| Exosomes are the cup-shaped phospholipid membrane nanovesicles with a diameter of 30 nm～150 nm.It carries a large number of biologically active molecules derived from host cells,such as nucleic acids,phospholipids,and proteins.In addition,the exosomes released by cancer cells have the specific genetic information and epigenetic modifications,which make them play an important role in cell communication,tumor migration and invasion.Exosomes are widely present in various biological fluids,especially in the blood.Therefore,exosomes have been considered as a reliable and noninvasive biomarkers source for liquid biopsy of cancer.Among them,the carried tumor-specific protein and microRNAs,which are both tumor biomarkers with great clinical application potential,have been used in the diagnosis and research of ovarian cancer,breast cancer,prostate cancer and other diseases.However,the current detection of exosomes are mainly based on the recognition between the antibodies and specific membrane proteins,and there are many disadvantages such as complicated antibody modification process,high price,and difficulty in storage,which limit its further application in clinical diagnosis.Functional nucleic acid,a type of special functions DNA sequence,can specifically bind to the target or exhibit excellent catalytic activity.It also has the unique advantage in the field of bioanalysis,eg.low-cost,easy synthesis and modification,and good chemical stability.Moreover,functional nucleic acids are easy to be combined with nanotechnology and nucleic acid signal amplification technology,making it an attractive application prospect in the field of high sensitivity,in-situ imaging and tracing.Inspired by the unique advantages of functional nucleic acids,based on the specific recognition of functional nucleic acids with tumor exosomal membrane proteins and miRNAs in exosomes,this thesis has been developed a series of novel functional nanoprobes for the highly specific and sensitive detection of exosomes by combination with nucleic acid amplification technology and nanotechnology.On this basis,the correlation between tumor exosomes-related biomarkers and tumor development process has been preliminary explored.The details are as follows:1.Structure-switching aptamer triggering hybridization displacement reaction for label-free detection of exosomes.Based on molecular recognition between DNA aptamer and exosome surface biomarker(protein tyrosine kinase-7),a novel activatable and label-free strategy was designed for highly sensitive and specific sensing of exosomes.In this work,the target exosomes trigger strand replacement reaction to form G-quadruplex,which resulted in an obvious fluorescence enhancement of N-methylmesoporphyrin IX due to the bonding between G-quadruplex and N-methylmesoporphyrin IX.Under the optimum experimental conditions,the linear range for exosomes was measured to be 5.0 ×108～5.0 × 1010 particles/mL and the detection limit(LOD)was calculated to be 3.4 × 108 particles/mL(3σ).This assay possessed high specificity to distinguish exosomes derived from different cell lines,and had been successfully validated in patient and healthy serum samples.Furthermore,the probe can effectively detect the exosomes in fetal bovine serum,indicating that the biological matrix has a negligible effect on this method.This developed label-free,convenient,and highly sensitive biosensor will offer a great opportunity for exosomes quantification in biological study and clinical application.2.Recognition triggered assembly of split aptamers to initiate a hybridization chain reaction for wash-free and amplified detection of exosomes.Highly sensitive and specific detection of exosomes in clinical samples is of great significance for cancer diagnosis and prognosis.Herein,we developed a facile split aptamer-based system for the amplified,specific,and wash-free detection of exosome in situ assisted by a target-induced hybridization chain reaction(HCR).In this design,the aptamer was split into two segments,and the trigger for initiating the HCR was also divided into two fragments.The split triggers can form an intact strand to initiate the HCR via specific recognition between the split aptamers and target exosomes,resulting in an amplified fluorescence signal.Moreover,the split aptamer-based design could efficiently reduce the background signal,leading to a higher signal-to-background ratio.This developed strategy showed high specificity and sensitivity with LOD of 2.08 × 105 particles/mL,and was successfully used to detect target exosomes in a complex bio-matrix.Moreover,this assay could distinguish liver cancer patients from healthy individuals with satisfactory results,indicating that this novel split aptamer-based HCR amplification system may provide a feasible exosomes sensing platform for early cancer diagnosis and precision medicine.3.The cascade amplification strategy based on entropy-driven catalytic reaction-power hybrid chain reaction for highly sensitive detection of exosomes.In order to improve the sensitivity,herein,we developed an enzyme-free,ultrasensitive and isothermal fluorescent biosensor for exosomes detection by combining the entropy-driven circuit reaction(EDCR)with a hybridization chain reaction(HCR).In the presence of target exosomes,two split triggers can form an intact trigger to initiates EDCR,and then the accumulated AS can continue to trigger and accelerate the next amplification reaction HCR,resulting in the amplified fluorescence signal and high signal-to-background ratio.Based on the proposed strategy,the developed fluorescent biosensor displayed a wide linear relationship in the range of 1.16 × 105～2.32 × 109 particles/mL with a LOD of 7.95 × 104 particles/mL.Moreover,this proposed method can be significant differences between healthy controls and liver cancer patients,indicating its great potential in early cancers diagnosis.4."Poong Ring" double-accelerated hairpin DNA nanostructures for in situ,ultra-fast,amplified detection and imaging of exosomal microRNAs.Exosomal microRNAs(miRNAs)are reliable and non-invasive biomarkers for the early diagnosis of cancer.However,it remains a formidable challenge to directly detect and image of miRNA due to the low abundance of miRNAs in exosomes.In this work,double-accelerated hairpin DNA nanostructures based on the FRET design were implemented for in situ,ultra-fast,amplified detection and imaging of miRNA-21 in exosomes.Compared with a regular DNA cascade reaction,the catalytic reaction initiated by target miRNA-21 in exosomes can be completed within the 20 min due to the "Poong Ring" effect.In addition,the FRET-based LHDCN nanoprobes also provide the ability to avoid false-positive signals and rapidly imaging and tracing exosomal miRNA through the employment of FRET readout,showing its great potential in clinical diagnostics. |
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