We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

中文翻译：

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利用四个正交单颗粒分析平台表征细胞外囊泡和人造纳米颗粒

我们比较了四种正交技术对细胞外囊泡（EV）和合成颗粒的大小，计数和表型分析。这些平台是：具有荧光的单粒子干涉反射成像传感（SP-IRIS），具有荧光的纳米粒子跟踪分析（NTA），微流阻脉冲传感（MRPS）和纳米流式细胞术测量（NFCM）。通过差异超速离心（dUC）或超滤（UF）组合将人T淋巴细胞H9（高CD81，低CD63）和原单核细胞系U937（低CD81，高CD63）的电动汽车从条件培养基（CCM）中分离出来和尺寸排阻色谱（SEC），并通过透射电子显微镜（TEM）和蛋白质印迹（WB）进行表征。还测试了具有已知尺寸和/或浓度的合成颗粒（二氧化硅和聚苯乙烯球）的混合物。MRPS和NFCM返回的颗粒计数相似，而NTA检测到的电动汽车的颗粒计数大约低一个数量级，但合成颗粒的计数却没有。SP-IRIS事件不能用于估计颗粒浓度。对于尺寸调整，SP-IRIS，MRPS和NFCM返回相似的尺寸轮廓，其中较小的尺寸占主导地位（按幂定律分布），但灵敏度通常下降到60 nm以下。NTA检测到众数直径大于100 nm的粒子。此外，SP-IRIS，MRPS和NFCM能够识别二氧化硅或聚苯乙烯纳米粒子混合物中四个不同大小的种群中的至少三个。最后，对于四跨素表型，SP-IRIS平台在荧光模式下能够检测同一颗粒上的至少两个标记，而NFCM可以检测CD81或CD63。根据这项研究的结果，我们可以得出有关现有单颗粒分析功能的结论，这些功能可能对EV生物标志物的开发和机理研究有用。