Quantifying Extracellular ROS Levels in Individual Pancreatic Islets using an Optical Sensor and Microfluidic Device

Abstract

The metabolism of pancreatic islet beta-cells is negatively regulated by reactive oxygen species (ROS), which is a toxic byproduct of the electron transport chain and excessive aerobic respiration. During oxidative stress it is evident that ROS levels increase and result in apoptotic signaling. Chronic oxidative stress is associated with beta-cell failure and type 2 diabetes. Oxidative stress increases expression of antioxidant enzymes as a protective mechanism that paradoxically reduces glucose stimulated insulin secretion. To remove ROS, the cell uses superoxide dismutase (SOD) an enzyme that catalyzes the conversion of superoxide into hydrogen peroxide and oxygen. The hydrogen peroxide is then converted into water and oxygen via glutathione peroxidase or catalase. Therefore, both hydrogen peroxide (H2O2) release and molecular oxygen (O2) consumption of islets can serve as proxies to measure conversion rates in the tissue. We have designed a microfluidic device to hold individual pancreatic islets separately in chambers optimized for live cell confocal imaging. This device puts islets very close to the planar slide and also collects islet effluent in a well-defined tail, thus offering us two positions to place a sensor: planar slide and solution. We will first modify the device by introducing a clear fluorescent sensor for oxygen directly beneath the islets. We are currently exploring RuII(bpy)3 [Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate as an optical sensor which has excitation and emission maxima at 450nm, and 600nm, respectively. The ruthenium compound will serve as a seed layer on the base of the device that will increase in fluorescence intensity when oxygen levels decrease. Furthermore, we are exploring putting the dye in solution and using other sensors such as palladium and platinum compounds that react to oxygen. This device will enable toxicity screening of living pancreatic islets and other types of cells.