An array of femtoliter wells for sensitive detection of copper using click chemistry

Sensitive detection of copper ion (Cu²⁺), which is of great importance for environmental pollution and human health, is crucial. In this study, we present a highly sensitive method for measuring Cu²⁺ in an array of femtoliter wells. In brief, magnetic beads (MBs) modified with alkyne groups were bound to the azide groups of biotin-PEG3-azide (bio-PEG-N₃) via Cu⁺-catalyzed click chemistry. Cu⁺ in the click chemistry reaction was generated by reducing Cu²⁺ with sodium ascorbate. Following the ligation, the surface of the MBs was modified with biotin, which could be labeled with streptavidin-β-galactosidase (SβG). The MBs complex was then suspended in β-galactosidase substrate fluorescein-di-β-d-galactopyranoside (FDG), and loaded into the array of femtoliter wells. The MBs sank into the wells due to gravity, and the resulting fluorescent product, generated from the reaction between SβG on the surface of the MBs and FDG, was confined within the wells. The number of fluorescent wells increased with higher Cu²⁺ concentrations. The bright-field and fluorescent images of the wells were acquired using an inverted fluorescent microscope. The detection limit of this assay for Cu²⁺ was 1 nM without signal amplification, which was 10³ times lower than that of traditional fluorescence detection assays.