DEVELOPMENT OF A QUARTZ NANOPIPETTE FOR INTRACELLULAR SUPEROXIDE SENSING
Engineering
CAMP/BME 281P
Reactive oxygen species (ROS), including superoxide radical anions are highly reactive oxygen-containing molecules. ROS’s are essential components in numerous biological functions, including cell signaling and immune responses. They are also generated as a natural byproduct in a wide variety of physiological pathways such as the electron transport chain in mitochondria. Because they are so reactive, it is vital that cells have superoxide-scavenging enzymes and other regulating enzymes that can catalyze the dismutation of superoxide radical anions into less damaging molecules in order to achieve ROS homeostasis. Otherwise, ROS overproduction can cause oxidative damage to proteins, cells, and tissues, which are associated with the pathogenesis of a range of neurodegenerative disorders, age-related diseases, and cancer. Understanding the relationship between superoxide and these disorders can help the development of innovative therapies for combating oxidative stress and neural-degeneration in cells. Although methods to quantify ROS already exist, they are indirect, destructive, ambiguous, and/ or incapable of performing real-time measurements in single cells. We have developed a novel technique for sensing superoxide radical anions using functionalized nanopipettes fabricated from single barrel quartz capillaries. The nanopipette undergoes a series of modifications on its inner surface, including the immobilization of cytochrome c, a hemeprotein associated with the electron transport chain in the mitochondria. Cytochrome c allows for the direct measurement of superoxide levels in the cell based on the redox reaction between the iron active site in cytochrome c and the generated superoxide radical anions. These electrochemical biosensors not only enables for the quantitative analysis of superoxide levels in a single cell, but also the monitoring of these levels over a period of time.
