Study Of New Biosensing Methods Based On Terminal Protection And Nanomaterials

The essential information on small molecules, nucleic acids, protein and enzymes obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. However, with the development of the scientific research, getting the dynamic information of these life processes sensitively and accurately in real time is still great challenge to analysts. Therefore, the development of strategies with high sensitivity, selectivity and accuracy is important for biomedical research and clinical diagnosis. In this thesis, a series of novel biosensing strategies based on terminal protection and nanomaterials were developed for small molecule, nucleic acid, proteins and enzyme activity detection, respectively. These results primarily proved that the proposed technologies were feasible, reliable and accurate. The detailed content was described as follows:In chapter2, Detection for tumor-specific biomarker on cell membranes is critical in the research of basic biological phenomena and applications of diagnose and therapy. Detection for tumor-specific biomarker on cell membranes is critical in the research of basic biological phenomena and applications of diagnose and therapy. Herein, we develop washing-free, simple and multiplexed detection strategies for specific proteins assay using small molecule-linked programmable DNA based on DNA terminal protection assay. DNA probes with small molecule ligand at3’end were programmed according to the signal readouts and programming DNA probes were used to specifically recognize the membrane-binding proteins under living cell conditions. The DNA probes were designed with three different signal readouts which were based on hemie DNAzyme, DNA-templated AgNCs and Quantitative Fluorescence PCR respectively for the purpose of detection, imaging and quantification. Nonspecific absorption DNA is degraded by Exo I and remains silent, ensuring highly specific detection. Folate receptor (FR) has been employed to demonstrate the feasibility of multiplexed detection strategies using small-molecule-linked programmable DNAs. The results reveal that DNA probes programmed can specifically recognize the membrane proteins and distinguish the cancer cells from health cells, and furthermore give the exact amounts of FR expressed on individual cancer cell membrane.In chapter3, Dephosphorylation of the3’termini of nucleic acids, catalyzed by various repair enzymes is important for cellular events, such as DNA replication, recombination. Here, using T4polynucleotide kinase phosphatase (T4PNKP) as a model target, a novel fluorescence nanosensor based on the FRET between dye labelled DNA and WS2nanosheets has been developed for monitoring the activity and inhibition of T4PNKP. In this assay, we designed a single-strand dye labelled probe that formed self-complementary structure at one end and with a3’-phosphoryl end that served as the substrate for T4PNKP. Once the phosphorylated probe was hydrolyzed by T4PNKP, the resulting probe with a3’-hydroxyl end was immediately elongated to form double-strand product by Klenow fragment polymerase (KF polymerase). WS2nanosheets was introduced to quench the fluorescence of the single-strand dye labelled probe without polymerase elongation. The dye labelled double-strand product preserves most of the fluorescence when mixed with WS2nanosheets. Because of the super quenching ability and the high specific surface area of WS2nanosheets, the as-proposed platform exhibits an excellent performance with wide linear range and low detection limit. Additionally, the effect of its inhibitors has also been investigated. The method not only provides a universal platform for monitoring activity and inhibition of DNA3’-phosphatases but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.In chapter4, SNP plays a significant regulatory role in drug programming,drug testing, in response to the identification of disease genes and in basic research on the biology. Graphene oxide has a strong adsorption function to single-stranded DNA molecule, has a broad fluorescence group quenching function, and does not quench the fluorescence of a single base. We constructed a simple and effective technique based on T7exonuclease enzyme, Single nucleic acid probe labeled fluorophore, and Graphene oxide for sensitivity SNP detection analysis. The proposed method is simple, rapid response and high sensitivity, the detection limit is down to lOpM.