{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T19:53:47Z","timestamp":1773690827916,"version":"3.50.1"},"reference-count":99,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2024,5,25]],"date-time":"2024-05-25T00:00:00Z","timestamp":1716595200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FCT\/MCTES (Portugal)","award":["PTDC\/EQU-EQU\/2223\/2021"],"award-info":[{"award-number":["PTDC\/EQU-EQU\/2223\/2021"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [N1112(OH)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs\u2019 biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.<\/jats:p>","DOI":"10.3390\/ijms25115766","type":"journal-article","created":{"date-parts":[[2024,5,27]],"date-time":"2024-05-27T10:12:27Z","timestamp":1716804747000},"page":"5766","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Towards Enhanced Tunability of Aqueous Biphasic Systems: Furthering the Grasp of Fluorinated Ionic Liquids in the Purification of Proteins"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5751-9933","authenticated-orcid":false,"given":"Sara F.","family":"Carvalho","sequence":"first","affiliation":[{"name":"LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, NOVA University Lisbon, 2829-516 Caparica, Portugal"}]},{"given":"Margarida H.","family":"Cust\u00f3dio","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, NOVA University Lisbon, 2829-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7166-6764","authenticated-orcid":false,"given":"Ana B.","family":"Pereiro","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, NOVA University Lisbon, 2829-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8648-7539","authenticated-orcid":false,"given":"Jo\u00e3o M. M.","family":"Ara\u00fajo","sequence":"additional","affiliation":[{"name":"LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, NOVA University Lisbon, 2829-516 Caparica, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,25]]},"reference":[{"key":"ref_1","unstructured":"Albertsson, P.A. (1986). Partition of Cell Particles and Macromolecules, Wiley."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4966","DOI":"10.1039\/c2cs35151j","article-title":"Aqueous biphasic systems: A boost brought about by using ionic liquids","volume":"41","author":"Freire","year":"2012","journal-title":"Chem. Soc. 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