Optical Probes Based On Signal Amplification For Sensitive Detection Of Small Extracellular Vesicle Surface Proteins

Small extracellular vesicles（sEVs）are bilayer enclosed membrane vesicles smaller than 200 nm in size secreted by living cells,which carry various bioactive molecules such as proteins,lipids,and nucleic acids from the parent cells.sEVs are widely present and stably circulating in various body fluids and can participate in many physiological and pathological processes.Tumor-derived sEVs carry specific tumor marker proteins and mi RNAs from maternal tumor cells,and have great potential for the early diagnosis,treatment evaluation and prognosis monitoring of cancer.However,sEVs are highly heterogeneous,and in the early stage of cancer,the expression levels of protein markers on sEVs are low.Therefore,how to precisely identify and detect protein markers on the surface of tumor sEVs with high sensitivity and specificity is a hot spot in current research.In this paper,we focused on the high specificity,high sensitivity detection and multiplex analysis of tumor sEV surface protein markers.Taking breast cancer（BC）and gastric cancer（GC）derived sEVs as research models,we chose aptamers and antibodies as specific identification units.And a series of novel signal amplification biosensing platforms were constructed in combination with signal amplification strategies based on functional DNA and nanomaterials.The research on the detection and clinical application of tumor sEV surface protein markers have been systematically investigated.The main contents are as follows:Chapter 1.OverviewThis chapter focused on the origin,function of sEVs and the application of sEVs in cancer detection.The isolation and characterization strategies of sEVs,and the research status and existing problems in sEVs-based cancer detection were discussed.Finally,the research content and significance of this work were expounded.Chapter 2.A lanthanide-activated signal amplification strategy for the ultrasensitive determination of surface proteins on tumor-derived sEVs and breast cancer identificationGiven the high heterogeneity of sEVs and the low abundance of sEV surface protein markers,we constructed a signal amplification biosensing platform based on Na Eu F₄ nanoparticles（NPs）dissociation-enhanced luminescence for the sensitive detection and multiplex analysis of surface protein markers on sEVs.BC is a highly heterogeneous cancer that still lacks specific biomarkers.The combined detection of sEV surface proteins provides a new non-invasive method for early diagnosis of BC.Three BC-associated proteins,epidermal growth factor receptor（Ep CAM）,epidermal growth factor receptor（EGFR）and programmed cell death-ligand 1（PD-L1）,were selected as the recognition sites of sEVs.And nanoprobes were prepared by the conjugation of antibodies corresponding to the three proteins with Na Eu F₄ NPs by amidation reaction.Tetraspanins（CD63）is ubiquitous on the surface of sEVs,therefore,we coated the bottom of the 96-well plate with anti-CD63 antibody as a capture platform,and a sandwich structure of anti-CD63/sEVs/Na Eu F₄ were formed finally.After adding luminescence enhancement solution（Triton X-100,β-naphthoyl trifluoroacetone（β-NTA）and trioctylphosphine oxide（TOPO）,p H=2.3）to the 96-well plate,Na Eu F₄NPs dissociated and formed a kind of strongly luminescent complex,which significantly amplified the luminescence signal.Meanwhile,the newly generated complexes had a millisecond luminescence lifetime that avoided background luminescence interference.The sensing platform could distinguish sEVs derived from different subtypes of breast cells and distinguish healthy people,early and late BC patients with high accuracy（90.5%）.Chapter 3.An ultrasensitive strand displacement signal amplification-assisted synchronous fluorescence assay for tumor-derived sEV surface proteins analysis and gastric cancer identificationDNA-based signal amplification methods are flexible in design and highly programmable.Synchronous fluorescence technique has the advantages of detecting multiple fluorescence signals simultaneously and improving the detection sensitivity.We developed a novel dual color DNA fluorescence signal amplification biosensing platform by combining toehold-mediated DNA strand displacement signal amplification with synchronous fluorescence technology,which enabled the simultaneous detection of multiple proteins on the surface of sEVs.Gastric cancer is one of the malignant tumors of the digestive tract,which has high mortality and low early diagnosis rate,so it is of great significance for the early diagnosis of gastric cancer.CD63 protein is ubiquitous on sEV surface,and the expression level of Mucin（MUC1）protein is related to the dynamic progression of gastric cancer.Therefore,we took sEVs derived from gastric cancer cells as a model,and selected CD63 and MUC1 proteins as the recognition sites of sEVs.The detection of sEV surface proteins was converted into single-stranded DNA（ss DNA）detection by the recognition of CD63 and MUC1aptamers,and then the signal amplification was achieved by DNA strand displacement reaction.And the DNA labeled with FAM and ROX dyes loaded on the signal probe were released into solution.The simultaneous determination of the two dyes could be realized by synchronous fluorescence spectroscopy,and then the simultaneous detection of the two proteins could be achieved.The detection limits（LOD）for sEVs by the detection of the two surface proteins were 67 particles/μL（CD63）and 37particles/μL（MUC1）,respectively.In addition,the sensing platform could detcct surface proteins of sEVs derived from different tumor cell lines.It was further used for the detection of clinical samples,which could distinguish gastric cancer patients and healthy donors with high accuracy（AUC=0.972）.This sensing platform provided new design ideas for sEVs-based cancer diagnosis.Chapter 4.An AND logic gate based on DNA cascade reaction activated CRISPR/Cas12a for ultrasensitive detection of tumor-derived sEVs and breast cancer identificationIn order to further improve the specificity and sensitivity of tumor sEV detection,we combined the hybrid chain reaction（HCR）and catalytic hairpin assembly（CHA）with the CRISPR/Cas12a system to construct a novel cascade signal amplification strategy.Based on this signal amplification strategy,a dual aptamer-assisted AND logic gate was developed for the specific and ultrasensitive detection of tumor sEVs.Firstly,Ep CAM and human epidermal growth factor receptor 2（HER2）aptamers recognized the sEV surface proteins and released complementary ss DNAwhich served as the inputs of the AND logic gate.The released ss DNA could form a ternary complex containing T1 structure with the help of a linker DNA,which converted the simultaneous recognition of the two sEV surface proteins into T1 sequence detection.The T1sequence was amplified into a duplex containing a specific Cas12a-cr RNA recognition sequence by the HCR-CHA reaction,thereby activating the trans-cleavage activity of the Cas12a protein,and achieving signal amplification,the LOD was 3.8×10³particles/m L.Compared with other AND logic based on signal amplification,our method had higher sensitivity.This strategy could specifically differentiate sEVs derived from different breast cell subtypes.In addition,the method could be used to detect sEVs in human serum samples and reliably distinguished BC patients from healthy donors（AUC=0.981）,and BC patients at different stages（AUC=0.950）,which had a good clinical application prospect.