Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
Anal Chem. 2009 Nov 15;81(22):9267-73. doi: 10.1021/ac901508x.
Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis (muFFE) with online laser-induced fluorescence detection (LIF) is reported. Mitochondrial electrophoretic profiles were acquired in less than 30 s. In comparison to macroscale FFE instruments, muFFE devices consumed approximately 100-fold less sample, used 10-fold less buffer, and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using muFFE were compared to those measured with a capillary electrophoresis instrument with laser-induced fluorescence detection (CE-LIF). There was high similarity between the two distributions with CE-LIF distribution being offset by 1.8 x 10(-4) cm(2) V(-1) s(-1) with respect to the microFFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. In comparison to CE-LIF, analysis of mitochondria using muFFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125 nL) and buffer (250 microL) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make microFFE a very attractive tool for the high-throughput analysis of organelle subpopulations as well as investigating the fundamentals of the electrophoretic mobility of biological particles.
本文报道了使用微流控自由电泳（muFFE）与在线激光诱导荧光检测（LIF）快速、连续地从大鼠成肌细胞中分离线粒体。线粒体的电泳图谱在不到 30 秒内即可获得。与宏观 FFE 仪器相比，muFFE 装置的样品消耗量减少了约 100 倍，缓冲液用量减少了 10 倍，所需的电场强度降低了 15 倍。使用 muFFE 测量的线粒体电泳迁移率分布与使用带有激光诱导荧光检测的毛细管电泳仪（CE-LIF）测量的分布进行了比较。两种分布具有高度相似性，CE-LIF 分布相对于 microFFE 分布偏移了 1.8 x 10(-4) cm(2) V(-1) s(-1)。我们假设这种偏移是由于两种技术中使用的电场强度不同造成的。与 CE-LIF 相比，使用 muFFE 分析线粒体大大缩短了分离时间，所需的分离电压更低，同时保持了低样品（125 nL）和缓冲液（250 μL）体积。这些特点以及为进一步表征收集分离细胞器部分的潜力，使微流控自由电泳成为分析细胞器亚群以及研究生物颗粒电泳迁移率基本原理的非常有吸引力的工具。
