Functional Nucleic Acid-based Biosensing Technology For Detecting Of Breast Cancer-derived Exosomes

Breast cancer-derived exosomes carry a variety of cell specific proteins,nucleic acids,and lipids,and can participate in cell communication,cell migration,angiogenesis and immune reaction.Therefore,the detection of breast cancer-derived exosomes is very important for the early diagnosis and prognosis monitoring of breast cancer.However,the detection of breast cancer-derived exosomes still faces challenges:on the one hand,in the early stage of breast cancer,the concentration of exosomes secreted by cancer cells in body fluid is very low,so how to avoid false negative signals caused by insufficient sensitivity;On the other hand,how to accurately detect breast cancer-derived exosomes in complex system.Herein,aiming at the above problems,this thesis has constructed a series of sensitive and accurate methods for the detection of breast cancer-derived exosomes.By using functional nucleic acid probes（including aptamer and DNA tetrahedron）as recognition elements and introducing nucleic acid amplification strategy,studies on the surface protein and mi RNA of breast cancer-derived exosomes have been investigated.The research contents are as follows:1.Sensitive detection of breast cancer-derived exosomes was critical to early diagnosis of breast cancer.Herein,a sensitive surface plasmon resonance（SPR）sensor was demonstrated for breast cancer-derived exosomes detection by aptamer with dual gold nanoparticle（Au NP）-assisted signal amplification.Dual nanoparticle amplification was achieved by controlled hybridization attachment of Au NPs resulting from electronic coupling between the Au film and Au NPs,as well as coupling effects in plasmonic nanostructures.By blocking the Au film surface with 11-Mercapto-1-undecanol,nonspecific adsorption of Au NPs onto the SPR chip surface was suppressed and regeneration of the SPR sensor was realized.This method was highly sensitive and we have achieved the limit of detection（LOD）down to 5.0×10³ exosomes/m L,which showed a 104-fold improvement in LOD compared to commercial ELISA.Furthermore,the SPR sensor could effectively detect the exosomes in 30%exosomes-depleted fetal bovine serum（UC FBS）.The work was expected to provide a new method for the early diagnosis of breast cancer.2.Developing a simple point-of-care testing（POCT）metod for breast cancer-derived exosomes detection was crucial to liquid biopsy.However,conventional POCT methods of exosomes often used single-readout mode,which were susceptible to external interference and undoubtedly affected the accuracy of detection.Herein,a colorimetric and photothermal POCT biosensor was demonstrated based on rolling circle amplification（RCA）mediated 2,2’-azinobis-（3-ethylbenzthiazoline-6-sulphonate）（ABTS）-H₂O₂ system for sensitive detection of breast cancer-derived exosomes.In the presence of target exosomes,RCA could be triggered and produced multiple units that hybridized to horseradish peroxidase（HRP）modified DNA probes.Colorless ABTS would be converted by HRP into the oxidized form with strong near-infrared absorbance.Then,colorimetric and photothermal responses were recorded by optical fiber amplifier and forehead thermometer,respectively,enabling hand-held detection.Moreover,Ep CAM aptamer was used as biorecognition element for MCF-7 exosomes,so that the POCT method could effectively detect the cancerous exosomes in 30%fetal bovine serum with lots of normal exosomes.Moreover,the biosensor could avoid the influence of the external environment,including surrounding light and temperature.The dual-readout POCT method was expected to provide a new method for accurate detection of breast cancer-derived exosomes.3.Accurate analysis of breast cancer-derived exosomes has great prospects in non-invasive breast cancer diagnosis.However,exosomes from different breast cancer cells are usually heterogeneous,which face challenges to sensitive and accurate identification of breast cancer-derived exosomes.Human epidermal growth factor receptor 2（HER2）overexpressed breast cancer has a higher risk of metastasis.Herein,we constructed an accurate detection method of breast cancer-derived exosomes by scattering based on proximity ligation using fluorescence spectrophotometer and flow cytometry.In this method,three aptamers were used as exosomes recognition elements,and gold nanoparticles enhanced the scattering signal.In addition,based on the separation and enrichment of magnetic beads,this method could directly detect exosomes in50%fetal bovine serum and detect exosomes inμL clinical serum samples.This work provided a new strategy for the detection of HER2 positive breast cancer-derived exosomes,and was expected to provide an effective tool for accurate early detection of breast cancer-derived exosomes.4.The detection of various exosomal mi RNAs could provide high-precision and accurate disease information,but conventional methods of exosomal mi RNAs faced the challenges of poor sensitivity and narrow dynamic range.Herein,a sensitive imaging ellipsometry sensor was introduced to improve sensitivity with a tunable detection range by terminus-regulated DNA hydrogelation.Tetrahedron DNA probes with complementary sequence to the target mi RNA were used as biorecognition elements to form DNA hydrogelation.This DNA hydrogelation was formed by templateindependent and isothermal amplification on the Au film.Due to its high dielectric constant,DNA hydrogelation structure could be used for improving the sensitivity of imaging ellipsometry significantly.Importantly,by changing the cycle of the DNA hydrogelation amplification,this strategy showed a tunable detection range from f M to n M for mi RNA with a limit of detection of 0.2 f M for let-7a,10 f M for mi R-375,and 40 p M for mi R-21.Furthermore,it also performed satisfactorily for the mi RNA sensing in 50%human serum and 50%human plasma.This work could detect mi RNAs with different abundances in breast cancer-derived exosomes,and was expected to provide effective information for accurate diagnosis of breast cancer.Taking breast cancer exosomes as the research object,in view of the shortcomings of sensitivity and accuracy of breast cancer exosomes analysis methods,functional nucleic acid biosensor technology was used to provide signal recognition elements and signal amplification strategies,and a series of methods with high sensitivity and accuracy were constructed for the detection of breast cancer exosomes.They were expected to provide powerful means for accurate diagnosis of breast cancer.