Molecular Interactions of Protein with TiO2 by the AFM-Measured

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Molecular interactions of protein with TiO2 by AFM measured adhesion force Yihui Dong, Rong An, Shuangliang Zhao, Wei Cao, Liangliang Huang, Wei Zhuang, linghong Lu, and Xiaohua Lu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02024 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

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Molecular interactions of protein with TiO2 by AFM measured adhesion force ∥

⊥

Yihui Dong,† Rong An,‡ Shuangliang Zhao,§ Wei Cao, Liangliang Huang, Wei Zhuang,† Linghong Lu† and Xiaohua Lu*† †

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech

University, Nanjing, 210009, P. R. China ‡

Herbert Gleiter Institute of Nanoscience, Nanjing University of Science &

Technology, Nanjing, 210094, P. R. China §

School of Chemical Engineering, East China University of Science and Technology,

Shanghai, 200237, P. R. China ∥

State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China

⊥

School of Chemical, Biological & Materials Engineering, University of Oklahoma, Norman, 73019, United States

ABSTRACT: Understanding the interactions between porous materials and biosystems is of great important in biomedical and environmental sciences. Upon Atomic Force Microscopy (AFM) adhesion measurement, a new experimental approach was presented here to determine the molecular interaction force between proteins and mesoporous TiO2 of various surface roughness. The interaction force between each protein molecule and pure anatase TiO2 surface was characterized by fitting the adhesion and adsorption capacity per unit contact area, and it was found that the adhesion forces were approximately 0.86 nN, 2.63 nN and 4.41 nN for lysozyme, myoglobin and BSA, respectively. Moreover, we reported that the molecular interaction force was independent of the surface topography of the material, but the protein type is a factor of the interaction. These experimental results on the molecular level cast helpful insights for stimulating model calculation and molecular simulation studies of protein interaction with surfaces. KEYWORDS: AFM; TiO2; molecular interaction; adhesion force; adsorption capacity.

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1. INTRODUCTION Protein interacting with substrate plays a pivotal role in engineering bio-surfaces and is of fundamental importance for the development of nanotechnology and nanomaterials design towards biological applications.1 The interaction of protein with the surface can generally be classified into three regions2, 3: weak, intermediate (typically 5 ng·cm-2~170 ng·cm-2)4 or strong, which was involved, respectively, for the

applications

of nonfouling

devices,5 protein separation6 and enzyme

immobilization.7. Understanding the interactions between porous materials and biomolecules are necessitated in biomedical and environmental sciences.1, 8, 9 It is largely recognized that the amount of adsorbed proteins provides a general standard for determining the strength of interaction between the protein and the related surface.10-13 For instance, the surface is regarded as a nonfouling one when the amount of adsorbed protein is less than 5 ng·cm-2.14, 15 To characterize the binding affinity for the separation of binary mixture of proteins, Huber and co-worker studied the competitive adsorption of proteins of three kinds in silica mesopores through the adsorption capacity.16 In the previous work, we developed a linear model to describe the relationship between the geometrical topography of mesoporous TiO2 and protein adsorption.4 The line is divided into three zones: nonfouling, protein separation and immobilization, according to the amount of adsorbed proteins on the surfaces. However, the adsorption amount is not always an indication of interaction strength. For instance, hydrogel, a perfect nonfouling materials with strong hydration property, can adsorb a large amount of small proteins due to its three-dimensional network structure.17 To achieve good results for the enzyme immobilization, the high adsorption capacity is needed together with the strong operation stability.18,

19

Moreover, during the adsorption from a physiological solution, many kinds of proteins compete for the binding sites on a nanomaterial surface.2,20-22 Some proteins diffuse rapidly and are adsorbed onto the nanomaterial surface in prior, producing a high adsorption amount in the initial stage.23 However, they can be replaced finally by other proteins with a stronger protein-surface interactions.2, 24 Therefore, the amount of adsorbed proteins is not adequate to determine the interaction between protein and


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surface. Meanwhile, many works focused on evaluating the interactions of proteins with biomaterial surfaces by using AFM-based technique.25-28 This method provides both dynamic and quantitative information of protein interaction at nanoscale.29,

