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However, their isolation and purification from complex biofluids remain technically challenging. Conventional methods, such as ultracentrifugation, suffer from low yield, long processing times, and contamination risks, while commercial isolation kits often lack scalability, offer limited purity, and are generally costly. Here, we present the development of a microfluidic platform, termed HIC-on-a-chip, which integrates miniaturized hydrophobic interaction chromatography (HIC) for the rapid, small-volume isolation and purification of EV-sized particles. This device enables improved separation of EVs from contaminating components through controlled hydrophobic interactions. Proof-of-concept experiments using conditioned media from MDA-MB-231 metastatic breast cancer cells demonstrated that the HIC-on-a-chip platform showed improved enrichment of small EV-sized subpopulations compared with a membrane-affinity commercial kit, which are often challenging to recover with conventional approaches. The platform achieved high apparent overall EV recovery, favorable selectivity, reduced processing time, and low sample-volume requirements while maintaining effective purification performance. While comprehensive biological validation and testing with complex clinical biofluids remain subjects for future work, these results underscore the potential of the HIC-on-a-chip as a promising alternative for EVs isolation and purification. Its versatility positions it as a valuable tool for future clinical and research applications, including cancer diagnostics, therapeutic monitoring, and biomarker discovery, with potential for integration into point-of-care workflows.<\/jats:p>","DOI":"10.1021\/acsabm.6c00841","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T19:02:00Z","timestamp":1788894120000},"page":"8540-8554","source":"Crossref","is-referenced-by-count":0,"title":["Hydrophobic Interaction Chromatography-on-a-Chip for Extracellular Vesicle Isolation and Purification"],"prefix":"10.1021","volume":"9","author":[{"given":"Diogo C.","family":"Fernandes","sequence":"first","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catarina M.","family":"Abreu","sequence":"additional","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Cl\u00e1udia","family":"Lima","sequence":"additional","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Castanheira","sequence":"additional","affiliation":[{"name":"International Iberian Nanotechnology Laboratory , Av. Mestre Jos\u00e9 Veiga s\/n , ,","place":["Braga, Portugal, 4715-330"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7645-2834","authenticated-orcid":true,"given":"Bego\u00f1a","family":"Espi\u00f1a","sequence":"additional","affiliation":[{"name":"International Iberian Nanotechnology Laboratory , Av. Mestre Jos\u00e9 Veiga s\/n , ,","place":["Braga, Portugal, 4715-330"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3041-0687","authenticated-orcid":true,"given":"Nuno M.","family":"Neves","sequence":"additional","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7052-8837","authenticated-orcid":true,"given":"Joaquim Miguel","family":"Oliveira","sequence":"additional","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7930-2535","authenticated-orcid":true,"given":"David","family":"Caballero","sequence":"additional","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4295-6129","authenticated-orcid":true,"given":"Rui L.","family":"Reis","sequence":"additional","affiliation":[{"name":"University of Minho , , Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark-Parque da Ci\u00eancia e Tecnologia, Rua Ave 1, Edif\u00edcio 1 (Sede), Barco , ,","place":["Guimar\u00e3es, Portugal, 4805-694"]},{"name":"ICVS\/3B\u2019s-PT Government Associate Laboratory , ,","place":["Braga\/Guimar\u00e3es, Portugal, 4805-694"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"issue":"1","key":"2026092100082578700_cit1","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1016\/j.blre.2012.12.002","article-title":"Extracellular vesicles in physiological and pathological conditions","volume":"27","author":"Yuana","year":"2013","journal-title":"Blood Rev."},{"issue":"4","key":"2026092100082578700_cit2","doi-asserted-by":"publisher","first-page":"877","DOI":"10.1038\/s12276-024-01209-y","article-title":"Regulation of cargo selection in exosome biogenesis and its biomedical applications in cancer","volume":"56","author":"Lee","year":"2024","journal-title":"Exp. 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