Affimer-enzyme-inhibitor switch sensor for rapid wash-free assays of multimeric proteins.

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, hCRP) and plant virus (cow pea mosaic virus, CPMV) 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 ELISA analysis confirms that the principle of an Affimer-enzyme-inhibitor switch provides a platform for point-of-care and in-field diagnostics. KEYWORDS: Protein switch, homogenous assay, wash-free diagnostics, protein engineering, Affimers, point-of-care, in-field, biosensor.

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