Diamond & Related Materials 38 (2013) 28–31

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Diamond & Related Materials

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Direct formation of amine functionality on DLC films andsurface 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 Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, Chinab Key Laboratory of Liquid Structure and Heredity of Materials, Ministry of Education, Shandong University, JiNan, 250061, China

⁎ Corresponding author.E-mail address: paul.chu@cityu.edu.hk (P.K. Chu).

0925-9635/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.diamond.2013.05.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 May 2013Received in revised form 13 May 2013Accepted 20 May 2013Available online 5 June 2013

Keywords:Diamond-like carbon(DLC)AmineProliferationCyco-compatibility

Hydrogenated diamond-like carbon (H-DLC)filmswere synthesized ona p-type siliconwafer using radio frequencyplasma composed of amixture of Ar and C2H2 (ratio of 7 to 28). NH3 plasma treatment was carried out to generatesurface-terminal amino groups. The treated surfaces were characterized by Raman scattering, atomic force micros-copy (AFM), water contact angle, and X-ray photoelectron spectroscopy (XPS). MC3T3-E1 mouse pre-osteoblastswere cultured on the samples, and cell morphology and proliferation were investigated to evaluate the cyto-compatibility. Cell–surface interactions were investigated by fluorescence microscopy in terms of spreading andproliferation. A cell count kit-8 (CCK-8 Beyotime) was employed to determine quantitatively the viable pre-osteoblasts. The formation of the amine functionality led to better cyto-compatibility on the DLC.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Doped diamond electrodes have been widely reported in bio-medical applications [1–6], but the practical use of diamond hasbeen hampered by its scarcity and high cost. Diamond-like carbon(DLC) has properties similar to diamond, for instance, high hard-ness, 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 uniqueproperties of DLC match the criteria of good biomaterials for appli-cations to orthopedics, cardiovascular implants, contact lenses, ordentistry [7–12].

To be more competitive with other biomaterials, a high func-tional surface addressing biological needs is crucial to the suc-cess of DLC. Recently, many techniques such as photochemicalreactions, radical reactions, plasma treatment, and electrochemi-cal reactions have been proposed [13,14]. The incorporation ofadventitious elements into DLC is a good means to alter its proper-ties [14]. For example, the biological reactions on DLC can be changedby alloying [15–17]. In this paper, we report the direct formation ofamine functionality on DLC by means of Ar /NH3 plasma treatmentand the biological responses before and after the treatment are investi-gated and compared.

rights reserved.

2. Experimental details

2.1. Preparation of DLC

DLC films (1–1.5 μm thick) were deposited on p-type silicon sub-strates (12 mm × 12 mm) by physical vapor deposition (PVD) in a con-ventional reactor. The fabrication conditions were as follows: substratetemperature = room temperature (RT), ratio of C2H2/Ar = 23/7, volt-age applied to the cathode = 10 kV, plasma power = 150 W, pres-sure = 6 × 10−2 Pa, and deposition time = 3 h. The silicon substrateswere ultrasonically washed with acetone, alcohol, and distilled waterfor 15 min sequentially and then dried before introduction into thedeposition chamber. Prior to DLC deposition, an argon plasma was trig-gered to clean the substrates for 15 min at a bias voltage of 1 kV toremove undesirable surface oxide and contamination.

2.2. Plasma treatment

Ammonia plasma treatment was carried out in the same PVD systemunder the following conditions: substrate temperature = room temper-ature; 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 chemicalcharacteristics of the carbon films. The Raman spectra were excitedby a 514-nm argon ion laser with a power of 30 mW at room

Fig. 1. AFM images of the as-deposited DLC surface.

Table 1Water contact angle of DLC surfaces with different terminations.

Surface Contact angle, θ(°)

As-deposited DLC 75 ± 2Plasma-treated DLC 50 ± 2

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

4.0x105

(a)

(b)

O 1sN 1s

C 1s

Inte

nsi

ty/ a

.u.

Binding energy/ ev

0

500

1000

1500

2000

2500

3000

3500

4000

4500

d

N 1s

Inte

nsi

ty/ a

.u.

Binding energy / eV

c

200 400 600 800

392 394 396 398 400 402 404 406

29M. Wang et al. / Diamond & Related Materials 38 (2013) 28–31

temperature, and the spectra in the range of 800–4000 cm−1 werefitted using Gaussian distributions. The surface morphology of filmswas characterized by atomic force microscopy (AFM), and X-ray photo-electron spectroscopy (XPS) was employed to determine the surfacecomposition after ammonia plasma treatment. The wetting propertieswere evaluated by measuring the water contact angles.

2.4. Cell cultures

Mouse MC3T3-E1 pre-osteoblasts were cultured in high-glucoseDulbecco'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 platesat a density of 6.7 × 104 cells per well for cell morphology observationand proliferation assay. After culturing for 24 h, the cells were rinsedwith PBS and fixed with 4% paraformaldehyde in a humidified atmo-sphere of 5% CO2 at 37 °C for 20 min. After fixing, the cells were thor-oughly rinsed with 2,4,6-trinitrobenzenesulfonic acid (TNBS) andthen stained with phalloidin and 4′,6-diamidino-2-phenylindole (DAPI)

1000 1500 2000 2500 3000 3500 4000

400

600

800

1000

1200

1400

1600

Inte

nsi

ty (

a.u

)

Raman shift (cm-1)

Fig. 2. Raman spectra of the as-deposited DLC.

sequentially prior to examination by fluorescence microscopy. A cellcount kit-8 (CCK-8 Beyotime) was employed to determine quantita-tively the viable pre-osteoblasts. The cells were cultured by renewingthe medium every day. After culturing for 1, 2, and 3 days, the samples

280 285 290 295

(f)

(e)

Inte

nsi

ty/ a

.u.

