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

Liu, Zhichao, Jing, Xia, Zhang, Sanjun, Tian, Yang
Analytical chemistry 2019 v.91 no.3 pp. 2488-2497
biocompatibility, calcium, cell death, copper, copper nanoparticles, cytotoxicity, detection limit, fluorescence, fluorescent dyes, formaldehyde, image analysis, ions, ligands, neurodegenerative diseases, neurons, polyethyleneimine, signal transduction, superoxide anion, water solubility
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.