Construction of a DNA-AuNP-based satellite network for exosome analysis

Abstract

As exosomes have been playing an increasingly important role in the diagnosis, treatment and prognosis of diseases, the analysis of exosome contents becomes more crucial. Therefore, the development of a cost-effective and simple exosome separation method that achieves high purity is urgently needed, and it is vital for further research in cancer. In this work, we constructed a DNA-AuNP-based satellite network which integrates low-speed centrifugal exosome isolation, detection and protein analysis. The rolling circle amplification (RCA) reaction is used to produce a long-chain DNA hairpin structure comprising a plurality of functional domains, such as CD63 aptamer sequences, linker sequences, and spacer sequences with complementary base pairs to form a hairpin structure. When the CD63 aptamers bind to exosomes, the hairpin structure changes its conformation, exposing the linker sequences (AuNP binding sequence). Then the probe on the surface of AuNPs combines with the long-chain DNA by the toehold-mediated strand displacement reaction, releasing the fluorescent labeled complementary probe as the detection signal and simultaneously forming the DNA-AuNP-based satellite network. Thus, exosomes can be isolated by low-speed centrifugation. The formation of the DNA-AuNP-based satellite network was confirmed by transmission electron microscopy and confocal fluorescence microscopy. In addition, we established a standard curve for exosome detection which showed good linearity of the fluorescence ratio vs. log(exosome concentration). LC/MS for protein profiling of the captured exosomes demonstrated that our method has potential application in the field of exosome research.