Article pubs.acs.org/Langmuir
Ultrathin Hybrid Films of Polyoxohydroxy Clusters and Proteins: Layer-by-Layer Assembly and Their Optical and Mechanical Properties You-Xian Yan,† Hong-Bin Yao,† Scott E. Smart,‡ Li-Bo Mao,† Wei Hu,† Shaotang Yuan,‡ Laurence Du-Thumm,‡ James G. Masters,‡ Shu-Hong Yu,*,† and Long Pan*,‡ †
Division of Nanomaterials & Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, Collaborative Innovation Center of Suzhou Nano Science and Technology, Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China ‡ Colgate-Palmolive Company, 909 River Road, Piscataway, New Jersey 08854, United States S Supporting Information *
ABSTRACT: The hierarchical assembly of inorganic and organic building blocks is an efficient strategy to produce high-performance materials which has been demonstrated in various biomaterials. Here, we report a layer-by-layer (LBL) assembly method to fabricate ultrathin hybrid films from nanometer-scale ionic clusters and proteins. Two types of cationic clusters (hydrolyzed aluminum clusters and zirconium-glycine clusters) were assembled with negatively charged bovine serum albumin (BSA) protein to form high-quality hybrid films, due to their strong electrostatic interactions and hydrogen bonding. The obtained hybrid films were characterized by scanning electron microscope (SEM), UV−vis, Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence (XRF), and X-ray diffraction (XRD). The results demonstrated that the cluster-protein hybrid films exhibited structural homogeneity, relative transparency, and bright blue fluorescence. More importantly, these hybrid films displayed up to a 70% increase in hardness and up to a 100% increase in reduced Young’s modulus compared to the pure BSA film. These hybrid clusterprotein films could be potentially used as biomedical coatings in the future because of their good transparency and excellent mechanical properties.
I. INTRODUCTION In the past few decades, various hybrid materials with welldefined morphologies and hierarchical structures have been fabricated via the biomimetic mineralization process.1−3 However, the size of these artificial hybrids obtained from biomineralization can be achieved only on the nano/microscale and it is hard to aquire high performance, which is far away from macroscale bulk biomaterials generated by biological systems. In order to prepare artificial bulk composite materials with bioinspired multiscale hierarchical structures, the assembly technique was integrated into the synthesis procedure.4,5 Layerby-layer (LBL) assembly is a feasible technique for organizing oppositely charged components including polyelectrolytes, clusters, and nanoparticles into multilayered structural films via alternating depositions.6−13 Various multilayer hybrid films with specific functionalities have been prepared by using suitable nanoscale objects as building blocks by this method.14 For example, nacrelike polymer/amorphous calcium carbonate−polymer multilayer hybrid films were prepared via layerby-layer assembly,15,16 but the quality of CaCO3−polymer hybrid films was unsatisfactory due to the weak interaction of calcium carbonate and polymers. The strong interaction of the organic and inorganic parts is expected in the fabrication of high-quality hybrid materials. © 2014 American Chemical Society
Recently, the interaction between positively charged inorganic clusters and negatively charged biomolecules has attracted much attention.17−20 Deschaume and co-workers studied the interactions of commercial hydrolyzed aluminum ion clusters (Al13‑mers and Al30‑mers) with model proteins (bovine serum albumin (BSA) and lysozyme) and explored the formation of bio−inorganic hybrids via the interactions between ion clusters and proteins.19,20 Their studies showed that the surface charge of aluminum ion clusters is strongly related to their electrostatic interactions with proteins. Our group also studied the morphological and structural transformation process of the water-soluble zirconium−glycine hybrid cluster (Zr6(O)4(OH)4(H2O)8(Gly)8)12+ (CP-2) in a BSA protein matrix,18 showing that the formed bioinorganic hybrid structures are a result of strong electrostatic interactions. Taking advantage of these strong interactions between inorganic clusters and proteins, their ordered assembly is a promising route to fabricating macro-scale hybrid films.21−25 Meanwhile, the construction of protein-based hybrid films is highly desired in bioactive devices.9−13,26 Several approaches Received: January 31, 2014 Revised: April 17, 2014 Published: April 22, 2014 5248
