Design,Synthesis And Properties Of Reaction-based Fluorescent Probes

Fluorescent probes have the advantages of low detection limit,ease of operation,real-time detection,and easily applied in vivo.It has been widely used in many fields,such as detection of pollutants and biological imaging.For a long time,researchers paid more attention to the detection of heavy metal ions,which is more mature in the field of fluorescent sensor,while literature related to detection of anions,amino acids or organic small molecules is not many.And most of them suffer some defects,such as low selectivity,poor sensitivity and so on.Herein,four kinds of reaction-based fluorescent probes specific to hypochlorite,biothiol and hydrazine were designed and synthesized.Moreover,sensing mechanism of the probes were further investigated by means of several analysis methods,and the probes were applied in cellular imaging.1.Synthesis and application of a ratiometric fluorescence probe towards hypochlorite.In view of the fact that phenanthroimidazole-based fluorescent probes has the advantages of high fluorescence quantum yield,good photochemical stability,but poor water solubility.A novel ratiometric probe(PIPH)specific to hypochlorite on the basis of the phenanthroimidazole derivative as fluorophores and N-N single bond in the acetylphenylhydrazine group as specific recognition site was successfully developed.Simultaneously,the proportion of organic solvent was reduced to 50% in our work.With the addition of hypochlorite,a nearly 20-fold variation in the fluorescence ratio(I/I0)was induced.Then through NMR spectra and high-resolution mass spectrometry,it was confirmed that PIPH was oxidized to PIBA.As the probe is based on a newly discovered recognition mechanism,its theoretical explanation is not perfect.Therefore,Gaussian 09 program package was used to conducte the theoretical calculation and DFT(density functional)was utilized to optimize the spatial structure of PIPH and PIBA,while TD-DFT(time-dependent density functional theory)calculation was carried out on both the ground(S0)and the first excited(S1)state.Theoretical calculations exhibited that fluorophore and recognition group is approximately coplanar in S1 state,inducing the intramolecular charge transfer(ICT)between the two groups.With the departure of phenylhydrazine group,the ICT process was blocked and a blue strong fluorescence was observed.Finally,cellular imaging verified that PIPH could be a useful molecular sensor to monitor hypochlorite in living cells.2.Synthesis and application of a ratiometric fluorescence probe towards biothoils.A novel fluorescence sensor(DNBS)on the basis of naphthalimide derivatives as fluorophores and 2,4-dinitrobenzenesulfonate moiety as specific recognition site was developed.The probe exhibits high sensitivity and selectivity towards cysteine(Cys),homocysteine(Hcy)and glutathione(GSH)relative to other natural amino acids.Especially,the detection limit(LOD)of Cys is 28.9 n M.Then through mass spectrometry,it was confirmed that the receptor unit was decomposed by biothiols.With the the departure of 2,4-dinitrobenzenesulfonate moiety,the donor excited photoinduced electron transfer(d-Pe T)process was blocked and a strong green fluorescence emission emerged.According to the celluar imaging,the probe could be applied to image ectogenic biothiols in living cells.3.Synthesis and application of a ratiometric fluorescence probe towards cysteine.To offset the drawback of the previous probes,a novel quinizarin-based probe(AQDA)bearing two acrylate moieties was developed.Owing to nucleophilic addition and specific intramolecular cyclization reaction,the probe displayed high selectivity towards cysteine(Cys)relative to other natural amino acids including homocysteine and glutathione.The maximal fluorescent intensity is 30-fold of the initial value in the presence of 5.0 equiv.Cys,and only 20% organic solvent is sufficient for in the detection.The recognition mechanism was further confirmed through the mass spectroscopy and proton nuclear magnetic resonance titration.With the the departure of acrylate moieties,the photoinduced electron transfer(PET)was blocked and a strong orange fluorescence was emitted.Simultaneously,the fluorescence enhancement mechanism was characterized by theoretical calculations,and experimental data were consistent with the theoretical results.Finally,through the addition of BSO(an inhibitor of the GSH biosynthesis)and NEM(a thiol blocking reagent)in cellular imaging,we demonstrate that AQDA possesses the capacity to detect endogenous Cys in living cells.4.Synthesis and application of a ratiometric fluorescence probe towards hydrazine.By a one-step reaction,a novel fluorescein-based sensor(FLB)bearing a 4-bromobutyrate moiety was developed.The recognition mechanism was confirmed through the mass spectroscopy and proton nuclear magnetic resonance titration.With the addition of hydrazine,the spirolactam ring was opened and a strong green fluorescence emerged.FLB has excellent water-solubility,and only 10% organic solvent was sufficient for the above detection.Moreover,the probe exhibited a high sensitivity and selectivity towards hydrazine.Other analytes,including 15 anions,10 cations and 9 small molecules,did not induce any obvious interference.In addition,the detection limit of FLB is 0.92 ppb,which is about 10 times lower than the maximum level(10 ppb)of hydrazine in drinking water permitted by the United States Environmental Protection Agency(EPA).Finally,He La cells were applied in cellular imaging.It was proved that FLB has potential capacity to detect hydrazine in vivo.