DESIGNED PROTEIN PORES AS COMPONENTS FOR BIOSENSORS

Background: There is a pressing need for new sensors that can detect a variety of analytes, ranging from simple ions to complex compounds an d even microorganisms. The devices should offer sensitivity, speed, re versibility and selectivity. Given these criteria, protein pores, remo deled so that their transmembrane conductances are modulated by the as sociation of specific analytes, are excellent prospects as components of biosensors. Results: Structure-based design and a separation method that employs targeted chemical modification have been used to obtain a heteromeric form of the bacterial pore-forming protein staphylococca l a-hemolysin, in which one of the seven subunits contains a binding s ite for a divalent metal ion, M(II), which serves as a prototypic anal yte. The single-channel current of the heteromer in planar bilayers is modulated by nanomolar Zn(II), Other M(II)s modulate the current and produce characteristic signatures. In addition, heteromers containing more than one mutant subunit exhibit distinct responses to M(II)s, Hen ce, a large collection of responsive pores can be generated through su bunit diversity and combinatorial assembly. Conclusions: Engineered po res have several advantages as potential sensor elements: sensitivity is in the nanomolar range; analyte binding is rapid (diffusion limited in some cases) and reversible; strictly selective binding is not requ ired because single-channel recordings are rich in information; and fo r a particular analyte, the dissociation rate constant, the extent of channel block and the voltage-dependence of these parameters are disti nguishing, while the frequency of partial channel block reflects the a nalyte concentration, A single sensor element might, therefore, be use d to quantitate more than one analyte at once, The approach described here can be generalized for additional analytes.