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Adsorption ability comparison of plasma proteins on amorphous carbon surface
Journal of Physics and Chemistry of Solids 73 (2012) 1331–1334
Contents lists available at SciVerse ScienceDirect
Journal of Physics and Chemistry of Solids journal homepage: www.elsevier.com/locate/jpcs
Adsorption ability comparison of plasma proteins on amorphous carbon surface Aoi Takeda a, Hiroki Akasaka a,n, Shigeo Ohshio a, Ikumi Toda a, Masayuki Nakano b, Hidetoshi Saitoh a a b
Department of Materials Science and Technology, Nagaoka University of Technology, 1603-1, Kamitomioka, Nagaoka, Niigata Pref. 940-2188, Japan Department of Chemical Science and Engineering, Tokyo National College of Technology, 1220-2, Kunugida Hachiouji, Tokyo 193-0997, Japan
a r t i c l e i n f o
abstract
Article history: Received 11 March 2012 Received in revised form 30 May 2012 Accepted 10 June 2012 Available online 16 June 2012
To understand why amorphous carbon (a-C:H) film shows antithrombogenicity, an adsorption ability of plasma proteins on a-C:H surface was investigated. Protein adsorption is the initial process of clot formation. The protein adsorption ability on a-C:H film surface was compared by the detection using the surface plasmon resonance (SPR) phenomenon to estimate the protein adsorption. The protein adsorption abilities of a fibrinogen (Fib) and a human g-globulin (HGG) were estimated by the SPR method using a multilayer structure of a-C:H/Au/Cr/glass. Although the adsorption of HGG for a-C:H was saturated at 32 mM in HGG concentration, the adsorption of Fib was not saturated under the detection limit of this method. These results indicated that the adsorption ability to the a-C:H film surface of Fib was higher than HGG. & 2012 Elsevier Ltd. All rights reserved.
Keywords: A. Amorphous materials A. Thin films A. Multilayers B. Plasma deposition D. Surface properties
1. Introduction Recently, hydrogenated amorphous carbon (a-C:H) films have been studied for their potential use as coatings of biochemical parts and medical materials [1–3]. Especially, some reports indicated that amorphous carbon (a-C:H) film shows antithrombogenicity [4,5]. In such studies, samples coated with an a-C:H film were placed in flowing blood, and the completely dried samples were examined by a scanning electron microscopy and were observed to count the blood clots [6,7]. In these studies, the evaluation process required a long time; moreover, bloods are in particular value and limited supply because they are collected from volunteers. Hence, a simple method of evaluating the biocompatibility of a-C:H in a short time is required. During clot formation adsorption on material surface, protein adsorption is the first step of clot formation on a material surface [8,9]. Hence, the amount of protein adsorption determines the amount of clot formations; thus, the evaluation of protein adsorption is necessary to understand clot formations. Especially, some reports indicated that protein adsorptions of the fibrinogen (Fib) and the human g-globulin (HGG) are the primary factors for clot formations [10]. We already reported a simple method of evaluating the protein adsorption in a short time such that the protein adsorption is detected on a-C:H films using surface plasmon resonance (SPR) phenomenon [11,12].
n
Corresponding author. E-mail address: [email protected] (H. Akasaka).
0022-3697/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpcs.2012.06.006
SPR is the resonance between a surface plasmon wave and an evanescent wave on a metal surface [13–18]. Metal film is deposited on a glass prism, and a laser is irradiated from the glass side and an evanescent wave penetrates through the metal film. At the same time, plasmon waves are excited at the reverse side of the film. When the wave numbers of the surface plasmon and evanescent waves are similar, SPR occurs and the intensity of the laser light reflected from the back side of the metal film markedly decreases. These waves depend on the laser incident angle; thus, the conditions required for SPR also depend on this angle. The SPR angle depends on the refractive index of the metal surface. Hence, the refractive index changes with a change in the configuration on the metal surface, leading to a shift in the SPR angle. Basically, the sensing area of this refractive index is less than 200 nm above the metal film; this distance is mainly determined by the penetration distance of the evanescent wave [17]. Thus, the system used to detect protein adsorption on a metal film surface is based on the concept of detecting the change in the refractive index due to the adsorption. Adsorption on a film surface may be detected when a a-C:H film with a thickness of a few nanometers is deposited on a metal. When protein adsorption site on the a-C:H surface is occupied by protein, SPR angle will not be increased by introduction of higher protein concentration solution to film surface. When the protein adsorption thickness exceeds the limit of detection of 200 nm, SPR shift is saturated against the deposited amount of proteins. From these SPR angle shift behavior against protein concentrations, adsorption ability of plasma proteins was investigated.
