Deprotonation-Driven Assembly of Copper Nanoclusters Boosts Electrochemiluminescence for Sensitive N -Acetyl-β- d -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 N -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 p -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.