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have been developed to prepare protein-based hybrid films including physical adsorption,27 bioaffinity immobilization,28 and Langmuir−Blodgett assembly.21 In addition, a number of protein-based high-quality hybrid films were prepared via layerby-layer assembly of oppositely charged protein molecules and nanoscale building blocks such as hemoglobin/clay nanoparticles,25 papain/clay minerals,22 heme proteins/polyelectrolytes/silica nanoparticles,24 and polyoxometalate/protein nanoparticles.23 These obtained hybrid films exhibited uniform nanostructures, interesting biosensing properties, electrochemical performance, and catalytic activity.15,16 However, highquality macroscale hybrid films constructed with polyoxohydroxy clusters and protein with a certain function and high performance are rarely reported. Moreover, the mechanical reinforcement effect of inorganic ion clusters in clusters/protein hybrid films has not been studied. To understand better the interactions of inorganic clusters with proteins and extend the applications of as-prepared hybrid materials, herein we report the fabrication and properties of robust polyoxohydroxy ion clusters−protein ultrathin hybrid films via the layer-by-layer assembly of BSA protein with Al30‑mer or CP-2 clusters. The layer-by-layer assembly process was monitored by UV−vis absorption spectra to show the homogeneous alternative adsorption of Al30‑mer or CP-2 clusters to BSA. The well-defined morphologies and structures of asprepared hybrid films were characterized by scanning electron microscopy (SEM) and small-angle X-ray diffraction. Moreover, Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence (XRF), and energy-dispersive Xrays (EDX) were used to characterize the components of the protein−cluster hybrid films. Optical properties were studied to show the transmittance and photoluminescence of the hybrid films. Finally, the nanoindentation tests were performed to study the nanoscale mechanical properties of the hybrid films. All of the results clearly show that the clusters acted not only as good building blocks but also as important inorganic binders which connect the protein to form hybrid films having structural homogeneity, relative transparency, and bright blue fluorescence properties. In this work, we also demonstrated that the mechanical properties of films were extensively enhanced with the introduction of polyoxohydroxy clusters into hybrid films. More interestingly, the reinforcement effects of clusters are related to their inherent structures and interactions with proteins.
Scheme 1. Schematic Illustration of the Layer-by-Layer Assembly of BSA/Cluster Hybrid Films
dipped into a 0.5 mg/mL Al30‑mer (or 0.5 mg/mL CP-2) solution for 10 min (3), and again rinsed with DIW twice for 1 min (4). This procedure was a single deposition cycle, and multilayered films with the desired number of layers were fabricated by repeating this cycle. The multilayered films were denoted as PDDA(BSA/Al30)n or PDDA(BSA/CP-2)n, where n is the number of layers in the film. Note that the last layer in all films was always BSA except in the samples for surface wettability measurements. An LBL procedure was also carried out with pure BSA for comparative purposes. Self-Evaporation Process to Fabricate a Thick, Pure BSA Film. The LBL assembly of pure BSA protein resulted only in a film with a thickness of several hundred nanometers, which was not thick enough for a nanoindentation test. A relatively thick, pure BSA film was prepared by evaporating approximately 15 mL of the 0.5 wt % BSA solution in a 55-mm-diameter × 15-mm-deep Petri dish with a cleaned glass slide in it, and it was subsequently dried at 80 °C for 48 h. Characterization. The UV−vis spectra were collected with a Shimadzu UV-2250. Zeta potential measurements were carried out by using a Beckman Coulter (Delsa Nano C). Scanning electron microscope (SEM) images were taken with a Zeiss Supra 40 scanning electron microscope at an acceleration voltage of 5 kV. Raman spectroscopy was carried out on a JY LABRAM-HR confocal laser micro-Raman spectrometer using Ar+ laser excitation with a wavelength of 514.5 nm. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were measured on a Thermo Nicolet 8700 at room temperature. The photoluminescence (PL) spectra of the samples were obtained on a Hitachi F-7000 PL at room temperature. X-ray powder diffraction (XRD) was carried out on a Japan Rigaku Dmax-γA X-ray diffractometer with Cu Kα radiation (λ = 1.54178 Å). An energy-dispersive X-ray (EDX) spectrum was taken with an FEI Sirion 200 scanning electron microscope with an INCA Oxford EDX system. X-ray fluorescence (XRF) analysis was carried out on a Shimadzu XRF-1800 analyzer using Rh Kα and Lα radiation with an excitation voltage of 40 kV and an electron current of 95 mA. Mechanical Measurements. Nanoindentation tests were performed using a Nano Indenter G200 system (Agilent Technologies). A Berkovich indenter was employed for the measurement based on a load-control method. A trapezoidal load profile was used, with 15 s loading and 10 s peak holding times. The thermal drift during nanoindentation was kept below 0.05 nm·s−1. Proper corrections for the contact area (calibrated with a fused quartz specimen), initial penetration depth, and instrument compliance were applied. To avoid the substrate effect on the mechanical property measurement of films, the maximum penetration depth during the tests was below 10−15% of the overall film thickness, and the peak load value was chosen to be