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Fig. 1. Schematic illustration of SPR measurement device with a-C:H/Au multilayer structure.
In this study, we attempted to compare the protein adsorption ability for fibrinogen (Fib) and a human g-globulin on the a-C:H surface through the detection of SPR on an a-C:H/metal multilayer structure device.
2. Experimental The multilayer device used for SPR detection is shown in Fig. 1. An optical glass (S-TIH 11) with a refractive index of 1.778 was chosen as the substrate for the SPR device. A gold (Au) layer was deposited on the glass by sputtering. Thickness of Au layer was approximately 50 nm. a-C:H films were synthesized on the Au/STIH 11 glass by radio-frequency sputtering from a graphite target. During a-C:H layer synthesis, pressure was maintained at 20 Pa and RF power was set at 150 W. The deposition duration for each a-C:H film layer was less than 30 min, because the film thickness should be less than 200 nm, which is the limit of detection in SPR measurement. The chemical bonding structures of a-C:H film were investigated using a Fourier transform infrared (FT-IR) spectrometer. The composition of the a-C:H and related films were determined by Rutherford back scattering (RBS) and elastic recoil detection analysis (ERDA) systems. The RBS and ERDA with MeV-He þ irradiation were performed. RBS and ERDA signals were simultaneously detected and ERDA technique was applied to determine the atomic fraction of H in the sample. g-globulin (HGG) and fibrinogen (Fib) were used as proteins in the protein adsorption tests. Generally, sizes of HGG and Fib are approximately 4 9–12 [19–20] and 6.5 45 nm2 [21], which also depend on pH around proteins. Hence, pH of the solution was maintained at pH7 which was adjusted by the buffer solution. The buffer used for the proteins was a commonly used phosphor buffer (PB). To prepare the PB solution, Na2HPO4 12H2O and NaH2PO4 2H2O (Wako Co. Ltd.) were mixed and the phosphate solution was coordinated to a concentration of 0.05 M using deionized water. The PB solutions were adjusted to pH 7. HGG-containing PB solutions were coordinated to HGG concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 mM, and Fib-containing PB solutions were coordinated to Fib concentrations of 0.05, 0.075, 0.1, 0.2, 0.4,
0.8 and 1.6 mM. The protein concentrations were dominated by the saturated concentration of the buffer solution for each protein. The Kretschmann configuration in which the phenomenon of total internal reflection is employed to achieve the resonant condition used for SPR detection [14]. A diode laser as a light source of 635 nm with the beam diameter to approximately 0.8 mm (LDU33-635-3: Sigma Koki) and a photodetector as detector for the intensity of the reflected light (Q82324-ADCE8250A: Advantest) were mounted on separate rotating stages controlled by stepping motor with a maximum resolution of 0.00251. The SPR profiles were obtained as functions of the reflectance of the laser beam and the incident angle of the beam. To investigate the adsorption of HGG or Fib, a flow cell made by silicon rubber with a volume of approximately 0.5 ml was attached on the a-C:H layer of the multilayer SPR device, and PB solutions with and without proteins were placed on the surface of the a-C:H layer in the SPR device using this flow cell. The experiment to estimate the adsorption of proteins on the a-C:H surface was performed as follows. First, the initial SPR angle was measured for only the PB solution injected into the flow cell. Next, the PB solution in the flow cell was replaced with a PB solution containing plasma proteins, and the SPR angle was measured. Then, the protein-containing solution was again replaced with the PB solution to remove nonadsorbed proteins and the resonance angle was measured. Flow rates for all injections were controlled at 2.0 ml/min by a syringe driver. To prevent pressure on the cell inside, solutions out of the exhaust was opened. When plasma proteins are adsorbed on the layer, the SPR angle will be larger than SPR angle at the initial injection of PB solution, because the multilayer index above the a-C:H layer increases upon the adsorption of proteins. These processes of protein injections and non-adsorbed removals were repeated on each protein concentration without the device change. Hence, results of protein adsorption tests are results caused by the integrated adsorption of proteins.
