다기능 나노복합체 기반의 신속하고 간편한 엑소좀 분리 및 검출

Abstract

Exosomes are extracellular vesicle secreted from cells that are enclosed by a phospholipid bilayer. The size of exosome is about 30 - 200 nm. They are found in most biological fluids including serum, urine, saliva, cerebrospinal fluid, and breast milk. Exosomes are containing a variety of functional components, including nucleic acid, proteins, lipid, and metabolites. Recently, exosomes have been great interest as promising biomarker for early detection of cancer. However, despite their potential importance, enrichment of exosome remains significant issues due to the lack of exosome isolation technology. Therefore, simple and rapid exosome isolation and detection methods are required. In this dissertation, we developed the metal oxide-based multifunctional nanocomposites, CaTiO3:Eu3+@Fe3O4, to quickly and easily isolate and detect exosome. The CaTiO3:Eu3+@Fe3O4 nanocomposites were synthesized by high-energy ball-milling (HEBM), and their crystal structure and surface morphology were investigated by X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). XRD peaks of the CaTiO3:Eu3+@Fe3O4 nanocomposites were good matches with the standard XRD peaks of the orthorhombic CaTiO3 phase without impurities. The morphology of the CaTiO3:Eu3+@Fe3O4 nanocomposites present spherical shape and agglomerates of tiny particles. We also investigated the magnetic property of the CaTiO3:Eu3+@Fe3O4 nanocomposites using vibrating sample magnetometer (VSM). The saturation magnetization of the CaTiO3:Eu3+@Fe3O4 nanocomposites was less than that of the Fe3O4 nanoparticles. However, it was strong enough for effective magnetic separation in cell culture media. The synthesized CaTiO3:Eu3+@Fe3O4 nanocomposites used in the isolation and the concentration of the exosomes through the binding of CaTiO3:Eu3+@Fe3O4 nanocomposites and hydrophilic phosphate head of the exosome phospholipids. Compared with the commercially available TiO2, the proposed CaTiO3:Eu3+@Fe3O4 nanocomposites achieved comparable. We demonstrate a Surface-enhanced Raman Scattering (SERS)-based immunoassay for the detection of exosomal biomarker CD81. The CaTiO3:Eu3+@Fe3O4 nanocomposites were utilized as the separation and enrichment of exosome. Gold nanorods (Au NRs) were modified with detection antibodies and antibody-conjugated Au NRs were labeled with 3, 3, diethylthiatricarbocyanine iodide (DTTC), as the SERS probes. A method combining magnetic separation and SERS was developed to detection of exosomal biomarker CD 81. As a result, the proposed SERS-based immunoassay was able to detect with high sensitivity the exosome. The results demonstrate the feasibility of the new strategy as a useful tool for exosome isolation and detection. Therefore, this technique might be a useful tool for the early diagnosis of disease.