3.2. Synthesis of Superparamagnetic Iron Oxide Nanoparticles

We used a co-precipitation method to synthesize the iron oxide (Fe₃O₄) nanoparticles which is based on Massart’s method [36,37], due to its simplicity and suitability for a variety of metal oxides [38]. To optimize the precipitation process, various influencing parameters were controlled. The control of size, shape, and nanoparticle properties during the co-precipitation method strongly depends on the types of salts, pH, temperature, supersaturation, mixing velocity (stirring), and Fe³⁺/Fe²⁺ molar ratio [39,40,41].

Typically, 2.2 g of FeCl₃·6H₂O and 0.8 g of FeCl₂·4H₂O with 2:1 molar ratio were dissolved in deionized (DI) water in a three-neck flask under vigorous stirring. Five grams of surfactant (dextran) were solubilized separately in deionized (DI) water in a beaker, and then added into the three-neck reaction flask. The reaction temperature was adjusted to 40 °C, to which 25% NH₃ solution as a precipitating medium was injected into the glass reactor under stirring. After 1 h reaction under the same conditions, a dark black solution was obtained, and stored in clean polypropylene plastic bottles. For labeling with a fluorescent marker, a stoichiometric amount of fluorescence dye was dissolved in DI water, and then a stoichiometric amount of iron oxide nanoparticles was introduced, followed by electronic shaking. The nanoparticles syntheses were probe-sonicated for 10 min. The overall reaction during the co-precipitation method is

Nanoparticle size was controlled/adjusted through a suitable choice of process and apparatus parameters during the synthesis.