3. Results and discussion The structure of the films was obtained by FT-IR spectroscopy and RBS/ERDA. On FT-IR spectrum the absorption at wave numbers in the range of 2800–3000 cm 1 in all the films was assigned to the stretching vibrational-mode of CH, CH2 and CH3 bonds. The absorption at wave numbers in the range of 1350– 1500 cm 1 in some films was assigned to the bending of CH2 and CH3 bonds. The hydrogen concentration obtained by RBS/ERDA were 29 at% for a-C:H films. From these results, a-C:H film surface was terminated by hydrogen. First, the adsorption of HGG on the a-C:H film surface was evaluated using the multilayer SPR device. The dependence of reflectivity on the laser incident angle, used to evaluate HGG adsorption on the a-C:H surface are shown in Fig. 2. Solid line (1) in Fig. 2 is the initial dependence when PB without HGG was placed on the a-C:H surface. From the attenuation of the reflected light, the SPR angle was determined to be 60.021 from line (1). After this initial measurement, a HGG-containing PB solution was injected onto the a-C:H surface. Then, to remove the nonadsorbed HGG from the a-C:H surface, PB solution was again injected onto the a-C:H surface, and the dependence of reflectivity on the laser incident angle was remeasured, as shown by line (2) in Fig. 2. The SPR angle obtained from this line was to be 60.641. The two dependences of reflectivity were compared with each other to detect the HGG adsorption on the surface of the a-C:H surface. The difference between the SPR angles obtained from lines (1) and (2) was 0.621; the SPR angle did not return to its original value for PB without HGG on the a-C:H surface. Thus, this shift of 0.621 suggests the adsorption
A. Takeda et al. / Journal of Physics and Chemistry of Solids 73 (2012) 1331–1334
Fig. 2. The dependence of reflectivity on the laser incident angle; used to evaluate HGG adsorption on a-C:H surface. (1) PB solution, and (2) 64.0 mM HGG-containing PB solution.
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Fig. 4. The dependence of reflectivity on the laser incident angle; used to evaluate Fib adsorption on a-C:H surface. (1) PB solution, and (2) 1.6 mM Fib-containing PB solution.
Fig. 5. SPR angle shifts obtained by Fib adsorption tests.
Fig. 3. SPR angle shifts obtained by HGG adsorption tests.
of HGG on the a-C:H film surface. The SPR angles obtained from the dependence of the reflectivity on the laser incident angle for different concentrations of HGG are plotted and labeled in numerical order in Fig. 3 in accordance with the experimental sequence. Plot (1) corresponds to the initial measurement for PB without HGG placed on the a-C:H surface. Plots with even numbers of (2), (4), (6), (8), (10), (12), (14), (16) and (18) are those for PB containing HGG concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 mM, respectively. Because the cell on the SPR device was charged by the injection of PB solution containing HGG in the previous operation, the cell contains both nonadsorbed HGG and adsorbed HGG. Plots with odd numbers of (3), (5), (7), (9), (11), (13), (15), (17) and (19) are obtained after the removal of non-adsorbed HGG by the injection of PB into the cell from the a-C:H surface. The SPR angles obtained from the dependence of reflectivity on the laser incident angle labeled with even numbers in Fig. 3 increased with HGG concentration. The SPR angles labeled with odd numbers also increased with HGG concentration. The difference between the SPR angles before and after the removal of non-adsorbed HGG slowly increases during the experimental sequence as the available sites for HGG adsorption on the a-C:H surface become filled to capacity with HGG. Although it was tiny variation in Fig. 3, saturation was shown at plot (17) because the relationship between duration and SPR angle deviated from the proportional line. These trends suggest that all the available sites on the a-C:H surface for HGG adsorption become occupied, because for SPR the angle obtained after the
