Colloids and Surfaces B: Biointerfaces 116 (2014) 383–388
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A comparative PM-IRRAS and ellipsometry study of the adsorptive behaviour of bovine serum albumin on a gold surface Mahdi Dargahi, Sasha Omanovic ∗ Department of Chemical Engineering, McGill University, 3610 University Street, Montréal, Québec H3A 0C5, Canada
a r t i c l e
i n f o
Article history: Received 18 January 2013 Received in revised form 12 December 2013 Accepted 16 December 2013 Available online 24 December 2013 Keywords: Serum albumin Protein adsorption PM-IRRAS/infrared spectroscopy Ellipsometry
a b s t r a c t Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and ellipsometry were used ex situ to investigate adsorption of bovine serum albumin (BSA) on a gold surface, in terms of the adsorption equilibrium and kinetics. The aim of the work was to examine if the two different techniques give similar/complementary results under the same experimental conditions employing the same protein/surface system, and thus validate the use of the techniques for the investigation of protein/surface interactions under the applied experimental conditions, in general. It was found that the adsorption of BSA on gold follows Type I isotherm, which can be described by the Freundlich isotherm. The initial BSA adsorption kinetics was found to be very fast, and the results were modelled using a two-step kinetic model. The first step represents reversible BSA adsorption that yields BSA adsorbed in a native configuration ( 1 ) that is not thermodynamically stable. The second step represents the irreversible transformation of this protein configuration into a thermodynamically stable surface-adsorbed configuration ( 2 ). It was found that the 2 / 1 ratio increased with time. Finally, the comparison of the results obtained by the two techniques showed a very good agreement. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The key role of spontaneous adsorption of proteins on various surfaces is well appreciated [1] and it is of primary importance in many practical applications such as the design of biocompatible medical materials (catheters, heart valves, stents, dental and bone implants, etc.) [2], drug carriers [3], biosensors [4], and in the food processing and pharmaceutical industry [5,6], among many others. A wide variety of experimental techniques have been used for the investigation of protein adsorption at various interfaces: X-ray photoemission electron microscopy (X-PEEM) [7], enzyme-linked immunosorbent assay (ELISA) [8,9], radiolabelling [10,11], ellipsometry [12–15], total internal reflection fluorescence (TIRF) [16,17], optical waveguide lightmode spectroscopy (OWLS) [18,19], surface plasmon resonance (SPR) [20,21], neutron reflectivity (NR) [22,23], electrochemical quartz crystal nanobalance (EQCN) [24–26], quartz crystal microbalance (QCM) [8,27–29], electrochemical impedance spectroscopy (EIS) [25,26,30], X-ray photoelectron spectroscopy (XPS) [31–33], time-of-flight static secondary ion mass spectroscopy (TOF-SIMS) [34,35], and IR-based techniques such as attenuated total reflection Fourier transform
IR (ATR-FTIR) [36,37]. Besides electrochemical techniques, our laboratory has also previously used polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) to study the interaction of bovine serum albumin (BSA) and fibrinogen with a 316LVM stainless steel surface [38,39]. Here we are extending our research further, to examine the interaction of BSA with a gold surface using two different experimental techniques, PM-IRRAS and ellipsometry, in terms of the adsorption equilibrium and kinetics. The aim of the work was to examine whether the two different techniques give similar/complementary results under the same experimental conditions employing the same protein/surface system, and thus validate the use of the techniques for the investigation of protein/surface interactions under the applied experimental conditions, in general. A gold surface was selected for this study due to its good stability and chemical inertness. Bovine serum albumin was selected as a model protein in this study, because it is the major constituent of blood serum [40]. 2. Materials and methods 2.1. Substrate
∗ Corresponding author. Tel.: +1 514 398 4273; fax: +1 514 398 6678. E-mail address: [email protected] (S. Omanovic). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.028
A gold-coated glass slide (2.54 cm × 2.54 cm, 5 nm chromium and 100 nm gold sputtered on one side of the glass slide – EMF Corporation) was used as the adsorption substrate. The slide was
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cleaned by sonication in acetone for 30 min and then rinsed successively with deionised water (Nanopure, resistivity 18.2 M cm), acetone, and ethanol. This was followed by drying with argon. A surface RMS roughness value measured by atomic force microscopy, after the surface preparation, was 1.2 ± 0.1 nm.
Bovine serum albumin, BSA (≥99%, Sigma–Aldrich, Cat. No. A0281) was used as received. The protein was dissolved in a phosphate-buffered solution pH 7.4. This solution was prepared by first dissolving 0.68 g of anhydrous, monobasic potassium phosphate (KH2 PO4 , 99.7%, Sigma–Aldrich, Cat. No. P5379) in 39.1 mL of 0.1 M sodium hydroxide (NaOH, prepared from standard 5 M concentrate; Fisher Scientific), followed by dilution with deionised water (Nanopure, resistivity of 18.2 M cm) to yield a final volume of 100 mL. For BSA equilibrium adsorption experiments, the adsorption was performed by immersing the gold slides in a solution with a desired protein concentration (0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15, 0.20, 0.30 and 0.40 g L−1 ) for 3 h, at which point the slides were quickly removed from the protein-containing solution and thoroughly rinsed with abundant deionised water and dried with argon. BSA adsorption kinetic experiments were performed by immersing gold slides in a solution containing a desired protein concentration (0.01, 0.06 and 0.20 g L−1 ) for a pre-determined length of time and then quickly removed and rinsed with abundant deionised water. Following protein adsorption and rinsing, the gold slides were dried using a gentle stream of argon and mounted first in the PMIRRAS sample holder for analysis, which was followed by analysis in an ellipsometer. All the experiments were performed at 22 ± 1 ◦ C and repeated at least three times. The corresponding standard deviation was calculated and presented on graphs as error bars, or explicitly indicated in tables. 2.3. Ex situ ellipsometry measurements Ellipsometric characterization of a protein layer adsorbed on a gold substrate was performed ex situ employing an imaging singlewave ellipsometer fitted with a He–Ne laser (658 nm). The angel of incidence was 45◦ . Protein layer thickness measurements were carried out under a four-zone nulling condition to correct the derivations caused by a possible instrumental misalignment. The results showed that there is an increase in the amplitude ratio, , and a decrease in phase difference, , which proved the presence of a surface-adsorbed protein layer. Then, the average thickness was calculated using the EP4Model v.1.0.0 software for a threephase model, taking the optical constant real part of refractive index (n) for the adsorbed ‘dry’ BSA layer as 1.53 [41,42]. The extinction coefficient (k) was estimated to be nearly zero [43]. The protein surface concentration, (mg m−2 ), was calculated employing the following equation [44]: =ı
M n2 − 1 Ar
n2 + 2
(1)
where ı (nm) is the ellipsometry-measured BSA layer thickness, M the molecular weight (g mol−1 ), and Ar is the molar refractivity (cm3 mol−1 ). For BSA, the ratio M/Ar = 4.12 g cm−3 . 2.4. Ex situ PM-IRRAS measurements PM-IRRAS was used to ex situ characterize a BSA layer adsorbed on a gold substrate. A liquid-nitrogen-cooled MCT detector was used in all experiments. The wavelength setting on a polarization
0.001
(d) (c)
R/R
2.2. Chemicals
Amide I
Amide II
(b)
(a)
1750
1650 Wavenumber / cm
1550
1450
-1
Fig. 1. PM-IRRAS spectra of BSA on a gold surface substrate recorded after 3 h of immersion of the substrate in a phosphate buffer solution containing various BSA concentrations: (a) 0, (b) 0.02, (c) 0.10 and (d) 0.40 g L−1 .
