Protein profiling and sizing of individual exosomes down to 40 nm for purity assessment and cancer diagnosis
演讲题目:Protein profiling and sizing of individual exosomes down to 40 nm for purity assessment and cancer diagnosis
厦门大学化学化工学院化学生物学系主任，厦门大学特聘教授，国家杰出青年科学基金获得者，国家“万人计划”科技创新领军人才。1986-1990年厦门大学科学仪器工程系本科；1990-1996年厦门大学/日本名古屋大学联合培养，获分析化学理学博士学位；1996-1997年美国佛罗里达大学化学系博士后；1997-2005年美国著名Los Alamos National Laboratory（洛斯阿拉莫斯国家实验室）生物科学部博士后、研究员（独立PI）。2005年8月起任职厦门大学化学化工学院化学系教授、博士生导师，2008年起担任化学生物学系创系系主任至今。主要从事生化分析与生物传感研究，主持国家自然科学基金国家重大科研仪器研制项目、杰出青年科学基金、科学仪器基础研究转款等项目，在J. Am. Chem. Soc., Angew. Chem. Int. Ed., ACS Nano, Anal. Chem.等国际杂志发表70多篇SCI论文，先后获得教育部新世纪优秀人才、国家杰出青年科学基金、厦门市五一劳动奖章、福建省高校领军人才、第十二届福建青年科技奖、科技部“创新人才推进计划中青年科技创新领军人才、首届中国女分析化学家奖、国家“万人计划”科技创新领军人才等奖项与称号。
演讲题目：Protein profiling and sizing of individual exosomes down to 40 nm for purity assessment and cancer diagnosis
演讲摘要： Exosomes have become the focus of rising interest due to their numerous functions in physiology and pathology, and their diagnostic and therapeutic potential. Because exosomes are highly heterogeneous in particle size and molecular composition, quantitative and multiparameter analysis at the single-particle level is of great importance. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, our laboratory has developed high-sensitivity flow cytometry (HSFCM) that allows light scattering detection of single silica nanoparticles and viruses as small as 24 nm and 27 nm diameter, respectively.[1,2] Here we report sensitive flow cytometric analysis of exosomes isolated from cancer cell culture supernatant and human platelet free plasma (PFP) through ultracentrifugation. Both the side scattering and multiple fluorescence signals emitted from single exosomes as small as 40 nm can be simultaneously detected with an analysis rate up to 10,000 particles per minute.
We first validated the purity of exosomes in isolates by measuring the particle concentration before and after the Triton X-100 treatment, which can lyse the membrane phospholipid bilayer of vesicles. It was found out that 85-90% and 70% of the particles are exosomes for isolates from cell culture supernatant and PFP, respectively. The other particulates can be ascribed to lipoproteins. Through this approach, we compared the purity of exosomes purified from different commercially available exosome isolation kits, including ExoQuick Kit (System Biosciences), Total Exosomes Isolation Kit (Life Technologies), qEV column (iZON), Ultrafiltration (Millipore) and exoEasy Maxi kit (Qiagen). Employing monodisperse silica nanoparticles (refractive index of 1.46) as the size reference standards, accurate size measurement of exosomes was achieved in minutes upon refractive index mismatch correction based on the Mie theory. The particle size distribution histogram not only exhibits comparable resolution with that of cryo-TEM measurement but is much faster and more statistically representative. Subpopulations of exosomes expressing CD9, CD63, and/or CD81 were quantified upon immunofluorescent staining. When HSFCM was used to analyze blood samples, a significantly elevated level of CD147-positive exosomes was identified in colorectal cancer patients compared to healthy controls (P < 0.001). HSFCM provides a sensitive and rapid platform for surface protein profiling and sizing of individual exosomes, which could greatly aid the understanding of exosome-mediated intercellular communication and the development of novel diagnostic and therapeutic strategies.
 S. Zhu, L. Ma, S. Wang, C. Chen, W. Zhang, L. Yang, W. Hang, J. P. Nolan, L. Wu, X. Yan*. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS Nano 2014, 8, 10998-11006.
 L. Ma, S. Zhu, Y. Tian, W. Zhang, S. Wang, C. Chen, L. Wu, X. Yan*. A label-free analysis of single viruses with a resolution comparable to that of electron microscopy and the throughput of flow cytometry, Angew. Chem. Int. Ed., 2016, 55, 10239-10243.