Synchronous Determination Of ATP And ROS By Employing Sequential Injection Analysis Lab-on-valve Chemiluminescence Technique

Adenosine disodium triphosphate （ATP） is the mediator of energy exchanges, which isan important index reflecting mitochondrial energy generation and utilization. Reactiveoxygen species （ROS） are physiological essential metabolites, but overproduction of ROS candamage cellular components such as proteins, lipids, and DNA. In the fine balancing act ofaerobic metabolism, mitochondrial oxidative phosphorylation accomplishes the reduction ofO₂to H₂O while maximizing ATP synthesis and maintaining ROS production to only theamounts required for microdomain cell signaling. Therefore, simultaneous determination ofATP and ROS are essential for evaluating the physiological functions. Owing to itscharacteristic advantages, e.g., automated sampling and analytical procedures, miniaturization,stability, short analysis time, low consumption of sample and reagents, and high sensitivity,SIA-LOV-CL system has already been a very useful tool in bioanalysis. Based on these, theaim of the present work was to simultaneously detect ATP and ROS with SIA–LOV-CLsystem, and the contents consist with the following parts:1. Development of SIA-LOV-CL method for synchronous determination of ATPand ROS.We firstly explored and optimized the factors influencing the determination of ROS andATP in SIA-LOV system using Luminol-chemiluminescence and Luciferase-bioluminescencerespectively. With a sample consumption of10μL and30μL, ATP and ROS were quantifiedwith absolute detection limit of6.810^-15mol and810^-15mol. When ATP and ROS co-existed,the simultaneous determination for them showed good performance with high sensitivity andselectivity. The RSD values of4.6%at100nmol·L^-1ROS and2.3%at5nmol·L^-1ATP wereachieved.This method could be applied in synchronous detection of ATP and ROS in Chang livercells with satisfactory results. The recoveries were in the range of99%^-104%for thedetermination of ATP, and the recoveries of ROS were in99%^-104%.2. Simultaneously determination of ATP and ROS in plant leavesThis part focused on the plant leaves sample pretreatment，including extraction methodand the elimination of interference. The results showed that TCA could efficiently extractATP and ROS in plant leaves, and the main influence, i.e., polyphenol, could be eliminated by PVP. The value of ATP and ROS in leaves showed the relativity with the growth age: thelevel of ATP in leaves performed agedness <youth <middle age, and ROS level was middleage <youth <agedness.