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Integrated Immunomagnetic Bead-Based Microfluidic Chip for Exosomes Isolation

机译:集成的基于免疫磁珠的微流控芯片用于外来体分离

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摘要

Exosomes are essential early biomarkers for health monitoring and cancer diagnosis. A prerequisite for further investigation of exosomes is the isolation, which is technically challenging due to the complexity of body fluids. This paper presents the development of an integrated microfluidic chip for exosomes isolation, which combines the traditional immunomagnetic bead-based protocol and the recently emerging microfluidic approach, resulting in benefits from both the high-purity of the former and the automated continuous superiority of the latter. The chip was designed based on an S-shaped micromixer with embedded baffle. The excellent mixing efficiency of this micromixer compared with Y-shaped and S-shaped micromixers was verified by simulation and experiments. The photolithography technique was employed to fabricate the integrated microfluidic chip, and the manufacturing process was elucidated. We finally established an experimental platform for exosomes isolation with the fabricated microfluidic chip built in. Exosomes isolation experiments were conducted using this platform. The distribution and morphology of the isolated exosomes were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Quantitative size analyses based on transmission electron micrographs indicated that most of the obtained particles were between 30 and 150 nm. Western blot analyses of the isolated exosomes and the serum were conducted to verify the platform’s capability of isolating a certain subpopulation of exosomes corresponding to specified protein markers (CD63). The complete time for isolation of 150 μL serum samples was approximately 50 min, which was highly competitive with the reported existing protocols. Experimental results proved the capacity of the established integrated microfluidic chip for exosomes isolation with high purity, high integrity, and excellent efficiency. The platform can be further developed to make it possible for practical use in clinical applications as a universal exosomes isolation and characterization tool.
机译:外泌体是健康监测和癌症诊断中必不可少的早期生物标记。进一步研究外泌体的先决条件是分离,由于体液的复杂性,这在技术上具有挑战性。本文介绍了用于外泌体分离的集成微流控芯片的开发,该芯片结合了传统的基于免疫磁珠的方案和最近出现的微流控方法,从而从前者的高纯度和后者的自动连续优势中受益。该芯片是基于带有嵌入式挡板的S型微混合器设计的。通过仿真和实验验证了该微型混合器与Y形和S形微型混合器相比出色的混合效率。采用光刻技术制造集成的微流控芯片,并阐明了制造工艺。我们最终使用内置的微流控芯片建立了用于外泌体分离的实验平台。利用该平台进行了外泌体分离实验。通过透射电子显微镜(TEM)和扫描电子显微镜(SEM)观察分离的外泌体的分布和形态。基于透射电子显微照片的定量尺寸分析表明,大多数获得的颗粒在30至150 nm之间。对分离的外泌体和血清进行了蛋白质印迹分析,以验证该平台分离与特定蛋白质标记(CD63)相对应的外泌体某些亚群的能力。分离150μL血清样品的完整时间约为50分钟,这与已报道的现有方案具有很高的竞争力。实验结果证明了所建立的集成微流控芯片具有高纯度,高完整性和高效率的外泌体分离能力。该平台可以进一步开发,使其有可能作为通用的外泌体分离和表征工具在临床应用中实际使用。

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