Synthesis of SiO2-Coated Fe3O4 Nanoparticles Using Ultrasound and Its Application in DNA Extraction from Formalin-Fixed, Paraffin-Embedded Human Cancer Tissues

摘要

SiO2-coated Fe3O4 nanoparticles (Fe3O4@SiO2 NPs) were successfully synthesized using ultrasound in order to extract DNA from cancer tissues for application in diagnostics. The core 10.7-nm-diameter Fe3O4 nanoparticles were synthesized by co-precipitation of Fe3+ and Fe2+ as reaction substrates and NH4OH as precipitant, then coated with a thin layer of amorphous silica by a modified Stober method. Further SiO2 coating using alkaline hydrolysis of tetraethyl orthosilicate in ethanol and water mixture was accelerated in the presence of a 37-kHz ultrasound, resulting in the NPs having different sizes of 14.5 nm (version M1), 24.4 nm (version M2), and 34.9 nm (version M3) with saturation magnetization values of 50.2 emu/g, 18.6 emu/g, 10.3 emu/g, respectively. Among the three Fe3O4@SiO2 NPs versions, the M1 NPs allowed extraction of DNAs from 10 mg formalin-fixed and paraffin-embedded (FFPE) tissues of nasopharyngeal carcinoma patients with the highest recovery of about 100-500 ng/mu l and good purity (A(260)/A(280): 1.8-1.9). The extracted DNAs could be used as templates for downstream amplification of 252-bp sequencing specifically for the Braf cancer biomarker gene using polymerase chain reaction (PCR), as well as detection of the pathogenic Epstein-Barr virus (EBV) and the human papilloma-virus (HPV) using real-time PCR. DNA extraction recoveries of both EBV and HPV using Fe3O4@SiO2 NPs M1 were significantly better that those using commercialized Fe3O4@SiO2 microbeads, as indicated by lower threshold cycles of all fluorescent signals including fluorescein amidite (FAM) dye representative for EBV infection, hexachlorofluorescein (HEX) dye representative for beta-globin (internal control), and SYBR Green dye representative for HPV infection in tested clinical samples from patients with nasopharyngeal carcinoma (NPC).