Functional Regulation Of Fluorescent Dyes And Their Application In Bioimaging

Fluorescent dyes or fluorescent probes are widely used for the detection and imaging of biological materials in biological systems because of their good biocompatibility and high selectivity.In particular,fluorescent dyes or fluorescent probes with special structural functions have provided important research value for the study of life activities,mainly because of their special functional groups and structures that can target subcellular organelles,biological macromolecules,organic small molecules and metal ions and other substances in biological systems for identification testing.Research and development on fluorescent probes have provided important guiding values for the development of functionalized fluorescent probes.Among them,the structural regulation of dyes plays a decisive role in the construction of functionalized dyes.In this thesis,4-bromo-1,8-naphthalene anhydride,quinone and 2,3,3-trimethylindole were selected as fluorescent precursors,and different functional groups were introduced to control their molecular structures to detect subcellular organelles（mitochondria and Platelets）and glutathione（GSH）and mitochondrial membrane potential（MMP）within the platelet,and preliminary study of its biological value of the cell level by experiments.（1）An example of a two-photon fluorescent probe（IQDC-L）specifically targeted to thrombin-mediated targeting of GSH for proportional detection and bioimaging applications for ultra-trace levels of GSH in platelets.In order to detect GSH in platelets,methylpiperazine was chosen as the localization group of the organelles to target platelets through thrombin-mediated action.In IQDC-L,two fluorophores,4-bromo-1,8-naphthalene anhydride and quinoid,which had excellent two-photon properties,are coupled by the sulfonyl group of the reaction site introduced with GSH.Fluorescence occured due to the energy of the two introduced fluorescent structures Resonance energy transfer（FRET）resulted in the cleavage of the"S-N"bond in sulfonamide during IQDC-L recognition of GSH,and the probe emitted different fluorescence signals at 520 and 592 nm.When the concentration of probe-bound GSH varies from2.0 to 65 nM,a very good linear scale-change pattern was present which allowed it to detect GSH changes in a proportional model.The experimental results showed that the lowest detection limit of probe molecules to GSH was 0.083 nM.More significantly,IQDC-L can be screened for PLT from peripheral blood without any interference under thrombin-mediated conditions.Therefore,IQDC-L can be used to screen PLT from peripheral blood while enabling real-time imaging of ultra-trace GSH.（2）Based on the different reaction mechanisms of sulfonamides at different pHs,an example of the use of sulfonamide as a reaction site for the detection of ultra-trace changes in the mitochondrial GSH of cancer cells by a ratio fluorescent probe（IQDC-M）was demonstrated.In order to detect GSH in mitochondria of cancer cells,the targeting of mitochondria to the organelles was selected by using triphenylphosphine.To achieve targeting mitochondria at the same time,change the molecular water/oil amphiphilicitytargetingmitochondriaincancercells.InIQDC-M,twofluorophore4-bromo-1,8-naphthalene anhydrides and quinone,which had excellent two-photon properties,are coupled by the sulfonyl group of the reactive site introducing GSH.Fluorescence resonance energy transfer of the energy introduced by the two fluorescent structures that caused the IQDC-M had fluorescence signals at520 and 592 nm during its identification of GSH,allowing it to detect trace changes in GSH with a proportional model.And the lowest detection limit of IQDC-M for the change of super-sensitivity of GSH was 2.02 nM that was much less than 1.0‰of endogenous GSH in living cells.More importantly,IQDC-M was able to target the identification of trace changes that occur in the mitochondria of cancer cells due to the appropriate water/oil amphipathicity（log P）that was provided by the structurally controlled IQDC-M.Thus,these molecular properties maked IQDC-M was the first two-photon fluorescent probe molecule to image and monitor mitochondrial GSH trace changes during cancer cell apoptosis.（3）An example of a molecular switch（hemicyanine derivative,TPP-CY）was designed as a fluorescence probe for real-time detection of trace changes in mitochondrial membrane potential（MMP）in living cells.In order to detect the mitochondrial membrane potential,triphenylphosphine was used to locate the organelles.To achieve targeting mitochondria and affecting the intramolecular"C-O"bond to achieve the probe fluorescence ratio detection at the same time.TPP-CY showed an excellent selectivity,in response to the conversion of TPP-CY to TPP-SP for the negative potential in the detection of MMPs,that it showed a significant change in fluorescence intensity over several seconds I₅₆₃/I₆₆₃.In addition,TPP-CY had very low cytotoxicity（at a concentration of 0.23μM）and very high sensitivity（minimum detection limit of MMP is-0.16 mV）.Therefore,TPP-CY can precisely monitor subcellular level changes in MMP and mitochondrial morphology during apoptosis with excellent selectivity and high resolution,andcould assess cell health by mitochondrial morphology at the subcellular level.Thus,it has potential application in the research of chemical biology and medical diagnosis of mitochondrial diseases.