Origin of Red Emission in Protein-Stabilized Copper Nanoparticles: Evidence for Cu ^I -Metallothionein-Like Cluster Formation

Nanoparticles (<b>NP</b>) produced in or added to biological milieu will spontaneously form a shell composed of biomolecules, most commonly proteins, around the <b>NP</b> core. The explicit interaction of the protein shell with the <b>NP</b> core remains poorly resolved, particularly for <b>NP</b> based on metals essential to life. Red emissive copper nanoclusters (<b>RCuNC</b>) serve as a synthetic model for the Cu⁰ <b>NP</b>-protein interface, and have been developed as biocompatible sensors, though the mechanism underlying their red emission is still unclear. Herein, we identify that the red emission from <b>RCuNC</b> does not originate from the Cu⁰ <b>NP</b> but instead from previously unidentified Cu^I-metallothionine (<b>MT</b>)-like clusters. Emission decay measurements, Cu^I-quantification assays, native polyacrylamide gel electrophoresis imaging experiments, and direct protein metalation with Cu^I identify at least two distinct populations of Cu that form during the reduction of Cu^II in the presence of proteins. Our findings reveal that approximately 47% of the total Cu in the as-prepared bovine serum albumin-stabilized <b>RCuNC</b> is present as Cu^I. Our results underscore the need for the scrutiny of the assignment of emitting species in copper-treated protein samples prepared under reducing conditions, while revealing the opportunity for the development of protein-based sensors with red-emitting embedded Cu^I-<b>MT</b>-like clusters.