细菌囊泡纯度、浓度及粒径分布的单颗粒水平定量表征  

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

作　　者：牛倩 田野 吴明凯 张宇豪 陈晨 颜晓梅[1] NIU Qian;TIAN Ye;WU Ming-Kai;ZHANG Yu-Hao;CHEN Chen;YAN Xiao-Mei(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)

机构地区：[1]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

出　　处：《分析化学》2021年第5期733-742,共10页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21934004,21627811);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。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.

关 键 词：细菌囊泡 纳米流式检测仪 密度梯度离心 纯度 浓度 粒径分布 

分 类 号：Q935[生物学—微生物学]