Construction Of A Ratio-Type Fluorescent Probe For Heptamethine Cyanines And Its Application In The Analysis Of Biological Enzyme Activity

In recent years,the organic fluorescent probes have attracted much attention due to their high sensitivity and spatiotemporal resolution.They are important tools for biological detection and imaging.Among them,heptamethine cyanine as a typical near-infrared fluorescent dye,is a good candidate for developing a chemical sensor because of its excellent photophysical properties,good biocompatibility,and low toxicity to biological systems.With the help of molecular recognition and specific organic reactions,research on cyanine dyes has flourished over the past few decades.In particular,cyanine-based fluorescent probes have been proven to be highly selective for specific analytes.In this dissertation,we use heptamethine cyanine as a fluorophore to modulate itsconjugated polymethyne?-electronic system by modifying different recognition groups,and design the following three near-infrared ratiometric-fluorescent probes:（1）We introduced a phosphate group on the meso site of Cy-O to obtain a near-infrared ratiometric fluorescent probe Cy-OP with mitochondrial specificity for highly sensitive and selective recognition of alkaline phosphatase（ALP）.Under the treatment of ALP,the probe Cy-OP showed excellent recognition performance,such as high selectivity,good sensitivity（low detection limit as low as 0.16 m U/m L）,short response time（within 30 minutes）,and large spectral shift with low fluorescence background.In addition,the emission ratio of enzyme residues(I_{616 nm}/I_{766 nm})is conducive to its application in complex biological systems.Therefore,we also successfully applied probe Cy-OP to the detection of ALP in living cells.（2）A near-infrared ratiometric fluorescent probe Cy-tyr based on heptamethine cyanine was designed to detect tyrosinase（TYR）activity.When TYR was co-incubated with the probe Cy-tyr,the phenolic hydroxyl group on the probe could be oxidized to o-benzoquinone in the presence of oxygen,and the resulting enzymatic product showed a strong H-aggregation tendency.Therefore,the maximum absorption of this probe shifted from 630 nm to 516 nm,with the reduction of near-infrared emission at 760 nm,and new red fluorescence appeared at 560 nm.The probe has a good signal-to-noise ratio and high sensitivity to TYR activity,and its LOD is 0.02 U·mL^-1.In addition,we successfully applied the probe to the imaging of endogenous TYR activity in B16 cells and other cells with low TYR expression,and the results showed that the probe Cy-tyr had no noise at all in the cells with low TYR expression.（3）Glutathione was modified on cyanine as the gamma-glutamyltransferase（GGT）cleavage site to obtain a near-infrared ratiometric fluorescent probe Cy-GGT for GGT activity detection.The Cy-GGT probe has good water solubility and adding GGT to its aqueous solution can make its fluorescence emission wavelength shift from 808 nm to616 nm（emission shift 192 nm）.The probe shows extremely high sensitivity（LOD of0.02 mU/mL）and selectivity for GGT activity detection,and has been successfully used for detection of GGT activity and fluorescence imaging in living cells and animals.The probe can effectively distinguish tumor cells from normal cells,which is expected to play a positive role in the early diagnosis and treatment of related cancers.