was observed. The importance of electrostatic forces on the adhesion process in the retraction curves is discussed in more detail in the next section. 3.6. Retraction curves
3.4. Adhesion forces between BSA coated tip and MMT surfaces Samples of MMT were prepared on stainless steel discs, as described above, in order to probe the interaction of the BSA coated AFM tip with the MMT surface. The morphology of a thin layer of MMT powder on the stainless steel is shown in Fig. 8. The average surface roughness on a scan size 100 m × 100 m and 1 m × 1 m was 125 ± 35 nm and 3.3 ± 0.8 nm respectively. Throughout this study, the force measurements were carried out through the flat part of the MMT surface with a scan size of 1 m × 1 m or 2 m × 2 m which effectively eliminates the effect of surface roughness on the force measurements. 3.4.1. Force comparison between the bare silicon nitride and BSA coated tip with the MMT surface Prior to measuring the interaction force between BSA coated tip and the MMT surface, we conducted force measurements between the cleaned, uncoated silicon nitride tip and the MMT surface and then compared to BSA coated tip–MMT forces at particular values of pH. Typical force–distance curves measured at pH 3.8 of uncoated tip–MMT and BSA coated tip–MMT are shown in Fig. 9. It was observed that the distance at which the tip broke away from the MMT surface for the BSA coated tip was much larger than that for a bare tip. The corresponding adhesion forces at different values of pH with the absence of ethanol are shown in Fig. 10. At pH values of 3.3 and 7, the BSA–MMT adhesion forces were about two-fold that of the forces between the bare tip and MMT surface. However, the adhesion forces between the tip and the MMT surface were similar at the pH 10 whether the tip was bare or BSA coated. The similarity of the adhesion forces between the bare and BSA coated tip with MMT surfaces at pH 10 is discussed in more detail in the following section.
The interactions between the MMT surface and BSA coated tip in the retraction curves as a function of the solution pH were examined. A series of force–distance curves obtained at different pH values ranging from 3.3 to 13.2 between BSA coated tip and the MMT surface are shown in Fig. 11b. The corresponding forces are shown in Fig. 12. For these measurements, no ethanol was included in the pH buffer solution. It was observed that pH values had a strong effect on the adhesion force of the BSA protein onto the MMT surfaces. Large adhesion force between the BSA coated tip and MMT surface was observed with a maximum value of 10.2 ± 0.7 nN at pH 4.6. The adhesion forces then gradually decreased with an increasing pH. A minimum adhesion force of 1.3 ± 0.1 nN was found at pH 12 and then the interaction became completely repulsive at pH 13.2. There are a number of possible interaction forces acting between the two surfaces. These include surface tension, van der Waals, hydrogen bonding, electrostatic double-layer force, and hydrophobic interaction. The sum of interaction forces between the BSA coated tip and the MMT surface in air was found to be 24.5 ± 3.2 nN. This was nearly three times the maximum adhesion force in solution (at pH 4.6). The higher value in air was mainly due to the liquid–air interfacial tension arising from the formation of a thin layer of water adsorbed on the tip and the sample surface. The significant reduction of measured force in the liquid environment suggests that these surface tension forces are eliminated when measuring under fluid environment, which is in good agreement with the literature [34]. Thus, surface tension force is not a component part of the adhesion of protein to MMT. In the current study, the zeta potential measurements in the previous section showed the amphoteric nature of the BSA protein. The isoelectric point of the BSA protein was observed at the
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Fig. 8. 2D and 3D AFM images of MMT surfaces at a scan size of (a) 100 m and (b) 5 m.
pH 5.0 approximately. Below this point of zero-charged, BSA was positively charged whereas it was negatively charged with the pH above pH 5.0. However, the MMT surface was negatively charged throughout the range of studied pH from 3.3 to 13.2. Thus, the nature of the interaction force between MMT and BSA depended mainly on the surface charge of BSA protein. When the pH was below the isoelectric point of the BSA protein, a high attractive force was observed between negatively charged MMT surface and positively charged BSA protein. The attraction force observed at the pH of 3.3, 3.8 and 4.6 were not short range van der Waals force for such a large distance of 50 nm (Fig. 11b), but were undoubtedly long range electrostatic forces. The increase of the positively surface charge of the BSA protein with pH values from 3.3 to 4.6 (Fig. 7) corresponded to the increase of the attractive forces as observed in Fig. 12.
Fig. 9. Force–distance curve measured at pH 3.8 with (a) a clean, bare silicon nitride tip and (b) with a tip coated with BSA.
