A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonuclease-mediated signal amplification.
A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonuclease-mediated signal amplification.
复制标题
DOI：
10.1039/c5an00516g
复制
发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Lianfang Zhang;Ting Hou;Haiyin Li;Feng Li
中科院分区：
文献类型：
--
作者：
Lianfang Zhang;Ting Hou;Haiyin Li;Feng Li
碱性磷酸酶（alkaline phosphatase，ALP）是一类催化多种底物去磷酸化的酶，是临床上最常用的酶之一，也是许多人类疾病的重要生物标志物。本文提出了一种基于单分子信标启动T7核酸外切酶辅助信号放大的ALP活性均相电化学生物传感方法。巧妙设计了一种3 ′-磷酸化5 ′-亚甲基蓝（MB）标记的发夹探针（HP）。在ALP存在下，HP发生去磷酸化，随后发生Klenow片段（KF）聚合酶催化的延伸和HP双链体茎的T7外切核酸酶催化的消化，释放MB标记的单核苷酸和触发DNA（tDNA）。然后，tDNA与另一个HP杂交，并启动随后的循环切割过程。因此，释放大量MB标记的单核苷酸肽，产生朝向ALP活性测定的显著放大的电化学信号。直接测量的检测限低至0.1 μ L（-1），这是可比的荧光方法和高达三个数量级低于以前报道的固定化为基础的电化学策略。除了高灵敏度和良好的选择性外，所提出的策略还具有简单和方便的优点，因为测定在均相溶液中进行，并且避免了复杂的电极修饰过程。因此，我们提出的均相电化学方法是一个理想的候选人ALP活性检测在生化研究和临床实践。
Alkaline phosphatase (ALP), a class of enzymes that catalyzes the dephosphorylation of a variety of substrates, is one of the most commonly assayed enzymes in routine clinical practice, and an important biomarker related to many human diseases. Herein, a facile and highly sensitive homogeneous electrochemical biosensing strategy was proposed for the ALP activity detection based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. One 3'-phosphorylated and 5'-methylene blue (MB) labeled hairpin probe (HP) is ingeniously designed. In the presence of ALP, the dephosphorylation of HP, the subsequent Klenow fragment (KF) polymerase-catalyzed elongation and T7 exonuclease-catalyzed digestion of the duplex stem of HP take place, releasing MB-labeled mononucleotides and the trigger DNA (tDNA). tDNA then hybridizes with another HP and initiates the subsequent cycling cleavage process. As a result, a large amount of MB-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ALP activity assay. A directly measured detection limit as low as 0.1 U L(-1) is obtained, which is comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy previously reported. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of simplicity and convenience, because the assay is carried out in the homogeneous solution phase and sophisticated electrode modification processes are avoided. Therefore, the homogeneous electrochemical method we proposed here is an ideal candidate for ALP activity detection in biochemical research and clinical practices.