30

To

comprehensively understand the biofouling resistance of the thin-film composite membrane, Ye et al. measured the probe−membrane interactions by using particle-functionalized AFM probe.31 Logan et al. also examined the adhesion force using AFM to understand the combination presence of proteins and polysaccharides.32 According to these works, AFM method is thought to be a better approach to determine the strength of interaction between protein and surface. It was known that the adhesion force measured by AFM is related to the contact area between the tip and sample.33 The AFM measurement of the adhesion force of the protein interacting with the samples was obtained through the proteins immobilization on self-assembled monolayers functionalized AFM tip.29 Then, the amount of the protein molecules adhered on the tip can affect the contact area between the protein and surface, further resulting in a different interaction even with the same substrate. However, it is difficult to obtain the amount of protein molecules on the tip which contact with the surface directly. In this study, we applied an experimental approach to obtain the molecular interaction force between proteins and TiO2 by performing AFM adhesion measurements. Mesoporous TiO2 materials with different geometrical topographies were prepared, and used as models to investigate the adhesion force and adsorption capacity of the lysozyme. The molecular lysozyme-TiO2 interaction was obtained by correlating the adhesion force with the adsorption capacity. Other two typical proteins, BSA and Myoglobin, were also studied to validate this approach. This work described an experimentally straightforward procedure for acquiring interaction force between a single protein molecule with the TiO2 surface. The results can not only cast help insights for the manipulation of this interaction for various applications, but also provide useful force field parameters for molecular simulations.


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2. EXPERIMENTAL SECTION Materials. Lysozyme (Molecule Weight, Mw: 14.4 kDa), Myoglobin (Mw: 17.6 kDa) and BSA (Mw: 66.5 kDa) were purchased from Bio Dee Bio Tech Co. Ltd. (Beijing, China), and used as received without further treatment or purification. 16-Mercaptohexadecanoic acid (HS(CH2)15COOH) (90%wt) was purchased from Sigma-Aldrich Trading Co. Ltd., (Shanghai, China). N,N-dimethyl formamide, triethylamine (99%wt) and trifluoroacetic anhydride (98%wt) were purchased from J&K Scientific Ltd., (Shanghai, China). Dichloromethane (99.5%wt) was purchased from Sinopharm Chemical Reagent Co. Ltd., (Shanghai, China).

Deionized water

was used in all the experiments. Preparation of Mesoporous TiO2. The mesoporous TiO2 was prepared using the soft-chemistry and template-free method.34, 35 The samples were prepared following the procedure: 1) Hydrous titanium dioxide (TiO2·nH2O) was mixed with K2CO3 (TiO2/K2O molar ratio was controlled at 1.9) and then sintered to 810 ºC at a heating rate of 5 oC·min-1 in an oven and maintained for 2 h. Then the K2Ti2O5 fibers were prepared. 2) The intermediate K2Ti2O5 was added to a 0.5 M HCl solution with vigorous stirring until the K+ ions were completely exchanged. The dried hydrated titanate H2Ti2O5 was filtered and washed with distilled water and thereafter dried at 60 ºC under vacuum for 2 h. 3) The dried hydrated titanate H2Ti2O5 was finally sintered at 300 ºC, 500 ºC, 600 ºC and 700 ºC respectively, at a heating rate of 2 ºC·min-1, held for 2 h. It was then naturally cooled to room temperature in the furnace. Finally, the mesoporous TiO2 with different mesopore geometries were obtained by the different calcination temperatures. These mesoporous TiO2 samples were named T300, T500, T600 and T700, respectively. Characterization. The morphology of mesoporous TiO2 samples was characterized by the field-emission scanning electron microscopy (FESEM; Hitachi S-4800). X-ray diffraction (XRD, Bruker D8, Cu-Kα radiation) and Raman spectrometer (Horiba Labram HR 800) were used to identify the crystal phases of the materials. Textural