removal of nonadsorbed HGG by PB becomes saturated at a HGG concentration of 32.0 mM. Next, the adsorption of Fib on the a-C:H film surface was evaluated using the same type of device. Fig. 4 shows the dependence of reflectivity on the laser incident angle used to evaluate Fib adsorption on the a-C:H surface. Solid line (1) in Fig. 4 is the initial dependence when PB without Fib is placed on the a-C:H surface. The SPR angle was determined from the attenuation of the reflected light to be 58.831 from line (1). After the initial measurement, a Fib-containing PB solution with a Fib concentration of 0.04 mM was injected onto the a-C:H surface. Then, to remove the nonadsorbed Fib from the a-C:H surface, PB solution was again injected onto the a-C:H surface, and the dependence of reflectivity on the laser incident angle was measured as shown by line (2) in Fig. 4. The SPR angle obtained from line (2) was to be 59.591. These dependences of reflectivity were compared to detect the Fib adsorption on the a-C:H surface. The difference between the SPR angles obtained from lines (1) and (2) was 0.761; the SPR angle did not return to its initial value for PB without Fib on the a-C:H surface, also indicating the adsorption of Fib on the a-C:H film surface. Using the same procedure as that for HGG, the SPR angles obtained from the dependence of the reflectivity on the laser incident angle for different concentrations of Fib are plotted and labeled in numerical order in Fig. 5 in accordance with the experimental sequence. Plot (1) corresponds to the initial measurement for PB without Fib placed on the a-C:H surface. Plots (2), (4), (6), (8), (10), (12), and (14) are those for PB containing Fib concentrations of 0.05, 0.075, 0.1, 0.2, 0.4, 0.8 and 1.6 mM, respectively. Plots (3), (5), (7), (9), (11), (13), and (15) are obtained after the removal of nonadsorbed Fib by the injection of PB into the cell from the a-C:H surface. The SPR angles
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labeled with all numbers in Fig. 5 increased with Fib concentration, indicating that the almost injected Fib adsorbed to the a-C:H surface. The amount of SPR angle shift of PBS of (3), (5), and (7) are larger than that before each test (2), (4), and (6). Hence, all the available sites for Fib adsorption were not occupied until the injection of 0.075 mM. Subsequently, The amount of SPR angle shifts of plot (8) for Fib-containing solution injection and (9) for PB solution injection are close to each other. The SPR angle plots labeled with the numbers of more than 10 was saturated with Fib concentration. It was indicated that the plot at (10) was the detection limit for this method because plots labeled with even and odd numbers showed same behavior, saturation. From these results, the adsorption ability was compared. The detectable space on these evaluations is approximately the same volume because the three dimensional detection limits was dominated by both the distance from the metal layer and the laser spot diameter which were same value on these measurement. On the evaluation for HGG adsorption on the a-C:H film, it was earlier than detection limit for this method that all the available sites on the a-C:H surface for HGG adsorption become occupied. On the other hand, the behavior in Fib adsorption test showed that the detection limit for this method is earlier than all the available sites occupied for Fib adsorption. It was indicated that the volume of adsorbed Fib is larger than the volume of adsorbed HGG. Hence, these results suggested that the adsorption ability of Fib for the a-C:H film surface was higher than that of HGG.
4. Conclusion The protein adsorption abilities of a Fib and a HGG were estimated by the SPR method using a multilayer structure of a-C:H/Au/Cr/glass. The adsorption of HGG for a-C:H was saturated at 32 mM in HGG concentration, and the adsorption of Fib was not saturated under the detection limit of this method. These results indicated that the adsorption ability to the a-C:H film surface of Fib was higher than HGG. References [1] D.A. LaVan, R.F. Padera, T.A. Friedmann, J.P. Sullivan, R. Langer, D.S. Kohane, In vivo evaluation of tetrahedral amorphous carbon, Biomaterials 26 (2005) 465–473.
