Study On The Construction And Properties Of Tyrosinase Electrochemical Biosensor

Tyrosinase,also known as polyphenol oxidase,is a copper-containing metalloenzyme widely present in animals,plants and microorganisms.It is the main catalyst for melanin formation.It has dual function catalysis.It can not only catalyze the hydroxylation reaction of monophenols or polyphenols without substituents,but also can be used in the electrochemical detection of tyrosinase.In the field of analysis,tyrosinase is often used to prepare electrochemical biosensor to detect a wide range of phenolic compounds based on the mechanism of tyrosinase.Therefore,in the aspects of environmental pollution detection,food ingredient detection and health care testing,the Tyrosinase biosensor has a wide application prospect and commercial application potential,which has aroused wide interest of researchers.Although the researchers have done some research on tyrosinase electrochemical biosensor,there are still some problems in the current tyrosinase electrochemical biosensor,such as inability to deal with complex real component detection,unstable detection performance and so on,which still need to be solved.Based on the defects of the present electrochemical biosensor for tyrosinase,the preparation of biosensor using two materials as the carrier of tyrosinase was studied in this paper:(1)Combined with other previous articles the design of a sensor using magnetic particles and chitosan in combination with Tyrosinase,a novel in-situ deposition of quaternary ferric oxide nanoparticles modified chitosan microspheres immobilized enzyme(Chitosan@Fe3O4)was prepared and the Tyrosinase Biosensor was prepared based on the magnetic glassy carbon electrode.The prepared Chitosan@Fe304 were characterized by scanning electron microscopy,IR and energy dispersive X-ray spectra.Magnetic Chitosan microspheres were determined to be 31.46emu·g-1,and the magnetic electrodes were immobilized.After comparing experiments with the conventional chitosan-coated magnetic-core material(Fe3O4@Chitosan)immobilization enzyme electrode,the electrode junction prepared by Chitosan@Fe3O4 modified Tyrosinase was validated by different electrochemical methods,which promoted the electronic transmission performance and had the-0.05V working potential.The pH,material addition,working voltage and temperature-were optimized so that the detection range of catechol of the biosensor was 2.64 to 84μM,sensitivity was 14.72μA·mM-1,the detection limit was 0.79μM(S/N=3)and Michaelis-Menten constant was 14.3μM.Its stability can be tested for multiple measurements with little relative deviation,and after a half-month preservation test it can still maintain 85%performance.Because of the low operating voltage,for the designed glucose,vitamin C,urea and hydrogen peroxide interferometer test,it is found that the prepared biosensor has good selective performance.Compared with the pure substrate test,the doped solution measured catechol content relative deviation is 1.7%,which is an effective electrochemical design method to reduce the substrate voltage.(2)The use of calcium carbonate nano-crystalline whisker as a template,through the use of dopamine polymerization coated calcium carbonate whiskers,carbonization wash demoulding version of the preparation of nitrogen-doped hollow porous carbon(NHPC).Nitrogen-doped hollow porous carbon of NHPC-1,NHPC-2 and NHPC-3 three different structures were prepared according to the different drilling steps,respectively,and characterized by scanning electron microscope,X-ray diffraction and BET adsorption test.The relationship between the properties of tyrosinase biosensor and material structure was studied by comparing the preparation electrodes of immobilized tyrosinase.By measuring the enzyme activity and electrochemical properties of the three materials after immobilization,it was found that the electrochemical properties of the electrode structure prepared by NHPC-2 combined with tyrosinase were the best,the activity of the enzyme was the highest,the Michaelis-Menten constant was 595μM,and the structural immobilization enzyme of NHPC-2 could promote the best preparation of tyrosinase.The biosensor was then prepared using NHPC-2 fixed tyrosinase for detection of catechol.Under the optimal conditions of determining pH,temperature and operating voltage,the biosensor prepared by NHPC-2 was obtained with a detection limit of 4.05μM(S/N=3),the sensitivity was 4.74μA·mM-1,detection range of catechol of the biosensor was 5.88 to 95.9μM.After 100 cyclic voltammograms,the stability of the sensor was still 96.5%strength.But its preservation performance decreased to 50%with the passage of half a month.The electrode tested the anti-interference performance of the sensor by dropping three electrochemically active substances of vitamin C,uric acid and dopamine,which the results showed that the dopamine is lowest interference and the vitamin C is highest interference.