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

Abstract

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 μM, 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 O₂^•--induced cytoplasmic Ca²⁺ burst and O₂^•--induced neuronal death possibly resulted from Ca²⁺ overload in the neurons.