Journal
ACS SENSORS
Volume 4, Issue 11, Pages 3014-3022Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.9b01574
Keywords
protein switch; homogenous assay; wash free diagnostics; protein engineering; Affimers; point-of-care; in-field; biosensor
Funding
- Medical Research Council, MRC [MR/N029976/1]
- MRC [MR/N029976/1] Funding Source: UKRI
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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 TEMI-beta-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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