Î²-SiC Composite Films for

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Controlled Surface Chemistry of Diamond/#-SiC Composite Films for Preferential Protein Adsorption Tao Wang, Stephan Handschuh, Yang Yang, Hao Zhuang, Christoph Schlemper, Daniel Wesner, Holger Schönherr, WenJun Zhang, and Xin Jiang Langmuir, Just Accepted Manuscript • DOI: 10.1021/la404277p • Publication Date (Web): 11 Jan 2014 Downloaded from http://pubs.acs.org on January 19, 2014

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Controlled surface chemistry of diamond/β-SiC composite films for preferential protein adsorption Tao Wang1, Stephan Handschuh-Wang2, Yang Yang3, Hao Zhuang1, Christoph Schlemper1, Daniel Wesner2, Holger Schönherr2, Wenjun Zhang3 and Xin Jiang1,* 1

Institute of Materials Engineering, University of Siegen, Paul-Bonatz-Str. 9-11, 57076 Siegen,

Germany, 2Physical Chemistry I, University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany, and 3Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong. * Corresponding Author Email: [email protected] Abstract Diamond and SiC both process extraordinary biocompatible, electronic and chemical properties. A combination of diamond and SiC may lead to highly stable materials e.g. for implants or biosensors with excellent sensing properties. Here we report on the controllable surface chemistry of diamond/β-SiC composite films and its effect on protein adsorption. For systematic and high-throughput investigations, novel diamond/β-SiC composite films with gradient composition have been synthesized using the hot filament chemical vapor deposition (HFCVD) technique. As revealed by scanning electron microscopy (SEM), the diamond/β-SiC ratio of the composite films shows a continuous change from pure diamond to β-SiC over a length of ~ 10 mm on the surface. X-ray Photoelectron Spectroscopy (XPS) and Timeof-flight secondary ion mass spectrometry (ToF SIMS) was employed to unveil the surface termination of chemically oxidized and hydrogen treated surfaces. The surface chemistry of the composite films was found to depend on diamond/β-SiC ratio and the surface treatment. As observed by confocal

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fluorescence microscopy, albumin and fibrinogen were preferentially adsorbed from buffer: After surface oxidation the proteins preferred to adsorb on diamond rather than on β-SiC, resulting in an increasing amount of proteins adsorbed to the gradient surfaces with increasing diamond/β-SiC ratio. By contrast, for hydrogen treated surfaces, the proteins preferentially adsorbed on β-SiC, leading to a decreasing amount of albumin adsorbed on the gradient surfaces with increasing diamond/β-SiC ratio. The mechanism of preferential protein adsorption is discussed by considering the hydrogen bonding of the water self-association network to OH-terminated surfaces and the change of the polar surface energy component, which was determined according to the van Oss method. These results suggest that the diamond/β-SiC gradient film can be a promising material for biomedical applications which require good biocompatibility and selective adsorption of proteins and cells to direct cell migration.

Keywords: protein adsorption, diamond/β-SiC composite film, gradient, surface chemistry

Introduction Biological tissues are suggested to interact with only the outermost atomic layers of an implant and consequently much research has been devoted to modify the surfaces of existing biomaterials in order to achieve more desirable biological integration.1,

2

One of the biggest challenges for integrating

microelectronics and biotechnology is the need to develop interfaces that are not only compatible with microelectronics processing methods, but also stable and selective when exposed to biological environments.3 Most of currently available substrates, such as gold, silicon, or glassy carbon can be biologically modified, but the degradation of the functionalized interfaces often prevents the development of long time stable integrated biological devices. Moreover, such materials cannot fulfill all desired properties like high chemical stability, possible biochemical surface modification and high


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reproducibility.3, 4 In this regard, diamond and SiC can be promising candidates for biomedical and biosensor applications due to their extraordinary biocompatible, electronic and chemical properties.5, 6 Diamond is famous of its combined properties of high hardness, high thermal conductivity, semiconductivity, chemical inertness, corrosion resistance and good biocompatibility. It has been utilized in chemical and biological sensors, DNA and protein chips, and electrodes for electrocatalytic reactions.7 In particular, diamond surfaces can be modified, producing DNA layers exhibiting higher stability than those on gold, silicon, and SiO2.3 Likewise, silicon carbide is also an attractive substrate for biosensing applications due to its unique electronic properties, mechanical robustness, chemical inertness, thermal stability, non-toxicity, and biocompatibility.8,

9

Recently, SiC based implantable

sensors, porous membranes for protein separation, microelectrode arrays, and micro-systems have been investigated.10-12 Due to the unique properties and different surface terminations of diamond and SiC,13 the combination of surface terminated diamond and SiC may lead to highly stable implants, which induce controlled, guided, and rapid healing or biosensors with excellent sensing properties. One possible way to achieve such a combination is the synthesis of diamond/β-SiC composite films, which have been successfully deposited before by microwave plasma and hot filament chemical vapor depositions (MWCVD and HFCVD).14, 15 The surface chemistry can be changed with different diamond/β-SiC ratios by adjusting the gas concentration during the deposition process. Moreover, gradient surfaces are utilized as a valuable materials research tool that allows one to perform systematic studies with continuously varying surface parameters within a single experiment. An entire library of information on materials’ behavior under different experimental conditions can be generated with minimal time and material.16-20 Furthermore, gradient surfaces are widely utilized to investigate interactions between proteins and surfaces and to determine the surface conditions for cell


