Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands
Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands
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DOI：
10.1021/acs.analchem.5b04449
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发表时间：
2016-08-02
影响因子：
7.4
通讯作者：
Carabelli, Valentina
中科院分区：
文献类型：
--
作者：
Picollo, Federico;Battiato, Alfio;Carabelli, Valentina
将微结构化石墨4 × 4多电极阵列嵌入单晶金刚石基底（4 × 4 μ G-SCD MEA）中，用于实时监测培养的嗜铬细胞和肾上腺切片的胞吐事件。目前的方法依赖于平行离子束光刻技术的发展，这保证了扩展阵列的时间有效的制造，具有可再现的电极尺寸。所报道的设备适合以高灵敏度和时间分辨率进行电流法和伏安法记录，通过同时从由200 μ m间隙隔开的16个矩形微电极（20 x 3.5 μ m（2））获取数据。利用阵列的几何形状，我们解决了以下具体问题：（0检测自发和KCl诱发的分泌同时从几个嗜铬细胞直接培养在设备表面上，（ii）解决的波形的不同子集的胞吐事件，和（iii）从肾上腺薄片监测量子分泌事件。自发释放的频率是低的（0.12和0.3 Hz，分别为肾上腺片和培养的细胞），并增加到0.9 Hz刺激后，与30 mM KCl在培养的细胞。尖峰振幅以及上升和衰减时间与碳纤维微电极测量的那些相当，并且允许识别与“完全融合”事件、“吻-跑”和“吻-留”胞吐相关的分泌事件的三个不同子集，确认该装置具有用于实时记录的足够的灵敏度和时间分辨率。该设备提供了显著的优势，通过允许同时记录原代细胞培养物或完整组织中的细胞群来缩短收集数据的时间。
A microstructured graphitic 4 x 4 multi electrode array was embedded in a single-crystal diamond substrate (4 x 4 mu G-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time-effective fabrication of extended arrays, with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20 x 3.5 mu m(2)) separated by 200 mu m gaps. Taking advantage of the array geometry we addressed the following specific issues: (0 detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, (ii) resolve the waveform of different subsets of exocytotic events, and (iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 and 0.3 Hz, respectively, for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated with "full fusion" events, "kiss-and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues.