Studies of metal ion binding by apo-metallothioneins attached onto preformed self-assembled monolayers using a highly sensitive surface plasmon resonance spectrometer.

The use of a flow-injection surface plasmon resonance (FI-SPR) spectrometer equipped with a bicell detector or a position-sensitive device for determining coordination of heavy metal ions (Cd(2+) and Hg(2+)) by surface-confined apo-metallothionein (apo-MT) molecules is described. To facilitate the formation of a compact MT adsorbate layer with a uniform surface orientation, MT molecules were attached onto a preformed alkanethiol self-assembled monolayer. The method resorts to the generation of apo-MT at the surface by treating the MT-covered sensor chip with glycine-HCl and the measurement of the apo-MT conformation changes upon metal ion incorporation. Domain-specific metal ion binding processes by the apo-MT molecules were observed. Competitive replacement of one metal ion by another can be monitored in real time by FI-SPR. The tandem use of an immobilization scheme for forming a sub-monolayer of MT molecules at the sensor surface and the highly sensitive FI-SPR instrument affords a low concentration detection level. The detection level for Cd(2+) (0.1 μM or 15 ppb) compares favorably with similar studies and the methodology complements to other well-established sensitive analytical techniques. The extent of metal incorporation by apo-MT molecules was also determined.