title: Determination of partition coefficients of biomolecules in a microfluidic aqueous two phase system platform using fluorescence microscopy.
abstract: 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.
authors.affiliation: 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.
authors.forename: D F C
authors.initials: DF
authors.lastname: Silva
authors.affiliation: 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.
authors.forename: A M
authors.initials: AM
authors.lastname: Azevedo
authors.affiliation: 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.
authors.forename: P
authors.initials: P
authors.lastname: Fernandes
authors.affiliation: Instituto de Engenharia de Sistemas E Computadores, Microsistemas e Nanotecnologias (INESC MN) and IN, Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.
authors.forename: V
authors.initials: V
authors.lastname: Chu
authors.affiliation: Instituto de Engenharia de Sistemas E Computadores, Microsistemas e Nanotecnologias (INESC MN) and IN, Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: J P
authors.initials: JP
authors.lastname: Conde
authors.affiliation: 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. Electronic address: rabarros@ist.utl.pt.
authors.forename: M R
authors.initials: MR
authors.lastname: Aires-Barros
mesh_terms: D007526:Isoelectric Point; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D014867:Water
chemical_list: D011108:Polymers; D014867:Water; D011092:Polyethylene Glycols
keywords: Aqueous two phase systems; Biomolecule separation; Microfluidic device; Partition coefficients