Preparation of chitosan-bound Fe3O4 nanoparticles
Fe3O4 nanoparticles were prepared by co-precipitation of the Fe3+and Fe2+ions by ammonia solution and treating under hydrothermal conditions [40]. Chemical precipitation was achieved at 25 °C under vigorous stirring by adding NH4OH solution. The precipitates were heated at 80 °C for 30 min, washed several times with water and ethanol, and then finally dried in a vacuum oven at 70 °C.
Since chitosan has no suitable functional groups to bind directly onto iron oxide nanoparticles, it was first carboxymethylated and then covalently bound onto iron oxide nanoparticles via carbodiimide activation. Chitosan was carboxymethylated according to the following method [41]. First, 3 g chitosan and 15 g sodium hydroxide were added into 100 ml of isopropanol/water (80/20) mixture at 60 °C to swell and alkalize for 1 h. Then, 20 ml of monochloroacetic acid solution (0.75 g ml−1 in isopropanol) was added into the reaction mixture in drops in 30 min. After reaction for 4 h at the same temperature, 200 ml of ethyl alcohol was added to stop the reaction. Finally, the solid was filtered, rinsed with ethyl alcohol to desalt and dewater, and dried in an oven at 50 °C.
The carboxymethyl chitosan (CM-chitosan) was conducted to bind on magnetic nanoparticles. First, 100 mg of Fe3O4 nanoparticles were added to 2 ml of buffer A (0.003 M phosphate, pH 6, 0.1 M NaCl). Then, the reaction mixture was sonicated for 10 min after adding 0.5 ml of carbodiimide solution (0.025 g ml−1 in buffer A). Finally, 2.5 ml of carboxymethyl chitosan solution (50 mg ml−1 in buffer A) was added and the reaction mixture was sonicated for 60 min. The chitosan-bound Fe3O4 nanoparticles were recovered from the reaction mixture by placing the bottle on a permanent magnet with a surface magnetization of 4000 G. The magnetic particles settled within 1–2 min and then were washed with water and ethanol.