Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.

细胞外膜囊泡（EMV）是生物分泌型脂质囊泡，在与人类疾病和细菌发病机理有关的细胞间通讯中起重要作用。正在研究它们在诊断，疫苗和生物技术中的可能用途。但是，现有方法存在许多问题。高速离心是一种广泛使用的收集EMV的方法，可能会导致结构伪影。免疫染色方法需要几个步骤，因此从分泌细胞中分离和检测EMV非常耗时。此外，使用这些方法检测EMV需要特异性和昂贵的抗体。为了解决这些问题，迫切需要开发一种简单，快速的培养基中EMV检测方法，而又不与分泌细胞分离。产生大量EMV的Shewanella vesiculosa HM13（包括高纯度的货物蛋白）作为模型。曲率敏感肽用于EMV检测工具。FAAV，一种源自分选神经蛋白1的肽，即使在复杂培养基中存在分泌细胞的情况下，也能选择性结合EMV。FAAV可以在几分钟内完全检测到EMV，而FAAV对蛋白酶的抗性使其可以承受在培养基中长时间使用。荧光/福斯特共振能量转移被用于开发一种方法来以高灵敏度检测EMV的数量变化。总体而言，我们的结果表明FAAV在培养基中原位EMV检测中的潜在适用性。