Single molecular multianalyte (Ca²⁺, Mg²⁺) fluorescent probe and applications to bioimaging

Intracellular signal transduction relies on spatial and temporal signal transmitter dynamics. To clarify the correlations of these transmitter molecules, multicolor-imaging has been widely used. However, in the case of applying multiple indicators in a cell, spectral overlap of the indicators prevents accurate quantitative analysis. Moreover, the invasive (toxic) effect, the localization, the metabolism, as well as photobleaching of these indicators complicate the situation. Here, we show that single-molecular multifluorescent probes can overcome these problems. While intracellular calcium plays a critical role as a signal transmitter and magnesium acts as a cofactor in many situations, the correlations between the two cations are now the main issue. We designed and synthesized a Ca²⁺-Mg²⁺ responsive multifluorescent probe, KCM-1. KCM-1 shows a spectral blue shift upon complexation to Ca²⁺ and a red shift to the presence of Mg²⁺. With data analyzed at different excitation wavelengths, the concentrations of Ca²⁺ and Mg²⁺ are simultaneously quantified. Furthermore, by using the AM-ester method, intracellular Ca²⁺ and Mg²⁺ concentrations are simultaneously imaged. Such a type of intracellular multiple analyte imaging by a single-molecular multifluorescent probe is successfully demonstrated for the first time.