Highly Sensitive Detection Of Exosome Based On Au@MnO₂ Nanoparticles Array Chip-Enhanced Fluorescence Effect

Exosomes are membranous vesicles derived from the late endosome secreted by cells,with the diameters about 30-150 nm.They are involved in the occurrence,invasion and metastasis of cancer.The biomolecular information they carried play great importance in cell communication.And these biomolecules can mediate the exchange of genetic material.Exosomes exist in many body fluids and have high tumor specificity,so they are expected to be the non-invasive tumor markers for early diagnosis of cancer.In this work,the array chips with enhanced fluorescence effect based on Au@MnO₂ nanoparticles（Au@MnO₂ NPs）were firstly develpoed.To amplify the fluorescent signal,the chain substitution reaction was triggered by exosomes to release reaction chain labeled with FAM.Then reaction chains were collected by magnetic separation and dropped on Au@MnO₂ NPs array chip for the second time signal amplification through enhanced fluorescence effect.Finally,a new method with high sensitivity was established for detecting exosomes.In the first chapter,the basic principles of metal-enhanced fluorescence（MEF）were briefly outlined.Furthermore,the factors affecting MEF and the methods of MEF biosensing were introduced.Afterward,the source,formation process,composition and functions of exosomes in biology and pathology were described.Moreover,the common methods for exosomes detection were presented.Finally,the ideas and research significance of this work were put forward.In the second chapter,the preparation and characterization of Au@MnO₂ NPs and Au@MnO₂ NPs array chip were discussed.Firstly,Au@MnO₂ NPs were obtained by growing MnO₂ nanolayers on the surface of Au NPs.The MnO₂ nanolayers were reduced by potassium permanganate in water bath.Then,the Au@MnO₂ NPs-enhanced fluorescent array chip was built via layer-by-layer electrostatic self-assembly technology.Lastly,the as-prepared Au@MnO₂ NPs and Au@MnO₂ NPs array chip were characterized by ultraviolet-visible spectroscopy,zeta analyzer,transmission electron microscopy,atomic force microscope and scanning electron microscope.In the third chapter,Au@MnO₂ NPs array chip with the enhanced fluorescence effect was used to detect exosomes.The design of chain substitution reaction and Au@MnO₂ NPs array chip with MEF effect can amplify the fluorescence signal for twice.The addition of exosomes initiated chain substitution reaction and released multiple reaction chains labeled with FAM,and then the released reaction chains were dropped onto the Au@MnO₂ NPs array chip for enhancing fluorescence.Exosomes were quantified via the intensity changes of fluorescence imaging.The experimental results show that linear range of exosomes is 8.5×10³-8.5×10⁵ exosomes μL^-1 and the limit of detection is 4.5×10³ exosomes μL^-1,which is much lower than most reported methods based on fluorescence signal amplification for exosomes detection.In addition,this method can be used to distinguish healthy people and breast cancer patients,demonstrating its potential application in real biological samples.