Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry , Northeast Normal University , 5268 Renmin Street , Changchun , Jilin Province 130024 , People's Republic of China.
Anal Chem. 2018 Mar 20;90(6):4071-4078. doi: 10.1021/acs.analchem.7b05353. Epub 2018 Feb 28.
A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with "egg-box" structure is triggered inside a capillary with releasing of Ca by changing the pH of the sol solution, which is accomplished in situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for online CE assays with immobilized enzymes, which can be widely applied in bioanalysis.
一种新型的毛细管电泳集成固定化酶反应器（CE-integrated IMER）是通过单步原位乙酰胆碱酯酶（AChE）介导的藻酸盐凝胶化和酶包封来开发的。通过 AChE 催化乙酰硫代胆碱的水解反应产生乙酸，在毛细管内改变溶胶溶液的 pH 值来引发具有“蛋盒”结构的藻酸盐凝胶化。AChE 和溶胶溶液中最初包含的任何其他酶（例如黄嘌呤氧化酶（XO））都被有效地包封在凝胶网络中，形成无需任何额外操作过程的 CE-integrated IMER。该方法便于根据用于 CE 分析的注入的底物使用相同的 IMER 分析不同种类的酶。溶胶混合物中约 68%的原始酶可以被包封，表明 CE-integrated IMER 具有高的载量能力。IMER 表现出优异的日内和日间稳定性以及批间重现性，这些特性表明所提出的 IMER 可用于准确的在线酶分析，具有可靠性。分析了固定化 AChE 和 XO 的酶活性和抑制作用，并将结果与使用游离酶的结果进行了比较。通过成功地将 IMER 应用于苹果汁样品中有机磷农药的检测，证明了该方法在实际样品分析中的潜在应用的可行性。该方法是一种用于在线 CE 分析的简单、高效和通用的固定化酶方法，可广泛应用于生物分析中。
