Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford , South Parks Road, Oxford OX1 3QZ, United Kingdom.
Nuffield Department of Clinical Neurosciences, University of Oxford , John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
Anal Chem. 2017 Mar 7;89(5):3184-3190. doi: 10.1021/acs.analchem.6b05037. Epub 2017 Feb 10.
Exosomes are both active in mediating intracellular communication and potentially present a potent cargo of disease biomarkers to an assay. The robust evaluation of exosomal markers could lead to a paradigm shift in clinical analysis and associated care. To date, much of this has been hindered by issues of sample preparation and assay signal-to-noise. We introduce here the use of ultrasensitive electrochemical impedance spectroscopy to quantify both external (tetraspanin) and internal (syntenin) exosome-specific markers. Associated exosome detection limits are 1.9 × 10 particles mL (equivalent to 320 aM or 9500 exosomes in 50 μL) for intact exosomes and 3-5 picomolar for internal exosomal syntenin levels with almost 5 decades of linear dynamic range. Sample preparation can be carried out by simple fine filtering of cell-conditioned medium prior to a non-NTA-determined (i.e., nanoparticle tracking analysis) exosome concentration analysis, lysing, and subsequent internal syntenin quantification. Such concentration-normalized dual-marker analysis can be used to define "analytical zones" in a manner which is then independent of absolute exosome concentration and sample preparation.
外泌体在介导细胞内通讯方面非常活跃，并且可能为检测提供大量疾病生物标志物的有效载荷。对外泌体标志物的有力评估可能会导致临床分析和相关护理的范式转变。迄今为止，这在很大程度上受到样品制备和检测信号噪声问题的阻碍。我们在这里介绍了使用超灵敏电化学阻抗谱来定量分析外泌体特异性的外部（四跨膜蛋白）和内部（衔接蛋白）标志物。相关的外泌体检测限为 1.9×10 个颗粒/mL（相当于 50μL 中完整外泌体的 320aM 或 9500 个外泌体），对于内部外泌体衔接蛋白水平的检测限为 3-5 皮摩尔，线性动态范围近 5 个数量级。样品制备可以通过在非 NTA 确定（即纳米颗粒跟踪分析）的外泌体浓度分析之前，用简单的精细过滤细胞条件培养基来完成，然后进行裂解，并随后对内源性衔接蛋白进行定量。这种浓度归一化的双标志物分析可以用于以一种不依赖于外泌体绝对浓度和样品制备的方式定义“分析区”。
