Interdisciplinary Nanoscience Center (iNANO), Faculty of Science and Technology , Aarhus University , Gustav Wieds Vej 14 , 8000 Aarhus C, Denmark.
ACS Sens. 2018 Oct 26;3(10):2104-2111. doi: 10.1021/acssensors.8b00662. Epub 2018 Oct 15.
Electrochemical enzyme-linked sandwich assays on magnetic beads (MBs) refer to one of the most sensitive approaches for analysis of nonamplified nucleic acid samples, with redox enzymes being routinely used as labels. Here, we report a sensitive and inexpensive electrochemical nucleic acid sandwich assay on MBs that exploits a hydrolytic enzyme cellulase as a label, while MBs are used for preconcentration and bioseparation of analyzed samples. Binding of target DNA or RNA to capture DNA-modified MB triggers sandwich assembly and its labeling with cellulase. Application of the assembled sandwich to the electrodes covered with insulating nitrocellulose films induces film digestion by the cellulase label and pronounced changes in the electrical properties of the electrodes, the extent of the changes being proportional to the concentration of the analyzed nucleic acids. Down to 1 amol of Lactobacillus brevis specific synthetic DNA and rRNA isolated from L. brevis cells could be detected in 1 mL samples in the overall from 2 to 3 h assay. The assay is universal and can be adapted for point-of-care-testing and for in-field environmental and microbiomic analysis of unamplified samples of any DNA/RNA sequences.
基于磁珠的电化学酶联夹心检测法是一种最灵敏的分析非扩增核酸样本的方法之一，通常使用氧化还原酶作为标记物。在这里，我们报告了一种基于磁珠的灵敏且廉价的电化学核酸夹心检测法，该方法利用水解酶纤维素酶作为标记物，同时磁珠用于分析样品的预浓缩和生物分离。目标 DNA 或 RNA 与捕获 DNA 修饰的磁珠结合触发夹心组装，并与纤维素酶标记。将组装好的夹心应用于覆盖有绝缘硝化纤维素膜的电极上，纤维素酶标记物诱导膜消化，并显著改变电极的电性能，变化程度与分析核酸的浓度成正比。在整个 2 到 3 小时的检测中，在 1 毫升样品中可以检测到低至 1 飞摩尔的来自短乳杆菌细胞的特异性合成短乳杆菌 DNA 和 rRNA。该检测方法具有通用性，可适用于即时检测以及对未经扩增的任何 DNA/RNA 序列的现场环境和微生物组学分析。
