Diamond & Related Materials 38 (2013) 28–31
Contents lists available at SciVerse ScienceDirect
Diamond & Related Materials
journal homepage: www.elsevier.com/locate/diamond
Direct formation of amine functionality on DLC films and
surface cyto-compatibility
Mei Wang a,b, Ying Zhao a, Ruizhen Xu a, Ming Zhang a, Ricky K.Y. Fu a, Paul K. Chu a,⁎
a
b
Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China
Key Laboratory of Liquid Structure and Heredity of Materials, Ministry of Education, Shandong University, JiNan, 250061, China
a r t i c l e
i n f o
Article history:
Received 2 May 2013
Received in revised form 13 May 2013
Accepted 20 May 2013
Available online 5 June 2013
Keywords:
Diamond-like carbon(DLC)
Amine
Proliferation
Cyco-compatibility
a b s t r a c t
Hydrogenated diamond-like carbon (H-DLC) films were synthesized on a p-type silicon wafer using radio frequency
plasma composed of a mixture of Ar and C2H2 (ratio of 7 to 28). NH3 plasma treatment was carried out to generate
surface-terminal amino groups. The treated surfaces were characterized by Raman scattering, atomic force microscopy (AFM), water contact angle, and X-ray photoelectron spectroscopy (XPS). MC3T3-E1 mouse pre-osteoblasts
were cultured on the samples, and cell morphology and proliferation were investigated to evaluate the cytocompatibility. Cell–surface interactions were investigated by fluorescence microscopy in terms of spreading and
proliferation. A cell count kit-8 (CCK-8 Beyotime) was employed to determine quantitatively the viable preosteoblasts. The formation of the amine functionality led to better cyto-compatibility on the DLC.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental details
Doped diamond electrodes have been widely reported in biomedical applications [1–6], but the practical use of diamond has
been hampered by its scarcity and high cost. Diamond-like carbon
(DLC) has properties similar to diamond, for instance, high hardness, low frictional coefficient, high wear and corrosion resistance,
chemical inertness, high electrical resistivity, infrared transparency,
high refractive index, biocompatibility, and surface smoothness,
and it is also cheaper to produce than diamond. In fact, the unique
properties of DLC match the criteria of good biomaterials for applications to orthopedics, cardiovascular implants, contact lenses, or
dentistry [7–12].
To be more competitive with other biomaterials, a high functional surface addressing biological needs is crucial to the success of DLC. Recently, many techniques such as photochemical
reactions, radical reactions, plasma treatment, and electrochemical reactions have been proposed [13,14]. The incorporation of
adventitious elements into DLC is a good means to alter its properties [14]. For example, the biological reactions on DLC can be changed
by alloying [15–17]. In this paper, we report the direct formation of
amine functionality on DLC by means of Ar /NH3 plasma treatment
and the biological responses before and after the treatment are investigated and compared.
2.1. Preparation of DLC
⁎ Corresponding author.
E-mail address: [email protected] (P.K. Chu).
0925-9635/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.diamond.2013.05.005
DLC films (1–1.5 μm thick) were deposited on p-type silicon substrates (12 mm × 12 mm) by physical vapor deposition (PVD) in a conventional reactor. The fabrication conditions were as follows: substrate
temperature = room temperature (RT), ratio of C2H2/Ar = 23/7, voltage applied to the cathode = 10 kV, plasma power = 150 W, pressure = 6 × 10−2 Pa, and deposition time = 3 h. The silicon substrates
were ultrasonically washed with acetone, alcohol, and distilled water
for 15 min sequentially and then dried before introduction into the
deposition chamber. Prior to DLC deposition, an argon plasma was triggered to clean the substrates for 15 min at a bias voltage of 1 kV to
remove undesirable surface oxide and contamination.
2.2. Plasma treatment
Ammonia plasma treatment was carried out in the same PVD system
under the following conditions: substrate temperature = room temperature; ratio of NH3/Ar = 3/10; pressure in the reactor = 6 × 10−2 Pa;
plasma power = 150 W; exposure time = 30 min.
2.3. Surface characterization
Raman scattering was performed to investigate the chemical
characteristics of the carbon films. The Raman spectra were excited
by a 514-nm argon ion laser with a power of 30 mW at room
M. Wang et al. / Diamond & Related Materials 38 (2013) 28–31
29
Table 1
Water contact angle of DLC surfaces with different terminations.
Surface
Contact angle, θ(°)
As-deposited DLC
Plasma-treated DLC
75 ± 2
50 ± 2
sequentially prior to examination by fluorescence microscopy. A cell
count kit-8 (CCK-8 Beyotime) was employed to determine quantitatively the viable pre-osteoblasts. The cells were cultured by renewing
the medium every day. After culturing for 1, 2, and 3 days, the samples
4.0x105
C 1s
3.5x105
Intensity/ a.u.
3.0x105
N 1s
O 1s
2.5x105
(b)
2.0x105
1.5x105
1.0x105
Fig. 1. AFM images of the as-deposited DLC surface.