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properties (BET surface area, pore size and pore volume) were assessed using N2 adsorption-desorption measurements (Micromeritics Tristar II 3020) at liquid N2 temperature, 77 K. Protein immobilization on self-assembled monolayers (SAMs) functionalized AFM tip. Firstly, AFM silicon nitride tips (radius=20 nm), Si3N4, coated with gold (Top-60 nm Au/Botton-15nm Cr) was immersed in a 1 mM HS(CH2)15COOH/ethanol 50 vol% solution and maintained under dark conditions in an incubator for 12 h. Then, the tip was washed three times with ethanol and dried using nitrogen. Secondly, the tips were immersed in a mixture of N,N-dimethyl formamide (9.58 mL), triethylamine (0.28 mL) and trifluoroacetic anhydride (0.14 mL) for approximately 20 min.29 Then, the tip was again washed with dichloromethane by three times, and dried using nitrogen. Finally, the tip was immersed in a 5 mg·mL-1 PBS lysozyme solution and washed with the PBS buffer solution and dried using nitrogen. AFM measurements. The surface roughness was measured using AFM (ICON, Bruker, USA) under the tapping mode. The images underwent second-order flattening using Nanoscope Analysis. Root-mean-square (RMS) roughness for each surface topography was calculated based on the average of 500 × 500 nm height scans collected from the 3~5 different positions of the samples. The adhesion forces were measured using AFM under the contact mode. The lysozyme modified tips with nominal spring constants between 0.05 and 0.5 N/m were used throughout the experiments and the scan rate was 1.00 Hz. The normal spring constant of the tip was calibrated using deflection sensitivity of supported cantilever to transform the normal load signals from volts (V) into the true normal load (N). The tips’ calibration using the thermal tune method was performed in air over a sapphire substrate at room temperature. The adhesion force between the lysozyme-modified tip and mesoporous TiO2 surfaces can be acquired as the force jump during retraction, which represents the pull-off force required to separate tip after contact. About 80 force-distance curves at the maximal adhesion force upon retraction were recorded at multiple randomly chosen spots.


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Protein adsorption assays. The adsorption capacity of lysozyme on mesoporous TiO2 was achieved by measuring the adsorption of lysozyme from the aqueous solution. 200 mg mesoporous TiO2 samples (T300, T500, T600 and T700, respectively) were added to a centrifuge tube containing 20 mL lysozyme solution with certain concentrations (varying from 0.2 mg·mL-1 to 2 mg·mL-1, a 0.01 M PBS at pH =7.2). During the adsorption process, the samples in the centrifuge tubes were kept in a water bath at 30 ºC and shaken at 180 rpm for 72 h. The lysozyme adsorption kinetics results showed that the adsorption time of 72 h is enough for the adsorption process (see Figure S1) to reach the equilibrium state.36, 37 Then, lysozyme adsorbed on the mesoporous TiO2 samples were separated by spinning the lysozyme-TiO2 mixtures in sealed centrifuge tubes at 8000 rpm for 10 min. The supernatant concentration of lysozyme was determined by measuring the lysozyme absorbance with a UV-vis spectrophotometer at λ=280 nm. The amount of lysozyme bound to the mesoporous TiO2 was calculated from the difference in the initial and final protein concentrations. The adsorption isotherms curves were drawn.

3. RESULTS AND DISCUSSION Mesoporous TiO2 Characterization. Mesoporous TiO2 calcinated at four different temperatures (300, 500, 600, and 700 °C) were denoted as T300, T500, T600 and T700, respectively. The surface morphology of these calcined products was examined by FESEM and AFM. The FESEM images shown in Figure 1 display the mesopore structure of these four samples. After sintering at 300 °C, a well-defined separated fibrous morphology was obtained (see Figure 1a). When the sintering temperature increased to 500 °C, mesopores with pore size of 9-12 nm were generated throughout the fibers to form the three-dimensional mesoporous architecture (see Figure 1b). At higher sintering temperatures of 600 °C and 700 °C, the interconnected network of mesopores was still observed and became larger (see Figure 1c,d). The AFM topographic 3D images of mesoporous TiO2 with different geometrical topographies are shown in Figure 2. There are many mathematical approaches to quantify surface


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roughness from AFM images, root mean squared roughness (RMS) is one of the more commonly reported measures of surface roughness from AFM images.38 It represents the standard deviation of the distribution of surface heights, so it is an important parameter to describe the surface roughness by statistical methods.39 The resulting average roughness (RMS) values of these samples were approximately 2.3 nm, 7.3 nm, 8.4 nm and 10.2 nm for T300, T500, T600 and T700, respectively (see Table 1). This shows that the surface roughness of mesoporous TiO2 increases with increasing calcination temperature. The surface area and pore distribution for those mesoporous TiO2 samples annealed at various temperatures were determined upon N2 adsorption– desorption isotherms, as displayed in Figure S2. The physical properties and structure parameters of mesoporous TiO2 are listed in Table S1. All four samples show a typical isotherm of type-IV, indicating the well-developed mesoporosity in these samples. As calcination temperature increases from 300 °C to 700 °C, the BET surface area of the samples decreases (see Table 1), while the average pore size (see Table S1) and surface roughness increase (see Table 1). From these results, simple control of the calcination temperature can be used to vary the pore geometry and the surface roughness of TiO2 nanostructures.

