Synthesis And Application Of Colorimetric And Fluorescent Probes For Detection Of Bioactive Small Molecule

Bioactive small molecules include reactive oxygen species,reactive nitrogen species,reactive sulfur species,reactive aldehydes and biothiols.Abnormal activity of small molecules in living system can lead to oxidative stress damage,which will eventually lead to various diseases such as diabetes,cancer,Alzheimer's disease and Parkinson's disease.Therefore,real-time monitoring of the distribution and dynamic changes of bioactive small molecules in living system can help us explore the mechanism of disease occurrence,which is of great significance for the early prevention and diagnosis of related diseases.However,active small molecules are highly reactive,unstable and it is difficult to be detected in complex environment.Therefore,it is urgent to design and synthesize probes with high sensitivity,high selectivity and fast response.Based on this,we designed and synthesized five different probes for the detection of H₂O₂,biothiol,cysteine?Cys?,NO and methylglyoxal?MGO?.Details of this paper are as follows:?1?Preparation of functionalized WS₂ nanocomposite for colorimetric detection of H₂O₂,GSH and GlucoseWe prepared a two-dimensional water-soluble WS₂-hemin nanosheet with one step sonication-assisted exfoliation method.The successful modification of hemin on WS₂nanosheet was confirmed by absorption spectroscopy,transmission electron microscopy,Fourier transform infrared spectroscopy and Raman spectroscopy.The content of hemin in the WS₂ nanosheet was about 18.9 wt%as confirmed by thermogravimetric analysis?TGA?.This nanocomposite possessed highly intrinsic peroxidase-like activity which can quickly catalyze oxidation the substrate3,3',5,5'-tetramethybenzichne?TMB?in the presence of H₂O₂ to produce a blue-colored solution at room temperature in 10 min.Furthermore,the nanosheet exhibited excellent catalytic activity over a wide range of temperature and pH.We further confirmed the catalytic mechanism that was due to the generation of hydroxyl radicals??OH?by using terephthalic acid as a probe.Based on the above principle,the nanosheet was employed to sensitively detect H₂O₂ and glucose.In addition,reduced substances such as GSH and Cys could reduce H₂O₂ and Ox-TMB,leading to bleaching of solution.Nanosheets was also used to sensitive detection of GSH and Cys.Finally,WS₂-hemin nanosheet was successfully used as a colorimetric biosensor for sensitive detection of glucose in FBS.?2?Synthesis and application of ratiometric fluorescent probe for rapid and sensitive detection of biothiolsWe designed and synthesized a rhodamine-coumarin acryloyl ester fluorescent probe RHA by amidation reaction for sensitive detection of biothiols.The probe consisted of two parts:one was Rhodamine B serving as a fluorescence reference,and the other was coumarin derivative as the responsive fluorophore with an acrylate group for biothiols recognition.Acryloyl ester could quench the fluorescence of coumarin HC by photoinduced electron transfer?PET?process and recognize biothiols by Michael addition and intramolecular cyclization reactions.The probe showed good ratiometric and sensitive response to biothiols,especially to cysteine?Cys?.The linear range was 1 to 10?M and the detection limit of this probe for cysteine was found to be 0.13??.More importantly,the probe showed rapid response to Cys within 10 min.The ratiometric fluorescent probe has been successfully applied for the determination of biothiols in fetal bovine serum samples and the result was in good agreement with that tested by Ellman method.?3?Synthesis and application of ratiometric fluorescent nanoprobe for highly selective detection of cysteineCys plays an important role in many physiological processes,including protein synthesis,cell redox balance and so on.However,it is difficult to discriminate Cys from homocysteine?Hcy?and glutathione?GSH?,due to their similar chemical structures and reactivity.Herein,we have developed a polymeric nanoprobe nanoHFA by self-assembly method,for ratiometric,highly selective,and sensitive detection of Cys based on reference unit 7-hydroxycoumarin-3-carboxylic acid?HC?and recognitionmoietyfluoresceinisothiocyanate?FITC?-acrylate?FITC-A?group-functionalized lipopolymer DSPE-PEG.The average size of nanoparticle was about 18 nm,which was determined by dynamic light scattering?DLS?and transmission electron microscopy?TEM?.The nanoprobe displayed excellent dispersibility,photostability and high selectivity to Cys over other biothiols.The nanoprobe has been successfully used to detection of Cys with a wide range of 5-80?M and the detection limit was 0.38?M.Moreover,we further used probe nanoHFA to detect Cu²⁺ions by the coordination of Cys and Cu²⁺.Finally,we developed a fluorescent strips for facile and visual detection of Cys and Cu²⁺respectively.The nanoprobe was successfully applied for the quantitative detection of Cys in fetal bovine serum and the test result was satisfactory.?4?A lysosome-targeted fluorescence probe for imaging nitric oxide in cells and tissue slices.In order to accurately monitor the concentration change of NO in lysosomes,we designed and synthesized a new chemotype probe MBTD by o-phenylenediamino?OPD?moiety as a NO-specific recognition unit and benzo-bis-thiadiazole as fluorescence unit,for fluorescence imaging of lysosomal NO.Compared with commercial probe Lysotracker Red,the morpholino group of the probe has good targeting to lysosome and the colocalization coefficient is 0.99.MBTD displayed high selectivity and sensitivity to NO and the detection limit was 14 nM.In addition,compared with commercial NO probe DAF-FM-DA,MBTD exhibited good photostability,the fluorescence remained stable for 6 min during continuous irradiation at 488 nm.MBTD has been successfully employed for fluorescence imaging of the NO in MCF-7 and HepG2 cells.In addition,reaction product possessed the property of pH response and was a potential pH imaging probe.More importantly,MBTD was able to detect the variation of lysosomal NO level in an acute liver injury mouse model under stimulation of LPS.In the presence of the NO synthetase inhibitor Ng-nitro-L-arginine methyl ester?L-NAME?,the concentration of NO was significantly reduced,and on the other hand,after the inflammatory mice were treated with the inflammatory drug nitrogen acetylcysteine?NAC?,the MBTD probe monitored treatment effect of NAC in situ by detecting changes in NO.We believe that MBTD probe provided a new method for early detection of NO-related diseases.?5?Near-infrared fluorescent probe for imaging MGO in vitro and in vivoMethylglyoxal?MGO?,as a highly reactive?-dicarbonyls,abnormal level of MGO is related to various diseases including Type 2 diabetes mellitus,neurodegenerative diseases,and cancers.However,fluorescence probe in near-infrared region for sensitive detection of MGO was still lacking at present.We designed and synthesized a near infrared?NIR?fluorescent probes based on o-phenylenediamine?OPD?as MGO recognition unit and benzothiadiazole as fluorescent group for real time and in situ imaging of MGO in vitro and in vivo.In the presence of MGO,probe MEBTD displayed greatly enhanced fluorescence in the range of 550 nm to 850 nm,implying the probe has deep tissue penetration,low fluorescence background,which effictively reduced the interference of auto-absorption and autofluorescence of living organisms.MEBTD has been successfully used for fluorescence imaging of endogenous and exogenous MGO in 4T1 cells.Moreover,the probe could distinguish the difference of GLO1 activity in different tumor cells and normal cells in the presence of glyoxalase inhibitors.We found 4T1 cells and MDA-MB-231 cells possess higher content of GLO1 by fluorescence imaging,flow cytometry and western blot analysis.Finally,MEBTD was used for endogenous MGO imaging of tumors in vivo and in tissue slices.These results indicated that the probe provided a new imaging tool for the diagnosis and prevention of MGO-related diseases.