Study On Highly Sensitive Optical Biosensors Based On Peroxidase Mimics And Nucleases

Optical biosensors provide significant advantages, such as simple, rapid response, homogeneous and high sensitivity. In this thesis, colorimetric and fluorescent detection methods were employed as the main test means. Based on the new ideas and new detection methods of small molecules, nucleic acid and protein, two kinds of amplified DNA detection sensors were developed by using peroxidase mimics and nucleases as signal amplification technology, A new detection platform of hydrogen peroxide was designed combining with the peroxidase mimetic activity of dendritic gold nanospheres. The main works are listed below:(1) Through the magnetic bead-based separation and enrichment technology, a simple and sensitive label-free colorimetric DNA detection platform was developed by using hybridization chain reaction strategy to bring numerous hemin/G-quadruplex tags on the magnetic bead for signal amplification. This simple system is highly sensitive and is able to detect as low as1pM target DNA with the colorimetric method. It avoids the use of any expensive instrument, dark room, expensive antibody and enzyme, or complicated process. The colorimetric detection approach will find its wide applications in real samples.(2) Nucleases, especially restriction/nicking endonucleases are DNA modifying agents that are increasingly being used in DNA diagnostics. One of the limitations for the use of restriction/nicking endonucleases in DNA diagnostics is the requirement for the target of interest to contain a specific sequence. Y-or junction probe (JP)-based detection platforms obviate the need for the target to contain a specific cognate sequence and are finding uses for the detection of nucleic acids as well as for the detection of single nucleotide polymorphism. However, the sensitivity of conventional Y-or junction probe (JP)-based detection platforms is not high enough. In this chapter, a new molecular beacon-based Y-shaped junction sensing system was developed based on an autocatalytic and nuclease-assisted target recycling amplification strategy for highly sensitive DNA detection. It could detect as low as0.1pM target DNA and show a strong capability to identify base-mismatched target DNA. Further, the isothermal amplification system was suitable for detecting different sequences of target DNA via adding a new hairpin. (3) In the present work,3D dendritic gold nanospheres (3D-DGNs) with tunable openings were prepared by a one-step, rapid, room temperature and seedless approach with the use of3-(4-aminophenyl) propionic acid as reductant. The morphology change of3D-DGNs from with a big opening to intact one could be easily and effectively controlled by changing amounts of anionic surfactant such as SDS or SDBS. The unique3D-DGNs also demonstrated an intrinsic peroxidase-like activity with a robust stability. The catalytic oxidation of3D-DGNs toward peroxidase substrate of TMB by H2O2was explored for the determination of H2O2with a good performance. The special morphology of currently prepared3D-DGNs with a large surface area may hold considerable potential in the applications of catalysis, biosensor platform fabrication, surface-enhanced raman scattering.