modulator was fixed at 1600 cm−1 . All samples were scanned for 500 scans, with a 3 cm−1 resolution and an aperture setting of 5 mm. The incident beam angle used in all experiments was 85◦ . 3. Results and discussion 3.1. BSA adsorption under equilibrium conditions PM-IRRAS was first used in this study to investigate the equilibrium of BSA adsorption on gold. When studying surface-adsorbed proteins using PM-IRRAS, the spectral region of interest includes amide bands, which are characteristic of all proteins. More particularly, those are Amide I (ca. 1600–1700 cm−1 ) and Amide II bands (ca. 1480–1580 cm−1 ). In this study, the analysis was focused on Amide I band due to its strong, well-defined signal, as well as due to the possibility of later extracting information on the secondary structure of adsorbed protein [38,39,45]. Fig. 1 shows a set of selected PM-IRRAS spectra of a gold surface following a three-hour immersion in phosphate buffer solutions containing increasing BSA concentrations. It should be noted that three hours of immersion was sufficient to reach equilibrium, since the kinetics experiments presented later in the text (Fig. 3) confirmed that the adsorption equilibrium was reached within one hour of immersion. In Fig. 1, the noticeable increase in the intensity of Amide I band indicates progressively greater surface concentrations of the protein. However, to better visualize this trend, the area under each Amide I band (i.e. its integrated intensity, AAmide I ) was determined and presented in Fig. 2(a) as a function of the bulk BSA concentration. Assuming that the integrated intensity is proportional to the protein surface concentration, the plot evidences that the BSA surface concentration increases with an increase in bulk BSA concentration, and then levels off into a plateau (constant surface concentration) at a bulk BSA concentration of ca. 0.3 g L−1 . The plot in Fig. 2(a) resembles a shape of a unimodal adsorption isotherm (Type I isotherm), and is in agreement with results on the adsorption of BSA on a range of surfaces [38,46–48]. The equilibrium of BSA adsorption on a gold surface was also studied by ellipsometry. The resulting plot is shown in Fig. 2(b). Similarly to Fig. 2(a), the plot resembles a shape of a unimodal (Type I) isotherm. The average surface BSA layer thickness (ı) increases with an increase in protein bulk concentration and reaches an average thickness of 4.3 ± 0.1 nm at the highest bulk protein concentration. However, given that the technique measures the average protein thickness, which depends on both the number
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385
or in a linear form: 0.20
log() = log(KF ) + n log([BSA]) AAmide I / cm
-1
0.15
where represents the fractional surface coverage by BSA, calculated either as = A/Amax (where = A/Amax (cm−1 ) represents an integrated intensity measured at a particular BSA bulk solution concentration and Amax is the maximum integrated intensity corresponding to a saturated BSA surface concentration), or as = ıi /ımax (where ıi (nm) represents an average BSA layer thickness at a particular BSA bulk solution concentration, and ımax is its maximum value at a saturated BSA surface concentration), KF (Ln mol−n ) is the Freundlich constant, n is a dimensionless parameter that varies between 0 and 1, and [BSA] (mol L−1 ) represents the bulk solution equilibrium BSA concentration. It should be noted that the Freundlich isotherm is a simple power equation, commonly used in engineering to scale up adsorption-based processes and also for comparative purposes. For the Freundlich isotherm to be considered valid for the system under the investigation, a plot of log() vs. log([BSA]) should yield a straight line with an intercept log(KF ) and slope n. The data in the inset to Fig. 2 demonstrate that a linear behaviour was obtained for the PM-IRRAS and ellipsometry adsorption data when presented in accordance with Eq. (3), confirming that the Freundlich isotherm can successfully describe the adsorption of BSA onto the gold surface under the experiment conditions applied in this work. The corresponding isotherm parameters are n = 0.139 ± 0.007 and KF = 113 ± 3 Ln mol−n .
0.10
0.05 (a) 0.00 (b)
4.0
5
2.0
3
1.9
2
-2
log( )
2.0
/ mg m
/ nm
4
2.1
3.0
1.8
1.0
1
1.7 -2.0
-1.5
-1.0
-0.5
0.0
log([BSA])
0.0
0 0.0
0.1
0.2
0.3
[BSA] / g L
0.4
0.5
-1
Fig. 2. Dependence of (a) the Amide I integrated intensity of adsorbed BSA and (b) average surface-adsorbed BSA layer thickness and BSA surface concentration on the BSA concentration in the bulk solution. The data were obtained respectively from PM-IRRAS spectra and ellipsometry measurements of BSA on a gold surface substrate recorded after 3 h of immersion of the substrate in a phosphate buffer solution containing BSA concentrations indicated on the plot and subsequent drying. The lines represent the corresponding Freundlich isotherm. The symbols are mean values of at least three measurements, while the error bars represent the corresponding standard deviation. Inset: Experimental data (symbols) from Fig. 2a and 2b presented in a linearized Freundlich isotherm form, Eq. (3). () PM-IRRAS and () ellipsometry measurements. The line represents the best fit employing Eq. (3).
of molecules on the surface and their actual dimension, the increase in Fig. 2(b) actually resembles the increase in BSA surface concentration, similarly to the behaviour in Fig. 2(a). The corresponding surface concentration values were calculated using Eq. (1), and are presented on the right axis of Fig. 2(b). Taking into account that that the highest average thickness of a BSA layer adsorbed on the gold surface was determined to be 4.3 ± 0.1 nm (Fig. 2(b)) and that this BSA layer is “dry” (argon-dried BSA surface layer), and then considering the dimensions of a BSA crystal [49] one can conclude that BSA reaches a monolayer surface configuration at the highest BSA bulk solution concentrations, and that the BSA molecule lays flat on the gold surface, under the experimental conditions employed (the BSA unit crystal thickness 4.5 nm [49]). Similar results were previously reported in the literature for adsorption of BSA on solid substrates when measured by ex situ ellipsometry [44,50–52]. As previously mentioned, the data in Fig. 2 display a shape of Type I isotherm. Consequently, several corresponding isotherms were tested. The best results were obtained when the Langmuir and Freundlich isotherms were applied. However, the former was dismissed due to the violation of the corresponding isotherm assumptions (reversible adsorption, no lateral interactions of BSA molecules adsorbed on the gold surface, equal energy of all Au surface sites). Hence, the Freundlich isotherm was selected for the further treatment of the data [53–55]: = KF [BSA]n
(3)
(2)
3.2. Kinetics of BSA adsorption It is often believed that the first few hours following the implantation of a medical device into the human body are very crucial in determining the host response to the implant. Since it is generally accepted that the initial adsorption of proteins from the biological fluids significantly influence the implant’s biocompatibility [56], it is of great interest to investigate the kinetics of protein adsorption in order to elucidate the time scale within which the biomaterial surface will be covered to varying degrees by proteins. Similarly, in cases where protein adsorption induces surface fouling of process equipment, such as in heat exchangers and filtration membranes in the dairy and in the pharmaceutical industry, it is of importance to have information on the kinetics of protein adsorption and consequently plan the cleaning steps. The suitability of PM-IRRAS and ellipsometry in studying the kinetics of protein adsorption was further investigated using BSA and gold as a model adsorption system. Three different concentrations of BSA in the bulk solution were used: 0.01, 0.06, and 0.20 g L−1 . From the equilibrium adsorption measurements presented in Fig. 2, it can be seen that the BSA concentrations of 0.01 and 0.06 g L−1 correspond to the rising portion of the adsorption isotherm, whereas the high concentration (0.20 g L−1 ) corresponds to the onset of the adsorption plateau. The same experimental procedure used in the equilibrium experiments was applied for the kinetic experiments, with the only difference being that independent variable was the immersion time of the gold substrate in the BSA solution (as opposed to the bulk protein concentration as an independent variable). Fig. 3a shows the dependence of integrated intensity of Amide I peak of surface-adsorbed BSA on time, while Fig. 3b shows the time dependence of average BSA surface layer thickness obtained by ellipsometry, respectively. The two plots demonstrate that at a constant BSA concentration in the bulk solution, the surface concentration of adsorbed BSA increases very rapidly immediately upon initiation of the adsorption process, and then gradually levels off into a plateau. The adsorption equilibrium is reached after
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0.20
AAmide I / cm-1
0.15
0.10
0.05 (a) 0.00 5.0
/ nm
4.0
Fig. 4. The kinetics of BSA adsorption on gold presented in terms of the dependence of relative surface coverage by BSA on adsorption time. The data were obtained by ellipsometry, in a phosphate buffer solution containing 0.20 g L−1 BSA. Symbols represent the experimental data and the solid line represents simulated data fitted using the adsorption kinetic model described by Eqs. (4)–(6) and the schematic representation in the inset to the figure. The symbols are mean values of at least three measurements. The corresponding standard deviation values, presented by error bars, are not visible since the largest value is 3.84. In the schematic representation of the BSA adsorption mechanism 1 represents surface coverage by BSA adsorbed in a thermodynamically unstable surface configuration. This BSA can either desorb back into the solution, or transform on the surface into a thermodynamically stable configuration, 2 .