[2] S.E. Rodil, R. Olivares, H. Arzate, S. Muhl, Properties of carbon films and their biocompatibility using in-vitro tests, Diamond Relat. Mater. 12 (2003) 931–937. [3] H. Matsuo, K. Kanasugi, Y. Ohgoe, K.K. Hirakuri, Y. Fukui, Biocompatibility of a-C:H film coating for synthetic vascular graft, Diamond Relat. Mater. 18 (2009) 1031–1034. [4] T. Hasebe, T. Ishimaru, A. Kamijo, Y. Yoshimoto, T. Yoshimura, S. Yohena, H. Kodama, A. Hotta, K. Takahashi, T. Suzuki, Effects of surface roughness on anti-thrombogenicity of diamond-like carbon films, Diamond Relat. Mater. 16 (2007) 1343–1348. [5] T. Saito, T. Hasebe, S. Yohena, Y. Matsuoka, A. Kamijo, K. Takahashi, T. Suzuki, Antithrombogenicity of fluorinated diamond-like carbon films, Diamond Relat. Mater. 14 (2005) 1116–1119. [6] R.K. Roy, H.W. Choi, J.W. Yi, M.-W. Moon, K.-R. Lee, D.K. Han, J.H. Shin, A. Kamijo, T. Hasebe, Hemocompatibility of surface-modified, silicon-incorporated, diamond-like carbon films, Acta Biomater. 5 (2009) 249–256. [7] V. Karagkiozaki, S. Logothetidis, A. Laskarakis, G. Giannoglou, S. Lousinian, AFM study of the thrombogenicity of carbon -based coatings for cardiovascular applications, Mater. Sci. Eng. B 15 (2008) 16–21. [8] J.D. Andrade, Surface and Interfacial Aspects of Biomedical Polymers, Plenum Press, New York, 1985. [9] S.C.H. Kwok, J. Wang, P.K. Chu, Surface energy, wettability, and blood compatibility phosphorus doped diamond-like carbon films, Diamond Relat. Mater. 14 (2005) 78–85. [10] T. Akaike, Bio-functional Material–Fundamental for Artificial Organs Tisuue Engineering and Regenerative Medicine, Corona Publishing Co., Ltd., 2005 (in Japanese). [11] H. Akasaka, A. Takeda, T. Suzuki, M. Nakano, S. Ohshio, H. Saitoh, Fibrinogen and lysozyme adsorption on amorphous carbon film surface detected by multilayer device from the back side of the film, Diamond Relat. Mater. 20 (2011) 213–216. [12] H. Akasaka, N. Gawazawa, T. Suzuki, M. Nakano, S. Ohshio, H. Saitoh, Evaluation of protein adsorption on hydrogenated amorphous carbon films by surface plasmon resonance phenomenon, Diamond Relat. Mater. 19 (2010) 1235–1239. [13] B. Liedberg, C. Nylander, I. Lundstrom, Biosensing with surface plasmon resonance–how it all started, Biosens. Bioelectron. 10 (1995), i-ix. [14] R.W. Wood, On a remarkable case of uneven distribution of light in a diffraction grating spectrum, Philos. Mag. 48 (1902) 396–402. [15] L. Rayleigh, Dynamical theory of the grating, Proc. R. Soc. London 79 (1907) 399. [16] U. Fano, The theory of anomalous diffraction gratings and of quasi-stationary waves on metallic surfaces (Sommerfeld’s waves), J. Opt. Soc. Am. 31 (1941) 213–222. [17] E. Kretschmann, Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflachenplasmaschwingugnen, Z. Phys. 241 (1971) 313–324. [18] S. Kishimoto, S. Ohshio, H. Akasaka, H. Saitoh, Detection of protein adsorption on silica surface using surface plasmon resonance sensor, Jpn. J. Appl. Phys. 47 (2008) 8106–8108. [19] P. Imlah, S.E. Brownlie, K.W. Head, H.S. McTaggart, J.G. McVie, Serum gamma globulin detection of IgG heavy chain in the serum and pig levels and the chain and light urine of cases of Hereditary Lymphosarcoma, Eur. J. Cancer 15 (1979) 1337–1349. [20] H. Remold, E. Zimmermann, Stability of structures of human g globulin, Arch. Biochem. Biophys. 120 (1967) 35–41. [21] Z. Andrei Budzynski, Fibrinogen and fibrin: biochemistry and pathophysiology, Crit. Rev. Oncol./Hematol. 6 (1986) 97–146.
Contents lists available at SciVerse ScienceDirect
Journal of Physics and Chemistry of Solids journal homepage: www.elsevier.com/locate/jpcs
Adsorption ability comparison of plasma proteins on amorphous carbon surface Aoi Takeda a, Hiroki Akasaka a,n, Shigeo Ohshio a, Ikumi Toda a, Masayuki Nakano b, Hidetoshi Saitoh a a b
Department of Materials Science and Technology, Nagaoka University of Technology, 1603-1, Kamitomioka, Nagaoka, Niigata Pref. 940-2188, Japan Department of Chemical Science and Engineering, Tokyo National College of Technology, 1220-2, Kunugida Hachiouji, Tokyo 193-0997, Japan
a r t i c l e i n f o
abstract
Article history: Received 11 March 2012 Received in revised form 30 May 2012 Accepted 10 June 2012 Available online 16 June 2012
To understand why amorphous carbon (a-C:H) film shows antithrombogenicity, an adsorption ability of plasma proteins on a-C:H surface was investigated. Protein adsorption is the initial process of clot formation. The protein adsorption ability on a-C:H film surface was compared by the detection using the surface plasmon resonance (SPR) phenomenon to estimate the protein adsorption. The protein adsorption abilities of a fibrinogen (Fib) and a human g-globulin (HGG) were estimated by the SPR method using a multilayer structure of a-C:H/Au/Cr/glass. Although the adsorption of HGG for a-C:H was saturated at 32 mM in HGG concentration, the adsorption of Fib was not saturated under the detection limit of this method. These results indicated that the adsorption ability to the a-C:H film surface of Fib was higher than HGG. & 2012 Elsevier Ltd. All rights reserved.
