Over the past 15-20 years, there has been growing interest and concern from the scientific and regulatory communities over the potential risks of emerging environmental contaminants (ECs). State-of-the-art techniques used for monitoring ECs do not provide the high spatial and temporal resolution measurements required to better understand and mitigate the risks. Immunoassays, which use antibodies to detect a target compound with high affinity, specificity and selectivity, partly address these limitations. However, the use of antibodies for the detection of small-sized, non-immunogenic 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, increased stability, and in vitro selection to ensure rapid discovery of binders against a wide range of targets. This study explores the potential of Affimers, a recent example of antibody mimetics, as suitable bioreceptors for the detection of small organic molecules. Methylene blue (MB), a redox-active molecule used as a fabric dye and diclofenac (DCF), an important environmental contaminant, were selected as the target compounds and Affimers against MB and DCF were developed by the BioScreening Technology Group, University of Leeds. The objectives of this project were to a) demonstrate that the developed Affimers can bind to the selected targets with very high affinity, b) assess their performance in the complexity of environmental water samples (selectivity), and c) investigate the potential of an Affimer-based assay for small molecule detection.
Target immobilisation for Affimer characterisation was achieved using long-chained alkanethiol linkers coupled with oligoethylene glycol (LCAT–OEG) and the immobilisation approach was evaluated through electrochemical measurements and infrared spectroscopy. Subsequently, binding between the immobilised targets and target-specific Affimers was quantified using quartz crystal microbalance with dissipation monitoring (QCM-D). Affimer affinity studies revealed Affimer dissociation constants (KD=13.7 nM and 73 nm for MB and DCF Affimers respectively) was comparable to that of high affinity antibodies. Furthermore, the high selectivity of MB-Affimers was demonstrated using limnetic water samples. Finally, an Affimer-based competition ELISA was demonstrated (LOD=75 nM), illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, non-immunogenic compounds. These findings are very promising, encouraging further research into Affimer-based assays and biosensors in order to introduce a novel, alternative path for rapid, on-site monitoring of contaminants in the environment.
