Study Of New Methods For Exosomes Analysis Via Biosensor-based Signal Amplification Strategy

Exosomes with a diameter of 30-200 nm are extracellular vesicles that can be secreted by various types of cells.Exosomes are widely distributed in biological fluids in the range of blood,urine,tears,saliva,spinal fluid,milk,semen,ascites and amniotic fluid.More and more studies have confirmed that exosomes are closely related to the process of diseases and are even involved in the origin and metastasis of tumors.Benefitting from the diverse sources and relatively simple sampling,exosomes,as a promising tumor marker for liquid biopsy,are expected to be applied in clinical disease diagnosis and prognosis.At present,nanoparticle tracking analysis（NTA）plays an important role in detecting the total concentration and particle size of purified exosomes.Enzyme labeled immunosorbent assay（ELISA）and Western blotting are common standard methods for the identification of proteins existing in exosomes.However,these methods suffer from low sensitivity,large sample consumption,poor specificity,and tedious operation,making accurate and highly sensitive analysis of exosomes a huge challenge.Since exosome is a vesicle containing all kinds of proteins,the analysis of exosome can be converted to the examination of the membrane and membrane protein of exosomes.By taking advantage of the specific combination between aptamers and membrane proteins,negative charge and hydrophobicity of the phospholipid bilayer of exosome membranes,the detection of exosomes can be converted into nucleic acids which bind to exosomes.The sensitivity of exosome detection can be greatly improved by the signal amplification techniques,such as nanomaterial technology,enzyme-mediated signal amplification,isothermal nucleic acid amplification,and the combination of these technologies.In this paper,in order to maximumly improve the sensitivity,specificity and simplicity of exosome detection,we have developed a variety of new signal amplification methods based on the synthesis of nanomaterials,enzyme-mediated nucleic acids signal amplification,and the combined utilization of multiple signal amplification strategy,providing the foundation for the detection of exosomes in complex clinical samples.The research work of this paper is present in the following parts:1.Signal amplification based on zirconium phosphate coordination chemistry between phospholipid membranesZr⁴⁺ion can bind strongly to phosphate group of the phospholipid membranes via electrostatic interactions.Considering that exosomes and liposomes are actually vesicles composed of phospholipid bilayer,we hypothesize that Zr⁴⁺can bind with both exosomes and liposomes through coordination chemistry.Thus,artificial vesicles encapsulating with many signaling molecules are combined on the surface of the exosomes,and a sensitive,label-free and cost-effective method for detection of exosomes has been proposed by making use of phosphate-Zr⁴⁺-phosphate interaction.This method takes advantage of magnetic bead for easy separation and liposomes for one-step signal release process,which avoids the involvement of expensive equipment and complicated signal amplification designs.Due to the direct interaction between zirconium ions and phospholipid membranes,the signal transformation of the biosensor is easily achieved without extra chemical modification.With liposomes as superior signal amplification elements in this method,the detection sensitivity for exosomes is improved,and the limit of detection is 7.6×10³particles/μL.Hence,a new method with advantages of sensitive,easy to operate,no modification probe has been developed for the detection of exosomes.This biosensor also shows good performance in the complex biological samples for the detection of exosomes.Moreover,it is the first time to detect exosomes by linking the phospholipids in both exosomes and liposomes with Zr⁴⁺,which may bring about inspiration in the construction of biosensor for biomedical research by the rational utilization of the intrinsic phospholipid of exosomes.2.Template-free enzyme-mediated multiple signal amplificationIn this work,exosomes are specifically captured by immunomagnetic beads modified with CD63 antibody and another MUC1 protein on the surface of exosomes is combined with the extended MUC1 aptamer(Apt _MUC1).Thus,the concentration of exosomes is positively correlated with the content of elongated Apt _MUC1,and we have designed triple signal amplification methods based on HCR reaction,DNase I cleavage,and TDT mediated amplification for the highly sensitive detection of exosomes.The scheme of design is that a large number of ds DNA stemed from HCR reaction triggered by aptamers are digested by endonuclease to form short nucleic acid chains,and then these short nucleic acid chains acting as primers are extended by Td T enzyme to generate signals.Due to the strategy of multiple signal amplification methods,this assay has achieved a highly sensitive analysis of exosomes with limit of detection as 10 particles/μL.The enzymes used in this method are independent of DNA template