II. EXPERIMENTAL SECTION Materials. BSA (bovine serum albumin, biotechnology grade) was purchased from Solon Ind. Pkwy, Ohio. Al30‑mers and CP-2 were synthesized as described in previous works.18,29−31 Sulfuric acid (98 wt %) and hydrogen peroxide (30 wt %) were purchased from Sinopharm Chemical Reagent Co. Ltd. Poly(diallyldimethylammonium chloride) (PDDA) (MW = 100 000−200 000 g/mol, 20 wt % aqueous solution) was purchased from Aldrich. All reagents were used as received without further purification. LBL Fabrication of Hybrid Films. The detailed assembly process is illustrated in Scheme 1. A glass substrate was first cleaned in freshly prepared piranha solution (2:1 concentrated 98 wt % H2SO4/30 wt % H2O2: this solution is dangerous in contact with organic matter) for 30 min and then washed thoroughly with deionized water (DIW) and dried with a gentle flow of nitrogen. The cleaned substrate was dipped into 0.1 wt % PDDA (MW = 100 000−200 000 Da) solution for 10 min, rinsed extensively with DIW, and dried with a gentle flow of nitrogen to endow positive charge to the surface of the glass substrate. Then, the PDDA-coated glass substrate was immersed in a 5 mg/mL solution of BSA for 10 min (1), rinsed with DIW twice for 1 min (2), 5249
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2 mN. The relatively thick hybrid films ((BSA/Al30)100 and (BSA/CP2)60) with a thickness of 5 μm were chosen for nanoindentation tests. The pure BSA film (about 6 μm) prepared by the self-evaporation process was used for mechanical measurement.
CP-2, the charge-to-size ratio of CP-2 cluster is 1.5 times larger than that of the Al30 cluster. The CP-2 cluster is ball-shaped, and has a higher level of symmetry than the Al30 cluster, which is shaped as an elongated capsule. Both clusters contain a large number of hydrogen bonding sites; Al30 contains all H−O−Al2 or H2−O-Al sites, while CP-2 has predominantly (>50%) H−N sites. BSA is a water-soluble protein with a molecular weight of 66 500 Da composed of 580 amino acid residues. BSA is a versatile carrier protein with amphiphilic, anionic, and cationic properties, which makes it an excellent material for the adhesion, nucleation, and growth of bio−inorganic materials.17,18,32 Fabrication of PDDA(BSA/Cluster)n Hybrid Films via Layer-by-Layer Assembly. The layer-by-layer assembly of BSA and ion clusters on a PDDA-coated glass substrate was carried out by a cycled operation (Scheme 1) as described in the Experimental Section. The glass substrates were first modified with PDDA to endow the surface of the substrate with positive charge to facilitate the adsorption of BSA. The zeta potentials of Al30‑mer and CP-2 clusters in solution (0.5 mg/mL) are 38.2 and 26.36 mV, respectively, while that of pure BSA in aqueous solution (5 mg/mL) is −30.52 mV, showing their opposite surface charges. In the LBL assembly process (Scheme 1), negatively charged BSA and positively charged ion clusters were alternately attached to the PDDA-modified substrate through an electrostatic attraction and hydrogen bonding. Examples of the obtained (BSA/cluster)20 hybrid films on PDDA-modified glass substrates (highly transparent) are shown in Figure 5a. The layer-by-layer assembly processes of BSA and clusters on glass substrates were monitored by UV−vis absorption spectra to confirm the homogeneous and consecutive assembly. As shown in Figure 1a, the UV−vis absorbance of (BSA/Al30)n
III. RESULTS AND DISCUSSION Structures and Characteristics of CP-2, Al30‑mers, and BSA. The structure of CP-2 ((Zr 6 (O) 4 (OH) 4 (H 2 O) 8 (Gly)8)12+) polyoxocation hybrid clusters contains a hexanuclear zirconium(IV) core in the form of an octahedron where the eight vertical edges of each of the respective complexes are bridged by the carboxylate groups from the glycine ligands.18,29 The Al30‑mer (Al2O8Al28(OH)56(H2O)2418+) is a large Kegginbased polyoxohydroxy cluster formed partially of hydrolyzed aluminum solutions upon thermal treatment or aging of the solutions.19,30,31 Both CP-2 clusters and Al30 mers are monodisperse, hydrophilic, positively charged in solutions, and small (ca. 1 nm for CP-2 clusters and 1−2 nm for Al30 Keggin ions). A detailed comparison of these clusters is shown in Table 1. While the Al30 cluster has a +6 higher charge than Table 1. Summary of the Relevant Properties of the Two Clusters properties
charge
Al30
18
CP-2
12
charge/sizea 3.179e+/ nm−3 4.654e+/ nm−3
shape
hydrogen bonds
outside atoms
capsule
104/108
O
ball
44
O, N
a
Size represents the occupied volume as calculated from respective CIF files using Material Studios.
