Highly sensitive glucose monitoring has potential applications in conditions where the glucose levels are below the detection limit of currently available technology. Examples include bioprocess monitoring of bacterial cultures and measurement of minute amounts of human interstitial fluid extracted by iontophoresis. Here we describe a ratiometric glucose sensor capable of measuring micromolar levels of glucose. This sensor is based on an E. coli glucose binding protein (GBP) labeled with two fluorophores. The L255C mutant of GBP was labeled with the environment-sensitive fluorophore, acrylodan, at the cysteine mutation and a long-lived metal ligand complex of ruthenium (ruthenium bis(2,2'-bipyridyl)-1, 10-phenanthroline-9-isothiocyanate) at the N-terminal. The acrylodan emission is quenched in the presence of glucose while the ruthenium emission remained constant, thereby serving as a reference. The sensitivity of the sensor is in the micromolar range (K(d) = 0.4-1.4 microM). Thus, it is possible to measure glucose concentrations at micromolar levels and higher (with dilution). Calculations of the fluorescence energy-transfer efficiency between acrylodan and ruthenium gave an approximate distance of 25 A between the two fluorophores, consistent with X-ray crystallographic data. The effect of temperature on glucose binding was measured and analyzed. Maximum signal changes and apparent binding constants increase with temperature. The enthalpy change for glucose binding as calculated from the apparent binding constants is approximately 43.1 kJ/mol. In addition to ratiometric measurements, the presence of the long-lived ruthenium metal ligand complex allows for low-cost modulation-based sensing.