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attachment, growth, proliferation, and phenotype changes for the development of medical devices and artificial organs.21-23 It has been suggested that in all biological processes, the first stage of implant integration is the surface adsorption of protein on the implant surface followed by cell attachment.24, 25 Thus, protein adsorption and adhesion play a prominent role in determining the biocompatibility and -integration of biomaterials. In order to systematically study the surface chemistry and biointerface properties of diamond/β-SiC composite films, novel diamond/β-SiC gradient surfaces with continuously varied surface chemistry have been synthesized and characterized in detail, as reported in this paper. The adsorption behavior of two important proteins, namely bovine serum albumin (BSA) and fibrinogen, was unraveled and showed an intimate relation to the local surface chemistry and composition of the diamond/β-SiC composite films, which provides useful clues for the future development of diamond/βSiC based biomaterials. Experimental Section Film Deposition. Gradient diamond/β-SiC composite films were synthesized by hot-filament chemical vapor deposition (HFCVD) with a special filament/substrate configuration. P-type (100) single crystallized Si wafers (thickness of 500 µm, single side polished, prime grade, Siegert Consulting e. K. Aachen, Germany) were used as substrates. Prior to film deposition, the substrates were immersed in piranha solution (H2SO4:H2O2 3:1) for 30 min followed by ultrasonically cleaning in distilled water for three times. Warning: Piranha solution is highly reactive and may explode upon contact with organic material, such as solvents. Extreme precautions must be taken at all times. Afterwards the samples were ultrasonically seeded with a 5 nm nanodiamond dispersion (0.05 wt% in water) for 30 min in order to enhance the diamond nucleation. The samples were then dried with flowing N2. The deposition was carried out at a constant gas pressure of 30 mbar. The flow rates of H2, CH4 and tetramethylsilane (TMS,


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1% TMS diluted in H2) were maintained at 500, 5 and 30 sccm. Two tantalum wires with a diameter of 0.6 mm were used as hot filaments (Chen Fei Baoji Nonferrous Metal Co. China, purity of 99.95%). The configuration of hot filament and substrate is shown in Figure 1. In general, the samples were positioned directly below the middle of the filaments to obtain homogenous films. However, in order to obtain gradient surface, the substrates were shifted partly away from the filaments as shown in Figure 1b. The filament temperature was around 2500°C, as measured by an optical pyrometer (IR-GAG CHINO, Mawi-therm, Monheim, Germany). The substrate temperature was between 880 (±20) to 766 (±20) °C along the length of substrate measured by thermocouples (Type K, nickel-chromium/nickel, TC Direct, Germany). Surface Treatment. After the deposition, two kinds of surface treatments were employed as follows. Oxidation. The film was first heated at 250°C for 30 min in an oxidizing mixture of concentrated H2SO4 and KNO3 in a beaker, and afterwards dipped into HF solution (HF/HNO3=1/15) in order to remove the SiO2 on the surface. The samples were finally ultrasonically washed three times in Milli-Q water (from a Direct-Q 8 system (Millipore) with a resistivity of 18.0 MΩ/cm) and dried with flowing N2. H-treatment. After oxidation, the samples were treated in HFCVD with H2 at a gas pressure of 10 mbar for 15 min. Protein Adsorption. Bovine serum albumin (BSA) conjugated with fluorescein isothiocyanate (FITC) was purchased from Sigma/Aldrich. FITC is coupled to the protein through the ε-amino group of the lysines of the albumin. The degree of substitution is 7 to 12 moles of FITC per mole of albumin, according to the producer. Fibrinogen from human plasma conjugated with Alexa Fluor 647 was purchased from Life Technologies GmbH. There are approximately 15 molecules of Alexa Fluor 647 dye for each fibrinogen molecule. All proteins were used as received. Albumin was diluted in 10 mM


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Tris buffer, which was previously adjusted to pH 7.0 by addition of hydrochloric acid. The final concentration of albumin was 1 mg/mL. Fibrinogen was diluted in 0.1 M sodium bicarbonate buffer with pH 8.3. The concentration of fibrinogen was 0.1 mg/mL. These different concentrations were chosen, because the concentration of albumin is known to be around 10 times more than fibrinogen in the human blood (albumin: 35-55 mg/ml and fibrinogen: 2-4 mg/ml).26 The gradient composite films with two different surface treatments were immersed in the albumin or fibrinogen solution, respectively, for 1 h. Subsequently, the samples were washed with buffer for 5 times and finally kept immersed in buffer. For various adsorption measurements, at least 3 samples were tested.

Figure 1. Schematic illustration of the hot filament-substrate configuration.