A maximum adhesion force was observed at the pH of 4.6 which which was close to the i.e.p. of BSA (5.0). The maximum forces observed close to protein i.e.p. in this study are consistent with other observations from the literature [2,35,36]. BSA protein comprises long chains of amino acid and has a low internal stability. Parts of these amino acids are hydrophobic and parts are hydrophilic. It has been reported that, near the isoelectric point, Coulomb attraction between oppositely charged residues in the protein promote the rearrangement of the molecules making the protein structure more compact [2,35–37]. This enhances the interactions between two hydrophobic molecules and thus reducing their undesirable interaction with water molecules. The soluble proteins then had a highly ordered protein structure with hydrophobic core and charged and polar side chains situated on the solvent-exposed surface where they interacted with surrounding molecules. Minimising the number of hydrophobic side
Fig. 10. Comparison of the adhesion force between the original tips (blue)/BSA coated tips (red) and MMT surfaces at specific pH values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Fig. 11. Force–distance curves obtained at different pH values between BSA coated tip and MMT surface with the absence of ethanol.
chains exposed to water resulted in a large entropy increase and a relatively small enthalpy effect [2]. Thus, Gibbs free energy Gads = Hads − TSads is more negative (where H, S, and T are the enthalpy, entropy and the absolute temperature respectively). In the current study this is demonstrated by a maximum adherence of BSA protein on the hydrophilic MMT surface at the pH near the isoelectric point. Thus, the maximum adhesion force observed at this particular pH was not electrostatic in nature but tied to the rearrangement of the protein structure. At pH above the isolectric point of approximately 5.0, both the MMT surface and BSA protein bear the same negative charge. The increase in surface negative charge of BSA with increasing pH values would cause an increase in repulsion of electronic double layers surrounding the BSA protein, and thus increase the potential energy barrier which hinders the approach to the MMT and then prevents attachment. Thus, a repulsion force between the MMT surface and BSA molecules was expected. However, the adhesion forces between the BSA and MMT surface at the pH range 5.6–10 took place as shown in Figs. 11b and 12. This suggested that the adhesion force between BSA and MMT surface was not solely due to electrostatic effects. BSA protein structure has a low internal stability. At a pH above the isoelectric point, the charge-charge interactions arose which easily affected the low internal stability structure of BSA protein.
This resulted in the expanding of the protein structure. As such, the hydrophobic amino acids of BSA protein come into contact with the siloxane of MMT surface and adhere to the MMT surface by hydrophobic interactions [13], resulting in the adhesion between the BSA and MMT surface even though charges of both BSA and MMT were the same. Thus, the adherence of BSA onto the MMT surfaces was driven by different types of forces: electrostatic, hydrophobic and structural rearrangement in the BSA protein.
Fig. 12. Adhesion force between the BSA coated tip and MMT surface at different pH values with the absence of ethanol.
Fig. 13. Force–distance curves obtained at different ethanol content between BSA coated tip and MMT surface at the pH 3.8.
3.6.1. Effect of ethanol concentration on the adhesion force Measurements were also performed to examine the influence of ethanol concentration on the interaction between the BSA and the MMT surfaces. It has been reported that most grape wines have an ethanol concentration of about 12% (by volume) [9]. Therefore, in this study, ethanol concentration ranging from 0 to 20% was examined. Fig. 13 shows a series of force–distance curves between
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hydrophobic interactions. The dependence of measured forces as a function of pH and ethanol concentrations indicates that the MMT can be used as a cation exchange material.
References
Fig. 14. Adhesion force between the BSA coated tips and MMT surfaces as a function of ethanol content at the pH of 3.8.
BSA coated tip and MMT surface obtained at different ethanol content. In these measurements, the pH was maintained at a constant value of 3.8. Strong attraction forces were observed in all cases. A large distance from 50 nm to 100 nm approximate indicated that attraction force was not due to van der Waals force. The corresponding forces are illustrated in Fig. 14. It was observed that there were no significant changes in adhesion force with the increase of ethanol concentration from 0% to 12%. However, the adhesion force increased sharply when the ethanol concentration increased from 16% to 20%. Zeta potential measurement in Section 3.3 confirmed that the negative surface charge of MMT unchanged. However, positive surface charge of BSA increased with the ethanol content, thus increasing the electrostatic force between the negatively charged MMT surface and positively charged BSA. It was also observed that MMT particles have a strong affinity with water, as shown in Fig. 5. In water, MMT swells significantly enabling intercalation of protein molecules. Sun et al. [38] and Archarandio et al. [28] proposed that the presence of ethanol enhances the swelling of MMT and separates the silicate layers of the MMT particles. This results in the expansion and broadening of the MMT channel which enhances the approach of larger proteins to adsorb on the MMT surface and facilitate adherence. That, at high concentration of ethanol, the binding capacity of MMT is improved for larger protein that otherwise would not fit. An increase of adhesion force between MMT surface and BSA protein as a function of ethanol content in this is consistent with a study of Achaerandio et al. [28] who found that adsorption capacity of BSA on the bentonite surface tended to increase with increasing ethanol concentrations. 4. Conclusions The adsorption of bovine serum albumin protein on the MMT surfaces was systematically examined using an AFM. The tip was modified by incorporation of thin films of BSA. The force curves were measured at different conditions of solution pH and ethanol content. It was observed that interaction between the BSA protein and MMT surfaces were dependent on solution pH and ethanol content where pH had a stronger effect on the adhesion force. Highest adhesion force occurred at the pH of 4.6, which is near to the isoelectric point of proteins. This maximum adhesion force at this point related to the structure rearrangement in the protein to form a more compact structure. The adhesion forces decreased linearly with both an increase and a decrease of the solution pH. At pH values lower than the isoelectric point of BSA protein, the adsorption of BSA molecules onto the MMT surface followed the behaviour of electrostatic occurring between the positively charged BSA and negatively charged MMT. At pH values higher than the isoelectric point, the adhesion force was found to be dominated by
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