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We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC50 values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.

A novel method utilizing the BD Oxygen Biosensor System has been developed to rapidly, simply, and accurately determine the growth rate of microorganisms in broth, with no needfor plate counts, standardized inocula, or technically difficult manipulations. The BD Oxygen Biosensor System incorporates an oxygen-sensitive material into the wells of standard Falcon microplates. The time response of this sensor monitored in afluorescence plate reader can be used to quantitate microbe growth. The method entails seeding a dilution series of microorganism onto the plate and reading at regular intervals for 3-10 h. As the organisms grow and consume oxygen, thefluorescence intensities increase over time to form a family of sigmoidal growth curves. A simple mathematical analysis of the time intervals between the curves yields the doubling time, which is independent of the initial concentration of organism. The method is ideally suited as a screening tool for assessing the impact of culture conditions, media composition, or added compounds on growth kinetics.

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