Detection method and application of extracellular vesicles and subtypes thereof in separation-free system
A system and detection probe technology, applied in biochemical equipment and methods, microbial determination/inspection, material excitation analysis, etc., can solve the problem of low sensitivity and achieve wide application prospects, simple operation, and high specificity.
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Embodiment 1
[0084] Example 1 Preparation of cell line extracellular vesicle standard solution
[0085] (1) Extraction of extracellular vesicles from cell lines
[0086] The cell culture medium was centrifuged at 3000g at room temperature to remove cells and large debris to obtain a supernatant; the obtained supernatant was centrifuged at 100,000g for 120 minutes, the supernatant was discarded to get the precipitate, and the PBS buffer with pH 7.4 was used. The pellet was resuspended, centrifuged again at 100,000g for 120 minutes, and the supernatant was discarded to collect the pellet. The pellet was resuspended in 100 μL of PBS buffer pH 7.4 to obtain cell line extracellular vesicles. The concentration of extracellular vesicles of the obtained cell line was measured by nanoflow, and then stored in aliquots at -80°C for the subsequent construction of a standard curve.
[0087] (2) Preparation of EVs-free plasma
[0088] Normal human plasma samples were centrifuged at 100,000 g overnigh...
Embodiment 2
[0091] Example 2 Construction of working curve of total EVs in tumor cell MCF7 detected by fluorescence polarization method
[0092] The EVs obtained in Example 1 were subjected to fluorescence polarization detection (principle such as figure 1 A). Will I biotin-Chol , II Fam-F , III Chol-C Three strands were mixed in the sample diluent at 1:1:1 to obtain a reaction solution with a final concentration of 2nM. Mix 50 μL of the reaction solution with 50 μL of standard solutions of MCF7 cell line EVs with different concentrations (the final concentration of EVs is 1.25×10). 2 pcs / μL, 1.25×10 3 pcs / μL, 1.25×10 4 pcs / μL, 1.25×10 5 pcs / μL, 1.25×10 6 pcs / μL, 1.25×10 7 1 / μL), react in the dark for 50 minutes at room temperature, then add 1 μL of 1 mg / mL streptavidin, and react in the dark for 10 minutes at room temperature. After the reaction, the reaction solution was added to a black microplate, and the fluorescence polarization value was measured with a multi-function mic...
Embodiment 3
[0094] Example 3 Construction of a working curve for detecting EVs expressing a specific protein in tumor cells MCF7 by fluorescence polarization
[0095] Ⅰ biotin-AptCD63 The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 CACCCCACCTCGCTCCCGTGACACTAATGCTA-3'.
[0096] The EVs obtained in Example 1 were subjected to fluorescence polarization detection (principle such as figure 1 B). Will I biotin-AptCD63 , II Fam-F , III Chol-C Three strands were mixed in the sample diluent at 1:1:1 to obtain a reaction solution with a final concentration of 2nM. Mix 50 μL of the reaction solution with 50 μL of different concentrations of MCF7 cell line EVs standard solution (the final concentration of EVs is 12.5 / μL, 1.25×10 2 pcs / μL, 1.25×10 3 pcs / μL, 1.25×10 4 pcs / μL, 1.25×10 5 pcs / μL, 1.25×10 6 pcs / μL, 1.25×10 7 1 / μL), react in the dark for 50 minutes at room temperature, then add 1 μL of 1 mg / mL streptavidin, and react in the dark for 10 minutes at room temperature. After the ...
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