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Surface Morphology and Topography Measurements. The surface morphology of the gradient composite films was examined using a Zeiss ultra55 Field Emission Scanning Electron Microscopy (FE-SEM, Carl Zeiss NTS GmbH, Germany). All measurements were performed with an operation voltage of 5 kV with the Inlens secondary electron detector.

Surface topography and roughness

information of the gradient films was acquired by tapping mode atomic force microscopy (AFM) on an Asylum Research MFP-3D AFM (Asylum Research, Santa Barbara, CA). Topography images were obtained with standard silicon cantilevers (Olympus, AC 160TS, nominal resonance frequency of 300 kHz) with a nominal tip radius of 7 nm. 10 × 10 µm2 scans with a pixel resolution of 2048 × 2048 were taken on each sample with a scan rate of 0.1 Hz and an amplitude and amplitude setpoint ratio of approx. 85 nm and 0.84, respectively. All data were acquired at room temperature and atmospheric pressure. X-ray Photoelectron Spectroscopy (XPS). High resolution X-ray photoelectron spectra were recorded at a pressure of 1~3 × 10-9 mbar with a VG ESCALAB 220i-XL ultrahigh vacuum (UHV) surface analysis system (VG Scientific, England), using an Al Kα monochromatic radiation of 1486.6eV. All XPS spectra were measured with a step of 0.050 eV using a pass energy of 20.0 eV. The data were collected at an angle 54.7°, as defined by the position of the detector relative to the normal of the sample surface. The measurement area of each sample was 500 µm2. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) Measurement. The surface termination of samples after the two different surface treatments was examined by using ToF-SIMS. The spectra were recorded using a ToF-SIMS IV instrument (ION-TOF GmbH, Münster, Germany). Negative secondary ion mass spectra were acquired over the mass range from m/z = 0 to 100 using Bi+ ions (target current 1.0 pA). The analysis area for each spectrum was 500 µm × 500 µm, and the acquisition time was set to 30 s.


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Contact Angle Measurement. Static contact angle measurements were performed on the gradient composite films after both treatments using the sessile drop method with an OCA 15plus instrument (Data Physics Instruments GmbH, Filderstadt, Germany) with Milli-Q water at ambient conditions. The static contact angle of surface treated pure diamond and SiC films were measured using the following liquids: Milli-Q water, diiodomethane (99%, Alfa Aesar), glycerol (99.6%, Acros Organics) and ethylene glycol (puriss. p.a., Reag. Ph. Eur., Riedel deHaen). Their surface tension components are obtained from the literature.27 Fluorescence Microscopy Measurement. The fluorescence microscopy data of samples exposed to labeled protein were measured at room temperature in buffer solutions (see above) using a Microtime 200 laser scanning fluorescence microscope (PicoQuant, Berlin, Germany) comprising an OLYMPUS IX-71 frame (Olympus, Hamburg, Germany), a commercial main optical unit (Microtime 200, PicoQuant, Berlin, Germany), a fiber coupling unit (FCU II, PicoQuant Berlin, Germany) and a data acquisition module (PicoHarp 300, PicoQuant, Berlin, Germany). The FITC and Alexa Fluor fluorophores were excited by pulsed lasers at a wavelength of 485 nm (pulse rate 20 MHz, LDH-D-C485, PicoQuant, Berlin, Germany) and 635 nm (pulse rate 20 MHz, LDH-P-C-635B, PicoQuant, Berlin, Germany) pulsed lasers, respectively. The emitted photons were detected with a Single-Photon Avalanche Diode detector (PD1CTC, Micro Photon Devices, Bolzano, Italy). Samples were held on a XYZ piezo controlled scanner. The gradient areas were measured by moving the sample in X direction in steps of 0.1 mm. For each area, fluorescence emission intensity images were taken at an area of 80 µm × 80 µm with a pixel resolution of 256 × 256. The zoomed-in areas of 10 µm × 10 µm were also analyzed with pixel resolutions of 512 × 512 or 256 × 256 to obtain the emission intensity of the dyes. Dwell times of 3 × 0.2 ms / pixel or 10 × 0.2 ms / pixel were utilized.


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Results and Discussions Figure 2 shows the continuous changes of the surface morphology (SEM), surface topography (AFM) and protein adsorption (fluorescence microscopy) on the gradient surface at distances of 1 mm to 7 mm away from the edge of the sample. The surface morphologies of the local areas of composite gradient films are shown in Figure 2a. Over a length of 7 mm the surface changed from pure β-SiC to pure diamond. The areas with bright contrast (i.e. with higher secondary electron emission intensity) correspond to diamond, whereas the areas with dark contrast correspond to β-SiC. At 1 mm, i.e. the area which was furthest away from the hot filaments, there was only β-SiC in the film. At 7 mm, which was nearest to the filaments, pure diamond crystallites were detected. With increasing distance between filaments and substrate, the gases were hence less activated for diamond growth, which resulted in a lower content of diamond in the film. Therefore the diamond/β-SiC radio gradually decreased along the length of the film with increasing distance between filaments and substrate. The surface topographies of the gradient film in the same areas are shown in Figure 2b. The rms (root mean square) roughness, determined at a scan size of 10×10 µm2 of local areas at 1 mm, 3 mm, 4 mm and 7 mm were 8.8 nm, 66.8 nm, 77.3 nm, 58.1 nm, respectively. Diamond crystallites were found to be about 250 nm higher than β-SiC. Accordingly, with increasing content of diamond in the diamond/β-SiC composite film, the roughness increased. For the pure diamond film there were no β-SiC islands, hence the roughness was reduced again.


