Three different synthesis methods w

Three different synthesis methods were used in preparingsurface engineered Fe3O4 MNP. The aqueous stable carboxyl functionalized Fe3O4 magnetic nanoparticles (carboxyl MNP) wereobtained by co-precipitation method. In a typical synthesis, 5.406 gof FeCl3, 6H2O and 1.988 g of FeCl2, 6H2O were dissolved in80 ml of water in a round bottom flask and temperature wasslowly increased to 70 ◦C in refluxing condition under nitrogenatmosphere with constant mechanical stirring at 1000 rpm. Thetemperature was maintained at 70 ◦C for 30 min and then 20 ml of25% ammonia solution was added instantaneously to the reactionmixture, and kept for another 30 min at 70 ◦C. Then, 4 ml aqueoussolution (0.3 gm/ml) of succinic acid (SA) was added and temperature was slowly raised up to 90 ◦C under reflux and reacted for60 min with continuous stirring. The obtained black coloured precipitates were then thoroughly rinsed with water and separatedfrom the supernatant using a permanent magnet.