Department of Chemistry, University of North Dakota, Grand Forks, ND 58202, USA.
Analyst. 2019 Oct 22;144(21):6231-6239. doi: 10.1039/c9an01283d.
The 3'-5' exonuclease enzyme plays a dominant role in multiple pivotal physiological activities, such as DNA replication and repair processes. In this study, we designed a sensitive graphene oxide (GO)-based probe for the detection of exonuclease enzymatic activity. In the absence of Exo III, the strong π-π interaction between the fluorophore-tagged DNA and GO causes the efficient fluorescence quenching via a fluorescence resonance energy transfer (FRET). In contrast, in the presence of Exo III, the fluorophore-tagged 3'-hydroxyl termini of the DNA probe was digested by Exo III to set the fluorophore free from adsorption when GO was introduced, causing an inefficient fluorescence quenching. As a result, the fluorescence intensity of the sensor was found to be proportional to the concentration of Exo III; towards the detection of Exo III, this simple GO-based probe demonstrated a highly sensitive and selective linear response in the low detection range from 0.01 U mL-1 to 0.5 U mL-1 and with the limit of detection (LOD) of 0.001 U mL-1. Compared with other fluorescent probes, this assay exhibited superior sensitivity and selectivity in both buffer and fetal bovine serum samples, in addition to being cost effective and having a simple setup.
3'-5'外切核酸酶在多种关键生理活动中发挥主导作用，如 DNA 复制和修复过程。在本研究中，我们设计了一种基于氧化石墨烯（GO）的灵敏探针用于检测外切核酸酶活性。在没有 Exo III 的情况下，荧光标记的 DNA 与 GO 之间的强π-π相互作用通过荧光共振能量转移（FRET）导致有效的荧光猝灭。相比之下，在存在 Exo III 的情况下，DNA 探针的荧光标记的 3'-羟基末端被 Exo III 消化，当引入 GO 时，荧光团从吸附中释放出来，导致荧光猝灭效率降低。结果，传感器的荧光强度与 Exo III 的浓度成正比；对于 Exo III 的检测，这种简单的基于 GO 的探针在低检测范围内（0.01 U mL-1 至 0.5 U mL-1）表现出高度灵敏和选择性的线性响应，检测限（LOD）为 0.001 U mL-1。与其他荧光探针相比，该测定法在缓冲液和胎牛血清样品中均表现出优异的灵敏度和选择性，此外还具有成本效益和简单的设置。