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Figure 2. (a) FE-SEM and (b) AFM topography images at different positions (1 mm, 3 mm, 4 mm and 7 mm away from the edge of the film) on a diamond/β-SiC composite gradient film. Confocal fluorescence images of (c) albuminFITC adsorbed on an oxidized gradient film, (d) albuminFITC adsorbed on a H-treated gradient film, and (e) fibrinogen

Alexa Fluor

adsorbed on an oxidized gradient film at

different positions.

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After deposition of the diamond/β-SiC composite gradient films, oxidation and H-treatment were performed to change the surface terminations, resulting in different wettabilities (Figure 5, 6) and protein adsorption abilities (Figure 2c, d, e). To evaluate the surface chemical composition and the species present at the surface, XPS spectra of β-SiC, diamond and diamond/β-SiC composite surfaces after oxidation (O-) and H-treatment (H-) were recorded. The result of the deconvolution of the spectra into different components and fitting parameters were collected in the Supporting Information. On oxidized surface, each surface Si atom is bound to one oxygen atom only (Si-O, 101.5 eV).28, 29 The SiO2 was removed after HF etching during the oxidation process. After H-treatment, there are 50% SiO and 6% silicon in a high oxidation state, i.e. SiO2 on the surface,28 rather than silicon atom bond to hydrogen. Bernhardt et al. have found that the surface of β-SiC was covered by a highly ordered monolayer of silicon dioxide after hydrogen plasma or etching in hydrogen flow.30 The arrangement of surface oxygen could also be changed, but the oxygen could not be replaced by hydrogen after Htreatment in our experiment. Except for these Si atoms, the remnant of the surface is occupied by C atoms. A C-O bond (283.7 eV) on the surface carbon atoms was found on both treated surface.28, 29 However, there is only Si3C-H (284.0 eV), one carbon atom is bound to three silicon atoms and one hydrogen atom, bound on the H-treated surface.31, 32 Accordingly, on the oxidized β-SiC surface, silicon atoms are bound to oxygen and the carbon atoms are also predominantly bound to oxygen. After Htreatment, silicon atoms are mainly bound to oxygen, and nearly half of carbon atoms bound to oxygen, another half bound to hydrogen (for detailed calculations, see Supporting Information). For the pure diamond surface, a survey scan shows that there is no detectable O signal on the H-treated surface, but 6.8% O atoms on the oxidized surface. On the oxidized diamond surface, sp3 bonded carbon in bulk (284.8 eV),33, 34 sp2 bonded carbon (283.7 eV),33 C-O-C ether bonds (285.9 eV) and C=O ketone bonds (288.0 eV)33 were detected. On the H-treated diamond surface, the strong peak at 284.2 eV is attributed to sp3 bonded carbon in bulk diamond. The weak peak at 285.0 eV can be ascribed to carbon atoms


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bonded in polyhydride configurations (-C-Hx, x ≥ 2) adsorbed on the surface.34 After H-treatment, the bonds of the sp2 C-C, C-O-C and C=O all are etched away due to H radicals in the gas-phase. The diamond/β-SiC composite films consist of diamond and β-SiC. The surface chemical state of the composite film is a combination of diamond and β-SiC. The fractional diamond coverage ( ) was at first introduced, which means the area percentage of diamond on the surface, determined by analyzing FE-SEM images using the software Scanning Probe Image Processor (SPIP, V5.0.7, Image Metrology A/S, Hørsholm, Denmark). For pure β-SiC and diamond, the values are 0 and 100%, respectively. Figure 3 shows the relative concentrations of certain chemical bonds with different , which summarizes the XPS results of diamond, β-SiC and the composite films. After oxidation, the concentration of Si-O and C-O (from β-SiC) decreases and the concentration of C-O-C increases with increasing . After H-treatment, the concentration of Si3C-H, Si-O and C-O (from β-SiC) bonds decreases, and the concentration of -C-Hx increases with increasing . Accordingly, a broad range of combinations of surface terminations can be obtained on diamond/β-SiC composite films after oxidation and H-treatment.


