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Abstract
Conventional immunoassays rely on antibodies that provide high affinity, specificity and selectivity against a target analyte. However, the use of antibodies for the detection of small-sized, non-immunogenic targets, such as pharmaceuticals and environmental contaminants presents a number of challenges. Recent advances in protein engineering have led to the emergence of antibody mimetics that offer the high affinity and specificity associated with antibodies but with reduced batch-to- batch variability, high stability and in vitro selection to ensure rapid discovery of binders against a wide range of targets. In this work we explore the potential of Affimers, a recent example of antibody mimetics, as suitable bio-receptors for the detection of small organic target compounds, here methylene blue. Target immobilisation for Affimer characterisation was achieved using long-chained alkanethiol linkers coupled with oligoethyleneglycol (LCAT-OEG). Using quartz crystal microbalance with dissipation monitoring (QCM-D), we determine the affinity constant, KD , of the methylene blue Affimer to be comparable to that of antibodies. Further, we demonstrate the high selectivity of Affimers for its target in complex matrices, here a limnetic sample. Finally, we demonstrate an Affimer-based competition assay, illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, non-immunogenic compounds.
Original language | English |
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Pages (from-to) | 3051-3058 |
Number of pages | 8 |
Journal | Analytical Chemistry |
Volume | 89 |
Issue number | 5 |
Early online date | 1 Feb 2017 |
DOIs | |
Publication status | Published - 7 Mar 2017 |
Bibliographical note
© 2017, American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details.Profiles
Projects
- 1 Finished
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Cutting Edge Approaches for Pollution Assessment in Cities
Boxall, A. B. A. (Principal investigator), Bate, I. J. (Co-investigator), Burns, C. J. (Co-investigator), Carslaw, N. (Co-investigator), Chesmore, D. (Co-investigator), Cowling, P. I. (Co-investigator), Johnson, S. D. (Co-investigator), Lewis, A. (Co-investigator), Reed, D. J. (Co-investigator), Thomas-Oates, J. E. (Co-investigator) & Timmis, J. (Co-investigator)
1/10/13 → 30/09/17
Project: Research project (funded) › Research