se9b01574_si_001.pdf (1.6 MB)
Affimer–Enzyme–Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins
journal contribution
posted on 2019-10-16, 21:16 authored by Hope Adamson, Modupe O. Ajayi, Emma Campbell, Erika Brachi, Christian Tiede, Anna A. Tang, Thomas L. Adams, Robert Ford, Alex Davidson, Matt Johnson, Michael J. McPherson, Darren C. Tomlinson, Lars J. C. JeukenRobust
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.