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Figure 3. Relative atom concentration versus fractional diamond coverage after (a) oxidiation and (b) H-treatment, respectively. Furthermore, ToF-SIMS was employed to analyze each surface on diamond and β-SiC samples after oxidation (O-) and H-treatment (H-) (Figure 4). The mass fragments of C¯, CH¯, O¯, OH¯, C2¯ and C2H¯ at m/z 12.000, 13.008, 15,995, 17.003, 24.000 and 25.008 were observed, respectively. The relative signal intensities were compared by computing I/I0, where I is the signal intensity observed for a certain mass fragment, I0 is the sum of all intensities calculated by adding all peak intensities of one spectrum. For diamond films, the relative fraction of OH and O at the oxidized surface is higher than at the Htreated surface, whereas the relative fraction of C, C2 and C-H bonds at the oxidized surface is much lower than on the H-treated surface. The XPS results also showed that there are C-O-C and C=O bonds on oxidized diamond, but no oxygen was detected on H-treated diamond. Recent studies also showed that diamond is H-terminated after hydrogen plasma treatment, but O-/OH-terminated after oxidation. 7, 35, 36

Both our results and the literature thus indicate that the surface of diamond is OH- and O-

terminated after oxidation, and the surface of diamond is H-terminated after H-treatment. For oxidized and H-treated β-SiC films, the relative fraction of C, C2 and C-H bonds is similar, whereas the relative fraction of O and OH on oxidized β-SiC film is more than that on H-treated β-SiC films. The XPS results indicated that there are Si-O and C-O bonds on both treated β-SiC, but Si3C-H only on the Htreated surface. Previous work also showed that HF etching of oxidized SiC leads to OH termination due to the inability of HF to remove the last oxygen layer at the oxide/SiC interface.28 Therefore the carbon and silicon on oxidized β-SiC surface are OH terminated. After H-treatment, there is not only OH termination on the surface, but also a fraction of C-H bond on β-SiC surface. The concentration of Si3C-H bond on SiC was calculated to be 21%, and the rest surface bonds are OH with concentration of 79%. The ToF-SIMS data is in full agreement with XPS data.


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Figure 4. ToF-SIMS spectra of pure diamond and β-SiC films after oxidation (O-) and H-treatment (H-). The variations of contact angle of water on the surfaces after oxidation and H-treatment are shown in Figure 5. From the β-SiC dominated surface to the diamond dominated surface, the contact angles on the oxidized samples changed from below 10° to 28° ± 2°. The surfaces were OH-terminated, resulting in a highly hydrophilic surface. On the other hand, the contact angles on the H2 treated gradient surfaces decreased from 90° ± 2° to 24° ± 1°. This result is similar to former work on films deposited by microwave plasma-enhanced chemical vapor deposition (MWCVD).13 The H-terminated diamond surface was hydrophobic. With increasing content of diamond in the composite film, the surface gradually changed from hydrophilic to hydrophobic because of the increasing fraction of H-terminated diamond and decreasing fraction of OH-terminated β-SiC. The wettability of diamond/β-SiC composite films hence strongly depends on the diamond/β-SiC content ratio.


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Figure 5. CCD camera images of water drops in contact angle measurements of gradient films after oxidation (a) and H-treatment (b) from pure β-SiC (left side) to pure diamond (right side). Because of the gradient nature of the surface chemical composition, the water drop moves to one side and expected contact angles change. The position-resolved contact angles could not be measured on the inhomogeneous gradient surface. Pure β-SiC, diamond and two homogenous composite films with certain fixed diamond/β-SiC ratios were synthesized for further contact angle and surface energy studies. measured from the FE-SEM data for pure β-SiC, diamond, and two homogenous composite films were 0, 100%, 31% and 68%, respectively. Figure 6a shows the plots of the water, diiodomethane and glycerol contact angles on the oxidized and H-treated composite films versus . For water there is a monotonic relationship of the contact angle with composition, which is shown in Figure 6a. Due to the patchy morphology of the two different components in the composite films, the Cassie equation37, 38 has been utilized to estimate independently as follows: cos cos 1 cos

(1)

where is the contact angle for water on the composite surface, is the contact angle on pure diamond with area fraction , and is the contact angle on pure SiC with area fraction 1-


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. For instance, for = 24° on an oxidized composite film, was calculated to be 34%, which is similar with value of determined from FE-SEM data of 31%. For = 84° on a Htreated composite film, was calculated to be 71% which is similar to the value of 68% from FE-SEM. For contact angles measured with diiodomethane and glycerol, similar values were obtained. Therefore, the SPIP measured value is approximately equal to the calculated with Cassie equation, implying that the main effect on is due to the chemical composition rather than roughness. These results suggest in accordance that the wettability varies with the diamond/β-SiC ratio on the surface due to different concentrations of surface bonds. The range of contact angles is different on oxidized and H-treated composite surface because of different surface terminations.


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Figure 6. Static contact angles versus (FE-SEM) measured on the composite film surfaces: (a) water, (b) diiodomethane and (c) glycerol.