5.0x104
0.0
200
temperature, and the spectra in the range of 800–4000 cm− 1 were
fitted using Gaussian distributions. The surface morphology of films
was characterized by atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) was employed to determine the surface
composition after ammonia plasma treatment. The wetting properties
were evaluated by measuring the water contact angles.
Mouse MC3T3-E1 pre-osteoblasts were cultured in high-glucose
Dulbecco's modified eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin in 75 cm2
tissue culture flakes and incubated in a humidified atmosphere of 5%
CO2 at 37 °C. The cells were seeded on each sample in 12-well plates
at a density of 6.7 × 104 cells per well for cell morphology observation
and proliferation assay. After culturing for 24 h, the cells were rinsed
with PBS and fixed with 4% paraformaldehyde in a humidified atmosphere of 5% CO2 at 37 °C for 20 min. After fixing, the cells were thoroughly rinsed with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and
then stained with phalloidin and 4′,6-diamidino-2-phenylindole (DAPI)
400
600
800
Binding energy/ ev
4500
N 1s
4000
3500
Intensity/ a.u.
2.4. Cell cultures
(a)
3000
2500
2000
d
1500
1000
c
500
0
392
394
396
398
400
402
404
406
Binding energy / eV
(e)
1600
(f)
Intensity/ a.u.
Intensity (a.u)
1400
1200
1000
800
600
400
280
1000
1500
2000
2500
3000
3500
Raman shift (cm-1)
Fig. 2. Raman spectra of the as-deposited DLC.
4000
285
290
295
Binding energy/ ev
Fig. 3. XPS survey spectra of (a) as-deposited and (b) NH2-DLC surfaces; high-resolution
XPS spectra of the N 1-s band of (c) as-deposited DLC and (d) NH2-DLC and highresolution XPS spectra of the C 1-s band of (e) as-deposited DLC and (f) NH2-DLC.
30
M. Wang et al. / Diamond & Related Materials 38 (2013) 28–31
2.4
N 1s
450nm
2.2
C-NH2
2.0
DLC-H
DLC-NH2
1.8
Absorbance
Intensity/ a.u.
1.6
N-H
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1day
396
398
400
402
404
2days
3days
Culture Time
Binding energy / eV
Fig. 6. Cell viability assays of cells incubated on H-DLC and NH2-DLC for 1, 2, and 3 days.
Fig. 4. High-resolution N 1-s XPS spectra of the NH2-DLC surface.
with the seeded cells were rinsed twice with sterile PBS and transferred to
fresh 12-well plates. The culture medium with 10% CCK-8 was added to
these samples, and after incubation for 4 h, the solution was aspirated
and the absorbance was measured at 450 nm using a spectrophotometer.
3. Results and discussion
Fig. 1 depicts a representative AFM image of the as-deposited DLC.
The surface consists of random and irregular grains (average diameter
around 40 nm). The root mean square (RMS) roughness and average
roughness (Ra) are quite small (approximately 1 nm), indicating a
smooth surface.
Fig. 2 shows the Raman spectra of the as-deposited DLC films. The G
peak at 1580 cm−1 and the D peak at 1350 cm−1 are consistent with
those in typical Raman spectra of DLC films [11]. The G band corresponds
to the stretching vibration of sp2 carbon, whereas the D band is attributed
to the breathing mode of sp2. The peaks around 2850 cm−1 are associated
with vibrational modes of CH, CH2, and CH3 groups, indicating covalent
bonding with the suitable functional groups.
Water contact angle measurements were used to examine the macroscopic evolution of the wetting properties of the DLC surface (as shown in
Table 1). The Raman spectra suggest that the as-deposited DLC is hydrogen terminated. This termination confers a hydrophobic characteristic
with a water contact angle of θ = 75 ± 2°. After ammonia plasma
modification, the contact angle decreases significantly to θ = 50 ± 2°,
indicating that the DLC becomes hydrophilic. It is possibly due to the
formation of surface amine groups.
X-ray photoelectron spectroscopy is used to determine the chemical
composition of the DLC surfaces before and after NH3 plasma treatment
as well as the nature of the chemical bonding associated with transformation on the surface. Fig. 3a displays the XPS survey spectra of the
as-deposited DLC surface. The spectrum is dominated by the C 1-s signal
at 283 eV from the substrate and a small peak due to oxygen O 1 s at
531 eV. The oxygen signal may originate from different sources, including
surface contamination, partial oxidation of the surface during storage and
handling, and interstitial oxygen on the DLC surface. After NH3 plasma
Fig. 5. Fluorescent images of osteoblasts: (a, c) as-deposited DLC surface and (b, d) NH2-DLC surface after culturing for 24 h.
M. Wang et al. / Diamond & Related Materials 38 (2013) 28–31
treatment, a new signal due to N 1 s at 399 eV is observed (Fig. 3b, c, d). It
is consistent with partial or full conversion of the hydrogenated surface
into amine-terminated DLC. Fig. 3(e, f) shows the high-resolution XPS
spectrum of the C 1-s peak before and after NH3 plasma treatment, illustrating that addition of nitrogen induces carbon phase variations.
