Herein, we report on fluorescent probes which enable the selective quantification of Na+ and K+ in water by fluorescence enhancement (FE) independent of the pH value. These fluorescent probes, so called fluoroionophores, consist of benzo‐crown‐ether derivatives as ionophores, a benzo‐15‐crown‐5 (cf. 5) or a benzo‐18‐crown‐6 (cf. 6), respectively, to selectively bind either Na+ or K+ and a 4,4‐difluoro‐1,3,5,7‐tetramethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) fluorophore. The fluoroionophore 5 shows a FE factor of 7.3 in the presence of 1000 mM Na+ and 6 of 4.7 upon addition of 250 mM K+ in an acidic environment. The dissociation constants (Kd) for 5+Na+ is 276 mM and for 6+K+ is 18 mM enabling the fluorometric determination of biological relevant Na+ or K+ levels. The fluorescence intensities of 5 and 6 were not impacted over a broad range of proton concentrations (pH stable from 3.5 to 9.5) and by the presence of other biologically relevant cations.

Herein, we represent cation‐responsive fluorescent probes for the divalent cations Zn2+, Mg2+ and Ca2+, which show cation‐induced fluorescence enhancements (FE) in water. The Zn2+‐responsive probes Zn1, Zn2, Zn3 and Zn4 are based on o‐aminoanisole‐N,N‐diacetic acid (AADA) derivatives and show in the presence of Zn2+ FE factors of 11.4, 13.9, 6.1 and 8.2, respectively. Most of all, Zn1 and Zn2 show higher Zn2+ induced FE than the regioisomeric triazole linked fluorescent probes Zn3 and Zn4, respectively. In this set, ZN2 is the most suitable probe to detect extracellular Zn2+ levels. For the Mg2+‐responsive fluorescent probes Mg1, Mg2 and Mg3 based on o‐aminophenol‐N,N,O‐triacetic acid (APTRA) derivatives, we also found that the regioisomeric linkage influences the fluorescence responds towards Mg2+ (Mg1+100 mM Mg2+ (FEF=13.2) and Mg3+100 mM Mg2+ (FEF=2.1)). Mg2 shows the highest Mg2+‐induced FE by a factor of 25.7 and an appropriate Kd value of 3 mM to measure intracellular Mg2+ levels. Further, the Ca2+‐responsive fluorescent probes Ca1 and Ca2 equipped with a 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid (BAPTA) derivative show high Ca2+‐induced FEs (Ca1 (FEF=22.1) and Ca2 (FEF=23.0)). Herein, only Ca1 (Kd=313 nM) is a suitable Ca2+ fluorescent indicator to determine intracellular Ca2+ levels.