3.0 2.0 1.0 (b) 0.0 0
20
40
60
80
100
120
140
Time / minute Fig. 3. (a) Amide I integrated intensity and (b) average surface-adsorbed BSA layer thickness obtained respectively from PM-IRRAS and ellipsometry measurements of BSA adsorbed on a gold surface at different times of adsorption. BSA bulk solution concentration was: () 0.01, () 0.06, and () 0.20 g L−1 . The symbols are mean values of at least three measurements, while the error bars represent the corresponding standard deviation.
ca. 40–60 min of adsorption, depending on the bulk solution BSA concentration. Taking into account the equilibrium results in Fig. 2(a) and (b), the kinetic data from Fig. 3a and b were converted into the corresponding time-dependent surface coverage data, and a representative curve is presented in Fig. 4 (main plot, symbols). In order to model the kinetic data, a one-step kinetic model was first used. However, this model was found not to satisfactorily describe the kinetics of BSA adsorption on gold. Therefore, a two-step kinetic model presented schematically in the inset to Fig. 4 was employed. In this model, the first adsorption step is reversible, and the protein’s surface conformation is assumed to resemble that one in the bulk solution, i.e. the native conformation. However, this surface conformation is not thermodynamically stable, and the protein molecule either desorbs (Step 1, reverse) or irreversibly adopts a more thermodynamically favourable surface conformation (Step 2) [14,57]. Now, expressing the surface concentration of BSA in terms of its relative surface coverage, , the two-step BSA adsorption kinetic model can be formulated as:
( 2 ) configuration, ka (M−1 min−1 ) is the adsorption constant, kd (min−1 ) is the desorption constant, and kf (min−1 ) is the transformation (surface rearrangement) constant. The experimental data (Fig. 4, symbols) were then fitted using the kinetic model in Eqs. (4)–(6), and the result is presented in Fig. 4 (main plot) as a solid line. A good agreement between the experimental and simulated data was obtained for the presented and the other two BSA bulk concentrations and for both techniques (six sets of data, three replicates). This indicates the applicability of the proposed model in describing the kinetics of BSA adsorption on gold under the experimental conditions employed. The corresponding kinetics constants are presented in Table 1. The comparative kinetic constant values obtained using the two techniques (Table 1) are very close. This again validates the approach used in treating the experimental data and the techniques for the investigation of adsorption of BSA on gold under the experimental conditions employed. The kinetic constant values in the table are also very close to those reported in literature, for the adsorption of human serum albumin on a biofunctionalized silicon surface [13]. Further, the table shows that the adsorption kinetic constant depends on the BSA bulk solution concentration in a regular Table 1 Kinetic parameters for the adsorption of BSA onto gold for various experimental conditions. The parameters were determined by fitting the experimental data obtained from PM-IRRAS and ellipsometry experiments. The values listed in the table are mean values and the corresponding standard deviation, obtained from at least three experiments. BSA (g L−1 )
d1 = ka [BSA](1 − 1 − 2 ) − (kd + kf )1 dt
(4)
d2 = kf 1 st
(5)
d d1 d2 = + = ka [BSA](1 − ) − kd 1 dt dt dt
(6)
where = 1 + 2 and 0 ≤ ≤ 1 is the fraction of surface covered by BSA in both the thermodynamically unstable ( 1 ) and stable
Ellipsometry ka × 10−5 (M−1 min−1 )
kd × 101 (min−1 )
kf × 102 (min−1 )
0.01 0.06 0.20
8.30 ± 0.00 3.66 ± 0.00 1.57 ± 0.00
1.39 ± 0.03 2.06 ± 0.04 0.74 ± 0.06
1.00 ± 0.24 1.08 ± 0.10 0.80 ± 0.05
PM-IRRAS 0.01 0.06 0.20
8.30 ± 0.00 3.66 ± 0.00 1.57 ± 0.00
1.42 ± 0.03 2.02 ± 0.02 0.82 ± 0.06
1.00 ± 0.24 1.05 ± 0.07 0.82 ± 0.06
100
1
80
0.8
1
60
387
0.6
2
/%
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1
0.4
40 2
0.2
20
0
0
0
0
10
20
30
40
50
60
10
20
70
Time / minute
manner; it decreases with an increase in BSA bulk solution concentration. This indicates that the determined adsorption constant is an apparent adsorption constant. Namely, the proposed kinetic model does not take into account intermolecular interactions of adsorbing BSA molecules with those that are already on the surface, but only the occupancy of the surface with the latter molecules. At higher BSA bulk solution concentrations, the substrate surface becomes covered by BSA faster, which increases the probability for the occurrence of the mentioned intermolecular interactions. Consequently, the apparent adsorption rate constant changes. The observed decrease in its value (Table 1) indicates that the intermolecular interactions are of a negative type, i.e. the already adsorbed BSA molecules “inhibit” further adsorption of BSA molecules from the solution. The proposed BSA adsorption kinetic model shown in the inset to Fig. 4 and Eqs. (4)–(6) shows that the substrate surface coverage by thermodynamically unstable ( 1 ) and stable ( 2 ) BSA changes with time. It would, thus, be interesting to examine the kinetics of this transformation. For that purpose, the modelled (simulated) kinetic data were deconvoluted into the two contributions ( 1 and 2 ), and presented in Fig. 5 (note that both PM-IRRAS and ellipsometry gave very similar results, as evidenced by the kinetic constant values in Table 1). The data in Fig. 5 demonstrate that the initial increase (within the first 5–10 min of adsorption) in total BSA surface coverage, , is mostly due to the formation of a thermodynamically unstable, reversibly bound BSA layer, 1 . The surface coverage with such bound BSA initially increases sharply and then reaches a maximum value, which occurs at earlier times at higher bulk solution concentrations (not shown). Then, it starts gradually decreasing. On the other hand the surface coverage with a thermodynamically stable, irreversibly bound BSA, 2 , gradually increases in the entire time interval studied. The results in Fig. 5 indicate that the transformation from the thermodynamically unstable ( 1 ) into the thermodynamically stable ( 2 ) adsorbed state/conformation of BSA is rather slow, and does not get completed within the time interval studied. A similar behaviour was previously reported in the literature for the kinetics of interaction of C-reactive protein (CRP) with a CRP antibody chemically immobilized on a gold surface [57]. Fig. 6 shows the values of the ratio of the coverage of the thermodynamically stable to the unstable surface state of BSA, for three different BSA bulk solution concentrations. The data demonstrate that with an increase in adsorption time, the relative surface ratio also increases. The trend can be approximated by a second-order polynomial function behaviour. Also, the values at a particular adsorption time are close for the three BSA bulk solution
40
50
60
70
Fig. 6. Time dependence of the ratio of surface coverage of thermodynamically stable to unstable adsorbed BSA ( 2 / 1 ) obtained at different BSA bulk solution concentrations: () 0.01, () 0.06, and () 0.20 g L−1 .