Figure 1. (a) UV−vis absorption spectra of (BSA/Al30) with different layer numbers. (b) Plot of UV−vis absorbance of peaks at 379 nm versus number of bilayer of BSA/Al30 on a glass substrate. (c) UV−vis absorption spectra of (BSA/CP-2) with different layer numbers. (d) Plot of UV−vis absorbance of peaks at 379 nm versus number of layer of BSA/CP-2 on a glass substrate. 5250
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Figure 2. FTIR (a) and Raman spectra (b) of different films for (1) a pure BSA film, (2) a BSA/Al30 hybrid film, and (3) a BSA/CP-2 hybrid film. The inset in (b) shows Raman spectra of two types of clusters.
Figure 3. Energy-dispersive X-ray (EDX) spectra of different films for (a) a pure BSA film, (b) a BSA/Al30 hybrid film, and (c) a BSA/CP-2 hybrid film.
hybrid films increased with the number of BSA/Al30 bilayers formed on the substrate, indicating that LBL assembly was carried out via electrostatic attraction and hydrogen bonding. In addition, the absorption intensity of hybrid films at 379 nm increased with consecutive cycles of the assembly (Figure 1b), which indicates that the deposition is a typical LBL assembly process. Furthermore, the UV−vis absorbance of (BSA/CP-2)n hybrid films (Figure 1c,d) also displays a monotonic increase with n, indicating a similar stepwise and regular film growth procedure via LBL assembly. As a control, a pure BSA film was also prepared by LBL assembly without using any ion clusters, and the LBL assembly process of BSA was monitored by UV− vis spectra as well. The UV−vis absorbance of BSA films also increased with the number of as-formed BSA layers due to the layer-by-layer adsorption of BSA through the hydrogen bonding interaction (Figure S1). However, the increasing rate
of UV−vis absorbance with deposition number was much lower than that of (BSA/Al30)n and (BSA/CP-2)n hybrid films, indicating that electrostatic interactions between ion clusters and BSA protein facilitated the LBL deposition process. FTIR and Raman Spectra of Hybrid Films. FTIR and Raman spectra were taken on these hybrid films to reveal their components. As shown in Figure 2a, the FTIR adsorption peaks at 3293, 2932, 1649, 1531, and 1393 cm−1 are assigned to the stretching vibration of −OH, the stretching vibration of −NH in amide A, the CO stretching vibration of amide I, the N−H bending mode of amide II, and the C−N stretching mode, respectively, which are fully consistent with the pure BSA.33,34 In the Raman spectra (Figure 2b), there is a dominant Raman peak at 2932 cm−1 along with other peaks at 1653, 1450, and 1332 cm−1 which are due to the presence of strong amide groups (I, III), CO in amino acid groups, and C−H 5251
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scissoring modes, respectively.35 In comparison, the clusters show no obvious Raman peak except for the N−H bending of CP-2 due to the glycine ligands (inset in Figure 2b). Both FTIR and Raman spectra provide clear evidence of the existences of protein in these hybrid films, indicating the participation of BSA in LBL assembly. EDX Spectra and XRD Patterns of Hybrid Films. Energy-dispersive X-ray (EDX) spectra (Figure 3) were further taken on these hybrid films to confirm their components. The signals of C, O, N, and S in EDX spectra in Figure 3 indicate the existence of BSA protein in all samples. The Al and Zr signals in EDX spectra of BSA/Al30 and BSA/CP-2 hybrid films (Figure 3b,c) confirmed the presence of Al30 and CP-2 clusters in the hybrid films, respectively, which indicates that the ion clusters were successfully adsorbed on the BSA layer during the LBL assembly process. Furthermore, the element analysis conducted by X-ray fluorescence (XRF) also confirmed this (see Table S1). XRD patterns (Figure S2) were also taken to show the phases of the films. The results indicate that all hybrid films are amorphous, which suggests that Al30‑mers and CP-2 species did not transform into other crystal phases such as aluminum hydroxide and zirconium hydroxide during the LBL assembly process. Furthermore, SAXRD (Figure S2b) confirmed the sandwichlike lamellar structure of the hybrid films induced by the LBL assembly. The basal reflection peaks in the low 2θ range of the as-obtained hybrid films indicated that the BSA/ Al30 and BSA/CP-2 hybrid films have periodic long-rangeordered structures with thicknesses of about 3.2 and 2.25 nm, respectively. Different from hybrid films, the pure BSA film exhibited no obvious small-angle diffraction due to the lack of ordered hybrid structure. This also means that the clusters were periodically assembled with BSA protein to form the sandwichlike lamellar hybrid structure. Morphologies and Microstructures of Hybrid Films. The morphologies of as-fabricated BSA/cluster hybrid films were studied by scanning electron microscopy (SEM). The cross-sectional SEM images show that the thickness of both hybrid films (PDDA/(BSA/Al30)20 and PDDA/(BSA/CP-2)20) is about 1 μm (Figure 4a,b), while the pure (BSA)20 film