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Based on these wettability data, the surface energy components of pure diamond, β-SiC and two homogenous composite films after oxidation and H-treatment were calculated by using the van Oss39 method (see Figure 7). The total surface free energy includes apolar Lifshitz-van der Waals and polar Lewis acid-base interactions. Figure 7a and 7b shows the surface energy components of oxidized and H-treated diamond, β-SiC and composite films versus , respectively. The difference of on all surfaces was not large. decreased slightly with increasing . The Lewis acid-base component is composed of two different surface tension parameters representing the electron-accepticity (acidic, ) and electron-donicity (basic, ).40 can be expressed as = 2( )1/2. The acidic component is given by the contribution of all positive charges resulting from the active site protonation. The basic component is given by negative charges resulting from the deprotonation of the active sites or lone electron pairs, which serve as a Lewis base. With increasing , the value decreased gradually on oxidized surface (Figure 7a), indicating a higher ability of electron donicity of the OH- terminated SiC surface to generate hydrogen bonding. On H-treated composite films (Figure 7b) the trend of the surface energy components versus was similar to oxidized composite films. decreased with increasing . Especially drastically decreased to zero at diamond-rich area (high ) because of the H-termination on diamond surface. On the β-SiC-rich surface (low ), the value was much higher than on the diamond-rich surface because of the increased fraction of OH bonds on the β-SiC surfaces. β-SiC processes enough lone pairs on the oxygens to function a H-bond acceptor and thereby facilitates hydrogen bonding. In summary, the surface free energy components gradually change with increasing both after oxidation and H-treatment. The polar component depends on the OH bonds on the surface.


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Figure 7. Surface energy components of diamond, β-SiC and composite films after (a) oxidation and Htreatment calculated by using the van Oss acid-base method. To study the protein adsorption behavior on the gradient films, confocal fluorescence microscopy was carried out after exposing the surfaces to fluorescently tagged albumin and fibrinogen. The relative amounts of protein adsorbed on the gradient films were determined by comparing the emission intensity in the fluorescence images. As the labeled protein molecules contain a nearly identical number of conjugated fluorophores and their quantum yields are approximately constant regardless of the protein conformation, the measured fluorescence emission intensities are proportional to the amount of adsorbed protein for a given area.41-44 Confocal fluorescence images of albuminFITC adsorbed on oxidized diamond/β-SiC composite gradient films from position 1 mm to 7 mm are shown in Figure 2c. At 1 mm, the fluorescence emission intensity was quite low, indicating a low amount of albumin adsorbed on pure β-SiC surface. At 3 mm, bright green spots (diameter ≈ 1.1 µm) were observed on the diamond/β-SiC composite film. The distribution and size of the bright green areas matched the diamond islands (diameter ≈ 1.0 µm) in the AFM and ≈ 0.8 µm in the SEM data), which are shown in Figure 2a, suggesting that albumin preferred to absorb on diamond crystallites. The bright green fluorescent areas were larger at 4 mm compared to 3 mm, i.e. they increased in size with increasing surface coverage of diamond in the film. Compared with the corresponding surface morphology (Figure 2a), the dark green


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areas matched the β-SiC areas, indicating less albumin was adsorbed on β-SiC. The pure diamond surface at 7 mm was covered by albumin with a high density. These data demonstrate that albumin was preferentially adsorbed on the diamond crystallites on the oxidized diamond/β-SiC composite gradient surface. Figure 2d shows confocal fluorescence images of albuminFITC adsorbed on a H-treated diamond/β-SiC composite gradient film from position 1 mm to 7 mm along the length of the film. At 1 mm, albumin was strongly adsorbed on pure β-SiC surface with high density. At 3 mm, the fluorescence intensity was lower on the diamond/β-SiC composite films than on pure β-SiC. Compared to the corresponding surface morphology (Figure 2a), the dark green areas, where almost no albumin was adsorbed, matched the areas of diamond crystallites, and the bright green areas, where albumin was adsorbed, matched the β-SiC. The amount of adsorbed protein on the surface was lower at 4 mm than at 3 mm, i.e. with increasing surface coverage of diamond. At 7 mm, there were nearly no proteins adsorbed on the pure diamond surface. This indicates that on H-treated diamond/β-SiC composite gradient films, albumin was preferentially adsorbed on the β-SiC crystallites, but not on diamond crystallites. The same behavior was observed for fibrinogen

Alexa Fluor

adsorbed on oxidized diamond/β-

SiC composite gradient films. Fig. 2e shows the confocal fluorescence images of fibrinogen

Alexa Fluor

adsorbed on an oxidized diamond/β-SiC composite gradient film from 1 mm to 7 mm. At 1 mm, there was a quite low intensity of fluorescence emission on β-SiC. At 3 mm, bright red spots could be recognized, which correspond to adsorbed proteins on the surface. The fluorescence emission intensity increased from 3 mm to 4 mm because of the higher content of diamond in the composite film. Compared to the corresponding surface morphology (Figure 2a), the bright red areas matched the diamond crystallites, indicating stronger adsorption of fibrinogen on the diamond crystallites. The dark red areas matched with the β-SiC covered areas, indicating that almost no protein was adsorbed on βSiC. At 7 mm, fibrinogen was strongly adsorbed on pure diamond with high density. It can be


