| Intracellular signal transduction molecules play important roles in regulating the physiological function of cells. Monitoring concentration change and location migration of these signal transduction molecules is important for revealing mechani sm of the transduction pathways and guiding the development of drugs for related diseases. In recent years, the emergence of new nanomaterials and the development of functional nucleic acids provide new design ideas and platforms for the construction of bi ological fluorescent probes. These probes are able to interact with variety of targets and be applied in live cell imaging. Based on the interaction between functional nucleic acid and its target, and the unique optical properties of nanomaterials, this do ctoral dissertation focuses the research hotspot of the important signal transduction molecules in cells and develops some multifunctional composite biological fluorescent probes for live cell imaging. The construction methods of the probes and detection mechanism are also discussed in the dissertation. In general, the developed novel biological fluorescent probes are able to be used for sensitive, in situ and real-time live cell imaging detection of corresponding signal transduction molecules. The detailed contents are described as follows:Adenosine triphosphate(ATP) is not only an energy substance of organisms, but also a signal molecule which can transfer information between nerve cells. As glial transmitter, the release of the ATP from the glial cells is involved in the physiological and pathological processes of the nervous system. It is very important to monitor the release of ATP for the diagnosis and prognosis of the related diseases. In chapter 2, based on the configuration changes of the aptamer w hen binding with ATP, a new type of functional nucleic acid probe was constructed and was used for anchoring on the cell membrane for the detection of ATP release. The aptamer was labeled with Cy3 and biotin at its 5’ end and 3’ end respectively. An ss DNA partially complementary with the aptamer was labeled with Cy5 at its 3’ end and used for hybridization with the aptamer. Hence, a hybrid chain with fluorescence energy transfer(FRET) signal was obtained. The biotinylated lipid can spontaneously localize i nto membranes at the low temperature and the functional nucleic acid biological fluorescent probes was anchored on the cell membrane via biotin-streptavidin(SA) complexation. When the cells necrosis, the ATP would be released from the cell and flew throug h the membrane. FRET effect was blocked by the configuration changes of the aptamer for the interaction between the ATP and the probe. Then the optical signal of the probe was changed, and the real-time study on live cell imaging has been performed for the release process of ATP. This method was simple, low cost and selective, and it was expected to be used in clinical research.Highly reactive oxygen species(h ROS) are important signaling molecules. Over accumulation of h ROS in cells induces oxidativestres s and is associated with many pathological conditions. In chapter 3, it was newly report that gold nanoclusters(Au NCs) synthesized using a glutathione template show a fluorescence quenching signal sensitively and selectively responsive to h ROS such as ?OH, HCl O, and ONOOin homogeneous solution, and the detection limit were 0.03 μM for ?OH, 0.5 μM for HCl O, 0.2 μM for ONOO-, respectively. To take advantage of this exciting finding and to apply the Au NCs for visualizing and monitoring h ROS in living cells, in chapter 4, we design a complex fluorescent probeas a novel nanosensor for live cell imaging of h ROS which was generated by cell under the stimulation of the outside environment. The encapsulated dye of the silica nanoparticle(The excitation and emission peaks are 435 nm and 405 nm respectively) was modified with SA and the Au NCs were conjugated to a cell penetration peptide(CPP) using the same method. The above dye-encapsulating silica nanoparticles and the Au NCs were linked together by an oligoarginine peptide via the streptavidin(SA)-biotin and a composite nanoprobe with a single excitation double emission property was formed. The CPP could improvethe efficiency of intracellular delivery which made the probe could be spontaneously into the cell. The probe was responsed to h ROS, the Au NCs with their fluorescence sensitively quenched and the silica nanoparticles acting as an internal reference. This method is good at selectivity and fast response, especially this design give a well-resolved, intensity-comparable signal, thus affording a high contrast in imaging applications and avoid the generation of false positive signal to the target. Compared with organic fluorescent probes, this probe with high biocompatibility and stability against photobleaching offers the advantages of real-time tracking ability in cell imaging.Cytochrome c is an important signal for cell apoptosis. The release of cytochrome c is the key point of the cell apoptosis, often defining the point of no-return in cell apoptosis. In chapter 5, based on the aptamer could be absorbed on the surface of the graphene oxide easily and the fluorescence of the dye was quenched, we developed a biological fluorescent probe based on graphene oxide-aptamer with the PEG molecular responded to the cytochrome c. Using chemical crosslinking agent EDC/suflo-NHS, the surface of graphene oxide was covalently cross-linked PEG molecules(the molecular weight was 1500) with two amino group, and modified folic acid molecules on the other side of the PEG, then adsorbed cytochro me c aptamer. Compared with the traditional graphene oxide probe, the water solubility of this nanocomposite probe is better, the sensitivity is higher, and the linear range of cytochrome c is wider. Subsequently, in chapter 6, we realized a biological fluorescent probe in the first time that enables direct fluorescence activation imaging of cytochrome c released from mitochondria to the cytoplasm in apoptotic cells. When the cell was alive, cytochrome c was located in the inner and outer membrane of mitoch ondria, and the probe was efficiently internalized by tumor cells into the cytoplasm by the folate mediated endocytosis. There was no fluorescent signal to be fined, because they were unable to contact with each other. When the cells were apoptosis in drug stimulation, the cytochrome c was released from mitochondria to the cytoplasm, which was associated with the binding of the nucleic acid and away from the graphene oxide, which led to the generation of the fluorescent signal, and the in situ detection of cytochrome c in the cell was realized. The release of cytochrome c was started after 15 min of stimulation by 1 μM staurosporine(STS, apoptosis inducing agent), and completed after 25 minutes. Compared with the traditional method of detecting the activity of Caspase family enzymes in the cell to reflect the apoptosis of the cells, this method was taken less time. At the same time, this method has good reproducibility, which is expected to be a commercial probe to detect cell apoptosis, and provide a reliab le and practical platform for apoptotic studies and catalyze the fundamentalinterrogations of cytochrome c mediated biology and the screening of anticancer drugs at the cellular level. |
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