prepared by LBL assembly exhibited only about a 230 nm thickness (Figure S3b), which is much lower than that of cluster/protein hybrid films. The rapidly growing thickness of the obtained hybrid films can reach several micrometers, indicating the success of LBL fabrication by using ion clusters as a bridging layer (Figure S4). It is also clear evidence that the clusters acted not only as building blocks but also as important inorganic binders connecting the protein to form a hybrid film. However, it is noted that the LBL assembly process has different average growth rates for the thickness of the two different hybrid films (ca. ∼50 nm/layer for the BSA/Al30 hybrid film and ∼80 nm/layer for the BSA/CP-2 hybrid film). SEM images (Figure 4) reveal approximately a 1 μm thickness for both CP-2 and Al30 hybrid films when n = 20. However, a 5 μm film is obtained with n = 60 for BSA/CP-2, but for BSA/ Al30, the number of layers is 100 to reach a similar thickness (Figure S4). The BSA/CP-2 film has a larger growth rate than the BSA/Al30 film in the LBL assembly process. The different growth rates of these films are probably due to the different interactions between the two types of clusters and proteins. The larger growth rate of the BSA/CP-2 layer indicates that the strong binding and preferred special configurations between CP-2 and BSA promote the deposition and layering process of BSA/CP-2.18 Furthermore, the top-view SEM images (Figure 4c,d) indicate that the surfaces of both hybrid films are continuous and uniform. The surface of the pure BSA film (Figure S3a) is very flat and smooth, while the surface of the BSA/CP-2 hybrid film is slightly fluctuating and the BSA/Al30 hybrid film has a more uneven surface. The differences in the surface smoothness of hybrid films are also due to the different interactions between the two types of clusters and proteins. Optical Properties of BSA/Cluster Hybrid Films. The finally obtained (BSA/cluster)20 hybrid films are transparent (inset in Figure 5a (1) and (2)). Accordingly, the optical transmittances of hybrid films were measured by a UV−vis spectrophotometer and are shown in Figure 5a. Interestingly, we note that the BSA/CP-2 hybrid film is more transparent than the BSA/Al30 hybrid film for the same number of layers. Furthermore, the BSA/Al30 hybrid film became translucent and eventually opaque when the number of layers was increased to hundreds (Figure S5b,c). The transmittance spectra show that the transmittance of these hybrid films decreased with an increasing numbers of BSA/cluster bilayers. As shown in Figure S5, the (BSA/CP-2)20 hybrid film has ∼95% optical transmittance in the visible light region, but the optical transmittance of (BSA/CP-2)40 and (BSA/CP-2)60 films decreases to ∼78 and 51%, respectively. The (BSA/Al30)20 hybrid film exhibits ∼80% optical transmittance, while (BSA/Al30)40 and (BSA/Al30)100 films show only ∼39 and 4% transmittance, respectively. These results also indicate that the smaller CP-2 clusters can form more homogeneous hybrid films with BSA through LBL assembly compared to the Al30 clusters. The photoluminescence of hybrid films was investigated as well. Blue fluorescence was observed when the films were under UV light irradiation (Figure S6). Figure 5b shows the fluorescence spectra of these films, which indicates that the hybrid films emitted light at around 437 nm upon excitation by 360 nm light, the same as that of the pure BSA protein film. These results show that the ion clusters in hybrid films did not affect the fluorescence of the BSA protein. Furthermore, the photoluminescence peak of hybrid films of both (BSA/Al30) and (BSA/CP-2) also increases with bilayer number n as shown in Figure S6, indicating that the fluorescence intensity of the
Figure 4. Cross-sectional and surface SEM images of different films. (a) Cross-sectional and (c) top-surface SEM image of the (BSA/ Al30)20 hybrid film, respectively. (b) Cross-sectional and (d) topsurface SEM image of the (BSA/CP-2)20 hybrid film, respectively. 5252
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Figure 5. Transmittance spectra (a) and fluorescence spectra (b) of different films. Insets in (a) are photographs of as-fabricated (1) PDDA(BSA/ Al30)20 and (2) PDDA(BSA/CP-2)20 hybrid films, respectively.
