A protein-based oxygen biosensor for high-throughput monitoring of cell growth and cell viability Maria Strianese a,1 , Gerhild Zauner a , Armand W.J.W. Tepper a , Luigi Bubacco b , Eefjan Breukink c , Thijs J. Aartsma d , Gerard W. Canters a , Leandro C. Tabares a, * a Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands b Department of Biology, University of Padua, 30121 Padua, Italy c Department of Chemical Biology and Organic Chemistry, Utrecht University, 3584 CH Utrecht, The Netherlands d Leiden Institute of Physics, Leiden University, 2300 RA Leiden, The Netherlands article info Article history: Received 12 August 2008 Available online 21 November 2008 Keywords: Biosensors Oxygen sensing Cell viability Antibiotic screening FRET abstract Fluorescently labeled hemocyanin has been previously proposed as an oxygen sensor. In this study, we explored the efficacy of this biosensor for monitoring the biological oxygen consumption of bacteria and its use in testing bacterial cell growth and viability of Escherichia coli, Pseudomonas aeruginosa, Para- coccus denitrificans, and Staphylococcus simulans. Using a microwell plate, the time courses for the com- plete deoxygenation of samples with different initial concentrations of cells were obtained and the doubling times were extracted. The applicability of our fluorescence-based cell growth assay as an anti- bacterial drug screening method was also explored. The results provide a proof-of-principle for a simple, quantitative, and sensitive method for high-throughput monitoring of prokaryotic cell growth and anti- biotic susceptibility screening. Ó 2008 Elsevier Inc. All rights reserved. During the past decade, remarkable advances have been made in the design of methods for determining growth and viability of living cells. Measurements of the growth of microorganisms play a key role in a wide variety of applications such as development and testing of antibiotics, preservation or fermentation conditions of food [1,2], and optimization of the conditions for cell culture [3]. Cellular growth is typically measured by monitoring the opti- cal density of a culture at 600 nm. However, this method does not account for the number of viable cells present in the culture; rather, it accounts for the total number of cells or cell detritus. To circumvent this limitation, cell-counting methods, such as microscopy and plate counting, are normally used. Although these methods can easily discriminate viable cells, they are not suitable for fast high-throughput screening [4]. In particular, in the com- monly used plate-counting method, the time needed for the for- mation of visible colonies is relatively long and also lacks sensitivity [5]. A similar problem occurs with the widely used disk diffusion method for testing antibiotic sensitivity, which is also time-consuming and, in some cases, not very accurate [6]. Automated instruments, such as electric cell counters and flow cytometers, can partially alleviate these problems, but they re- quire not only sophisticated and expensive equipment but also significant technical expertise [7]. These reasons motivate the development of faster and larger scale methods to measure cell growth and viability. Monitoring cellular metabolic activity has been recently proposed as a valid alternative to the assessment of cell viability and growth [5,8]. Oxygen is one of the key sub- strates in aerobic systems; therefore, the ability to consume oxy- gen is a good indicator of cellular metabolic activity and so can be correlated to cell viability and growth. Different methods for oxy- gen detection have been reported, and for a long time the Clark- type electrode has been considered as the method of choice for monitoring the respiration of cells [9]. During the past few years, new methods based on fluores- cence-detected oxygen consumption have been developed for use in high-throughput systems. The BD BioSensor, commercial- ized by BD Bioscience, was the first reported example. This method uses 96- or 384-well plates containing a ruthenium- based fluorescent compound immobilized in an oxygen-perme- able silicon matrix [8]. In the presence of oxygen, the fluores- cence of the ruthenium compound is quenched; thus, an increase in the fluorescence signal indicates oxygen depletion. This system has been shown to be suitable for determining growth and viability of a variety of bacteria, mammalian cells, and fungi [8]. Phosphorescent porphyrin dyes [10,11] and a plat- inum complex [5] embedded in polymers have also been re- ported as oxygen-sensing materials. These studies have evolved 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.11.017 * Corresponding author. Fax: +31 715274349. E-mail address: lctabares@chem.leidenuniv.nl (L.C. Tabares). 1 Current address: Department of Chemistry, University of Salerno, 84081A Fisciamo, Salerno, Italy. Analytical Biochemistry 385 (2009) 242–248 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio