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.
Highlights
A general assay platform for rapid detection of diverse analytes is needed to underpin development of new point-of-care and in-field diagnostics
Approaches based on split enzyme complementation can suffer from low in vitro stability and limited activity recovery.[11−13] Target-driven modulation of bioluminescent resonant energy transfer (BRET) has been predominantly used for small molecule and antibody sensing.[14−20] Synthetic allosteric switches often give subtle, unpredictable signal changes, and so are hard to generalize for varied targets.[10,21,22]
We introduced restriction sites to enable simple sensor reconfiguration, and this modified between TEM1-β-lactamase (BLA)−BLIP anti-HA
Summary
A general assay platform for rapid detection of diverse analytes is needed to underpin development of new point-of-care and in-field diagnostics. We utilize Affimer affinity reagents, a class of non-immunoglobulin binding protein, based on a cystatin scaffold with two variable target binding regions.[31−33]. They are efficiently selected against diverse targets by phage display to provide high affinity binders with exquisite specificity, enabling timely development of new sensors.[32−34]
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