Keywords: A. Amorphous materials A. Thin films A. Multilayers B. Plasma deposition D. Surface properties
1. Introduction Recently, hydrogenated amorphous carbon (a-C:H) films have been studied for their potential use as coatings of biochemical parts and medical materials [1–3]. Especially, some reports indicated that amorphous carbon (a-C:H) film shows antithrombogenicity [4,5]. In such studies, samples coated with an a-C:H film were placed in flowing blood, and the completely dried samples were examined by a scanning electron microscopy and were observed to count the blood clots [6,7]. In these studies, the evaluation process required a long time; moreover, bloods are in particular value and limited supply because they are collected from volunteers. Hence, a simple method of evaluating the biocompatibility of a-C:H in a short time is required. During clot formation adsorption on material surface, protein adsorption is the first step of clot formation on a material surface [8,9]. Hence, the amount of protein adsorption determines the amount of clot formations; thus, the evaluation of protein adsorption is necessary to understand clot formations. Especially, some reports indicated that protein adsorptions of the fibrinogen (Fib) and the human g-globulin (HGG) are the primary factors for clot formations [10]. We already reported a simple method of evaluating the protein adsorption in a short time such that the protein adsorption is detected on a-C:H films using surface plasmon resonance (SPR) phenomenon [11,12].
n
Corresponding author. E-mail address: [email protected] (H. Akasaka).
0022-3697/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpcs.2012.06.006
SPR is the resonance between a surface plasmon wave and an evanescent wave on a metal surface [13–18]. Metal film is deposited on a glass prism, and a laser is irradiated from the glass side and an evanescent wave penetrates through the metal film. At the same time, plasmon waves are excited at the reverse side of the film. When the wave numbers of the surface plasmon and evanescent waves are similar, SPR occurs and the intensity of the laser light reflected from the back side of the metal film markedly decreases. These waves depend on the laser incident angle; thus, the conditions required for SPR also depend on this angle. The SPR angle depends on the refractive index of the metal surface. Hence, the refractive index changes with a change in the configuration on the metal surface, leading to a shift in the SPR angle. Basically, the sensing area of this refractive index is less than 200 nm above the metal film; this distance is mainly determined by the penetration distance of the evanescent wave [17]. Thus, the system used to detect protein adsorption on a metal film surface is based on the concept of detecting the change in the refractive index due to the adsorption. Adsorption on a film surface may be detected when a a-C:H film with a thickness of a few nanometers is deposited on a metal. When protein adsorption site on the a-C:H surface is occupied by protein, SPR angle will not be increased by introduction of higher protein concentration solution to film surface. When the protein adsorption thickness exceeds the limit of detection of 200 nm, SPR shift is saturated against the deposited amount of proteins. From these SPR angle shift behavior against protein concentrations, adsorption ability of plasma proteins was investigated.
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A. Takeda et al. / Journal of Physics and Chemistry of Solids 73 (2012) 1331–1334
Fig. 1. Schematic illustration of SPR measurement device with a-C:H/Au multilayer structure.
In this study, we attempted to compare the protein adsorption ability for fibrinogen (Fib) and a human g-globulin on the a-C:H surface through the detection of SPR on an a-C:H/metal multilayer structure device.
2. Experimental The multilayer device used for SPR detection is shown in Fig. 1. An optical glass (S-TIH 11) with a refractive index of 1.778 was chosen as the substrate for the SPR device. A gold (Au) layer was deposited on the glass by sputtering. Thickness of Au layer was approximately 50 nm. a-C:H films were synthesized on the Au/STIH 11 glass by radio-frequency sputtering from a graphite target. During a-C:H layer synthesis, pressure was maintained at 20 Pa and RF power was set at 150 W. The deposition duration for each a-C:H film layer was less than 30 min, because the film thickness should be less than 200 nm, which is the limit of detection in SPR measurement. The chemical bonding structures of a-C:H film were investigated using a Fourier transform infrared (FT-IR) spectrometer. The composition of the a-C:H and related films were determined by Rutherford back scattering (RBS) and elastic recoil detection analysis (ERDA) systems. The RBS and ERDA with MeV-He þ irradiation were performed. RBS and ERDA signals were simultaneously detected and ERDA technique was applied to determine the atomic fraction of H in the sample. g-globulin (HGG) and fibrinogen (Fib) were used as proteins in the protein adsorption tests. Generally, sizes of HGG and Fib are approximately 4 9–12 [19–20] and 6.5 45 nm2 [21], which also depend on pH around proteins. Hence, pH of the solution was maintained at pH7 which was adjusted by the buffer solution. The buffer used for the proteins was a commonly used phosphor buffer (PB). To prepare the PB solution, Na2HPO4 12H2O and NaH2PO4 2H2O (Wako Co. Ltd.) were mixed and the phosphate solution was coordinated to a concentration of 0.05 M using deionized water. The PB solutions were adjusted to pH 7. HGG-containing PB solutions were coordinated to HGG concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 mM, and Fib-containing PB solutions were coordinated to Fib concentrations of 0.05, 0.075, 0.1, 0.2, 0.4,
