Department of Chemistry, Chungnam National University, Daejeon, Republic of Korea.
Department of Chemistry, Chungnam National University, Daejeon, Republic of Korea; Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru, 81310 Johor, Malaysia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Aug 1;1228:123828. doi: 10.1016/j.jchromb.2023.123828. Epub 2023 Jul 17.
In recent years, extracellular vesicles (EVs) have gained attention for their potential as biomarkers for the early diagnosis and treatment of various diseases. Traditionally, EV isolation has relied exclusively on ultracentrifugation. However, alternative enrichment methods such as size-exclusion chromatography (SEC) and polyethylene glycol-based precipitation have been introduced. This study utilized SEC as a characterization tool to assess the efficiency of EV isolation. Urinary EVs isolated from human urine using centrifugation (40,000 × g) were analyzed using an SEC column with a pore size of 1000 Å, an inner diameter of 7.8 mm, and a length of 300 mm. The EVs were detected sequentially using UV (280 nm) and fluorescence (λex/em = 550 nm/565 nm); the EVs were observed at approximately 6 min, while the proteins were observed at approximately 12 min. The repeated centrifugation enrichment steps resulted in an increase in EV peaks and a decrease in protein peaks. SEC analysis of the enriched EV samples confirmed that a four-cycle repetition of centrifugation is necessary for successful EV enrichment and removal of non-EV proteins from 40 mL of human urine.
近年来，细胞外囊泡 (EVs) 因其作为各种疾病早期诊断和治疗的生物标志物的潜力而受到关注。传统上，EV 的分离完全依赖于超速离心。然而，已经引入了替代的富集方法，如大小排阻色谱 (SEC) 和基于聚乙二醇的沉淀。本研究将 SEC 用作表征工具来评估 EV 分离的效率。使用离心（40,000×g）从人尿中分离的尿 EVs 使用孔径为 1000Å、内径为 7.8mm 和长度为 300mm 的 SEC 柱进行分析。EVs 依次使用 UV（280nm）和荧光（λex/em = 550nm/565nm）进行检测；EVs 在大约 6 分钟时观察到，而蛋白质在大约 12 分钟时观察到。重复的离心富集步骤导致 EV 峰增加和蛋白质峰减少。富集 EV 样品的 SEC 分析证实，从 40mL 人尿中成功富集 EV 并去除非 EV 蛋白需要重复离心四次。
