Anderson Will, Lane Rebecca, Korbie Darren, Trau Matt
†Centre for Personalized NanoMedicine, ‡Australian Institute for Bioengineering and Nanotechnology, and §School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD Australia.
Langmuir. 2015 Jun 16;31(23):6577-87. doi: 10.1021/acs.langmuir.5b01402. Epub 2015 Jun 1.
Size distribution and concentration measurements of exosomes are essential when investigating their cellular function and uptake. Recently, a particle size distribution and concentration measurement platform known as tunable resistive pulse sensing (TRPS) has seen increased use for the characterization of exosome samples. TRPS measures the brief increase in electrical resistance (a resistive pulse) produced by individual submicrometer/nanoscale particles as they translocate through a size-tunable submicrometer/micrometer-sized pore, embedded in an elastic membrane. Unfortunately, TRPS measurements are susceptible to issues surrounding system stability, where the pore can become blocked by particles, and sensitivity issues, where particles are too small to be detected against the background noise of the system. Herein, we provide a comprehensive analysis of the parameters involved in TRPS exosome measurements and demonstrate the ability to improve system sensitivity and stability by the optimization of system parameters. We also provide the first analysis of system noise, sensitivity cutoff limits, and accuracy with respect to exosome measurements and offer an explicit definition of system sensitivity that indicates the smallest particle diameter that can be detected within the noise of the trans-membrane current. A comparison of exosome size measurements from both TRPS and cryo-electron microscopy is also provided, finding that a significant number of smaller exosomes fell below the detection limit of the TRPS platform and offering one potential insight as to why there is such large variability in the exosome size distribution reported in the literature. We believe the observations reported here may assist others in improving TRPS measurements for exosome samples and other submicrometer biological and nonbiological particles.
在研究外泌体的细胞功能和摄取时，对外泌体的大小分布和浓度进行测量至关重要。最近，一种称为可调电阻脉冲传感（TRPS）的粒度分布和浓度测量平台在表征外泌体样品方面的应用越来越多。TRPS测量单个亚微米/纳米级颗粒穿过嵌入弹性膜中的尺寸可调亚微米/微米级孔时产生的电阻短暂增加（电阻脉冲）。不幸的是，TRPS测量容易受到系统稳定性问题的影响，即孔可能被颗粒堵塞，以及灵敏度问题，即颗粒太小以至于在系统的背景噪声中无法被检测到。在此，我们对外泌体TRPS测量中涉及的参数进行了全面分析，并证明了通过优化系统参数来提高系统灵敏度和稳定性的能力。我们还首次分析了系统噪声、灵敏度截止极限以及外泌体测量的准确性，并给出了系统灵敏度的明确定义，该定义表明了在跨膜电流噪声范围内能够检测到的最小颗粒直径。我们还比较了TRPS和冷冻电子显微镜对外泌体大小的测量结果，发现大量较小的外泌体低于TRPS平台的检测限，这为文献中报道的外泌体大小分布存在如此大差异的原因提供了一个潜在的见解。我们相信这里报道的观察结果可能有助于其他人改进外泌体样品以及其他亚微米生物和非生物颗粒的TRPS测量。
