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Transparent Electrode Materials for Simultaneous Amperometric Detection of Exocytosis and Fluorescence Microscopy. 同时用于胞吐和荧光显微镜安培检测的透明电极材料。
Pub Date : 2012-01-01 DOI: 10.4236/jbnb.2012.322030
Kassandra Kisler, Brian N Kim, Xin Liu, Khajak Berberian, Qinghua Fang, Cherian J Mathai, Shubhra Gangopadhyay, Kevin D Gillis, Manfred Lindau

We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12-17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.

我们已经开发并测试了透明微电极阵列,能够同时对氧化分子进行安培测量,并通过电极进行荧光成像。表面图案微电极由三种不同的导电材料制成:氧化铟锡(ITO),沉积在ITO上的氮掺杂类金刚石(DLC),或玻璃衬底上的非常薄(12-17 nm)金薄膜。将装载溶酶追踪器绿色或吖啶橙染料的染色质细胞置于电极顶部,用全内反射荧光(TIRF)显微镜对囊泡进行荧光成像,同时用表面图案电极测量单个囊泡释放的儿茶酚胺的安培峰值。由这三种材料制成的电极能够以高分辨率检测安培信号。出乎意料的是,ITO电极记录的电流峰值只有DLC或金电极检测到的振幅的一半左右,电荷的一半左右,这表明ITO电极在测量量子儿茶酚胺释放方面不如金电极或DLC电极敏感。在不同的分析物和不同的电极材料存在的情况下,通过计时电流测量证实了ITO电极的低灵敏度。
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引用次数: 41
MEASUREMENT of Protein 53 Diffusion Coefficient in Live HeLa Cells Using Raster Image Correlation Spectroscopy (RICS). 用光栅图像相关光谱(RICS)测定活HeLa细胞中蛋白53的扩散系数。
Pub Date : 2010-10-01 DOI: 10.4236/jbnb.2010.11004
Sungmin Hong, Ying-Nai Wang, Hirohito Yamaguchi, Harinibytaraya Sreenivasappa, Chao-Kai Chou, Pei-Hsiang Tsou, Mien-Chie Hung, Jun Kameoka

We have applied Raster Image Correlation Spectroscopy (RICS) technique to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. HeLa cells expressing Green Fluorescent Protein (GFP) tagged p53 were incubated with and without DNA damaging agents, cisplatin or eptoposide, which are widely used as chemotherapeutic drugs. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. Together, our results demonstrated that RICS is able to detect the protein dynamics which may be associated with protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening.

我们应用光栅图像相关光谱(RICS)技术表征了DNA损伤剂处理前后活细胞中p53蛋白的动态变化。表达绿色荧光蛋白(GFP)标记p53的HeLa细胞与DNA损伤剂、顺铂或表topo苷(广泛用作化疗药物)一起或不一起孵育。然后通过RICS测定GFP-p53的扩散系数,药物治疗后GFP-p53的扩散系数明显降低,而未经药物治疗的GFP-p53的扩散系数则没有明显降低。提示这些药物诱导p53与其他蛋白或DNA相互作用。总之,我们的研究结果表明,RICS能够检测活细胞中可能与蛋白质-蛋白质或蛋白质- dna相互作用相关的蛋白质动力学,并可能对药物筛选有用。
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引用次数: 12
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Journal of Biomaterials and Nanobiotechnology
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