Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection
Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection
复制标题
DOI：
10.1021/ac061652u
复制
发表时间：
2007-01-15
影响因子：
7.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Zhu, Cuiru;He, Xinya;Dovichi, Norman J.
在二维毛细管电泳中，样品在第一维毛细管中通过筛分电泳进行分离。级分周期性地通过界面转移到第二维毛细管中，其中组分通过胶束电动毛细管电泳进一步解析。以前的仪器采用一对毛细管来分析单个样品。我们现在报告一个多路复用系统，允许平行分离五个样品。将样品注入五个第一维度毛细管中，将馏分通过界面转移到5个第二维度毛细管中，并在五毛细管鞘流比色皿中通过激光诱导荧光检测分析物。该仪器产生的检测限为940 +/- 350 yoctomoles的3-（2-呋喃甲酰基）喹啉-2-甲醛标记的胰蛋白酶抑制剂在一维分离;检测限降低了3.8倍的二维分离。采用毛细管筛分电泳（CSE）和胶束电泳毛细管色谱（MECC）技术，从肺癌（A549）细胞制备的匀浆中获得了二维毛细管电泳表达指纹图谱。平均分辨131个斑点，信噪比大于10。将高斯表面拟合到每个电泳图中的一组20个点。以高斯函数的标准偏差表示的平均光斑宽度在CSE维度中为2.3 +/-0.7次转移，在MECC维度中为0.46 +/-0.25秒。光斑位置的标准偏差在CSE维度中为1.8 +/- 1.2次转移，在MECC维度中为0.88 +/- 0.55 s。容量为300。
In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.