Design and applications of methods for fluorescence detection of iron in biological systems

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.