Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai, PR China.
Biosens Bioelectron. 2011 Sep 15;27(1):178-82. doi: 10.1016/j.bios.2011.06.047. Epub 2011 Jul 7.
In this work we have developed a novel electrochemical biosensor for the detection of alkaline phosphatase (AP) by the use of two complementary DNA probes (DNA 1 and DNA 2) coupled with λ exonuclease (λ exo). Firstly, the 5'-phosphoryl end of DNA 1 is dephosphorylated by AP. Then DNA 1 hybridizes with DNA 2, previously modified on a gold electrode surface. In this double-strand DNA, DNA 2 strand will be promptly cleaved by λ exo with its phosphoryl at the 5' end. After the DNA 2 strand is completely digested, DNA 1 will be released from the double strands and then hybridizes with another DNA 2 strand on the electrode surface, thus the cycle of the release of DNA 1 and the digestion of DNA 2 continues. Since the DNA probes may absorb hexaammineruthenium(III) chloride, the electrochemical species, and the removal of the DNA 2 strand from the electrode surface will result in the decrease of the detected electrochemical signal, which is initially activated by AP, an electrochemical biosensor to assay the activity of AP is proposed in this work. This method may have a linear detection range from 1 to 20 unit/mL with a detection limit of 0.1 unit/mL, and the detection of the enzymatic activity in complex biological fluids can also be realized.
在这项工作中，我们开发了一种新颖的电化学生物传感器，用于通过使用两种互补的 DNA 探针（DNA1 和 DNA2）与 λ 外切核酸酶（λexo）结合来检测碱性磷酸酶（AP）。首先，AP 将 DNA1 的 5'-磷酸基团去磷酸化。然后，DNA1 与先前修饰在金电极表面的 DNA2 杂交。在这种双链 DNA 中，DNA2 链将被 λexo 迅速切割，其 5'端带有磷酸基团。DNA2 链完全消化后，DNA1 将从双链中释放出来，然后与电极表面上的另一个 DNA2 链杂交，从而释放 DNA1 和消化 DNA2 的循环继续进行。由于 DNA 探针可能会吸附六氨合钌（III）氯化物，电化学物质，并且 DNA2 链从电极表面的去除会导致检测到的电化学信号减少，这最初是由 AP 激活的，因此在这项工作中提出了一种用于测定 AP 活性的电化学生物传感器。该方法的线性检测范围为 1 至 20 单位/mL，检测限为 0.1 单位/mL，并且可以实现复杂生物流体中酶活性的检测。
