Electrochemiluminescence Analysis Of Exosomes Derived By Breast Cancer Cells And Their Surface Proteins

Breast cancer is the most common malignancy with the highest incidence rate（30%）and mortality（15%）.To improve the survival rate of cancer patients,cancer surveillance is imperative during the treatment of breast cancer,as well as recurrence after cure.However,the common pathological diagnosis often requires not only clinical information,but also qualified,adequate and complete biopsy or tissue specimens.The sampling may cause great physical and mental damage to patients.Exosomes,nanoscale bilayer vesicles actively secreted by cells,are widely found in almost all body fluids,including blood,urine,breast milk,sweat,tears,etc.Almost all cells can release exosomes under both physiological and pathological conditions,and tumor-derived exosomes play critical roles in tumor development.Exosomes carry a large number of nucleic acids,proteins,lipids and other bioactive substances,and many of their properties have been proved to be similar or closely related to their parent cells.Therefore,exosomes are more accessible than tumor samples and have the potential to be used as markers of non-invasive surveillance.Thus,the development of methods that can detect the composition of tumor-derived exosomes,such as protein types and expression levels,is necessary to further promote the application of exosomes in cancer surveillance.Recently,various analysis techniques have been used to detect exosomal composition.Western blot（WB）,flow cytometry and enzyme-linked immunosorbent assays（ELISA）can determine the biological characteristics and protein compositions of exosomes,but they are time-consuming and laborious,needing a large number of samples.Therefore,they are impractical for clinical application.Surface plasmon resonance（SPR）,mass spectrometry（MS）,nuclear magnetic resonance and surface enhanced raman scattering（SERS）are efficient and accurate analytical techniques,and can analyze a small amount of samples.However,they commonly require expensive instruments and professional operations.Electrochemiluminescence（ECL）analyzes the chemiluminescence caused by electrochemical reaction of luminescent substance on electrode surface.Thus,it combines the advantages of electrochemical and chemiluminescent biosensors,such as high sensitivity,high stability,low cost,easy operation and easy automation.Also,ECL does not need the stimulation of external light source and has low background signal.Therefore,electrochemiluminescence methods have been proposed for the analysis of proteins on the surface of breast cancer cells-derived exosomes with the further application in actual serum samples.1)Graphite-like carbon nitride nanosheets（g CN NSs）were prepared by high temperature calcination and ultrasonic stripping,and then combined with ruthenium pyridine(Ru（bpy）₃²⁺)modified gold nanoparticles（Au@Ru NPs）to form a composite nanomaterials--Au@Rug CN NSs.Based on the electrochemiluminescence resonance energy transfer（ECL-RET）between Ru（bpy）₃²⁺and g CN NS_S,the signal amplification was realized.The modification of 4-mercaptophenylboric acid（MPBA）on Au@Ru-g CN NSs,obtaining MPBA-Au@Ru-g CN nanoprobes,enabled the recognition of glycoproteins on the surface of exosomes.Thus,a highly sensitive electrochemiluminescence sensor was constructed to analyze MCF-7exosomes and the expression of glycoprotein on the surface.It exhibited linear responses in the range of 100 to 1×10⁷ exosomes/m L with a limit of detection（LOD）of 58 exosomes/μL（S/N=3）.2)Luminol@Au-aptamer nanoprobes（lum@Au-apt NPs）were prepared by reducing chlorauric acid with luminol,and Ru（bpy）₃²⁺@Si O₂-aptamer nanoprobes（Ru@Si O₂-apt NPs）were obtained by reverse microemulsion method.A potential-resolved electrochemiluminescence aptasensor was developed with these two nanoprobes to detect simultaneously MUC1 and HER2 proteins on the surface of exosomes by blocking the ECL-RET interaction between different probe molecules using physical space segmentation.It can distinguish the subtle differences in the expression of different exosome proteins and be applied to the detection of exosome in actual serum samples,which has the potential for clinical application.