Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments

• Published in: Analytical chemistry (Washington) Vol. 91; no. 12; pp. 7631 - 7638
• Main Authors: Islam, Jiaul, Riley, Blake T, Fercher, Christian, Jones, Martina L, Buckle, Ashley M, Howard, Christopher B, Cox, Rosalind P, Bell, Toby D. M, Mahler, Stephen, Corrie, Simon R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.06.2019
• Subjects: Amino acids; Antibodies; Antigens; Assaying; Binding sites; Chemistry; Detection limits; Emission; Epidermal growth factor receptors; Fluorescence; Immunosensors; Microfluidics; Polarity; Solid phases; Variation; Wavelength
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b00445

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.