Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.
Bioelectrochemistry. 2019 Dec;130:107325. doi: 10.1016/j.bioelechem.2019.06.010. Epub 2019 Jun 28.
In order to understand how energy metabolism adapts to changes in neuronal activity it is imperative to perform direct measurements of the flux of glucose (and other metabolites) in brain tissue. Metabolic studies using brain slice preparations are attractive due to the controllability of recording conditions, absence of anesthetic interference and refined animal experimental protocols. In this work, taking advantage of the small size and versatility of carbon fiber microelectrodes (CFMs), we aimed to develop an amperometric glucose microbiosensor suitable for glucose measurement in brain slices. Potentiostatic- or galvanostatic-driven platinum electrodeposition was used to improve the analytical properties of CFMs towards detection of hydrogen peroxide. The platinized CFMs served as platform for the development of glucose microbiosensors through the immobilization of glucose-oxidase (GOx) by cross-linking with glutaraldehyde in the presence of BSA. Selective glucose measurements were attained by modifying the electrode with a permselective layer of meta-phenylenediamine and by integrating a null sensor. The in vitro characterization studies support the good analytical features of the CFM/Pt-based microbiosensors to reliably measure glucose in brain tissue. The ex vivo experiments in rodent hippocampal slices validated their suitability to measure evoked changes in extracellular glucose. This approach, encompassing the use of null sensor to cross-check the selectivity on a moment-to-moment basis, allowed us to provide the temporal and quantitative profile of extracellular glucose changes in hippocampal slices following a spreading depolarization event. Overall, these results support the potential of these microbiosensors to be used as a valuable tool to investigate the complex nature of glucose utilization in brain tissue linked to neuronal activation both in physiological and pathological conditions.
为了了解能量代谢如何适应神经元活动的变化，必须对脑组织中葡萄糖（和其他代谢物）的通量进行直接测量。由于记录条件可控、没有麻醉干扰以及动物实验方案得到了改进，因此使用脑片制备进行代谢研究具有吸引力。在这项工作中，我们利用碳纤维微电极 (CFMs) 的体积小和多功能性的优势，旨在开发一种适用于脑切片中葡萄糖测量的安培葡萄糖微生物传感器。恒电位或恒电流驱动的铂电沉积用于改善 CFMs 对过氧化氢检测的分析性能。铂化 CFMs 作为平台，通过在 BSA 存在下与戊二醛交联固定葡萄糖氧化酶 (GOx)，开发了葡萄糖微生物传感器。通过用间苯二胺的选择性层对电极进行修饰，并集成一个零传感器，实现了选择性葡萄糖测量。体外特性研究支持基于 CFM/Pt 的微生物传感器可靠测量脑组织中葡萄糖的良好分析特性。在啮齿动物海马切片的离体实验中，验证了它们适合测量细胞外葡萄糖的诱发性变化。这种方法包括使用零传感器来实时检查选择性，使我们能够提供海马切片中细胞外葡萄糖变化的时间和定量特征，以响应扩展去极化事件。总的来说，这些结果支持这些微生物传感器有潜力成为研究与神经元激活相关的脑组织中葡萄糖利用的复杂性质的有价值工具，无论是在生理还是病理条件下。
