Adsorption Behavior of Lysozyme at Titanium OxideâWater Interfaces

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Interfaces: Adsorption, Reactions, Films, Forces, Measurement Techniques, Charge Transfer, Electrochemistry, Electrocatalysis, Energy Production and Storage

The adsorption behavior of lysozyme at titanium oxide-water interfaces Yury Forov, Michael Paulus, Susanne Dogan, Paul Salmen, Christopher Weis, Tobias Gahlmann, Andreas Behrendt, Christian Albers, Mirko Elbers, Wiebke Schnettger, Simon Egger, Elena Zwar, Heinz Rehage, Irena Kiesel, Thomas Riedl, and Metin Tolan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00280 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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The adsorption behavior of lysozyme at titanium oxide-water interfaces Yury Forov,∗,† Michael Paulus,† Susanne Dogan,† Paul Salmen,† Christopher Weis,† Tobias Gahlmann,‡ Andreas Behrendt,‡ Christian Albers,† Mirko Elbers,† Wiebke Schnettger,¶ Simon Egger,§ Elena Zwar,§ Heinz Rehage,§ Irena Kiesel,k Thomas Riedl,‡ and Metin Tolan† †Fakultät Physik / DELTA, Technische Universität Dortmund, 44221 Dortmund, Germany ‡Institute of Electronic Devices, University of Wuppertal, 42119 Wuppertal, Germany ¶Physikalische Chemie I-Biophysikalische Chemie, Fakult¨ at f¨ ur Chemie und Chemische Biologie, TU Dortmund, Dortmund 44227, Germany §Physikalische Chemie II, TU Dortmund, 44227 Dortmund, Germany kPhysikalische Chemie I-Biophysikalische Chemie, Fakult¨ at f¨ ur Chemie und Chemische Biologie, TU Dortmund, Dortmund 44221, Germany E-mail: [email protected] Phone: +49 (0)231 7558519. Fax: +49 (0)231 7553657

Abstract We present an in-situ X-ray reflectivity study of the adsorption behavior of the protein lysozyme on titanium oxide layers under variation of different thermodynamic parameters, such as temperature, hydrostatic pressure and pH-value. Moreover, by varying the layer thickness of the titanium oxide layer on a silicon wafer, changes in the adsorption behavior of lysozyme were studied. In total, we determined less adsorption

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on titanium oxide compared to silicon dioxide, while raising the titanium oxide layer thickness causes stronger adsorption. Furthermore, the variation of temperature from 20 ◦ C to 80 ◦ C yields an increase in the amount of adsorbed lysozyme at the interface. Additional measurements with variation of the pH-value of the system in a region between pH 2 and pH 12 show that the surface charge of both the protein and the titanium oxide have a crucial role in the adsorption process. Further pressure dependent experiments between 50 and 5000 bar show a reduction of the amount of adsorbed lysozyme with rising pressure.

Introduction The adsorption of proteins at solid-liquid interfaces is a frequently studied phenomenon in current research. 1–4 Since proteins take part in several biological processes and are an important component for human life, a lot of effort has been put into the investigation of protein structure and function in different environments. 5,6 Especially in the human body, where the thermodynamic properties are not constant, proteins are in permanent interaction with different kinds of interfaces. Moreover, protein adsorption and desorption plays an important role in a wide range of applications, ranging from food industry to medicine. 7–9 Even though the adsorption process of proteins at solid-liquid interfaces is frequently studied, usually silicon surfaces are used as substrates. 1,10 However, the adsorption process depends on surface properties and studies have thus to be extended to other materials. Here, titanium oxides are interesting materials because of their importance for technical and medical applications. 11–14 There are numerous parameters that control adsorption behavior of proteins at solid-liquid interfaces. 15–17 Since the adsorption process is driven by the entropy and enthalpy of the system and electrostatic interactions between the proteins and the surface, effects of a variety of parameters on the adsorption behaviour of proteins have to be evaluated. 18 In this study, we present the investigation of the adsorption behavior of the protein lysozyme 2