It is expected that the sp3 C-H bonds are terminated with NH2
groups. The formation of the amine functionality on the DLC is confirmed by N 1-s spectra obtained by high-resolution XPS (Fig. 4). The
broad signal at 399.2 eV, which can be deconvoluted into two bands
at 398.6 and 399.6 eV, can be ascribed to the N-H and C-NH2 functional
groups, respectively [18].
Fig. 5 displays the fluorescent microscopy images of cells cultivated
for 24 h on the DLC with different surface termination. More cells attach
to the ammonia plasma-treated DLC, and they also spread better compared to the untreated surfaces. Fig. 6 shows the cell viability at different
days. The cell number increases with time and there are more cells on
NH2-DLC surface. The cell attachment and spreading appear to be related
to the surface wettability. Cells on the hydrogenated surfaces attach
more slowly and weakly and spread less, but on the other hand, the hydrophilic samples (amine-terminated) induce better cell adhesion and
proliferation.
4. Conclusions
H-DLC is deposited on silicon by PVD using a mixture of C2H2 /Ar
(23/7). The H-DLC surface is converted to an amine-terminated one by
ammonia plasma treatment. Fluorescent images of the cultured osteoblasts reveal more osteoblasts and better spreading on the amine functionalized surface after culturing for 24 h. The cell viability assay shows
that osteoblast proliferation is also improved on the plasma-treated
DLC surface after culturing for 1, 2, and 3 days. In summary, the amine
functionalized DLC shows better cyto-compatibility.
Prime novelty statement
In this paper, we report the direct formation of amine functionality on
DLC by means of Ar /NH3 plasma treatment and the biological responses
31
before and after the treatment are investigated and compared. We found
the amine functionalized DLC shows better cyto-compatibility.
Acknowledgments
This work was financially supported by the Hong Kong Research
Grants Council (RGC) (general research funds no. 112212), the City University of Hong Kong Applied Research Grant (ARG) (no. 9667066), the
National Nature Science Foundation of China (no. 51002090), the Outstanding Young Scientist Research Award Fund of Shandong Province
(no. BS2010CL028), and the Hong Kong Scholars Program sponsored by
The Society of Hong Kong Scholars.
References
[1] J.M. Halpern, S. Xie, G.P. Sutton, B.T. Higashikubo, C.A. Chestek, H. Lu, H.J. Chiel,
H.B. Martin, Diamond Relat. Mater. 15 (2006) 183.
[2] S. Xie, G. Shafer, C.G. Wilson, H.B. Martin, Diamond Relat. Mater. 15 (2006) 225.
[3] Y.L. Zhou, J.F. Zhi, Electrochem. Commun. 8 (2006) 1811.
[4] A. Härtl, E. Schmich, J.A. Garrido, J. Hernando, S.R. Catharino, S. Walter, P. Feulner,
A. Kromka, D. Steinmüller, M. Stutzmann, Nat. Mater. 3 (2004) 702.
[5] W. Yang, J.E. Butler, J.N. Russell, R.J. Hamers, Langmuir 20 (2004) 6778.
[6] K. Stolarczyk, E. Nazaruk, J. Rogalski, R. Bilewicz, Electrochem. Commun. 9 (2007) 115.
[7] A.H. Lettingtom, Proc. R. Soc. A 342 (1993) 287.
[8] R. Lappalainen, H. Heinonen, A. Antla, S. Santavirta, Dimond Relat. Mater. 7 (1998) 482.
[9] V.M. Tiainen, Diamond Relat. Mater. 10 (2001) 153.
[10] M.I. Jones, I.R. McColl, D.M. Grant, K.G. Parker, T.L. Parker, J. Biomed. Mater. Res.
52 (2000) 413.
[11] J. Robertson, Mater. Sci. Eng. R Rep. 37 (4–6) (2002) 129.
[12] G. Dearnaley, J.H. Arps, Surf. Coat. Technol. 200 (2005) 2518.
[13] C. Ronning, Appl. Phys. A 77 (2003) 39.
[14] R. Hauert, Diamond Relat. Mater. 12 (2003) 583.
[15] A. Dorner-Reisel, C. Schurer, C. Nischan, O. Seidel, E. Muller, Thin Solid Films 420
(2002) 263.
[16] A. Schroeder, G. Francz, A. Bruinink, R. Hauert, J. Mayer, E. Wintermantel, Biomaterials
21 (2000) 449.
[17] I. De Scheerder, M. Szilard, H. Yanming, J. Invasive Cardiol. 12 (8) (2000) 389.
[18] H.S. Biswas, J. Datta, D.P. Chowdhury, A.V.R. Reddy, U.C. Ghosh, A.K. Srivastava,
N.R. Ray, Langmuir 26 (22) (2010) 17413.