Toward Microbioreactor Arrays: A Slow-Responding Oxygen Sensor for Monitoring of Microbial Cultures in Standard 96-Well Plates (original) (raw)
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Biotechnology and Bioengineering, 2003
Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k L a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k L a values in one commercial reader were about 10 to 40 h −1. k L a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k L a values in a 96-well microtiter plate. The resulting values were about 130 h −1 , which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h −1. Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.
Biotechnology and Bioengineering, 2004
For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were F 5.1 mm Hg and À 3.8 mm Hg at the bioreactor outlet, and F 19 mm Hg and À 18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.
A novel integrated optical dissolved oxygen sensor for cell culture and micro total analysis systems
Technical Digest. MEMS 2002 IEEE International Conference. Fifteenth IEEE International Conference on Micro Electro Mechanical Systems (Cat. No.02CH37266), 2002
Production of accurate microscale oxygen sensors has been conventionally attempted using amperometric methods, which prove to be difficult to miniaturize, mainly due to the requirement of a bulky reference electrode. This paper details the design and fabrication process for an optically based oxygen sensor using an oxygen-sensitive fluorescent dye combined with microfabricated Cytop1 waveguides that are monolithically integrated into the
Journal of Microbiological Methods, 2006
High-throughput screening (HTS) assays based upon fluorometric detection of oxygen consumption in microtiter plates were primarily developed for applications in drug discovery and ecotoxicology but have recently been adopted for use in microbial community-level physiological profiling assays (CLPP). The widespread use of oxygen biosensor systems for CLPP applications has, however, been hindered by the relatively high cost of oxygen biosensor reagent systems and limited access to microplate fluorometer instrumentation platforms. The ability to recycle and reuse oxygen biosensor system plates would expand their utilization for CLPP assays and other research applications in microbial ecology. Here, the efficacy and cost effectiveness of multiple procedures for sterilization of Oxygen Biosensor System™ (OBS; BD Biosciences) plates for reuse was evaluated. OBS plates were sterilized using ethylene oxide, ultraviolet radiation, and bleach treatments, then evaluated for biosensor response and plate life-cycle performance. Of the sterilization methods tested, ethylene oxide sterilization was most effective based on its low cost, high sterilization efficacy, and minimal impact upon OBS plate response.
2018
The quantity of organic pollutants present in wastewater is classically evaluated by measuring the quantity of dissolved oxygen during five days; it is quantified by the so called BOD5 parameter (Biological Oxygen Demand) [1]. This work constitutes the first step of an overall strategy targeting to improve the monitoring of BOD5. We focus on the development of a microsystem approach allowing monitoring the O2 consumption induced by the biodegradation process of organic matter. To evaluate the organic pollutants concentration, we use Escherichia coli as bacterial indicator, confined in a PDMS-glass chip. Their metabolic activity in presence of organic molecules is deduced from their oxygen consumption. These measurements are ensured by optical sensors present in each of the five instrumented chambers of the chip. The results show that the microsystem approach is suitable to measure simultaneously different concentrations of organic load, and that it is possible to reduce the analysis...
Phase fluorometric sterilizable optical oxygen sensor
Biotechnology and Bioengineering, 1994
We report here on a low-cost, optical oxygen sensor as an attractive alternative to the widely used amperometric Clark-type oxygen electrode for measuring dissolved oxygen tensions in cell cultures and bioreactor. Our sensor is based on the defferential quenching of the fluorescence lifetime of chromophore in response to the partial pressure of oxygen. This is measured as a phase shift in fluorescence emission from the chromophore due to oxygen quenching when excited by an intensity modulated beam of light. In this article we demonstrate the advantages of lifetime-based optical methods over both intensity based optical methods and amperometric electrodes. Our sensor is particularly suitable for measuring dissolved oxygen in bioreactors. It is autoclavable, is free of maintenance requirements, and solvents the problems of long-term stability, calibration drifts, and reliable measurement of low oxygen tensions in dense microbial cultures that limit the utility of Clark-type elcectordes. © 1994 John Wiley & Sons, Inc.
Innovative Food Science & Emerging Technologies, 2005
An instrument was fabricated to measure the real-time response of a bioluminescent construct of Escherichia coli to ozone treatment. The bioluminescent output from E. coli inoculated on nutrient agar plates was measured with a photomultiplier tube coupled to a lock-in amplifier and the ozone concentration was calculated from absorption measurements from a 254 nm source. Data from the two measurement systems were acquired in real-time via an oscilloscope, captured to a PC for analysis and displayed on the graphical user interface. The ozone and bioluminescence measurements were done in two different chambers to isolate the measurement systems and to prevent any adverse effects of the UV radiation on the bacteria. The real-time data showed the instantaneous changes in the bioluminescence output with increasing ozone concentration. The dark current from plastic petri dishes both with and without agar was measured and a peak to peak variation of 0.10 dB in the signal was observed; this was negligible compared to the bioluminescent signal. D Industrial relevance: A real time monitoring system for microbial viability would be of relevance for pathogenic and spoilage microorganisms as well as probiotics. This paper demonstrates the potential to use a bioluminiscent microorganism to evaluate the effectiveness of decontamination systems and possibly leading to their optimization.
Biochemical oxygen demand sensor arrays
2015
Biochemical oxygen demand (BOD) is one of the most widely utilized parameters in water quality evaluation. BOD as a parameter illustrates the amount of organic compounds susceptible to biochemical degradation in the water. The BOD test lasts for at least 5 -7 days or even up to 21 days. An incubation time this long is not acceptable for monitoring purposes or system control. In order to shorten the BOD measurement time, a multitude of biosensors have been proposed. Unfortunately, BOD biosensors have several limitations, such as short lifetime, limited substrate range, precision etc. Some of those limitations can be overcome by using microbial sensor-arrays. Such bioelectronic tongues can achieve the much wider substrate range usually attributed to multiculture sensors and still maintain the long lifetime of a single culture sensor. This is achieved by separating different cultures from each other in the array and using the signals of separate sensors to produce summarised informatio...