泊洛沙姆 188 作为润湿剂用于微流控电阻脉冲传感测量细胞外囊泡。

Poloxamer-188 as a wetting agent for microfluidic resistive pulse sensing measurements of extracellular vesicles.

机构信息

Amsterdam Vesicle Center, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Laboratory of Experimental Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

出版信息

PLoS One. 2024 May 2;19(5):e0295849. doi: 10.1371/journal.pone.0295849. eCollection 2024.

DOI:10.1371/journal.pone.0295849

PMID:38696491

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11065227/

Abstract

INTRODUCTION

Microfluidic resistive pulse sensing (MRPS) can determine the concentration and size distribution of extracellular vesicles (EVs) by measuring the electrical resistance of single EVs passing through a pore. To ensure that the sample flows through the pore, the sample needs to contain a wetting agent, such as bovine serum albumin (BSA). BSA leaves EVs intact but occasionally results in unstable MRPS measurements. Here, we aim to find a new wetting agent by evaluating Poloxamer-188 and Tween-20.

METHODS

An EV test sample was prepared using an outdated erythrocyte blood bank concentrate. The EV test sample was diluted in Dulbecco's phosphate-buffered saline (DPBS) or DPBS containing 0.10% BSA (w/v), 0.050% Poloxamer-188 (v/v) or 1.00% Tween-20 (v/v). The effect of the wetting agents on the concentration and size distribution of EVs was determined by flow cytometry. To evaluate the precision of sample volume determination with MRPS, the interquartile range (IQR) of the particles transit time through the pore was examined. To validate that DPBS containing Poloxamer-188 yields reliable MRPS measurements, the repeatability of MRPS in measuring blood plasma samples was examined.

RESULTS

Flow cytometry results show that the size distribution of EVs in Tween 20, in contrast to Poloxamer-188, differs from the control measurements (DPBS and DPBS containing BSA). MRPS results show that Poloxamer-188 improves the precision of sample volume determination compared to BSA and Tween-20, because the IQR of the transit time of EVs in the test sample is 11 μs, which is lower than 56 μs for BSA and 16 μs for Tween-20. Furthermore, the IQR of the transit time of particles in blood samples with Poloxamer-188 are 14, 16, and 14 μs, which confirms the reliability of MRPS measurements.

CONCLUSION

The solution of 0.050% Poloxamer-188 in DPBS does not lyse EVs and results in repeatable and unimpeded MRPS measurements.

摘要

简介

微流控电阻脉冲感应 (MRPS) 通过测量单个 EV 通过孔时的电阻，可以确定细胞外囊泡 (EV) 的浓度和粒径分布。为了确保样品流过孔，样品需要含有润湿剂，如牛血清白蛋白 (BSA)。BSA 使 EV 保持完整，但偶尔会导致不稳定的 MRPS 测量。在这里，我们旨在通过评估泊洛沙姆 188 和吐温 20 来找到一种新的润湿剂。

方法

使用过期的红细胞血库浓缩液制备 EV 测试样品。将 EV 测试样品在 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 或 DPBS 中稀释，其中含有 0.10% BSA（w/v）、0.050% 泊洛沙姆 188（v/v）或 1.00% 吐温 20（v/v）。通过流式细胞术确定润湿剂对 EV 浓度和粒径分布的影响。为了评估 MRPS 中样品体积确定的精度，检查了粒子通过孔的传输时间的四分位距 (IQR)。为了验证含有泊洛沙姆 188 的 DPBS 可产生可靠的 MRPS 测量结果，检查了 MRPS 在测量血浆样品时的重复性。

结果

流式细胞术结果表明，与泊洛沙姆 188 相比，吐温 20 中的 EV 大小分布与对照测量值（DPBS 和 DPBS 中含有 BSA）不同。MRPS 结果表明，与 BSA 和吐温 20 相比，泊洛沙姆 188 提高了样品体积确定的精度，因为在测试样品中 EV 传输时间的 IQR 为 11 μs，低于 BSA 的 56 μs 和吐温 20 的 16 μs。此外，含有泊洛沙姆 188 的血液样本中粒子传输时间的 IQR 为 14、16 和 14 μs，这证实了 MRPS 测量的可靠性。