| Small extracellular vesicles(sEVs)are nanoscale vesicles(30-200 nm)secreted by most types of cells and are present in large numbers of body fluids,including saliva,semen,blood,cerebrospinal fluid,and urine.It facilitates intercellular communication by transporting molecular cargo(e.g.,nucleic acids and proteins)from the donor cell to the recipient.Due to high stability,specificity and wealth of information from body fluids,sEVs have emerged as one of the most sought-after disease markers in recent years and play an important role in cancer diagnosis and prognosis.The abundance of biomarkers on sEVs is very low during the early stage of cancers,which poses a great difficulty for the detection of sEVs.Therefore,signal amplification strategies are needed to improve the sensitivity of sEVs detection.Among them,nucleic acids-based signal amplification has become one of the most widely used strategies due to their programmability,simple synthesis,high biocompatibility,and excellent signal amplification effect.Based on reaction kinetics,nucleic acid signal amplification strategies can be categorized into linear and exponential nucleic acid signal amplification strategies.Compared to other linear signal amplification approaches,enzyme-driven primer exchange reaction(PER)and catalytic hairpin assembly(CHA)are programmable,flexible in design,easy to operate,and have robust signal amplification effects.These techniques can be employed independently or in cascade to generate exponential signal amplification for the detection of surface proteins of sEVs.It is essential to ensure meet the need for high specificity and accuracy for sEVs testing to be used in cancer diagnosis and treatment.However,the complex composition of the body fluid matrix and the phenotypic heterogeneity of surface proteins of sEVs make single marker detection results unreliable.DNA logic gates utilize the principle of base complementary pairing to transmit multiple biological signals to the logic circuit with precision and specificity.These gates can generate a Boolean-operated signal,which can effectively avoid "false positive" or "false negatives" results.Based on the aforementioned,we design two polymerase-driven signal amplification techniques that enable the generation of amplified fluorescent signals in either linear or exponential degrees,following logic-gated analysis of biosignals from multiple proteins on the surface of sEVs.The system has been applied for discriminating mother cell in breast cell line-derived sEVs and breast cancer(BC)identification.The details of the study are as follows:Chapter 1.OverviewThis chapter presents an overview of the concept,classification,composition,function,isolation and enrichment of sEVs and their application in liquid biopsies.It also specifies the nucleic acid signal amplification strategies currently in use and the application of DNA bio-logical gates in sEVs detection.Finally,the chapter outlines the research significance and content of this thesis.Chapter 2.Polymerase-driven logic signal amplification for detection of surface proteins of sEVs and breast cancer identificationIn this chapter,a polymerase-driven logical signal amplification system(PLSAS)was developed to simultaneously detect two kinds of surface proteins on sEVs with high sensitivity and BC identification.The strategy uses the mucin 1(MUC1)protein and epithelial cell adhesion molecule(EpCAM),which are enriched on tumor-derived sEVs,as signal inputs.And it performs logic operations and signal amplification with PER to output fluorescent signals.These can construct two biological computational modules of OR and AND logic gates.Among them,the OR logic gate can provide a highly sensitive detection of sEV,and the detection limits of sEVs are 24 particles/μL and 58 particles/μL when MUC1 or EpCAM protein is used as a single signal input,respectively.While the AND logic gate has high discrimination for BC patients,the area under the curve(AUC)for receiver operating characteristic(ROC)analysis of serum assays from BC-positive samples and healthy individuals was 0.981.Chapter 3.Polymerase-driven exponential signal amplification for simultaneous detection of multiple surface proteins on sEVs and breast cancer identificationBased on the previous chapter,we designed a polymerase-driven primer catalyzed cycle(PCC)circuit that spontaneously implements PER and CHA mutual catalysis for "AND" detection of two proteins on sEVs.This strategy chose the EpCAM protein and human epidermal growth factor receptor 2(HER2)as models,which are over expressed on the sEVs of BC patients.In the PCC signal amplifier,the primer P of PER and trigger T of CHA are cyclically regenerated by only four hairpins to achieve exponential signal amplification.PCC greatly increases the sensitivity,reduces the limit of detection of sEVs to 8 particles/μL.And it improves AUC to 0.992 for ROC analysis of BC-positive patients,and also enables the differentiation of BC patients in early and late stages. |