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concluded that for oxidized diamond/β-SiC composite gradient films, fibrinogen preferred to adsorb on diamond crystallites rather than β-SiC crystallites. Figure 8 shows the fluorescence emission intensities of albuminFITC adsorbed on oxidized and Htreated gradient surfaces along the length of the samples. The intensity represents the mean fluorescence intensity, which was calculated as arithmetic mean of the intensities at all pixels in fluorescence images of identical size (80 µm × 80 µm) and a pixel resolution of 512 x 512 or 256 × 256. Figure 8a shows the plot of fluorescence emission intensities versus position from 1 mm to 7 mm. From 1 mm to 7 mm, the intensities gradually increased on the oxidized gradient surface, but decreased on the H-treated gradient surface, indicating that an increasing amount of albumin was adsorbed on the oxidized gradient surface but decreasing amount of albumin adsorbed on the H-treated surface. It is known from Figure 2a that the diamond/β-SiC radio gradually increased from 1 mm to 7 mm. The plot of diamond fractional coverage ( ) and β-SiC fractional ( ) coverage derived from FE-SEM versus position is shown in Figure 8b. Both the fluorescence intensity data and the fractional surface coverages show corresponding sigmoid trends. Figure 8c shows a plot of fluorescence intensity on an oxidized surface versus (FE-SEM). A linear relationship between intensity and diamond coverage was observed. The scaled intensities were calculated from the experimental values using equation (2):

!!%

# !% # 100%


(2)

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Figure 8. (a) Fluorescence emission intensities of albuminFITC adsorbed on oxidized and H-treated gradient film versus different positions. (b) Fractional diamond coverage of gradient surface at different positions calculated from FE-SEM. Fluorescence emission intensities of albuminFITC adsorbed on (c) oxidized and (d) H-treated gradient film versus diamond coverage. Scaled lines correspond to equation (2). The increasing amount of albumin adsorption on the oxidized surface depends on the increasing surface coverage of diamond. Figure 8d shows the plot of fluorescence intensity on the H-treated surface versus (SEM). Also here a linear dependence of fluorescence intensity vs. (SEM) was obtained, which was calculated from the experimental values using equation (2). Thus the


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increasing amount of albumin adsorption on H-treated surface depends linearly on the decreasing surface content of diamond and increasing surface content of β-SiC. The mechanism of the preferential protein adsorption is a complex process involving electrostatic interactions, hydrogen bonding, van der Waals interactions, and hydrophobic interactions between protein and surface.45 The isoelectric points of albumin and fibrinogen are 4.7 and 5.5, respectively.46, 47 At pH 7 and 8, the net charge of free albumin and fibrinogen in aqueous buffer is thus negative.48-50 The electrostatic interactions between negatively charged protein and the anionic O-/OH-terminated diamond surface is expected to be repulsive, however, there was a high amount of protein on it (compare Figure 2c). The Debye length has been calculated as 4 nm for 10 mM Tris buffer at pH 7.0 (detailed information is shown in the Supporting Information). When the distance between protein and gradient surface is larger than the Debye length, there would be low to no ionic interaction effect. These features underline the relative short range of the electrostatic interactions. Under these conditions van der Waals interaction and hydrogen bonding were analyzed by the surface energy components in Figure 7. After H-treatment, the proteins prefer to adsorb on the OH/H-terminated β-SiC rather than on the Hterminated diamond. The polar component %& and were analyzed to be comparatively higher on βSiC surfaces than on diamond due to more OH-termination. The oxygen in surface OH bonds on the βSiC surface possesses lone pairs, which are acceptors for hydrogens from the N-H groups in proteins to result in hydrogen bonding. This hydrogen bonding could be much easier generated on OH-terminated β-SiC surfaces rather than on apolar H-terminated diamond surfaces, resulting in higher amount of protein adsorbed on β-SiC than diamond. Moreover, the higher electron-donicity ( ) on OHterminated β-SiC implies a pronounced ability to donate electrons and facilitate hydrogen bonding, which results in the preferential adsorption of protein on this surface. On the other hand, both OHterminated β-SiC and O/OH-terminated diamond are polar after oxidation. The values of %& and


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are higher on β-SiC than on diamond (see Figure 7b). More proteins were expected to be adsorbed on βSiC. In contrast, protein preferred to adsorb on diamond. Since the protein/surface interactions occurred in water, the role of water cannot be ignored. Water self-associates through hydrogen bonding to establish a network.24 The anionic-hydrophilic surfaces bearing relatively weak Lewis-base functional groups like hydroxyl resist protein adsorption by hydrogen-bonding to water so strongly that protein cannot displace interphase water and enter the ! is the overall free energy of protein adsorption from adsorbed state. Vogler suggested that the ∆(

purified aqueous solution follows the basic rule:24 ! ! ! ! ∆( ∆G*+,-./*.012 344325

∆G,3*+,-651.7

∆G1753-6251.7

(3)

