Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification
Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification
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DOI：
10.1021/acs.analchem.6b02421
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发表时间：
2016-11-01
影响因子：
7.4
通讯作者：
Tosar, Juan Pablo
中科院分区：
文献类型：
--
作者：
Doldan, Ximena;Fagundez, Pablo;Tosar, Juan Pablo
细胞外囊泡（EV），即外泌体和微囊泡，是细胞间通讯途径的重要介质。由于EV可以在各种生物流体中检测到，并且包含一组特定的生物标志物，这些生物标志物使人联想到其亲本细胞，因此它们在临床诊断中显示出巨大的前景，因为EV分析可以在微创液体活检中进行。然而，仍然需要可靠、快速和具有成本效益的方法来确定这些指标，特别是如果打算进行分散分析。在这项研究中，我们开发了一种电化学生物传感器，其工作与1.5 μ L的样品体积，可以检测低至200外来体每微升，与跨越近4个数量级的线性范围。该传感器是特异性的，并且容易区分外泌体和含有1000倍过量的微泡的样品中的微泡。显示了检测真实的样品（稀释的血清）中的外来体的能力。这是通过将兔抗人CD 9抗体固定在金基底上并使用针对CD 9的单克隆抗体检测捕获的外来体来实现的。信号放大大概是从多个检测抗体结合到每个捕获的囊泡表面的事实中获得的。加入辣根过氧化物酶（HRP）偶联抗IgG抗体后，基于3，3 '，5，5'-四甲基联苯胺（TMB）的电化学还原进行检测。这种电流型生物传感器可以很容易地纳入未来的小型化和半自动装置的EV测定。
Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 mu L sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3',5,5'-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.