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

Fluorescent calcium ion (Ca2+) sensing and imaging have become an essential technique for investigation of signaling pathways of Ca2+ and understanding the role of Ca2+ in neurodegenerative disease. Herein a copper nanocluster (CuNC)-based ratiometric fluorescent probe was developed for real-time sensing and imaging of Ca2+ in neurons, in which a specific Ca2+ 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 Ca2+ concentration in the range of 2-350 mu M, and a detection limit down to 220 +/- 11 nM was achieved. In addition, the response time of the present probe for Ca2+ 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 Ca2+ increase in various parts of neurons was different. Moreover, it was found O-2(center dot-)-induced cytoplasmic Ca2+ burst and O-2(center dot-)-induced neuronal death possibly resulted from Ca2+ overload in the neurons.