Deprotonation-Driven Assembly of Copper Nanoclusters Boosts Electrochemiluminescence for Sensitive <i>N</i> -Acetyl-β- <scp>d</scp> -glucosaminidase Biosensing

The development of efficient and stable electrochemiluminescence (ECL) emitters remains a major challenge in practical sensing applications. Although copper nanoclusters (CuNCs) have attracted increasing attention due to their low cost and molecule-like electronic structures, their ECL activity is often limited by disordered aggregation and weak radiative emission. In this study, we report a deprotonation-driven interfacial assembly strategy for CuNCs, leading to a remarkable enhancement in the ECL performance. Using 4,6-diamino-2-mercaptopyrimidine (DAMP) as a multifunctional reductant and capping ligand, disordered aggregates (CuNCs_acid) formed under acidic conditions undergo structural evolution in response to pH, transforming into highly ordered nanosheets (CuNCs_base) in mildly alkaline media. This transformation is driven by the deprotonation of amino groups, which strengthens the interligand hydrogen bond networks and promotes π-π stacking, ultimately yielding compact structures enriched in Cu(I) species. The ordered assemblies effectively suppress nonradiative relaxation and lower the onset potential, leading to an ECL enhancement of nearly 3 orders of magnitude compared to that of CuNCs_acid. Benefiting from these features, a highly sensitive ECL biosensor for <i>N</i>-acetyl-β-d-glucosaminidase (NAG) detection in human urine was constructed employing CuNCs_base as emitters. The signal is generated by the enzyme catalytic production of <i>p</i>-nitrophenol (PNP), which perturbs the hydrogen-bond-directed assembly of CuNCs_base, causing an activity-dependent decrease in the ECL signal. By establishing a pH-modulated assembly strategy to boost ECL emission, this study opens new avenues for the rational design of high-performance, CuNC-based bioanalytical luminophores.