0.8 and 1.6 mM. The protein concentrations were dominated by the saturated concentration of the buffer solution for each protein. The Kretschmann configuration in which the phenomenon of total internal reflection is employed to achieve the resonant condition used for SPR detection [14]. A diode laser as a light source of 635 nm with the beam diameter to approximately 0.8 mm (LDU33-635-3: Sigma Koki) and a photodetector as detector for the intensity of the reflected light (Q82324-ADCE8250A: Advantest) were mounted on separate rotating stages controlled by stepping motor with a maximum resolution of 0.00251. The SPR profiles were obtained as functions of the reflectance of the laser beam and the incident angle of the beam. To investigate the adsorption of HGG or Fib, a flow cell made by silicon rubber with a volume of approximately 0.5 ml was attached on the a-C:H layer of the multilayer SPR device, and PB solutions with and without proteins were placed on the surface of the a-C:H layer in the SPR device using this flow cell. The experiment to estimate the adsorption of proteins on the a-C:H surface was performed as follows. First, the initial SPR angle was measured for only the PB solution injected into the flow cell. Next, the PB solution in the flow cell was replaced with a PB solution containing plasma proteins, and the SPR angle was measured. Then, the protein-containing solution was again replaced with the PB solution to remove nonadsorbed proteins and the resonance angle was measured. Flow rates for all injections were controlled at 2.0 ml/min by a syringe driver. To prevent pressure on the cell inside, solutions out of the exhaust was opened. When plasma proteins are adsorbed on the layer, the SPR angle will be larger than SPR angle at the initial injection of PB solution, because the multilayer index above the a-C:H layer increases upon the adsorption of proteins. These processes of protein injections and non-adsorbed removals were repeated on each protein concentration without the device change. Hence, results of protein adsorption tests are results caused by the integrated adsorption of proteins.
3. Results and discussion The structure of the films was obtained by FT-IR spectroscopy and RBS/ERDA. On FT-IR spectrum the absorption at wave numbers in the range of 2800–3000 cm 1 in all the films was assigned to the stretching vibrational-mode of CH, CH2 and CH3 bonds. The absorption at wave numbers in the range of 1350– 1500 cm 1 in some films was assigned to the bending of CH2 and CH3 bonds. The hydrogen concentration obtained by RBS/ERDA were 29 at% for a-C:H films. From these results, a-C:H film surface was terminated by hydrogen. First, the adsorption of HGG on the a-C:H film surface was evaluated using the multilayer SPR device. The dependence of reflectivity on the laser incident angle, used to evaluate HGG adsorption on the a-C:H surface are shown in Fig. 2. Solid line (1) in Fig. 2 is the initial dependence when PB without HGG was placed on the a-C:H surface. From the attenuation of the reflected light, the SPR angle was determined to be 60.021 from line (1). After this initial measurement, a HGG-containing PB solution was injected onto the a-C:H surface. Then, to remove the nonadsorbed HGG from the a-C:H surface, PB solution was again injected onto the a-C:H surface, and the dependence of reflectivity on the laser incident angle was remeasured, as shown by line (2) in Fig. 2. The SPR angle obtained from this line was to be 60.641. The two dependences of reflectivity were compared with each other to detect the HGG adsorption on the surface of the a-C:H surface. The difference between the SPR angles obtained from lines (1) and (2) was 0.621; the SPR angle did not return to its original value for PB without HGG on the a-C:H surface. Thus, this shift of 0.621 suggests the adsorption
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Fig. 2. The dependence of reflectivity on the laser incident angle; used to evaluate HGG adsorption on a-C:H surface. (1) PB solution, and (2) 64.0 mM HGG-containing PB solution.
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Fig. 4. The dependence of reflectivity on the laser incident angle; used to evaluate Fib adsorption on a-C:H surface. (1) PB solution, and (2) 1.6 mM Fib-containing PB solution.
Fig. 5. SPR angle shifts obtained by Fib adsorption tests.
Fig. 3. SPR angle shifts obtained by HGG adsorption tests.
