与脂蛋白颗粒 (LP) 相比，细胞外囊泡 (EV) 在血液中的浓度要低得多（5-6 个数量级）。由于 LP 和 EV 在尺寸和密度上重叠，因此隔离高纯度 EV 是一项重大挑战。虽然当前使用尺寸表达色谱 (SEC) 和密度梯度 (DG) 的两步顺序 EV 分离工艺可实现高纯度，但 DG 中耗时的超速离心 (UC) 步骤阻碍了工作流程效率。本文介绍了一种经过优化的磁珠试剂 LipoMin，它采用糖胺聚糖 (GAG) 进行功能化，可作为第二步过程中 LP 去除的快速替代方案，仅需约 10 分钟。我们评估了 LipoMin 对两种样品类型的功效：(a) 通过尺寸排阻色谱分离的 EV 级分 (SEC LipoMin) 和 (b) 通过超速离心获得的沉淀 (UC LipoMin)。工作流程非常简单，涉及与 LipoMin 孵育 10 分钟，然后对耗尽 LP 且含有 EV 的上清液进行磁力分离。酶联免疫吸附测定 (ELISA) 结果显示，LipoMin 从 SEC EV 级分中去除了 98.2% ApoB，与 SEC DG 两步过程中 DG 的 LP 去除能力相当。重要的是，EV 产量（CD81 ELISA）保持在 93.0%，Western blot 分析证实关键的 EV 标记物，flotillin 和 CD81 没有受到损害。将重组 EV (rEV)（一种 EV 参考标准品）添加到 SEC EV 级分中，回收了 89% 的 CD81 蛋白。对于 UC LipoMin，ApoA1 降低了 76.5%，同时保留了 90.7% 的 CD81。值得注意的是，经 SEC LipoMin 和 UC LipoMin 处理的结直肠癌 (CRC) 和阿尔茨海默病 (AD) 样本均显示出相关 EV 和临床标志物的清晰表达。 LipoMin 试剂的工作流程仅需 10 分钟（与传统方法相比节省了 96% 的时间），为 DG 消除 LP 提供了快速高效的替代方案，为简化的 SEC LipoMin 两步 EV 分离流程铺平了道路。

Extracellular vesicles (EVs) are present in blood at much lower concentrations (5-6 orders of magnitude) compared to lipoprotein particles (LP). Because LP and EV overlap in size and density, isolating high-purity EVs is a significant challenge. While the current two-step sequential EV isolation process using size-expression chromatography (SEC) followed by a density gradient (DG) achieves high purity, the time-consuming ultracentrifugation (UC) step in DG hinders workflow efficiency. This paper introduces an optimized magnetic bead reagent, LipoMin, functionalized with glycosaminoglycans (GAGs), as a rapid alternative for LP removal during the second-step process in about 10 minutes. We evaluated LipoMin's efficacy on two sample types: (a) EV fractions isolated by size exclusion chromatography (SEC + LipoMin) and (b) the pellet obtained from ultracentrifugation (UC + LipoMin). The workflow is remarkably simple, involving a 10 min incubation with LipoMin followed by magnetic separation of the LP-depleted EV-containing supernatant. Results from enzyme-linked immunosorbent assay (ELISA) revealed that LipoMin removes 98.2% ApoB from SEC EV fractions, comparable to the LP removal ability of DG in the SEC + DG two-step process. Importantly, the EV yield (CD81 ELISA) remained at 93.0% and Western blot analysis confirmed that key EV markers, flotillin and CD81, were not compromised. Recombinant EV (rEV), an EV reference standard, was spiked into SEC EV fractions and recovered 89% of CD81 protein. For UC + LipoMin, ApoA1 decreased by 76.5% while retaining 90.7% of CD81. Notably, both colorectal cancer (CRC) and Alzheimer's disease (AD) samples processed by SEC + LipoMin and UC + LipoMin displayed clear expression of relevant EV and clinical markers. With a 10 min workflow (resulting in a 96% time saving compared to the traditional method), the LipoMin reagent offers a rapid and efficient alternative to DG for LP depletion, paving the way for a streamlined SEC + LipoMin two-step EV isolation process.