Development of a novel latent electrochemical molecular substrate for the real-time monitoring of the tumor marker aminopeptidase N in live cells, whole blood and urine

Sakthivel Kumaravel; Guo-Rong Luo; Sheng-Tung Huang; Hsin-Yi Lin; Chun-Mao Lin; Yu-Chieh Lee

doi:10.1016/j.bios.2022.114049

Development of a novel latent electrochemical molecular substrate for the real-time monitoring of the tumor marker aminopeptidase N in live cells, whole blood and urine

Biosens Bioelectron. 2022 May 1:203:114049. doi: 10.1016/j.bios.2022.114049. Epub 2022 Feb 3.

Authors

Sakthivel Kumaravel¹, Guo-Rong Luo², Sheng-Tung Huang³, Hsin-Yi Lin⁴, Chun-Mao Lin⁵, Yu-Chieh Lee⁶

Affiliations

¹ 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. Electronic address: kumaravelchemist@gmail.com.
² 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.
³ 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. Electronic address: ws75624@ntut.edu.tw.
⁴ Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, Taipei, 106, Taiwan.
⁵ Department of Biochemistry and Molecular Cell Biology, Taipei Medical University, Taipei, 110, Taiwan.
⁶ Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, 110, Taiwan.

PMID: 35134686
DOI: 10.1016/j.bios.2022.114049

Abstract

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^-1 and a low limit of detection of 23.18 pg mL^-1. 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.

Keywords: Aminopeptidase N; Electrochemical substrate; Tumor cells surface protein monitoring; Urinary APN; Whole blood assay.

MeSH terms

Biomarkers, Tumor
Biosensing Techniques*
Body Fluids*
CD13 Antigens
Humans
Neoplasms* / diagnosis

Substances

Biomarkers, Tumor
CD13 Antigens