Silva D F C, Azevedo A M, Fernandes P, Chu V, Conde J P, Aires-Barros M R
Instituto de Engenharia de Sistemas E Computadores, Microsistemas e Nanotecnologias (INESC MN) and IN, Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; iBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; iBB, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
J Chromatogr A. 2017 Mar 3;1487:242-247. doi: 10.1016/j.chroma.2016.12.036. Epub 2016 Dec 15.
Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in molecular solubility in each of the two immiscible phases. However, ATPS use has been limited due to the difficulty in predicting the behavior of a given biomolecule in the partition environment together with the empirical and time-consuming techniques that are used for the determination of partition and extraction parameters. In this work, a fast and novel technique based on a microfluidic platform and using fluorescence microscopy was developed to determine the partition coefficients of biomolecules in different ATPS. This method consists of using a microfluidic device with a single microchannel and three inlets. In two of the inlets, solutions containing the ATPS forming components were loaded while the third inlet was fed with the FITC tagged biomolecule of interest prepared in milli-Q water. Using fluorescence microscopy, it was possible to follow the location of the FITC-tagged biomolecule and, by simply varying the pumping rates of the solutions, to quickly test a wide variety of ATPS compositions. The ATPS system is allowed 4min for stabilization and fluorescence micrographs are used to determine the partition coefficient.The partition coefficients obtained were shown to be consistent with results from macroscale ATPS partition. This process allows for faster screening of partition coefficients using only a few microliters of material for each ATPS composition and is amenable to automation. The partitioning behavior of several biomolecules with molecular weights (MW) ranging from 5.8 to 150kDa, and isoelectric points (pI) ranging from 4.7 to 6.4 was investigated, as well as the effect of the molecular weight of the polymer ATPS component.
双水相系统（ATPS）通过探索各种生物分子在两个不混溶相中的分子溶解度差异，为其选择性分离提供了巨大潜力。然而，由于难以预测给定生物分子在分配环境中的行为，以及用于确定分配和萃取参数的经验性和耗时技术，ATPS的应用受到了限制。在这项工作中，开发了一种基于微流控平台并使用荧光显微镜的快速新颖技术，以确定生物分子在不同ATPS中的分配系数。该方法包括使用具有单个微通道和三个入口的微流控装置。在其中两个入口中，加载含有形成ATPS的组分的溶液，而第三个入口则注入用超纯水制备的带有异硫氰酸荧光素（FITC）标记的目标生物分子。使用荧光显微镜，可以追踪FITC标记的生物分子的位置，并且通过简单地改变溶液的泵送速率，可以快速测试各种ATPS组成。ATPS系统有4分钟的稳定时间，然后使用荧光显微照片来确定分配系数。所获得的分配系数与宏观ATPS分配的结果一致。这个过程允许仅使用几微升材料对每种ATPS组成更快地筛选分配系数，并且适合自动化操作。研究了几种分子量（MW）范围为5.8至150kDa、等电点（pI）范围为4.7至6.4的生物分子的分配行为，以及聚合物ATPS组分分子量的影响。
