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Objective: The overarching goal of this project is the development of nanoscale sensors capable of monitoring single cell communication by specifically detecting ATP as it is released from astrocyte cells. The methodology we develop here should be able to transcend from single cell measurements to in vivo measurements and should be adaptable to a wide range of biomolecular targets.

Over the past decade, adenosine triphosphate (ATP) has emerged as an important signaling molecule particularly in the central nervous system providing possible functions related to information processing, memory formation, sleep homeostasis, gene expression and neurological disorders. There has been recent evidence suggesting that astrocytes – non-neuronal glial cells – play a vital role in intercellular communication in the nervous system via the release of ATP as well as glutamate and D-serine. Several questions still remain, however, about ATP signaling from astrocytes such as 1) what is the general release mechanism of ATP, 2) what activities control this release and finally, 3) what is the role of ATP signaling in vivo. Currently, there are no good methods to directly measure ATP release and furthermore the translation of these methodologies to the in vivo setting has been left unmet. As such, we set out to develop the analytical methodology to address ATP signaling from astrocytes in experimental conditions ranging from cultured single cells to conditions in the central nervous system of a living animal. Achievement of this goal provides an analytical methodology to probe the physiological role of ATP and how this changes in respond to various disease states.

Electrochemical aptamer-based (E-AB) sensors, employing oligonucleotide aptamers that bind specific targets, represent a new class of biosensor that can specifically detect a wide range of biomolecular targets regardless of their intrinsic activity. For example, E-AB sensors have been described against a number of targets including small molecules, proteins and inorganic ions. Sensor signaling is predicated on specific target binding induced changes in the conformation or flexibility of the electrode-bound, redox-tagged DNA aptamer. These changes thus change the efficiency with which electrons can transfer between the redox tag and electrode and can be measure voltammetrically. As such E-AB sensors are reagentless and reusable and are capable of detecting targets in complex sample matrices including whole blood and cellular lysates. Coupling the promising attributes of E-AB sensors with nanometer-scale electrodes, we will develop sensors that will achieve the sensitivity and resolution (both spatial and temporal) needed to specifically detect ATP release from single astrocyte cells.