细菌囊泡纯度、浓度及粒径分布的单颗粒水平定量表征

引用本文: 牛倩, 田野, 吴明凯, 张宇豪, 陈晨, 颜晓梅. 细菌囊泡纯度、浓度及粒径分布的单颗粒水平定量表征[J]. 分析化学, 2021, 49(5): 733-742. doi: 10.19756/j.issn.0253-3820.211054 shu

Citation: NIU Qian, TIAN Ye, WU Ming-Kai, ZHANG Yu-Hao, CHEN Chen, YAN Xiao-Mei. Quantitative Characterization of Purity, Concentration and Size Distribution of Bacterial Membrane Vesicles at Single-Particle Level[J]. Chinese Journal of Analytical Chemistry, 2021, 49(5): 733-742. doi: 10.19756/j.issn.0253-3820.211054 shu

细菌囊泡纯度、浓度及粒径分布的单颗粒水平定量表征

厦门大学化学化工学院化学生物学系, 谱学分析与仪器教育部重点实验室, 化学生物学福建省重点实验室, 厦门 361005

通讯作者: 颜晓梅,E-mail:xmyan@xmu.edu.cn

基金项目:
国家自然科学基金项目（Nos.21934004，21627811）和国家基础科学人才培养基金项目（No.J1310024）资助。

摘要: 细菌囊泡（Bacterial membrane vesicles，BMVs）是细菌在生长过程中分泌的纳米尺度脂质小泡，其携载核酸、蛋白质，特别是毒力因子和免疫调节因子等，进行种间、种内以及与宿主的信息交流和物质交换。BMVs不仅影响多种生物过程，而且在疫苗和抗癌药物研发方面显示出重要的应用价值。但是，由于BMVs具有高度的个体差异性、粒径微小且内容物稀少，其纯化和表征均面临严峻挑战。基于本研究组自行研制的纳米流式检测装置（Nano-flow cytometer，nFCM）可在单颗粒水平对纳米粒子进行高灵敏、高通量分析的独特优势，本研究结合差速离心和密度梯度离心对BMVs进行分离纯化，建立了BMVs的纯度、浓度及粒径分布的单颗粒水平定量表征方法。采用nFCM对密度梯度离心后密度层中BMVs的纯度进行快速评估，可获得纯度>90%的BMVs样品。此外，基于以上单颗粒表征方法，本研究发现，革兰氏阴性菌大肠杆菌（E.coli）O157：H7与革兰氏阳性菌金黄色葡萄球菌（S.aureus）分泌BMVs的浓度、密度和粒径存在较大差异，从相同数目细菌中提取到的E.coli O157：H7 BMVs的浓度比S.aureus BMVs的浓度高一个数量级；E.coli O157：H7 BMVs的密度约为1.10 g/mL，粒径分布中位值为83.5 nm，而S.aureus BMVs的密度约为1.06 g/mL，粒径分布中位值为64.9 nm。本研究建立了一种在单颗粒水平对BMVs纯度、浓度及粒径分布进行表征的方法。nFCM有望为BMVs更深层次的生物学功能研究提供有力的技术支持。

关键词:

English

Quantitative Characterization of Purity, Concentration and Size Distribution of Bacterial Membrane Vesicles at Single-Particle Level

Departmentof Chemical Biology, MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China

Corresponding author: YAN Xiao-Mei, xmyan@xmu.edu.cn

Received Date: 22 January 2021
Revised Date: 23 March 2021
Fund Project:
Supported by the National Natural Science Foundation of China (Nos.21934004, 21627811) and the National Fund for Fostering Talents of Basic Science (No.J1310024).

Abstract: Bacterial membrane vesicles (BMVs) are nanostructures shed naturally by most bacterial species. BMVs carry nucleic acids, proteins, especially virulence factors and immune regulatory factors for the inter- and intra-species communication, showing great potential in the development of vaccines and anticancer drugs. However, the purification and characterization of BMVs remain challenging due to their large heterogeneity, nanoscale particle size, and low contents of cargo molecules. Based on the unique advantages such as the laboratory-built nano-flow cytometer (nFCM) in the sensitive and rapid analysis of individual nanoparticles, the purity, concentration and size distribution of BMV preparations were measured in this work. The experimental results demonstrated that the components in each density layer after density gradient centrifugation could be detected by nFCM to obtain BMVs samples with a high purity of more than 90%. BMVs secreted by Gram-positive bacteria S.aureus and Gram-negative bacteria E.coli O157:H7 were significantly different in density, which mainly presented in the second (ρ=1.06 g/mL) and fourth (ρ=1.10 g/mL) fractions of the Optiprep density gradient layer respectively. The median particle sizes of BMVs derived from Gram-positive (S.aureus) and Gram-negative (E.coli O157:H7) bacteria were 64.9 nm and 83.5 nm, respectively. In conclusion, the study presented here reported the development of an efficient method to characterize the purity, concentration, and size distribution of BMVs at the single-particle level. nFCM was expected to provide powerful technical support for the biological function study of BMVs.

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沈阳化工大学材料科学与工程学院沈阳 110142

细菌囊泡纯度、浓度及粒径分布的单颗粒水平定量表征

通讯作者: 颜晓梅,E-mail:xmyan@xmu.edu.cn

English

Quantitative Characterization of Purity, Concentration and Size Distribution of Bacterial Membrane Vesicles at Single-Particle Level

Corresponding author: YAN Xiao-Mei, xmyan@xmu.edu.cn

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

细菌囊泡纯度、浓度及粒径分布的单颗粒水平定量表征

通讯作者: 颜晓梅,E-mail:xmyan@xmu.edu.cn

English

Quantitative Characterization of Purity, Concentration and Size Distribution of Bacterial Membrane Vesicles at Single-Particle Level

Corresponding author: YAN Xiao-Mei, xmyan@xmu.edu.cn

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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通讯作者: 陈斌, bchen63@163.com

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