Self-reporting Cu clusterzyme acting as an ultra-small luminescent laccase for the catalytic detection of neurotransmitter epinephrine.

Mimicking the structure of natural laccase, we designed and synthesized a Cu-clusterzyme with Cu atom as the catalytic center, histidine and cysteine as the recognition unit and auxiliary fluorescence unit, respectively (denoted as Cys-His-CuNCs). Cys-His-CuNCs integrate the dual properties of laccase-like catalysis and fluorescence-sensing, and can simultaneously catalyze and detect the neurotransmitter epinephrine (EP). Cys-His-CuNCs exhibit a 44-fold higher catalytic activity than laccase. Enhanced fluorescence of Cys-His-CuNCs caused by EP reaction can be used to detect as low as 1 nM EP in urine, with a linear concentration range of 1-20 nM, The limit of detection of EP by a visual colorimetry is 10 nM. The accuracy of this method was verified by the classical LC-MS analysis. The calculations using density functional theory (DFT) reveals the molecular mechanism of EP oxidation. The valence-state transition of Cu in Cys-His-CuNCs was found to play an important role in its catalysis for the reaction of EP. The Cu0/Cu2+ redox pair initiates the oxidation of phenolic hydroxyl groups of EP, generating intermediates such as adrenochrome and adrenoquinone that eventually convert into fluorescent product 3, 4-dihydroxyphenethylacetone (3,4-DHAP). The increased Cu+ and fluorescent products contribute to the fluorescence enhancement of Cys-His-CuNCs. This work demonstrates the potential of Cu-clusterzymes acting as low-cost, ultra-small versatile nanozymes for visual biosensing in real-time.