Synthesis And Application Of Fluorescent Probes For Detection Of Alkaline Phosphatase

Alkaline phosphatase(ALP)is the most common and crucial subclass,as it regulates biofunctions in the biogeochemical cycle of phosphorus.ALP not only facilitates the hydrolysis of monoesters of phosphoric acid but also participates in transphosphorylation reaction in the presence of high concentrations of phosphate acceptors.ALP behaves important roles in protein cyclic metabolism of phosphorylation and dephosphorylation in cells.It is well documented that the increase of endogenous ALP activity is closely related to the proliferation and differentiation of osteoblasts and bone mineralization.Moreover,accumulating clinical manifestations suggest that the aberrant elevation of serum ALP level and activity is associated with many diseases,such as breast cancer,prostatic cancer,liver dysfunctions,intestinal diseases,bone diseases,and diabetes.Alkaline phosphatase(ALP)is one kind of enzyme widely distributed in bodies of water,and it has the capability of nonspecifically catalyzing the phosphoryl esters hydrolysis,making the organic phosphorus bioavailable for phytoplankton.Large amounts of data in literature indicated that,the dissolved reactive phosphorus(DRP)is closely associated with water body eutrophication.When DRP was in deficiency states,phytoplankton induced the increase of ALP to change organophosphorus into DRP;on the contrary,the high-level DRP inhibited ALP expression rapidly.With the fast development of social economy,the heavy metals and other pollutants from the human are input into the marine environment,causing persistent and harmful effects on all kinds of animals and plants and microorganisms in the ocean.Large amounts of data in literature indicated that various metal ions have different effects on the activity of ALP.Reduction of primary productivity of seawater under heavy metal pollution is needed to be understood.We report two fluorescent probes for the detection of alkaline phosphatase activity based on phosphate groups as response site.It is not only applied to various cells and mouse models,but also investigated the correlation between ALP activity of seawater samples and the occurrence of red tide.The main work of this paper is as follows:(1)Based on the development of published fluorescent probes for detecting ALP activity,we designed a near infrared fluorescent probe QcyP to detect ALP activity,and explained the response mechanism of ALP detection.The photophysical properties of the probe QcyP,enzyme kinetics,and the stability and selectivity of the probe and the potential quantitative ability of the probe under different pH values were also investigated.(2)The bioimaging of probes in cells was investigated by confocal microscopy.The results showed that the probe has the function of locating mitochondria,and it can be used as a tool for detecting ALP in mitochondria.After that,the levels of endogenous ALP activity of different cells were quantitatively analyzed and sorted,and the reliability of the method was proved by flow cytometry and Western blotting.The ALP activity of HepG2 tumor bearing mice was evaluated by small animal imaging system based on the near infrared fluorescence characteristics of the probe.The results showed that the activity of ALP in serum and tumor region of the HepG2 tumor bearing mice was much higher than that of the normal and inhibitor mice.(3)Based on the near infrared characteristics of the probe,the QcyP was applied to the sea water of the red tide,and the relationship between the red tide and the activity of ALP in the sea water was analyzed.(4)Inspired by the above work,in order to exclude the interference of acid phosphatase,we designed and synthesized a highly selective fluorescence probe CyP based on pH value for detecting ALP activity,and explained the response mechanism of ALP detection.The photophysical properties of the probe CyP,enzyme kinetics,the stability under different pH values,selectivity of the probe and the potential quantitative ability of the probe are investigated.The proteins in Chlorella were extracted,and the effects of various metal ions on Chlorella and Escherichia coli ALP activity were analyzed.