Xianhua Chen1,2, Weilun Pan1, Bo Li1, Lei Zheng1. 1. Department of Clinical Laboratory, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. 2. Department of Clinical Laboratory, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou 545007, China.
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 AptEpCAM-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 AptEpCAM-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.
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 cancerMDA-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 AptEpCAM-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 cancerMDA-MB-231 cell-derived exosomes expressed abundant membrane protein CD63. Polyacrylamide gel electrophoresis indicated that AptEpCAM-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.
Entities:
Keywords:
catalytic hairpin assembly; exosome; magnetic separation method; membrane proteins
Authors: Marcel I Ramirez; Maria G Amorim; Catarina Gadelha; Ivana Milic; Joshua A Welsh; Vanessa M Freitas; Muhammad Nawaz; Naveed Akbar; Yvonne Couch; Laura Makin; Fiona Cooke; Andre L Vettore; Patricia X Batista; Roberta Freezor; Julia A Pezuk; Lívia Rosa-Fernandes; Ana Claudia O Carreira; Andrew Devitt; Laura Jacobs; Israel T Silva; Gillian Coakley; Diana N Nunes; Dave Carter; Giuseppe Palmisano; Emmanuel Dias-Neto Journal: Nanoscale Date: 2018-01-18 Impact factor: 7.790
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