Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.
Department of Electrical Engineering, Stanford University, Stanford, CA, 94305, USA.
Nat Commun. 2023 Jul 13;14(1):4192. doi: 10.1038/s41467-023-39772-z.
Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3-4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved-in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two-pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our 'EVROS' strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 µl sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion.
精准医学需要高度可扩展的多重生物标志物定量方法，能够准确描述患者的生理状况。然而，当前的分子检测方法通常仅限于灵敏度动态范围跨越 3-4 个数量级，而人类血浆蛋白质组的实际生理动态范围跨越了 10 个数量级以上。当前的方法依赖于样品分割和差分稀释来弥补这种不匹配，但这些措施极大地限制了可实现的重现性和可扩展性——特别是，非线性稀释的影响会极大地混淆多重分析的分析。我们在这里描述了一种双重策略，用于平衡多重分析面板中每个分析物产生的信号，从而能够同时定量检测跨越广泛浓度范围的靶标。我们将我们的“EVROS”策略应用于邻近连接测定，并证明了在未稀释的人血清中浓度跨度从低飞摩尔到中纳摩尔的四个分析物的同时定量。在这个初步的演示中，我们在单个未稀释的 5μl 人血清样本中实现了跨越七个数量级的动态范围，突出了以高度多重化的方式实现对各种分析物的灵敏、准确检测的机会。
