Bifunctional red-emitting copper-doped carbon dots as fluorescent probes and peroxidase nanozymes for dual-mode ampicillin detection with theoretical insights

Cu, B, N, S co-doped CDs with Cu–N₂O₂ sites were hydrothermally synthesized. • Cu-CDs function as both a red-emitting fluorophore and a peroxidase nanozyme in a single nanostructure. • The ·O 2 − generated by the nanozyme oxidizes TMB, quenching fluorescence and producing color. • AMP strongly adsorbs to the Cu site, scavenges ·O 2 − , and restores the fluorescence. • The dual-mode platform detects AMP at LODs of 0.78 μM (fluorescence) and 0.62 μM (colorimetry). Integrating optical signaling and enzymatic catalysis into a single nanostructure offers a promising route for biosensing. This work presents bifunctional copper-doped carbon dots (Cu-CDs) that serve simultaneously as red-emitting fluorescent probes (emission at 602 nm, quantum yield 15.84%) and as peroxidase-mimicking nanozymes for dual-mode detection of ampicillin (AMP). Hydrothermal treatment of 2,5-diaminotoluene sulfate and 3-aminophenylboronic acid with Cu 2+ produces spherical Cu-CDs (2.68 ± 0.15 nm) featuring well-defined Cu–N 2 O 2 coordination sites. Electron paramagnetic resonance spectroscopy and radical scavenging experiments confirm that the Cu–N 2 O 2 sites catalyze H 2 O 2 to generate of superoxide radicals (·O 2 − ), which oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to coloured oxTMB. The oxTMB absorbs strongly at 674 nm, spectrally overlapping with the Cu-CDs emission and quenching it through an inner filter effect-dominated static quenching mechanism. Density functional theory calculations reveal a catalytic cycle mediated by a Cu 2+ /Cu + redox couple and further demonstrate that AMP adsorbs much more strongly (−2.425 eV) than TMB (−1.578 eV) on the Cu–N 2 O 2 site. Consequently, AMP suppresses oxTMB formation not by homogeneous radical scavenging but through surface-adsorption-driven competitive inhibition, leading to fluorescence recovery and a decrease in absorbance at 674 nm. This dual-readout platform quantifies AMP over 1–150 μM (fluorometric, LOD 0.78 μM) and 1–80 μM (colorimetric, LOD 0.62 μM). It exhibits excellent selectivity against common ions, amino acids and various antibiotics, including structurally similar β-lactams (penicillin G, penicillin V, amoxicillin, cephalexin). Validation with real water samples and pharmaceutical preparations (ampicillin capsules and injections) gives recoveries of 90.40–109.20%. The integrated probe provides a robust self-validating strategy for environmental monitoring and pharmaceutical quality control. Download: Download high-res image (172KB) Download: Download full-size image