Cell Imaging Studies Based On Conjugated Polymer PDBPTBT Nanoparticles And Tricyanofuran Fluorescent Probes

Fluorescence probes have a wide range of applications in molecular detection,cell labeling and in vivo imaging studies because of their advantages of simple operation,high resolution,high sensitivity and rapid analysis.Fluorescence probes with red emission as well as good light stability,excellent biocompatibility and low damage to biological tissue are highly desirable in biosensing and bioimaging.It provides useful tools for diagnosis and therapeutic treatment for important disease such as,cancer.Based on congugated polymer fluorescent nanoparticles(CPNs)and fluorescent molecules that used tetracyano dihydrofuran as electron acceptor,it is very promising to develop a photo-stable,biocompatible,and red emitted fluorescent probes for cell imaging as well as specific marker for tumor.My thesis included the following works:1.Study on Cell Imaging of Red Fluorescent Nanoparticles Based on Conjugated PolymersIn this paper,a donating electronic structural unit building dibenzopyran(DBP)has been developed which has a cyclic pyran ring between two benzene rings.Since the ringing of the cyclized pyran ring and the alkoxyl chain helps to reduce the tight packing in the solid phase,a similar design is proposed in this paper to help produce bright red fluorescent CPNs.The red fluorescent conjugate polymer PDBPTBT was synthesized by Suzuki polycondensation using benzothiadiazines and DBP,which are mainly substituted by alkoxy chains,showing a large absorption rate,effective dark red emission and high brightness fluorescence in organic solvents and the quantum yield is as high as 72%.In this paper,CPNs were prepared by two commonly used amphiphilic polymer nonionic surfactants plutonic-F127(F127)and poly(styrene-co-maleic anhydride)(PSMA)rapid precipitation conjugate polymer PDBP TBT.The method is very easy to handle and has good reproducibility and the resulting CPNs are fairly stable in aqueous solution,which no precipitation and aggregation phenomena are observed for several months after preparation.In addition,the quantum yield of the obtained conjugated polymer nanoparticles was 36.8%-51.8%and the particle size was 10.2-36.8 nm.In order to further keep CPNs in the cells,P3/PSMA CPNs were modified by EDC catalyzed coupling with 4-aminobutyric acid ethyl ester hydrochloride and the ester groups were introduced on the surface of CPNs increasing its binding to the cell membrane and applied to cell imaging and long-term track of mammalian cells.Experiments show that cell culture can be performed for about one week after 4 hours of cell culture.Thus,the P3/PSMA CPNs have successfully applied for long periods of cell tracking.2.Near-Infrared Fluorescence Probe Based on Tetracyano Dihydrofuran(TCF)for Endogenous Nitroreductase(NTR)in Cell ImagingNTR is a marker for tumor cell and hypoxia,thus it is important to develop method for its detection.By using TCF as electron acceptor and amine or hydroxyl substituted benze ring as electron donator,a photo-stable and red emitted fluorescence molecule can be developed.A red fluorescent probe based on TCF as electron acceptor was designed and synthesized.The probe have nitro group as a strong fluorescence quenching group,which makes the probe have very weak fluorescence.NTR can catalyze the reduction of nitro groups in a wide range of nitroaromatic compounds to amino groups in the presence of reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate(NADPH).In the cells,NADPH is present as an electron donor and the endogenous NTR reduces the nitro group in the probe molecule to amino,then,the nature of the electron donor gro up changes the distribution of the electron cloud in the molecule to form a p-? conjugated system.The resulting push-pull effect causes the fluorescence in the probe molecule to recover.In this paper,we further studied the effect of reaction time of the fluorescence intensity of the probe with the NTR reaction system in vitro,as well as the selectivity and sensitivity of the system.The studies provide the foundation for in vivo imaging applications.Endogenous NTR detection was studied by using the NTR inhibitor cultured cells as blank.The results demonstrated the high selectivity of NTR detection.The probe not only the easily prepared,but also has low damage to biological tissue.Moreover,the method is simple,high sensitive,high selective.Combined with fluorescence confocal microscopy,real-time observation and detection of NTR in different kinds of cells can be realized.The probe can also be used for the screening ofNTR inhibitors.