Construction Of Reactive Small Molecule Fluorescence Probes For Biomarker Detection

Early detection,diagnosis,and treatment are crucial for effectively managing serious diseases,including diabetes and cancer.Abnormal expression of biomolecules such as reactive sulfur,reactive oxygen species,proteins,and enzymes make them useful biomarkers for specific diseases.Currently,clinical biomarker monitoring methods include magnetic resonance imaging（MRI）,nuclear imaging（including positron emission tomography（PET）and single photon emission computed tomography（SPECT））,and fluorescence imaging（FI）.However,MRI lacks sufficient specificity and sensitivity for biomarkers with low abundance.Moreover,even though nuclear imaging has high sensitivity,their poor spatial resolution and high cost limit their effectiveness.In contrast,the fluorescence detection method offers high sensitivity,low detection limit,non-invasiveness,and high imaging contrast,enabling in-situ nondestructive detection and real-time imaging in micro-nano space.Therefore,developing biomarker-responsive fluorescent probes has become a research hotspot.Based on the above background,my doctoral works mainly focused on the construction of fluorescent small molecular probes and their applications in imaging and monitoring biomarkers in cells and mice.The main contents are as follows:1.A novel turn-off fluorescence probe,ML-NAP-DPPEA,was proposed for highly selective imaging of peroxynitrite（ONOO^-）produced byexogenous and endogenous stimuli in living cell lysosomes.The probe ML-NAP-DPPEA was based on the naphthyl imide fluorophore connected with aminomorpholine and 2-（diphenylphosphine）ethylamine group.ML-NAP-DPPEA had excellent properties of high selectivity,low cytotoxicity,low detection limit（17.6 n M）,and non-interference to ONOO^-.With the addition of ONOO-,the secondary amine（NH）in the probe ML-NAP-DPPA was oxidized to the electron-absorbing group（HN→O）,thus quenching the fluorescence of ML-NAP-DPPA.The success of intracellular lysosome imaging indicated that the probe can be used to further study the biological function and pathological effect of ONOO^-in subcellular structure.2.A new near-infrared emissivity ratio fluorescent probe DCM-PA(I_{640 nm}/I_564nm)was designed and synthesized to detect the activity of Vanin-1 in vitro and in vivo.DCM-PA had the advantages of a short response time（30 minutes）,high selectivity,and low sensitivity（DL=0.69 ng/m L）.In addition,DCM-PA had been applied to imaging cells and mice,and the results showed that the probe DCM-PA had an undeniable potential in monitoring Vanin-1 activity in situ,which was conducive to further study the role of Vanin-1 in physiology and pathology.To our best knowledge,this probe DCM-PA may be the first near-infrared ratio(I_{640 nm}/I_564nm)fluorescence probe reported to monitor the activity of Vanin-1 in vivo.In addition,hunger-induced up-regulation of this enzyme confirmed the inevitable relationship between diabetes and abnormal expression of Vanin-1.3.A near-infrared（NIR）ratio emission fluorescence probe TMN-PA was developed for the detection of Vanin-1 activity in living cells and the fluorescence imaging of up-regulated expression of Vanin-1 caused by hunger in mice.TMN-PA can quickly（within 15 minutes）respond to Vanin-1,and the detection limit was 0.37ng/m L.In addition,we first studied the Michaelis constant Km of Vanin-1 catalytic probe hydrolysis and explored the relationship between different probe Km and their performance（detection time,minimum detection limit）.For Vanin-1,the smaller the Km of the probe,the greater the affinity between the probe and the enzyme,and the better the performance of the probe.4.The turn-on fluorescence probe BODIPY-vinylfuran of H₂S was synthesized by the reaction of 8-methyl-BODIPY with the functional group 5methylfuraldehyde.In the presence of H₂S,the furan ring was hydrolyzed and opened,and the nucleophilic addition reaction between H₂S and the carbon-carbon double bond of vinyl furan further occured,enhancing the fluorescence.Due to the additional reaction of the carbon-carbon double bond with H₂S,theπ-πconjugate structure of furan and BODIPY was destroyed,resulting in the maximum emission peak shift from 535 nm to 496 nm The sensor BODIPY-vinylfuran showed a fluorescence intensity increase of more than 150 times,a low detection limit（99.4n M）,and excellent performance for H₂S specific recognition.In addition,BODIPY-vinylfuran had low cytotoxicity and good biocompatibility.Cellular fluorescence imaging further proved that BODIPY-vinylfuran had high responsiveness to both endogenous and exogenous H₂S,and can be used to recognise cancer cells CT-26.5.The first near-infrared ratio emission fluorescence probe TMN-Hexs was designed and synthesized to detect Hexs activity in vitro and in vivo.The probe TMN-Hexs had the ability to quickly and specifically recognize Hexs with a detection limit of 0.0046 U/m L.Additionly,the probe TMN-Hexs had the advantage of a large Stokes shift（approximately 200 nm）.The probe TMN-Hexs was successfully applied to distinguish common cells from colon cancer cells CT-26.