Threonine Phosphorylation of an Electrochemical Peptide-Based Sensor to Achieve Improved Uranyl Ion Binding Affinity

We have successfully designed a uranyl ion (U(VI)-specific peptide and used it in the fabrication of an electrochemical sensor. The 12-amino acid peptide sequence, (n) DKDGDGYIpTAAE (c), originates from calmodulin, a Ca(II)-binding protein, and contains a phosphothreonine that enhances the sequence's affinity for U(VI) over Ca(II). The sensing mechanism of this U(VI) sensor is similar to other electrochemical peptide-based sensors, which relies on the change in the flexibility of the peptide probe upon interacting with the target. The sensor was systematically characterized using alternating current voltammetry (ACV) and cyclic voltammetry. Its limit of detection was 50 nM, which is lower than the United States Environmental Protection Agency maximum contaminant level for uranium. The signal saturation time was ~40 min. In addition, it showed minimal cross-reactivity when tested against nine different metal ions, including Ca(II), Mg(II), Pb(II), Hg(II), Cu(II), Fe(II), Zn(II), Cd(II), and Cr(VI). Its reusability and ability to function in diluted aquifer and drinking water samples were further confirmed and validated. The response of the sensor fabricated with the same peptide sequence but with a nonphosphorylated threonine was also analyzed, substantiating the positive effects of threonine phosphorylation on U(VI) binding. This study places emphasis on strategic utilization of non-standard amino acids in the design of metal ion-chelating peptides, which will further diversify the types of peptide recognition elements available for metal ion sensing applications.