Design And Application Of Fluorescent Probe With Function Of Detecting Reducing Species

In complex living systems,reducing species maintain the normal physiological processes of cells by regulating the relative balance of redox states in organisms.Reduced coenzyme I(NADH,nicotinamide adenine dinucleotide)is an important electron transport carrier in cells and plays an important role in the electron transport process of the respiratory chain and energy metabolism.Small biomolecule thiols,such as:cysteine(Cys),homocysteine(Hcy),glutathione(GSH)and hydrogen sulfide(H₂S),play a role in maintaining the dynamic balance of the redox state of the biological system and the normal operation of the body.Therefore,monitoring the relative content of reducing species in organisms has important physiological significance.In recent years,fluorescent probe molecules are favored by the majority of researchers due to their rapid response,high sensitivity,trace detection and easy operation.Their combination with fluorescent imaging technology can explore the pathogenesis of various diseases at the molecular level.Based on this,the thesis takes reduced coenzyme I and thiols as detection targets,and carries out the following work:In chapter 2,using cyanine dye as the parent structure and introducing quinolinium salt as recognition site,based on the intramolecular charge transfer(ICT)mechanism,fluorescent probes DPMQL1 and DPMQL2 with high selective recognition for NADH were designed and synthesized.Spectral test results show that after the above probes interact with NADH,the ultraviolet absorption spectra were red-shifted to 528 nm and 572 nm,respectively,and the color of the solution changes significantly.At the same time,the fluorescence signal intensity was significantly enhanced(31-fold and 12-fold,respectively),and the detection limits were as low as0.36 n M and 4.5 n M,respectively.In addition,the probe has a large Stokes shift(103nm and 92 nm,respectively),which makes it possible to reduce background interference in biological imaging applications.Cell imaging experiments show that the probe can achieve fluorescence imaging detection of intracellular NADH,and the cell morphology is good,indicating that the probe has low biotoxicity,good biocompatibility and cell membrane permeability.In Chapter 3,using NBD-Cl as the recognition site for thiols,fluorescent probes NBD-RH1,NBD-DPM and NBD-RH2 that selectively recognize thiols(Cys/Hcy/GSH/H₂S)were designed and synthesized.Spectral test results show that the probe NBD-RH1 can use ultraviolet absorption spectroscopy to detect GSH,and fluorescence emission spectroscopy to detect Cys/Hcy,so as to distinguish the detection of Cys/Hcy and GSH according to the difference in spectrum.At the same time,the detection limits for Cys/Hcy were 5.0 n M/3.96 n M,and the pseudo-first order reaction rate constants were k _Cys=0.17121 s^-1 and k _Hcy=0.02807 s^-1.The probe NBD-DPM has good selectivity and high detection sensitivity for Cys/Hcy.The detection limits were 17.9 n M/18.2 n M,and the pseudo-first order reaction rate constants for its interaction with Cys/Hcy were k _Cys=0.00541 s^-1,k _Hcy=0.00076 s^-1.The probe NBD-RH2 showed high selectivity for H₂S,with a detection limit of 18.5n M.The three probes can achieve fluorescent imaging detection of thiols in living cells due to their low cytotoxicity and good membrane permeability.