Selim et al. Nanoscale Research Letters 2011, 6:528 http://www.nanoscalereslett.com/content/6/1/528 insulin also acts as a growth factor, inducing cell prolif- eration [18,19]. It has been previously shown by research groups [18-21] that immobilized insulin stimu- lates cell growth more actively than free insulin. There- fore, introduction of PEG-insulin conjugate onto the surface of Q-dots through chemical bonding may confer the combined advantage of PEG and insulin. Introduc- tion of PEG onto the surface of nanoparticles protects against unwanted agglomeration, makes them more bio- compatible, and decreases their nonspecific intracellular uptake. On the contrary, insulin grafted onto the distal end of the PEG chain can enhance cells growth. In this study, mercaptoacetic acid-coated cadmium sulfide nanoparticles (CSNPs), typical semiconductor Q- dots, were synthesized in aqueous medium by the arrested precipitation method at room temperature. Then, PEG with amino groups at both ends was reacted with carboxyl groups of CSNPs (PCSNPs) in order to introduce amino groups to the surface as well as to enhance biocompatibility. Finally, insulin was immobi- lized on the surface of PCSNPs (ICSNPs) to promote cell growth and further enhance biocompatibility. The surface properties of CSNPs and ICSNPs were charac- terized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, transmission electron microphotography (TEM), and dynamic light scattering (DLS). Finally, human fibroblasts were cultured in the presence of nanoparticles to evaluate cell proliferation and cytotoxicity. Experimental Preparation of mercaptoacetic acid-coated CdS quantum dots (CSNPs) Water-soluble CSNPs were synthesized by following a previously published method [6]. Briefly, carboxyl-stabi- lized CSNPs were synthesized by arrested precipitation at room temperature in aqueous solution using mercap- toacetic acid as the colloidal stabilizer. Nanocrystals were prepared from a stirred solution of 0.0456 g of CdCl2 (5 mM) in 40 ml of pure water. The pH was low- ered to 2 with mercaptoacetic acid and then raised to 7 with 1 N NaOH. The mixture was deaerated by N2 bub- bling for about 30 min, after which 40 ml of freshly pre- pared 5 mM Na2S (0.0480 g of Na2S in 40 ml of water) was added to the mixture with rapid stirring. The solu- tion turned yellow shortly after the sulfide addition due to the formation of CSNPs (Scheme 1a in Additional file 1. CSNPs were separated from reaction by-products (sodium salt) via precipitation by the addition of acetone (4 ml of acetone per milliliter of nanocrystal solution). The precipitate was then isolated by centrifugation and dried in a freeze dryer. The prepared powder CSNPs were finally redispersed in water to obtain a clear colloi- dal solution with excellent stability (zeta potential, Page 2 of 9 -66.65 mV). The free carboxylic acid groups of the pre- pared CSNPs are suitable for covalent coupling with the primary amino groups of various biomolecules. Immobilization of insulin on the surface of CSNPs Immobilization of insulin on CSNPs was performed in two steps. First, CSNPs were reacted with amino-termi- nated polyethylene glycol (PEG) to introduce amine groups on their surface. For this, CSNPs (0.2 g) were dis- solved in aqueous solution (20 ml) containing 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC) and stirred for 4 h to activate the carboxylic acid groups on the sur- face. Then, an excess amount of amine-terminated PEG was added to the solution, which was stirred for 24 h to obtain PEG-grafted CSNPs (PCSNPs) (Scheme 1b in Additional file 1). An excessive amount of PEG was used to suppress the crosslinking reaction on the surface and keep free amine groups at one end of the PEG chain after the reaction [20]. Prepared PCSNPs were isolated via repeated centrifugation and finally dried in a freeze dryer. In the second step, insulin was immobilized on the surface of PCSNPs as follows: insulin was dissolved in phosphate buffer solution (2 mg/ml, pH 7.4) followed by the addition of a small amount of 0.1 N HCl. Then, 2% w/v water-soluble EDC and NHS were added to the solu- tion, which was incubated at 4°C for 5 h to activate the carboxylic acid groups of the chain. Then, PCSNPs (5 mg/ml) were suspended in phosphate buffer solution (pH 7.4) with vortexing. This PCSNP suspension was mixed with the insulin aqueous solution and stirred gently overnight at room temperature to obtain insulin- immobilized PCSNPs (ICSNPs), as shown in Scheme 1 in Additional file 1. ICSNPs were isolated by repeated cen- trifugation and stored in phosphate-buffered saline (PBS) at pH 7. All conjugation reactions, unless otherwise noted, were carried out in the dark under a N2 ambient environment. Surface characterization Fourier transform infrared (FT-IR) spectra were obtained using a JASCO FT-IR 300E spectrometer (JASCO Inc., Easton, MD, USA) at a resolution of 4 cm- 1. Dried samples were ground with KBr powder and compressed into pellets for FT-IR examination. The samples were prepared by dropping the diluted nano- particles on carbon-coated grids, followed by natural drying; then, the samples were observed by a transmis- sion electron microphotograph (Philips CM 200 TEM; applied operation voltage, 120 kV; Philips Inc, Berlin, Germany. The hydrodynamic diameter and size distribu- tion were determined by DLS by means of a standard laboratory-built light scattering spectrometer equipped with a BI 90 particle sizer (Brookhaven Instruments Corp., Holtsville, NY, USA). It had a vertically polarized