Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn2+ and Ca2+ in cells

Abstract

Fluorescent dyes are widely used in the detection of labile (free or exchangeable) Zn²⁺ and Ca²⁺ in living cells. However, their specificity over other cations and selectivity for detection of labile vs. protein-bound metal in cells remains unclear. We characterized these important properties for commonly used Zn²⁺ and Ca²⁺ dyes in a cellular environment. By tracing the fluorescence emission signal along with UV-Vis and size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) in tandem, we demonstrated that among the dyes used for Zn²⁺, Zinpyr-1 fluoresces in the low molecular mass (LMM) region containing labile Zn²⁺, but also fluoresces in different molecular mass regions where zinc ion is detected. However, FluoZin™-3 AM, Newport Green™ DCF and Zinquin ethyl ester display weak fluorescence, lack of metal specificity and respond strongly in the high molecular mass (HMM) region. Four Ca²⁺ dyes were studied in an unperturbed cellular environment, and two of these were tested for binding behavior under an intracellular Ca²⁺ release stimulus. A majority of Ca²⁺ was in the labile form as tested by SEC-ICP-MS, but the fluorescence from Calcium Green-1™ AM, Oregon Green® 488 BAPTA-1, Fura red™ AM and Fluo-4 NW dyes in cells did not correspond to free Ca²⁺ detection. Instead, the dyes showed non-specific fluorescence in the mid- and high-molecular mass regions containing Zn, Fe and Cu. Proteomic analysis of one of the commonly seen fluorescing regions showed the possibility for some dyes to recognize Zn and Cu bound to metallothionein 2. These studies indicate that Zn²⁺ and Ca²⁺ binding dyes manifest fluorescence responses that are not unique to recognition of labile metals and bind other metals, leading to suboptimal specificity and selectivity.