Applications Of Highly Sensitive Functional Fluorescent Nucleic Acid Probes In Biochemical Analysis

The essential information on small molecules, nucleic acids, proteins and cells obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. Functional fluorescent nucleic acid probe,whose functions are beyond the conventional genetic roles of nucleic acids, had attracted researchers'increasing attention. However, how to get higher sensitivity, increase its ability to tolerate any interference from complex environment, get the dynamic data of these life processes sensitively and accurately in real time are still great challenges to analysts.In this dissertation, a series of functional fluorescent nucleic acid probes based on molecule recognization and signal amplification technique have been developed for small molecule, nucleic acid, protein and cancer cell detection, respectively. The main researches of this dissertation are summarized as follows.(1) An aptasensor for cocaine amplified detection based on a strand-displacement polymerization reaction and fluorescence resonance energy transfer (FRET) has been developed. Herein, the acceptor fluorophore (Cy5) is labelled at the 3'end of the hairpin probe, which contains the cocaine aptamer; and the donor fluorophore (FAM) is labelled at the 5'end of the primer. When the aptamer binds to cocaine and undergoes a conformational change, the hairpin structure opened. Then the primer anneals with the exposed stem of the hairpin probe. Following this, the primer will extend to form a complementary DNA of the hairpin probe in the presence of dNTPs/polymerase. The newly duplex will push the donor and acceptor in close proximity, resulting in FRET. Meanwhile, the target cocaine is displaced by the primer extension. To renew the cycle, the displaced free cocaine binds to another aptamer, making the signal amplification realized. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent.(2) A nucleic acid probe for highly sensitive DNA detection based on pyrene excimer and hybridization chain reaction (HCR) amplification has been developed. Two hairpin probes have been labeled two pyrene molecules at both ends, where the pyrene molecules are separated each other because of the sticky end. When the target DNA triggers HCR, there will be produce a long nicked duplex, allowing the formation of numerous pyrene excimers. Sensitive detection of target DNA in buffer and complex biological sample is achieved through steady-state fluorescence assay and time-resolved fluorescence measurement. The limit of detection for DNA is 250 fM in buffer.(3) An aptasensor for sensitive lysozyme detection in human serum based on pyrene excimer and competition reaction has been developed. Herein, a dual-pyrene labeled hairpin probe and label-free lysozyme aptamer coexist in solution. In the absence of target, the aptamer hybridizes with part of the hairpin probe to form a duplex, resulting in two pyrene molecules spatially separated. However,in the presence of target, the dual-pyrene labeled probe is gradually displaced from the aptamer by the target, subsequently forming a hairpin structure, this brings the two pyrene moieties into close proximity and allows the formation of an excimer. Detection of target lysozyme in buffer and human serum is achieved through steady-state fluorescence assay and time-resolved fluorescence measurement. The detection limit of this method for lysozyme is 200 pM in buffer. Finally, the success of ATP detection suggests the generality of the strategy.(4) A nucleic acid probe for highly sensitive target cell detection based on molecular beacon and nicking endonuclease signal amplification has been developed. A ssDNA , which can open the molecular beacon, is hybridized with aptamer to form a duplex. The formation of the aptamer/ssDNA complex inhibits the competitive hybridization of molecular beacon/ssDNA unless triggered by target cells. In the presence of the specific target cancer cells, the aptamer binds to the target cell and forms an aptamer/cell complex, resulting in the disassembly of the original duplex and release of the ssDNA. The released ssDNA sequence contains the nick endonuclease recognition site. One ssDNA hybridizes with one molecular beacon and then the nick endonuclease makes a nick in the beacon strand. After nicking, the molecular beacon is cleaved and the ssDNA can be reused for next cycle of cleavage. In this way, each ssDNA can go through many cycles, resulting in cleavage of many beacons, achieving a signal amplification. The detection limit of this method is 200 cells/mL and the specificity of this approach is excellent.(5) An angiogenin-mediated cell-internalized aptamer probe for improve the efficiency of photodynamic therapy (PDT) has been developed. A Ce6-conjugated, angiogenin aptamer was sythesized and characterized, which can deliver the photosensitizer into target cells mediated by angiogenin. After irradiation by an appropriate light, the Ce6 active the surrounding oxygen molecules to form reactive oxygen species, that ultimately leads to tumor cell death. The results showed high efficiency of PDT.