Furtwangen University, Institute of Precision Medicine, Jakob-Kienzle-Straße 17, 78054, Villingen-Schwenningen, Germany; Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Biochimie. 2019 Mar;158:233-237. doi: 10.1016/j.biochi.2019.01.014. Epub 2019 Jan 25.
There is a great need for fast, simple and precise diagnostic assays capable of direct quantification of biomarkers in complex biological matrices. Yet, the commonly used techniques such as ELISA/Immunoassays are tedious and involve various steps e.g. blocking, washing and signal development. Moreover, most of these assays have very limited ability of detecting small molecules and have hardly any multiplexing capabilities. The gold standard and alternative, mass-spectrometry, however, depends upon expensive hardware and is incompatible with point of care (POC) diagnostics. As opposed to POC assays for proteins or larger targets where variable formats are readily available. Here, we present a simple, versatile and fast one-step assay for detecting a small molecule, ethanolamine as example. The assay makes use of commonly available qPCR machines to detect target-concentration dependent shifts in the melting temperatures of aptamer beacons. The method allows detection of ethanolamine in the low nM range without requiring tedious elaboration of assay conditions as required for molecular beacons at room temperature. If generalizable, it may change the situation of small molecule assays significantly.
非常需要快速、简单和精确的诊断检测方法，能够直接定量分析复杂生物基质中的生物标志物。然而，常用的技术，如 ELISA/免疫测定，繁琐且涉及各种步骤，例如阻断、洗涤和信号开发。此外，这些检测方法中的大多数对小分子的检测能力非常有限，几乎没有任何多重检测能力。然而，金标准和替代方法，即质谱法，依赖于昂贵的硬件，并且与即时检测（POC）诊断不兼容。与针对蛋白质或更大目标的 POC 检测方法不同，这些方法中易于获得各种格式。在这里，我们提出了一种简单、通用且快速的一步检测小分子的方法，以乙醇胺为例。该检测利用了常用的 qPCR 机器，检测适配体信标的熔点随目标浓度的变化。该方法可在低纳摩尔范围内检测乙醇胺，而无需像室温下的分子信标那样繁琐地精心设计检测条件。如果具有普遍性，它可能会显著改变小分子检测的情况。