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on titanium oxide coated silicon wafers as a function of temperature, pressure and pH-value variation as well as the variation of the oxides layer thickness by X-ray reflectivity (XRR). Lysozyme is a well known and characterized protein which is stable under a broad range of thermodynamic conditions. As this study focuses on the interaction between proteins and interfaces under variation of thermodynamic conditions structural changes of the protein structure in solution are not desirable. Thus, lysozyme is an ideal sample for this work. The variation of the thermodynamic parameters was partially extended from physiological to extreme conditions since protein adsorption also appears in industrial processes, where high temperatures or high pressures are used, e.g. in high pressure food processing where pressures up to 6000 bar are reached. 19 XRR is a powerful tool to investigate in-situ structural properties of a multilayer system with ˚ angstrom resolution. 20,21 In an XRR experiment, the incident beam is specularly reflected at the sample’s interface and the reflected intensity is measured as a function of incident angle θ. Usually, the reflected intensity is shown as a function of momentum transfer q . Due to the scattering geometry, only the component qz perpendicular to the sample surface contributes to the momentum transfer and thus can be described by qz =

4π λ

sin θ

with the wavelength λ of the used radiation. The reflectivity R(qz ) is given in the first Born approximation by 2 Z dρ(z) 1 iq z e z dz R(qz ) = RF (qz ) ρ∞ − ρsolution dz

(1)

with the Fresnel reflectivity RF (qz ) of a smooth interface, the difference between the bulk electron density of the substrate and the solution ρ∞ − ρsolution and the vertical electron density profile (EDP) ρ(z). 22,23

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Experimental Section Materials and Setup: Silicon wafers with a native silicon dioxide layer with a thickness of 1 nm and a roughness of 3 ± 1 ˚ A provided by Wacker Siltronic (Burghausen, Germany) were used as substrates. Subsequently, titanium oxide layers were deposited on top of the silicon wafer by atomic layer deposition (ALD), 24,25 obtained from TiCl4 and H2 O. With this method, we obtained layer thicknesses of 50 ˚ A , 60 ˚ A , 80 ˚ A (each layer with a roughness of 4 ˚ A, respectively) and 115 ˚ A (roughness: 6 ˚ A). X-ray diffraction reveals that the layers have an amorphous structure. 26 AFM images taken from the sample’s surface (one example is shown in the supporting information) show a complete surface coverage of the titanium oxide. Prior to use, the wafers were cut into 10 × 19 mm2 pieces and cleaned for 5 minutes in an ultrasonic bath and rinsed with ultrapure water. Lysozyme was purchased from Sigma-Aldrich (Taufkirchen, Germany) as lyophilized powder. This protein is obtained from hen egg white and can be described as a prolate ellipsoid 3 with dimensions of 30 × 30 × 45 ˚ A . 27 The isoelectric point (IEP) of lysozyme is 11. 28

Lysozyme was solved in aqueous buffer solution yielding a protein concentration of 1 mg/mL which is the same as used in a silimiar study on silicon. 10 In case of temperature dependent measurements, 10 mM phosphate buffer with a pH-value of 7 was used, while for pressure dependent measurements we chose pressure stable 20 mM BisTris 29 buffer with a pH-value of 7.1. At this pH the zeta potential of the titanium oxide layers is slightly negative if TiO2 is used as a reference. For pH-dependent measurements lysozyme was dissolved in ultrapure water, with the pH value adjusted by the addition of HCl or NaOH. A custom made PTFE sample cell with polyimide windows was used for temperature and pH dependent measurements with a sample volume of 10 mL. The temperature of the cell was controlled by a continuous flow cooling system. In case of pressure dependent measurements, a sample cell presented by Wirkert et al. 30 was used. This cell allows pressures up to 5 kbar. The experiments were performed at beamline BL9 of the synchrotron radiation source DELTA (Dortmund, Germany) 20 with a photon energy of 27 keV and beamline ID31 of the 4