of HGG on the a-C:H film surface. The SPR angles obtained from the dependence of the reflectivity on the laser incident angle for different concentrations of HGG are plotted and labeled in numerical order in Fig. 3 in accordance with the experimental sequence. Plot (1) corresponds to the initial measurement for PB without HGG placed on the a-C:H surface. Plots with even numbers of (2), (4), (6), (8), (10), (12), (14), (16) and (18) are those for PB containing HGG concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 mM, respectively. Because the cell on the SPR device was charged by the injection of PB solution containing HGG in the previous operation, the cell contains both nonadsorbed HGG and adsorbed HGG. Plots with odd numbers of (3), (5), (7), (9), (11), (13), (15), (17) and (19) are obtained after the removal of non-adsorbed HGG by the injection of PB into the cell from the a-C:H surface. The SPR angles obtained from the dependence of reflectivity on the laser incident angle labeled with even numbers in Fig. 3 increased with HGG concentration. The SPR angles labeled with odd numbers also increased with HGG concentration. The difference between the SPR angles before and after the removal of non-adsorbed HGG slowly increases during the experimental sequence as the available sites for HGG adsorption on the a-C:H surface become filled to capacity with HGG. Although it was tiny variation in Fig. 3, saturation was shown at plot (17) because the relationship between duration and SPR angle deviated from the proportional line. These trends suggest that all the available sites on the a-C:H surface for HGG adsorption become occupied, because for SPR the angle obtained after the
removal of nonadsorbed HGG by PB becomes saturated at a HGG concentration of 32.0 mM. Next, the adsorption of Fib on the a-C:H film surface was evaluated using the same type of device. Fig. 4 shows the dependence of reflectivity on the laser incident angle used to evaluate Fib adsorption on the a-C:H surface. Solid line (1) in Fig. 4 is the initial dependence when PB without Fib is placed on the a-C:H surface. The SPR angle was determined from the attenuation of the reflected light to be 58.831 from line (1). After the initial measurement, a Fib-containing PB solution with a Fib concentration of 0.04 mM was injected onto the a-C:H surface. Then, to remove the nonadsorbed Fib from the a-C:H surface, PB solution was again injected onto the a-C:H surface, and the dependence of reflectivity on the laser incident angle was measured as shown by line (2) in Fig. 4. The SPR angle obtained from line (2) was to be 59.591. These dependences of reflectivity were compared to detect the Fib adsorption on the a-C:H surface. The difference between the SPR angles obtained from lines (1) and (2) was 0.761; the SPR angle did not return to its initial value for PB without Fib on the a-C:H surface, also indicating the adsorption of Fib on the a-C:H film surface. Using the same procedure as that for HGG, the SPR angles obtained from the dependence of the reflectivity on the laser incident angle for different concentrations of Fib are plotted and labeled in numerical order in Fig. 5 in accordance with the experimental sequence. Plot (1) corresponds to the initial measurement for PB without Fib placed on the a-C:H surface. Plots (2), (4), (6), (8), (10), (12), and (14) are those for PB containing Fib concentrations of 0.05, 0.075, 0.1, 0.2, 0.4, 0.8 and 1.6 mM, respectively. Plots (3), (5), (7), (9), (11), (13), and (15) are obtained after the removal of nonadsorbed Fib by the injection of PB into the cell from the a-C:H surface. The SPR angles
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labeled with all numbers in Fig. 5 increased with Fib concentration, indicating that the almost injected Fib adsorbed to the a-C:H surface. The amount of SPR angle shift of PBS of (3), (5), and (7) are larger than that before each test (2), (4), and (6). Hence, all the available sites for Fib adsorption were not occupied until the injection of 0.075 mM. Subsequently, The amount of SPR angle shifts of plot (8) for Fib-containing solution injection and (9) for PB solution injection are close to each other. The SPR angle plots labeled with the numbers of more than 10 was saturated with Fib concentration. It was indicated that the plot at (10) was the detection limit for this method because plots labeled with even and odd numbers showed same behavior, saturation. From these results, the adsorption ability was compared. The detectable space on these evaluations is approximately the same volume because the three dimensional detection limits was dominated by both the distance from the metal layer and the laser spot diameter which were same value on these measurement. On the evaluation for HGG adsorption on the a-C:H film, it was earlier than detection limit for this method that all the available sites on the a-C:H surface for HGG adsorption become occupied. On the other hand, the behavior in Fib adsorption test showed that the detection limit for this method is earlier than all the available sites occupied for Fib adsorption. It was indicated that the volume of adsorbed Fib is larger than the volume of adsorbed HGG. Hence, these results suggested that the adsorption ability of Fib for the a-C:H film surface was higher than that of HGG.
4. Conclusion The protein adsorption abilities of a Fib and a HGG were estimated by the SPR method using a multilayer structure of a-C:H/Au/Cr/glass. The adsorption of HGG for a-C:H was saturated at 32 mM in HGG concentration, and the adsorption of Fib was not saturated under the detection limit of this method. These results indicated that the adsorption ability to the a-C:H film surface of Fib was higher than HGG. References [1] D.A. LaVan, R.F. Padera, T.A. Friedmann, J.P. Sullivan, R. Langer, D.S. Kohane, In vivo evaluation of tetrahedral amorphous carbon, Biomaterials 26 (2005) 465–473.
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