Novel potentiometric sensors based on polysulfone immobilized metallothioneins as metal-ionophores

We show here the use of immobilized metal-binding biomolecules for metal analysis by using novel potentiometric sensors. To this end and as a model, Ag+-ISEs were developed using polysulfone matrix embedding metallothioneins as ionophores (mouse MT1 (P1) or sea urchin SpMTA (P2)). Polysulfone, a porous polymer that was not used until the present in potentiometric biosensors, has the advantage of being compatible with biological materials. Also, the phase inversion procedure allows protein incorporation into the membrane with minima alterations, since it always remains in the aqueous phase. Construction of these biosensors required small amounts of protein; they can be dry-stored and have long lifetimes. They exhibited linear responses with slopes of ca. 61 mV per decade within the 10−5 to 10−2 M Ag+ concentration range, detection limits of about 10−5 M, and worked in the 2-to-8 pH range. Except for Hg2+, the Pb2+, Zn2+, Cd2+, Cu2+ cations do not interfere with Ag+ determination. Significantly, different affinities of Pb2+ and Zn2+ towards P1- and P2-ISE were found, in good correlation with the higher affinity of these cations towards SpMTA than to MT1. Consequently, the distinct metal-binding features of each MT are conserved and determine the differential properties of their biosensors. These results open a broad range of possibilities for the use of proteins as ionophores in what could be considered a new type of potentiometric biosensor if their response mechanism is taken into account.