Exosomes purification based on PEG coated Fe3O4

• Main Authors: Pei-Yu Chao, 巢沛禹
• Other Authors: Ming Chang
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/02074383761495791195

碩士 === 中原大學 === 機械工程研究所 === 104 === Cancer also known as malignant tumors, is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body, for many years, cancer is a disease of the highest mortality, over 100 cancers affect humans. When cancer metastasis, cancer cells secretes a kind of microvesicles called “Exosome” to create the microenvironment at the destination. So, if we can detect the quantity of exosomes in human body, perhaps have the opportunity to check out the cancer at an early stage. Exosomes appears in biological fluids like blood, but it is impurity that contain lots of protein like globulin and albumin, will reduce the accuracy of liquid biopsy. So this study focuses on exosome isolation with magnetic nanoparticles. The use of chemical co-precipitation method to synthesize Polyethylene glycol(PEG)-coated Fe3O4 nanoparticles, the surface of the Fe3O4 has the reticular structure of PEG, can produce space crowing effect to make protein precipitated from the original location. The particles are uniformly mixed with serum, let particles catch protein, then use magnet to separate the particles and the supernatant, the supernatant contain pure exosomes. In order to use the protein precipitation, the purification of exosomes can be achieved. The diameter of PEG-coated Fe3O4 nanoparticles is about 20 nm, and the agglomerates size is about several hundred nanometers. The use of FESEM, XRD and FTIR to check the PEG is indeed coated on the surface of Fe3O4 nanoparticles. In the experiment of protein isolation, through ELISA to detect, the particles can decline the protein concentration to 39.9% of stock solution. To confirm exosomes are still in the supernatant, the result of Nanosight shows the supernatant content 1.6x1010 particles/ml the size of 30-200nm inclusions, same as the stock solution, SDS-PAGE shows the CD63 exosomes band (~53kD) both are equally show in supernatant and stock solution clearly, the immunoglobulin and albumin band (160~188kD and ~66kD) are shown in stock solution but disappear in supernatant, the result shows the exosome still in the supernatant but the other impurity protein are filtered. As a result, the PEG-coated Fe3O4 nanoparticles indeed can catch the protein and retain the exosome, this is a practical method in exosome isolation.