Wavelength-dependent fluorescent immunosensors via incorporation of polarity indicators near the binding interface of antibody fragments

Jiaul Islam, Blake T. Riley, Christian Fercher, Martina L. Jones, Ashley M. Buckle, Christopher B. Howard, Rosalind P. Cox, Toby D.M. Bell, Stephen Mahler, Simon R. Corrie

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Abstract

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.

Original languageEnglish
Pages (from-to)7631-7638
Number of pages8
JournalAnalytical Chemistry
Volume91
Issue number12
DOIs
Publication statusPublished - 18 Jun 2019

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