We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

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Shake flasks are ubiquitous in cell culture and fermentation. However, conventional devices for measuring oxygen concentrations are impractical in these systems. Thus, there is no definitive information on the oxygen supply of growing cells. Here we report the noninvasive, nonintrusive monitoring of dissolved oxygen (DO) in shake flasks using a low-cost optical sensor. The oxygen-sensitive element is a thin, luminescent patch affixed to the inside bottom of the flask. The sensitivity and accuracy of this device is maximal up to 60% DO, within the range that is critical to cell culture applications. By measuring actual oxygen levels every 1 or 5 min throughout the course of yeast and E. coli fermentations, we found that a modest increase in shaker speed and a decrease in culture volume slowed the onset of oxygen limitation and reduced its duration. This is the first time that in situ oxygen limitation is reported in shake flasks. The same data is unattainable with a Clark type electrode because the presence of the intrusive probe itself changes the actual conditions. Available fiber optic oxygen sensors require cumbersome external connections and recalibration when autoclaved.

An optical assay for glucose is described based on the luminescence decay time of a long wavelength dye (Cy5) which can be excited with currently available red laser diodes. Concanavalin A was covalently labeled with Cy5 which served as the donor in an assay based on fluorescence resonance energy transfer (FRET). The acceptor was Malachite Green which was covalently linked to insulin which served as a carrier protein. To provide binding affinity for ConA Malachite Green insulin was also covalently labeled with maltose (MIMG). Binding of Cy5ConA to MIMG resulted in a decreased intensity and decay time of Cy5 as observed by time-correlated single photon counting. Glucose was detected by competitive displacement of MIMG from Cy5ConA, resulting in increased intensity and decay time. This glucose assay has several features which can result in practical real world assays for glucose. The long absorption wavelength of Cy5 allows excitation with red laser diodes, which can be readily pulsed or amplitude-modulated for time-domain or frequency-domain decay time measurements. Additionally, decay times can be measured through skin using long wavelength excitation and emission, suggesting the possibility of an implanted glucose sensor. And finally, the assay affinity and reversibility can in principle be adjusted by controlling the extent and type of sugar labeling of the carrier protein.

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.