College of Pharmacy, Seoul National University, Seoul, 08826, South Korea.
Department of Chemistry, Seoul National University, Seoul, 08826, South Korea.
Mikrochim Acta. 2019 Mar 4;186(4):211. doi: 10.1007/s00604-019-3317-9.
A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2-3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors. Graphical abstract A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.
一种基于氧化石墨烯（GO）的经济高效、自动化条带测试已被开发用于筛选内切酶 EcoRV 的抑制剂。该方法涉及使用 GO 和 EcoRV 的 DNA 底物，该底物包含一个用于与 GO 结合的 ssDNA 区域和一个荧光素 amidite（FAM）标记的 dsDNA。所有成分都通过喷墨打印在一张羊皮纸上。ssDNA 区域与 GO 表面结合并固定，从而使 FAM 的荧光被猝灭。然后将羊皮纸条与含有 EcoRV 的样品一起孵育，导致酶解，dsDNA 从 GO 上分离。结果，在反应点产生绿色荧光。可以在存在和不存在作为 EcoRV 抑制剂的金精三羧酸的情况下测量酶活性。与标准微孔板测定相比，该方法的优点是所需试剂少 2-3 个数量级。因此，它是一种基于 GO 的 EcoRV 酶抑制剂筛选的有效平台。
