From Synthesis to Application: Functionalized Magnetic Nanoparticles as a Simple and Reliable Tool for Nucleic Acid Purification

Magnetic nanoparticles (MNPs) are widely used for nucleic acid (NA) extraction, but their performance strongly depends on the synthesis and surface functionalization. In this work, we applied a design of experiments (DoE) approach to optimize the coprecipitation synthesis of iron oxide nanoparticles, identifying NH₄OH flow rate and reaction temperature as the key factors. Under optimal conditions (5.5 mL min^-1, 65 °C), Fe₃O₄ nanoparticles were coated with SiO₂ and subsequently functionalized with (3-aminopropyl)-triethoxysilane (Fe₃O₄@SiO₂-APTES). The resulting nanoparticles (∼12 nm) were stable and magnetically responsive and provided efficient NA binding. Their performance in NA extraction was validated by RT-qPCR, yielding Ct values (20-25 for S, ORF, and N genes) comparable to those of both silica column and commercial magnetic bead methods. These results demonstrate that DoE is an effective strategy for tailoring nanoparticle synthesis and highlight Fe₃O₄@SiO₂-APTES as a simple, cost-effective, and reliable material for nucleic acid purification in routine laboratory applications.