Research On Enzyme Immobilized On Magnetic Complex Nanoparticles And Hydrogel Microspheres Aggregates

Numerous biological parameters in the human body are important indicators for the observation,diagnosis and treatment of many diseases.Cholesterol concentration is used as an important reference index for cardiovascular and cerebrovascular diseases.At the same time,rapid and sensitive detection of blood glucose content has important clinical significance for the diagnosis and treatment of diabetic patients.As a new technology,fiber optic biosensing technology is the combination of fiber optic sensing technology and biological sensing technology.Based on its rapid development,fiber optic biosensing technology has attracted wide attention in the many fields including clinical medicine,microelectronics and materials science.Enzyme based fiber optic biosensor is one of the important branches of fiber optic biosensors.Magnetic particles are used to immobilize the biological enzyme,which is beneficial to their enrichment and separation from the reaction system.This immobilized enzyme can be used repeatedly.If the fluorescent indicator is introduced into the magnetic particles,the enzyme catalysis effect can arrive at the fluorescent indicator directly,which will be advantageous to the miniaturization of sensors and improve their response rate and accuracy.With the development of science and technology,the detection of single parameter can not meet the need of practical application,people need to develop two parameters and multi parameters fiber optic biosensors,and one of their key technologies is to develop the new sensing materials which have“on-off”property for enzyme catalysis.IfN-isopropylacrylamide?NIPAAm?isused to prepareintelligent hydrogel microspheres which has the lower critical solution temperature?LCST?of35°Cand the biological enzyme immobilized on fluorescent nanoparticles are combined into the microspheres,the swelling and shrinking process of the microspheres can be controlled by changing temperature.When the hydrogel swells,it presents the network structure.The substance to be measured can pass through the network to contact with the enzyme and the biochemical reaction will take place.When the hydrogel shrinks,the substance to be detected can not contact with enzyme,the reaction is stopped.Therefore,we can realize the intelligent control of detection by changing temperature.The research above will lay a good foundation for the development of multi parameters fiber optic biosensors.The main research results of this thesis are in the following:?1?Themagneticfluorescentcore-shellstructurednanoparticles,Fe₃O₄@SiO₂?F?@meso-SiO₂ nanoparticles,were prepared and characterized.Cholesterol oxidase?COD?was immobilized on their surface to form Fe₃O₄@SiO₂?F?@meso-SiO₂@COD nanoparticles.The optimal immobilization conditions were achieved with APTES concentration of 2.5%?v/v?,GA concentration of 2.0%?v/v?,CODamount of 10 mg in 15 mg carrier and pH of 7.0.The Fe₃O₄@SiO₂?F?@meso-SiO₂@COD nanoparticles showed maximal catalytic activity at pH7.0 and 50°C.The thermal,storage and operational stability of COD were improved greatly after its immobilization.Since Fe₃O₄@SiO₂?F?@meso-SiO₂@COD nanoparticles contained Ru?bpy?₃Cl₂ and were optical sensitive to oxygen in solution,it might be used as the sensing material and has the application potential in multiparameter fiber optic biosensor based on enzyme catalysis and oxygen consumption.?2?The immobilized enzyme-hydrogel microsphere aggregates were prepared and their enzyme catalysis“on-off”property was studied.The optimal synthesis conditions were achieved with NIPAAm amount of 150mg,APS amount of 300?L and BIS amount of 30mg.The immobilized enzyme-hydrogel microsphere aggregates showed maximal catalytic activity at pH7.0 and 30°C.The aggregates might be used as one sensing element for multi parameters fiber optic biosensors.?3?A fiber glucose sensor based on immobilized enzyme-hydrogel microspheres aggregates were designed and fabricated.The linear relationship between the relative phase change and glucose concentration is defined by the equation of y=0.0102+0.000808x and R²=0.99604.The detection range of the sensor is50-700mg/mL,and the response time is 50s,which demonstrates that the detection efficiency of the optical fiber sensing system is good.