| Chapter 1:The basic principles of molecular fluorescence, fluorescent probes, the relative influences and classification were introduced in brief. Based on these principles mentioned above, the research development of calcium ion (Ca2+) probes was introduced. Because most of reported Ca2+ probes are intensity-based probes, thus internal calibration of Ca2+, the experimental corrections of sample thickness variability or differential concentration of probe within the cell could not be created. Between excitation-and emission-ratiometric probes, the latter is more desirable than the former in proper detection of Ca2+since excitation ratiometric probe need to set two excitation wavelengths simultaneously, and most of fluorescence spectrometer could not satisfy this demand. In the commercial Ca2+ probes, only Indo-1 and Indo-5F could perform emission ratio measurements as a function of Ca concentration. Unluckily, Indo-1 and Indo-5F require UV excitation, and their Kd are relatively small (0.23 and 0.47 μmol/L respectively) so that their applications in cells and tissues are quite limited. Herein, we presented the new design of ratiometric emission probes for Ca2+ with low toxicity, large Stokes shift and large Kd.Chapter 2:1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) as ionophore, and 2-(4’-ethoxyphenyl)-5-(4’-methylphenyl)-1,3,4-oxadiazole as fluorophore were introduced and joined, forming the Ca2+ probe OB based on ICT, and its molecular structure of OB was demonstrated by 1H NMR,13C NMR, mass spectrometry and elemental analysis.Chapter 3:1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA)as ionophore, and two 2-(4’-ethoxypheny1)-5-(4’-methylphenyl)-1,3,4-oxadiazoles as fluorophores were introduced and coupled, forming the Ca2+ probe OBO based on ICT, and its molecular structure of OBO was demonstrated by 1H NMR,13C NMR, mass spectrometry and elemental analysis.Chapter 4:The spectra of OB and OBO with Ca2+ were studied. The fluorescence spectra showed that OB (F490/F582) and OBO (F490/F594) were probes for Ca2+ based on ratiometric emission, OB and OBO were all highly sensible and selective for Ca2+ over other bivalent metal ions in cellular environment or in analog cellular environment. The optical condition for the application of probe OB or OBO was pH 7.0-7.5, and the stoichiometric ratio for OB-Ca2+or OBO-Ca2+ complex was 1:1. The dissociation constants were 0.56±0.08μmol/L for OB-Ca2+ and 2.25±0.47 μmol/L for OBO-Ca2+, illustrating OB is high affinity probe for Ca2+, OBO is low affinity probe for Ca2+. Moreover, their dissociation constants (Kd) are all larger than that of Indo-land Indo-5F. Furthermore, OB and OBO could monitor Ca2+ in cytosol in living HUVEC and showed the changes of Ca2+ concentration by the fluorescence imaging under confocal scanning laser microscopy in resting and dynamic states.Chapter 5:Since Cd2+ ionic radius(0.96 A) closely approximates Ca2+ ionic radius (1.12 A), therefore, Ca2+ was replaced with Cd2+ and the spectric properties were studies. The fluorescence spectra of OB-Cd2+ or OBO-Cd2+ presented a vastly different from that of OB-Ca2+ or OBO-Ca2+ based on ICT (Intramolecular Charge Transfer). OB was the probe with ratiometric emission (F530/F670) for Cd2+, while OBO was only intensity-based probe for Cd2+ with the small blue shift in emission spectra.OB and OBO were all highly sensible and selective for Cd2+ with or without other divalent metal ions, including Ca2+ion. The optimal pH was 7.0 for OB or OBO. The stoichiometric ratio for OB-Cd2+ or OBO-Cd2+ complex was 1:1, dissociation constants were 0.59±0.009μmol/L for OB-Cd2±and 0.116±0.027μmol/L for OBO-Cd2+. Finally, OB was employed to monitor the Cd2+ in the practical samples, while OBO was used to detect Cd2+ in cytosol and confocal fluorescence images were presented in MCF-7 cells, the two results were satisfactory.Chapter 6:Summary and outlook. According to the current problems of fluorescence probes, some advice was presented, and the directions of research in this area were introduced in the near future. |
