Affimer-Enzyme-Inhibitor Switch Sensor for Rapid Wash-free Assays of Multimeric Proteins

Area of Science:

• Biotechnology
• Biosensor Technology
• Molecular Diagnostics

Background:

• Rapid point-of-care and in-field diagnostics are crucial for timely decision-making across various sectors.
• Current diagnostic strategies often lack broad applicability for diverse targets.
• Enzyme-switch sensors offer a promising approach by directly linking target binding to signal generation.

Purpose of the Study:

• To develop a broadly applicable enzyme-inhibitor switch sensor platform.
• To demonstrate the sensor's utility for quantifying diverse multimeric analytes.
• To validate the sensor's performance for point-of-care and in-field diagnostic applications.

Main Methods:

• Engineered an enzyme-inhibitor switch by inserting Affimer binding proteins between TEM1-β-lactamase and its inhibitor.
• Designed the switch architecture so that target binding disrupts the enzyme-inhibitor complex.
• Quantified multimeric target analytes in biological samples (serum, plasma, leaf extracts).

Main Results:

• Achieved rapid (minutes), simple (wash-free), and sensitive (picomolar) quantification of targets.
• Successfully targeted a therapeutic antibody (Herceptin), a protein biomarker (human C-reactive protein), and a plant virus (cow pea mosaic virus).
• Demonstrated applications in therapeutic drug monitoring, health diagnostics, and plant pathogen detection.

Conclusions:

• The Affimer-enzyme-inhibitor switch provides a robust platform for point-of-care and in-field diagnostics.
• The sensor exhibits batch-to-batch reproducibility and shelf-life stability.
• Assay performance is consistent with validated enzyme-linked immunosorbent assay (ELISA) methods.