Selim et al. Nanoscale Research Letters 2011, 6:528 http://www.nanoscalereslett.com/content/6/1/528 incident light of 514.5 nm supplied by an argon ion laser (Lexel laser, model 95; Cambridge Lasers Labora- tories Inc., Fremont, CA, USA. To investigate the crystal structure of CSNPs and bare CdS, XRD (RA/FR-571, Enraf Nonius, Deift, The Netherlands was used. The result was also compared with Joint Committee on Pow- der Diffraction Standards (JCPDS) file no. 10-454 to confirm whether or not any impurity phase exists in the CSNPs. The surface chemical composition was analyzed by electron spectroscopy for chemical analysis (ESCA, ESCA LAB VIG microtech, Mt 500/1, etc., East Grin- stead, UK) with MgK a at 1, 253.6 eV and 150 W of power at the anode. A survey scan spectrum was taken, and the surface elemental compositions relative to the carbon were calculated from the peak heights, taking into account atomic sensitivity. The zeta potential is a very useful way of evaluating the stability of any colloi- dal system. In this study, the zeta potential was mea- sured with a NicompTM 380 Zeta Potential (ZLS, Tokyo, Japan) employing the electrophoretic light scat- tering technique and using double-distilled water as a diluent. In vitro cell behavior MRC-5 human fibroblast cells (ATCC CCL, 171) were used in this experiment. Cells were routinely cultured at 37°C in a humidified atmosphere of 5% CO2 (in air) in a 75-cm2 flask containing 10 ml of Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin G sodium. The medium was changed every 3 days. For subculture, the cells were washed twice with PBS and incubated with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.25% trypsin, 1 mM EDTA) for 10 min at 37°C to detach the cells. The cells were washed twice by centrifugation and resuspended in DMEM media containing quantum dot nanoparticles, including CSNPs, PCSNPs, and ICSNPs (particle concentration, 0.2 mg/ml) for reseeding and growing in culture flasks. The cell density was fixed at 1 × 105 cells/ml. Cell morphologies were observed under a phase contrast microscope (Nikon Eclipse TS100, Tokyo, Japan) at pre- determined time intervals. The proliferation of fibroblasts cultured in the absence and presence of CSNPs, PCSNPs, and ICSNPs was determined by colorimetric immunoassay based on the measurement of 5-bromo-2-deoxyuridine (BrdU), which was incorporated during DNA synthesis [22,23]. BrdU enzyme-linked immunosorbent assay (ELISA; Roche Molecular Biochemicals, Mannheim, Germany) was per- formed according to the manufacturer’s instructions. Briefly, after 48 h of cell culture with CSNPs, PCSNPs, and ICSNPs in 24-well plates, the BrdU-labeling solu- tion was added to each well and allowed to incorporate Page 3 of 9 into the cells for an additional 20 h in a CO2 incubator at 37°C. Subsequently, the supernatant in each well was removed by pipetting. The cells were then washed twice with PBS and treated with 0.25% trypsin-EDTA (Gibco, Invitrogen, Tulsa, OK, USA) and harvested by centrifu- gation at 1, 000 rpm for 15 min. The harvested cells were mixed with a FixDenat solution to fix the cells and denature the DNA, followed by further incubation for 30 min. Subsequently, diluted anti-BrdU peroxidase (dilution ratio = 1:100) was added, and the cells were kept at 20°C for 120 min. After the removal of unbound antibody conjugates, 100 μl of substrate solution was added. The resulting mixture was allowed to stand for 20 min, and the reaction was completed by adding 1 M H2SO4 solution. The solution was then transferred to a 96-well plate and measured within 5 min at 450 nm with a reference wavelength of 690 nm using an ELISA plate reader. Cytotoxicity To evaluate the cytotoxicity of Q-dots, the cells were separately cultured in a dish containing CSNPs, PCSNPs and ICSNPs and in a polystyrene culture dish alone. For qualitative observation, Live/Dead fluorescent staining with a LIVE/DEAD Cytotoxicity Kit (Biovision research products, Mountain view, CA 94043 USA) was used. Briefly, fibroblasts (3 × 104 cell/well) were seeded in a microplate with 1 ml of media containing CSNPs, PCSNPs, and ICSNPs (particle concentration = 0.1 mg/ ml) without nanoparticles. After 2 and 4 days of incuba- tion, the media were removed and the cells washed gently with PBS. Then, 0.3 ml of staining solution (pre- pared by mixing calcein-AM and propidium iodide with staining buffer at a concentration specified by Molecular Probes) was added to each well, and the plate was kept in an incubator for 15 min. Then, calcein/propidium iodide solution was removed, and the cells were washed once again with PBS. Finally, cells were viewed using a fluorescence microscope (FV-300, Olympus Co., Tokyo, Japan) coupled with a digital camera (FV-300, Olympus Co.). Live cells show green fluorescence images and dead cells show red images. Statistical analysis The cell viability experiment was performed in triplicate, and the results are expressed as mean ± standard devia- tion. Student’s t test was employed to assess statistical significant difference of the results. Results and discussion Characterization of surface-modified CdS nanoparticles The X-ray diffraction spectra of CSNPs and bare CdS are shown in Figure 1. It was observed that the number and positions of peaks of CSNPs (Figure 1a) matched