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Direct Exosome Quantification via Bivalent-Cholesterol-Labeled DNA Anchor for Signal Amplification
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-11-21 00:00:00 , DOI: 10.1021/acs.analchem.7b03919 Fang He 1 , Hui Liu 2 , Xinggang Guo 2 , Bin-Cheng Yin 1 , Bang-Ce Ye 1, 3
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-11-21 00:00:00 , DOI: 10.1021/acs.analchem.7b03919 Fang He 1 , Hui Liu 2 , Xinggang Guo 2 , Bin-Cheng Yin 1 , Bang-Ce Ye 1, 3
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Exosomes, as an important subpopulation of extracellular vesicles (EVs), play an important role in intercellular communications in various important pathophysiological processes, especially cancer-related. However, reliable and convenient quantitative methods for their determination are still technically challenging. In this study, we developed an efficient and direct method by combining immunoaffinity and lipid membrane surface modification into a single platform for specific isolation and accurate quantification of exosomes. Exosomes are specifically captured by immunomagnetic beads, and then a bivalent-cholesterol (B-Chol)-labeled DNA anchor with high affinity is spontaneously inserted into the exosome membrane. The rationally designed sticky end of the anchor acts as the initiator for the subsequent horseradish peroxidase (HRP)-linked hybridization chain reaction (HCR) for signal amplification. Detection is based on the color change of HRP-catalyzed H2O2-mediated oxidation of 3,3′,5,5′- tetramethyl benzidine (TMB), which can be conveniently observed by the naked eye and monitored by UV–vis spectrometry. This proposed method enables sensitive detection of 2.2 × 103 exosomes per microliter with a relative standard deviation of <5.6%, with 100-fold higher sensitivity compared to conventional ELISA. We believe that our assay has considerable potential as a routine bioassay (cost-efficient, reliable, and easy to operate) for the accurate quantification of exosomes in clinical samples.
中文翻译：
通过二价胆固醇标记的DNA锚直接外泌体定量信号放大
外泌体作为细胞外囊泡（EVs）的重要亚群，在各种重要的病理生理过程中，尤其是与癌症相关的细胞间通讯中，发挥着重要作用。但是，可靠，方便的定量方法用于测定仍在技术上具有挑战性。在这项研究中，我们通过将免疫亲和力和脂质膜表面修饰结合到单个平台中来进行特异性分离和外泌体的准确定量，从而开发了一种有效而直接的方法。用免疫磁珠特异性捕获外泌体，然后将具有高亲和力的二价胆固醇（B-Chol）标记的DNA锚自发插入外泌体膜中。锚的合理设计的粘性末端可作为随后的辣根过氧化物酶（HRP）连接的杂交链反应（HCR）的引发剂，以进行信号放大。检测基于HRP催化的H的颜色变化2 O 2介导的3,3'，5,5'-四甲基联苯胺（TMB）的氧化，可以方便地用肉眼观察并通过紫外可见光谱法进行监测。与传统的ELISA相比，该方法可灵敏地检测每微升2.2×10 3个外泌体，相对标准偏差<5.6％，灵敏度高100倍。我们相信我们的测定作为常规生物测定（具有成本效益，可靠且易于操作）的潜力非常大，可以对临床样品中的外泌体进行准确定量。
更新日期：2017-11-22
中文翻译：
通过二价胆固醇标记的DNA锚直接外泌体定量信号放大
外泌体作为细胞外囊泡（EVs）的重要亚群，在各种重要的病理生理过程中，尤其是与癌症相关的细胞间通讯中，发挥着重要作用。但是，可靠，方便的定量方法用于测定仍在技术上具有挑战性。在这项研究中，我们通过将免疫亲和力和脂质膜表面修饰结合到单个平台中来进行特异性分离和外泌体的准确定量，从而开发了一种有效而直接的方法。用免疫磁珠特异性捕获外泌体，然后将具有高亲和力的二价胆固醇（B-Chol）标记的DNA锚自发插入外泌体膜中。锚的合理设计的粘性末端可作为随后的辣根过氧化物酶（HRP）连接的杂交链反应（HCR）的引发剂，以进行信号放大。检测基于HRP催化的H的颜色变化2 O 2介导的3,3'，5,5'-四甲基联苯胺（TMB）的氧化，可以方便地用肉眼观察并通过紫外可见光谱法进行监测。与传统的ELISA相比，该方法可灵敏地检测每微升2.2×10 3个外泌体，相对标准偏差<5.6％，灵敏度高100倍。我们相信我们的测定作为常规生物测定（具有成本效益，可靠且易于操作）的潜力非常大，可以对临床样品中的外泌体进行准确定量。
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