Development Of Highly Sensitive Electrochemical DNA And Biosensors

In this thesis, according to signal amplification and enzymatic amplificationstrategies. we built several electrochemical biosensing platforms, realized the sensitivedetection of DNA fragments and UDG. The main contents are listed below:1. In this work, a very simple, label-free, isothermal and ultrasensitiveelectrochemical DNA biosensor has been developed based on an autocatalytic andexonuclease III (Exo III)-assisted target recycling amplification strategy. A duplexDNA probe constructed by the hybridization of a quadruplex-forming oligomer with amolecular beacon is ingeniously designed and assembled on the electrode asrecognition element. Upon sensing of the analyte nucleic acid, the strand of molecularbeacon in the duplex DNA probe could be stepwise removed by Exo III accompaniedby the releasing of target DNA and autonomous generation of new secondary targetDNA fragment for the successive hybridization and cleavage process. Simultaneously,numerous quadruplex-forming oligomers are liberated and folded intoG-quadruplex-hemin complexes with the help of K+and hemin on the electrodesurface to give a remarkable electrochemical response. It could only obtain a detectionlimit of10fM.2. we developed a high sensitive electrochemical biosensor based onuracil-DNA glycosylase (UDG) treatment, endonuclease IV cleavage and usingterminal transferase (TdTase)-mediated extension for the detection of the UDG activity.A stem-loop DNA probe was assembled on the electode. Upon UDG treatment, uracilbase was removed to generate an AP site that can be cleaved by endonuclease Ⅳ(Endo Ⅳ). This cleavage produces a new primer probe with3’ hydroxyl end that caninitiate no template extension reaction in the presence of dUTP (dUTP-biotin) and TdTase. The extended biotin could conjugate with the streptavidin-alkalinephosphatase (SA-ALP). ALP catalyzes conversion of electrochemically inactive1-naphthyl phosphate (1-NP) into an electrochemically active phenol for thegeneration of an amplified electrochemical signal. It could only obtain a detection limitof2mU/mL.3. Based rolling circle amplification technology and Exo III-assited recyclingamplification technology, We have desiged a homogeneous, highly sensitive newmethod to detect nucleic DNA. The target DNA probes connect the padlockoligonucleotide probe to induce rolling circle amplification. resulting in periodiclong-chain DNA sequences. We design a DNA probe which is stem-loop structure and3’ end labled with ferrocene, it hybridized with periodic long-chain DNA to triggerexonuclease III auxiliary cleaving cycle, and release the ferrocene-labeled electroactivemononucleotide. Due to its less negative charge and small size, diffuses easily to thenegative ITO electrode, resulting in an increased electrochemical signal. It could onlyobtain a detection limit of0.5pM.