SERS Probe For The Detection Of Reactive Oxygen Species Produced In Cells By Electrostimulus

Electrostimulus（ES）is an important therapeutic method for diseases caused by abnormal intracellular electrical activity.At the same time,electrostimulus can induce apoptosis of cells,which may be a way of tumor treatment.At present,there are no relevant studies on whether changes in intracellular reactive oxygen species（ROS）levels can be induced in the process of electrostimulus,and what effects will be brought by the simultaneous application of conventional antioxidant S and electrostimulus therapy.Bioelectric field and current exist widely in cells and play an important role in regulating various biological processes.Disorders in the electrical activity of cells can cause various diseases,such as epilepsy,Parkinson’s disease,heart disease and so on.Electrostimulus（ES）has become an effective treatment for regulating disrupted electrical activity.Compared with drug therapy,ES is a fast and controlled method,which can avoid the disadvantages of chemical drugs,that is,the longer action time and uncontrollable side effects caused by exogenous substances entering cells.There is no relevant literature to discuss whether there is a dynamic change in ROS production in cells during the process of electrostimulus leading to apoptosis.In addition,in the treatment of ES,whether adjuvant therapy with some antioxidant drugs will interfere with tumor treatment also requires in-situ evaluation of ROS responsive probes.Surface-enhanced Raman spectroscopy（SERS）has been widely used in cell analysis due to its unique fingerprint information,high sensitivity,simple operation and almost nondestructive to biological samples.The Raman signal of ROS is weak,so indirect SERS technology can be used to detect ROS.one is based on the chemical reaction between Raman reporter molecules（such as p-aminophenylthiophenol and 4-mercaptoboric acid）and ROS,resulting in the change of Raman signal for sensing;The other is a regulation method based on SERS physical enhancement mechanism,that is,a strategy of etching silver shells by ROS and thinning the silver shells to weaken SERS signals.Due to the short lifespan,low physiological concentration and high reactivity of ROS.Therefore,sensitivity is critical.In order to detect and quantify reactive oxygen species,SERS technology and electrochemical technology were combined to design core-shell probes to study ROS in the electrochemical process.The main research contents are as follows:（1）A m PEG-modified core-shell probe was designed and used to detect ROS produced during electrostimulus.Its sensing mechanism is to use ROS to etch the silver shell and make the silver shell thin,resulting in the SERS signal weakening and forming ROS turn-off detection method.The probe was co-incubated with cells and was internalized into cells.The cells were then electrostimulus stimulated for different periods of time;Finally,the signal of SERS probe was detected by Raman spectroscopy.The experimental results showed that SERS signal weakened with the extension of electric stimulation time,indicating that the amount of ROS produced in cells increased significantly.In addition,two antioxidation inhibitors,ascorbic acid（AA）and glutathione（GSH）,were studied when combined with electrical stimulation.It was observed that they could eliminate ROS produced by electrical stimulation.This study developed a spectrometric method for intracellular ROS detection and suggests a combination of drugs for abnormal oxidative stress and tumor therapy.（2）A core-shell probe with organelle targeting was designed and used to detect ROS produced during electrostimulus.Its sensing mechanism is to use ROS to etch the silver shell and make the silver shell thin,resulting in the SERS signal weakening and forming ROS turn-off detection method.We demonstrated the organelle targeting of the probe using FITC modified targeted peptide fluorescence kit,and detected the ROS produced in the organelle during the electrostimulus process.The experimental results showed that SERS signal weakened with the extension of electrostimulus time,indicating that the amount of ROS produced in cells increased significantly.It revealed that ROS would be generated in cells during electrical stimulation,which has great potential for exploring the changes caused by abnormal organelle microenvironment in the process of electrostimulus by exploring organelle targeting probes combined with SERS technology.