Reactive Oxygen Species Electrochemcial Sensors And Their Application In Cell Biology Studies

Reactive oxygen species (ROS) is a reactive oxygen-containing compound, and a normal product formed during aerobic metabolism. Oxidation and antioxidant enzymes are in dynamic equilibrium state, cellular metabolism is require reactive oxygen species under normal circumstances, such as low concentrations of reactive oxygen species can promote cell growth. But when the body’s imbalance so that increased production of reactive oxygen species can lead to apoptosis, even cause cell damage and cell death. Currently, the method is detection reactive oxygen species often use the traditional biological methods, it is indirect evaluation the level of reactive oxygen species, this method has inadequate the time-consuming and testing costs higher. With the rapid development of biology, biological electrochemical sensor as the earliest and most in-depth is a branch of chemical sensors, and it has advantages of a detection specificity, convenience and low cost. Electrochemical sensors have played a very important significance for analysis and detection of biological molecules, obtain the life processes in the chemical and biological information, understand the relationship between their structure and function of biological molecules and describe the mechanism of life activity for the effective diagnosis and treatment of diseases. Since the cells was produced excessive reactive oxygen species within intracellular, it will be destroy oxidation reduction balance, finally lead to cell apoptosis and death. In this study, we choose the molecule inhibitors of cancer cells (PLX4032) and antioxidants (Tocopherol, Eriodictyol) for reaserch cancer cells release reactive oxygen species. For the first time, we were choose electrochemical biosensor for quantitative detection reactive oxygen species. Electrochemical sensors with high sensitivity, good stability and short time-consuming. The same results was obtained from electrochemical method and traditional fluorescent molecular probes staining. Therefore, reactive oxygen electrochemical sensors was provided a new method for understand mechanisms of cell death and apoptosis induced by oxidative damage, which has a potential application in high-throughput screening of anticancer drugs. The main findings are as follows:1. Involvement of superoxide and nitric oxide in BRAFV600E inhibitor PLX4032-induced growth inhibition of melanoma cellsThe BRAFV600E inhibitor PLX4032 (Vemurafenib) is an FDA-approved new drug for the treatment of metastatic melanomas, which specifically inhibits the RAS/MEK/ERK signaling pathway to control cell proliferation and adhesion. Superoxide (O2-) is the major free radical that contributes to the pathogenesis of many diseases such as Alzheimer’s disease, myocardial infarction and atherosclerosis. Nitric oxide (NO) acts as an important signaling molecule in many physiological and pathological processes. However, no study has been carried out to investigate the role of intracellular oxidative balance in PLX4032-induced tumor growth inhibition. Herein, for the first time, superoxide (O2-) and nitric oxide (NO) generated from PLX4032-challenged melanoma cells were monitored using electrochemical sensors and conventional fluorescein staining techniques. Impacts of superoxide dismutase (SOD) and NG-Monomethyl-Larginine, Monoacetate Salt (L-NMMA), a nitric oxide synthase inhibitor, were also examined to demonstrate the specificity of ROS/NO generation and its biological consequences. PLX4032 specifically triggers production of O2- and NO from BRAFV600E mutant A375 cells. In addition, PLX4032 treatment could decrease the mitochondrial membrane potential in A375BRAFV600E cells. The results suggest that PLX4032 can selectively cause ROS production and depolarization of mitochondrial membranes, potentially initiating apoptosis and growth inhibition of PLX4032-sensitive cells.2. Label-free electrochemical sensor to investigate effect of Tocopherol and Eriodictyol on superoxide generation following UV irradiationUV irradiation stimulated generation of free radical species is associated with skin carcinogenesis. Superoxide anion (O2-) is one of the key radical species that is involved in UV irradiation induced cellular damages. Particularly, the protective efficacy of an unknown anti-oxidant compound can be evaluated by analyzing the production of O2-from treated cells. Vitamin E is a fat-soluble vitamin, a hydrolyzate thereof as tocopherol, is one of the most important antioxidant. In this study, for the first time, a carbon nanotube(CNT)/DNA@Mn3(PO4)2 composite functionalized glass carbon electrode was applied for quantitative determination of O2- generation from melanoma cell A375 following UV irradiation (UVR) i.e., UV, UVA and UVB. In addition, the protective efficacy of the representative anti-oxidants, tocopherol was evaluated by quantifying O2- production in anti-oxidant pre-treated cells. Electrochemical sensors directly quantified O2- induced cathode current changes and the results showed:for melanoma A375 cells, tocopherol is more capable for rebating UVA induced O2-, but it has weak scavenging for UVB induced O2-. The success of label-free electrochemical quantification of O2- from UVR irradiated human skin cells demonstrates its potential application for high-throughput screening of anti-oxidation compound. In summary, we have successfully fabricated label-free electrochemical sensors. The label-free electrochemical quantification of O2- from UVR irradiated human skin cells demonstrates, and it has a significance potential application for high-throughput screening of anti-oxidation compound.