School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore, 639798, Singapore.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
Small. 2022 Feb;18(6):e2104470. doi: 10.1002/smll.202104470. Epub 2022 Jan 5.
Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease-specific biomolecular cargoes and importance in cell-cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50-200 nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20 min) replica molding technique, a fritless microfluidic SEC device (μSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young's modulus ≈1 GPa) for high pressure (up to 6 bar) sample processing. Controlled on-chip nanoliter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50 nm), albumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, EVs are directly isolated from undiluted human platelet-poor plasma using μSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent μSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics.
细胞外囊泡 (EVs) 因其具有疾病特异性生物分子货物,并且在细胞间通讯中具有重要作用,因此被认为是下一代诊断生物标志物。EV 样品制备的一个主要瓶颈是从生物样品中的内源性蛋白质中低效且费力地分离纳米级 EV(≈50-200nm)。本文报道了一种基于尺寸排阻色谱 (SEC) 原理的独特微流控平台,用于 EV-蛋白质的分离。使用新型快速(≈20min)复制成型技术,使用硫醇-烯聚合物(UV 胶 NOA81,杨氏模量≈1GPa)制造无 frit 的微流控 SEC 器件(μSEC),用于高压(高达 6bar)样品处理。首先使用改进的 T 型接头注射器演示了在片纳米升样品塞的受控注入(600nL),具有快速的流量切换响应时间(<1.5s)。使用荧光纳米颗粒(50nm)、白蛋白和乳腺癌细胞(MCF-7)衍生的 EVs 验证了器件性能。作为临床应用的概念验证,直接使用 μSEC 从未稀释的人血小板贫乏血浆中分离 EVs,并基于纳米颗粒粒子分析(NTA)、Western blot 和流式细胞术分析,显示出 EVs 和蛋白质之间的明显洗脱曲线。总体而言,这种光学透明的 μSEC 可以很容易地自动化,并与 EV 检测分析集成,用于 EV 制造和临床诊断。