A Tyrosinase Responsive Near-infrared “Turn-on” Fluorescent Probe And Its Application In Melanoma Imaging

Melanoma is a highly malignant and fast-evolving cutaneous cancer,early detection of melanoma via analyzing the biomarker level in vivo decreases the likelihood of reoccurrence and mortality.Moreover,tyrosinase is a cytoplasmic melanocyte differentiation protein that causes the formation of melanin by mediating tyrosine to some of the quinones.Because of its overexpression in melanoma cells,tyrosinase used as an independent biomarker for melanoma diagnosis and prognosis.This paper designs and synthesizes a tyrosinase responsive near-infrared“Turn-on”fluorescent probe.We have synthesized a kind of near-infrared fluorescent dyes 9-(bis(3-methoxy-3-oxopropyl)amino)-5H-benzo[?]phenoxazin-5-iminium.The fluorescent probe uses the modified Nile Blue for fluorescent groups.First of all,the structure of amino through urea linkage connects 4-aminophenol which is the substrate of tyrosinase.Because of intramolecular charge transfer,urea linkage quenches the fluorescence of the modified Nile Blue.In the presence of tyrosinase,the fluorescent probe molecules trigger group is oxidized to diphenol,then a series of electron transfer and hydrolysis reaction follows.At last it releases the modified Nile blue to restore fluorescence,so as to achieve the role of imaging.The main conclusions are as follows:(1)We confirmed the chemical structure of the intermediate products and the final product through ~1H NMR and HR-MS.(2)The ultraviolet visible light absorption spectrum and fluorescence spectrum tests show that the fluorescence probe has good responsiveness to tyrosinase.The reaction of fluorescent probe with tyrosinase is dependent on time and concentration.As the reaction time increases,the fluorescent intensity increases gradually and reaches saturation after 60 min.The concentration of fluorescent intensity and tyrosinase is linear in low concentration.Moreover,the reaction of fluorescent probe with tyrosinase is stable under simulated physiological condition,and the fluorescent probe has specific selectivity to tyrosinase and is not affected by other substances.In the presence of tyrosinase,the fluorescent probe fractures urea linkage and releases fluorescent dyes to restore fluorescence.(3)The fluorescent probe has almost no cytotoxicity on B16F10 cells that are overexpressed tyrosinase,indicating that the biocompatibility of the fluorescent probe that we designed and synthesized is better.It has time and concentration dependence to detect cellular tyrosinase,and the low fluorescence of HeLa cells further proves that tyrosinase can effectively activate fluorescence probe to restore fluorescence.Our fluorescent probe can effectively image tyrosinase in the cells.(4)The fluorescent probe permeates into zebrafish mainly through the skin and digestive tract,mediated by endogenous tyrosinase oxidation reaction,and can detect zebrafish endogenous tyrosinase activity in the body.(5)The fluorescent probe selectively detect melanoma by imaging overexpression tyrosinase in melanoma.The fluorescent probe can image early melanoma through detection of tyrosinase activity,and can track the transfer of melanoma.Among them,lung is the main transfer target of malignant melanoma.In summary,we have designed and synthesized a tyrosinase responsive near-infrared“Turn-on”fluorescent probe.It could detect the activity of tyrosinase,and provides a new method for the early diagnosis of melanoma.