Study Of Novel Methods For Detection Of Breast Cancer-Derived Exosomes

Tissue biopsies are the gold standard for molecular evaluation,but their invasiveness and limited sampling challenge clinical management,especially in the face of tumor spatial heterogeneity and temporal evolution.As a non-invasive biomarker for diagnosis and prognosis,exosome can reveal its biological function and its relationship with disease and has great potential in clinical diagnosis.Nevertheless,establishing of a simple and quantitative method for exosome detection is still a challenging task.Currently,most of the methods usually involve labor-intensive procedures,require lengthily quantitative protocols and expensive laboratory infrastructures,which delay the diagnosis and treatment,and greatly limits their wide application.In this paper,novel methods for the analysis of breast cancer-derived exosomes were proposed,which were simple,fast,accurate and sensitive.Furthermore,exosomal nucleic acids were also investigated.The major contents of the thesis were as follows1.A Novel Method for Detection of Exosomes Based on Mussel-Inspired Surface ChemistryIn this work,we reported a novel approach for exosome quantification using pH test paper via HRP-mediated promotion of mussel-inspired surface engineering and reagent-free functionalization of urease molecules.Urease can hydrolyze urea into ammonia and carbon dioxide,and simultaneously raise the pH value of the solution.By establishing the relationship between exosome recognition and the change of pH value of the sensing solution,we can directly employ the low-cost,widely used and commercially available pH test paper to quantitatively analyze exosomes.The pH-responsive bioassay enables sensitive detection of exosomes with a detection of limit down to 4.46×103 particles/?L and can be successfully applied for determination of exosomes in clinical specimens.The versatility and reliability of our sensing platform may open up opportunities towards cancer diagnostics in paper-based point-of-care testing(POCT).2.A Novel Method for Detection of Exosomes Based on Membrane Fusion-Activated Cascade Signal AmplificationIn this work,we proposed a novel method for label free analysis of exosomes based on membrane fusion-activated cascade signal amplification.Cationic liposomes containing horseradish peroxidase(HRP)and phospholipase A2(PLA2),are tethered to negatively charged exosomes via electrostatic interactions allowing efficient content mixing and transfer of HRP and PLA2 molecules for the subsequent stage.Upon membrane fusion,PLA2 is activated and hydrolyzes the phospholipid comportments of exosomes to release HRP molecules.Through a sequential cascade of membrane fusion,lipase degradation and HRP-assisted oxidation,the concentration of exosomes can be directly determined by measuring the absorbance chance.Its performance may satisfy the requirements for direct,rapid,sensitive cancer diagnosis.It also provides a novel mode of signal transduction for exosome detection,signifying its great potential in clinical diagnostics3.A Novel Method for Detection of Exosomal Nucleic Acids Based on Cell Membrane-Biointerfaced Gold NanoshellsIn this work,we fabricated a plasmon-enhanced fluorescence probe for miRNAs assay through gold nanoshells(AuNSs)biointerfacing by erythrocyte membrane clocking,which not only enables tailor-engineered signal amplification but also exhibits anti-interference property.As a consequence,the assay enables direct and absolute analysis of miR-141 in complex media without any form of sample preparation or enzymatic amplification,which vastly simplifies the detection procedure.Experimental results reveal that the assay permits ultrasensitive quantification of miR-141,with a detection of limit down to 33.9 aM,and shows a high selectivity for discriminating miR-200 family members.More importantly,the assay enables robust miRNA analysis in complex medium.Its performance satisfies the requirements for direct,rapid,sensitive and specific early diagnosis of cancer,signifying its great potential in clinical diagnostics.