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.
Subject(s)
Humans , Blotting, Western , Exosomes , Magnetic Phenomena , Membrane Proteins , Sensitivity and SpecificityABSTRACT
Isothermal nucleic acid amplifications, as powerful as polymerase chain reaction but functioning at a constant temperature, are considered to be very promising technique in achieving point-of-care gene diagnostics. However, until now, their practical applications are still seriously lagged by the bad reliability resulting from the problems such as false positive amplification and low signal amplitude. In this work, a universal transduction method in which any sequence ( including loop-mediated isothermal amplification products) could be transduced via a hairpin transducer into a catalyst of a well-engineered circuit (catalytic hairpin assembly, CHA) was established. Because CHA circuit could amplify tens to hundreds fold with especially high sequence specificity, it could provide both accuracy and high amplitude for sequence detection. And for a new targeting sequence, the only sequence needed to be changed was the hairpin transducer. Due to the importance of the transducer, we provided and verified a universal designing rule-set to guarantee the transducing efficiency ( signal to background ratio) of the transducer. Transducers designed following this rule set were then proved to be very efficient in detecting pathogen gene targets. As less as near single molecule ( 20 copies ) of pathogen genes could be detected with significant fluorescent and electrochemical signals.