Small-molecule Fluorescent Probe With High Sensitivity For Detecting Reactive Sulfur And Its Application In Biochemical Analysis

Cysteine?Cys?,homocysteine?Hcy?and glutathione?GSH?are important small-molecule biothiols in human body,and participate in many physiological and pathological activities.Their intracellular level is closely related to human health,which is hence considered as an important parameter of physical health.Therefore,it is of vital importance to make an accurate evaluation for them,and there have been many reports on fluorescent probes for biothiol detection so far.However,most probes are difficult to effectively distinguish between Cys,Hcy,GSH or other reactive sulfur substances?RSSs?,and their emission wavelength is mainly located in the ultraviolet region with short Stokes shift?<100 nm?.Therefore,it remains tough to elaborate probes with supreme selectivity for target thiols avoiding any potential interference from other RSSs.For example,Cys is an important thiol-containing amino acid and is closely related to cell growth.Cysteine of an abnormal content in body could indicate a variety of diseases.It is important to design fluorescent probes for the selective detection and imaging of intracellular Cys.Most of the reported fluorescent probes for Cys could be affected by RSSs such as Hcy and GSH.Under the aforementioned considerations,this thesis focuses on a series of issues such as selectivity,intrinsic background,and improvement on the probe structures.The following work was thereby carried out:?1?In order to improve the selectivity to Cys,a new fluorescent probe?ABT-MVK?based on ESIPT process was designed and synthesized.This probe utilizes a typical ESIPT dye?HBT?as a fluorophore,an acrylate group as an ESIPT blocker and a Cys recognition unit.Upon the sensing process,Cys can quickly?especially in aqueous buffer?cleave the acrylate,so that the ESIPT process and the fluoresence is restored,giving rise to a large Stokes shift of?225 nm,much larger than the reported literatures.This probe has excellent selectivity for Cys,with little interference from other RSSs?including Hcy,GSH,Na₂S or NaHSO₃?.Through the concentration linear curve,the detection limit of this probe for Cys is calculated to be19 nM,which is relatively low.It has been successfully applied to the biological imaging of Cys in living cells,indicating its great potential in physiological study.In order to improve the detection sensitivity and reduce the fluorescence background interference,we designed and synthesized a hydrogen bond-driven fluorescent probe?DEASA?based on the dual mechanism of ESIPT and ICT coupling for detection of Cys.The acrylate group,an ESIPT blocking agent,is introduced as a recognition unit for Cys.The double bond on the probe could readily react with Cys,followed by a rapid cyclization and recovery of the ESIPT process with an significant enhancement in fluorescence.This probe has little response to the other RSSs,enabling specific detection for Cys.The detection limit was calculated to be 36.9 nM through the concentration linear curve.Due to the good biocompatibility,it has been successfully applied to the fluorescence imaging of Cys in Sk-Br-3 cells,capable of monitoring the change of Cys in Sk-Br-3 cells in real time.?3?In order to reduce the inherent background and enhance the output signal to improve the detection sensitivity,we selected an organic framework without a fluorescence chromophore under the detection condition,which combined with the analytes to generate a new fluorescent group.The micelles formed by surfactants can provide an amphiphilic surface to enhance the reaction between hydrophilic and hydrophobic substrates.The hydrophobic cavity can not only protect the fluorescent substance but also amplify the fluorescent reponse.Therefore,free background and enhanced output signal will become an important factor to improve the detection sensitivity.We designed a zero-background fluorescent probe?SA?for the detection of GSH.SA contains two reaction sites,?,?-unsaturated carbonyl units and aldehyde groups to combine with GSH,and CTAB micelles could accelerate the cyclization of SA and GSH,and enhance the fluorescence signal of the luminescent product.The detection scheme has high selectivity and sensitivity for GSH in aqueous buffer,and the good detection performance for SA makes it suitable for fluorescence imaging of GSH in live cells,animal tissue sections at different depths,and quantitative detection in food.It provides a new detection tool for GSH by fluorophore-free probes.