Zhang Jiarui, Zhao Wenbo, Zhang Wei, Liu Yizhou, Qin Yang, Zhang Wenkai, Zhou Zhiyuan, Zhou Yujie, Wang Hongbo, Xiao Xianjin, Wu Tongbo
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
State Key Laboratory of NBC Protection for Civilian, Beijing, 102205, China.
Anal Chim Acta. 2020 Oct 23;1135:64-72. doi: 10.1016/j.aca.2020.08.040. Epub 2020 Aug 27.
Alkaline phosphatase (ALP), which converts the phosphate group (-PO) in the substrate to the hydroxyl group (-OH), is a useful tool in the biological analysis, a good indicator of dissolved inorganic phosphorus levels and an important biomarker for several diseases. In conventional designs for ALP detection, both the interferent with a -PO and the target with a -OH will go into the sensing path and give out the undesired background and the desired signal respectively. This limited the sensitivity of the method and required the complicated design to achieve a satisfying limit of detection (LOD) of ALP. Here, we provided a new sensing strategy for ALP detection design. We designed a path-choice-based biosensor with two DNA tracks in which ALP works as the switch to guide the reaction path of lambda exonuclease (λ exo). The path-choice character enlarged the difference between signal and background by separating the interferent removing path and the target sensing path. The substrate preference of ALP and λ exo was studied to optimize the structure of DNA tracks. The path-choice-based biosensor achieved simple, fast (30 min), sensitive (LOD 0.014 U L) and selective detection of the activity of ALP. The method has been applied to detect the activity of ALP in cell lysates, which shows the potential application in ALP-related biological research.
碱性磷酸酶（ALP）可将底物中的磷酸基团（-PO）转化为羟基（-OH），是生物分析中的一种有用工具，是溶解无机磷水平的良好指标，也是多种疾病的重要生物标志物。在传统的ALP检测设计中，含有-PO的干扰物和含有-OH的靶标都会进入传感路径，分别产生不需要的背景信号和所需信号。这限制了该方法的灵敏度，并且需要复杂的设计才能实现令人满意的ALP检测限（LOD）。在此，我们提供了一种用于ALP检测设计的新传感策略。我们设计了一种基于路径选择的生物传感器，其具有两条DNA轨道，其中ALP作为开关来引导λ外切核酸酶（λ exo）的反应路径。通过分离干扰物去除路径和靶标传感路径，路径选择特性扩大了信号与背景之间的差异。研究了ALP和λ exo的底物偏好性以优化DNA轨道的结构。基于路径选择的生物传感器实现了对ALP活性的简单、快速（30分钟）、灵敏（LOD 0.014 U L）和选择性检测。该方法已应用于检测细胞裂解物中ALP的活性，这表明其在与ALP相关的生物学研究中的潜在应用。