sequence,avoiding the complex nucleic acid sequence design.The processes of exosomes capture and signal amplification require the simultaneous presence of two kinds of membrane proteins on exosomes,which improves the specificity of the method.In addition,by taking advantage of the simple and easy separation of magnetic beads,the assay holds the capacity to detect exosomes in complex biological samples with high sensitivity and effectiveness,which indicates that the proposed method has excellent anti-interference ability and provides potentials for the clinical application of exosomes.3.Spherical nucleic acids-based cascade signal amplificationSpherical nucleic acids（SNAs）are a type of nanomaterial with a small spherical core functionalized with dense and highly oriented oligonucleotides.Compared with free nucleic acids,SNAs have higher binding affinity with their complementary nucleic acid chains,protein receptors or enzymes.SNAs have been synthesized by many kinds of templates,such as gold particles,coordination polymer particles,and liposomes.Similar to liposomes,exosomes are also composed of lipid bilayer membrane and can be remolded by membrane surface engineering.Therefore,we have proposed a strategy of exosomes-templated SNAs and enzyme-mediated cyclic signal amplification based on the binding,growth,and incision of DNA for universal and highly sensitive detection of exosomes.Namely,aptamers are used to specifically capture exosomes,and then the hydrophobic phospholipid bilayer of exosomes binds to the cholesterol-modified single-strand DNA based on the hydrophobic interaction,which can initiate the single-strand DNA-based the signal amplification reaction.In this proposed strategy,there is a triple signal amplification:1)one exosome can bind with a number of primer strands;2)one primer strand of SNAs can be extended by Td T and then act as template strands to digest numerous probe（probe A）;3)truncated probe A as a new primer triggers the circle of the second signal amplification progress.Therefore,under the assistance of Td T,Exo III,probe A,and primer strand,the highly sensitive detection of exosomes can be achieved without complicated nucleic acids sequence design,and the limit detection is 44 particles/μL.Meanwhile,exosomes are captured based on the specific recognition between aptamers and membrane proteins,and the signal is amplified based on the hydrophobic interaction between cholesterol group and exosomes membrane,which synergistically endow the biosensor with high specificity and low signal background.With simple centrifugation,filtration,and dilution of clinical serum samples to remove cell debris and other large extracellular vesicles,the proposed method can distinguish malignancy colorectal cancer patients from the control groups without the high-speed centrifugation,which offers an alternative solution in the diagnosis of cancer in clinical samples.4.Fabrication of an aptamer-coated liposome complex and terminal deoxynucleotidyl transferase-mediated signal amplificationIn this work,we have proposed a new and efficient method for the detection and profiling of exosomes with high sensitivity,cost-effectiveness,and easy operation process in small samples.We have first fabricated cationic1,2-dioleoyl-3-trimethylammoniumpropane（DOTAP）liposomes with aptamers absorbed via self-assembly for targeting exosomal membrane proteins.Aptamers with large numbers of negative charges will combine with DOTAP liposomes due to electrostatic interaction,resulting in no recognition or extension by terminal deoxynucleotidyl transferase（Td T）because of the steric hindrance between Td T and liposomes.However,in the presence of exosomes,the weaker and nonspecific combination between aptamers and liposomes is superseded by the stronger and specific binding between aptamers and exosomes,making the free 3′-OH of aptamers to be available to Td T,which will trigger the polymerization of aptamers to form multiple G-quadruplex structures under the condition of a d GTP-rich pool.The G-quadruplex can give rise to strong fluorescence in the presence of thioflavin T（Th T）,and the fluorescence intensity is relevant to the abundance of exosomal membrane proteins.The proposed strategy takes advantage of the electrostatic interaction between negative-charge DNA and positive-charge liposomes,and the resulting steric hindrance renders the Td T enzyme inaccessible to the aptamers.Therefore,without complex design and labels on aptamers,the proposed method achieves highly sensitive and cost-effective detection for exosomes with one step of Td T-mediated signal enlargement.In addition,all experiments are conducted in one-pot due to the rational design without separation procedures,so this method is beneficial to mass sample analysis simultaneously.The proposed method has been employed to interrogate various membrane proteins of different cancer cell-derived exosomes by using a panel of corresponding aptamers and obtained a heat map to describe the profiling of exosomes.Thus,patterns that differentiate the heterogeneity of different exosomes can be eventually obtained.