Preparation And Characterization Of Miniaturized Enzyme Electrode For ROS Detection

Reactive Oxygen Species（ROS）are partially reduced metabolic products of oxygen with highly reducing ability,which participate in regulating biological processes such as growth,development,and differentiation of organisms.Under pathological conditions,increased or accelerated ROS levels can activate other disease pathways.H₂O₂is one form of ROS in the body.Quantitative analysis of H₂O₂can help us understand the concentration and duration of ROS production at specific locations in vivo and explain various biochemical reaction mechanisms.Currently,methods for detecting H₂O₂include spectroscopy,chromatography,and titration,which are difficult to achieve in situ detection within living organisms.Microelectrodes have small size,high sensitivity and mass transfer efficiency,and can be implanted to achieve real-time detection in living organisms.Therefore,it is necessary to develop microelectrodes that can specifically and quantitatively detect H₂O₂.Among the many microelectrodes used for H₂O₂detection,microenzyme electrodes can achieve specific recognition of substrate in complex environments.However,poor enzyme conductivity and deep-buried active centers within the enzyme molecule make it difficult for the electrons generated by the enzyme reaction effectively are transfered to the electrode surface and converted into a quantifiable signal in the form of output current.In this study,a system was designed enhance the efficient of electron transfer between the Catalase（CAT）and the surface of miniaturized platinum electrode to improve the detection ability of the microenzyme electrode for H₂O₂.Compared with the development of a series of miniaturized Catalase electrodes,continuous monitoring of H₂O₂content in animals was ultimately achieved.The specific research contents are as follows:（1）Based on the excellent conductivity and adsorption properties of carbon nanotubes MWCNTs,a miniaturized Catalase electrode was constructed using MWCNTs as an electron transport medium and an effective and stable method for preparing miniaturized enzyme electrodes was developed.It was found that the HNO₃/H₂O₂treatment system greatly improved the dispersion of MWCNTs and increased the surface carboxyl group content to 3.26%,laying a foundation for covalent binding to the electrode surface.The amount of immobilized Catalase on the MWCNTs reached 0.48 g/g,and the activity recovery rate increased to 46%.In the electrode performance test,the miniaturized Catalase electrode had a fast electron transfer efficiency,a detection range from 0.02 to 8 m M,a low detection limit of 10.2μM,a response time of 1.8 s,a sensitivity of 215μA m M^-1cm^-2,good stability,and a decrease in current of only 1.5%after 100 h.（2）Based on the excellent conductivity and biocompatibility of polyaniline（PANI）,as well as the good conductivity and high specific surface area of MWCNTs and gold nanoparticles（Au NP）,a miniaturized Catalase electrode was constructed using PANI/MWCNTs/Au NP as the electronic transmission medium.A uniform PANI network can be formed by scanning aniline in an electrolyte containing carbon nanotubes for 8 cycles at a CV of-0.2-1.0 V at a rate of 20 mv/s;MWCNTs with a concentration of 0.4 mg/m L could form a uniform network structure and meet the optimal sensitivity and noise level.In the performance test,the PANI/MWCNTs/Au NP/CAT-Pt had a wide linear detection range from 0.25μM to 200 m M,a low detection limit of10 n M,a response time of 2 s,and a sensitivity of 784.3μA m M^-1cm^-2.Recovery rate of sample addition was between 96%and 102%.Based on its excellent detection ability,an effective method for detecting trace amounts of H₂O₂in biological systems was created.（3）The miniaturized Catalase electrode was constructed using polypyrrole as the electronic transmission medium through various polymerization methods based on the excellent conductivity of polypyrrole in neutral p H.The surface microstructure and detection performance of enzyme electrode were optimized by adjusting the electrochemical reaction parameters in the process of pyrrole polymerization.The polymerization of pyrrole and the doping of Catalase occur simultaneously in the Catalase electrode prepared by one-step polymerization,resulting in a unified structure where spherical enzyme protein nanoparticles were coated on the surface of the conductive material polypyrrole.In the electrode performance test,the electrode showed excellent detection ability for H₂O₂with a linear detection range of 0.025-350m M,a detection limit of 2μM,a response time of only 3.6 s,and a sensitivity of 230.5μA m M^-1cm^-2.Recovery rate of sample addition was between 98%and 102%,and the stability was excellent,with a decrease in current of only about 2%after 100 hours.A stable miniaturized enzyme electrode preparation method was developed.（4）Based on a miniaturized Catalase enzyme electrode constructed with PANI/MWCNTs/Au NP as electron transfer mediators,a miniaturized three-electrode system was prepared to achieve quantitative detection and real-time analysis of H₂O₂in animal body fluids and tissues.The miniaturized three-electrode system don’t require pretreatment for detecting H₂O₂content in rat blood,and the detection is convenient,with a detection value of 2.32μM（RSD=7.37%）.In the detection of H₂O₂content in rat liver tissues,the detected H₂O₂content was approximately 8-13.4μM.This system can track the changes in H₂O₂content during the infusion of trace amounts of exogenous H₂O₂,and monitor the dynamic level of H₂O₂in real-time.The H₂O₂content calibrated by the enzyme electrode system is consistent with the actual H₂O₂content.This study provides a feasible method to construct an efficient electron transfer system between enzyme molecules and electrode surfaces,enabling in situ detection of H₂O₂in biological systems,which is helpful for understanding the content and variation of H₂O₂in organism tissues and organs.