| Summary: | 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.
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