| The detection of tumor biomarkers plays a crucial part in cancer diagnosis and prognosis.Recently,liquid biopsy,a new star of cancer detection,has been reported to be able to detect biomarkers that carry information on tumor development and progression.It involves the extraction of biofluids from the patient and the analysis of tumorrelated biomolecules,such as cell-free tumour RNA(ctRNA),cell-free tumor DNA(ctDNA),circulating tumor cells(CTCs)and exosomes.Exosome as a prognostic biomarker displays many advantages over ctRNA,ctDNA,and CTCs.Firstly,tumor-related exosomes have higher concentrations than ctRNAs,CTCs and ctDNA in biofluids.Secondly,specific exosomes secreted by tumor cells contain a variety of key bioactive molecules.Thirdly,these bioactive molecules are protected from external proteases and other enzymes by the exosomal lipid bilayer.Due to their small size,distinct biogenesis,and heterogeneous marker expression,isolation and detection of cancerous exosomes remain challenging.For example,antibodies are expensive,which increase the cost of detection.The current methods for the detection of exosomes can't distinguish tumor-derived exosomes and normal cell-derived exosomes owning the lack of specificity,and require a series of procedures of isolation.Low sensitivity has limited its application.Therefore,a growing need for sensitive and reliable methods to enrich and detect tumor-derived exosomes specifically.In this,aiming at developing new methods for tumor-derived exosomes detection with specificity and sensitivity,aptamer functionalized microbeads combined with hybridization chain reaction(HCR)strategies have been developed.The detailed description is listed as follows:1.Leukemia exosome detection based on PTK7 aptamer functionalized microbeads enrichment and HCR signal amplificationA leukemia exosomes detection platform was built by using PTK7 Aptamer functionalized beads to enrich leukemia cell derived extracellular vesicles and combining hybridization chain reaction initiated by CD63 aptamer-trigger.The results of electrophoresis and atomic force microscope characterization indicated the feasibility of HCR.Flow cytometry and fluorescence microscopy image showed that PTK7 Aptamer functional microbeads could capture leukemic exosomes specifically.By comparative experiments,including optimize the time of capture and HCR,thebest experiment conditions of our method have been determined.The experiment results demonstrate that our method have performed good effect of signal amplification and good linear correlation in the range of 100-4 000 pg/?L.Meanwhile,our method also can be successfully used for the detection of target exosomes in diluted serum samples,providing a promising platform for early clinical diagnosis of leukemia.2.A universal tumor-derived exosome detection platform based on CD63 aptamer functionalized microbeads enrichment and HCR signal amplificationOwing to the drawbacks of tedious and time-consuming caused by the single target detection,we herein develop a universal platform for the detection of multiple tumor-derived exosome,where the functionalized microbead as capturing element,aptamer of tumor cell as a guide,and HCR product as a signal transduction and amplification unit.Firstly,the recognition effect of tumor aptamer for tumor-derived exosomes were investigated using tumor-derived exosomes enriched by CD63 aptamer functional microspheres.And then,aptamers,which have better recognition effect for tumor-derived exosomes,were selected as the guide of the signal unit.In this process,a series of signal units labeled with different fluorescent molecules have been constructed through aptamer-trigger induced HCR,whereas the aptamer moieties can guide the signal unit to target tumor-derived exosomes.Furthermore,our novel approach,as a universal platform,could be extended to detect other tumor-derived exosomes by changing aptamer sequence.Meanwhile,this work provides a new method for the simultaneous detection of multiple tumor-derived exosomes with high specificity. |
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