期刊
ANALYTICAL CHEMISTRY
卷 91, 期 12, 页码 7631-7638出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.9b00445
关键词
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资金
- Australian Research Council's Centre of Excellence in BioNano Science [CE140100036]
- Monash University's Interdisciplinary Research Fund
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.
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