Intrinsically photoluminescent hydrogels to measure peptides‑copper binding affinities

NH₂ decorated intrinsically photoluminescent hydrogels (IPH-NH₂) were functionalized with the addition of various peptides via EDC/NHS coupling method. These peptidic devices bind copper with binding affinities depending on surface functionalization. Particularly, fluorescence analysis of copper titrations, alongside the determination of quenching efficiency and lifetime measurements, allowed to assess binding constants and to elucidate the underlying binding mechanism. Various peptides, having the same copper binding amino acidic residues (GHK) but different chain lengths, were tested and it was found that increasing the distance of the GHK sequence from the IPH-NH₂ surface resulted in a decrease in the binding constant, as well as a reduction in quenching efficiency, whereas the binding mechanism remained unchanged as indicated by lifetime measurements. This method not only provides binding constants for peptides immobilized on biosensor surfaces or pre-fabricated devices without altering their structure, but also contributes to the optimization of biosensor design, tailoring it to its intended application.