concentrations. This indicates that the BSA bulk solution concentration, i.e. the corresponding BSA surface coverage (Fig. 3) does not influence the kinetics of the BSA surface transformation (from 1 to 2 ).
3.3. Comparison of PM-IRRAS and ellipsometry results One of the major goals of this work was to investigate whether two completely different experimental techniques, PM-IRRAS and ellipsometry, could reliably be used to investigate adsorption of a protein on a solid surface, i.e. would they yield similar adsorption data/parameters. The analyses presented in the previous sections of the paper demonstrate that the two techniques indeed gave very close adsorption data, thus not only validating the suitability of their parallel/complementary use, but also the approaches used to treat the experimental data. To more closely compare the two techniques, the corresponding equilibrium BSA surface coverage values are presented in Fig. 7 (circles). It is evident that the experimental points are very close to the 45◦ line, demonstrating that the two techniques yield similar adsorption coverage values (R2 = 0.924). Further, the same conclusion could be made from the kinetic results shown in the same figure (triangles). Thus, the results in Fig. 7 demonstrate that PM-IRRAS and ellipsometry can be reliably and comparatively used to study adsorption of proteins on solid reflective surfaces, at least under the experimental conditions employed in this work.
100
by PM-IRRAS / %
Fig. 5. Time dependence of () the total surface coverage by BSA () obtained by adsorption on a gold surface immersed in a phosphate buffer solution containing 0.20 g L−1 BSA, deconvoluated into the contribution of the coverage by the thermodynamically () unstable ( 1 ), and () stable ( 2 ) BSA. The data were obtained by modelling the PM-IRRAS experimental data using Eqs. (4)–(6).
30
Time / minute
90 80 70 60 50 50
60
70
80
90
100
by Ellipsometry / % Fig. 7. BSA surface coverage values obtained by PM-IRRAS and ellipsometry illustrating the agreement between the two techniques. Data from () equilibrium and () kinetic BSA adsorption measurements are presented. The latter refers to the BSA bulk solution concentration of 0.20 g L−1 . Symbols represent experimental data, whereas the solid 45◦ line represents the equality line.
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4. Conclusions PM-IRRAS and ellipsometry were used to ex situ investigate the adsorption of bovine serum albumin (BSA) on a gold surface. The adsorption process yields a uni-modal (Type I) adsorption isotherm, which can be modelled using the Freundlich isotherm. The kinetic measurements demonstrate that the BSA adsorption process reaches equilibrium after 40–60 min, depending on the protein bulk concentration. The BSA adsorption kinetics follows a two-step model; the first step represents the reversible BSA adsorption, while the second step represents the irreversible transformation of BSA into a thermodynamically stable configuration. The results presented demonstrate that PM-IRRAS and ellipsometry show a very good general agreement, and can thus be reliably and comparatively used to study adsorption of proteins on solid reflective surfaces. Acknowledgments The authors gratefully acknowledge the financial support from the Natural Science and Engineering Research Council of Canada (NSERC) and, the Fonds de recherche du Québec – Nature et technologies (FRQ NT). References [1] C.V. Gruian, E. Simon, S. Simon, Biochim. Biophys. Acta: Proteins Proteom. 1824 (2012) 873–881. [2] M. Gagliardi, Mater. Sci. Eng. C 32 (2012) 2445–2451. [3] M. Jansch, P. Stumpf, C. Graf, E. Rühl, R.H. Müller, Int. J. Pharm. 428 (2012) 125–133. [4] Y. Masuda, T. Ohji, K. Kato, M. Ajimi, M. Bekki, S. Sonezaki, Thin Solid Films 519 (2011) 5135–5138. [5] K. Nagase, J. Kobayashi, A. Kikuchi, Y. Akiyama, H. Kanazawa, T. Okano, Biomaterials 32 (2011) 619–627. [6] A. Noisuwan, Y. Hemar, B. Wilkinson, J.E. Bronlund, Carbohydr. Polym. 84 (2011) 247–254. [7] B.O. Leung, A.P. Hitchcock, R.M. Cornelius, J.L. Brash, A. Scholl, A. Doran, J. Electron Spectrosc. Relat. Phenom. 185 (2012) 406–416. [8] L. Guicai, S. Xiaoli, Y. Ping, Z. Ansha, H. Nan, Solid State Ionics 179 (2008) 932–935. [9] Y. Yin, K. Fisher, N.J. Nosworthy, D. Bax, R.J. Clarke, D.R. McKenzie, M.M.M. Bilek, Thin Solid Films 520 (2012) 3021–3025. [10] C.C. Barrias, M.C.L. Martins, G. Almeida-Porada, M.A. Barbosa, P.L. Granja, Biomaterials 30 (2009) 307–316. [11] L.D. Unsworth, H. Sheardown, J.L. Brash, Biomaterials 26 (2005) 5927–5933. [12] T. Berlind, P. Tengvall, L. Hultman, H. Arwin, Acta Biomater. 7 (2011) 1369–1378. [13] T.M. Byrne, S. Trussler, M.A. McArthur, L.B. Lohstreter, Z. Bai, M.J. Filiaggi, J.R. Dahn, Surf. Sci. 603 (2009) 2888–2895. [14] D.K. Goyal, A. Subramanian, Thin Solid Films 518 (2010) 2186–2193. [15] M.A. McArthur, T.M. Byrne, R.J. Sanderson, G.P. Rockwell, L.B. Lohstreter, Z. Bai, M.J. Filiaggi, J.R. Dahn, Colloids Surf. B 81 (2010) 58–66. [16] J.E. Klinth, R. Larsson, P.O. Andersson, K.N. Ekdahl, Biosens. Bioelectron. 21 (2006) 1973–1980. [17] M. Lundin, U.M. Elofsson, E. Blomberg, M.W. Rutland, Colloids Surf. B 77 (2010) 1–11.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A comparative PM-IRRAS and ellipsometry study of the adsorptive behaviour of bovine serum albumin on a gold surface Mahdi Dargahi, Sasha Omanovic ∗ Department of Chemical Engineering, McGill University, 3610 University Street, Montréal, Québec H3A 0C5, Canada
a r t i c l e
i n f o
Article history: Received 18 January 2013 Received in revised form 12 December 2013 Accepted 16 December 2013 Available online 24 December 2013 Keywords: Serum albumin Protein adsorption PM-IRRAS/infrared spectroscopy Ellipsometry
