Catalytic Hairpin Assembly-Based Electrochemical Biosensor For MicroRNA Assay

Electrochemical sensors have attracted researcher more and more attention due to their advantages of highly sensitivity,fast response,low-cost,simple operation,easily miniaturization,and suitability for molecular diagnosis.The electrochemical biosensors combine electrochemistry,biology and physics,and its electrochemical signals can reflect the concentration of substances.In the past decades,electrochemical biosensors have been widely applied for the detection of small molecules(e.g.,glucose),nucleic acids,and proteins.Despite their high sensitivity,it is difficult for them to achieve accurate measurement.The introduction of different signal amplification strategies into electrochemical biosensors can greatly improve the detection sensitivity.Catalytic hairpin assembly(CHA)is an enzyme-free amplification method that is triggered by specific sequence microRNA,with the advantages of mild reaction conditions,simple operation,high efficiency,and good specificity.And DNA-templated nanoparticles exhibit distinct advantages of non-toxicity,low cost and ease of preparation,and may be in situ synthesized as environment-friendly nano-dyes for signal transduction.In this thesis,we integrate catalytic hairpin assembly with metal nanoparticle in-situ growth technology which can embed metal nanoparticles in the ds DNA groove to achieve double signal amplification.MicroRNAs belong to a class of small(about 22 nucleotides),endogenous,non-coding RNA molecules and they may function as sequence-specific post-transcriptional regulators.These highly conserved single-stranded RNAs modulate gene expression by binding to specific ribosomes,thereby determining the amino acid sequence of the protein product expressed by the gene,and transferring genetic information from DNA to protein,and their expression levels are associated with many human diseases including cancers.Therefore,sensitive detection of microRNAs in cancer cells is essential to biomedical research and early clinical diagnosis.In this thesis,we demonstrate the construction of a low background electrochemical biosensor with the capability of tandem signal amplification for sensitive microRNA assay based on microRNA-activated CHA of heteroduplex-templated copper nanoparticles.The target mi RNA-21 can trigger the strand displacement assembly of two hairpin substrates,resulting in the cyclic reuse of target microRNA-21.The alkaline phosphatase(ALP)catalyzes the hydrolysis of a substrate ascorbic acid 2-phosphatase(AAP),producing a reductive product ascorbic acid(AA)whose catalysis reduction may result in the in situ formation of CuNPs with ds DNA as the template for the generation of an amplified electrochemical signal.The proposed electrochemical biosensor exhibited excellent analytical performance with a wide linear detection range from 10 f M to 10 n M and a detection limit down to 1.1 f M.Moreover,this tandem amplification biosensor is capable of detecting microRNA extracted from cells in living cells and the detection limit down to 3 cells.Notably,there is no strict requirement for the sequenceof the ds DNA template,facilitating the design of different biosensors for the detection of various biomarkers.Therefore,it has great potential for biomedical research and disease management.