The Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165, PR China.
Biosens Bioelectron. 2013 Dec 15;50:351-5. doi: 10.1016/j.bios.2013.06.064. Epub 2013 Jul 5.
In this work, based on the fact that pyrophosphate (PPi) could regulate the activity of Zn(2+)-dependent DNAzyme, we for the first time report a fluorescence turn-on sensing system for alkaline phosphatase (ALP) with improved sensitivity via nonprotein-enzymatic signal amplification. A catalytic and molecular beacon (CAMB) design was employed to further improve its sensitivity. Taking advantage of the strong interactions between PPi and the Zn(2+), the cofactor Zn(2+) was caged, and the DNAzyme activity was effectively inhibited. The introduction of ALP, however, could catalyze the hydrolysis of PPi and release free Zn(2+), resulting in the activation of DNAzyme to catalyze the cleavage of the molecular beacon substrate with a remarkable increase of fluorescent signal. These optimized designs together allow a high sensitivity for ALP, with a detection limit of 20 pM observed, much lower than previously reported methods. It has also been used for detection of ALP in human serum with satisfactory results, demonstrating its potential applications in clinical diagnosis.
在这项工作中，基于焦磷酸根（PPi）能够调节 Zn(2+)-依赖性 DNA 酶活性这一事实，我们首次报道了一种通过非酶促信号放大提高灵敏度的荧光开启型碱性磷酸酶（ALP）传感系统。采用催化和分子信标（CAMB）设计进一步提高其灵敏度。利用 PPi 与 Zn(2+) 之间的强相互作用，将辅助因子 Zn(2+) 封闭，从而有效抑制 DNA 酶活性。然而，引入 ALP 可以催化 PPi 的水解并释放游离的 Zn(2+)，从而激活 DNA 酶，使其能够切割分子信标底物，荧光信号显著增强。这些优化设计共同实现了对 ALP 的高灵敏度检测，检测限低至 20 pM，明显低于先前报道的方法。该方法还用于人血清中 ALP 的检测，结果令人满意，表明其在临床诊断中有潜在的应用价值。