a b s t r a c t Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and ellipsometry were used ex situ to investigate adsorption of bovine serum albumin (BSA) on a gold surface, in terms of the adsorption equilibrium and kinetics. The aim of the work was to examine if the two different techniques give similar/complementary results under the same experimental conditions employing the same protein/surface system, and thus validate the use of the techniques for the investigation of protein/surface interactions under the applied experimental conditions, in general. It was found that the adsorption of BSA on gold follows Type I isotherm, which can be described by the Freundlich isotherm. The initial BSA adsorption kinetics was found to be very fast, and the results were modelled using a two-step kinetic model. The first step represents reversible BSA adsorption that yields BSA adsorbed in a native configuration ( 1 ) that is not thermodynamically stable. The second step represents the irreversible transformation of this protein configuration into a thermodynamically stable surface-adsorbed configuration ( 2 ). It was found that the 2 / 1 ratio increased with time. Finally, the comparison of the results obtained by the two techniques showed a very good agreement. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The key role of spontaneous adsorption of proteins on various surfaces is well appreciated [1] and it is of primary importance in many practical applications such as the design of biocompatible medical materials (catheters, heart valves, stents, dental and bone implants, etc.) [2], drug carriers [3], biosensors [4], and in the food processing and pharmaceutical industry [5,6], among many others. A wide variety of experimental techniques have been used for the investigation of protein adsorption at various interfaces: X-ray photoemission electron microscopy (X-PEEM) [7], enzyme-linked immunosorbent assay (ELISA) [8,9], radiolabelling [10,11], ellipsometry [12–15], total internal reflection fluorescence (TIRF) [16,17], optical waveguide lightmode spectroscopy (OWLS) [18,19], surface plasmon resonance (SPR) [20,21], neutron reflectivity (NR) [22,23], electrochemical quartz crystal nanobalance (EQCN) [24–26], quartz crystal microbalance (QCM) [8,27–29], electrochemical impedance spectroscopy (EIS) [25,26,30], X-ray photoelectron spectroscopy (XPS) [31–33], time-of-flight static secondary ion mass spectroscopy (TOF-SIMS) [34,35], and IR-based techniques such as attenuated total reflection Fourier transform
IR (ATR-FTIR) [36,37]. Besides electrochemical techniques, our laboratory has also previously used polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) to study the interaction of bovine serum albumin (BSA) and fibrinogen with a 316LVM stainless steel surface [38,39]. Here we are extending our research further, to examine the interaction of BSA with a gold surface using two different experimental techniques, PM-IRRAS and ellipsometry, in terms of the adsorption equilibrium and kinetics. The aim of the work was to examine whether the two different techniques give similar/complementary results under the same experimental conditions employing the same protein/surface system, and thus validate the use of the techniques for the investigation of protein/surface interactions under the applied experimental conditions, in general. A gold surface was selected for this study due to its good stability and chemical inertness. Bovine serum albumin was selected as a model protein in this study, because it is the major constituent of blood serum [40]. 2. Materials and methods 2.1. Substrate
∗ Corresponding author. Tel.: +1 514 398 4273; fax: +1 514 398 6678. E-mail address: [email protected] (S. Omanovic). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.028
A gold-coated glass slide (2.54 cm × 2.54 cm, 5 nm chromium and 100 nm gold sputtered on one side of the glass slide – EMF Corporation) was used as the adsorption substrate. The slide was
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cleaned by sonication in acetone for 30 min and then rinsed successively with deionised water (Nanopure, resistivity 18.2 M cm), acetone, and ethanol. This was followed by drying with argon. A surface RMS roughness value measured by atomic force microscopy, after the surface preparation, was 1.2 ± 0.1 nm.
Bovine serum albumin, BSA (≥99%, Sigma–Aldrich, Cat. No. A0281) was used as received. The protein was dissolved in a phosphate-buffered solution pH 7.4. This solution was prepared by first dissolving 0.68 g of anhydrous, monobasic potassium phosphate (KH2 PO4 , 99.7%, Sigma–Aldrich, Cat. No. P5379) in 39.1 mL of 0.1 M sodium hydroxide (NaOH, prepared from standard 5 M concentrate; Fisher Scientific), followed by dilution with deionised water (Nanopure, resistivity of 18.2 M cm) to yield a final volume of 100 mL. For BSA equilibrium adsorption experiments, the adsorption was performed by immersing the gold slides in a solution with a desired protein concentration (0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15, 0.20, 0.30 and 0.40 g L−1 ) for 3 h, at which point the slides were quickly removed from the protein-containing solution and thoroughly rinsed with abundant deionised water and dried with argon. BSA adsorption kinetic experiments were performed by immersing gold slides in a solution containing a desired protein concentration (0.01, 0.06 and 0.20 g L−1 ) for a pre-determined length of time and then quickly removed and rinsed with abundant deionised water. Following protein adsorption and rinsing, the gold slides were dried using a gentle stream of argon and mounted first in the PMIRRAS sample holder for analysis, which was followed by analysis in an ellipsometer. All the experiments were performed at 22 ± 1 ◦ C and repeated at least three times. The corresponding standard deviation was calculated and presented on graphs as error bars, or explicitly indicated in tables. 2.3. Ex situ ellipsometry measurements Ellipsometric characterization of a protein layer adsorbed on a gold substrate was performed ex situ employing an imaging singlewave ellipsometer fitted with a He–Ne laser (658 nm). The angel of incidence was 45◦ . Protein layer thickness measurements were carried out under a four-zone nulling condition to correct the derivations caused by a possible instrumental misalignment. The results showed that there is an increase in the amplitude ratio, , and a decrease in phase difference, , which proved the presence of a surface-adsorbed protein layer. Then, the average thickness was calculated using the EP4Model v.1.0.0 software for a threephase model, taking the optical constant real part of refractive index (n) for the adsorbed ‘dry’ BSA layer as 1.53 [41,42]. The extinction coefficient (k) was estimated to be nearly zero [43]. The protein surface concentration, (mg m−2 ), was calculated employing the following equation [44]: =ı
M n2 − 1 Ar
n2 + 2
(1)
where ı (nm) is the ellipsometry-measured BSA layer thickness, M the molecular weight (g mol−1 ), and Ar is the molar refractivity (cm3 mol−1 ). For BSA, the ratio M/Ar = 4.12 g cm−3 . 2.4. Ex situ PM-IRRAS measurements PM-IRRAS was used to ex situ characterize a BSA layer adsorbed on a gold substrate. A liquid-nitrogen-cooled MCT detector was used in all experiments. The wavelength setting on a polarization
0.001
(d) (c)
R/R
2.2. Chemicals
Amide I
Amide II
(b)
(a)
1750
1650 Wavenumber / cm
1550
1450
-1
Fig. 1. PM-IRRAS spectra of BSA on a gold surface substrate recorded after 3 h of immersion of the substrate in a phosphate buffer solution containing various BSA concentrations: (a) 0, (b) 0.02, (c) 0.10 and (d) 0.40 g L−1 .