on the measured mechanical properties of the films, a relatively thick hybrid film ((BSA/Al30)100 hybrid film or (BSA/CP-2)60 hybrid film) was used for mechanical measurements to ensure that the maximum penetration depth during the tests was below 10−15% of the overall film thickness. A load-control method was employed to identify the hardness of as-prepared films, and the value of the maximum applied force was chosen to be 2 mN. The typical load−displacement curves of these films are shown in Figure 7. In the first stage, all the
hybrid films is highly dependent on the amount of BSA in hybrid films. Surface Wettability of BSA/Cluster Hybrid Films. The surface wettability of hybrid films has been measured to identify the influence of clusters on the surface affinity of hybrid films (Figure 6). It was observed that the surface of the pure BSA
Figure 6. Water contact angle measurements for different films. (A) the pure BSA film; (B, C) (BSA/Al30)20 hybrid films with the outermost layer of BSA and Al30 clusters, respectively; and (D, E) (BSA/CP-2)20 hybrid films with the outermost layer of BSA and CP-2 clusters, respectively.
Figure 7. Representative load−displacement nanoindentation curves of different films.
film exhibits hydrophilicity, and the contact angle is about 72°. When the top surface layer of films was BSA, both (BSA/ Al30)20 and (BSA/CP-2)20 hybrid films exhibit hydrophilicity with a slight increase in the contact angle (about 77 and 74°, respectively). The increase in the contact angles is due to the rougher surfaces of hybrid films.36 When the top surface layer of a hybrid film was covered with clusters, the contact angle further increased to a higher value (about 83.3° for the (BSA/ Al30)20 film and 81.7° for the (BSA/CP-2)20 film). This means that the cluster layer shows more hydrophobicity than the BSA layer due to the rougher surface and more hydrophobic functional groups (Figure S8). Mechanical Properties of BSA/Cluster Hybrid Films. With LBL assembly with organic components, the inorganic building blocks (i.e., nanoparticles, nanosheets, and nanowires)37−41 show an impressive reinforcement effect in asfabricated hybrid materials. However, the reinforcement effect of inorganic ion clusters in hybrid films is still not clear. Here, we measured the mechanical properties of these hybrid films by the nanoindenter. To avoid the influence of the glass substrate
displacements increased with increasing loading force. When the loading force reached 2 mN, the displacements of the pure BSA film, BSA/CP-2 film, and BSA/Al30 film were about 485, 440, and 370 nm, respectively. It is clear that the penetration depth in BSA/cluster films is smaller than that of the pure BSA film, which suggests that the BSA/cluster films are harder than the pure BSA film. Of the two, the BSA/Al30 film is the mechanically harder material. In the second stage, with the peak load holding a specific period, the plats of displacement lasted about 16, 11, and 9 nm for the pure BSA film, the BSA/CP-2 film, and the BSA/Al30 film, respectively. These results indicated that it is more difficult to go deeper into BSA/ cluster films than into pure BSA films under the same load due to their greater hardness. In the third stage, the slope of the unloading indentation segment for the BSA/cluster films is more pronounced than that for the pure BSA film, suggesting a larger modulus for the BSA/cluster hybrid films. Furthermore, the load−displacement curves also suggested that in order to reach the same depth (for example 200 nm), larger load forces were required for BSA/cluster films than for the pure BSA film 5253
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(about 0.38, 0.46, and 0.64 mN for the pure BSA film, the BSA/ CP-2 film, and the BSA/Al30 film, respectively), which also indicated that the BSA/cluster films are much harder than the pure BSA film. The hardness and reduced Young’s modulus of different films obtained from the nanoindentation measurements are summarized in Table 2, indicating that the ion clusters exhibited an obvious reinforcement effect in the mechanical properties of hybrid films.
