A new type of nanopore sensor design is reported for a reagent-less electrochemical biosensor with no analyte “tagging” by fluorescent molecules, nanoparticles, or other species. This sensor design involves immobilization within Au-coated polycarbonate nanopores of bacterial periplasmic binding proteins (bPBP), which undergo a wide amplitude hinge-twist motion upon ligand binding. Ligand binding thus triggers a reduction in the effective thickness of the immobilized protein film, which is detected as an increase in electrolyte conductivity (decrease in impedance) through the nanopores.

This new sensor design is demonstrated for glucose detection using a cysteine-tagged mutant (GGR Q26C) of the galactose/glucose receptor (GGR) protein from the bPBP family. The GGR Q26C protein is immobilized onto Au nano-islands that are deposited within the pores of commercially available nanoporous polycarbonate membranes.

Results are presented for glucose concentrations ranging from 40-200 ìM and for pore diameters of 10, 30 and 100 nm. As expected, the sensitivity to glucose is reduced at concentrations much greater than the equilibrium ligand binding constant, about 0.2 ìM for the soluble form. The binding constant can be increased into the physiological range through residue substitution near the ligand binding pocket. The greatest sensitivity to glucose is found for the smallest pore diameter (10 nm).