modulator was fixed at 1600 cm−1 . All samples were scanned for 500 scans, with a 3 cm−1 resolution and an aperture setting of 5 mm. The incident beam angle used in all experiments was 85◦ . 3. Results and discussion 3.1. BSA adsorption under equilibrium conditions PM-IRRAS was first used in this study to investigate the equilibrium of BSA adsorption on gold. When studying surface-adsorbed proteins using PM-IRRAS, the spectral region of interest includes amide bands, which are characteristic of all proteins. More particularly, those are Amide I (ca. 1600–1700 cm−1 ) and Amide II bands (ca. 1480–1580 cm−1 ). In this study, the analysis was focused on Amide I band due to its strong, well-defined signal, as well as due to the possibility of later extracting information on the secondary structure of adsorbed protein [38,39,45]. Fig. 1 shows a set of selected PM-IRRAS spectra of a gold surface following a three-hour immersion in phosphate buffer solutions containing increasing BSA concentrations. It should be noted that three hours of immersion was sufficient to reach equilibrium, since the kinetics experiments presented later in the text (Fig. 3) confirmed that the adsorption equilibrium was reached within one hour of immersion. In Fig. 1, the noticeable increase in the intensity of Amide I band indicates progressively greater surface concentrations of the protein. However, to better visualize this trend, the area under each Amide I band (i.e. its integrated intensity, AAmide I ) was determined and presented in Fig. 2(a) as a function of the bulk BSA concentration. Assuming that the integrated intensity is proportional to the protein surface concentration, the plot evidences that the BSA surface concentration increases with an increase in bulk BSA concentration, and then levels off into a plateau (constant surface concentration) at a bulk BSA concentration of ca. 0.3 g L−1 . The plot in Fig. 2(a) resembles a shape of a unimodal adsorption isotherm (Type I isotherm), and is in agreement with results on the adsorption of BSA on a range of surfaces [38,46–48]. The equilibrium of BSA adsorption on a gold surface was also studied by ellipsometry. The resulting plot is shown in Fig. 2(b). Similarly to Fig. 2(a), the plot resembles a shape of a unimodal (Type I) isotherm. The average surface BSA layer thickness (ı) increases with an increase in protein bulk concentration and reaches an average thickness of 4.3 ± 0.1 nm at the highest bulk protein concentration. However, given that the technique measures the average protein thickness, which depends on both the number
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385
or in a linear form: 0.20
log() = log(KF ) + n log([BSA]) AAmide I / cm
-1
0.15
where represents the fractional surface coverage by BSA, calculated either as = A/Amax (where = A/Amax (cm−1 ) represents an integrated intensity measured at a particular BSA bulk solution concentration and Amax is the maximum integrated intensity corresponding to a saturated BSA surface concentration), or as = ıi /ımax (where ıi (nm) represents an average BSA layer thickness at a particular BSA bulk solution concentration, and ımax is its maximum value at a saturated BSA surface concentration), KF (Ln mol−n ) is the Freundlich constant, n is a dimensionless parameter that varies between 0 and 1, and [BSA] (mol L−1 ) represents the bulk solution equilibrium BSA concentration. It should be noted that the Freundlich isotherm is a simple power equation, commonly used in engineering to scale up adsorption-based processes and also for comparative purposes. For the Freundlich isotherm to be considered valid for the system under the investigation, a plot of log() vs. log([BSA]) should yield a straight line with an intercept log(KF ) and slope n. The data in the inset to Fig. 2 demonstrate that a linear behaviour was obtained for the PM-IRRAS and ellipsometry adsorption data when presented in accordance with Eq. (3), confirming that the Freundlich isotherm can successfully describe the adsorption of BSA onto the gold surface under the experiment conditions applied in this work. The corresponding isotherm parameters are n = 0.139 ± 0.007 and KF = 113 ± 3 Ln mol−n .
0.10
0.05 (a) 0.00 (b)
4.0
5
2.0
3
1.9
2
-2
log( )
2.0
/ mg m
/ nm
4
2.1
3.0
1.8
1.0
1
1.7 -2.0
-1.5
-1.0
-0.5
0.0
log([BSA])
0.0
0 0.0
0.1
0.2
0.3
[BSA] / g L
0.4
0.5
-1
Fig. 2. Dependence of (a) the Amide I integrated intensity of adsorbed BSA and (b) average surface-adsorbed BSA layer thickness and BSA surface concentration on the BSA concentration in the bulk solution. The data were obtained respectively from PM-IRRAS spectra and ellipsometry measurements of BSA on a gold surface substrate recorded after 3 h of immersion of the substrate in a phosphate buffer solution containing BSA concentrations indicated on the plot and subsequent drying. The lines represent the corresponding Freundlich isotherm. The symbols are mean values of at least three measurements, while the error bars represent the corresponding standard deviation. Inset: Experimental data (symbols) from Fig. 2a and 2b presented in a linearized Freundlich isotherm form, Eq. (3). () PM-IRRAS and () ellipsometry measurements. The line represents the best fit employing Eq. (3).
of molecules on the surface and their actual dimension, the increase in Fig. 2(b) actually resembles the increase in BSA surface concentration, similarly to the behaviour in Fig. 2(a). The corresponding surface concentration values were calculated using Eq. (1), and are presented on the right axis of Fig. 2(b). Taking into account that that the highest average thickness of a BSA layer adsorbed on the gold surface was determined to be 4.3 ± 0.1 nm (Fig. 2(b)) and that this BSA layer is “dry” (argon-dried BSA surface layer), and then considering the dimensions of a BSA crystal [49] one can conclude that BSA reaches a monolayer surface configuration at the highest BSA bulk solution concentrations, and that the BSA molecule lays flat on the gold surface, under the experimental conditions employed (the BSA unit crystal thickness 4.5 nm [49]). Similar results were previously reported in the literature for adsorption of BSA on solid substrates when measured by ex situ ellipsometry [44,50–52]. As previously mentioned, the data in Fig. 2 display a shape of Type I isotherm. Consequently, several corresponding isotherms were tested. The best results were obtained when the Langmuir and Freundlich isotherms were applied. However, the former was dismissed due to the violation of the corresponding isotherm assumptions (reversible adsorption, no lateral interactions of BSA molecules adsorbed on the gold surface, equal energy of all Au surface sites). Hence, the Freundlich isotherm was selected for the further treatment of the data [53–55]: = KF [BSA]n
(3)
(2)
3.2. Kinetics of BSA adsorption It is often believed that the first few hours following the implantation of a medical device into the human body are very crucial in determining the host response to the implant. Since it is generally accepted that the initial adsorption of proteins from the biological fluids significantly influence the implant’s biocompatibility [56], it is of great interest to investigate the kinetics of protein adsorption in order to elucidate the time scale within which the biomaterial surface will be covered to varying degrees by proteins. Similarly, in cases where protein adsorption induces surface fouling of process equipment, such as in heat exchangers and filtration membranes in the dairy and in the pharmaceutical industry, it is of importance to have information on the kinetics of protein adsorption and consequently plan the cleaning steps. The suitability of PM-IRRAS and ellipsometry in studying the kinetics of protein adsorption was further investigated using BSA and gold as a model adsorption system. Three different concentrations of BSA in the bulk solution were used: 0.01, 0.06, and 0.20 g L−1 . From the equilibrium adsorption measurements presented in Fig. 2, it can be seen that the BSA concentrations of 0.01 and 0.06 g L−1 correspond to the rising portion of the adsorption isotherm, whereas the high concentration (0.20 g L−1 ) corresponds to the onset of the adsorption plateau. The same experimental procedure used in the equilibrium experiments was applied for the kinetic experiments, with the only difference being that independent variable was the immersion time of the gold substrate in the BSA solution (as opposed to the bulk protein concentration as an independent variable). Fig. 3a shows the dependence of integrated intensity of Amide I peak of surface-adsorbed BSA on time, while Fig. 3b shows the time dependence of average BSA surface layer thickness obtained by ellipsometry, respectively. The two plots demonstrate that at a constant BSA concentration in the bulk solution, the surface concentration of adsorbed BSA increases very rapidly immediately upon initiation of the adsorption process, and then gradually levels off into a plateau. The adsorption equilibrium is reached after
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0.20
AAmide I / cm-1
0.15
0.10
0.05 (a) 0.00 5.0
/ nm
4.0
Fig. 4. The kinetics of BSA adsorption on gold presented in terms of the dependence of relative surface coverage by BSA on adsorption time. The data were obtained by ellipsometry, in a phosphate buffer solution containing 0.20 g L−1 BSA. Symbols represent the experimental data and the solid line represents simulated data fitted using the adsorption kinetic model described by Eqs. (4)–(6) and the schematic representation in the inset to the figure. The symbols are mean values of at least three measurements. The corresponding standard deviation values, presented by error bars, are not visible since the largest value is 3.84. In the schematic representation of the BSA adsorption mechanism 1 represents surface coverage by BSA adsorbed in a thermodynamically unstable surface configuration. This BSA can either desorb back into the solution, or transform on the surface into a thermodynamically stable configuration, 2 .
