基于免疫-超声分离技术的全血样本中目标外泌体的特异性分离、提纯和分析

Detection and analysis of exosomes has gained popularity in both life sciences and clinical diagnosis research. To isolate and purify target exosomes from biological samples at high purity and high recovery is an important prerequisite for exosomes-related research. Due to their extremely small size (diameter 40-120 nm), and overlapping in size with other EVs such as microvesicles, exosomes are difficult to isolate and purify. Conventional size-based methods for isolating exosomes, such as ultracentrifugation and filtering, suffer from the disadvantages in low purity and cumbersome and tedious operation, also these methods cannot specifically isolate target exosomes that are related to diseases. Immunoaffinity-based methods such as using magnetic beads with particular antibodies could achieve high specificity and purity, however have low recovery and require additional biological sample pretreatment steps which may introduce contamination and bias. Microfluidics technologies are capable of achieving higher purity and recovery, and shortening the processing time, and thus have been the major force in developing next-generation exosomes isolation technologies. This project proposes a novel immuno-acoustic sorting technology that combines the advantages of highly specific capturing from immunoaffinity and noncontact cell manipulation capability from acoustofluidics, achieved specific isolation, purification, and analysis of target exosomes from whole blood samples without any sample pretreatment steps, and applied this technology to exosomes research of clinical samples from breast cancer patients. The detailed procedures of the proposed method include acoustofluidic plasmapheresis to remove blood cells, and then microparticles with coated antibodies bind with target exosomes in the blood plasma by immunoaffinity, the exosomes-microparticle conjugates are hereby captured, purified and collected for further analysis by acoustofluidics. The proposed immuno-acoustic sorting technology facilitates surface biomarker-based specific isolation through acoustofluidic technology, and holds the promise as a new way of biological sample processing with benefits such as simple operation, good extensibility, and broad applications.

外泌体的检测及分析是近年来生命科学和临床诊断领域的研究热点。将高纯度的目标外泌体从生物样本中分离出来并高效回收是开展外泌体研究的重要先决条件。由于外泌体体积非常小（粒度40-120 nm），其分离和纯化比较困难。常用的超速离心、过滤等基于体积的方法存在分离纯度低、操作复杂费时等缺陷，且没有特异性。基于磁珠免疫吸附的方法特异性强、纯度高，但回收率低，且需要额外的样本预处理环节。微流控技术具有能够取得更高的性能并缩短操作时间等优势，是下一代外泌体分离技术的主要来源。本项目提出一种新型的免疫-超声分离技术，将免疫吸附的特异性与超声微流控的非接触式细胞操控能力结合起来，实现了不需要样本预处理的全血样本中目标外泌体的特异性分离、提纯与分析，并将其应用于临床乳腺癌患者的外泌体研究。具体研究方案是在芯片上集成超声微流控模块进行全血样本中的血浆置换，然后进行抗体微球与血浆中外泌体的特异性结合，接着通过声场力将外泌体-微球复合物从血浆中分离出来并进行清洗、回收。本项目所构建的免疫-超声分离技术能够利用超声场力实现基于表面生物标志物的特异性分离，提供了一种新的生物样本处理范式，并将其应用于临床研究。

细胞外泌体的检测及分析是近年来生命科学和分子诊断领域的研究热点，将高纯度的目标外泌体从生物样本中分离出来并高效回收是分析研究外泌体的重要先决条件。目前常用的外泌体分离方式包括超高速离心、过滤法、化学沉淀法、亲和沉淀法。它们分别存在分离和纯化时间较长，外泌体会有损坏，富集效率较低等问题。超声场力是一种非接触式的场作用力，与细胞体积成正比，这些特点使其被广泛应用于基于体积的细胞分离。我们提出一种新型的免疫-超声分离技术，将抗体微球免疫吸附的特异性与超声微流控的非接触式操控有机结合，实现了针对全血样本的目标外泌体的特异性分离、清洗和回收。该方法实现了无需任何预处理的情况下对全血样本中的目标外泌体进行直接、简便的分离方法，初步实现从病患全血样本中高效提取疾病相关的外泌体并进行分析，为基于外泌体的液体活检与疾病早期诊断和预后监测开辟新的路径。