Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips.

Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of phi X174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 microns) have been explored. By use of channels with an effective length of only 3.5 cm, separations of phi X174 Hae II DNA fragments from approximately 70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQ alpha alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips. IMAGES:

本研究采用光刻掩模与化学刻蚀技术，在平面玻璃基板上制备毛细管电泳阵列（capillary electrophoresis arrays）芯片。首先在玻璃基板上刻蚀出光刻定义的通道图案，随后将刻蚀后的基板与另一块玻片热压键合，从而形成毛细管通道。利用该芯片通道内填充的羟乙基纤维素（hydroxyethyl cellulose）筛分基质，可对φX174 Hae III DNA限制性酶切片段实现高分辨率电泳分离。DNA片段在电泳运行缓冲液中通过染料完成荧光标记，并通过激光激发式共聚焦荧光检测系统完成信号采集与检测。本研究系统探究了电场强度变化、进样操作流程，以及分离与进样通道尺寸（范围30至120微米）对分离效果的影响。当采用有效长度仅为3.5厘米的通道时，对长度约70至1000碱基对的φX174 Hae II DNA片段的分离仅需120秒即可完成。本研究同时实现了聚合酶链式反应（PCR）扩增的人类白细胞抗原DQα（HLA-DQ alpha）等位基因的高速片段长度分析。该项工作确立了毛细管阵列电泳芯片上实现高速、高通量DNA分离的技术方法。图像：