A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux
A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux
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DOI：
10.1016/j.jneumeth.2010.03.001
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发表时间：
2010-05-30
影响因子：
3
通讯作者：
Porterfield, D. M.
中科院分区：
文献类型：
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作者：
McLamore, E. S.;Mohanty, S.;Porterfield, D. M.
由于缺乏足够的工具来直接监测生理条件下的生物活性通量，神经递质运输动力学的量化受到阻碍。研究神经递质释放/摄取的传统技术需要从非选择性电记录中推断，具有侵入性/破坏性，和/或时间分辨率较差。电化学生物传感器的最新进展增强了生理/病理生理条件下神经递质浓度的体外和体内检测。酶促生物传感器与性能增强材料（如碳纳米管）的使用一直是许多这些进步的主要焦点。然而，由于相对较低的灵敏度、过多的漂移/噪声、低信噪比以及无法量化突触传递过程中的快速神经化学动力学，这些技术并未被用作主流神经科学研究工具。一种被称为自我参照的传感技术克服了许多这些问题，并允许对生物物理运输进行非侵入性量化。这项工作提出了一种自参考碳纳米管修饰谷氨酸氧化酶生物传感器，用于监测神经/神经元细胞附近的谷氨酸通量。基础谷氨酸的浓度与其他体内和体外测量值相似。该生物传感器采用自参考（振荡）模式测量电刺激时神经细胞附近的净谷氨酸通量。增产前，平均内流为33.9±6.4 fmol cm(-2) s（-1）。刺激后立即发生谷氨酸外排，并在50-150 fmol cm(-2) s（-1）范围内摄取谷氨酸。三- β -苯氧天冬氨酸抑制了摄取，复制细胞的平均表面通量(1.1 +/- 7.4 fmol cm（-2) s(-1)）显著低于未抑制的细胞。该技术对于研究动态生理条件下与神经传递相关的神经病理状况具有重要价值。(C) 2010 Elsevier B.V.版权所有
Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average influx was 33.9 +/- 6.4 fmol cm(-2) s(-1)). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50-150 fmol cm(-2) s(-1) range. Uptake was inhibited using threo-beta-benzyloxyaspartate, and average surface flux in replicate cells (1.1 +/- 7.4 fmol cm(-2) s(-1)) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions. (C) 2010 Elsevier B.V. All rights reserved.