Chem. SCI.: fluorescent benzochalcone probe for ultra micro specific real-time imaging of the dynamic changes of neutrophils in zebrafish

Neutrophils are the key components of mammalian nonspecific immune response, which have the functions of digestion, phagocytosis and sterilization There are a lot of neutrophils in their cytoplasm These neutrophils are closely related to the physiological function and growth and development of neutrophils, so they are concerned by researchers However, up to now, there is no specific fluorescent living dye to monitor the dynamics of neutrophil particles in vivo Recently, Romain Duval, the fifth University of Paris and Emma Colucci Guyon, the Institut Pasteur of France, designed and synthesized a series of benzochalcone fluorescent probes based on the research goal of monitoring the dynamic changes of neutrophil particles HAB was identified as the first small molecule tracer of neutrophil particles in the larva of grouper through phenotype screening The dynamic changes of HAB stained neutrophils during phagocytosis were also recorded Relevant results were published in Chem SCI (DOI: 10.1039/c8sc05593a) As we all know, the organic fluorescence group is usually a push-pull electron system By extending the conjugation system, the molecules can have the ability of effective intramolecular charge transfer in excited state Chalcone itself has no fluorescence properties The electron donor aromatic ring (b) forms a conjugated structure with alkene ketone, and the arylcarbonyl group (a) acts as an electron withdrawing group in chalcone structure The author selected 3-aminobenzochalcone as the best fluorescence group to construct the fluorescence molecule, and expected that the molecule could show the maximum fluorescence intensity and red shift effect (Figure 1) (source: chem SCI.) the author first synthesized a series of fluorescence probes 3-8, 12 and 14 (Figure 2a) based on the structure of chalcone By analyzing the optical properties of probe 3 - 8, we found that probe 8 has the highest fluorescence quantum yield (Figure 2b) Compared with probes 12 and 14, the fluorescence quantum yield of probe 8 is significantly increased, which is due to the strong electron donor group of naphthalene ring, which can modify the fluorescence group in chalcone structure, and the carbonyl group has a permanent alkenolation effect when the conjugation mode is extended Therefore, the fluorescence of probe 8 is stronger than probe 12, and the quantum yield is increased by 20 times (Figure 3) Based on the good photophysical properties of probe 8, the author named it HAB and used it in the subsequent bioassay (source: chem SCI.) (source: chem SCI.), the author then studied the solvent effect of HAB (Figure 4) HAB and its unsubstituted analogues 6 showed significant solvation and color rendering, almost no fluorescence in water, biological buffer and alcohol, moderate fluorescence in polar aprotic solvent (such as ethyl acetate), and strong fluorescence in nonpolar solvent (such as toluene) (Figure 4A and 4B) With the increase of BSA concentration, the red shift of fluorescence spectrum and the fluorescence enhancement at 578 nm (Figure 4d-f) were observed The authors conjecture that, although the fluorescence of HAB is weak in the biological buffer, the fluorescence of HAB increases obviously after it connects with the target protein in the biological system The results showed that the fluorescence of HAB cytosol and free form membrane was weak or negligible in the cell chamber, and strong when the target combined (source: chem SCI.) in order to verify that HAB can mark the neutrophil particles in zebrafish embryo and larva, the author has carried out in vivo imaging experiment of zebrafish The results showed that there was weak fluorescence in the ventral tail region of zebrafish embryo after fertilization for 32H With the growth of zebrafish, the fluorescence increased gradually, because macrophages and neutrophils also grew (Figure 5) Based on the above experimental results, the authors speculate that HAB can label these two types of cells and accumulate in the cells as the cells grow (source: chem SCI.) the author further explored the labeling effect of HAB on neutrophils In this paper, we constructed a transgenic zebrafish larva model containing mcurry protein, which was mainly enriched in macrophages and showed red fluorescence After treated with HAB, the green fluorescence of HAB did not overlap with the red fluorescence of mcurry protein; in contrast, the red fluorescence of mcurry protein and HAB were mainly enriched in neutrophils The results showed that HAB could differentiate cell types and mainly label neutrophils (Figure 6a-l) Subsequently, the author further confirmed the labeling effect of HAB on neutrophils by means of DIC microscope, and the neutrophils are constantly moving in the cytoplasm (Figure 6m-p) (source: chem SCI.) next, the dynamic changes of neutrophils in phagocytosis were studied by HAB We found that under the phagocytic effect of yeast polysaccharide, HAB labeled neutrophils gathered in the phagosome containing particles (Figure 7), which is similar to the movement of Sudan black B (sb) labeled neutrophils containing myeloperoxidase HAB targets serine protease in neutrophils through reversible electrophilic inhibition Finally, HAB was applied to the staining of peripheral blood in vivo, and it was found that HAB had good selectivity to neutrophils of lymphocytes, monocytes, erythrocytes and platelets (Figure 9) (source: chem SCI.) (source: chem SCI.): in this paper, the author designed and synthesized a probe HAB based on benzochalcone, and applied it to monitor the dynamic changes of neutrophil particles in living neutrophils, providing a new monitoring method for the research of human physiology and pathology.