Microchip DNA electrophoresis with automated whole-gel scanning detection.
Microchip DNA electrophoresis with automated whole-gel scanning detection.
复制标题
具有自动全凝胶扫描检测功能的微芯片 DNA 电泳。
DOI：
10.1039/b811033f
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发表时间：
2008
期刊：
影响因子：
6.1
通讯作者：
Ugaz,VictorM
中科院分区：
文献类型：
--
作者：
Lo,RogerC;Ugaz,VictorM
凝胶电泳在小型生物分析系统中继续发挥着重要作用，无论是作为一种独立的技术，还是作为集成芯片实验室诊断的关键组成部分。大多数微芯片电泳的实现采用终点线检测方法，其中荧光标记的分析物被观察到，因为它们迁移通过一个固定的检测点附近的分离通道的末端。但是，在同时实现最大分辨率（通常通过使用更长的分离通道来实现）和最大可研究分析物的尺寸范围（其中较短的分离距离减少了最慢的分析物到达检测器所需的时间）之间可能存在权衡。在这里，我们展示了小型化格式如何为采用替代检测方案提供新的机会，这些方案可以通过引入自动化全凝胶扫描检测系统来帮助解决这些问题，该系统可以沿着整个微通道连续监测基于微芯片的DNA凝胶电泳的进展。这使得在分离的早期阶段，在它们经历显著的扩散加宽之前，可以有选择地观察较小的更快的移动碎片，同时允许在运行的后期观察较大的较慢的移动碎片，当它们可以更好地解决时，而不需要它们行进整个分离通道的长度。全凝胶扫描还提供了电泳过程的连续和详细的图像，允许在单个实验中快速准确地测量与DNA迁移现象相关的基本物理参数（例如，迁移率，扩散展宽）。使用终点线方法实现这些功能具有挑战性，并且可以设想一个能够在广泛的凝胶基质材料和操作条件下快速筛选分离性能的平台，甚至允许在单个自校准实验中同时执行分离和基质表征步骤。
Gel electrophoresis continues to play an important role in miniaturized bioanalytical systems, both as a stand alone technique and as a key component of integrated lab-on-a-chip diagnostics. Most implementations of microchip electrophoresis employ finish-line detection methods whereby fluorescently labeled analytes are observed as they migrate past a fixed detection point near the end of the separation channel. But tradeoffs may exist between the simultaneous goals of maximizing resolution (normally achieved by using longer separation channels) and maximizing the size range of analytes that can be studied (where shorter separation distances reduce the time required for the slowest analytes to reach the detector). Here we show how the miniaturized format can offer new opportunities to employ alternative detection schemes that can help address these issues by introducing an automated whole-gel scanning detection system that enables the progress of microchip-based gel electrophoresis of DNA to be continuously monitored along an entire microchannel. This permits flexibility to selectively observe smaller faster moving fragments during the early stages of the separation before they have experienced significant diffusive broadening, while allowing the larger slower moving fragments to be observed later in the run when they can be better resolved but without the need for them to travel the entire length of the separation channel. Whole-gel scanning also provides a continuous and detailed picture of the electrophoresis process as it unfolds, allowing fundamental physical parameters associated with DNA migration phenomena (e.g., mobility, diffusive broadening) to be rapidly and accurately measured in a single experiment. These capabilities are challenging to implement using finish-line methods, and make it possible to envision a platform capable of enabling separation performance to be rapidly screened in a wide range of gel matrix materials and operating conditions, even allowing separation and matrix characterization steps to be performed simultaneously in a single self-calibrating experiment.