Figure 1.

FESEM images for mesoporous TiO2 subjected to different sintering

procedures: (a) T300, (b) T500, (c) T600, and (d) T700.


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Figure 2. AFM 3D topographic images of mesoporous TiO2: (a) T300, (b) T500, (c) T600, (d) T700. Table 1. BET surface area, surface roughness and isoelectric point of mesoporous TiO2 prepared by calcination at different temperatures.

a

Samples

STa, m2·g-1

RMSb, nm

IEPc

T300

237.9േ0.52

2.3േ0.37

4.50

T500

89.9േ0.40

7.3േ0.34

4.78

T600

53.6േ0.26

8.4േ0.21

4.49

T700

29.2േ0.21

10.2േ0.44

4.53

ST: BET surface area, m2·g-1; bRMS: Root-mean-square roughness, nm, values

presented as an average ± standard deviation (~3-5 total topography maps from the surfaces); cIEP: Isoelectric point. The crystalline phases existing in the mesoporous TiO2 were examined using XRD and Raman experiments, as shown in Figure 3. In XRD patterns, the peaks at the 2 theta of 25.3°, 37.8° and 48.0° represent the diffraction from the anatase (101), anatase (004) and anatase (200) facets, respectively (see Figure 3a). The samples exhibit the characteristic bands at the frequency of 144.6 cm-1, 397.7 cm-1, 515.7 cm-1


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and 640.9 cm-1, marked with solid square in Figure 3b. These bonds are recognized to represent the pure anatase TiO2 nanocrystals.40 Both XRD and Raman results indicate the existence of pure anatase crystal phase in all these mesoporous TiO2. Thus, we do not have to consider the effect of the difference in crystal phases on the protein-TiO2 interaction.

Figure 3. (a) XRD patterns and (b) Raman spectra of mesoporous TiO2 prepared by calcination at different temperatures. AFM adhesion measurement. AFM is proved to provide quantitative information on protein adhesion on surfaces.29 The adhesion force is determined by the vertical force required to pull the AFM lysozyme-tip from the surface of mesoporous TiO2. This force is related to the contact area between the tip and the sample.41 Here, the lysozyme adhered on the tip is a fixed protein cluster and the adhesion forces of lysozyme on TiO2 are shown in Figure 4. To obtain statistical significance, 80 positions were measured and the results were averaged (see Table 2). The distribution of the adhesion forces were shown in Figure S3. Meanwhile, different surface charges can result in different electrostatic interactions, which plays an important role in protein interaction with surface.42, 43 From the Zeta potential titration curves of these four TiO2 samples (see Figure S4), the isoelectric points (IEPs) of T300, T500, T600 and T700 are approximately 4.50, 4.78, 4.49 and 4.53, respectively (see Table 1). These results showed that the IEPs of these four samples are almost the same, which indicated that the surface charge of these different samples are basically the same


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when at a fixed electrostatic condition. Different surface structures, such as geometrical morphologies and surface roughness, were utilized to regulate the protein interaction by adjusting the contact area between adsorbed molecules and the surface.8, 44-47

Figure 4 shows that when the pore size and the surface roughness is smaller than

the size of the protein (such as the lysozyme on T300), the contact area between the lysozyme-modified tip and the surface is restricted, leading to a lower adhesion force. When the pore size and the surface roughness of the mesoporous TiO2 increase, the contact area also increases, further leading to a larger adhesion force. Hence, the distinct adhesion force delivers the information that protein-surface adhesion largely depends on the contact area between protein and the mesoporous TiO2. Table 2. Adhesion force between lysozyme-tip and mesoporous TiO2 with various geometrical morphologies.

FAd, nN d

T300

T500

T600

T700

4.6േ0.65

9.5േ1.32

12.2േ2.53

20.3േ2.95

FA, adhesion force, nN. Values presented as an averageേstandard deviation (over 80

force-distance curves).

