Biosensor Amplifying Technique Research Based On Nucleic Acid Probe Conformational Switch

With a combinatorial method called in vitro selection or systematic evolution ofligands by exponential enrichment (SELEX), short synthetic DNA and RNA sequencesknown as aptamers have been selected as ligands to bind analytes. Aptamer can bindtarget with high specificity. The emergence of aptamers have broken the opinion thatthe aptamer just was the carrier of store and convey genetic information. Aptamer canbind protein, polypeptide, organic matter and metal ions, and so on. Compared withantibodies, aptamers have high specility and affinity, convenient prepration and goodgenerality for detecting a broad range of analytes.Just because of the unique merit, aptamer can be used as molecular recognitionelement in biosensor system. Aptamer sensor is an device which can recognizemolecule continuously and reversibly. The principle of aptamer sensors is that whenthe targets pass through the receiver with the function of molecular recognition, theyare captured on the receiver and can interact with biological molecule and the energycan be transferred at the same time, the energy may be exported in the form of light orelectricity when they pass through the transducer, the electronic system can amplifythe signal and exhibit the results. We can quantitatively and qualitatively assay targets.These sensor systems are sensitive, rapid and selective. However, the use ofconformational switch based aptamer sensing methods has been limited in furthersensitivity improvement. In order to analyze the target of low concentration with highsensitivity, we have to introduce signal amplification into the sensor system design. Inthe following three segments, different amplificative strategies were adopted in assaysystems.(2) In chapter2, an electrochemical aptasensor based on Klenow fragment (KF)polymerase reaction that combines the aggregation of ferrocene-functionalizedoligonucleotides has been developed successfully for cocaine detection. In thepresence of cocaine, the recognition probe changed its hairpin conformation into thetripartite complex. The aptamer-cocaine complex gave a3’-single-stranded tailsequence complementary to the surface-tethered capture probe. In KF polymerasereaction, the recognition probe served as a template for the extension, and the captureprobe as the primer. The ferrocene-appended oligonucleotide incorporated into thenewly synthesized complementary probe leads to an amplication of theelectrochemical response. (3) In chapter3, we developed a label-free self-locked bifunctionaloligonucleotide probe (signaling probe) for the detection of different disease markersin parallel. Two signal enhancement techniques based on isothermal circular strand-displacement polymerization reaction, cyclical nucleic acid strand-displacementpolymerization (CNDP) and cyclical common (nonnucleic acid) target-displacementpolymerization(CCDP), were employed to implement the amplification assay for p53gene and PDGF-BB, respectively. In the presence of the targets, the developedbifunctional probe is expected to fold into a three-way helix junction, the well-knownsecondary structure motif, to bind to its target protein, dehybridizing the self-complementary stem1that releases the segment complementary to primer. In this case,the primer can hybridize to the bifunctional probe, initiating the isothermal circularpolymerization reaction. When being excited by a common monochromatic lightsource, the SGI that intercalate into the double stranded DNAs, bifunctionalprobe/polymerization products, can emit the enhanced fluorescence， Lead to theamplication of singal. This interrogating platform confirmed the effectiveness ofisothermal polymerization in common biosensing systems without evolving anychemical modification, exhibits the design flexibility, convenience, simplicity andcost-effectiveness.(4) In chapter4, we developed a electrochemical sensor for DNAdetection,which is based on HCR amplication. The target in the experiment not onlycan bing with the capture probe, but its extention can also hybridize with tigger probe.The trigger probe can trigger the HCR reaction, then the biotin labeled in theamplication products bind with streptavidin-alkaline phosphatase(SA-ALP), Theactivity of the immobilized enzyme was voltammetrically determined by measuringthe amount of1-naphthol generated after5min of enzymatic dephosphorylation of1-naphthyl phosphate. So only in the presence of the target, these reaction can take place.The results revealed that the sensor showed a sensitive response to complementarytarget sequences, In addition, the sensing system could discriminate thecomplementary sequence from mismatched sequences.