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synchrotron radiation source ESRF (Grenoble, France) with a photon energy of 70 keV. 31 The reflected intensity was recorded by a two-dimensional resolving detector. In order to determine the diffusely scattered radiation, transverse diffuse scans were recorded simultaneously to the reflectivity scan. During each series, we took a reflectivity measurement of the pure water-wafer system first, then removed the water and replaced it with the lysozyme solution. During this procedure the wafers were constantly kept under wet conditions. At BL9, each scan took 30 minutes while at ID31, each scan took 4 minutes. After alignment, the lateral position of the sample was slightly shifted. At least two scans were performed to investigate time and beam damage effects. Data analysis First, the raw data was normalized and background corrected by the subtraction of the transverse diffuse scan. Subsequently, the electron density profiles were determined by minimizing the mean square deviation between the reflectivity data and a reflectivity profile, which was calculated by Parratt’s recursive algorithm 32 in combination with the effective density model, 22 using a least-squares fitting routine. The electron density profiles contain information about the layer thickness d, electron density ρ and interfacial roughness σ of each layer of the model system. Detailed information on the extraction of these parameters is presented by Tolan. 22 In this study, we modeled the reference sample system (no protein present) with silicon as substrate material with a silicon dioxide layer on top, followed by a titanium oxide layer and water. For lysozyme solution measurements, one layer for adsorbed lysozyme was added on top of the titanium oxide layer. A fit to the data with more layers instead of a single slab for the protein, which yields frequently a better adaption of the fit, 6 caused no improvements. Due to this, we decided to keep the model system as simple as possible. However, this approach causes slight derivations in the mid-q range of the reflectivity curves. Since we expect no changes in the substrate material during protein adsorption, we kept the substrate parameters obtained from the reference measurements fixed within the corresponding measurement series. Only the parameters of the lysozyme layer were changed during each series. Since increasing pressure changes the

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electron density in water significantly, this parameter was also optimized during the analysis of a pressure series. With the electron density profiles of the pure water measurements ρref (z) and the lysozyme solution electron density profiles ρ(z), the volume fraction profile φ(z) along the surface normal can be calculated via φ(z) =

ρ(z) − ρref (z) ρlys − ρwater

(2)

3 where ρlys is the electron density of lysozyme (0.49 e− /˚ A , this value is used to keep a

comparability between this study and Kiesel et al. 10 being calculated using the program package chimera on the basis of a PDB file without a hydration shell. Other studies propose 3 a lower electron density value 6 ) and ρwater the electron density of water (e.g. 0.334 e− /˚ A at

ambient conditions). 10,33 φ(z) indicates the lysozyme portion in the adsorption layer which is assumed to be a composite of lysozyme and water. The adsorbed amount of lysozyme is then given by m Γ= v

Z

φ(z)dz

(3)

with the molar mass m (14.3 kg/mol) and molar volume v (9348 m3 /mol) of lysozyme. 27 Note, that Γ represents the minimum amount observable by XRR. 6

Results and discussion X-ray reflectivity measurements of a temperature series are shown in figure 1 a). The reflectivities were normalized by the Fresnel reflectivity of a smooth silicon-water interface. The strong oscillations occur from the 60 ˚ A thick titanium oxide layer. By inserting the lysozyme solution, both the minima positions and shapes vary slightly. In order to highlight the changes caused by the adsorption of the protein, temperature dependant reflectivity data compared to the measurements at 20 ◦ C is shown in the supporting information. As shown in figure 1 b) the increase of temperature results in a stronger absorbance of lysozyme on

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the titanium-oxide layer. Here an increase of electron density and layer thickness becomes visible. For a better discussion of the results, volume fraction profiles and the adsorbed 10 2

1.1 a) 1

10 0 0.9

0.8 (z) [e-/Å3]