3.0 2.0 1.0 (b) 0.0 0
20
40
60
80
100
120
140
Time / minute Fig. 3. (a) Amide I integrated intensity and (b) average surface-adsorbed BSA layer thickness obtained respectively from PM-IRRAS and ellipsometry measurements of BSA adsorbed on a gold surface at different times of adsorption. BSA bulk solution concentration was: () 0.01, () 0.06, and () 0.20 g L−1 . The symbols are mean values of at least three measurements, while the error bars represent the corresponding standard deviation.
ca. 40–60 min of adsorption, depending on the bulk solution BSA concentration. Taking into account the equilibrium results in Fig. 2(a) and (b), the kinetic data from Fig. 3a and b were converted into the corresponding time-dependent surface coverage data, and a representative curve is presented in Fig. 4 (main plot, symbols). In order to model the kinetic data, a one-step kinetic model was first used. However, this model was found not to satisfactorily describe the kinetics of BSA adsorption on gold. Therefore, a two-step kinetic model presented schematically in the inset to Fig. 4 was employed. In this model, the first adsorption step is reversible, and the protein’s surface conformation is assumed to resemble that one in the bulk solution, i.e. the native conformation. However, this surface conformation is not thermodynamically stable, and the protein molecule either desorbs (Step 1, reverse) or irreversibly adopts a more thermodynamically favourable surface conformation (Step 2) [14,57]. Now, expressing the surface concentration of BSA in terms of its relative surface coverage, , the two-step BSA adsorption kinetic model can be formulated as:
( 2 ) configuration, ka (M−1 min−1 ) is the adsorption constant, kd (min−1 ) is the desorption constant, and kf (min−1 ) is the transformation (surface rearrangement) constant. The experimental data (Fig. 4, symbols) were then fitted using the kinetic model in Eqs. (4)–(6), and the result is presented in Fig. 4 (main plot) as a solid line. A good agreement between the experimental and simulated data was obtained for the presented and the other two BSA bulk concentrations and for both techniques (six sets of data, three replicates). This indicates the applicability of the proposed model in describing the kinetics of BSA adsorption on gold under the experimental conditions employed. The corresponding kinetics constants are presented in Table 1. The comparative kinetic constant values obtained using the two techniques (Table 1) are very close. This again validates the approach used in treating the experimental data and the techniques for the investigation of adsorption of BSA on gold under the experimental conditions employed. The kinetic constant values in the table are also very close to those reported in literature, for the adsorption of human serum albumin on a biofunctionalized silicon surface [13]. Further, the table shows that the adsorption kinetic constant depends on the BSA bulk solution concentration in a regular Table 1 Kinetic parameters for the adsorption of BSA onto gold for various experimental conditions. The parameters were determined by fitting the experimental data obtained from PM-IRRAS and ellipsometry experiments. The values listed in the table are mean values and the corresponding standard deviation, obtained from at least three experiments. BSA (g L−1 )
d1 = ka [BSA](1 − 1 − 2 ) − (kd + kf )1 dt
(4)
d2 = kf 1 st
(5)
d d1 d2 = + = ka [BSA](1 − ) − kd 1 dt dt dt
(6)
where = 1 + 2 and 0 ≤ ≤ 1 is the fraction of surface covered by BSA in both the thermodynamically unstable ( 1 ) and stable
Ellipsometry ka × 10−5 (M−1 min−1 )
kd × 101 (min−1 )
kf × 102 (min−1 )
0.01 0.06 0.20
8.30 ± 0.00 3.66 ± 0.00 1.57 ± 0.00
1.39 ± 0.03 2.06 ± 0.04 0.74 ± 0.06
1.00 ± 0.24 1.08 ± 0.10 0.80 ± 0.05
PM-IRRAS 0.01 0.06 0.20
8.30 ± 0.00 3.66 ± 0.00 1.57 ± 0.00
1.42 ± 0.03 2.02 ± 0.02 0.82 ± 0.06
1.00 ± 0.24 1.05 ± 0.07 0.82 ± 0.06
100
1
80
0.8
1
60
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0.6
2
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1
0.4
40 2
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0
0
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20
30
40
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60
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20
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Time / minute
manner; it decreases with an increase in BSA bulk solution concentration. This indicates that the determined adsorption constant is an apparent adsorption constant. Namely, the proposed kinetic model does not take into account intermolecular interactions of adsorbing BSA molecules with those that are already on the surface, but only the occupancy of the surface with the latter molecules. At higher BSA bulk solution concentrations, the substrate surface becomes covered by BSA faster, which increases the probability for the occurrence of the mentioned intermolecular interactions. Consequently, the apparent adsorption rate constant changes. The observed decrease in its value (Table 1) indicates that the intermolecular interactions are of a negative type, i.e. the already adsorbed BSA molecules “inhibit” further adsorption of BSA molecules from the solution. The proposed BSA adsorption kinetic model shown in the inset to Fig. 4 and Eqs. (4)–(6) shows that the substrate surface coverage by thermodynamically unstable ( 1 ) and stable ( 2 ) BSA changes with time. It would, thus, be interesting to examine the kinetics of this transformation. For that purpose, the modelled (simulated) kinetic data were deconvoluted into the two contributions ( 1 and 2 ), and presented in Fig. 5 (note that both PM-IRRAS and ellipsometry gave very similar results, as evidenced by the kinetic constant values in Table 1). The data in Fig. 5 demonstrate that the initial increase (within the first 5–10 min of adsorption) in total BSA surface coverage, , is mostly due to the formation of a thermodynamically unstable, reversibly bound BSA layer, 1 . The surface coverage with such bound BSA initially increases sharply and then reaches a maximum value, which occurs at earlier times at higher bulk solution concentrations (not shown). Then, it starts gradually decreasing. On the other hand the surface coverage with a thermodynamically stable, irreversibly bound BSA, 2 , gradually increases in the entire time interval studied. The results in Fig. 5 indicate that the transformation from the thermodynamically unstable ( 1 ) into the thermodynamically stable ( 2 ) adsorbed state/conformation of BSA is rather slow, and does not get completed within the time interval studied. A similar behaviour was previously reported in the literature for the kinetics of interaction of C-reactive protein (CRP) with a CRP antibody chemically immobilized on a gold surface [57]. Fig. 6 shows the values of the ratio of the coverage of the thermodynamically stable to the unstable surface state of BSA, for three different BSA bulk solution concentrations. The data demonstrate that with an increase in adsorption time, the relative surface ratio also increases. The trend can be approximated by a second-order polynomial function behaviour. Also, the values at a particular adsorption time are close for the three BSA bulk solution
40
50
60
70
Fig. 6. Time dependence of the ratio of surface coverage of thermodynamically stable to unstable adsorbed BSA ( 2 / 1 ) obtained at different BSA bulk solution concentrations: () 0.01, () 0.06, and () 0.20 g L−1 .