Figure 4. Schematic representations of the adhesion force between the lysozyme-tip and mesoporous TiO2 with various geometrical morphologies and surface roughness. Although we correlate the adhesion with contact area from the above results, the


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quantitative relationship is still lacking and worth exploring. The contact area is suggested

to

be

estimated

through

the

combination

of

Hertz

and

Johnson-Kendall-Roberts (JKR) theories (equation (1)). In the conceptual model, the spheres are pressed into contact with a normal load, which results in a contact radius.48

ߨa2=ߨ(RFo/E)2/3

(1)

Where R represents the tip radius, a represents the radius of a contact area with the surface (then ߨa2 represents the contact area between tip and surface), E is Young’s modulus of the tip, and Fo represents a given loading force. We define Sc as the contact area between protein and TiO2 surface calculated by equation (2): Sc=π(RpFA/Ep)2/3

(2)

where Rp (nm) represents the radius of the cluster protein modified on the tip (was estimated as about 40 nm in this study); Ep represents Young's modulus of lysozyme molecules (~ 0.5 GPa49); FA represents the adhesion force (nN). The contact areas (Sc, m2) of the lysozyme interacting with T300, T500, T600 and T700 are 1.61×10-16 m2, 2.62×10-16 m2, 3.09×10-16 m2 and 4.34×10-16 m2, respectively. Then, we established the linear relationship between FA and Sc (see Figure 5). A linear regression is performed and the relationship is FA=5.78Sc-5.21(R2=0.99). Then we can obtain the adhesion force of lysozyme on unit contact area of TiO2. The FA/Sc values for T300, T500, T600 and T700 are 2.85×1016 nN·m-2, 3.63×1016 nN·m-2, 3.95×1016 nN·m-2 and 4.68×1016 nN·m-2, respectively. The difference in these values of FA/Sc indicates the different number of the lysozyme molecules contacting with the TiO2. Still, to determine the protein-TiO2 interaction, we should know the number of protein molecules contacting with the surface.


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Figure 5. The adhesion force vs. the contact area between lysozyme and mesoporous TiO2 with various geometrical morphologies. The dashed line is the guide to the eyes. Adsorption isotherms. To obtain the number of protein molecules contacting with the surface, we first investigated the adsorption isotherms of lysozyme on mesoporous TiO2. The adsorption capacity at constant temperature has been described by Langmuir and Freundlich isotherm models.50 The Langmuir model assumes the monolayer adsorption on a homogeneous surface and the interaction between adsorbed protein molecules is neglected.51 The Freundlich isotherm model generally describes equilibrium adsorption on heterogeneous surfaces. The linear form of Langmuir isotherm model and Freundlich isotherm model were given by equation (3)52 and (4),53 respectively:

Ce C 1 = + e qe bqm qm

(3)

1 (4) ln qe = ln K F + lnCe n where qe (mg·g-1) and Ce (mg·mL-1) represent the equilibrium amount of adsorbed

protein per unit mass and equilibrium concentrations of protein, respectively; qm (mg·g-1) is the maximum monolayer adsorption capacity; b (mL·mg−1) is a Langmuir adsorption constant related to the affinity of the binding sites; n (mL·g-1) is the Freundlich parameter and KF is the heterogeneity factor. The data collected from isothermal adsorption of lysozyme are plotted in certain


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ways to fit the Langmuir and Freundlich models (see Figure 6). The regressed isotherm parameters are summarized in Table 3. The results show that the Langmuir model seems to be a better fit (R2=0.97~1) than Freundlich model (R2=0.68~0.88). Additionally, the different slopes (1/qm) in Figure 6a indicates different adsorption capacities of the lysozyme adsorbed on TiO2 with various geometrical morphologies. This is due to that the various geometrical morphologies provided different surface area for the adsorption of lysozyme, and thus resulted in the different adsorption capacity (qm). Since the lysozyme is mono-layer adsorbed onto these TiO2 surface, we can further estimate the amount of adsorbed protein on unit area of surface.

Figure 6. Lysozyme adsorption data plots on mesoporous TiO2 with different geometrical morphologies, to fit (a) Langmuir and (b) Freundlich models. Table 3.

Adsorption isotherm constants for lysozyme adsorption onto mesoporous

TiO2 with various geometrical morphologies. Sample Isotherm model

Langmuir

Freundlich

Parameters T300

T500

T600

T700

b(mL·mg-1）

41.19

40.90

39.28

37.01

qm(mg·g-1)

87.01

67.20

56.34

37.39

R2

0.971

0.991

0.988

0.993

KF(mL·g-1)

221.41

90.02

66.69

44.70

1/n

0.455

0.281

0.242

0.214

R2

0.782

0.866

0.877

0.686


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Figure 7 shows the maximum amount of adsorbed lysozyme vs. the BET surface area of mesoporous TiO2. We can see the relationship between qm and ST, is not linear. This shows that not all the surface areas are effective to adsorb by lysozyme, though the adsorption conform to the Langmuir model. Such as T300, which possessed the largest BET surface area among the four samples, it deviated from the linearity obviously.

