Hahn Thomas, O'Sullivan Ciara K, Drese Klaus S
Fluidics and Simulation, Institut für Mikrotechnik Mainz GmbH, Carl Zeiss Strasse 18-20, 55129 Mainz, Germany.
Anal Chem. 2009 Apr 15;81(8):2904-11. doi: 10.1021/ac801923d.
In electrokinetic trapping (EKT), the electroosmotic velocity of a buffer solution in one area of a microfluidic device opposes the electrophoretic velocity of the analyte in a second area, resulting in transport of DNA to a location where the electrophoretic and electroosmotic velocities are equal and opposite and DNA concentrates at charged nanochannels. The method does not require an optical plug localization, a considerable advantage as compared to preconcentration techniques previously presented. In the work reported here, the trapping process is preceded by a field-amplification in the sample reservoir to reduce trapping time, as field-amplified EKT is shown to be an effective technique to preconcentrate samples from larger volumes. A theoretical model explaining the principle of field-amplified EKT considers different ionic strengths and cross-sectional areas in the microchip segments. The model is supported by experimental data using nucleic acids and fluorescein as sample analytes. An incorporated poly(ethylene terephthalate) (PET) membrane provides anion exclusion due to a negatively charged surface. A fluidic counter flow supports the trapping process in 100 nm pores due to anion exclusion. An analysis of Joule heating gives evidence that temperature gradient focusing effects are negligible and charge exclusion is responsible for trapping. The theoretical model developed and experimentally demonstrated can be exploited for the preconcentration of cell free fetal DNA circulating in maternal plasma and other rare nucleic acid species present in large sample volumes.
在电动捕获（EKT）中，微流控装置一个区域内缓冲溶液的电渗速度与另一个区域内分析物的电泳速度相反，导致DNA传输到电泳速度和电渗速度大小相等、方向相反的位置，并且DNA在带电纳米通道处浓缩。该方法不需要光学塞定位，与之前提出的预浓缩技术相比，这是一个相当大的优势。在本文报道的工作中，捕获过程之前在样品池中进行场放大以减少捕获时间，因为场放大EKT被证明是一种从更大体积样品中预浓缩样品的有效技术。一个解释场放大EKT原理的理论模型考虑了微芯片各段中不同的离子强度和横截面积。该模型得到了使用核酸和荧光素作为样品分析物的实验数据的支持。内置的聚对苯二甲酸乙二酯（PET）膜由于表面带负电而提供阴离子排斥作用。由于阴离子排斥，流体逆流支持在100nm孔隙中的捕获过程。对焦耳热的分析表明温度梯度聚焦效应可忽略不计，电荷排斥是捕获的原因。所开发并通过实验证明的理论模型可用于预浓缩母血中循环的游离胎儿DNA以及大量样品中存在的其他稀有核酸种类。
