Tehran University of Medical Sciences Acta Biochimica Iranica 0001-5261 1 3 2023 12 01 139 144 Hamidesadat Enayati Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran. Javad Mohammadnejad Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran. Alireza Nikfarjam MEMS and NEMS Laboratory, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran 2023 12 21 Objectives: The aim of this study was to design an enzyme immunoassay based on a modified ELISA with high sensitivity for detecting digoxin. Methods: The first step in development involved the conjugation of digoxin to the HRP (Horse Radish Peroxidase) enzyme via sodium metaperiodate oxidation. Surface modification, and thus assay modification, was achieved by the covalent immobilization of an anti-digoxin monoclonal antibody on a functional plate using 3-ATPES (3-aminopropyltriethoxysilane). Results: The developed ELISA demonstrated superior sensitivity (0.026μg/ml) and lower variability in measurements repeated throughout a day, compared to a conventional ELISA (0.051μg/ml). This assay detected the exact amount of digoxin. The sensitivity and specificity of the modified ELISA surpassed other methods, and measurements were performed within a few hours.  Conclusion: An efficient ELISA kit was produced, characterized by its affordability, ease of learning, and absence of a hand-washing step.   https://abi.tums.ac.ir/index.php/abi/article/view/52 https://abi.tums.ac.ir/index.php/abi/article/download/52/27