Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
Analyst. 2022 Mar 28;147(7):1348-1356. doi: 10.1039/d2an00145d.
Ethanolamine is an important analyte for environmental chemistry and biological sciences. A few DNA aptamers were previously reported for binding ethanolamine with a dissociation constant () as low as 9.6 nM. However, most of the previous binding assays and sensing work used either immobilized ethanolamine or immobilized aptamers. In this work, we studied three previously reported DNA sequences, two of which were supposed to bind ethanolamine while the other could not bind. Isothermal titration calorimetry revealed no binding for any of these sequences. In addition, due to their guanine-rich sequences, thioflavin T was used as a probe. Little fluorescence change was observed with up to 1 μM ethanolamine. Responses within the millimolar range of ethanolamine were attributed to the general fluorescence quenching effect of ethanolamine instead of aptamer binding. Finally, after studying the adsorption of ethanolamine to gold nanoparticles (AuNPs), we confirmed the feasibility of using AuNPs as a probe when the concentration of ethanolamine was below 0.1 mM. However, no indication of specific aptamer binding was observed by comparing the three DNA sequences for their color changing trends. This work articulates the importance of careful homogeneous binding assays using free target molecules.
乙醇胺是环境化学和生物科学的重要分析物。先前已有少数 DNA 适体被报道可与乙醇胺结合，解离常数（）低至 9.6 nM。然而，大多数先前的结合分析和传感工作都使用固定化的乙醇胺或固定化的适体。在这项工作中，我们研究了三个先前报道的 DNA 序列，其中两个被认为可以结合乙醇胺，而另一个则不能。等温滴定量热法显示这些序列都没有结合。此外，由于其富含鸟嘌呤的序列，我们使用了噻唑橙 T 作为探针。即使添加高达 1 μM 的乙醇胺，也几乎观察不到荧光变化。在乙醇胺的毫摩尔范围内的响应归因于乙醇胺的一般荧光猝灭效应，而不是适体结合。最后，在研究了乙醇胺在金纳米粒子（AuNPs）上的吸附后，我们证实了当乙醇胺浓度低于 0.1 mM 时，使用 AuNPs 作为探针是可行的。然而，通过比较三个 DNA 序列的颜色变化趋势，我们没有观察到特定适体结合的迹象。这项工作阐明了使用游离靶分子进行仔细的均相结合分析的重要性。