Binding energy/ ev

Fig. 3. XPS survey spectra of (a) as-deposited and (b) NH2-DLC surfaces; high-resolutionXPS spectra of the N 1-s band of (c) as-deposited DLC and (d) NH2-DLC and high-resolution XPS spectra of the C 1-s band of (e) as-deposited DLC and (f) NH2-DLC.

396 398 400 402 404

Inte

nsi

ty/ a

.u.

N-H

Binding energy / eV

N 1s

C-NH2

Fig. 4. High-resolution N 1-s XPS spectra of the NH2-DLC surface.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Ab

sorb

ance

4

50n

m

1day 2days 3days

Culture Time

DLC-H

DLC-NH2

Fig. 6. Cell viability assays of cells incubated on H-DLC and NH2-DLC for 1, 2, and 3 days.

30 M. Wang et al. / Diamond & Related Materials 38 (2013) 28–31

with the seeded cellswere rinsed twicewith sterile PBS and transferred tofresh 12-well plates. The culture medium with 10% CCK-8 was added tothese samples, and after incubation for 4 h, the solution was aspiratedand the absorbancewasmeasured at 450 nmusing 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 diameteraround 40 nm). The root mean square (RMS) roughness and averageroughness (Ra) are quite small (approximately 1 nm), indicating asmooth surface.

Fig. 2 shows the Raman spectra of the as-deposited DLC films. The Gpeak at 1580 cm−1 and the D peak at 1350 cm−1 are consistent withthose in typical Raman spectra of DLC films [11]. The G band correspondsto the stretching vibration of sp2 carbon, whereas the D band is attributedto thebreathingmodeof sp2. The peaks around2850 cm−1 are associated

Fig. 5. Fluorescent images of osteoblasts: (a, c) as-deposited DLC

with vibrational modes of CH, CH2, and CH3 groups, indicating covalentbonding with the suitable functional groups.

Water contact anglemeasurementswere used to examine themacro-scopic evolution of thewetting properties of theDLC surface (as shown inTable 1). The Raman spectra suggest that the as-deposited DLC is hydro-gen terminated. This termination confers a hydrophobic characteristicwith a water contact angle of θ = 75 ± 2°. After ammonia plasmamodification, the contact angle decreases significantly to θ = 50 ± 2°,indicating that the DLC becomes hydrophilic. It is possibly due to theformation of surface amine groups.

X-ray photoelectron spectroscopy is used to determine the chemicalcomposition of the DLC surfaces before and after NH3 plasma treatmentas well as the nature of the chemical bonding associated with transfor-mation on the surface. Fig. 3a displays the XPS survey spectra of theas-depositedDLC surface. The spectrum is dominated by the C 1-s signalat 283 eV from the substrate and a small peak due to oxygen O 1 s at531 eV. The oxygen signalmay originate fromdifferent sources, includingsurface contamination, partial oxidation of the surface during storage andhandling, and interstitial oxygen on the DLC surface. After NH3 plasma

surface and (b, d) NH2-DLC surface after culturing for 24 h.

31M. Wang et al. / Diamond & Related Materials 38 (2013) 28–31

treatment, a new signal due toN 1 s at 399 eV is observed (Fig. 3b, c, d). Itis consistent with partial or full conversion of the hydrogenated surfaceinto amine-terminated DLC. Fig. 3(e, f) shows the high-resolution XPSspectrum of the C 1-s peak before and after NH3 plasma treatment, illus-trating 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 con-firmed by N 1-s spectra obtained by high-resolution XPS (Fig. 4). Thebroad signal at 399.2 eV, which can be deconvoluted into two bandsat 398.6 and 399.6 eV, can be ascribed to the N-H and C-NH2 functionalgroups, respectively [18].

Fig. 5 displays the fluorescent microscopy images of cells cultivatedfor 24 h on the DLCwith different surface termination. More cells attachto the ammonia plasma-treated DLC, and they also spread better com-pared to the untreated surfaces. Fig. 6 shows the cell viability at differentdays. The cell number increases with time and there are more cells onNH2-DLC surface. The cell attachment and spreading appear to be relatedto the surface wettability. Cells on the hydrogenated surfaces attachmore slowly and weakly and spread less, but on the other hand, the hy-drophilic samples (amine-terminated) induce better cell adhesion andproliferation.

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 byammonia plasma treatment. Fluorescent images of the cultured osteo-blasts reveal more osteoblasts and better spreading on the amine func-tionalized surface after culturing for 24 h. The cell viability assay showsthat osteoblast proliferation is also improved on the plasma-treatedDLC surface after culturing for 1, 2, and 3 days. In summary, the aminefunctionalized DLC shows better cyto-compatibility.

Prime novelty statement

In this paper,we report the direct formation of amine functionality onDLC bymeans of Ar /NH3 plasma treatment and the biological responses

before and after the treatment are investigated and compared.We foundthe amine functionalized DLC shows better cyto-compatibility.

Acknowledgments

This work was financially supported by the Hong Kong ResearchGrants Council (RGC) (general research funds no. 112212), the City Uni-versity of Hong Kong Applied Research Grant (ARG) (no. 9667066), theNational Nature Science Foundation of China (no. 51002090), the Out-standing Young Scientist Research Award Fund of Shandong Province(no. BS2010CL028), and the Hong Kong Scholars Program sponsored byThe Society of Hong Kong Scholars.

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