Figure 7. The maximum amount of adsorbed lysozyme vs. the BET surface area of mesoporous TiO2 with various geometrical morphologies. The dashed line is the guide to the eyes. We further presented the pore-size distribution of these mesoporous TiO2, as shown in Figure 8a. The pore size calculated by BJH method was the average pore size, as summarized in Table S1. These results show that there are still pores smaller than the lysozyme, though the average pore size is larger than the lysozyme (3×3×4.5 nm3). The BET surface area created by these smaller pores are not accessible to the lysozyme. According to the fact, we deducted the BET surface area provided by smaller pores (< 4.5 nm) and obtained the effective surface area (S’T, m2·g-1).54 The resulted effective surface areas are shown in Figure 8b. The value of S’T is largely decreases because of the existence of much small pores in T300 (ST→S’T, 237.9 m2·g-1→142.7 m2·g-1). With the increase of calculation temperature, the pore size increases, and the number of smaller pores decreases. Thus, the effective surface area is expected to hardly change at high temperature, such as T600 and T700 (ST→S’T,


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53.6 m2·g-1→52.9 m2·g-1 for T600, 29.2 m2·g-1→29.2 m2·g-1 for T700).

Figure 8. (a) Pore-size distribution curves of these mesoporous TiO2 samples calcinated by different temperatures. (b) The maximum amount of adsorbed lysozyme vs. the effective surface area of these mesoporous TiO2 with various geometrical morphologies. Insets: Effective surface area of T300, T500, T600 and T700. The dashed line is the guide to the eyes. Then we re-established the relationship between the effective surface area (S’T) and the adsorption capacity of lysozyme, qm, as shown in Figure 8b. It is found that qm increases linearly with increasing effective surface area of mesoporous TiO2. The slope is 0.413 and the correlation coefficient (R2) is 0.98. Together with the fact that lysozyme adsorption is mono-layer style (see Figure 6), the exact adsorption capacity of lysozyme per unit area can be obtained. According to Figure 7b, the qm/S’T for T300, T500, T600 and T700 are 0.61 mg·m-2, 0.77 mg·m-2, 1.06 mg·m-2 and 1.28 mg·m-2, respectively. Actually, the different values of the amount of adsorbed lysozyme per unit area (qm/S’T) for these four samples represent the different number of proteins contacting with the surfaces. Combination of Adhesion and Adsorption. Lastly, we calculated the adhesion force of one lysozyme molecule interacting with the surface, according to the correlation between the adhesion force (FA/Sc) and the amount of adsorbed lysozyme per unit area (qm/S’t). The linear relationship between qm/S’t and FA/Sc in Figure 9a indicates that the interaction strength between one lysozyme molecule and mesoporous TiO2 with


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various geometries is a constant value. Here, the unit of qm is “mg” and we converted the unit to be same as Num./S’T , divided by the molecule weight (kDa=14400 g·mol-1) of lysozyme and multiplied by Avogadro's number (NA=6.02 × 1023 mol-1). The Num./S’T for T300, T500, T600 and T700 are 2.55×1016 per one/m2, 3.24×1016 per one/m2, 4.45×1016 per one/m2 and 5.36×1016 per one/m2, respectively. In Figure 9b, the slope represents the adhesion force of one lysozyme molecule interacting with TiO2 surface. The value equals to 0.86 nN/per molecule. It is also worth to note that the interaction strength of one protein molecule interacting with a particular material is a fixed value, though the surface topography changes when it is prepared in different condition.

