Real-Time Monitoring Of ROS Secreted By Mouse Macrophages Ana-1 By Nanomaterial Sensitized Electrochemiluminescence

Reactive oxygen species（ROS）,including superoxide anion free radicals（O2·^-）,hydroxyl free radicals（·OH）and hydrogen peroxide（H₂O₂）etc.,are closely related to the processes of aging,disease,immune metabolism,and cell signal transduction.Therefore,the real-time quantitative detection of ROS in organisms has important scientific research value.However,to monitor the trace level and short life of ROS secreted from cells,the real-time quantification is a technological challenge.To date,many methods have been attempted,including fluorescence,chemiluminescence,electrochemistry and so on.But these technologies still have some shortcomings,like high cost and cumbersome operation.Therefore,it is of great significance to design and develop a sensitive,convenient,and efficient method.Electrochemiluminescence（ECL）is expected to become a powerful tool for ROS detection due to its high sensitivity,low cost,low detection limit and good temporal and spatial controllability.In this study,two excellent ECL cytosensors based on indium tin oxide（ITO）glass pre-functionalized with gold nanoparticles（AuNPs）or AuNPs/titania nanotubes（TiO₂NTs）nanocomposites were prepared for real-time detecting the ROS released from macrophages stimulated by drugs.The subsequent modification of poly-Llysine（PLL）promoted cell adhesion while further improving the ability to resist disturbance and the biosensing interface’s biocompatibility.In the first part of the study,AuNPs were prepared by citric acid reduction method with a particle size of 18～20nm,and then AuNP-functionalized sensor（Sensor 1）was constructed using PLL.In the second part of the work,TiO₂NTs were prepared by hydrothermal method,and gold nanoparticles（AuNPs）were uniformly loaded around TiO₂NTs by Nafion.AuNPs/TiO₂NTs composite nanomaterials were obtained,then loading PLL and cells for electrochemiluminescence detection（Sensor 2）.For AuNP-functionalized electrodes,the result for the H₂O₂ assay demonstrated a good linear response from 0.01 μM to 0.2 μM and a limit of detection down to 8.9 nM（R²=0.994）.After capitalizing on the synergetic effect of AuNPs and TiO₂NTs,the AuNP/TiO₂NT-based biosensor displayed better sensitivity toward H₂O₂ detection with a linear response from 5 nM to 0.1 μM and a lower limit of detection（LOD）of 2 nM（R²=0.998）.On this basis,we changed the cell density and stimulated cells with different drugs to detect the production of ROS,indicating the good applicability of the biosensors.