Synthesis Of Nucleophilic Substitution Reactive Fluorescent Probes And Their Application In Bioanalysis

Fluorescence analysis has the advantages of micro,real-time,trace,automation,high efficiency and selectivity,high accuracy and sensitivity.It has been widely used in life science,environment,food science and other fields,and is a kind of important and effective chemical analysis means.Fluorescence probes combine molecular recognition and fluorescence analysis techniques.Due to their good biocompatibility,fluorescence probes can enter the organism for real-time in situ detection,and have become one of the important tools for fluorescence analysis and detection.Bioactive substances play an important and unique role in human health,and their overexpression is often closely related to some diseases.Therefore,effective,accurate and real-time detection of these active substances is important for biomedical research and disease diagnosis.Although fluorescent probes have been widely used in the analysis and detection of bioactive substances,there are still some problems to be solved,such as poor selectivity,low sensitivity and limitations of application in vivo.Based on this,several fluorescent probes based on nucleophilic substitution reaction were designed and synthesized in this paper,and their interactions with Bioactive substance—biological mercaptan,enzyme and hydrogen polysulfide were studied in detail,and a highly sensitive detection method for these biomolecules was established.The specific work is as follows:1.The types and recognition mechanism of fluorescent probes,the design,synthesis and application of small organic molecular fluorescent probes,the important physiological significance and analysis of bioactive substances-mercaptan,hydrogen polysulfide and enzymes are reviewed.The research ideas and directions of this paper are proposed.2.Modulating an in situ fluorogenic reaction for the label-free ratiometric detection of biothiolsCd Te quantum dots stabilized by mercaptopropionic acid(MPA-Cd Te)were prepared.A fluorescence ratio method for biothiol detection was designed based on the internal filtration effect of the fluorescence reaction products of dopamine and rescinol with MPA-Cd Te.This method can quickly detect thiol in 40 minutes,with high sensitivity and good selectivity.The detection limit of fluorescence experiment for Cys is 0.6?M.This ratiometric analysis strategy was further applied to detect biothiols in human serum samples,good recovery results(98.6%–104%)and reasonable RSD(1.1%–1.5%)were obtained.3.An turn-on fluorescent probe based on BODIPY for the specifically detecting of Cys over Hcy and GSHA novel fluorescent probe HO-BODIPY-S was designed and prepared with phenylthiophenol as the recognition group and BODIPY as the fluorophore,which was used for the specific detection of Cys.Its chemical structure was confirmed by ¹H and ¹³C NMR spectroscopy and high-resolution mass spectroscopy(HRMS).When the probe was used for the detection of biothiols,it showed an obvious response to Cys,and the fluorescence intensity increased by 12 times in 40 minutes.In the response time of Cys,Hcy and GSH almost did not respond.The detection limit of the probe for Cys was calculated to be 61 n M.The response mechanism of the probe was confirmed by high-resolution mass spectrometry,and the probe has been successfully utilized to image Cys in living cells.4.A fluorescent probe based on NBD was used to detect leucine aminopeptidase A novel fluorescent probe NBD-S-Leu was designed and prepared by connecting a dipeptide with NBD as a fluorescent group.When the probe is exposed to LAP,the amide bond in the dipeptide cleaves and generates free amino groups,resulting in a clear green fluorescence.The fluorescence intensity showed a good linear relationship with the concentration of LAP(0-0.10 U m L^-1).The limit of detection for LAP was50.35 ng m L^-1,and the maximum reaction rate V_maxwas 15.19?m s^-1.The sensing mechanism was fully supported by TD-DFT calculations and HPLC-HRMS analysis.Furthermore,NBDS-Leu has been effectively utilized to image endogenous LAP activity in various types of living cells and zebrafish models.the endogenous LAP distribution in different living cells and zebrafish organs were clearly observed from more than one angle.5.A fluorescent probe based on fluorescein is used for the detection and fluorescence imaging of hydrogen polysulfideA fluorescent probe NAFI-H₂S_nwith novel molecular structure,using 2-(benzoyl sulfide)benzoate as the recognition unit and half-naphthofluorescein as the fluorophore was presented.The structure of the probe was proved by NMR and high resolution mass spectrometry.The probe itself has no fluorescence emission,the fluorescence intensity is enhanced by about 1000 times in a rapid response of 10 min with H₂S_n.the detection limit is 16 n M.The probe could be utilized to image exogenous and endogenous H₂S_n,including those generated by LPS stimulation in living A549 cells and zebrafish models.