The Construction Of DNA Nano-self-assembly Structure And Its Application In Signal Amplification Biosensing And Imaging Analysis

With numerous advantages such as sequence designability,simple synthesis,diversity and good biocompatibility,DNA has been exploited for the construction of a variety of DNA nanostructures,which are widely applied in the fileds of biosensing,bioimaging and biomedicine.In this paper,multiple novel DNA nanostructures are constructed based on DNA self-assembly technology with the combination of fluorescence,electrochemical,colorimetric,photothermal analytical methods,which are applied for the sensitive,specific detection and imaging analysis of miRNA,pH and exosomes.This thesis carried out the following three works:1.Target-triggered dynamic hairpin assembly for in situ signal amplification imaging analysis of miRNA in living cellsA target-triggered dynamic hairpin assembly?DHA?strategy is proposed for the construction of DNA nanostructures,realizing the signal amplification detection of miRNA-21 and breast cancer susceptibility gene1?BRCA1?as well as in situ imaging analysis of miRNA in living cells.Compared with the traditional catalytic hairpin assembly technology,this work designs a"spacer"structure at the loop of hairpin to effectively prevent the target miRNA being replaced during the assembly process,therefore generates DNA nanobrush structure.In this cases,in the absence of the target miRNA,hairpin DNA can coexist in a metastable state in solution;Once miRNA is introduced in the solution,toehold-mediated strand displacement reaction is triggered to construct DNA nanobrush under isothermal conditions,realizing the sensitive detection of miRNA with a detection limit of 1.88 nM.The proposed DHA strategy has good specificity and can distinguish single-base mismatched target miRNA.The strategy is further applied to the fluorescence imaging of miRNA-21 in living cells,achieving the monitoring of miRNA-21 expression levels in different cells.In addition,highly sensitive and specific detection of BRCA1 is realized based on the bilateral DHA triggered by long DNA,which has broad application prospects in the fields of biosensing,bioimaging,and early diagnosis of cancer.2.Preparation of pH-sensitive DNA assembly based on rolling circle amplification for construction of electrochemical dual-signal biosensorA pH-sensitive DNA assembly is prepared based on the rolling circle amplification?RCA?technology,which is used to construct electrochemical dual-signal biosensor for highly sensitive detection of pH in a trace biological sample.Long single-stranded DNA?ssDNA?rich in pH-responsive sequences is synthesized by RCA technology,and immobilized on magnetic beads by amide reaction.Then the ssDNA hybridizes with short-stranded DNA?L0?to generate DNA assembly with alternating single and double strand,of which the double-stranded portion provides a large number of binding sites for doxorubicin?DOX?.The pH-responsive sequence in the DNA assembly folds into i-motif structure as pH decreases,resulting the melting of double-stranded region,and releasing L0 and DOX at the same time.The supernatant is collected after magnetic separation.Meanwhile,graphene quantum dots?GQDs?are used to modify the glassy carbon electrode and the capture probe is immobilized on GQDs through amide reaction.After supernatant dropping onto the electrode,DOX is adsorbed on the electrode surface,and L0 can trigger the catalytic hairpin assembly to capture the methylene blue?MB?-modified signal probe onto the electrode surface,achieving the sensitive detection of pH in trace samples by detecting the electrochemical signals of DOX and MB with a detection range of 4.0?7.0.This biosensor is further applied to the detection of pH in actual samples,such as urine.3.Encapsulation of nanozyme based on rolling circle amplification self-assembled DNA flower structure for the construction of exosome dual-modes sensorA novel method for colorimetric and photothermal dual-modes detection of exosomes is proposed based on nanoenzymes-encapsulated RCA self-assembled DNA flower structure.The complementary sequence of the exosome aptamer is designed into the circular template,achieving the amplification of the exosome aptamer through the RCA reaction.Then,the graphene quantum dot nanozyme/3,3',5,5'-tetramethylbenzidine?GQDzyme/TMB?-encapsulated micron-sized DNA flower structure is successfully prepared.Another sequence of exosomes aptamer is immobilized on the bottom of the 96-well plate to capture exosomes through the specific recognition between the exosomes and aptamer,which can further combine with nanoenzymes-encapsulated DNA flower structure to form an“aptamer-exosomes-DNA flower”sandwich structure.In the presence of hydrogen peroxide?H₂O₂?,GQDzyme catalyzes the oxidation of TMB.The oxidized TMB not only shows color change,but also exhibits photothermal effect driven by near-infrared laser,contributing to the sensitive and specific detection of exosomes in colorimetric and photothermal dual-modes.