High-Affinity Binding of Cadmium Ions by Mouse Metallothionein Prompting the Design of a Reversed-Displacement Protein-Based Fluorescence Biosensor for Cadmium Detection

Abstract

Reported in this study are the experimental design and results of a protein-based biosensor for the detection of the cadmium in water using a reversed-displacement format. This reversed-displacement biosensor methodology has successfully measured cadmium in water by direct injection, eliminating the need for preconcentration or pretreatment of samples. A column containing Chelex resin saturated with Zn2+ and a rodhamine-labeled metallothionein (MT) comprised the assay reactive chamber. In fact, MT are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper, and mercury. Since the affinity for cadmium is higher than zinc and mercury, we used this intrinsic feature of MT to develop a fluorescence biosensor. The rodhamine-labeled MT was incubated with the Zn2+-saturated Chelex resin until binding equilibrium was reached. Under a constant flow, samples containing cadmium were introduced into the flow stream displacing the rodhamine-labeled MT. Limits of detection were lower than 0.5 microM for cadmium in water. Importantly, the addition of 1.0 microM Cu2+, 1.0 microM Zn2+, 1.0 microM Mg2+, or 1.0 microM Ca2+ did not cause the displacement of the rodhamine-labeled MT, indicating that the presence of these ions do not affect the specificity of the biosensor. Furthermore, we also demonstrated that the reversed-displacement format can be used to screen water samples containing cadmium, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.