Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, 106, Taiwan; Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, Taipei, 106, Taiwan.
Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, 106, Taiwan; Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, Taipei, 106, Taiwan.
Biosens Bioelectron. 2022 May 1;203:114049. doi: 10.1016/j.bios.2022.114049. Epub 2022 Feb 3.
Aminopeptidase N (APN/CD13) plays an important role in the growth and metastasis, of tumor, and is a potential biomarker for the post-treatment surveillance of cancer reoccurrence and progression of various malignancies. Thus, we have designed and prepared a convenient and ultrasensitive APN-targeting activity-based ratiometric electrochemical molecular substrate (Ala-AFC) for direct real-time monitoring of APN activity in biosamples. The APN in our experiment was used to hydrolyze the alanine moiety of the Ala-AFC probe and, as a result of this hydrolysis, realize concomitantly a cascade reaction to unmask the electrochemical reporter N-alkylated amino ferrocene (AAF). The Ala-AFC probe exhibited high sensitivity with a wide detection range of 0.05-110 ng mL and a low limit of detection of 23.18 pg mL. The electrochemical signals were found to be distinctly specific for APN and free of interference from other electroactive biological species. Furthermore, the Ala-AFC probe was employed to monitor and quantify, in real-time, the activity of APN in tumor cells, whole blood, and urine. In addition, the results of our direct electrochemical quantifications of the amount of APN in whole blood and urine were found to be consistent with the results of the use of commercially available fluorometric assay kits to sense APN in serum and urine. Thus our approach shows promise as a point-of-care tool for cancer diagnostics and post-treatment surveillance of cancer reoccurrence.
天冬氨酰蛋白酶 N（APN/CD13）在肿瘤的生长和转移中起着重要作用，是各种恶性肿瘤治疗后监测癌症复发和进展的潜在生物标志物。因此，我们设计并制备了一种方便、灵敏的 APN 靶向活性比比率电化学分子底物（Ala-AFC），用于直接实时监测生物样品中的 APN 活性。实验中使用 APN 水解 Ala-AFC 探针中的丙氨酸部分，通过这种水解，实现级联反应，暴露出电化学报告分子 N-烷基化氨基二茂铁（AAF）。Ala-AFC 探针具有高灵敏度，检测范围为 0.05-110ng mL，检测限低至 23.18pg mL。电化学信号明显特异性地针对 APN，不受其他电活性生物物质的干扰。此外，该探针用于实时监测和定量肿瘤细胞、全血和尿液中的 APN 活性。此外，我们直接电化学定量全血和尿液中 APN 含量的结果与使用市售荧光测定试剂盒检测血清和尿液中 APN 的结果一致。因此，我们的方法有望成为癌症诊断和治疗后癌症复发监测的即时护理工具。