Figure 9. The adhesion force of lysozyme interacting with TiO2 surfaces per unit area vs. (a) the amount of adsorbed lysozyme per unit area and (b) number of adsorbed lysozyme molecules per unit area. The dashed lines are the guide to the eyes. To further verify this approach, other two model proteins, BSA and myoglobin were investigated. We measured both the equilibrium adsorption amounts of these two proteins onto mesoporous TiO2 surfaces and the adhesion force by AFM (see Table S2). All of the experimental condition and process were the same as those in the lysozyme. The results of FA/S’T as a function of Num./S’T, for these two proteins are shown in Figure 10. Interestingly, for the same proteins, the data points lie on the same straight lines. From the figure, we can also deduce the interaction strength of one protein molecule interacting with the mesoporous TiO2 surface, resulted from the


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linear fashion for both cases. The slopes corresponded to different proteins are different. The adhesion force of one protein molecule with TiO2 surface is about 0.86 nN, 2.63 nN and 4.41 nN for lysozyme, Myoglobin and BSA, respectively (see Table 4). These differences might mainly because of the different properties of the proteins, such as the dimensions, molecular weight, isoelectric point (pI) (see Table 4). All of these factors of proteins might contribute to the molecular interaction. Based on the above, it was found that the way to determine the molecular interaction force is universal for the different proteins. It is also admitted that the pH condition, has important impact on protein-surface interaction due to the effect of the pH-induced surface charge of protein. This would be taken into consideration in the future, about how does pH condition affect the interaction strength of one protein molecule interacting with the mesoporous TiO2 surfaces. Meanwhile, separation different proteins that have the same or similar pI/Mw is a hot topic and attracts considerable attention. In the next step, the study of the several types of proteins with one fixed feature (such as pI and Mw) is meaningful for controlling the interaction in the fields of protein separation.

Figure 10. The adhesion force of protein (BSA, Myoglobin) interacting with mesoporous TiO2 surface per unit area vs. the number of adsorbed protein molecules per unit area. The dashed lines are the guide to the eyes.


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Table 4. Parameters of Lysozyme, Myoglobin, BSA and the adhesion force of each one protein molecule with TiO2 surface. Dimensions, nm3

Molecule Weight, kDa

pI

One protein molecule-TiO2, nN

Lysozyme

3.0×3.0×4.5

14.4

10.0

0.86

Myoglobin

2.9×3.6×6.4

17.6

7.2

2.63

BSA

4.0×4.0×14.0

66.5

4.8

4.41

4. CONCLUSIONS In this study, a series of mesoporous TiO2 with various geometrical morphologies and surface roughness were prepared, and they were used as substrate samples to access the protein adsorption capacity and the adhesion force with materials. The AFM measurements showed that the adhesion force is related to the contact area between protein and surfaces. Based on the derivation of Hertz and JKR theories, the relationship between the protein adhesion interacting with TiO2 surface featuring distinct surface roughness and the contact area can be established. The better applicability of the Langmuir model over Freundlich model suggests that the mesoporous TiO2 surfaces are energetically homogeneous for lysozyme adsorption. According to the Langmuir isotherm model, a well-linear relationship between the amount of adsorbed proteins and the effective surface area was established. Then the interactions of a single protein molecule interacting with different TiO2 surfaces were obtained through the combination of the amount of adsorbed protein per unit area with the adhesion force per unit area. We also found that the adhesion force of one particular protein molecule interacting with TiO2 surface remain constant, and its value is only related to the type of protein molecule while independent of the geometrical topographies of the material. In future experimental studies, it would be interesting to examine the effects of pH condition and the distinct surface chemistry on molecular protein-surface interaction. These experimental findings on the molecular level cast helpful insights for material design towards biological applications and will stimulate model calculations and simulations of protein interaction with surfaces.


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ASSOCIATED CONTENT Supporting Information Figure S1: Adsorption kinetics of lysozyme on mesoporous TiO2 (T300~T700); Figure S2: N2 adsorption-desorption isotherms and pore-size-distribution curves of mesoporous TiO2 (T300~T700); Table S1: Structural parameters of mesoporous TiO2 (T300~T700) measured by BET; Figure S3: The distribution of the adhesion forces (measured at ~80 different positions) of lysozyme on mesoporous TiO2 (T300~T700); Figure S4: Zeta potential titration curves of mesoporous TiO2 (T300~T700); Table S2: The adsorption capacity and adhesion force of BSA and Myoglobin onto mesoporous TiO2 (T300~T700). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID 0000-0001-9244-6808 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by the National 973 Key Basic Research Development Planning Program (2013CB733500), the Major Research Plan of the National Natural Science Foundation of China (91334202), the National Natural Science Foundation of China (21506090 ). R. A. acknowledges the support from the National Natural Science Foundation of China (21606131) and the Fundamental Research Funds for the Central Universities (30916011351) at Nanjing University of Science & Technology.


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For Table of Content Only


Molecular Interactions of Protein with TiO2 by the AFM-Measured

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