Novel Methods Based On Dynamic DNA Framework For The Detection Of Exosome-related Biomarkers

Exosomes are small extracellular vesicles with lipid bilayer（30-100nm in diameter）released by cells into the biofluids and contain bioactive materials such as DNA,RNA,protein,and so on.Since exosomes are remarkably stable in biofluids,such as plasma and urine,and can be isolated for clinical evaluation even in the early stages of the disease,they have become of tremendous interest in biomarker research.Exosome-related biomarkers have quickly become adopted in the clinical field,for instance,the first exosome RNA based prostate cancer test has been included in the National Comprehensive Cancer Network guidelines for early prostate cancer detection.However,the current methods for the detection of exosome-related biomarkers suffer from several issues,including overlong testing time,complicated operation,low sensitivity.Thus,developing new sensing strategies that can simple,rapid,and sensitive detection of exosome-related biomarkers remains urgent requirement.Dynamic DNA structures that are assembled by DNA materials are designed to reconfigure upon recognition of external moieties,such as nucleic acids,antigens,or ligands,for performing various task.Such stimuli-responsive devices offer new chance to develop methods for the detection of exosome-related biomarkers.This paper integrated the latest research results of DNA nanoscience,bioanalysis,clinical laboratory diagnostics,and so on.Also,using the aptamer technology,tyramine signal amplification technology,catalytic hairpin self-assembly technology,etc.,a variety of new methods for exosome-related biomarkers detection were developed by combining with the surface plasma resonance（SPR）sensing platform and fluorescence analysis platform,respectively.This study not only provides a new theory and technical support for the detection of exosome-related markers,but also provides a new platform for the diagnosis of clinical diseases.This study mainly includes the following two parts:1.Surface plasmon resonance biosensor for exosome detection based on reformative tyramine signal amplification activated by molecular aptamer beaconHuman epidermal growth factor receptor 2（HER2）-positive exosomes play an extremely important role in the diagnosis and treatment options of breast cancers.Herein,based on the reformative tyramine signal amplification（TSA）enabled by molecular aptamer beacon（MAB）conversion,a label-free surface plasmon resonance（SPR）biosensor was proposed for highly sensitive and specific detection of HER2-positive exosomes.The designed MAB probe contained HER2 aptamer region and G-quadruplex DNA（G4 DNA）.The exosomes were captured by the HER2aptamer region,which enabled the exposure of the G4 DNA that could form peroxidase-like G4-hemin.In turn,the formed G4-hemin catalyzed the deposition of plentiful tyramine-coated gold nanoparticles（Au NPs-Ty）on the exosome membrane with the help of H₂O₂,generating a significantly enhanced SPR signal.In the reformative TSA system,the horseradish peroxidase（HRP）as a major component was replaced with nonenzymic G4-hemin,bypassing the defects of natural enzymes.Moreover,the dual-recognition of the surface proteins and lipid membrane of the desired exosomes endowed the sensing strategy with high specificity without the interruption of free proteins.As a result,this developed SPR biosensor exhibited a wide linear range from 1.0×10⁴ to 1.0×10⁷ particles/m L.Importantly,this strategy was able to accurately distinguish HER2-positive breast cancer patients from healthy individuals,exhibiting great potential clinical application.2.Catalytic hairpin assembly-induced DNA PX structures joint for rapid and stable detection of exosomal miRNAExosomal miRNAs are potential biomarkers for the early diagnosis of tumors with high clinical transformation.In this part,a new fluorescence sensing method based on catalytic hairpin self-assembly（CHA）-induced DNA PX joint was developed for the detection of exosomal miRNA with high speed and stability.The substrates H1 and H2 were respectively connected to the DNA PX structure to form the H1-PX-H1 and H2-PX-H2DNA nanostructures.In the presence of exosomal miRNA,CHA reaction occurred between hairpins,leading to forming DNA nano train owing to the connection of numerous PX structures and finally releasing fluorescence signal.The designed hairpin-PX structures were significantly more stable than the free hairpin owing to the protection of protein enzyme-resistant PX structure.The integration between isothermal amplification technology and static DNA PX structure endowed the developed sensing method with strong stability and high sensitivity with linear range from 10 p M to100n M.By comparing the mutated target at different sites,we found that the strategy was better able to identify the target whose mutation sites were in the region that hybridized with the toehold of the substrate,which contributed to the design of highly specific recognition probes based on the site of mutation in the target.In addition,this strategy could accurately distinguish patients with non-small cell lung cancer（NSCLC）from healthy individuals,showing potential clinical translational value.