Design, Synthesis And Application Of Fluorescence Probes In Nucleic Acid Detection Based On TPE And GFP Chromophore

Nucleic acids are the genetic material of life and essential in the growth, inheritance and variation of all forms of life. The detection of these molecules plays a crucial role in clinical diagnosis, gene expression analysis and biomedical studies. Biosensors relying on fluorescence in DNA detection have attracted great attention because of their high sensitivity, relatively rapid and easy operation approaches. However, most classic DNA fluorescent dyes mainly bind DNA with secondary structures by intercalation or groove binding, thus, many of them do not show significant fluorescence response for ssDNA without secondary structures. To sense ssDNA effectively and conveniently, some new fluorescent dyes based on electrostatic interaction were developed. But the practical application of these ionic probes is limited because of their low sensitivity.In this thesis, a series of new fluorescent materials based on restricted intramolecular rotation (RIR) were designed, synthesized and applied in oligonucleotide detection and cell imaging, the results are organized as follows:1. Firstly, we briefly introduced the basic principles of the fluorescence and several classic fluorescent sensing mechanisms, which are reviewed emphatically on the basis of the mechanism of restricted intramolecular rotation. Then, the recent rapid development of TPE and GFP chromophore were described. Secondly, we summarized the development of fluorescent probes for nucleic acid detection. According to different detection modes and luminescence mechanisms, the probing molecules were divided into classical small molecules, water-soluble conjugated polymers and TPE probes. Thirdly, we introduced the fluorescent stain used in DNA gel staining. Finally, the design strategies of the thesis are outlined.2. A new amino-functionalized TPE derivative was designed, synthesized and used as simple, universal and highly sensitive dyes for the detection of nucleic acids in both water and gel matrix. By the aid of the pure cis-and trans-configuration of TPE isomers, we demonstrated the significant differences of Z/E isomers in DNA detection and nucleic acid stain for the first time. The cis-configuration of dye showed much higher sensitivity than its trans-isomer. This should be contributed to the neighbouring group participation (NGP), which is easier to lead to the restriction of intramolecular rotaion (RIR). The ultra-low detection limits and universaly superiority compared to EB make it promising in real applications. 3. A series of tetraphenylethene (TPE) derivatives with different number of amino group were designed and synthesized. The TPE derivatives were used in the detection of nucleic acids in both water and gel matrix. Contrastive studies revealed that the number of binding groups play an important role in the detection of DNA. Among the TPE derivatives studied, TPE-2with two amino binding groups showed the highest sensitivity.4. A series of tetraphenylethene (TPE) derivatives with different length of hydrophobic side chain were designed and synthesized. The TPE derivatives were used in the detection of nucleic acids in both water and gel matrix. The results revealed that the length of hydrophobic linking arm and the configuration of TPE molecule play important roles on the sensitivity for nucleic acid detection. With the increase in the number of hydrophobic linking arms, the cis-and trans-isomer of TPE-5and TPE-6is better than the isomer of TPE-2, respectively. Among the TPE derivatives studied, TPE-5showed the highest sensitivity. Z-TPE-5is able to stain dsDNA with the lowest amount of1ng and exclusively stain40ng of short Oligonucleotide with only10nt.5. A series of tetraphenylethene (TPE) derivatives with different substitution degrees of amino group were designed and synthesized. The TPE derivatives including cis-and trans-isomers were used in the detection of nucleic acids in both water and gel matrix. Contrastive studies revealed that the sensitivity become lower with increasing of substitution degree of amino group, because the steric effect plays a more important role than electrostatic effect in the interaction between TPE derivatives and DNA.6. A series of Green Fluorescent Protein (GFP) chromophore derivatives were designed, synthesized and applied in DNA detection. The results showed that the fluorescence of p-BDI-6with DPA group functionalized in each side chain could be turned on by interaction with DNA in the presence Zn+. Though the singly functionalized p-BDI-2-与p-BDI-3, p-BDI-4and p-BDI-5could interact with DNA by electrostatic interaction and coordination, fluorescence of GFP chromophore derivatives can not be turned on. The contrastive studies proved that GFP chromophore derivatives could be used in the detection based on the explanation that the fluorescence are turned on when the configurations are locked by substrates.7. A series of GFP chromophore derivatives with amino groups were designed, synthesized and applied in DNA detection in organic solvent. The results show that there are a spectral blue shift of200nm of m-BDI-3and m-BDI-4with addition of DNA in DMF solvents, indicating that the interaction with the DNA can change the polarity of the environment around GPF chromophore derivatives. Besides, it was further confirmed that the coordination effect between DPA-Zn and DNA is significantly stronger than the electrostatic interaction between quaternary ammonium salt groups and DNA.