Yang Yupeng, Metem Prattakorn, Khaksaran Mohammad Hadi, Sahu Siddharth Sourabh, Stridfeldt Fredrik, Görgens André, Zhang Shi-Li, Dev Apurba
Division of Solid-State Electronics, Department of Electrical Engineering, The Ångström Laboratory, Uppsala University, SE-751 03 Uppsala, Sweden.
Division of Applied Electrochemistry, Department of Chemical Engineering, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden.
ACS Omega. 2024 Dec 18;9(52):51022-51030. doi: 10.1021/acsomega.4c05492. eCollection 2024 Dec 31.
Extracellular vesicles (EVs) are nanoparticles encapsulated with a lipid bilayer, and they constitute an excellent source of biomarkers for multiple diseases. However, the heterogeneity in their molecular compositions constitutes a major challenge for their recognition and profiling, thereby limiting their application as an effective biomarker. A single-EV analysis technique is crucial to both the discovery and the detection of EV subpopulations that carry disease-specific signatures. Herein, a plasmonic nanohole array is designed for capturing single EVs and subsequently performing fluorescence detection of their membrane proteins by exploiting plasmonic amplification of the fluorescence signal. Unlike other reported methods, our design relies on an exclusive detection of single EVs captured inside nanoholes, thus allowing us to study only plasmonic effects and avoid other metal-induced phenomena while leveraging on the proximity of emitters to the plasmonic hotspots. The method is optimized through numerical simulations and verified by a combination of atomic force, scanning electron microscopy, and fluorescence microscopy. Fluorescence enhancement is then estimated by measuring the CD9 expression of small EVs derived from the human embryonic kidney (HEK293) cell line and carefully considering the spatial distribution of emission and excitation intensities. Fluorescence intensities of immunostained EVs show a moderate overall enhancement of intensity and follow the intensity trend predicted by simulation for nanohole arrays with different nanohole periods. Moreover, the number of observed EVs in the best-performing nanohole array increases by more than 12 times compared with EVs immobilized on a reference substrate, uncovering a vast amount of weakly fluorescent EVs that would remain undetected with the regular fluorescent method. Our nanohole array provides a basis for a future platform of single-EV analyses, also promising to capture the signature arising from low-expressing proteins.
细胞外囊泡（EVs）是包裹在脂质双层中的纳米颗粒，是多种疾病生物标志物的优质来源。然而，其分子组成的异质性对其识别和分析构成了重大挑战，从而限制了它们作为有效生物标志物的应用。单EV分析技术对于发现和检测携带疾病特异性特征的EV亚群至关重要。在此，设计了一种等离子体纳米孔阵列，用于捕获单个EV，随后通过利用荧光信号的等离子体放大对其膜蛋白进行荧光检测。与其他报道的方法不同，我们的设计依赖于对捕获在纳米孔内的单个EV的独家检测，从而使我们能够仅研究等离子体效应，并在利用发射体与等离子体热点的接近性时避免其他金属诱导的现象。该方法通过数值模拟进行了优化，并通过原子力显微镜、扫描电子显微镜和荧光显微镜的组合进行了验证。然后通过测量源自人胚肾（HEK293）细胞系的小EV的CD9表达，并仔细考虑发射和激发强度的空间分布，来估计荧光增强。免疫染色EV的荧光强度总体上显示出适度的增强，并遵循模拟预测的不同纳米孔周期的纳米孔阵列的强度趋势。此外，与固定在参考底物上的EV相比，性能最佳的纳米孔阵列中观察到的EV数量增加了12倍以上，揭示了大量用常规荧光方法无法检测到的弱荧光EV。我们的纳米孔阵列为未来的单EV分析平台提供了基础，也有望捕获低表达蛋白产生的特征。
