Regulating Glucose and pH, and Monitoring Oxygen in a Bioreactor

The oxygen-monitoring system is designed to satisfy special requirements for noninvasiveness, sterilizability, compactness, light weight, and nontoxicity to the cells nourished by the solution. The oxygen sensor exploits dynamic quenching of fluorescence by oxygen molecules. The sensor includes a capillary tube of inside diameter 2 mm, lined with a 0.1-mm-thick sensing layer of oxygen-sensitive fluorescent dye, and titanium oxide encapsulated in a gas-permeable, ion-impermeable silicone rubber. The sensing layer is overlaid with a black shielding layer of carbon black encapsulated in silicone rubber. The solution of interest flows through the tube, and oxygen from the solution permeates the silicone-rubber layers. The degree of permeation (and, hence, the rate of quenching of fluorescence) is reversible; that is, it varies along with the concentration of dissolved oxygen.

The dye is tris(4,7-diphenyl-1,10- phenanthroline)ruthenium(ll)chloride [Ru(dpp)3Cl2], which exhibits a high quantum yield (30 percent) of red (wavelength 626 nm) fluorescence with a lifetime of 5.34 ms after irradiation with blue light. A pulsed light-emitting diode (LED) of wavelength 465 nm is used as the radiation source. Operation in pulse mode minimizes the dye-bleaching effect that could occur if the LED were to irradiate the dye continuously during long-term monitoring. A longwavelength- pass filter is used to remove the blue LED light reflected and scattered by the irradiated capillary tube and its contents. The remaining light — predominantly the red fluorescence — is detected by a photodiode. Image

The pulsed output of the photodiode is digitized and processed to determine the concentration of dissolved oxygen in terms of the partial pressure of oxygen (pO2). This determination is made by use of the Stern-Volmer equation for the intensity and fluorescence lifetime of oxygen-quenched fluorescence of a luminescent dye:

I0/I= t0/t = 1 + Ksv(pO2),

where I0 and I are the intensities of fluorescence in the absence and presence of oxygen, respectively; t0 and t are the fluorescence-decay lifetimes in the absence and presence of oxygen, respectively; and Ksv (the Stern-Volmer constant) depends on details of the sensor design and construction. The sensor is calibrated by use of a phosphate-buffered saline solution containing a known elevated concentration of oxygen. The oxygen sensor was tested for over 180 days in a perfused rotating-wall bioreactor, using a single, initial calibration for the entire experiment.

This work was done by Melody M. Anderson and Neat R. Pellis of Johnson Space Center, and Antony S. Jeevarajan, Thomas D. Taylor, Yuanhang Xu, Frank Gao of Wyle Laboratories. For further information, contact the Johnson Innovative Technology Partnerships Office at (281) 483-3809.

This invention is owned by NASA, and a patent application has been filed. Inquiries concerning nonexclusive or exclusive license for its commercial development should be addressed to the Patent Counsel, Johnson Space Center, (281) 483-0837. Refer to MSC-23473/504/13/54.

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