2.3. Synthesis of Iron Oxide Magnetic Nanoparticles Coated with Gold (Fe3O4/Au MNPs)

Magnetic nanoparticles composed of Fe₃O₄ and gold nanohybrid material (Fe₃O₄/Au MNPs), in particular, were synthesized via co-precipitation method according to previous reported methods with slight modification [25,26]. Briefly, 25 mL aqueous solution containing of 0.8 M FeCl₃, 0.4 M FeSO₄, 40 mM HCl was added drop wise into 1.5 M NaOH (250 mL) solution under vigorous stirring using a non-magnetic stirrer. Black Fe₃O₄ MNPs formed immediately, which were separated by NdFeB magnet and washed three times with DI water. The Fe₃O₄ MNPs were centrifuged for 15 min at 9000 rpm. Finally, the Fe₃O₄ MNPs were dispersed in DI water and stored at 4 °C for further use. To coat the Fe₃O₄ MNPs with gold nanoparticles, 5 mL aliquot of the Fe₃O₄ MNPs and 1.5 mL of 0.2 M NH₂OH·HCl was added into 75 mL of 0.01 M TMAOH aqueous solution. The solution was heated at 80 °C under vigorous stirring. Then, 40 mg of HAuCl₄ in 40 mL deionized water was added into the mixture drop by drop, followed by 100 mL of 15 mM sodium citrate solution was added drop wise. The color of the mixture changed from black to reddish brown gradually. The mixture was heated for another 1 h and then cooled to room temperature. The formed Fe₃O₄/Au MNPs were collected by a magnet, followed by centrifuged twice with deionized water and ethanol. Finally, the Fe₃O₄/Au MNPs was dried at 70 °C in a vacuum oven for 1 h.

For carboxylation steps, 2-mercaptoethanol and 12-mercaptododecanoic acid (ME-MDDA) was self-assembled on Fe₃O₄/Au MNPs surface by the well-known gold-thiol (Au-S) chemistry. Then, 10 mg of the Fe₃O₄/Au MNPs were suspended in 1 mM ethanolic ME-MDDA and incubated for 24 h at room temperature. The carboxylated nanoparticles were washed several times with water and dispersed in 0.01 M PBS solution with pH 7.4.