Laboratory of Quality & Safety Risk Assessment for Citrus Products (Chongqing), Ministry of Agriculture, Citrus Research Institute, Southwest University, Chongqing 400712, China; National Citrus Engineering Research Center, Chongqing 400712, China.
Laboratory of Quality & Safety Risk Assessment for Citrus Products (Chongqing), Ministry of Agriculture, Citrus Research Institute, Southwest University, Chongqing 400712, China; National Citrus Engineering Research Center, Chongqing 400712, China.
Talanta. 2017 Sep 1;172:171-175. doi: 10.1016/j.talanta.2017.05.041. Epub 2017 May 17.
Herein, we are presenting an approach that utilizes the λ exonuclease (λ exo) cleavage reaction in combination with the formation of G-quadruplex, thereby providing a label-free fluorometric tool for simply and accurately determining alkaline phosphatase (ALP) activity and inhibition. A hairpin probe (HP) with 5'-phosphoryl termini and 3'-end containing a G-rich region, is designed. Taking advantage of the efficient enzyme reactions, namely the λ exo cleavage reaction, the G-rich DNA fragment is released from HP and folds into a stable G-quadruplex in the presence of potassium ions, thus greatly enhance the fluorescence of N-methyl mesoporphyrin IX (NMM) (a specific G-quadruplex binder). However, in the presence of ALP, the 5'-phosphoryl of the HP is dephosphorylated. The yielding 5'-hydroxyl end product hampers the λ exo cleavage reaction. HP maintains its stem-loop structure. Thus the formation of the G-quadruplex is prohibited, and this results in weak fluorescence of NMM. The fluorescence intensity exhibits a linear correlation to ALP concentration in the range of 1-50 U/L with a detection limit of 0.75 U/L. Additionally, inhibition effect of sodium orthovanadate has also been investigated. This study offers a simple yet sensitive method for ALP activity assay.
在此，我们提出了一种利用 λ 核酸外切酶（λ exo）切割反应与 G-四链体形成相结合的方法，从而提供了一种简单、准确测定碱性磷酸酶（ALP）活性和抑制的无标记荧光工具。设计了具有 5'-磷酸基团末端和 3'-末端含有富含 G 区的发夹探针（HP）。利用高效的酶反应，即 λ exo 切割反应，在钾离子存在下，富含 G 的 DNA 片段从 HP 中释放出来并折叠成稳定的 G-四链体，从而大大增强了 N-甲基 mesoporphyrin IX（NMM）（一种特异性 G-四链体结合物）的荧光。然而，在存在 ALP 的情况下，HP 的 5'-磷酸基团被去磷酸化。产生的 5'-羟基末端产物阻碍了 λ exo 切割反应。HP 保持其茎环结构。因此，G-四链体的形成受到抑制，这导致 NMM 的荧光较弱。荧光强度与 ALP 浓度在 1-50 U/L 范围内呈线性相关，检测限为 0.75 U/L。此外，还研究了偏钒酸钠的抑制作用。本研究提供了一种简单而灵敏的 ALP 活性测定方法。
