Design, Synthesis Of Novel Near-infrared Fluorescent Probes Based On Silicon-rhodamine And Their Application In Biological Imaging

In recent years,fluorescent probes have become a hotspot in the field of analytical chemistry,which are powerful tools for observing cell morphology and studying cell physiological or pathological processes.Fluorescence imaging based on fluorescent probe has the advantages of high sensitivity,high spatial and temporal resolution,non-destructive sample preparation and in situ real-time detection,which can meet the needs of cell biological analysis and detection.However,most of fluorescent probes reported usually have short-wavelength excitation and emission(?_ex<600 nm and?_em<650 nm).These probes have limited practical applications,which suffered from a variety of problems,including small penetration depth of biological samples,photobleaching phenomenon,phototoxicity to biosamples,and interference from autofluorescence in vivo.Near-infrared fluorescent probes with long-wavelength excitation and emission wavelengths(?_ex>600 nm and?_em>650 nm),can solve the above problems,which can minimize photodamage to biological samples,increase tissue penetration depth,and minimize interference from background autofluorescence to obtain excellent imaging performance in vivo.Among them,cyanine dye is the most widely used.However,photostability of it is poor,which is difficult to meet the needs of long-term imaging and obtaining stable fluorescence signals.Si-rhodamine,with an excitation wavelength at more than 600 nm and an emission wavelength at about 680 nm,is a kind of near-infrared fluorescent dyes with excellent photophysical properties,which has been widely used in fluorescence detection and imaging.Especially,compared with the traditional near-infrared cyanine dye,Si-rhodamine has an excellent photostability,which is in favor of obtaining stable and persistent fluorescence signals in long-term fluorescence imaging process.For these reasons,two near-infrared fluorescent probes based on Si-rhodamine were designed andsynthesized for respectively imaging hypochlorous acid and pH in lysosome.The contents are as follows:?1?In the second chapter,a lysosome-targetable near-infrared fluorescent probe,Lyso-NIR-HClO,was synthesized for the first time for hypochlorous acid.Lyso-NIR-HClO has a high sensitivity and selectivity to hypochlorous acid,with a linear range from 5.0×10^-8 to 1.0×10^-5M and a detection limit of2.0×10^-8M in vitro,which has been successfully applied to real-time detection of HClO in MPO/H₂O₂/Cl^-system.Meanwhile,Lyso-NIR-HClO showed excellent imaging performance,such as low background fluorescence interference,stable and persistent fluorescence signal,and large depth of tissue imaging.In addition,Lyso-NIR-HClO has been successfully applied for imaging of endogenous and exogenous HClO in lysosome.Last,we successfully applied the probe to visualize endogenous HClO during lysosome-involved inflammatory response including bacteria-infected cells and inflamed mouse model,which proved that Lyso-NIR-HClO may be expanded to monitoring of molecular events associated with inflammation and study on role of lysosome in the inflammatory response.?2?In the third chapter,a lysosome-targetable near infrared pH fluorescence probe based onSi-rhodamine,Lyso-NIR-pH,was designed and synthesized,which has been successfully applied for dynamic monitoring of pH in lysosome.Si-rhodamine and morpholine groups were respectively choose as the fluorophore and the lysosome-targetable group of the probe Lyso-NIR-pH.Lyso-NIR-pH has specific a selectivity and a good reversibility for pH.In the response range of pH 3-7,the fluorescence intensity of Lyso-NIR-pH at 675 nm increased by more than 1400 times,and the pKa value was 4.63.At the same time,Lyso-NIR-pH has an excellent lysosome-targetable ability and a photostability,which can meet the needs of long-term dynamic monitoring of pH in lysosome.Finally,Lyso-NIR-pH has been successfully applied to real-time monitoring of the changes of lysosomal pH induced by chloroquine and apoptosis.These results indicate the practical application value of Lyso-NIR-pH in biological system.