Real time monitoring of glutamate flux in neurons by using a self referencing carbon nanotube based biosensor
Abstract
Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. Traditional measurement techniques for studying glutamate release and uptake are limited to concentration measurements (as opposed to flux), require inferences from pharmacological manipulation, or suffer from poor temporal and spatial resolution. In this work we present the construction, evaluation and application of a novel CNT modified glutamate microsensor to detect glutamate flux around neuronal clusters. Characterization of the sensor performance such as detection limit, response time, stability, linear range of operation and sensitivity was performed. We further investigated the underlying electrochemistry behind CNT based sensing and low potential detection of peroxide. The microsensor was finally operated in an advanced sensing modality called self-referencing mode that enabled selective monitoring of glutamate flux. The self-referencing technique converts a static concentration sensor into dynamic flux sensor by vibrating between two positions within the analyte gradient, thereby providing direct quantification of direction and magnitude of transport. The biosensor measured the local glutamate release (efflux) and subsequent uptake (influx) of glutamate near small clusters of neurons and astrocytes in culture following potassium-evoked stimulation. Efflux of glutamate took place immediately following stimulation, and was always followed by a net influx. This uptake was blocked by the inhibitor, threo-β-benzyloxyaspartate (TBOA), which blocks excitatory amino acid transporters (EAAT), indicating that the observed influx was due to classic glutamate uptake mechanisms. To our knowledge this is the first direct observation of temporally resolved glutamate efflux and influx from small numbers of neural cells in culture.
Degree
M.S.B.M.E.
Advisors
Rickus, Purdue University.
Subject Area
Neurosciences|Analytical chemistry|Biomedical engineering
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