A Copper Nanocluster-Based Fluorescent Probe for Real-Time Imaging and Ratiometric Biosensing of Calcium Ions in Neurons

Fluorescent calcium ion (Ca ²⁺ ) sensing and imaging have become an essential technique for investigation of signaling pathways of Ca ²⁺ and understanding the role of Ca ²⁺ in neurodegenerative disease. Herein a copper nanocluster (CuNC)-based ratiometric fluorescent probe was developed for real-time sensing and imaging of Ca ²⁺ in neurons, in which a specific Ca ²⁺ ligand with two formaldehyde groups was synthesized and further conjugated with polyethylenimine (PEI) to form a new ligand molecule for the synthesis of CuNCs. Meanwhile, water-soluble Alex Fluor 660 NHS ester was immobilized onto CuNCs as a reference element. The developed ratiometric fluorescence nanoprobe demonstrated a good linearity with Ca ²⁺ concentration in the range of 2-350 μM, and a detection limit down to 220 ± 11 nM was achieved. In addition, the response time of the present probe for Ca ²⁺ was found to be less than 2 s with good stability and high selectivity. Taking advantage of the low cytotoxicity and good biocompatibility of the developed nanoprobe, it was discovered that the histamine-induced cytoplasmic Ca ²⁺ increase in various parts of neurons was different. Moreover, it was found O ₂ ^- -induced cytoplasmic Ca ²⁺ burst and O ₂ ^- -induced neuronal death possibly resulted from Ca ²⁺ overload in the neurons.