College of Chemistry, Zhengzhou University, Zhengzhou, 450001, PR China.
College of Chemistry, Zhengzhou University, Zhengzhou, 450001, PR China.
Anal Chim Acta. 2020 Mar 1;1100:40-46. doi: 10.1016/j.aca.2019.12.008. Epub 2019 Dec 5.
In this work, a dual amplified signal enhancement approach based on coupling deoxyribozyme (DNAzyme)-driven bipedal DNA walkers (BDW) and terminal deoxynucleotidyl transferase (TdT)-mediated DNA elongation signal amplifications has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. In presence of thrombin, the BDW complex, which is comprised from the target thrombin and two DNAzyme-containing probes, can exhibit autonomous cleavage behavior on the surface of the substrate DNA (SD) modified electrode, and remove the cleaved DNA fragment from the electrode surface. Subsequently, the TdT can catalyze the elongation of the SD with free 3'-OH termini and formation of many G-quadruplex sequence replicates with the presence of 2'-deoxyaguanosine-5'-triphosphate (dGTP) and adenosine 5'-triphosphate (dATP) at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a "signal-on" and completely label-free fashion. Under optimized conditions, the response peak current was linear with the concentration of thrombin in the range from 0.5 pM to 100000 pM with detection limit of 0.31 pM. This research provides us a sustainable idea for the hyphenated multiple amplification strategies and a stable and effective method for the detection of protein biomarkers.
在这项工作中，开发了一种基于双信号增强的方法，该方法基于脱氧核酶（DNAzyme）驱动的双足 DNA walker（BDW）和末端脱氧核苷酸转移酶（TdT）介导的 DNA 延伸信号放大，用于在人血清中高灵敏和无标记电化学检测凝血酶。在凝血酶存在的情况下，BDW 复合物（由靶标凝血酶和两个包含 DNAzyme 的探针组成）可以在修饰电极的底物 DNA（SD）表面上表现出自动切割行为，并从电极表面去除切割的 DNA 片段。随后，TdT 可以催化 SD 的延伸，在存在 2'-脱氧鸟苷-5'-三磷酸（dGTP）和腺苷 5'-三磷酸（dATP）的情况下，形成许多 G-四链体序列的复制，摩尔比为 6:4。这些 G-四链体序列结合血红素，并以“信号开启”和完全无标记的方式产生灵敏检测凝血酶的急剧放大电流响应。在优化条件下，响应峰电流在 0.5 pM 至 100000 pM 的凝血酶浓度范围内呈线性关系，检测限为 0.31 pM。这项研究为杂交多重扩增策略提供了一个可持续的思路，并为蛋白质生物标志物的检测提供了一个稳定有效的方法。