10 -2 R/RF 10 -4

0.7

TiOx

0.6

Si SiO2

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0.5 10 -6 0.4

10 -8

0

0.1

0.2

0.3

0.4

0.3

0.5

0

q z [Å -1]

50

100 z [Å]

150

200

Figure 1: a) X-ray reflectivity data of a temperature series at ambient pressure. Open circles represent the measured data and solid lines represent the fit to the data. The color scale corresponds to the legend in b). Lysozyme 80 ◦ C 2 is the second measurement at 80 ◦ C repeated one hour after the first measurement. The spectra are shifted on the y-scale for better visualization. b) Electron density profiles of the temperature series shown in a). amount of lysozyme were calculated via equations 2 and 3. The volume fraction profiles of the temperature series are shown in figure 2 a). At 20 ◦ C, there is an adsorbed lysozyme monolayer with 45 ˚ A thickness on the titanium oxide surface, since only 14% of the surface ˚3 , an adsorption is covered with lysozyme. As the dimension of lysozyme is 30 × 30 × 45 A with the long axis perpendicular to the sample surface seems to be preferred. The figure also shows that in the temperature range between 20 ◦ C and 60 ◦ C there is almost no change in the surface coverage. Close to the denaturation temperature of lysozyme (71 ◦ C) a change of the lysozyme layer’s shape becomes visible. We observe an increase of layer thickness which goes in hand with a decrease in the layer’s density. While the overall surface coverage decreases from 15.8% at 60 ◦ C to 10.9% at 70 ◦ C, a slight increase of adsorbed amount of 7


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lysozyme (see figure 2 b)) is observed. This behavior can be explained by a partial unfolding of the protein yielding lower electron densities and larger layer thicknesses. Due to this, a lower surface coverage is observed, since the surface coverage depends on the maximum density in the protein layer. The slight increase of Γ also points to an ongoing adsorption of proteins from the bulk phase. As the proteins tend to aggregate at high temperatures, a further increase of the temperature to 80 ◦ C leads to an even higher amount of adsorbed lysozyme. However, even at 80 ◦ C the adsorbed amount of lysozyme is still smaller than on silicon dioxide as observed in previous studies. 10 Interestingly, as shown in figure 3, the titanium oxide layer thickness has an impact on lysozyme adsorption. A thicker titanium oxide layer causes more lysozyme adsorption. Here, the titanium oxide layer thickness dependence might give a hint that additional Van der Waals interactions play an important role. 5 In order to get an estimation of the Van der Waals contributions, we calculated the non retarded Hamaker constant for two different sample geometries. First, the interaction between titanium oxide with lysozyme via water was analyzed. For this purpose, we calculated the Hamaker constant 34 p p p p Aijk = ( Aii − Akk ) · ( Ajj − Akk )

(4)

where Aii = 15.3 · 10−20 J is the Hamaker constant of titanium dioxide, 35 Akk = 3.7 · 10−20 J the Hamaker constant of water 34 and Ajj = 13.8 · 10−20 J the Hamaker constant of lysozyme. In the last case we used values taken from Gripon et al., 36 where the interaction between lysozyme molecules in water was discussed. Here, Ajj is determined by the use of equation 4 and the corresponding water value. This approach yields Aijk = 3.6 · 10−20 J which indicates an attractive interaction. To estimate the effect of the silicon below the titanium oxide layer we calculated the Hamaker constant for silicon interacting with lysozyme via titanium dioxide. Here, Aii = 18.5 · 10−20 J for silicon was used. 37 This rough estimation yields a Hamaker constant of −8 · 10−22 J indicating a slight repulsive interaction. Thus, we conclude

