Constructions And Applications Of Highly Sensitive Analytical Methods For Tyrosinase And Alkaline Phosphatase Activity

Tyrosinase（TYR）,as a copper-containing oxidase,can catalyze the oxidation of tyrosine or tyrosine to dopamine（DA）,and further oxidize to dopaquinone,thereby triggering the formation of melanin.Melanin plays a key role in the development of melanoma and has an important relationship with the diagnosis of various diseases.In addition,DA,as an important catecholamine neurotransmitter,plays an important role in many tissues of human body.At the same time,alkaline phosphatase（ALP）plays an important role in many cases,and abnormal levels of its enzyme content can cause or predict many diseases.Therefore,it is very important to develop simple,fast and highly sensitive new detection methods for DA,TYR and ALP.In this paper,the fluorescence detection method is used.First,based on the specific reaction of TYR to phenolamines,a catalyst is introduced to accelerate the reaction to construct a simple and sensitive enzyme DA and TYR detection method.Secondly,a new fluorescent probe for TYR detection and a new near-infrared fluorescent probe for simultaneous detection of TYR and ALP activities were constructed.The main research contents are as follows:（1）By using benzoyl peroxide to promote in situ fluorescence reaction between resorcinol and dopamine,a simple,cost-effective,highly selective and sensitive dopamine concentration and tyrosinase activity determination method was established,in which BPO was introduced in the system to accelerate dopamine oxidation,thereby realizing a faster reaction with resorcinol to form the fluorescent substance.This assay enables the measurement of unlabeled fluorescence readings,and the fluorescence intensity signal of the product is basically dependent on the concentration of dopamine and the activity of tyrosinase.In the determination of dopamine concentration,the concentration has a good linear relationship in the range of 4.0 n M to 1.2?M,R²=0.9951,and the detection limit is as low as 0.8 n M,and it was successfully applied to measuring dopamine in human urine samples.In addition,a fluorescent method for determining the tyrosinase activity was further established based on the enzymatic oxidation of tyramine to dopamine,the detection limit is 0.0013 U ml^-1,the method was used to determine tyrosinase in calf serum and screening of tyrosinase inhibitors.（2）Using 3-bromomethylphenol as the recognition group of TYR,it was connected to the fluorescent dye hyprocoumarin through a nucleophilic substitution reaction to construct a new fluorescent probe for TYR detection.Under the catalytic oxidation of TYR,the recognition part of the probe was oxidized to o-benzoquinone,and then a cleavage reaction occurs,the recognition part is removed to liberate the fluorophore,so that the fluorescence of the product is restored,and the enhancement of the fluorescence intensity is directly related to the activity of TYR.In the detection of TYR activity,it has high sensitivity and wide linear range,LOD is 0.2 U ml^-1,and it is applied to the determination of tyrosinase activity in human serum and cell imaging,compared with the standard colorimetric method,it is proved to be feasible and practical.This provides a feasible strategy for the detection of endogenous TYR activity in biological systems.（3）First,the dye methylene blue was used as a fluorophore to react with tyramine to synthesize the dianiline urea bond compound MBOH,and then a near-infrared fluorescent probe MBP containing a phosphate group was synthesized in the presence of POCl₃and H₂O,which was used to simultaneously detect the activity of ALP and TYR.The fluorescence of this probe is quenched due to the phosphate recognition group,after adding ALP and TYR,the enzyme cascade reaction occurs,ALP can first catalyze the cleavage of the phosphate group to the phenolic substance MBOH,and then oxidize it to o-benzoquinone through TYR,the urea bond was further hydrolyzed,and the recognition group was removed to restore fluorescence at 680 nm.ALP and TYR were detected by the change of fluorescence intensity.Under the optimal conditions,the detection of ALP and TYR activity by the probe MBP can realize quantitative analysis of dual-readout（fluorescence and colorimetry）,and has a good linear relationship,a wide linear range,and high sensitivity,the detection of ALP and TYR activity,LOD can be as low as 0.005 U l^-1and 0.005 U ml^-1.