A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG–Salt Aqueous Two‐Phase Systems for Rapid Screening Applications

“…In contrast, unexpectedly, for the higher DoL of 2.6, the tagged IgG was indeed observed to diffuse for the PEG‐rich phase (>1), despite the still significant difference in magnitude between both scales. Regarding the difference between the nL‐ and mL‐scales, these can be explained by the cross‐contamination of fluorescence between each of the co‐flowing phases at the nL‐scale, as previously demonstrated by Brás et al On the other hand, concerning the unexpected hindered diffusion for the IgG conjugate with the lower DoL, it is hypothesized that without the addition of PEG to improve the solubility of the dye during conjugation, the IgG molecule can be labeled by dye aggregates instead of single molecules of fluorophore (in agreement with the results shown in Figure D). These aggregates of BODIPY dyes were previously reported to have different photochemical properties, exhibiting a red‐shifted photoluminescence at 650 nm in aqueous environments, thus justifying the underestimation of DoL when determined at the expected absorption/emission maximum of the dye.…”

“…When performed in microchannels, ATPE is characterized by shorter partition times due to short diffusion distances, combined with low volumes of reagents and analytes, having the potential of being low cost, disposable, and portable . Recently, several publications demonstrated the use of microfluidic screening tools for the bioseparation of target molecules in ATPE, typically requiring the preconjugation of the target molecule with a fluorophore. However, the effect of the additional conjugation step on the partition, relative to the nonconjugated molecule, has not yet been systematically evaluated.…”

Separation and purification of biomacromolecules based on microfluidics

“…35 Purification of biomacromolecules like therapeutic proteins, antibodies, enzymes and nucleic acids mostly requires several steps of extraction and polishing, which are typically comprising filtration, centrifugation, membrane technologies, and various types of chromatography, hence yielding high downstream processing costs. 36 Liquid-liquid extractions using non-denaturing solvent systems are gaining increased attention as a cost-effective alternative. 36,37 Liquid-liquid microextraction LLE comprises the mass transfer of a solute from the feed, i.e.…”

“…36 Liquid-liquid extractions using non-denaturing solvent systems are gaining increased attention as a cost-effective alternative. 36,37 Liquid-liquid microextraction LLE comprises the mass transfer of a solute from the feed, i.e. a liquid containing the molecule of interest, towards the extraction in a solvent immiscible with the feeding phase.…”

“…41,65 The authors pointed out several advantages for these to be used within a microfluidic device, namely their faster thermodynamic equilibrium, higher polarity difference between the phases and their lower viscosity. Polymer-salt ATPS have been applied on the extraction of BSA, 51,52,55,62 β-galactosidase, 52 GFP, 36,51,52 glutathione S-transferase (GST), 52 genomic DNA, 52 IgG, 36,51,54,66 protein A, 51 insulin, 51 α-amylase 67 and bacteriorhodopsin 53 (summary of all reports available in Table 4). Singh and collaborators 52 were the pioneers to apply polymer-salt ATPS into a microfluidic device.…”