Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai 200237 , China.
Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences , Zhejiang University of Technology , Hangzhou 310014 , Zhejiang , China.
Anal Chem. 2018 Jul 3;90(13):8072-8079. doi: 10.1021/acs.analchem.8b01187. Epub 2018 Jun 20.
Exosomes, a class of small extracellular vesicles, play important roles in various physiological and pathological processes by serving as vehicles for transferring and delivering membrane and cytosolic molecules between cells. Since exosomes widely exist in various body fluids and carry molecular information on their originating cells, they are being regarded as potential noninvasive biomarkers. Nevertheless, the development of convenient and quantitative exosome analysis methods is still technically challenging. Here, we present a low-cost assay for direct capture and rapid detection of exosomes based on a copper-mediated signal amplification strategy. The assay involves three steps. First, bulk nanovesicles are magnetically captured by cholesterol-modified magnetic beads (MB) via hydrophobic interaction between cholesterol moieties and lipid membranes. Second, bead-binding nanovesicles of exosomes with a specific membrane protein are anchored with aptamer-modified copper oxide nanoparticles (CuO NPs) to form sandwich complexes (MB-exosome-CuO NP). Third, the resultant sandwich complexes are dissolved by acidolysis to turn CuO NP into copper(II) ions (Cu), which can be reduced to fluorescent copper nanoparticles (CuNPs) by sodium ascorbate in the presence of poly(thymine). The fluorescence emission of CuNPs increases with the increase of Cu concentration, which is directly proportional to the concentration of exosomes. Our method allows quantitative analysis of exosomes in the range of 7.5 × 10 to 1.5 × 10 particles/μL with a detection of limit of 4.8 × 10 particles/μL in biological sample. The total working time is about 2 h. The assay has the potential to be a simple and cost-effective method for routine exosome analysis in biological samples.
外泌体是一类小细胞外囊泡，通过充当细胞间膜和胞质分子的转移和传递载体，在各种生理和病理过程中发挥重要作用。由于外泌体广泛存在于各种体液中，并携带其来源细胞的分子信息，因此它们被认为是潜在的非侵入性生物标志物。然而，开发方便和定量的外泌体分析方法在技术上仍然具有挑战性。在这里，我们提出了一种基于铜介导的信号放大策略的低成本外泌体直接捕获和快速检测方法。该测定法包括三个步骤。首先，通过胆固醇修饰的磁性珠（MB）与脂质膜之间的疏水相互作用，通过磁性捕获大量纳米囊泡。其次，具有特定膜蛋白的结合珠的外泌体与适配体修饰的氧化铜纳米颗粒（CuO NPs）锚定，形成三明治复合物（MB-外泌体-CuO NP）。第三，所得的三明治复合物通过酸解溶解，将 CuO NP 转化为铜(II)离子（Cu），在存在多（胸腺嘧啶）的情况下，铜(II)离子被抗坏血酸钠还原为荧光铜纳米颗粒（CuNPs）。CuNPs 的荧光发射强度随 Cu 浓度的增加而增加，与外泌体的浓度成正比。我们的方法允许在 7.5×10 到 1.5×10 个颗粒/μL 的范围内对外泌体进行定量分析，在生物样本中的检测限为 4.8×10 个颗粒/μL。总工作时间约为 2 小时。该测定法有可能成为生物样品中外泌体常规分析的简单且具有成本效益的方法。
