CD9、CD63 和 CD81 四跨膜蛋白的并行 SPR 和 QCM-D 定量分析：确定从人肺癌细胞中分离的细胞外囊泡浓度的简单而灵敏的方法,Analytical Chemistry

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CD9、CD63 和 CD81 四跨膜蛋白的并行 SPR 和 QCM-D 定量分析：确定从人肺癌细胞中分离的细胞外囊泡浓度的简单而灵敏的方法
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-06-12 , DOI: 10.1021/acs.analchem.3c00772
Agata Kowalczyk ₁ , Aleksandra Gajda-Walczak ₁ , Monika Ruzycka-Ayoush ₂ , Alicja Targonska ₃ , Grazyna Mosieniak ₃ , Maciej Glogowski ₄ , Anna Szumera-Cieckiewicz ₅ , Monika Prochorec-Sobieszek ₅ , Magdalena Bamburowicz-Klimkowska ₂ , Anna M Nowicka ₁ , Ireneusz P Grudzinski ₂

Affiliation

四跨膜蛋白，包括 CD9、CD63 和 CD81，是跨膜生物标志物，在调节癌细胞增殖、侵袭和转移以及质膜动力学和蛋白质运输中发挥着至关重要的作用。在这项研究中，我们开发了简单、快速且灵敏的免疫传感器，以使用四跨膜蛋白作为生物标志物来测定从人肺癌细胞中分离的细胞外囊泡（EV）的浓度。我们采用表面等离子共振（SPR）和耗散石英晶体微天平（QCM-D）作为检测器。使用蛋白 A 传感器芯片 (SPR) 或不使用放大器修饰金晶体的半胱胺层 (QCM-D)，将靶向 CD9、CD63 和 CD81 的单克隆抗体在受体层中垂直定向。SPR 研究表明，EV 与抗体的相互作用可以通过二态反应模型来描述。此外，正如 QCM-D 研究所证实的，EV 对四跨膜蛋白单克隆抗体的亲和力按以下顺序降低：CD9、CD63 和 CD81。结果表明，所开发的免疫传感器具有稳定性高、分析范围宽（6.1×10^{4 个}颗粒·mL ^–1至 6.1 × 10 ^{7 个}颗粒·mL ^–1，检测限低（0.6–1.8）× 10 ^{4 个}颗粒·mL ^–1。使用 SPR 和 QCM-D 检测器获得的结果与纳米颗粒跟踪分析之间非常一致，表明所开发的免疫传感器可以成功应用于临床样本。

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Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells

Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs’ affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 10⁴ particles·mL^–1 to 6.1 × 10⁷ particles·mL^–1, and a low detection limit (0.6–1.8) × 10⁴ particles·mL^–1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

更新日期：2023-06-12

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