Design,Synthesis And Application Of Fluorescent Probes For Biothiols

Biothiols,commonly referred to as cysteine（Cys）,homocysteine（Hcy）and glutathione（GSH）,play crucial roles in human physiological and pathological processes.In the physiological system,the metabolism and transportation of these sulfhydryl-containing compounds are closely related to a series of enzymes and proteins.The endogenous concentrations of these sulfhydryl compounds can determine the functional status of the corresponding enzymes and proteins,and their abnormal concentrations are associated with some diseases.Therefore,it is very important to detect and monitor these sulfhydryl compounds in biological samples to understand their roles in physiological and pathological processes.Fluorescence analysis technology is a powerful optical technology,which has attracted more attention because of its simple operation,real-time detection,high sensitivity,good selectivity,non-invasive,high spatial resolution and other advantages.Rare earth complexes have unique luminescence characteristics,including narrowband emission,large Stokes shift and long fluorescence lifetime.Carbon quantum dots（CQDs）are novel nanomaterials with sizes less than 10nm,which have excellent optical specificity and biocompatibility.These advantages make rare earth complexes and carbon quantum dots ideal fluorophores for constructing fluorescent probes.Therefore,three new fluorescent probes were designed and synthesized in this paper,which were based on displacement reaction,nucleophilic aromatic substitution reaction and Michael addition cyclization reaction,respectively,and had established high selectivity and sensitivity detection methods for biothiols and applications in biological samples and biological imaging.The main research contents are as follows:In chapter two,a novel red-emitting Eu³⁺-DTPA-bis（AMC）complex with large Stokes shift（288 nm）was synthesized for the detection of Cu²⁺,and a new Eu³⁺-DTPA-bis（AMC）/Cu²⁺system is used for high sensitivity and high selectivity detection of biothiols（Cys/Hcy/GSH）.The possible detection mechanism was verified by UV-vis,high-resolution mass spectrometry,and the fluorescence decay curve.The experimental parameters,including the solution p H,the incubation time,the concentration ratio of Eu³⁺-DTPA-bis（AMC）to Cu²⁺and biothiols concentration,were optimized.Under the optimal conditions,it shows a good linear relationship between the concentration（0-10μmol/L）of Cu²⁺and the fluorescence intensity of Eu³⁺-DTPA-bis（AMC）,with a low detection limit of 0.065μmol/L.The linear range and the limit of detection of the Eu³⁺-DTPA-bis（AMC）/Cu²⁺system for Cys/Hcy/GSH were 2.5-22.5/5-45/5-50μmol/L and 0.11/0.07/0.05μmol/L,respectively.Surprisingly,the concentration of Eu³⁺-DTPA-bis（AMC）/Cu²⁺can significantly affect the selectivity of the sensing system to biothiols（Cys/GSH/Hcy）.When the concentration of the Eu³⁺-DTPA-bis（AMC）/Cu²⁺system is 10.0μΜ,it could recognize biothiols（Cys/GSH/Hcy）from other substances,but when the concentration is as low as 3.33μmol/L,it could further specifically distinguish Cys from Hcy/GSH.Owing to the high anti-interference characteristics,accuracy and specificity,the sensing system was well applied to the detection of Cu²⁺in actual environmental samples and Cys in biological and food samples,including FBS,urine,milk,beverage,fresh juice with the satisfactory recoveries from 96.20 to 106.80%.In order to further selectively recognize Cys,Hcy and GSH,a new dual excitation and dual emission fluorescent probe,blue emission carbon quantum dot-oxy-7-nitrobenzo-2-oxa-1,3-oxadiazole（b-CQDs-O-NBD）was designed and synthesized for the detection of biothiols and H₂S in chapter 3.The blue carbon quantum dots（b-CQDs-OH）(λ_{ex/em maxima}=377 nm/467 nm)was synthesized by a microwave-assisted method using hydroquinone as the carbon source.Through the reaction of the phenolic hydroxyl group of the blue carbon quantum dots and4-chloro-7-nitrobenzo-2-oxa-1,3-diazole（NBD-Cl）,the b-CQDs-O-NBD was synthesized.Due to the static quenching（SQ）and the photo-induced electron transfer（PET）from b-CQDs-OH to NBD,the fluorescence of the b-CQDs-O-NBD is very weak.After adding biothiols and H₂S,the ether bond of the b-CQDs-O-NBD was broken through nucleophilic substitution reaction,which led to the release of the b-CQDs-OH（blue channel is turned on）and the generation of a new compound NBD-thiols（green channel）.The b-CQDs-O-NBD as a probe shows the high selectivity and sensitivity for Cys/Hcy/GSH/Na HS（LOD of 0.24μmol/L/0.21μmol/L/0.11μmol/L/0.18μmol/L）over other aminos and ions by blue channel.The newly formed compounds from NBD and Cys/Hcy have strong fluorescence under 470 nm wavelength light excitation（green channel is turned on）,while the formed compounds from NBD and GSH/Na HS have no fluorescence（no green channel）,so the b-CQDs-O-NBD can discriminate Cys/Hcy（LOD:0.07μmol/L/0.06μmol/L）from GSH/Na HS.In addition,the b-CQDs-O-NBD shows low toxicity and can be applied to the detection of Cys in fetal bovine serum and imaging in living cells.To further improve the specificity of biothiol detection,a novel carbon quantum dot-O-acryloyl chloride（g-CQDs-O-Acryl）fluorescent probe was synthesized in Chapter4 for the specific recognition of Cys.First,green fluorescent emitting carbon quantum dots with rich phenolic hydroxyl groups on the surfacewas synthesized by solvothermal method.Next,acryloyl groups were linked to carbon quantum dots through ester bonds,and the green fluorescence of carbon quantum dots（g-CQDs-OH）was quenched by the synergistic effect of static quenching（SQ）and photoinduced electron transfer（PET）.Acryloyl groups on the probe g-CQDs-O-Acryl can undergo successive Michael addition and cyclization reactions with biothiols,resulting in the release of carbon quantum dots（g-CQDs-OH）and the recovery of green fluorescence at 525 nm.Compared with other biological thiols（Hcy and GSH）,the probe reacts with Cys about 10 times faster than Hcy/GSH,and has high selectivity for Cys.Under the optimized conditions,the fluorescence intensity of the probe showed a good linearity with the concentration of Cys in the range of 0-16μmol/L,and the detection limit was 0.095μmol/L.Due to the probe’s high sensitivity,fast fluorescence response（10 min）and low toxicity,it was successfully applied to the imaging of Cys in A549 cells,zebrafish and mouse epidermis.