School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi'an 710021, China.
NHC Key Laboratory of Food Safety Risk Assessment, Food Safety Research Unit (2019RU014) of Chinese Academy of Medical Science, China National Center for Food Safety Risk Assessment, Beijing 100021, China.
Anal Chem. 2021 Feb 16;93(6):3217-3225. doi: 10.1021/acs.analchem.0c04732. Epub 2021 Feb 1.
The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction has drawn increasing attention in the field of analytical science. However, the poor stability of Cu(I) usually hinders not only the simplicity of the click reaction but also its applications in precise analyses. Therefore, the development of a nanocatalyst containing stable Cu(I) is of great significance for broadening the application of CuAAC-based assays. Herein, inspired by the active center structure of natural multicopper oxidases (MCOs), we successfully prepared a novel nanocatalyst containing abundant stable Cu(I) as an artificial "clickase" (namely, CCN) by using glutathione to stabilize Cu(I). The stability and enzyme-like catalytic activity in the CuAAC reaction of the prepared CCN clickase were studied, and the catalytic mechanism of the CCN clickase-mediated CuAAC reaction between 3-azide-7-hydroxycoumarin (Azide 1) and propargyl alcohol (Alkyne 2) was also revealed. Compared with the existing solid CuO nanocatalysts used in CuAAC-based assays, CCN clickases exhibited plenty of superior properties (including high stability, excellent catalytic activity, no requirements of dissolution and reducing agents/radical initiator during the detection, well-defined porosities benefiting the substrate diffusion, and good biocompatibility), which can greatly increase the reaction efficiency and shorten the detection time. Encouraged by these remarkable performances, CCN clickases were used as labels to establish a new catalytic click fluorescence immunoassay for foodborne pathogens. Notably, the proposed CCN clickase-based immunoassay exhibited high analytical performances for the quantification of in the linear range of 10-10 CFU/mL with a limit of detection as low as 11 CFU/mL. The developed method has also been used in the determination of in food samples, showing its great potential in the detection of foodborne pathogens.
铜（I）催化的叠氮化物 - 炔烃环加成（CuAAC）点击反应在分析科学领域引起了越来越多的关注。然而，Cu（I）的稳定性差不仅阻碍了点击反应的简单性，也阻碍了其在精确分析中的应用。因此，开发含有稳定 Cu（I）的纳米催化剂对于拓宽基于 CuAAC 的测定的应用具有重要意义。在这里，受天然多铜氧化酶（MCOs）活性中心结构的启发，我们成功地制备了一种新型纳米催化剂，该纳米催化剂含有丰富的稳定 Cu（I），用作人工“点击酶”（即 CCN），并使用谷胱甘肽稳定 Cu（I）。研究了制备的 CCN 点击酶在 CuAAC 反应中的稳定性和酶样催化活性，并揭示了 CCN 点击酶介导的 3-叠氮-7-羟基香豆素（叠氮化物 1）和丙炔醇（炔烃 2）之间的 CuAAC 反应的催化机制。与用于基于 CuAAC 的测定的现有固体 CuO 纳米催化剂相比，CCN 点击酶具有许多优越的性能（包括高稳定性、优异的催化活性、在检测过程中无需溶解和还原剂/自由基引发剂、明确的有利于底物扩散的孔隙率和良好的生物相容性），这可以大大提高反应效率并缩短检测时间。受到这些出色性能的鼓舞，CCN 点击酶被用作标签，建立了一种新的用于食源性病原体的催化点击荧光免疫分析。值得注意的是，所提出的基于 CCN 点击酶的免疫分析对于在 10-10 CFU/mL 的线性范围内定量 表现出高分析性能，检测限低至 11 CFU/mL。该方法还已用于食品样品中 的测定，显示出其在食源性病原体检测中的巨大潜力。
