Multicolor Upconversion Nanoprobes Based on a Dual Luminescence Resonance Energy Transfer Assay for Simultaneous Detection and Bioimaging of [Ca2+]i and pHi in Living Cells

Graphical By taking advantage of a lack of spectral overlapping and single NIR excitation wavelengths, sandwich-structured multicolor upconversion nanoparticles (SWUCNPs) were used to establish a dual luminescence resonance energy transfer (LRET) nanoprobe with special recognition ligands. The quenched fluorescence would be recovered after the detachment and/or absorbance change of the attached ligands (see scheme) to investigate the interplaying roles of [Ca 2+ ] i and pH i . Intracellular [Ca 2+ ] i and pH i have a close relationship, and their abnormal levels can result in cell dysfunction and accompanying diseases. Thus, simultaneous determination of [Ca 2+ ] i and pH i can more accurately investigate complex biological processes in an integrated platform. Herein, multicolor upconversion nanoparticles (UCNPs) were prepared with the advantages of no spectral overlapping, single NIR excitation wavelengths, and greater tissue penetration depth. The upconversion nanoprobes were easily prepared by the attachment of two fluorescent dyes, Fluo-4 and SNARF-4F. Based on the dual luminescence resonance energy transfer (LRET) process, the blue and green fluorescence of the UCNPs were specially quenched and selectively recovered after the detachment and/or absorbance change of the attached fluorescent dyes, enabling dual detection. Importantly, the developed nanoprobe could successfully be applied for the detection of [Ca 2+ ] i and pH i change in adenosine triphosphate (ATP) and ethylene glycol tetraacetic acid (EGTA) stimulation in living cells.