Adamson, H, Ajayi, MO, Campbell, E orcid.org/0000-0001-9564-9408 et al. (10 more authors) (2019) Affimer–Enzyme–Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins. ACS Sensors, 4 (11). pp. 3014-3022. ISSN 2379-3694
Abstract
Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an “active” enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme–inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-β-lactamase and its inhibitor protein, such that target binding disrupts the enzyme–inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer–enzyme–inhibitor switch provides a platform for point-of-care and in-field diagnostics.
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Copyright, Publisher and Additional Information: | © 2019 American Chemical Society. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. | ||||
Keywords: | Protein switch; homogenous assay; wash-free diagnostics; protein engineering; Affimers; point-of-care; in-field; biosensor | ||||
Dates: |
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Institution: | The University of Leeds | ||||
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Synthetic Biology (Leed) |
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Depositing User: | Symplectic Publications | ||||
Date Deposited: | 16 Oct 2019 13:23 | ||||
Last Modified: | 07 Jun 2023 14:00 | ||||
Status: | Published | ||||
Publisher: | American Chemical Society | ||||
Identification Number: | https://doi.org/10.1021/acssensors.9b01574 | ||||
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