Preparation Of Magnetic Metal Nano-mimic Enzyme And Its Detection Of Dopamine

Dopamine（DA）is an important neurotransmitter in the brain.Numerous diseases occur as a result of abnormal dopamine levels in the brain.Thus,accurate and fast detection of DA content in blood will be useful to the diagnosis of clinical diseases.Methods of DA detection mainly include HPLC-electrochemical method,capillary electropHoresis method and so on.Although these methods can achieve sensitive detection of DA,most of them are deficient in expensive instruments and equipment,high cost,long analysis time and tedious operation process.Hence,research on a novel,low-cost and simple detection of DA concentration has important application value for physiological function research and clinical diagnosis.ObjectiveLook for a method that is selective,convenient,and quick to detect dopamine.In order to improve the dopamine existing detection methods,there are disadvantages such as poor selectivity,complicated operation,and high equipment requirements.Methods1.The magnetic metal nanoparticle mimic enzyme Fe₃O₄@MnO₂ was prepared by hydrothermal synthesis method.And the nanoparticles were characterized by IR,TEM,SEM and elemental analysis.2.To explore the oxidation activity of magnetic metal Nano-mimic enzyme Fe₃O₄@MnO₂ with different oxidation substrates{3,3’,5,5’-Tetramethylbenzidine TMB,2,2’-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ABTS）},including the optimal temperature,pH,and buffer and other conditions.3.To investigate the relationship between the containment of quantitative magnetic Fe₃O₄@MnO₂ nanoparticles mimic enzyme activity and DA concentration under optimized temperature,pH,with TMB and ABTS as oxidation substrate.The linear range and detection limit of DA were determined by linear regression equation.4.The linear range of dopamine was determined by HPLC method,and the content of dopamine injection was determined by the experimental method and HPLC method to verify the reliability of the method.5.The experimental method was used to determine the amount of dopamine secreted by PC12 cells（rat adrenal medullary pheochromocytoma）and the amount of dopamine in human blood.Results1.The Fe₃O₄@MnO₂ was successfully prepared and was characterized by IR,TEM,SEM and elemental analysis.2.The high activity of Nano-mimetic enzyme was confirmed by TMB ABTS,When ABTS as the oxidizing substrate,Fe₃O₄@MnO₂ oxidation optimum temperature of 80℃,the optimal buffer is citrate buffer,the optimum pH 2.0.Taking TMB as the oxidation substrate,Fe₃O₄@MnO₂ oxidation of optimum temperature of 40℃,the optimal buffer is acetic acid sodium acetate buffer,the optimum pH 4.5.3.When ABTS as the oxidized substrates,the linear range of dopamine detected was5-125nM and the detection limit was 5nM.When TMB as the oxidized substrates,the linear range of dopamine detected was 40-900nM and the detection limit was 40nM.4.The HPLC method measures the linear range of dopamine at 10-200μg/ml.The concentration of 0.025mg/ml dopamine injection was determined by HPLC and experimental methods.The concentration of 0.0273mg/ml dopamine injection was determined by HPLC,and the concentration of 0.0205mg/ml was determined by experimental methods.p<0.01,the results were statistically significant.5.The amount of dopamine secreted by PC12 cells and the content of dopamine in human blood were successfully detected by this method.The average single cell secreted0.5 fmol of dopamine,and the concentration of dopamine in the blood was about 30 nM.The results were consistent with those reported in the literature.ConclusionIn this study,the magnetic metal nanoparticle mimic enzyme Fe₃O₄@MnO₂ was successfully prepared.The magnetic metal nano-mimetic enzyme was proved to have high enzymatic activity by using ABTS and TMB as oxidized substrates,and the optimal pH value,the optimal buffer and the optimal temperature of Fe₃O₄@MnO₂ oxidation substrate were explored.The linear range and detection limit of dopamine detection with different oxidative substrates were successfully determined,and the feasibility and reliability of the method were confirmed by comparison with HPLC.By this method,the amount of dopamine secreted by PC12 cells and the content of dopamine in human blood were successfully detected.Compared with the traditional detection method,the method has the advantages of low cost and good feasibility,and can be better applied to the rapid detection of dopamine in clinical blood samples.