Construction and application of a magnetic and catalytic hairpin assembly-based platform for detecting dual membrane proteins on exosomes.
10.12122/j.issn.1673-4254.2019.12.09
- Author:
Xianhua CHEN
1
;
Weilun PAN
1
;
Bo LI
1
;
Lei ZHENG
1
Author Information
1. Department of Clinical Laboratory, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
- Publication Type:Journal Article
- Keywords:
catalytic hairpin assembly;
exosome;
magnetic separation method;
membrane proteins
- MeSH:
Blotting, Western;
Exosomes;
Humans;
Magnetic Phenomena;
Membrane Proteins;
Sensitivity and Specificity
- From:
Journal of Southern Medical University
2019;39(12):1453-1460
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct a magnetic and catalytic hairpin assembly-based platform for detection of dual membrane proteins on exosomes.
METHODS:Exosomes in supernatant of breast cancer MDA-MB-231 cell culture were separated, purified and characterized. Super-resolution imaging and Western blotting were performed to confirm the expression of the membrane protein CD63 on the exosomes. Polyacrylamide gel electrophoresis was used to verify the combination of Apt-T and exosomes. Fluorescence experiments were carried out to test the feasibility of CHA nucleic acid sequence, optimize the reaction conditions, and determine the specificity of the detection platform.
RESULTS:Super-resolution imaging and Western blotting showed that breast cancer MDA-MB-231 cell-derived exosomes expressed abundant membrane protein CD63. Polyacrylamide gel electrophoresis indicated that Apt-T could recognize and bind to exosomes. The results of specificity test showed that the signal-to-noise ratio of the detection platform was 1.10±0.01 for detecting normal human breast epithelial cell-derived exosomes, and was 2.09±0.08 for breast cancer cell-derived exosomes.
CONCLUSIONS:Magnetic and catalytic hairpin assembly-based detection platform allows simultaneous detection of two membrane proteins expressed on exosomes and identification of the expressions of membrane proteins on exosomes from different sources.
