Construction And Analytical Application Of Self-assembled Fluorescent Probes Based On Cyclodextrin Host-guest Recognition

Self assembly, as one of the important means to obtain functional m aterials, is generally refers to the process that assembled elements spontaneously form specific structures through the weak bond interactions. Compared with covalent binding, self assembly can avoid multi-step synthesis and complex purification in the pro cess of fabrication. Furthermore, the self assembled structure via weak bond interaction has adaptive capability in response to external stimuli, which make s it possible to design and construct stimuli-responsive molecular devices and functional nanostruct ures for biological/chemical information acquisition or controlled drug release. Among various weak bond interactions under the definition of self assembly, host-guest interactions based on macrocyclic molecules have attracted a lot of interest. Through such host-guest inclusion, two or more assembled elements can be integrated together in a facile and reversible manner, providing vast possibilities for the design and fabrication of functional devices. In this dissertation, owing to the excellent advantages such as good biocompatibilities and easy fabrication, cyclodextrins have been used for the design and fabrication of a series of novel self-assembled fluorescent probes, and their applications in nucleic acid detection, cellular imaging, drug delivery and so on have also been studied. The main researches of this dissertation are summarized as follows.1. Based on the β-cyclodextrin/azobenzene(β-CD/Az) reversible host-guest inclusion and nucleic acid(DNA) self-assembly, we have developed a new strategy for fabrication of molecular beacon conformational switch. The conformational switch is designed as a triple stem molecular beacon through spontaneous hybridization of three DNA strands:(1) a hairpin DNA modified with a fluorophore and a quencher;(2) a 5’-azobenzene modified DNA;(3) a 3’-β-CD modified DNA. Owing to the formation of stable host-guest inclusion complex(β-CD/trans-Az) under normal temperature, TMB1 is in the locked state and emits weak fluorescence even in the presence of targets. However, TMB1 can be activated to hybridize with target DNA and emit strong fluorescence, due to the photo-dissociation of the β-CD/trans-Az complexes by UV light induced trans-to-cis isomerization of Az moieties. In addition, TMB1 can reversibly revert to the locked state by thermal regeneration mode(55 oC) and emit weak fluorescence, since heating can induce the release of target from duplex and the cis-to-trans conversion of Az moieties. The results show that TMB1 could be used for reversible control of DNA hybridization through photoactivated and thermal regeneration mode. In addition, the self-assembled functional modification of the non-recognition region in the probe can avoid labeling multiplex markers on a single-strand DNA, as well as avoid unfavorable impact on the binding of the probe/target complex. Furthermore, we believe that this strategy would provide a new concept for the design of functionalized nucleic acid probes with stimuli-responsive performances.2. Based on host-guest inclusion interactions between adamantane derivatives of fluorescent dyes and β-cyclodextrin polymer(poly-β-CD), we have developed a self-assembled supramolecular nanoprobe(SSNP) for ratiometric fluorescence measurement of p H values in cells. SSNP could be formulated by the self assembly of following assembled elements:(1) poly-β-CD, selected as the nanoprobe backbone;(2) Derivative dyes, p H-sensitive adamantane-labeled fluorescein(Ad-F);(3) p H-insensitive adamantane-labeled rhodamine B(Ad-R),used as the reference dye for ratiometric p H response. By mixing these components at a certain ratio in aqueous phase, the SSNP with a diameter of about 30 nm could be formulated. The nanoprobe with excellent advantages such as easy fabrication, good photostability and biocompatibility, has been successfully applied to measure intracellular p H with a range of 4-8 and estimate p H fluctuations associated with different stimuli in cells. In addition, this SSNP can be easily tunable of dyes ratios to obtain optimal ratiometric p H response. Moreover, this research work provides a modular design strategy for the construction of functionalized nanoprobes for other targets just by replacing the respective indicator dyes with relevant indicators.3. On the basis of previous research, we synthesized the adamantane-labeled aptamers(Ad-Sgc8c), and furtherly constructed the targeted self-assembled supramolecular nanoprobes(T-SSNP) for specific recognition and killing of tumor cells. T-SSNP could be formulated by the self assembly of the following assembled elements:(1) poly-β-CD, as the nanoprobe backbone;(2) Ad-Sgc8 c, as the recognition element;(3) adamantane derivatives of fluorescent dyes(or antitumor drugs). The results showed that T-SSNP with a diameter of about 70 nm could specifically recognize CEM cells. The flow cytometry analysis showed that the signal-to-noise ratio of T-SSNP containing Ad-F was 7.4, which was about 3 times than that of the free probe(FAM-Sgc8c). Interestingly, if the fluorescent dyes and adamantane are marked on the aptamer(Ad-Sgc8c-FAM), nanoparticles could not be formulated by mixing the Ad-Sgc8c-FAM and poly-β-CD, and this mixture also could not improve the signal-to-noise ratio of cell recognition. Furthermore, T-SSNP containing the antitumor drug(doxorubicin, Dox) instead of fluorescent dyes exhibited significant selective cytotoxicity toward CEM cells, and the results also indicated its capability of targeted drug delivery.4. The poly-β-CD used in the above research works belongs to the main chain-shaped polymer and is electrically neutral. If we use hanging bell-shaped β-cyclodextrin polymer with negative charge instead of the main chain-shaped poly-β-CD, the targeted cells recognition of SSNP will be improved because of the following reasons:(1) Hanging bell-shaped β-cyclodextrin polymer containing β-CD monomers with good degree of freedom can be better integrated with the guest molecules;(2) Because the surface of tumor cells is usually with negative, negatively charged polymers can reduce the nonspecific signal of cell recognition. Considering these, we synthesized a hanging bell-shaped β-cyclodextrin polymer using polyacrylic acid as a matrix(PAA-β-CD). Then we furtherly constructed a targeted self-assembled supramolecular nanoprobe using PAA-β-CD as the backbone(T-SSNP?) in which the PAA-β-CD can integrate with adamantane derivatives of fluorescent dyes(or antitumor drugs) and the Ad-Sgc8 c can be used for targeted cell recognition. The results show that T-SSNP? can specifically recognize CEM cells and the signal-to-noise ratio of T-SSNP? containing Ad-F increases to 8.3. In addition, compared with T-SSNP, the selective toxicity of T-SSNP? improved significantly. Meanwhile, the above work also demonstrates that the self-assembly preparation procedure of nanoprobes is flexible.5. Based on the self-assembled structures of fluorescent conjugated polymer(cationic poly-phenylene acetylene, PPE-N+) and aptamer, a facile and sensitive strategy for cell detection has been developed. After the formation of PPE-N+/Sgc8 c self-assembled structure via electrostatic interactions between positively charged PPE-N+ and negatively charged Sgc8 c aptamer, PPE-N+ configuration changes and its fluorescence intensity decreases. However, strong fluorescence is observed in the presence of target CEM cells, because Sgc8 c can specifically bind with CEM cells leading to the PPE-N+ in the free state. The experimental results indicate that the PPE-N+/Sgc8 c self-assembled conformational probe exhibits a good specificity to CEM cells and a detection limit of 500 cells/m L. This method does not need the fluorescent label of aptamer and has advantages of simple operation and good versatility. This self-assembled method canbe used for other target detection just by replacing the aptamer.