School of Life Sciences, Central South University, Changsha, 410013, China.
School of Life Sciences, Central South University, Changsha, 410013, China.
Mol Cell Probes. 2019 Feb;43:29-33. doi: 10.1016/j.mcp.2018.12.003. Epub 2018 Dec 17.
In this paper, we propose a fluorometric approach for the highly sensitive detection of inorganic pyrophosphatase (PPase) based on G-quadruplex-thioflavin T (ThT). In the absence of PPase, Cu can coordinate with pyrophosphate (PPi) to generate a Cu/PPi complex. Then the G-rich sequence folds into the G-quadruplex structure, which can combine with ThT to generate a remarkable fluorescent signal. In the presence of PPase, the coordinated compound can be destroyed by the PPase catalyzed hydrolysis of PPi into inorganic phosphate (Pi). The subsequent release of Cu can compete with ThT to induce a tighter G-quadruplex structure, causing the release of ThT and a sharp fluorescence decrease. Based on this mechanism, a facile and quantitative strategy for PPase detection was developed. The fluorescence intensity of the system shows a linear relationship with the PPase activities in the range of 0.5-30 U/L with a detection limit as low as 0.48 U/L. The proposed strategy for fluorescence spectrometric PPase detection is convenient, cost effective, and sensitive. This can be utilized to evaluate the inhibition effect of NaF on PPase as well as diagnose PPase-related diseases.
在本文中，我们提出了一种基于 G-四链体-硫代黄素 T（ThT）的荧光法来高灵敏检测无机焦磷酸酶（PPase）。在没有 PPase 的情况下，Cu 可以与焦磷酸盐（PPi）配位生成 Cu/PPi 配合物。然后富含 G 的序列折叠成 G-四链体结构，与 ThT 结合生成显著的荧光信号。在存在 PPase 的情况下，配合物可以被 PPase 催化水解 PPi 为无机磷酸盐（Pi）所破坏。随后释放的 Cu 可以与 ThT 竞争，诱导更紧密的 G-四链体结构，导致 ThT 的释放和荧光急剧下降。基于此机制，开发了一种简便、定量的 PPase 检测策略。该体系的荧光强度与 0.5-30 U/L 范围内的 PPase 活性呈线性关系，检测限低至 0.48 U/L。该荧光光谱法检测 PPase 的策略简便、经济高效且灵敏。这可用于评估 NaF 对 PPase 的抑制作用以及诊断与 PPase 相关的疾病。
