Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.
Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.
复制标题
胞外单个囊泡和单分子检测的双数字化检测。
DOI：
10.1002/advs.202303619
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发表时间：
2023-11
期刊：
影响因子：
15.1
通讯作者：
Ko, Jina
中科院分区：
文献类型：
--
作者：
Reynolds, David E.;Pan, Menghan;Yang, Jingbo;Galanis, George;Roh, Yoon Ho;Morales, Renee-Tyler T.;Kumar, Shailesh Senthil;Heo, Su-Jin;Xu, Xiaowei;Guo, Wei;Ko, Jina
关键词：
细胞外小泡(EVS)已成为疾病诊断生物标志物的重要来源。然而，由于EV样本携带的生物标记物的表达水平较低，以及它们复杂的物理和生物学特性，目前的EV诊断方法面临着巨大的挑战。在这里，开发了一种高灵敏的双数字分析，允许从单个电动汽车中绝对定量单个分子。由于单一电动汽车的蛋白质相对丰度较低，因此集成了酪胺信号放大(TSA)以增加用于评估的荧光信号读数。通过集成的微流控技术，成功地展示了该技术对单辆电动汽车的分割能力，证明了该技术的数字分割能力。然后应用该设备检测来自黑色素瘤细胞系的单个EV中的单个PD-L1蛋白，发现每个EV中有≈2.7分子表达，表明该系统可用于描述治疗反应、转移状态和肿瘤进展的重要预后和诊断癌症生物标志物。准确量化单个EV稀有丰度的蛋白质分子的能力将有助于理解EV的异质性，并发现EV亚型作为新的生物标志物。这项微流控技术展示了执行双数字化验的能力，允许从单个EV中检测单个PD-L1蛋白分子。准确定量单个EV的稀有蛋白分子的能力有助于理解EV的异质性和发现新的EV亚型生物标记物。
Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD‐L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers. This microfluidic technology demonstrates the ability to perform a double digital assay, allowing for the detection of single PD‐L1 protein molecules from individual EVs. The ability to accurately quantify rare protein molecules from individual EVs sheds light on the understanding of EV heterogeneity and the discovery of new EV subtype biomarkers.
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DOI：
10.1126/science.aau6977
发表时间：
2020-02-07
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Kalluri R;LeBleu VS
通讯作者：
LeBleu VS
影响因子：
17.1
作者：
Lee K;Fraser K;Ghaddar B;Yang K;Kim E;Balaj L;Chiocca EA;Breakefield XO;Lee H;Weissleder R
通讯作者：
Weissleder R
影响因子：
0.3
作者：
Akama, Kenji;Shirai, Kentaro;Suzuki, Seigo
通讯作者：
Suzuki, Seigo
影响因子：
4.6
作者：
Marcoux G;Duchez AC;Cloutier N;Provost P;Nigrovic PA;Boilard E
通讯作者：
Boilard E
影响因子：
4.1
作者：
Min J;Son T;Hong JS;Cheah PS;Wegemann A;Murlidharan K;Weissleder R;Lee H;Im H
通讯作者：
Im H