Properties And Application Of Fluorescent Probes For Active H₂S And Cys

Visualization analysis based on fluorescent probe molecules has become a very effective method for physiological and pathological study of life processes.The active sulfur species represented by hydrogen sulfide（H2S）and cysteine（Cys）and the heavy metal copper ion(Cu²⁺)play a vital role in biological activities and physiological functions.For example,the interaction between H2S and Cu SO4 can inhibit the expression of copper transporter ATP7A and cause intracellular accumulation of Cu²⁺,resulting in Cu²⁺dependent neurotoxicity.Cys plays as an indispensable role in protein or enzyme synthesis,and its deficiency in our body can lead to various disorders including stunting,skin lesions,hair depigmentation,lethargy and liver damage.H₂S is formed by Cys through the catalytic reaction of cythioneβ-synthetase（CBS）and cythioneγ-lyase（CSE）,which plays an important role in smooth muscle relaxation,nerve conduction regulation and insulin release regulation.Therefore,the development of novel fluorescent molecules that can detect H2S,Cys and Cu²⁺is of great practical significance for the in-depth understanding of biological physiology and pathogenicity.However,most fluorescent probes for detecting active sulfides and Cu²⁺have the following shortcomings:1.The emission wavelength is located in the visible region;2.There is only one target detection analyte;3.The detection process is prone to interference from other mercaptan compounds.Therefore,the development of fluorescent probes capable of detecting H2S,Cys and Cu²⁺is of great practical significance for the study of cell physiology and pathogenesis.Based on this,three kinds of fluorescence probes were developed in this paper,including dual-function H2S and Cu²⁺fluorescence probes,near-infrared（NIR）Cys fluorescence probes and dual-function NIR H2S and Cys fluorescence probes,and their optical properties,physical and chemical properties,reaction mechanism and application were studied.The research content mainly includes the following three aspects:（1）A double fluorophore double response probe RH-NP was constructed with naphthalimide and Rhodamine.Introducing azide groups on naphthalimide as specific recognition sites for H2S;The specific ring-opening of Rhodamine was used to identify Cu²⁺.The UV and fluorescence properties of RH-NP response to H₂S and Cu²⁺were investigated in DMF/PBS（9/1,v/v）solvent system.On the one hand,RH-NP emits green fluorescence at 560 nm in response to H2S excitation at 400 nm,and the color of the solution changes from colourless to green;On the other hand,when excited at 530 nm,RH-NP responds to Cu²⁺by emitting red fluorescence at630 nm,and the solution color changes from colourless to red.In addition,the response mechanism of RH-NP with H₂S and Cu²⁺was proposed and verified by means of NMR and mass spectrometry.Finally,RH-NP was successfully applied to detect exogenous H₂S and Cu²⁺in cells.（2）The NIR fluorescence probe DTRN was synthesized from dicyanisophorone derivatives,which was bonded with acrylyl ester group for Cys specific recognition.The ultraviolet and fluorescence properties of DTRN response to Cys were studied.At 500 nm excitation,DTRN emitted near infrared fluorescence at 718 nm in response to Cys,and the color of the solution changed from colorless to red.In addition,DTRN can distinguish between Cys and Hcy using time-varying fluorescence signals without interference from GSH.The response mechanism of DTRN with Cys and Hcy was proposed and verified by NMR and mass spectrometry.Finally,the probe was successfully applied to the intracellular detection of Cys.（3）A NIR bi-functional fluorescence probe Cy-NP was constructed with naphthalimide and cyanine.Introducing azide groups on naphthalimide as specific recognition sites for H2S;The UV and fluorescence properties of Cy-NP responded to H2S and Cys were studied by using the interaction between carbon and nucleophile in cyanine center as the Cys reaction site.In DMF/PBS（1/1,v/v）solvent system,on the one hand,Cy-NP emitted green fluorescence at 560 nm in response to H2S excitation at 450 nm,and the color of the solution changed from colourless to green;On the other hand,Cy-NP at 640 nm in response to Cys（undisturbed by Hcy and GSH）emits NIR fluorescence at 760 nm,and the color of the solution changes from colourless to blue.In addition,the effects of different addition orders of H2S and Cys on the fluorescence changes of Cy-NP were studied.The response mechanism of Cy-NP with H2S and Cys was proposed and verified by means of mass spectrometry and DFT calculation.Finally,the probe was successfully applied to detect the concentration of H2S released by raw meat decay and to detect exogenous H2S and Cys in cells.