Extracellular Cu2+ Pools And Their Detection: From Current Knowledge To Next-Generation Probes

Copper (Cu) is an essential micronutrient for most organisms and serves mainly as a redox-active catalytic centre in enzymes cycling between Cu+ and Cu2+. Membrane transporters and shuttles are involved to bring and insert Cu into these enzymes and to control tightly the copper metabolism, whose failure can lead to severe diseases. The main oxidation state intracellularly is Cu+, whereas Cu2+ is mainly found in extracellular fluids. A basic approach to investigate Cu metabolism in vivo and in cellulo contemplates the use of luminescent (mostly fluorescent) or magnetic resonance imaging (MRI)-active probes. Here, we focus on sensors of the Cu2+ state. First, Cu metabolism and speciation are revised, focusing on the main extracellular fluids (blood plasma, urine, cerebrospinal fluid, synaptic cleft, milk, saliva, sweat) and cell culture media, and then highlighting the notion of exchangeable Cu2+ pool. Indeed, in contrast to bulk Cu measurements, sensors can only detect Cu2+ that is labile and thermodynamically accessible. Thus, the kinetics and thermodynamics of the exchangeable pools determine the quantity of Cu2+ that can be measured and influence the design of potential sensors. The study of the best-known exchangeable Cu2+ pool in blood plasma, i.e. serum albumin, shows that a sensor might need a sub-femtomolar affinity for Cu2+ to compete with endogenous Cu2+ ligands. The selectivity of the probe for Cu2+ is also discussed, in particular against Zn2+, which is much more available than Cu2+ in extracellular fluids (e.g. at least 10(6) times in the blood). Finally, the analysis of the literature on luminescent and MRI-active Cu2+ sensors applied in extracellular media shows indeed how challenging such measurement is, and that none of the sensors reported to date convincingly and specifically detects Cu2+ in a biological system. Indeed, when considering all the sought parameters, i.e. thermodynamics and kinetics of the Cu2+-sensor, the specificity towards Cu2+, the reversibility, the sensitivity of the luminescent or MRI response and hence the required sensor concentration, it becomes clear that this is a huge challenge and that we stand just at the dawn of this field. (C) 2020 Elsevier B.V. All rights reserved.