Synthesis of Organic Two-photon Absorption Materials and Their Applications in Biological Fluorescent Probes and Photopolymerization
|Keywords||two-photon DNA probe cysteine probe Two-photon initiated photopolymerization radical mechanism|
The process of the two-photon absorption (TPA) has many features, such as absorbing light of longer wavelength with lower energy but emitting shorter wavelength, high three-dimensional spatial selectivity and high penetration depth. So the research about the organic TPA materials and their applications has attracted great attention from domestic and foreign scholars and made great progress. Recently, two-photon microscopes are commercially avialable and become common tools for biologist. Consequently, researchs on two-photon biological fluorescent probe is becoming focus. Compared with common and confocal microscopy, two-photon fluorescence microscopy offers a number of advantages, including improved resolution of three-dimension imaging, increased penetration depth and decreased photodamage. Because the research on two-photon fluorescent probe is lag behind, the wide application of TPM in biology is limited. So finding two-photon fluorescent probe with large two-photon excited fluorescence action cross-section, high fluorescence quantum yield and sensitivity has important theoretical and practical significance. Because of the high spatial selectivity "point" polymerization ability and good penetration of irradiation light, two-photon initiated photopolymerization (TPIP) is another research focus about the application of organic TPA materials. TPIP of vinyl monomers was first found in 1999, and then it has attracted a lot of interest due to its emerging applications, such as optical switch, photonic crystal, micromachine and three-dimensional microfabrication. Researchers generally agree that the mechanism of TPIP is radical polymerization, but the radical mechanism has not been proved. Therefore, thorough comprehension of the TPIP mechanism is of vital significance to design high-efficiency initiator, improve the total efficiency and decrease the threshold intensity for photopolymerization.In this thesis, two types of biological fluorescent probes were synthesized by Heck, Vilsmeier and Knoevenagel reaction. We took the carbazole, indole, pyrrole as parent substance and synthesized five pyridinium compounds which have good water-soluble and two-photon properties as DNA probes, and aldehyde was introduced into conjugated system of carbazole to synthesize three cysteine probes. Synthetic route is simple and easy to operate. The target compounds were characterized by NMR, elemental analysis and mass spectra.In this thesis, we detailedly analyzed the UV absorption, one-photon fluorescence and two-photon fluorescence spectra in various solvent of TMPC, THEPC, BMPI, BHEPI and BMPP, calculated the fluorescence quantum yield (Φ), two-photon absorption cross-sections (δ) and two-photon excited fluorescence action cross-sections (Φxδ. The results show that the fluorescence performance of TMPC, THEPC, BMPI, BHEPI is better in organic solutions than in aqueous environment, which means that the four compounds may be turn-on DNA probes having low fluorescence in cytoplasm and high emission when binding to DNA. And the fluorescence property of BMPP is best in five compounds, although the fluorescence performance has little difference between organic and aqueous solvent.DNA binding mechanism of TMPC, THEPC, BMPI, BHEPI and BMPP was investigated by titration experiments. The possible interaction modes between these derivatives and ctDNA may be intercalation into DNA base pairs at a lower concentration of DNA and aggregation on the surface of the DNA helix at a higher concentration. In the presence of DNA, the fluorescence intensity and peak positions of TMPC, THEPC, BMPI, BHEPI had obvious changes, and two-photon fluorescence intensity could be respectively enhanced 22,34,43 and 50-fold, while there was no big change of BMPP. The binding constant of TMPC, THEPC, BMPI, BHEPI and BMPP were obtained by the one-photon fluorescence titration and Scatchard equation, and the value is 2.7x106,5.79x106,2.06×106,2.26x106 and 4.36×105 L-mol-1, respectively. Based on the two-photon fluorescence changes, the optimal excitation wavelengths of probes after binding to DNA were gained by changing the excitation wavelength from 740 to 800 nm, and it is 780 nm for TMPC and THEPC and 790 nm for BMPI, BHEPI and BMPP.Φ,δandΦ×δin the presence of DNA were calculated, the value ofΦ×δare 27.6,33.32,2.3,2.18 and 28.11 GM, and increase 14,25,34,37 and 3-fold, whileΦ×δof DAPI (a commercial DNA probe) is only 0.16 GM. The five derivatives also exhibited high photostability in the presence of DNA. Therefore, they are potential two-photon DNA probes. The strictly staining experiments in fixed SiHa cells showed that clear Two-photon fluorescence images of the nuclei in cells accurately labeled with TMPC and THEPC could be obtained without any background noise and image distortion, so TMPC and THEPC should be valuable DNA probes for practical application in biological imaging.Preliminary studies of the reaction between cysteine and probes and their spectral properties were discussed in thesis. The spectral properties of 9E-PCAC,9E-ASCAC and 9E-HSCAC in benzene, ethanol and water show that they have good sensitivity to the environment, the fluorescence intensity andΦhave great changes in different solvent. In ethanol, in the presence of cysteine, the fluorescence intensity greatly increased, while unchanged in PBS buffer, so they may be environmental sensitivity and turn-on cysteine probes. For 9E-PCAC in ethanol, the maximum absorption and fluorescence emission peaks had near 100 nm red-shift,Φbecame 8.23%, at same time, 9E-PCAC have perfect two-photon performance:ΦandΦ×δbecame 227.55 and 18.14 GM, so 9E-PCAC could be used to probe cysteine by the changes of peak position and fluorescence intensity. After reaction with cysteine, the peak positions of 9E-ASCAC and 9E-HSCAC did not change, but fluorescence intensity enhanced 42 and 120-fold, so they could be excellent turn-on one-photon fluorescence cysteine probes. They all exhibited specific selectivity to cysteine in selectivity experiments and immunity to homocysteine and glutathione interference. The pH also could not affect their fluorescence and applications in biological cells. The strictly staining experiments in living SiHa cells showed that 9E-PCAC,9E-ASCAC and 9E-HSCAC could enter living cells and wide-field images were gained, especially, clear Two-photon fluorescence image to display the distribution of cysteine could be gotten by 9E-PCAC. Therefore, 9E-PCAC,9E-ASCAC and 9E-HSCAC should have practical application value to probe cysteine, especially 9E-PCAC could be a valuable cysteine probe for two-photon biological imaging.We also used the organic TPA materials DBMB as initiator, fabricated large periodic spacing microstructures of 15-200μm by 20×long working distance objective and mobile platform under the 800 nm laser excitation. At the same time, we discussed the TPIP mechanism of BDBAS/TMPTMA system. We real-time captured the free radical in BDBAS/TMPTMA system by ESR technology and first confirmed the radical mechanism of TPIP. The UV absorption, one-photon and two-photon fluorescence spectra showed that energy transfer occurred between BDBAS and TMPTMA, while there was no radical signal when initiator was alone, so the energy transfer between initiator and monomer should be the necessary condition to produce free radicals under the two-photon irradiation.In this thesis, two types of two-photon fluorescent probes, the large periodic spacing microstructures fabricated by TPIP and the TPIP mechanism were systematically studied, and some innovative results were obtained, laying the foundation for the further study on organic two-photon materials in the application of biological fluorescent probes and photopolymerization.