a 100% increase in the reduced Young’s modulus compared to the pure BSA film. The results have demonstrated the potential application of inorganic clusters as reinforcement fillers in novel hybrid materials. These hybrid films assembled from inorganic clusters and protein could be potentially used as biomedical coatings in the future because of their good transparency and excellent mechanical properties.
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samples
reduced Young’s modulus (GPa)
hardness (GPa)
pure BSA film BSA/CP-2 film BSA/Al30 film
8.9 ± 1.0 12.9 ± 1.2 17.8 ± 3.0
0.40 ± 0.03 0.47 ± 0.06 0.69 ± 0.14
ASSOCIATED CONTENT
S Supporting Information *
Table 2. Summary of the Mechanical Properties of Films Measured by Nanoindentation
UV−vis absorption spectra, XRD patterns, SEM images, fluorescence spectra, and photographs of hybrid films. This material is available free of charge via the Internet at http:// pubs.acs.org.
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Of the two hybrid films, this reinforcement effect is particularly prominent in the Al30/BSA hybrid film, where the Young’s modulus increased 101% and the hardness increased 70% from those of the pure BSA film, compared to a 45% increase in the modulus and an 18% increase in the hardness seen in the CP-2/BSA hybrid film. Furthermore, the changes in mechanical properties are not solely dependent on the alternating LBL deposition of protein, which is accomplished in both hybrid films, but is more related to the specific interactions between the clusters and protein. As addressed in the discussion of structure and properties of clusters above (Table 1), the higher charge of Al30 cluster implies that there exist stronger binding interactions with BSA than with the CP-2 cluster, resulting in a tighter and more closely bound hybrid film. It was predicted that Al30 binds to BSA so that the secondary structure of the protein is not greatly altered (as in chelation) but leads to clear morphological changes on the material’s surface.20 The CP-2 cluster, however, is greatly dispersed among BSA molecules, and its inability to alter the morphology of BSA layers indicates the homogeneity of the CP-2/BSA interaction. This is also confirmed in the optical properties of the hybrid films. It is possible that the smaller, higher-charge-density CP-2 particles have sufficient inter-repulsive forces to prevent penetration into certain parts of the protein. The relative ratio of cluster and BSA further confirmed this conjecture. The elemental analysis conducted by X-ray fluorescence (XRF) revealed that the ratio of Al/S is larger than that of Zr/S (see Table S1), where sulfur is attributed to BSA, showing that the relative content of Al30 in the BSA/Al30 film is much larger (∼28-fold) than that of CP-2 in the BSA/CP-2 film, while the Al30 concentration in the starting solution is lower than that of CP-2. (See the theoretical calculation in the SI.) However, significant differences in the resulting mechanical properties indicate the viability and potential of various inorganic clusters for LBL-assembled hybrid films.
AUTHOR INFORMATION
Corresponding Authors
*Fax: + 86 551 63603040. E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China (grants 2012BAD32B05-4, 2010CB934700, 2013CB933900, and 2014CB931800), the National Natural Science Foundation of China (grants 91022032, 91227103, 21061160492, and J1030412), and the Chinese Academy of Sciences (grant KJZD-EW-M01-1).
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IV. CONCLUSIONS We demonstrated that two types of cationic clusters (hydrolyzed aluminum clusters and zirconium-glycine clusters) can be assembled with negatively charged bovine serum albumin (BSA) protein to form high-quality hybrid films due to their strong electrostatic interactions and hydrogen bonding. The cluster−protein hybrid films have structural homogeneity, relative transparency, and bright blue fluorescence. These hybrid films exhibit up to a 70% increase in hardness and up to 5254
dx.doi.org/10.1021/la500434a | Langmuir 2014, 30, 5248−5255
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