Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made possible the measuring of cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor are coated with a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.
Bibliographical notePublisher Copyright:
© 2015 Journal of Visualized Experiments.
- Issue 104
- Molecular biology
- Purinergic signaling