Single Probe for Imaging and Biosensing of pH, Cu²⁺ Ions, and pH/Cu²⁺ in Live Cells with Ratiometric Fluorescence Signals

Abstract

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu(2+), to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu(2+), and pH/Cu(2+) with different ratiometric fluorescent signals. First, 2,2',2″-(2,2',2″-nitrilotris(ethane-2,1-diyl)tris((pyridin-2-yl-methyl)azanediyl))triethanethiol (TPAASH) was designed for specific recognition of Cu(2+), as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu(2+), and pH/Cu(2+) excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu(2+) against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic-organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu(2+) and in understanding the interplaying roles of Cu(2+) and pH in live cells by ratiometric multicolor fluorescent imaging.

Fluorescent nanoprobes for sensing and imaging of metal ions: Recent advances and future perspectives

“…Moreover, the whole-cell permeability of the complexes and the preserved ion-selective FRET in cells make these complexes effective for multicolor intracellular sensing of Hg 2+ . Very recently, Han et al [139] developed a single fluorescent probe based on the combination of AuNCs, FITC, and 7-diethylaminocoumarin-3-carboxylic acid with ratiometric fluorescence signals. This NC-based nanoprobe showed high selectivity, sensitivity, and accuracy for Cu 2+ , and was successfully applied in intracellular imaging and sensing of pH and Cu 2+ , as well as used in ROS regulation of pH and Cu 2+ changes in macrophage cells.…”

Electronic structure engineering and biomedical applications of low energy-excited persistent luminescence nanoparticles

“…76 Up to now, scientists have already developed several uorescent probes for uorophotometry, all of which play crucial roles in selective biodetection. [71][72][73][74][75][77][78][79] However, in complex biological environments, these probes are typically affected by strong background uorescence generated by biological substrates. 80 Due to the need of sustained excitation, it will signicantly reduce the sensitivity and limit of detection.…”

Luminescent Metal Nanoclusters for Potential Chemosensor Applications

“…Biswas et al employed Au NCs containing polymeric microcapsules to investigate the intracellular ratiometric fluorescence biosensing response to hydrogen peroxide [80]. As in the case of organic fluorescent probes, Au NC-based probes also acted as pH-sensitive probes in chemo-and bio-sensor investigations [81,82]. For example, Wu et al reported a dual-emission fluorescent probe consisting of BSA-Au NCs and fluorescein-5-isothiocyanate (FITC) as temperature and pH sensors [83].…”