The Study On Exosome Detection Method Of Breast Cancer Based On G-quadruplex

Breast cancer is the most common malignant tumor among women.Early diagnosis and treatment of breast cancer is of great significance to increase the survival of patients.In addition to the normal serum markers such as special proteins and nucleic acids,there are also some micro-scale nanoscale vesicles in the peripheral blood of breast cancer patients.It has been found that these nanoscale vesicles can be used as biomarkers for the early diagnosis of breast cancer.Exosomes are small lipid-encapsulated extracellular vesicles,containing tumor proteins,mRNAs and other tumor-specific molecular markers.It transmits molecular messengers between cells,participates in signal transduction between tumor cells and microenvironment,and changes the phenotype of receptor cells.Exosomes has clinical application value as a biomarker for liquid biopsy in the next generation of cancer diagnosis and management.Exosomes are secreted vigorously on the surface of tumor cells and can be isolated from body fluids for diagnosis and treatment of tumors.Therefore,exosomes as biomarkers have great potential in monitoring blood or urina-based diagnosis,prognosis,and cancer.So far,there have been many reports on exosomes,but convenient and low-cost detection methods still need to be developed.Based on this,we put forward two kinds time-saving and low-cost detection methods of exosomes,one of which is using G-quadruplex DNA peroxidase mimicase to achieve the high sensitivity of exosome detection,and the second method uses the irregular tetrahedral DNA structure as the scaffold,connects the natural enzyme and the artificial enzyme(DNA peroxidase mimic enzyme),and realizes the exosome detection through the enzyme cascade signal amplification.The main research contents of this paper are as follows:1.Detection of breast cancer-derived exosomes using the horseradish peroxidase-mimicking DNAzyme as an aptasensorWe have de'veloped a sensitive,simple and low-cost colorimetric aptasensor by designing a hairpin-like structure,which combined the highly specific MUC1 aptamer with a hemin/G-quadruplex for detection of breast cancer exosomes.The hemin/G-quadruplex toward H2O2 reduction were used to generate evidently strong colorimetric response owing to acting as a HRP-mimicking DNAzyme.The aptasensor is regarded as an "on-off" type switch,which strictly control the process of reaction responding to the existence or not of exosomes.In our study,associated exosome detection limits are 3.94×105 particles/mL,which showed a higher sensitivity compared to commercial ELISA.Our method not only exhibited the advantages of convenient and time-saving,with few instruments used,but also can be easily observed by naked eyes of the signal generation.Furthermore,the proposed strategy can well differentiate breast cancer patients from healthy individuals,demonstrating potential application to analysis of clinical specimens.2.Tetrahedral scaffold structure combined with multi-enzyme cascade amplification system was used for the detection of breast cancer exosomes.As a kind of DNA scaffold structure,tetrahedron avoids the disadvantages of poor controllability and poor biocompatibility of sclerotia skeleton compared with traditional scaffold.Based on the rigidity and controllability of the tetrahedron,we modified the tetrahedron side length,and designed two sides of the tetrahedron as special single-chain functional sequences,which were paired with the closed chain containing the protein adaptor by base complement.This functional sequence includes two parts:one is the complementary sequence of exosome protein adapter.The other one is rich in the simulated DNA enzyme sequence of guanine.When exosomes are not present,the probe fail to perform peroxidase simulation;Conversely,when the exosome sample is added,the exosome combines with aptamer,and the close-chain is separated from the tetrahedron.Then,the probe is exposed to the sequence of the simulated enzyme.Under the action of hemin,the guanine-rich simulated DNA enzyme sequence forms the spatial structure of the hemin/G-quadruplex,and plays the role of the simulated HRP peroxidase.The formation of Hemin/G-quadruplex not only makes the sensor switch but also enables spatial proximity between natural protease molecules and HRP-mimicking enzymes.After the enzymatic hydrolysis reaction,H2O2 accumulates in high concentration in the space around hemin/G-quadruplex,thus achieving the enzymatic cascade amplification effect.This strategy lays the foundation for the development of new and reliable scaffold materials and the construction of new biosensors,which is of great significance for early cancer diagnosis and sensing analysis.