! is the free energy (gain) of the hydrophobic effect that expels protein from where ∆G*+,-./*.012 344325 ! solution to recover hydrogen bonds among water molecules, ∆G,3*+,-651.7 is the energy cost of ! displacing interphase water by adsorbing protein, and ∆G1753-6251.7 is the free energy (gain) of protein-

protein and protein-surface interactions, which depends in some way on the chemistry of the adsorbent surface. Figure 9 shows a simplified schematic of protein adsorption on oxidized and H-treated diamond/βSiC composite films and the fluorescence emission intensities versus surface chemistry on the gradient film. After oxidation, the surface of SiC is covered with hydroxyl groups which tightly bind the H2O network to the surface. The H2O network hinders proteins to reach the surface of SiC. Figure 9b shows that there was less protein adsorbed on the oxidized gradient surface with increasing OH (on SiC) concentration. Figure 9b, c, e and f were plotted using the concentration of surface bonds versus from Figure 3 and the fluorescence emission intensities versus from Figure 8c, d. On the other hand, the diamond surface is terminated with OH and O groups and possesses lower


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%& and than OH-terminated SiC. The hydrogen bonding between O and the H2O network is weaker than between OH and the H2O network, resulting in a weaker binding of the H2O network to the surface. Thus the protein can displace the interphase water easier and interact with O and OH terminations on the diamond surface. Figure 9c shows that there was more protein adsorbed on the oxidized gradient surface with increasing C-O-C (on diamond) concentration, suggesting that the displacement of ! ! interphase water needs less energy at the diamond surface (lower ∆G,3*+,-651.7 ). Therefore, ∆( is

lower on diamond than on SiC, resulting in preferential protein adsorption on diamond. On the H-treated composite film, proteins prefer to adsorb on SiC. As discussed above, the Hterminated diamond surface is apolar. Hydrogen bonding with the protein or the H2O network cannot occur on this surface, thus no adsorption of protein occurs. On the other hand, the surface of SiC consists of both OH and H bonds. On this SiC surface the H2O network is bound to the surface through hydrogen bonding (see Figure 9d). However, the H2O network above the H-treated SiC is weaker bound to the surface than above the oxidized SiC surfaces. Figure 9e, f shows that there was more protein adsorbed on the H-treated gradient surface with increasing OH (on SiC) and Si3C-H concentration, ! ! suggesting that the Si3C-H lowered ∆G,3*+,-651.7 . Finally, ∆( is lower on SiC than on diamond,

resulting in preferential protein adsorption on SiC.


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Figure 9. Proposed schematic of protein adsorption on (a) oxidized and (d) H-treated diamond/β-SiC composite films. Fluorescence emission intensities of albuminFITC adsorbed on (b, c) oxidized and (e, f) H-treated gradient film versus different concentration of surface bonds. The concentration of –OH consist of Si-O and C-O concentration on β-SiC.

Conclusions Diamond/β-SiC composite gradient films were synthesized by careful manipulation of the sample position during the HFCVD process. The diamond/β-SiC ratio gradually changed from pure diamond to pure β-SiC along the length of the sample. X-ray Photoelectron Spectroscopy and Time-of-ﬂight secondary ion mass spectrometry measurements revealed that β-SiC is OH-terminated after oxidation, but OH-/H-terminated after H-treatment, while diamond is O-/OH-terminated after oxidation, but Hterminated after H-treatment. The surface chemistry could be controlled by different diamond/β-SiC ratios and surface treatments. According to confocal fluorescence microscopy, proteins were


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preferentially adsorbed corresponding to the distribution of diamond and β-SiC in the composite film, thus proteins preferred to adsorb on diamond surfaces rather than β-SiC after oxidation. On the other hand, proteins preferentially adsorbed on β-SiC surfaces rather than on diamond after H-treatment. The mechanism of preferential protein adsorption was discussed by considering the hydrogen bonding of the water self-association network to OH-terminated surfaces and the change of the polar surface energy component, which was determined together with the other thermodynamic surface free energy components according to the van Oss method, respectively, on the basis of static contact angle measurements of various probe liquids. This work shows that diamond/β-SiC composite gradient film can be used to control the protein adsorption, which is important for biomedical applications, such as materials for implants to direct cell migration and substrates for biosensors. Acknowledgment. We would like to thank Qiang Su and Dr. Gilbert Nöll of the Department Chemistry-Biology, University of Siegen, for helpful discussions regarding protein adsorption protocols. The authors are indebted to Prof. Bernd Wenclawiak of the Analytical Chemistry, University of Siegen for access to the ToF-SIMS equipment. We gratefully acknowledge financial support from China Scholarship Council, the Deutsche Forschungsgemeinschaft (DFG grant no. INST 221/87-1FUGG), the European Research Council (ERC grant to HS, ERC grant agreement No. 279202) and the University of Siegen. Supporting Information Available. XPS spectra, detailed XPS parameters, FE-SEM morphology and deposition parameters of the homogenous composite film measured by XPS and contact angles, histograms of fluorescence emission intensities in Figure 2, fluorescence emission intensities of FibrinogenAlexa Fluor adsorbed on oxidized and H-treated diamond and β-SiC films as well as the calculation of Debye length are shown in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.


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Î²-SiC Composite Films for

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