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that in the used sample geometry the silicon substrate appears to be repulsive, which might result in a reduced adsorption at low titanium oxide layer thicknesses. To explain the weak adsorption in comparison to the silicon dioxide surface, surface charge effects have to be taken into account, since one of the key factors in the adsorption process are electrostatic interactions. The experiments on silicon dioxide and titanium oxide were performed at a pH-value of 7±0.2. The IEP of silicon dioxide is at a pH of 2.0, 38 while titanium oxide has its IEP around a pH value of 5, 39 resulting in a stronger surface charge of silicon dioxide at the experimental conditions and thus in a stronger attraction of lysozyme offering an IEP of 11. To investigate the adsorption behavior of lysozyme as a function of titanium oxide surface charge, pH-value dependent measurements were performed. In Figure 4 a) the volume fraction profiles of lysozyme on titanium oxide as a function of the pH-value on a 60 ˚ A thick titanium oxide layer are shown. Almost no lysozyme adsorbs at pH-values below 5, since both the titanium oxide surface and lysozyme are positively charged. At a pH-value of 7, which is higher than the pH-value at the IEP of titanium oxide, the titanium oxide surface is negatively charged and thus lysozyme adsorbs equally to what was shown in figure 2. The highest adsorbed amount occurs at a pH-value of 9.8, where titanium oxide is strongly negatively charged while lysozyme still carries a positive charge. 40 Even though the pH-value difference for titanium oxide to the IEP is 4, there is still less lysozyme adsorbing than on silicon dioxide at a pH-value of 7. Consequently, the different adsorption behavior on silicon dioxide and titanium oxide is not only occurring from surface charge differences and thus has to be described by structural differences. 41 Raising the pH-value further to 12 leads to less adsorption on titanium oxide. This pH-value is beyond the IEP of lysozyme and thus lysozyme and the titanium oxide surface are not oppositely charged anymore. Finally, pressure dependent measurements on titanium oxide coated silicon wafers were performed. The results show a desorption behavior of lysozyme with rising pressure (see figure 5). This effect is almost completely reversible by releasing pressure (see 5 a)). The

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a)

20°C 35°C 50°C 60°C 70°C 80°C 80°C 2

φ(z) [%]

20 15 10 5 0 0

Γ [mg/m2]

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20

20 b) 17.5 15 12.5 10 7.5 5 2.5 0 10 20

40

60 80 z [Å]

100

120

TiOx 6 nm TiOx 11.5 nm SiO 2

30

40

50 60 T [°C]

70

80

90

Figure 2: a) Lysozyme volume fraction profiles of the temperature series shown in figure 1. b) Adsorbed amount of lysozyme on titanium oxide. The green symbols correspond to the data shown in a) and figure 1, while the blue symbols correspond to a measurement on a thicker titanium oxide layer. Moreover, to compare the results, the adsorbed amount of lysozyme on silicon dioxide as previously published 10 is shown in black. The errorbars of the data points are smaller than the data points themselves (0.3 mg/m2 ). effect of pressure on proteins in solution is frequently discussed in literature. It was shown that high pressure has a impact on protein - protein interactions, protein activity, folding and hydration and on the formation of protein dimers and oligomers. 42–47 Studies on protein adsorption under high hydrostatic pressure are rare. 30,48 In general, the application of high pressure transfers the system under investigation into a state with reduced volume. This can cause a destabilisation or denaturation of proteins, which might go in hand with an increased adsorption due to the interaction between hydrophobic parts of the protein and the surface. Moreover, the volume of proteins hydration shell and its density is changed 45 resulting in an 10


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40

a)

50 Å 50 Å 60 Å 60 Å 80 Å 115 Å

φ(z) [%]

30 20 10 0 0

20

40 z [Å]

60

80

60 80 100 TiOx layer thickness [Å]

120

2.5 2 Γ [mg/m2]

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1.5 1 0.5 0 40

Figure 3: a) Lysozyme volume fraction profiles as a function of titanium oxide layer thickness at ambient conditions. b) Adsorbed amount of lysozyme on titanium oxide for layers of different thicknesses. The errorbars were estimated by variation of lysozyme layer fit parameters. overall reduction of the systems volume. As a consequence, a completely hydrated protein in bulk solution might be preferred compared to a weakly bound protein offering a defective hydration shell towards the substrate. As lysozyme is very pressure stable this scenario might explain the observed desorption.

