Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles.
Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles.
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DOI：
10.1021/acsami.1c14506
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发表时间：
2021-10
影响因子：
9.5
通讯作者：
Seonhwa Lee;B. Crulhas;S. Šuvakov;S. Verkhoturov;D. Verkhoturov;M. Eller;H. Malhi;V. Garovic;E. Schweikert;Gulnaz Stybayeva;A. Revzin
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作者：
Seonhwa Lee;B. Crulhas;S. Šuvakov;S. Verkhoturov;D. Verkhoturov;M. Eller;H. Malhi;V. Garovic;E. Schweikert;Gulnaz Stybayeva;A. Revzin
细胞外囊泡（EV）是由多种细胞分泌的脂质双层颗粒。EV携带亲本细胞的分子信息，并为早期疾病诊断提供了相当大的希望。本文描述了一种通用的策略，用于EV表面蛋白的多重免疫传感，重点是表面标志物CD 63，CD 81，nephrin和podocin来证明这一概念。这种传感策略需要用两种类型的抗体功能化金纳米颗粒（AuNPs），然后用金属离子（Pb 2+或Cu 2+）进行标记。金属离子充当氧化还原报告物，在Pb 2+和Cu 2+的电化学氧化期间分别在-0.23 V和0.28 V（vs Ag/AgCl）处产生独特的氧化还原峰。在工作电极上捕获EV，然后用免疫探针和方波伏安法标记，产生与EV浓度和EV表面标记物表达水平成比例的氧化还原电流。重要的是，免疫探针的金属离子标记使得能够从同一电极同时检测两个EV表面标记。我们证明了来自尿EV的CD 63/CD 81或podocin/nephrin表面标志物的双重检测。NP-激活的免疫测定对CD 63-和CD 81-表达的EV的灵敏度分别为2.46 × 105个粒子/mL（或40.3 pg/mL）和5.80 × 105个粒子/mL（或47.7 pg/mL），线性范围为4个数量级。podocin和nephrin的检测限分别为3.1和3.8 pg/mL。在未来，可以通过扩展用于AuNP的氧化还原标记的金属离子库来增加多路复用的能力。
Extracellular vesicles (EVs) are lipid bilayer particles secreted from various cells. EVs carry molecular information of parent cells and hold considerable promise for early disease diagnostics. This paper describes a general strategy for multiplexed immunosensing of EV surface proteins, focusing on surface markers CD63, CD81, nephrin, and podocin to prove the concept. This sensing strategy entailed functionalizing gold nanoparticles (AuNPs) with two types of antibodies and then tagging with metal ions, either Pb2+ or Cu2+. The metal ions served as redox reporters, generating unique redox peaks at -0.23 and 0.28 V (vs Ag/AgCl) during electrochemical oxidation of Pb2+ and Cu2+, respectively. Capture of EVs on the working electrode, followed by labeling with immunoprobes and square wave voltammetry, produced redox currents proportional to concentrations of EVs and levels of expression of EV surface markers. Importantly, metal-ion tagging of immunoprobes enabled detection of two EV surface markers simultaneously from the same electrode. We demonstrated dual detection of either CD63/CD81 or podocin/nephrin surface markers from urinary EVs. The NP-enabled immunoassay had a sensitivity of 2.46 × 105 particles/mL (or 40.3 pg/mL) for CD63- and 5.80 × 105 particles/mL (or 47.7 pg/mL) for CD81-expressing EVs and a linear range of four orders of magnitude. The limit of detection for podocin and nephrin was 3.1 and 3.8 pg/mL, respectively. In the future, the capacity for multiplexing may be increased by extending the repertoire of metal ions used for redox tagging of AuNPs.