concentrations. This indicates that the BSA bulk solution concentration, i.e. the corresponding BSA surface coverage (Fig. 3) does not influence the kinetics of the BSA surface transformation (from 1 to 2 ).
3.3. Comparison of PM-IRRAS and ellipsometry results One of the major goals of this work was to investigate whether two completely different experimental techniques, PM-IRRAS and ellipsometry, could reliably be used to investigate adsorption of a protein on a solid surface, i.e. would they yield similar adsorption data/parameters. The analyses presented in the previous sections of the paper demonstrate that the two techniques indeed gave very close adsorption data, thus not only validating the suitability of their parallel/complementary use, but also the approaches used to treat the experimental data. To more closely compare the two techniques, the corresponding equilibrium BSA surface coverage values are presented in Fig. 7 (circles). It is evident that the experimental points are very close to the 45◦ line, demonstrating that the two techniques yield similar adsorption coverage values (R2 = 0.924). Further, the same conclusion could be made from the kinetic results shown in the same figure (triangles). Thus, the results in Fig. 7 demonstrate that PM-IRRAS and ellipsometry can be reliably and comparatively used to study adsorption of proteins on solid reflective surfaces, at least under the experimental conditions employed in this work.
100
by PM-IRRAS / %
Fig. 5. Time dependence of () the total surface coverage by BSA () obtained by adsorption on a gold surface immersed in a phosphate buffer solution containing 0.20 g L−1 BSA, deconvoluated into the contribution of the coverage by the thermodynamically () unstable ( 1 ), and () stable ( 2 ) BSA. The data were obtained by modelling the PM-IRRAS experimental data using Eqs. (4)–(6).
30
Time / minute
90 80 70 60 50 50
60
70
80
90
100
by Ellipsometry / % Fig. 7. BSA surface coverage values obtained by PM-IRRAS and ellipsometry illustrating the agreement between the two techniques. Data from () equilibrium and () kinetic BSA adsorption measurements are presented. The latter refers to the BSA bulk solution concentration of 0.20 g L−1 . Symbols represent experimental data, whereas the solid 45◦ line represents the equality line.
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4. Conclusions PM-IRRAS and ellipsometry were used to ex situ investigate the adsorption of bovine serum albumin (BSA) on a gold surface. The adsorption process yields a uni-modal (Type I) adsorption isotherm, which can be modelled using the Freundlich isotherm. The kinetic measurements demonstrate that the BSA adsorption process reaches equilibrium after 40–60 min, depending on the protein bulk concentration. The BSA adsorption kinetics follows a two-step model; the first step represents the reversible BSA adsorption, while the second step represents the irreversible transformation of BSA into a thermodynamically stable configuration. The results presented demonstrate that PM-IRRAS and ellipsometry show a very good general agreement, and can thus be reliably and comparatively used to study adsorption of proteins on solid reflective surfaces. Acknowledgments The authors gratefully acknowledge the financial support from the Natural Science and Engineering Research Council of Canada (NSERC) and, the Fonds de recherche du Québec – Nature et technologies (FRQ NT). References [1] C.V. Gruian, E. Simon, S. Simon, Biochim. Biophys. Acta: Proteins Proteom. 1824 (2012) 873–881. [2] M. Gagliardi, Mater. Sci. Eng. C 32 (2012) 2445–2451. [3] M. Jansch, P. Stumpf, C. Graf, E. Rühl, R.H. Müller, Int. J. Pharm. 428 (2012) 125–133. [4] Y. Masuda, T. Ohji, K. Kato, M. Ajimi, M. Bekki, S. Sonezaki, Thin Solid Films 519 (2011) 5135–5138. [5] K. Nagase, J. Kobayashi, A. Kikuchi, Y. Akiyama, H. Kanazawa, T. Okano, Biomaterials 32 (2011) 619–627. [6] A. Noisuwan, Y. Hemar, B. Wilkinson, J.E. Bronlund, Carbohydr. Polym. 84 (2011) 247–254. [7] B.O. Leung, A.P. Hitchcock, R.M. Cornelius, J.L. Brash, A. Scholl, A. Doran, J. Electron Spectrosc. Relat. Phenom. 185 (2012) 406–416. [8] L. Guicai, S. Xiaoli, Y. Ping, Z. Ansha, H. Nan, Solid State Ionics 179 (2008) 932–935. [9] Y. Yin, K. Fisher, N.J. Nosworthy, D. Bax, R.J. Clarke, D.R. McKenzie, M.M.M. Bilek, Thin Solid Films 520 (2012) 3021–3025. [10] C.C. Barrias, M.C.L. Martins, G. Almeida-Porada, M.A. Barbosa, P.L. Granja, Biomaterials 30 (2009) 307–316. [11] L.D. Unsworth, H. Sheardown, J.L. Brash, Biomaterials 26 (2005) 5927–5933. [12] T. Berlind, P. Tengvall, L. Hultman, H. Arwin, Acta Biomater. 7 (2011) 1369–1378. [13] T.M. Byrne, S. Trussler, M.A. McArthur, L.B. Lohstreter, Z. Bai, M.J. Filiaggi, J.R. Dahn, Surf. Sci. 603 (2009) 2888–2895. [14] D.K. Goyal, A. Subramanian, Thin Solid Films 518 (2010) 2186–2193. [15] M.A. McArthur, T.M. Byrne, R.J. Sanderson, G.P. Rockwell, L.B. Lohstreter, Z. Bai, M.J. Filiaggi, J.R. Dahn, Colloids Surf. B 81 (2010) 58–66. [16] J.E. Klinth, R. Larsson, P.O. Andersson, K.N. Ekdahl, Biosens. Bioelectron. 21 (2006) 1973–1980. [17] M. Lundin, U.M. Elofsson, E. Blomberg, M.W. Rutland, Colloids Surf. B 77 (2010) 1–11.
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