Conclusion Within this study we show X-ray reflectivity data of the adsorption behavior of lysozyme on titanium oxide surfaces. We investigated the adsorption behavior as a function of temper-

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a)

pH 2 pH 3.5 pH 4.9 pH 7.2 pH 9.8 pH 11.9

φ(z) [%]

20 15 10 5 0 0

3

Γ [mg/m2]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20

40

60 z [Å]

80

100

b)

2

1

0 2

4

6

8

10

12

pH

Figure 4: a) Lysozyme volume fraction profiles of a pH-value series at ambient conditions on a 60 ˚ A thick titanium oxide layer. b) Adsorbed amount of lysozyme on titanium oxide as a function of pH-value. The errorbars were estimated by variation of lysozyme layer fit parameters. The red line is a guide to the eye to illustrate the behavior of adsorbed amount of lysozyme as a function of pH. ature, pressure, pH-value and titanium oxide layer thickness. In accordance with a former study using silicon dioxide surfaces, we see a slight adsorption of lysozyme on titanium oxide, which increases as a function of temperature from 20 ◦ C to 60 ◦ C. After almost reaching the denaturation temperature of lysozyme around 71 ◦ C, the adsorbed amount of lysozyme increases strongly. However, even beyond the denaturation temperature, the adsorbed amount of lysozyme is still smaller than on silicon dioxide. Since the adsorption process is partially driven by electrostatic interactions, the surface charge of titanium oxide was varied by a change of the pH-value of the system. The results show that below a pH-value of 5 almost no lysozyme adsorbs on titanium oxide, because both titanium oxide and lysozyme 12


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φ(z) [%]

15

a)

50 bar 1000 bar 2000 bar 3000 bar 4000 bar 5000 bar 50 bar down

10

5

0 0

2.5

20

b)

40 z [Å]

60

80

50 Å 60 Å 60 Å 80 Å

2 Γ [mg/m2]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.5 1 0.5 0 0

1000

2000 3000 p [bar]

4000

5000

Figure 5: a) Lysozyme volume fraction profiles of a pressure series at ambient temperature on a 60 ˚ A thick titanium oxide layer. b) Adsorbed amount of lysozyme on titanium oxide as a function of pressure. The errorbars were estimated by variation of lysozyme layer fit parameters. are positively charged at these conditions. The further increase of the pH-value results in a higher adsorbed amount of lysozyme until the IEP of lysozyme is reached. However, even at a pH-value of 9.8, the adsorbed amount of lysozyme on titanium oxide is smaller than on SiO2 . These results indicate that not only surface charge differences, but also structural differences have to be taken into account. Therefore, titanium oxide layer thickness dependent measurements were performed. The results show that with rising layer thickness a higher amount of lysozyme adsorbs at the interface. Additionally, the Hamaker constant for the sample geometry was calculated, leading to the conclusion that the silicon substrate appears to be repulsive, resulting in a reduced adsorption of lysozyme at thin titanium oxide layers.

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Moreover, pressure dependent measurements were performed and show a desorption behavior of lysozyme with rising pressure, being almost completely reversible by releasing pressure. These results indicate that under hydrostatic pressure lysozyme prefers to be completely hydrated in solution instead of having a defective hydration shell towards the substrate. Thus, desorption of lysozyme at the interface is preferred leading to a smaller adsorbed amount of lysozyme at the titanium oxide surface. The presented results point out that the adsorption process of lysozyme depends strongly on the interfacial properties of the interface lysozyme adsorpbs on.

Acknowledgement The authors thank DELTA and ESRF machine groups for providing synchrotron radiation. This work is supported by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. CW thanks the BMBF projects (05K13PE2 and 05K16PE1) within FSP-302 for financial support.

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Adsorption Behavior of Lysozyme at Titanium OxideâWater Interfaces

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